©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Novel Proteins of the Phosphotransferase System Encoded within the rpoN Operon of Escherichia coli
ENZYME IIA AFFECTS GROWTH ON ORGANIC NITROGEN AND THE CONDITIONAL LETHALITY OF AN era MUTANT (*)

(Received for publication, September 7, 1994)

Bradford S. Powell (1) (2) Donald L. Court (1)(§) Toshifumi Inada (2)(¶) Yoshikazu Nakamura (2) Valerie Michotey Xuewen Cui Aiala Reizer Milton H. Saier Jr. Jonathan Reizer

From the  (1)Laboratory of Chromosome Biology, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201 and the (2)Department of Tumor Biology, The Institute of Medical Science, The University of Tokyo, P. O. Takanawa 108, Japan (3)Department of Biology, University of California at San Diego, La Jolla, California 92093-0116

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two rpoN-linked DeltaTn10-kan insertions suppress the conditionally lethal era allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by era. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIA and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIA (ptsN) and NPr (npr). Purified IIA and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate:sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIA to the unknown process regulated by the essential GTPase Era.


INTRODUCTION

A connection between nitrogen and carbon utilization has been recognized for over 25 years (Ullmann et al., 1969; Contesse et al., 1969), yet the molecular mechanisms linking these two assimilatory processes remain poorly defined. Recent computational studies led to the proposal that proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (^1)may provide such a link (Reizer et al., 1992a; Saier and Reizer, 1994). Specifically, a gene coding for a suspected PTS protein, now designated IIA (nitrogen-related Enzyme IIA), was identified within the operon containing rpoN, encoding the alternate sigma factor ^N of RNA polymerase. The work reported here supports the proposal that the PTS participates in nitrogen control by characterizing IIA and a second PTS protein encoded within the rpoN operon designated NPr (nitrogen-related HPr). We also provide evidence for a functional connection between these new PTS constituents and growth regulation by the Era protein.

The PTS mediates the uptake and concomitant phosphorylation of many carbohydrates in bacteria (for review see Postma et al., 1993; Saier and Reizer, 1994). Several phosphoryl transfer proteins catalyze the relay of phosphate from phosphoenolpyruvate (PEP) to an incoming sugar, and some proteins of the PTS contain more than one phosphoryl-transfer domain. Proteins containing these domains are functionally classified into two groups. Enzyme I and HPr comprise the soluble energy-coupling PTS proteins and function to transfer phosphate from PEP to the sugar-specific phosphoryl carrier proteins, the Enzyme II complexes that are localized to the inner membrane. These complexes consist of three (or four) proteins or domains designated IIA, IIB, IIC (and sometimes IID) (Saier and Reizer, 1992, 1994), with phosphate being passed sequentially from PEP to Enzyme I, HPr, IIA, IIB, and finally to the incoming sugar which is transported across the membrane via IIC. Each Enzyme II translocates a specific sugar or subset of sugar substrates. HPr and Enzyme IIA are also central regulatory proteins. Enzyme IIA of the Escherichia coli glucose-specific PTS (IIA) participates in global regulation of carbohydrate transport through the processes of inducer exclusion and catabolite repression (Saier, 1989). In Gram-positive bacteria this central regulatory role is fulfilled by HPr which becomes phosphorylated on a seryl residue (Ser) by an ATP-dependent, metabolite-activated kinase (Reizer et al., 1993a; Ye et al., 1994a, 1994b; Deutscher et al., 1994). Thus, the PTS plays a key regulatory role with respect to carbon metabolism in both Gram-negative and Gram-positive bacteria.

^N, the generalized name for the product of the rpoN gene (Merrick, 1993; Ishihama, 1993), is central to nitrogen metabolism because it is required for transcription of genes needed for nitrogen assimilation and fixation. ^N binds to a class of promoters characterized by the minimal core recognition sequence GG(-24)/GC(-12), and with the assistance of an enhancer protein that is specific for each regulon (e.g. NtrC), it recruits RNA polymerase for transcriptional initiation (for reviews see: Dixon, 1984; Kustu et al., 1986; Magasanik and Neidhardt, 1987; Merrick, 1988; Kustu et al., 1989; Magasanik, 1982, 1993). With respect to nitrogen regulation, many studies have revealed a hierarchy of sensory and regulatory proteins whose cascade of reversible protein modifications converge on the enhancer protein to control its net state of phosphorylation and hence its capacity to activate ^N-initiated transcription. To date, ^N-dependent regulons have been identified that encode more than 50 proteins affecting diverse physiological functions including nitrogen assimilation, nitrogen fixation, adaptation of cellular respiration to anaerobiosis, use of unusual carbon sources, photosynthesis, developmental switches, and adjustments to symbiotic and virulent growth. The mechanisms of ^N-dependent gene regulation are best understood for the control of glutamine synthetase and other nitrogen assimilatory pathways, but even within this purview, recent evidence for an alternate mode of NtrC activation (Schneider et al., 1991; McCleary et al., 1993) shows that ^N-related physiological regulation is not yet fully understood.

The era gene of E. coli encodes a GTP-binding protein with relatively high GTPase activity (Chen et al., 1990). Several investigations have established that Era is essential for cell growth (Inada et al., 1989; Takiff et al., 1989; Lerner and Inouye, 1991; March et al., 1992), but its cellular role remains enigmatic. To identify a physiological system in which Era may function, a temperature-sensitive allele of the era gene was isolated and characterized (Inada et al., 1989; Inada, 1992) and was employed for selection of secondary site suppressor mutants. This paper reports the discovery and characterization of a class of era suppressor genes that map within the rpoN operon. The data presented herein suggest a relationship between processes affected by Era and the new PTS protein IIA encoded within the rpoN operon. The first genetic interconnection between Era and another multiprotein cellular system is thus established. In addition, we provide evidence suggesting that IIA acts to regulate utilization of poor nitrogen sources by a novel but as yet unknown mechanism.


EXPERIMENTAL PROCEDURES

Bacterial Strains, Bacteriophage, and Plasmids

Relevant bacterial strains, bacteriophages, and plasmids used in this study are listed in Table 1, and some are also diagramed in Fig. 1. All in vivo tests employed lacZ strains derived from the E. coli K-12 prototroph W3110, except where specifically stated. The W3110 derivative BSP301 was used for era tests, and the derivative WJW45 was used for all betagalactosidase assays and defined nutrient plating tests. Many intermediate stages of plasmid cloning, phage isolation, and in vivo recombinations employed strain MC4100, while routine plasmid DNA propogation employed strain DH5alpha. The heat-sensitive allele of era, a double mutant previously designated era era::DeltaTn10 (Inada et al., 1989) and herein referred to as era, was transferred to strains by P1 transduction selecting for tetracycline resistance. The two suppressors of era called ersB1::kan and ersB2::kan were transferred into strains via P1 transduction with selection for their kanamycin resistance (Km^R) markers. All in vitro and in vivo DNA manipulations with plasmids and bacteriophages and P1 employed standard published procedures (Sambrook et al., 1989; Miller, 1992) unless stated otherwise.




Figure 1: Physical and genetic maps and clones of the rpoN region of E. coli.A, restriction enzyme sites are displayed below the physical map with current coordinate values (Rudd, 1992) shown above. Position 3362 kb (71.95 min) corresponds to 70.2 min on the old map (Bachmann, 1990). This map corrects the Kohara map (Kohara et al., 1987) for the number and relative positions of sites for PstI, PvuII, and KpnI as descibed in the text. B, map of the region determined by nucleotide sequence and drawn to scale with the physical map in A. The left end of the sequence corresponds to the Sau3AI site of Kohara 523 cloned into the BamHI site of the phage (Kohara et al., 1987). The approximate locations of two promoters as discussed in the text are indicated (P1 and P2). Open boxes represent the extent of genes with names designated as discussed in the text. Bold vertical arrowheads mark the locations of insertions for DeltaTn10::kan transposons (ersB1 and ersB2) and kanamycin cassette (npr::kan). C, the extent of DNA contained on several rpoN region clones is indicated by solid lines. All end points bordering the left hand edge stop at the EcoRI site within Kohara clone 523 and contain E. coli bacterial DNA extending clockwise from the Sau3AI site. pBP2 extends to the KpnI site, and pIT149 extends to the following PvuII site with right hand end points indicated by parentheses. Parentheses in clone pBP131 indicate the sequence deleted between the BglII and HindIII sites. pBP25 extends rightward to an unsequenced PstI site. Bold arrowheads indicate the presence of the corresponding DeltaTn10::kan transposons or portions thereof.



All lysogenic strain sets used for biological tests were made in the following sequential manner: 1) the parental strain was infected with purified phage, and lysogens were isolated; 2) these lysogenic strains were screened for single copy number and proper integration of prophage as described below; and 3) other chromosomal genetic markers were introduced by P1 transduction with selection for the newly introduced antibiotic resistance. Strains carrying the glnA-lacZ fusion prophages gln101 and gln105 (gifts from L. Reitzer; Backman et al., 1981; Schneider et al., 1991), used for assaying activities of the full glnAp1p2 and minimal glnAp2 promoters, were constructed similarly to ensure that all strains were otherwise isogenic.

Aliquots of the Kohara set of phages (National Institute of Genetics, Mishima, Japan) were amplified for storage and subsequent cloning using E. coli strain C600 as host. Recombinant derivatives of Kohara phages carrying the Km^R markers of ersB1 and ersB2 were generated in vivo by marker rescue from the host chromosome using the procedure given below. derivatives of plasmid-borne lacZ fusions used for transcriptional analyses, and complementation tests were constructed by recombination into the vector BDC531 as described below.

Plasmids pBP25 and pIT141 were derived from phage 523ersB2::kan and 523ersB1::kan by cloning PstI or BamHI fragments, respectively ( Table 1and Fig. 1), and both were used for sequencing the DeltaTn10 insertion junctions. All other plasmids used for structural and functional analyses were derived from Kohara phage 523 DNA subcloned on plasmids pBP2 or pIT149, whose construction employed the EcoRI and KpnI sites, and the EcoRI and PvuII (partial digest) sites, respectively (Fig. 1). pIT167 is an HincII to BamHI subclone into pUC119, and pBP120 is an HincII to PvuI subclone into pUC18 which expresses only ptsN from the lac promoter. Plasmid pBP130 expressing rpoN was derived from pBP124 (see below) by cutting with Bsu36I, and then filling in recessed ends with Klenow before ligation. Plasmid pBP131 expressing ptsN was made similarly but using BglII and HindIII to delete a 0.6-kb internal fragment from rpoN. Both of these plasmids retain the natural rpoN promoter and most probably also express orf95. Fusions of rpoN region fragments to the lacZ reporter gene were constructed using the operon fusion vector pRS415 (Simons et al., 1987). As illustrated in Fig. 1, three separate constructs placed lacZ either at the BamHI site within rpoN (pBP125) or at the second BamHI site within orf284 (pBP123 and pBP124). Plasmid pBP124 carries bacterial sequences including complete genes for orf185, orf251, rpoN, orf95, and ptsN. Plasmid pBP123 contains the internal 2.4-kb BamHI fragment which carries complete sequences for only the orf95 and ptsN genes. Plasmids pJRNtr and pJRNPr for overexpression of proteins IIA and NPr from ptsN and npr, respectively, are described in detail below.

Disruptions of the npr Gene or the Entire rpoN Operon by Marker Exchange

The npr gene was disrupted by in vivo marker exchange using the E. coli strain ATCC47002 (JC7623) essentially as described by Balbas et al.(1993) except for the selection of Km^R donated from plasmid pBP126 which carries the npr gene having a kanamycin cassette inserted into its NsiI site. The resultant npr::kan strain was used for preparation of P1 lysate used for subsequent transfer into experimental strains. A complete rpoN operon deletion mutation was made similarly using plasmid pBP127 which carries a kanamycin cassette substitution of the 2.15-kb NsiI fragment overlapping all five genes between rpoN and npr. Plasmids pBP126 and pBP127 were not used elsewhere for biological testing and are not listed in Table 1.

Bacteriological Media and Phenotypic Plating Tests

Nitrogen-free phosphate-buffered medium ``W'' was used for plate tests of nitrogen regulation essentially as described (Smith et al., 1971). It was modified as described herein for tests of nitrogen-limiting carbon repression. W salts medium contains per liter: 10.5 g of K(2)HPO(4), 4.5 g of KH(2)PO(4), and 0.241 ml of 1 M MgSO(4). For preparation of plates, Bacto-agar (1% w/v, final concentration) was autoclaved separately in water before adding W salts plus nitrogen and/or carbon sources from filter-sterilized concentrated stock solutions prior to pouring plates. To test for the glutamine control (Gln phenotype), W salts medium containing glucose and glutamine, each at 0.2% (w/v, final concentration) was used for the nitrogen-limiting condition. (NH(4))(2)SO(4) was added to this medium to a final concentration of 0.4% for the nitrogen excess condition as prescribed (Smith et al., 1971). To test for the second tier of nitrogen regulation (Ntr phenotype), glutamine in W salts glucose medium was replaced by other poor sources of nitrogen such as individual amino acids or the nucleoside, adenosine. Growth tests were also performed simultaneously on similar medium but lacking glucose and/or ammonium sulfate. Nitrogen-limiting carbon repression tests were routinely performed on W salts + alanine (0.2%) plates with other carbon substrates, such as sugars or intermediates of the tricarboxylic acid cycle, added either during plate preparation or afterwards by spreading them onto the plates from stock solutions (2-20%). The final concentrations of these supplements were 0.2-0.4%, and if introduced by spreading the plates were left at room temperature for 12 h before use in order to allow for uniform diffusion of supplements. Amino acids, carbon substrates, and other chemicals (Aldrich, Sigma) were adjusted to pH 7 if necessary (e.g. for arginine HCl) before filter sterilization. All tests for nitrogen and carbon source utilization were performed at 32 °C. Colony size and morphology was monitored every 6-12 h over a period of 3-7 days. Complementation tests of era heat sensitivity employed LB medium as reported previously (Inada et al., 1989) or low salt LB medium which contains per liter: 10 g of food grade agar (Taito Kaiso Products Co., Tokyo, Japan), 2.5 g of NaCl (Wako Pure Chemical Inductries LTD., Tokyo, Japan), 10 g of Bacto Tryptone (Difco), and 1 g of Yeast Extract (Difco). Tests were performed simultaneously at 32, 37, 40, and 42 °C, monitoring colony appearance over a period of 3 days. Antibiotics including sodium ampicillin (100 µg/ml), tetracycline (25 µg/ml), and kanamycin sulfate (50 µg/ml) were used at 2-4-fold lower concentrations when drug-resistant genes were present in single copy number.

Marker Rescue onto Kohara Phages

The DeltaTn10-kan transposons of ersB1::kan and ersB2::kan were transferred from the bacterial chromosome onto Kohara phages via homologous recombination by selecting Km^R transductants of W3110() using standard procedures. For isolation of recombinant phage, mixed lysates prepared from kanamycin-resistant multi-lysogens were titered on W3110, from which clear plaques (Kohara phage are cI-) were purified and rescreened to confirm kanamycin resistance. These purified plaques were then used to prepare lysate stocks for sequence analysis and storage.

Transfer of lacZ Fusions from Plasmid to -Phage

Plasmid-borne operon fusions to lacZ were recombined onto vectors for use in single copy number as follows. Strains containing the plasmids of interest were infected with BDC531 by spotting 15 µl of lysate onto a lawn of the host strain. During phage propagation, rare double cross-over events occur between the phage and plasmid that replace the ``stuffer'' supF marker of the phage with all DNA from the plasmid contained between bla (Ap^R) and lacZ. These recombinant phage were identified as blue plaques from the mixed lysates by titering onto a LacZ host in the presence of the indicator dye 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Recombinant lysogens were isolated by plating cells from the centers of blue plaques onto LB ampicillin, and spontaneous lysates from these cells were prepared from cleared supernatants of LB cultures grown to saturation. After a second round of blue plaque purification, stock lysates were prepared for storage and further use. Derivatives of these phage were made by infecting the -borne lacZ fusions into each of the ersB1::kan and ersB2::kan hosts and directly selecting for recombinant lysogens which had picked up the ersB marker by plating on LB kanamycin. The recombinant phage were isolated from the purified lysogen as described above, plaque purified, and amplified on a strain containing its respective ersB1::kan or ersB2::kan chromosomal marker to avoid the appearance of wild-type recombinants.

Confirmation of Single Copy Integration by PCR

During strain constructions, single copy number integrants were confirmed by PCR using three primers which abut the bacterial and prophage attachment sites: 1) a bottom strand primer within the int gene: 5`-actcgtcgcgaaccgctttc-3`; 2) a top strand primer to the left of attP: 5`-tttaatatattgatatttatatcattttacgtttctcgttc-3`; and 3) a top strand primer to the left of E. coli attB: 5`-gaggtaccagcgcggtttgatc-3`. Freshly grown small sized (<1-mm diameter) colonies were suspended in 100 µl of water, washed once, and used without delay. Reactions were performed with 10 µl of cell suspension using 200 mM nucleotide triphosphates and an equimolar mix (250 nM each) of all three primers in a final volume of 50 µl. Single integrants produced a single fragment of 501 base pairs ( int and attB primers nos. 1 and 3) while multiple integrants produced this fragment plus one of 379 base pairs ( int and attP primers nos. 1 and 2; data not shown). In this assay the 379-base pair fragment also appears weakly as an artifact in older cultures of single lysogens because of the spontaneous release of free phage.

DNA Sequencing

Plasmids and phage diagramed in Fig. 1were the sources of mutant and wild-type DNA for nucleotide sequence determination. To find a point of orientation near the transposon insertions, sequencing commenced on the ersB1::kan subclone pIT141 and the ersB2::kan subclone pBP25 using universal and reverse pUC primers. Additional primers were designed and employed progressively during the generation of new sequence data since no corresponding E. coli sequence flanking rpoN was then available in the EMBL or Genbank databases. Nucleotide sequences were determined using temperature cyclers (Atto Industries, Tokyo, Japan and Cetus Perkin-Elmer) and cycle sequencing modifications of the dideoxy-chain termination method as prescribed by the manufacturers of the reagents (Life Technologies, Inc., Promega, Madison, WI, Applied Biosystems, Inc., Thousand Oaks, CA). The entire sequence of both top and bottom strands was determined and is contained in the Genbank database under accession number U12684. Oligonucleotide primers were synthesized on an Applied Biosystems DNA Synthesizer (Applied Biosystems, Inc., Thousand Oaks, CA) and were either end labeled with [-P]ATP (Amersham) or used directly for the fluorescent incorporation reaction (Applied Biosystems, Inc., Thousand Oaks, CA).

Cloning of ptsN and Purification of the IIA Protein

A DNA fragment containing the gene encoding IIA (ptsN) was amplified by PCR using plasmid pMM18 (gift from M. Merrick) as template DNA. The top strand primer contained a KpnI site followed by an NdeI site within the initiation codon (underlined) of ptsN, 5`-atcttaggtacccatatgacaaataa-3`. The bottom strand primer was complementary to the 5`-region of orf284 and contained a SalI site, 5`-acgtccgtcgacgatcatcag-3`. Amplification was performed in a Hybaid thermal reactor (Hybaid Ltd., Teddigton, Middlesex, United Kingdom) in a reaction mixture consisting of 10 mM Tris-HCl buffer (pH 8.3), 50 mM potassium chloride, 1.5 mM MgCl(2), 0.01% (w/v) gelatin, each of the deoxynucleoside triphosphates at a concentration of 195 µM, each of the primers at a concentration of 1 µM, 5 ng of the template DNA, and 2.5 units of AmpliTaq DNA polymerase in a total volume of 50 µl. The amplification mixture was overlaid with 50 µl of mineral oil and subjected to 20 cycles of amplifications as follows: samples were incubated for 1 min at 94 °C to denature the DNA and for 1 min at 30, 35, 40, 45, and 55 °C, to anneal and extend the annealed primers. The PCR-amplified DNA was ligated to pCRII (Invitrogen, San Diego, CA), and the NdeI-SalI fragment encompassing the complete ptsN gene was then excised from this plasmid and cloned between the NdeI and SalI sites of the overexpression vector pRE1 (Reddy et al. 1989) to create plasmid pJRNtr.

IIA of E. coli was purified from crude extracts of E. coli MZ1/pJRNtr using SDS-PAGE to monitor the isolation of the IIA protein (17.9 kDa) during column chromatography. Cells were grown, collected by centrifugation, washed, and then ruptured as described previously (Reizer et al., 1989, 1992b). A crude extract derived from cells harvested from 12 liters of a logarithmic culture was dialyzed against (TDP buffer) 20 mM Tris-HCl buffer (pH 7.2) containing 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride and then loaded onto a DEAE-Sephacel column (2.6 cm times 13 cm; 50-ml bed volume) which was pre-equilibrated with the same buffer. The column was washed with 200 ml of TDP buffer, and proteins were then eluted using a linear salt concentration gradient (1000 ml; 0-0.5 M NaCl) in TDP buffer. Fractions (10 ml) were collected and assayed for IIA using SDS-PAGE. IIA eluted at about 0.1 M NaCl. Fractions containing IIA were pooled and concentrated using ultrafiltration with an Amicon YM-10 filter. The concentrated material was loaded onto a column of Sephadex G-75 (3.6 cm times 85 cm) pre-equilibrated with 0.1 M NaCl in TDP buffer. Proteins were eluted with the same buffer, and fractions (5.2 ml) containing IIA were pooled. This purification protocol yielded nearly homogeneous preparations of IIA as estimated by SDS-PAGE. Some preparations employed a third step to eliminate minor protein contaminants. This step included dialysis against TDP buffer and purification with a second DEAE-Sephacel column (10-ml bed volume). The column was washed with 50 ml of TDP buffer, and proteins were eluted using a linear salt concentration gradient (400 ml, 0-0.2 M NaCl). IIA eluted in fractions (4 ml each) containing 0.07-0.12 M NaCl and was shown to be more than 95% pure based on SDS- and nondenaturing- PAGE (see below).

Cloning of the npr Gene and Purification of the NPr Protein

A DNA fragment containing the NPr encoding gene (npr) was amplified by PCR using the phage 7E3(522) (Kohara et al., 1987) as the template DNA and the following two oligonucleotide primers: 1) 5`-ggaaaaaggtaccatatgaccgtcaagc-3`, a 5` primer containing a KpnI site followed by an NdeI site in the initiation codon (underlined) of npr; and 2) 5`-gtcaaagtcgacaagattaatcttcatc-3`, a 3` primer introducing a novel SalI site four nucleotides downstream of the translational stop codon of npr (orf90). The amplification reaction was performed for 20 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min. The amplified DNA was ligated to pCRI I, excised from this plasmid using NdeI and SalI, and then cloned between the NdeI and SalI sites of the overexpression vector pRE1 (Reddy et al., 1989) to generate pJRNPr.

NPr of E. coli was purified from crude extracts of E. coli MZ1/pJRNPr with initial cell harvesting and lysis as described for IIA above. A crude extract from 12 liters of a logarithmic culture was dialyzed against 20 mM Tris-HCl buffer (pH 7.2) containing 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol (TDPG buffer) and then loaded onto a column of DEAE-Sephacel (2.6 cm times 13 cm, 50-ml bed volume) which had been pre-equilibrated with the same buffer. The column was washed with 200 ml of TDPG buffer, and proteins were then eluted with a linear salt concentration gradient (1 liter, 0-0.5 M NaCl) in TDPG. Fractions (10 ml) were collected and assayed by SDS-PAGE for the presence of NPr which eluted at about 0.2 M NaCl. Fractions containing NPr were pooled, and the proteins were precipitated using 80% ammonium sulfate. The precipitated proteins were then dissolved in TDPG buffer and dialyzed (3-4 h) against the same buffer. In more recent purification protocols, proteins were concentrated by ultrafiltration (Amicon Corp.; YM-2 filter) rather than by ammonium sulfate. The concentrated material was loaded onto a Sephadex G-50 (superfine) column (2 times 85 cm, 270-ml bed volume) pre-equilibrated with 0.1 mM NaCl in TDPG buffer, and proteins were eluted with the same buffer. Fractions (2.5 ml) were collected, and those containing NPr (fraction 56-68) were pooled. This two-step purification protocol yielded a nearly homogeneous preparation (>95% pure) of NPr as determined by nondenaturing and SDS-PAGE (see below). In some cases, a third purification step was employed to eliminate minor protein contaminants. This step included dialysis against TDPG buffer followed by fractionation on a DEAE-Sephacel column (1.4 cm times 8.5 cm, 10-ml bed volume) which was developed with a salt concentration gradient (400 ml, 0-0.35 M NaCl) in TDPG.

Purification of Enzyme I, HPr, and Enzyme IIA Proteins

Enzyme I and HPr of E. coli and HPr and Enzyme IIA of Bacillus subtilis were overproduced and purified as described previously (Reizer et al., 1989, 1992b). Protocols for the overproduction and purification of the fructose-inducible diphosphoryl transfer protein (DTP) of Salmonella typhimurium will be described elsewhere.

beta-Galactosidase Assays

beta-Galactosidase activities were determined using method A of Miller(1992), and results were averaged from at least three independent experiments. W salts medium was used for measuring the activities of the glnA promoters under conditions of limiting versus excess nitrogen as described above. Precultures were grown in excess nitrogen medium to mid-log phase, equally divided, washed twice in the new medium, and assayed after a 2-h induction period. LB medium was used for assaying promoter activities of rpoN and rnc-era operon fusions.

Immunoblotting

The relative amount of Era protein present in different strains was analyzed by immunoblotting with rabbit polyclonal anti-Era antibodies (gift from B. Johnstone and B. Simons) according to protocols of the manufacturers of the gel and electrotransfer apparatus (Bio-Rad) and the peroxidase substrate detection system (Kirkegaard & Perry Laboratories, Gathersburg, MD). Strains were grown in LB medium at 32 °C to OD of 0.3. One ml of cells was chilled on ice, harvested, washed once in normal saline, and resuspended in 200 µl of SDS-loading buffer. Total proteins were separated on 10% SDS-PAGE in samples of 2 and 8 µl volumes of the loading buffer.

Computer-aided Structure Analyses

DNA sequences were aligned and assembled using the GCG package from the University of Wisconsin (Devereux et al., 1984), the DNA Strider program (Mark, 1988), and the Sequence Navigator program (Applied Biosystems, Inc., Thousand Oaks, CA). Database searches and sequence analyses were performed using GCG, DNA Strider, and the DNASYSTEM package (Smith, 1988). Comparison scores (expressed in standard deviations, S.D.) were calculated using the RDF2 program (Pearson and Lipman, 1988) with 100 or 200 shuffles of the sequences compared. A value of 6 S.D. is suggestive of homology whereas values of 9 S.D. or greater establish homology (Doolittle, 1986). Construction of phylogenetic trees and estimation of the relative evolutionary distances among members of a protein family were as described by Reizer and Reizer(1994) using the progressive alignment method of Feng and Doolittle(1990).

Other Methods

Routine PCR amplifications for plasmid and strain analyses were as specified by the manufacturers of the reagents (Promega, Madison, WI, Takara, Tokyo) except as described above. In vitro S30 transcription-translation was used according to the manufacturers of the [S]methionine isotope and labeling kit (Amersham Japan LTD., Tokyo). Polyacrylamide gel electrophoresis of PTS proteins (SDS- and nondenaturing-PAGE) was performed as described previously (Reizer et al., 1989, 1992b). [P]PEP was prepared by the method of Matoo and Waygood(1983). Proteins labeled with [P]PEP were separated by SDS-PAGE and detected by autoradiography as described previously (Reizer et al., 1984, 1989). PTS-dependent sugar phosphorylation assays were performed essentially as described previously (Reizer et al., 1989, 1992b). Assay mixtures (50 or 100 µl final volume) contained 2.5 mM MgCl(2), 25 mM KF, 50 mM potassium phosphate buffer (pH 7.4), 25 µM [^14C]sugar (specific activity 5 µCi/µmol), 5 mM PEP, purified protein constituents of the PTS, and either crude extracts or washed membranes as indicated. Reaction mixtures were incubated at 37 °C for 30 min and assayed for [^14C]sugar-P using ion-exchange columns to separate phosphorylated from free sugar (Kundig and Roseman, 1971). Protein was determined by the Lowry method(1951) with bovine serum albumin as the standard protein.


RESULTS

Isolation, Mapping, and Subcloning of Two era Suppressor Mutants Linked to rpoN

One of our strategies to search for extragenic suppressors of the heat-sensitive era mutant (Inada et al., 1989; Inada, 1992) employed the ``hop'' transposon mutator 1105 (Way et al., 1984) to generate DeltaTn10-kan gene disruption mutations in the E. coli chromosome. Kanamycin-resistant transductants were screened for survival at 42 °C, and three of 20 independent candidate disruptions were found to be unlinked to era. These could be crossed back into the era strain to confer heat resistance, thus confirming them as second site suppressors of era (Inada, 1992). This paper concerns the characterization of two of these suppressors, named ersB1 and ersB2 for era suppressor numbers B1 and B2. ersB1::kan and ersB2::kan were initially mapped at low resolution by P1 transduction into the ordered set of DeltaTn10-linked genetic mapping strains (Singer et al., 1989) and scoring Km^R transductants for loss of the tetracycline marker. The ersB2 mutation was found to cotransduce with the markers zgi-203::DeltaTn10 and zha-6::DeltaTn10 with linkage frequencies of 36 and 54%, respectively. Tests on the ersB1 mutant gave similar results, thereby showing that both mutations were localized to the 72-min region of the chromosome (Rudd, 1992; 69 min by Bachman, 1990).

Although both mutations were mapped to the same general location, the ersB2 mutation was a weaker suppressor of era. Their general locations were confirmed, and the associated transposon insertions were simultaneously cloned by recombination of the kanamycin markers onto Kohara (Kohara et al., 1987) phage containing chromosomal DNA fragments of this region. Only lysates derived from clones 7E3(522) and 3G10(523) conferred Km^R when crossed with the ersB mutants. Both mutations were therefore shown to reside within the overlap between these two Kohara clones and specifically between the physical map coordinates of 3361 and 3370 kb (Rudd, 1992). This conclusion concurred with previous Southern hybridization data which placed both ersB mutants on a 3.4-kb PstI chromosomal DNA fragment (Inada, 1992).

Fine Structure Mapping of Mutants and Nucleotide Sequencing of the rpoN Operon

The exact locations of both ersB mutants were determined by nucleotide sequencing across their transposon insertion junctions. Each transposon was found to lie within the coding region of different genes in the rpoN region ( Fig. 1and Fig. 2). The ersB2 transposon was inserted within the rpoN gene itself, while the ersB1 transposon disrupted the 5` terminus of an open reading frame (orf163) whose sequence was originally seen to be most similar to the fructose and mannitol-specific IIA protein domains of the PTS (Reizer et al., 1992a).


Figure 2: Nucleotide sequence for a portion of the rpoN operon indicating the ersB2::kan transposon insertion mutation in rpoN and the ersB1::kan transposon insertion mutation in ptsN. The junctions containing the nine nucleotide duplications caused by the transposon insertion events are underlined. The complete sequence of the 5.56-kb Sau3AI-PvuII fragment overlapping minute 72 of the E. coli chromosome and containing the rpoN operon is deposited in the Genbank data library under accession number U12684.



The junction of the ersB2 transposon appeared at codon 415 of rpoN with the 9-base duplication caused by the transposon insertion event partially overlapping the HindIII site (Fig. 2). Inspection of the 5` junction revealed that this insertion had fused the rpoN gene to the transposon outer border creating a truncated protein in which the last 62 amino acids of ^N are predicted to be replaced by the fusion carboxyl terminus SDESPIDPYQNH.

The junction of the ersB1::kan transposon was located beyond rpoN immediately after the fifth codon of orf163 (Fig. 2). Based on the biochemical activity of the protein produced from the wild-type gene, we have renamed orf163 as ptsN and its encoded protein as IIA (see below). This DeltaTn10-kan transposon inserted in the same orientation as did ersB2, and both mutations were determined to be partially polar on transcription of distal genes as discussed below. The nucleotide sequence between these two transposon insertions as well as regions 5` and 3` to rpoN were determined since, at the time, no sequence in the public domain was available outside of the E. coli rpoN gene. This sequence agrees with other E. coli sequences except the following positions. We observed disagreement with Sasse-Dwight and Gralla(1990) at our nucleotide numbers 1770 where we insert a C, and between 1786-1787 where we delete the T; disagreement with Imaishi et al. (1993, accession no. D12698) at the same positions listed above as well as at nucleotide numbers 2494 (G in place of A), between 3844-3845 (we delete TC), and 3861 (G in place of T). We observed agreement over the extent of the overlapping sequence with Jones et al. (1994, accession no. Z27094). Thus, we have defined the entire 5.56-kb Sau3A1 to PvuII fragment overlapping minute 72 on the current physical map of the E. coli chromosome (Rudd, 1992). Our sequence data extend in both directions previously reported sequence entries and disagrees with the restriction enzyme site map of Kohara et al.(1987) in three places (Fig. 1). We find no PvuII site between BamHI at 3364.7 kb and KpnI at 3365.2 kb, and within the newly defined orf185 we find two PstI sites and one PvuII site. These sites were confirmed by analysis of restriction enzyme digests of plasmid pBP2 and its derivatives (data not shown).

A total of seven open reading frames have been identified (Fig. 1). Protein products predicted by these genes (given in parentheses) have the following calculated molecular masses, in sequential order from 5` to 3`: (1) ORF185 (orf185), 20,114 Da; (2) ORF251 (orf251), 26,772 Da; (3) ^N (rpoN), 53,956 Da; (4) ORF95 (orf95), 10,743 Da; (5) IIA (ptsN, orf163), 17,948 Da; (6) ORF284 (orf284), 32,471 Da; and (7) NPr (npr, orf90), 9,803 Da. Proteins of approximate molecular weights in agreement with these predicted sizes have been observed by SDS-polyacrylamide gel electrophoresis of [S]methionine-labeled proteins synthesized in vitro by the S30 coupled transcription-translation system using plasmid pBP2 (data not shown). The IIA and NPr proteins encoded by ptsN and npr have been overexpressed, characterized and respectively named as described below. The calculated mass for ^N differs slightly from that previously predicted (Sasse-Dwight and Gralla, 1991; Imaishi et al., 1993) due to a frameshift that changes six codons yielding the following difference in predicted amino acid sequence beginning with codon 98: SGTSGD (previously SAPAVT). Cutting by FokI at a recognition site overlapping one of these sequence discepancies confirms that the sequence presented here is correct.

Database Comparisons for Proteins Encoded by Each Gene in the rpoN Operon: ORF185

The first gene in the putative operon is orf185. The deduced protein product ORF185 contains no cataloged motifs (Bairoch, 1992) and shares no significant similarity with proteins in the current databases (SWISSPROT version 27 and PIR version 39).

ORF241

orf241 is predicted to encode an ATP-binding protein, homologous to other such protein constituents of ABC-type transporters. This family of proteins provides both membrane-associated and cytoplasmic functions (Higgins, 1992). Some of the closest protein relatives are LivG, LivF, and MalK of E. coli and S. typhimurium, BraF and BraG of Pseudomonas aeroginosa, and NodI of Rhizobium meliloti, R. leguminosarum and Bradorizobium japonicum, all exhibiting similarity scores with ORF241 of 30-40 S.D.

RpoN (^N)

Nineteen rpoN genes have been fully sequenced, and the inferred protein sequences were multiply aligned (data not shown). The alignments of ^N as well as ORF241, IIA, ORF284, and NPr with their homologous proteins are available upon request. (^2)An average similarity plot was derived from the multiple alignment of the ^N homologs (Fig. 3A). At the NH(2) termini of these proteins are glutamine- and leucine-rich regions of almost 50 residues in length which exhibit a high degree of sequence similarity. This amino-terminal region is followed by a second region of approximately 110 residues that exhibits low sequence conservation and contains large gaps in the multiple alignment. The third region of approximately 400 residues is well conserved and contains notable helix-turn-helix and ``RpoN box'' motifs. These motifs are the most highly conserved sequences in the proteins of this family (see Fig. 3A). The two signature sequences (Bairoch, 1992) of this ^N family are shown in Fig. 3A, and the phylogenetic tree of these proteins is presented in Fig. 3B. The topology of the tree corresponds roughly to the phylogeny of the organisms from which the proteins were sequenced. Note that the ^N proteins of B. subtilis, R. capsulatus, and R. sphaeroides cluster on the same branch of the tree. Interestingly, the poorly conserved spacer region between region I and region III is virtually absent from the proteins of B. subtilis, R. capsulatus, R. sphaeroides, and B. japonicum RpoN1 (data not shown) indicating that region II is not essential for general ^N function.


Figure 3: Average similarity plot (A) and phylogenetic tree (B) of the 19 sequenced RpoN (^N) proteins. In A, a sliding window of 20 residues was used for calculation of the similarity score. The average similarity score along the entire sequence is provided by the dashed line. The two signature sequences of this protein family (Bairoch, 1992) are provided above the plot and correspond to the conserved helix-turn-helix region and the (RpoN box), respectively (Merrick 1993). Residues in brackets represent alternative possibilities at a particular position. Any amino acid at a position in which the residue is not specified is denoted by X. In B, relative evolutionary distances are provided adjacent to the branches. Construction of the phylogenetic tree was as described by Reizer and Reizer(1994). Abbreviations used and references to the published sequences are as follows: Pseudomonas putida (P. putida; Kohler et al., 1989; Inouye et al., 1989); Pseudomonas aeruginosa (P. aeruginosa; Jin et al., 1994); Azotobacter vinelandii (A. vinelandii; Merrick et al., 1987); Klebsiella pneumoniae (K. pneumoniae; Merrick and Gibbins, 1985; Merrick and Coppard, 1989); Salmonella typhimurium (S. typhimurium; Popham et al., 1991); Escherichia coli (E. coli; Sasse-Dwight and Gralla, 1990; Imaishi et al., 1993); Thiobacillus ferrooxidans (T. ferrooxidans; Berger et al., 1990); Acinetobacter calcoaceticus (A. calcoaceticus; Ehrt et al., 1994); Alcaligenes eutrophus (A. eutrophus; Warrelmann et al., 1992); Bacillus subtilis (B. subtilis; Debarbouille et al., 1991); Rhodobacter capsulatus (R. capsulatus; Jones and Haselkorn, 1989; Alias et al., 1989); Rhodobacter spheroides (R. spheroides; Meijer, 1992); Caulobacter crescentus (C. crescentus; Brun and Shapiro, 1992); Azorhizobium caulinodans (A. caulinodans; Stigter et al., 1993); Bradyrhizobium japonicum (B. japonicum, RpoN1 and RpoN2; Kullik et al., 1991); Rhizobium meliloti (R. meliloti(1); Ronson et al., 1987; R. meliloti(2); Shatters et al., 1989). The two RpoN sequences of R. meliloti, reported by Ronson et al.,(1987) and by Shatters et al.(1989) are 86.5% identical over 511 residues and both were included in the tree.



ORF95

Eleven orf95-like genes in various bacterial species have been sequenced. They encode homologous proteins that exhibit no significant sequence similarity with other proteins in the current databases (SWISSPROT version 27.0; PIR version 39.0) except for the E. coli ORF113 (13-21 SD) which is encoded immediately upstream of pheA (Hudson and Davidson, 1984; Gavini and Davidson, 1990). These 12 proteins were multiply aligned, and an average similarity plot was derived (Fig. 4A). It can be seen that these proteins exhibit highest sequence similarity in their NH(2)-terminal and COOH-terminal regions. The central region of approximately 30 residues exhibits low conservation due to multiple insertions and deletions, although 6 residues (Met, Ile, Gly, Ala, Asp, and Tyr) in this region are positionally conserved in all but one member of this protein family.


Figure 4: Average similarity plot (A) and phylogenetic tree (B) of the 11 members of the sequenced ORF95 family. Figure presentation is as described in the legend to Fig. 3. The signature sequence for this protein family is provided above the similarity plot. References to the published sequences are as follows: ORF113 of Acinetobacter calcoaceticus (A. calcoaceticus, Ehrt et al., 1994); ORF102 of Pseudomonas putida (P. putida, Inouye et al., 1989; Kohler et al., 1989); ORF104 of Rhizobium meliloti (R. meliloti, Ronson et al., 1987); ORF107 of Azotobacter vinelandii (A. vinelandii, Merrick et al., 1987; Merrick and Coppard, 1989); ORF103 of Pseudomonas aeruginosa (P. aeruginosa, Jin et al., 1994); ORF95 of Klebsiella pneumoniae (K. pneumoniae, Merrick and Coppard, 1989); ORF95 of Salmonella typhimurium (S. typhimurium, Popham et al., 1991); ORF95 of Escherichia coli (E. coli, Jones et al., 1994; Imaishi et al., 1993; this study); ORF203 of Bradyrhizobium japonicum (B. japonicum, Kullik et al., 1991); ORF130 of Alcaligenes eutrophus (A. eutrophus, Warrelmann et al., 1992); ORF113 of Esherichia coli (E. coli, Gavini and Davidson, 1990; Hudson and Davidson, 1984); ORF78 of Thiobacillus ferrooxidans (T. ferrooxidans, Berger et al., 1990).



The phylogenetic tree of the ORF95 proteins is presented in Fig. 4B. The branching order of this tree generally follows that of the RpoN tree. Note that the E. coli ORF113 is no more distant from the other members of the family than the latter are from each other.

The hypothetical 13.6-kDa protein (ORF117) in the div region of B. subtilis and the spinach 30 S ribosomal protein (SWISSPROT identifiers P28368 and P19954) previously proposed to be homologous (Merrick, 1993) are not included in the tree shown in Fig. 4for the following reasons. Although ORF117 exhibits significant similarity (14 S.D.) to the carboxyl terminal region of ORF203 of B. japonicum, it is not similar to the NH(2)-terminal region of this protein which is the region that is homologous to the other protein members of this family. Furthermore, the spinach 30 S ribosomal protein shows limited similarity (up to 9 S.D.) to only a few proteins of this family.

IIA (ORF163)

The protein encoded by ptsN, hereafter referred to as IIA, proved to be homologous to a small family of PTS Enzymes IIA specific for fructose and mannitol (Reizer et al., 1992a). The average similarity plot for these proteins is shown in Fig. 5A while the phylogenetic tree of this protein family is presented in Fig. 5B. The multiple alignment of IIA with all homologous IIA proteins revealed that IIA exhibits particularly strong residue conservation around the recognized phosphorylation site in IIA (data not shown but see Reizer et al., 1992a, 1994b). As shown by the signature sequence presented in Fig. 5A, 3 amino acids including the phosphorylatable histidyl residue (Reiche et al., 1988; van Weeghel et al., 1991) are fully conserved. Significantly, a second histidyl residue (His in IIA(Eco)) is conserved in all proteins of this family except IIA (Reizer et al., 1994b). This histidine is postulated to play a catalytic role in the phosphoryl transfer reaction (Reizer et al., 1992a). Three other residues (Arg, Gly, and Lys in IIA(Eco)) are conserved in all but one member of this family.


Figure 5: Average similarity plot (A) and phylogenetic tree (B) of the 15 members of the IIA family. Figure presentation is as described in the legend to Fig. 3. Abbreviations and references to the published sequences are as follows: IIA, the mannitol-specific Enzyme IIA of Staphylococcus carnosus (Sca) (Fischer et al., 1989), Enterococcus faecalis (Efa) (Fischer et al., 1991), Streptococcus mutans (Smu) (Honeyman and Curtis, 1992), and Escherichia coli (Eco) (Lee and Saier, 1983). IIA, the fructose-specific Enzyme IIA protein domain of E. coli (Eco) (Reizer et al., 1994a), Salmonella typhimurium (Sty) (Geerse et al., 1989), and Rhodobacter capsulatus (Rca) (Wu et al., 1990). IIA, the fructose-like Enzyme IIA of E. coli (Eco) (Reizer et al., 1994b). IIA, the cryptic mannitol Enzyme II of E. coli (Eco) (Sprenger, 1993). IIA, the COOH-terminal IIA protein domain of the Enzyme I-IIA fusion protein of E. coli (Eco) (Blattner et al., 1993; Saier and Reizer, 1994). IIA, the IIA protein encoded within the RpoN operon of E. coli (Eco) (this study; Imaishi et al., 1993; Jones et al., 1994), K. pneumoniae (Kpn) (Merrick and Coppard, 1989), P. aeruginosa (Pae) (Jin et al., 1994), P. putida (Ppu) (Inouye et al., 1989; Kohler et al., 1994), and B. japonicum (Kullik et al., 1991).



The phylogenetic tree of all currently known members of this family (Fig. 5B) reveals five clusters of proteins which correlate in function to the extent known: 1) mannitol-specific protein-domains, 2) fructose-specific protein-domains, 3) the cryptic mannitol (Cmt) IIA protein, 4) the IIA protein and the IIA protein-domain (both of unknown sugar specificity), and 5) the IIA proteins. Positions of the proteins within this last cluster correlate with the approximate phylogenies of the corresponding organisms.

ORF284

orf284 of E. coli encodes a protein (ORF284) that is homologous (96% identity over a stretch of 193 residues; 86 S.D.) to the corresponding protein product of the partially sequenced orf (ORF > 193) residing downstream of ORF162 in the Klebsiella pneumoniae rpoN operon (Merrick and Coppard, 1989). It also exhibits high similarity (S.D. > 25) with the partially sequenced open reading frame (ORF>39) located downstream of the ptsN homologue of P. aeruginosa and P. putida (Jin et al., 1994; Kohler et al., 1994). No statistically significant similarity of ORF284 to other protein(s) in the current databanks was detected. Nevertheless, examination of these sequences (ORF284, ORF>193, and ORF>39) with the PROSITE motif library (Bairoch, 1992) revealed that they all contain a glycine-rich region located between residues 8 and 15, i.e. GRSGSGKS, that matches the phosphate binding loop of numerous ATP- and GTP-binding proteins (Walker et al., 1982; Saraste et al., 1990). It should be noted, however, that the corresponding signature pattern, i.e. (AG)XGK(ST), is also present in proteins that do not bind ATP or GTP.

NPr(ORF90)

ORF90, hereafter called NPr, is homologous to a large family of proteins comprising the HPr and HPr-like proteins of the PTS which all function in generalized energy-coupling phosphoryl transfer (Reizer et al., 1993b). The comparison of NPr with other members of this family shows percent identities that range from 25 to 41% over stretches of 71-88 residues with similarity scores of 10-18 S.D. (data not shown). The average similarity plot for these proteins (Fig. 6A) shows that all members of the HPr family share amino acid sequences that are well conserved around two characterized sites: the catalytic histidyl residue His in all characterized HPr proteins (His in NPr), and the regulatory seryl residue Ser in HPrs of Gram-positive bacteria (Ser in NPr) (Fig. 6A). The two signature sequences shown in Fig. 6A differ from those proposed previously (Reizer et al., 1993b; Zhu et al., 1993) due to the inclusion of new members of the family. In addition to the absolute conservation of 4 residues in the two signature sequences (Gly, His, Arg, and Ser), Gly in NPr is fully conserved in all HPr proteins and protein domains.


Figure 6: Average similarity plot (A) and phylogenetic tree (B) of 15 completely sequenced HPr and HPr-like proteins. Figure presentation is as described in the legend to Fig. 3. Note that inclusion of the new members of this family required that our previously proposed signature sequences (Reizer et al., 1993b), which include the regions of the catalytic histidine residue (His) and the conserved serine (Ser) be modified to the signature sequences shown in panel A. Abbreviations are as indicated in the legend of Fig. 3Fig. 4Fig. 5. The HPr-like protein, which is encoded within the operon, is designated NPr. The HPr-like protein domain of the DTP of E. coli and S. typhimurium, and the multiphosphoryl transfer protein of R. capsulatus are denoted (Fru.). References of published sequences are as follows: S. carnosus (Eisermann et al., 1991), S. aureus (Reizer et al., 1988), B. subtilis (Reizer et al., 1988; 1989; Gonzy-Treboul, 1989), S. salivarius (Gagnon et al., 1992), E. faecalis (Deutscher et al., 1986), S. typhimurium (Powers and Roseman, 1984; Byrne et al., 1988), E. coli (De Reuse et al., 1985; Saffen et al., 1987), K. pneumoniae (Titgemeyer et al., 1990), A. eutrophus (Pries et al., 1991), R. capsulatus (Fru.) (Wu et al., 1990), S. typhimurium (Fru.) (Geerse et al., 1989), E. coli (Fru.) (Orchard and Kornberg, 1990; Reizer et al., 1994a), E. coli (NPr) (this study; Jones et al., 1994), M. capricolum (Zhu et al., 1993), S. mutans (Boyd et al., 1994).



The phylogenetic tree of the HPr family (Fig. 6B) reveals four main clusters. The Gram-positive bacterial proteins comprise a single cohesive group with the HPr of Mycoplasma capricolum comprising a deep branch emanating from the base of this group. Three sequenced fructose (Fru)-inducible protein domains, which all occur within larger proteins encoded by fructose catabolic operons, comprise the second cluster. The Gram-negative enteric bacterial HPrs comprise the third cluster. NPr is distant from all HPr-like proteins except the HPr-like protein of Alcaligenes eutrophus. These two proteins together comprise the fourth cluster. Interestingly, this nearest relative to NPr is believed to function in the regulation of poly-beta-hydroxybutyrate metabolism (Pries et al., 1991). This close relationship of NPr(Eco) with HPr(Aeu) may suggest a regulatory role for NPr.

Transcriptional Organization of the rpoN Operon

The transcriptional activities of rpoN region fusions to the lacZ reporter gene were measured in order to generally locate the rpoN promoter and to determine whether the ersB transposon mutations affect expression of the rpoN operon. LacZ-operon fusions constructed on plasmids (see Fig. 1C) were transferred to phage and placed into isogenic strains in single copy number.

In the wild-type strain, constructs that fused sequences upstream of rpoN to lacZ (BP125 and BP124) resulted in the highest levels of beta-galactosidase activity (Table 2). The lower expression of the longer fusion BP124 (46%) as compared to that of the shorter fusion BP125 may indicate the presence of a weak intervening transcriptional terminator, or may simply result from the difference in fusional junctions between the two constructs. Computer-assisted analyses revealed no obvious rho-independent terminators in the intervening region.



In comparison with these fusions, an internal BamHI fragment which excludes the 5` region of the rpoN gene (BP123) retained only 27% of the transcriptional activity of BP124 (Table 2). Thus, the strongest promoter (P1) upstream of orf284 must be upstream of the rpoN BamHI site. However, a much weaker promoter (P2) is also likely to exist between rpoN and orf284 because the activity of BP123 is more than 10 times that of the vector control BP100 (Table 2). Therefore, we predict that all four distal genes are primarily expressed from a promoter upstream of rpoN. Neither promoter has been physically determined in E. coli, but a transcription start has been mapped in the homologous region of R. meliloti 65 nucleotides upstream from the rpoN AUG start site (Albright et al., 1989) corresponding in position to a promoter proposed by Jones et al.(1994) based on cannonical sequences. A downstream promoter of positioning similar to the P2 proposed here has been identified in the P. aeruginosa rpoN operon (Kohler et al., 1994). No evidence reported to date unequivocally localizes the rpoN promoter of E. coli or address the possibility of transcription from another promoter upstream.

Operon fusions BP123, BP124, and BP125 were placed into the genetic backgrounds of mutants ersB1, ersB2, and glnF208::Tn10 to determine whether rpoN itself or the downstream genes alter transcription of the rpoN operon. Since transcriptional activities of all of the fusions were unaffected by these mutations (see legend to Table 2), neither ^N nor IIA appear to function as autogenous transcriptional regulators under these conditions. These data support previous reports suggesting that rpoN is expressed constitutively (Castano and Bastarrachea, 1984). These tests also indicate that the ^N-like consensus binding site, oriented leftward within the ptsN gene at nucleotides 3676-3663, does not affect transcription of the operon under the conditions used.

To examine polarity of the DeltaTn10-kan transposons on distal transcription, variants of the BP124 and BP123 fusions were constructed which contain the ersB1 (BP124.1, BP123.1) and ersB2 (BP124.2, BP123.2) transposon mutations. By examining BP123, BP123.1 and BP123.2, it became clear that the ersB1 insertion is downsteam of promoter P2, while ersB2 is upstream of it. Only the former mutation significantly diminished expression of the orf284-lacZ fusion of BP123. Both ersB insertions caused polarity in BP124 with ersB2 appearing to be less polar. However, when the relative level of P2 promoter activity (27%, Table 2) was subtracted from that of BP124.2 (51%), the polarity effects of ersB1 (5%) and ersB2 (14%) proved to be more comparable. This effect might be expected since the DeltaTn10-kan elements are inserted in the same orientation. While the mutations were shown to exert a significant polar effect, these data also indicate that the ersB2::kan insertion only partially blocked transcription of orf284, and presumably of ptsN as well. This fact could explain the weaker suppression of era by ersB2::kan relative to ersB1::kan.

The ersB Suppressor Mutations Do Not Affect Expression of Era

Two criteria were used to test whether the rpoN operon disruptions affect the expression of the era gene. First, the transcriptional activities of rnc operon fusions to lacZ were measured, and second the total cellular concentration of Era protein was compared between wild-type and suppressor strains. Since the era gene is transcriptionally and translationally coupled to the first gene of the operon rnc (Takiff et al., 1989; Chen et al., 1990), we used rnc operon fusions to indirectly monitor the transcription of era. derivatives of two plasmid-borne fusions of the rnc leader region to lacZ (pCF110, pCF120) (^3)were moved into six strains representing all combinations of one rpoN operon allele (rpoN, ersB1::kan, or ersB2::kan) together with one era allele (era or era). betaGalactosidase activities did not differ significantly between any of the strains (data not shown). Therefore, the suppressors appear not to affect the transcription of the rnc operon under the conditions tested.

Western blot analysis performed on total protein extracts from the same strains (see ``Experimental Procedures'') showed that a band comigrating with previously purified Era (Chen et al., 1990) appeared with equal intensity in all extracts (data not shown). Therefore the suppressors did not alter the steady state level of Era protein. Interestingly, the primary era mutation itself did not noticeably affect steady state Era levels either, which lessened the possibility that proteases play a role in era suppression.

Suppression of the conditional lethality of another eradefective mutant, rnc40::DeltaTn10, was also tested. In the rnc40::DeltaTn10 mutant, normal RNaseIII-dependent transcriptional regulation is severed by the placement of a DeltaTn10 transposon in the transcribed leader region which renders era expression dependent upon the tetracycline inducible tetA promoter within the transposon. Without added tetracycline, the levels of Era fall below a threshold value necessary for normal growth (Takiff et al., 1989). Since ersB1::kan specifically allowed tetracycline independent growth at 42 °C, the conclusion that rnc-dependent regulation of era transcription is not involved is supported. This also suggests that the suppression phenotype is not allele specific (see ``Discussion''). In addition, in a separate experiment involving an era-curing system, (^4)the ersB mutations did not allow growth of cells completely lacking the era gene. Thus the ersB mutants do not function to bypass the era requirement.

Complementation Tests of ersB Suppressor Mutants

Complementation tests using multicopy clones of the rpoN region were used to determine the identity of the protein whose absence promoted suppression of the era phenotype. Table 3shows that plasmids containing the wild-type ptsN gene (pBP123, pBP124, pBP131) caused both of the temperature-resistant double mutants, i.e.eraersB1 and eraersB2, to regain temperature sensitivity. Since none of these plasmids express genes downstream of ptsN and two of them contain only orf95 and ptsN, suppression of the era phenotype by the ersB1::kan insertion is most probably due to loss of ptsN. Interestingly, the pUC18-derived plasmid pBP120, which expresses very high levels of only IIA from the lac promoter, caused all wild-type strains tested to die at high temperature (data not shown). Plasmid pBP130, which expresses rpoN (but not ptsN) from its own promoter, caused all strains including the wild-type to grow more slowly at all temperatures tested. We also observed this growth inhibition with another rpoN clone, pTH7 (a gift from F. Clavie-Martin and B. Magasanik, data not shown), as have others (e.g. Sasse-Dwight and Gralla, 1991), and so the apparent weak complementation associated with multicopy expression of rpoN (Table 3, pBP130) does not allow us to clearly implicate or refute a role by ^N in the temperature sensitivity of era. We note that pBP124, the parental plasmid of pBP130 which expresses ptsN along with rpoN, did not cause the low temperature growth defect, suggesting that the stoichiometry of rpoN to ptsN is important to this general growth inhibition phenotype. The lack of suppression of era by an npr::kan disruption (Fig. 1, data not shown) further indicates that the suppressive effect of the ersB transposon insertions was not due to polarity on npr expression. As shown later, a 2-fold change in ptsN dosage has noticeable effects on cell growth under certain conditions. Therefore, it appears that the major suppression effect of both disruptions is to decrease expression of ptsN. In total, these data suggest that heat sensitivity caused by era is assisted by the product of ptsN, IIA, and that suppression is accomplished by loss of Enzyme IIA activity.



ersB1 Does Not Affect Classical Nitrogen Control by ^N

We investigated the effects of ersB1::kan and ersB2::kan on ^N-nitrogen control by phenotypic growth tests and by measuring the inducibility of the glnA promoter under nitrogen stress. Since the final result of ^N-dependent expression in this regard is to allow the cell to use poor nitrogen sources, the mutants were first assayed phenotypically by measuring colony growth on media containing any one of several poor sources of nitrogen. Table 4shows that in the presence of glucose, the well-characterized rpoN mutant, glnF208::Tn10, required glutamine for growth and could not utilize alternative sources of poor nitrogen, e.g. histidine, glutamate, arginine, lysine, and aspartate. Table 4thus defines the Gln and Ntr phenotypes of an rpoN mutant (Magasanik, 1982). The ersB2::kan mutant exhibited a similar growth pattern and is therefore also Gln and Ntr, as would be expected since the transposon interrupted the rpoN coding sequence. However, after an extended incubation time, leaky growth of the ersB2::kan mutant on alanine was observed although none was observed for the glnF208::Tn10 mutant. The two rpoN (glnF) disruptions therefore are not phenotypically identical. By contrast, the ersB1::kan mutant grew on plates containing glutamine, alanine, aspartate, glutamate, lysine, or arginine as a sole source of nitrogen (see Table 4, column 3). Similar results were observed using adenosine in place of alanine as the nitrogen source (data not shown). Therefore, the ptsN- strain carrying the ersB1::kan mutation is phenotypically both Gln and Ntr (Table 4). While growth was positive for the ersB1 strain on various poor sources of nitrogen, colony morphology differed between this mutant and the wild-type strain in the presence of added glucose. First, on media containing glucose plus either glutamine, aspartate, or alanine as a source of nitrogen, the wild-type strain grew better than did the ersB1 mutant. Second, in the presence of glucose and one of the poorer nitrogen sources tested (glutamate, lysine, arginine, histidine, proline, and adenosine) the ersB1 mutant strain acquired a mucoid morphology (Table 4, column 3 for H, E, K, and R; data not shown for proline or adenosine). This mucoidy could be suppressed by the presence of excess nitrogen in the form of an ammonium salt ( Table 4columns 4 for H, E, K, and R). In contrast, ersB1 cells grown on plates containing glucose and one of the better organic nitrogen sources (glutamine, alanine, aspartate, or serine) did not acquire the mucoid morphology ( Table 4column 3, Q, A, and D; data not shown for serine). Thus by the standard definition, the ersB1 mutant appears to be normal for standard nitrogen control since it does not require glutamine, but its general physiology clearly differs from that of the wild-type strain under nitrogen stress.



Nitrogen regulation was also investigated quantitatively at the molecular level by measuring the inducibility of the glutamine synthetase (glnA) promoter under conditions of nitrogen starvation. It is well established that nitrogen deprivation activates ^N-dependent initiation of transcription at the p2 promoter of the glnA operon (see reviews listed in Introduction). Table 5shows that the glnAp2 promoter is inducible in the wild-type and ersB1::kan strains but not in the rpoN mutants, ersB2::kan and glnF208::Tn10. beta-Galactosidase values and induction ratios are comparable with published data (Schneider et al., 1991). Under nitrogen stress conditions, the absolute values obtained for the ersB1 mutant were not quite as high as those obtained for the wild-type, but were nevertheless similarly inducible for both the minimal and the full glnA promoter fusions (Table 5). From these data we again conclude that the ersB1 disruption does not affect ^N-dependent induction of glutamine synthetase under these conditions.



The possibility of a role for glutamine regulation in suppression of the era phenotype was further checked by transducing a glnA::kan null mutation (gift from L. Reitzer) into the era strain and looking for an effect on temperature sensitivity. The absence of a noticeable effect (data not shown) demonstrates that suppression of the era phenotype by disruptions in the rpoN operon does not involve glutamine regulation.

The Effect of ersB1 on Catabolite Repression

During tests for Gln and Ntr phenotypes on limiting nitrogen media (Table 4), we noticed that alanine, as sole carbon and nitrogen source (Table 4, alanine, column 1), allowed both wild-type and ersB1 strains to grow well. However, addition of glucose gave rise to slight growth inhibition of the wild-type strain and slightly stronger inhibition of the ersB1 strain (Table 4, alanine, column 3). This inhibition was largely relieved by the presence of excess NH(4) (Table 4, alanine, column 4).

To better characterize this apparent nitrogen-related carbon repression, growth tests were performed on W-alanine medium supplemented with different carbon substrates. Several external carbon sources tested caused growth inhibition relative to growth in the absence of added carbon (Table 6). All of the tricarboxylic acid cycle intermediates tested, including succinate, citrate, and fumarate, caused the strongest inhibition of growth of the ersB1 mutant relative to the wild-type strain. Galactose and glycerol also strongly inhibited growth while other sugars including glucose caused a milder inhibition. On all carbons tested, this defect was complemented by phage 124 which carries rpoN, orf95 and ptsN (Table 6). Plasmid pBP120, which expresses only ptsN, also relieved this growth defect at 32 °C (data not shown). Interestingly, in the presence of an additional carbon source even the wild-type strain carrying phage BP124 grew better than the BP100 control lysogen (Table 6). In every case, loss of ptsN (ersB1::kan) accentuated this inhibitory effect, and diploidy (BP124) reduced the effect. As had been seen previously for glucose, the growth defect caused by other carbon sources tested was largely reversed by the addition of excess NH(4) (data not shown). These data implied that the added carbon sources interfered with the assimilation of nitrogen from alanine and that alanine metabolism is limiting for growth. Many of these tests were also performed using W-adenosine medium with no apparent difference in results (data not shown), indicating that the carbon-induced growth defect is not peculiar to alanine metabolism. Thus, ptsN and possibly other genes of the rpoN operon appear to facilitate utilization of organic nitrogen sources in an environment of multiple carbon sources.



To investigate whether metabolism of the added carbon source was required to observe this effect, we examined the effect of the nonmetabolizable glucose analog, methyl-alpha-glucopyranoside (alphaMG). Unexpectedly, this sugar analog inhibited growth identical to that by glucose (Table 6). While growth on W-alanine plus any added carbon source theoretically required that only nitrogen be extracted from alanine, growth with added alphaMG required the use of both carbon and nitrogen from alanine. However, since growth with alphaMG was indistinguishable from that with glucose, and worse than that on W-alanine alone, we question whether glucose is being metabolized under these conditions. Although not tested, we can think of no reason why the same logic might not also apply to the poorer carbon sources (e.g. succinate). It has not escaped our notice that citrate, which is just as inhibitory as succinate (Table 6), is not normally ultilized as a carbon source by E. coli. Interestingly, the poorer carbon sources had a greater inhibitory effect than did the sugars, an effect that appears to oppose the hierarchy of classical carbon repression.

We suspected that the nature of this repression might involve known regulation by the PTS because both ptsN and npr encode proteins homologous to established proteins of the PTS. For this reason we tested whether this novel nitrogen-related carbon repression is relieved by cyclic AMP (cAMP) or enhanced by alphaMG as would be predicted if standard catabolite repression were involved. Surprisingly, neither 5 mM cAMP nor 0.2% alphaMG noticeably altered the growth inhibition of ersB1 by glucose, fructose, mannose, or succinate (others not tested). While these data do not rule out the possible involvement of IIA and other components of the PTS-mediated catabolite repression system, they strongly suggest that the previously understood mechanisms of control are not directing this phenotype under these conditions.

Independence from the established mechanism of catabolite repression was also supported for era-related temperature sensitivity by the absence of an effect on era by added cAMP, glucose, or alphaMG. Disruptions of the adenylate cyclase (cya) gene or of the general PTS genes (ptsH, ptsI, and crr) did not have an effect either. As described under ``Experimental Procedures,'' these studies were performed with low salts LB medium since the era mutant strain used does not show temperature sensitivity on the minimal media used.

Overproduction, Purification, and Characterization of IIA

As shown in Fig. 7A, IIA represents about 20% of the total protein of E. coli MZ1/pJRNtr after temperature induction. Following a purification protocol that consisted of (a) ion-exchange chromatography on a DEAE-Sephacel column, and (b) gel filtration on a Sephadex G-75 column (see ``Experimental Procedures''), a nearly homogeneous IIA was obtained. The purified IIA migrated on SDS-polyacrylamide gels as a single band with an apparent molecular mass of 18 kDa in agreement with the molecular mass of 17,948 Da calculated from the deduced amino acid sequence of the protein. Nevertheless, two additional bands, one relatively intense (40 kDa) and the other faint (60 kDa), were detected in SDS-polyacrylamide gels when the protein samples were not boiled before loading the gels and mercaptoethanol was omitted from the sample buffer (see Fig. 7B). This observation leads to the possibility that IIA is oligomeric.


Figure 7: Electrophoretic analyses of IIA showing (A) the protein profile on SDS-polyacrylamide gel of the various steps of IIA purification; (B) the monomeric, dimeric, and trimeric species of the purified IIA (SDS-polyacrylamide gel), and (C) the mobility of the purified IIA on a non-denaturing gel before and after phosphorylation. Panel A, the purification steps and the amounts of proteins are as follows: crude extract, 60 µg, lane 1; DEAE-Sephacel pool, 40 µg, lane 2; Sephadex G-75 pool, 11 µg, lane 3; second DEAE-Sephacel pool, 22 µg, lane 4. Positions of molecular mass markers (in kDa) are indicated on the left. Panel B, 18 µg of purified IIA in SDS sample buffer lacking mercaptoethanol and without boiling (compare to lanes 3 and 4 in panel A). Panel C, samples contained the following amounts of PTS proteins: Enzyme I, 1.5 µg; HPr, 3 µg, and IIA, 7.5 µg. The phosphorylation reaction (at 37 °C for 30 min; 20 µl final volume) contained 50 mM Tris-HCl buffer (pH 7.2), 2 mM dithiothreitol, 5 mM MgCl(2), 5 mM PEP, and the indicated proteins as shown below the corresponding lanes.



Two Coomassie Blue bands were apparent when the purified IIA was analyzed by nondenaturing-PAGE (Fig. 7C). Addition of PEP, Enzyme I, and HPr (but not any two) increased the amount of the faster migrating band at the expense of the slower migrating band. This observation suggests that the faster band might be a phosphorylated derivative of IIA whereas the slower band may correspond to the free form of this protein (see Fig. 7C). Similar purification of a phosphorylated derivative of an Enzyme IIA, the xylitol-specific Enzyme IIA of Lactobacillus casei has been reported (London and Hausman, 1983).

An autoradiogram showing the phosphorylation of IIA by purified Enzyme I and HPr in the presence of [P]PEP is presented in Fig. 8. In lane 1, Enzyme I and HPr were incubated with [P]PEP, and only these two proteins were labeled. As shown, in lane 2, IIA was readily phosphorylated in the presence of Enzyme I and HPr. When HPr was replaced with the purified DTP of the S. typhimurium fructose PTS (Sutrina et al., 1988; Geerse et al., 1989), IIA was also phosphorylated (compare lanes 2-4). As expected for a histidyl phosphorylated protein, the P-labeled IIA was labile under acidic conditions but stable under alkaline conditions (data not shown).


Figure 8: Phosphorylation of IIA by HPr or DTP. The SDS-polyacrylamide gel autoradiogram shows P-labeled derivatives of the following PTS proteins: Enzyme I (0.5 µg) and HPr (1 µg), lane 1; IIA (4 µg), Enzyme I and HPr, lane 2; DTP (5 µg) and Enzyme I, lane 3; Enzyme I, DTP and IIA, lane 4. The phosphorylation reaction was as described in the legend to Fig. 7except that 2.5% glycerol was included in the phosphorylation mixture, and 0.5 mM [P]PEP (specific activity 5,000 counts/min/nmol) was used.



In other experiments IIA was P-labeled, purified, and subsequently tested for phosphoryl transfer. It was shown to readily transfer its phosphoryl group both to purified HPr and to purified DTP in the absence of Enzyme I (data not shown). These results establish that IIA is reversibly phosphorylated by either HPr or the FPr domain of DTP as predicted (Reizer et al., 1992a).

IIA was examined with respect to its potential activity in the mannitol and fructose phosphotransferase systems. Complementation assays for IIA were performed with a chromosomally deleted mtlA mutant (LGS322; Grisafi et al., 1989) harboring the plasmid pGJ9-Delta137 which encodes a carboxyl terminally truncated (residues 1 to 480) and inactive permease lacking the hydrophilic IIA protein domain. Extracts of this strain were readily complemented for mannitol phosphorylation by a crude extract of LGS322 bearing a plasmid-encoded site-specific mutant of Enzyme II in which the cysteyl phosphorylation site in IIB was replaced with histidine (C384H; Weng et al., 1992). By contrast, addition of purified IIA (13 µM) failed to complement the carboxyl terminally truncated mannitol permease. Similarly, the purified DTP of the fructose PTS (up to 6.8 µM) did not complement the IIA-deleted permease when mannitol phosphorylation was assayed. These results demonstrate that although IIA and IIA are homologous to IIA, neither protein is capable of complementing IIA under the conditions used.

Similar complementation analyses were performed with the fructose PTS. Fructose was readily phosphorylated by membranes of S. typhimurium strain LJ4031 (fruB::kan fruR::Tn10) bearing a disrupted IIA domain of DTP following addition of purified Enzyme I (0.8 µg) and DTP (1.8 µg). By contrast, addition of purified Enzyme I and HPr (2 µg) or Enzyme I, HPr, and IIA (6 µg) to membranes derived from this strain failed to restore fructose phosphorylation. Since HPr readily phosphorylated IIA ( Fig. 7and Fig. 8), we conclude that IIA cannot substitute for the IIA domain of DTP in the fructose PTS in spite of the sequence similarity exhibited by these proteins. This fact was also demonstrated genetically since a plasmid expressing ptsN (pBP120) did not complement the carboxyl terminally truncated mtlA or fruB::kan mutants of E. coli for specific sugar fermentation (data not shown).

Overproduction, Purification, and Characterization of NPr

NPr, the product of the npr (orf90) gene and a putative nitrogen-related HPr, was overproduced and purified using the protocols described under ``Experimental Procedures.'' Fig. 9A, lanes 1-3, shows the protein profile of the two-step purification scheme which yielded an apparently homogeneous protein. For comparative purposes, the relative mobilities in SDS-PAGE, of the E. coli HPr (lane 4) and the B. subtilis HPr (lane 5) are also shown. The purified NPr migrated as a single band with an apparent molecular mass of 13 kDa, somewhat more slowly than E. coli HPr. In contrast, when these three homologous proteins were examined on nondenaturing gels (Fig. 9B), each appeared to exhibit a distinctive rate of migration. E. coli HPr migrated most slowly, B. subtilis HPr migrated more rapidly, and NPr migrated most rapidly. In agreement with their respective observed mobilities in the non-denaturing gel (Fig. 9B), the calculated pI values for NPr, HPr of E. coli, and HPr of B. subtilis are 4.02, 5.50, and 4.52, respectively. Interestingly, the mobility of NPr in non-denaturing gels was greater than those of HPr(P) of E. coli and B. subtilis (data not shown).


Figure 9: Electrophoretic analysis of NPr. A, an SDS-polyacrylamide gel showing the protein profile of the various steps of NPr purification; and B, a nondenaturing gel comparing the migratory behavior of NPr and HPrs. Panel A, the purification steps and the amounts of protein loaded were as follows: lane 1, crude extract, 68 µg; lane 2, DEAE-Sephacel pool, 32 µg; lane 3, Sephadex G-50 pool, 40 µg; lane 4, purified HPr of E. coli, 6 µg; lane 5, purified HPr of B. subtilis, 4 µg; lane 6, molecular mass standards (kDa). Panel B, lanes 1-3, purified NPr (4 µg), HPr of E. coli (5 µg), and HPr of B. subtilis (6 µg), respectively.



An autoradiogram showing the phosphorylation of NPr by PTS proteins, i.e. Enzyme I, IIA, IIA, and DTP, is presented in Fig. 10A. As expected, no phosphorylated derivatives of NPr or HPr were detected when these proteins were incubated with [P]PEP alone (lanes 2 and 3, respectively). By contrast, a faint NPr-P band was apparent upon inclusion of Enzyme I in the phosphorylation reaction (lane 4). The amount of NPr (4 µg) in lane 4is 2-fold lower than the amount used in the control (8 µg, lane 2). The labeled NPr band in lane 4 is therefore not due to nonspecific labeling of this protein. Increasing the amount of NPr led to comparable increases in phosphorylated NPr (lane 4, 4 µg; lane 6, 8 µg; and lane 7, 16 µg). NPr was labeled even further when IIA was included (lane 9), and interestingly this was accompanied by a simultaneous reduction of the radioactivity in IIA-P (compare lanes 8 and 9). NPr was also readily phosphorylated by DTP (lane 11), most likely due to phosphoryl transfer from the IIA domain of this protein. The recombinant IIA of B. subtilis (Sutrina et al., 1990) also catalyzed NPr phosphorylation (lane 13). These data establish that NPr can serve as a phosphoryl acceptor from either Enzyme I or from the sugar-specific IIA proteins or protein domains, and they clearly demonstrate that NPr possesses the following two properties: its phosphorylation by Enzyme I is significantly lower than that of HPr or DTP (compare lanes 4 and 5), and the balance of phosphotransfer between NPr and Enzymes IIA including IIA, IIA, and IIA appears to favor the back reaction with net transfer of label from IIA to NPr (compare lane 4 with lanes 9, 11, and 13).


Figure 10: Phosphorylation of NPr by Enzyme I, IIA, DTP, and IIA. Panel A, the autoradiogram (a composite of SDS-polyacrylamide gels) shows P-labeled protein products that were obtained by phosphorylation reactions containing the indicated PTS proteins for the following lanes: lane 1, Enzyme I (1.4 µg); lane 2, NPr(8 µg); lane 3, HPr (4 µg); lane 4, NPr (4 µg) and Enzyme I; lane 5, Enzyme I and HPr; lane 6, NPr (8 µg), Enzyme I, and HPr; lane 7, NPr (16 µg), Enzyme I, and HPr; lane 8, IIA (4 µg), Enzyme I, and HPr; lane 9, NPr (4 µg), Enzyme I, HPr, and IIA; lane 10, DTP (5 µg) and Enzyme I; lane 11, NPr (4 µg), Enzyme I, and DTP; lane 12, IIA of B. subtilis (1.8 µg), Enzyme I, and HPr; lane 13, NPr (4 µg), IIA of B. subtilis, Enzyme I, and HPr. Panel B, an autoradiogram of nondenaturing gel showing the P-labeled proteins that were obtained by the following phosphorylation reactions: lane 1, NPr (4 µg); lane 2, Enzyme I (1.4 µg) and NPr; lane 3, Enzyme I, NPr, and HPr (0.25 µg); lane 4, Enzyme I, NPr, HPr, and IIA of B. subtilis (0.8 µg); lane 5, Enzyme I and HPr (1.25 µg). Reaction mixtures with unspecified amounts of the indicated proteins contained the amounts of proteins indicated above for previous lanes. The phosphorylation reaction was as described in the legend to Fig. 8except that [P]PEP (0.5 mM; specific activity 50,000 counts/min/nmol) was used in all reaction mixtures including the controls (lanes 2 and 3 in panel A and lane 1 of panel B).



These conclusions were further supported by electrophoretic analyses of similar phosphorylation reactions on a nondenaturing polyacrylamide gel that allowed clear resolution of NPr from HPr (Fig. 10B). As such, a relatively faint NPr-P band was apparent upon phosphorylation of NPr by Enzyme I in the absence or presence of HPr (lanes 2 and 3, respectively). Note that comparable phosphorylation of NPr and HPr required 16-fold more NPr (4 µg) than HPr (0.25 µg). By contrast, inclusion of the B. subtilis IIA protein in the phosphorylation reaction conspicuously increased phosphorylation of NPr (lane 4), which clearly surpassed the extent of phosphorylation of HPr (compare lane 4 with lanes 3 and 5).

Finally, functional complementation analyses performed with the mannitol and glucose PTS demonstrated that NPr (40 µM) could not replace HPr for the phosphorylation of mannitol and methyl alpha-glucoside (data not shown). Altogether, these results serve to characterize the phosphoryl transfer properties of the two new PTS proteins, NPr and IIA, as summarized schematically in Fig. 11.


Figure 11: Schematic summary of data from phosphorylation experiments in Fig. 8and Fig. 10drawn in context with the standard PTS phosphorelay. Arrows indicate the net direction of flux of phosphate transfer through components of the system from PEP to the sugar substrate. The dashed arrow denotes the low rate of phosphoryl transfer from Enzyme I to NPr. Abbreviations (and function): PEP, phosphoenoylpyruvate (high energy phosphate donor); Enzyme I, first general energy coupling protein; HPr, second general energy coupling protein; IIA, Enzyme IIA of a PTS permease; IIB, Enzyme IIB of a PTS permease; IIC, transmembrane channel of a PTS permease; NPr, HPr-like protein of the rpoN operon; IIA, the IIA-like protein of the rpoN operon; sugar-P, PTS substrate after phosphorylation and concomitant transport across the inner membrane. The sugar-specific Enzyme II complexes comprise domains A-C, and sometimes D, and are often arranged modularly throughout various PTS permeases (Saier and Reizer, 1994).




DISCUSSION

This paper describes the characterization of two new PTS proteins and the discovery of a genetic association between the previously unrelated operons encoding the transcription factor ^N and the essential GTPase Era. Reported herein are: 1) the nucleotide sequence of 5.56 kb of DNA including seven genes in the rpoN region of E. coli; 2) evidence suggesting that five genes beginining with rpoN are cotranscribed; 3) analyses of the seven predicted proteins and the evolutionary relationships with homologous proteins; 4) biochemical evidence demonstrating that two of the proteins encoded within this operon, IIA and NPr, are indeed new protein constituents of the PTS, and 5) data demonstrating that IIA affects temperature-sensitive growth of E. coli era mutants and functions to facilitate the assimilation of nitrogen, derived from organic sources such as alanine, particularly in the presence of preferable carbon sources.

We determined the complete nucleotide sequence of the rpoN region so as to identify and characterize genes affected by two transposon insertion mutations that alleviate the high temperature lethality of an aberrant era allele. Sequence spanning DNA from the BamHI/Sau3AI end of the Kohara phage clone 523 to a PvuII site residing 5.56 kb away, near minute 72 of the current E. coli chromosomal map, was found to contain seven genes oriented in the clockwise direction including rpoN and two PTS-like genes designated ptsN and npr (Fig. 1). Whereas some rpoN genes, such as that in Bradyrhizobium japonicum (Kullick et al., 1991), appear to be negatively autoregulated, we find no evidence for autogenous regulation in E. coli either by rpoN or by ptsN (Table 2). Using lacZ fusion constructions as promoter probes, our data support the proposal that rpoN and the following four genes are cotranscribed by a promoter located upstream of the rpoN translation start site ( Table 2and data not shown). This delimitation accommodates the promoter proposed by Jones et al.(1994). The exact location of this promoter as well as the possibility that transcription into rpoN can occur from the upstream genes awaits further investigation.

Although the two transposon mutations that suppress era interrupt two different genes of the rpoN operon, polarity studies (Table 2) and complementation analysis (Table 3) implicate one gene, ptsN, as playing a major role in suppression. This conclusion was further supported by examination of two additional rpoN region disruptions that separately interrupted only the last gene (npr::kan) or deleted the entire rpoN operon [Delta(rpoN-npr)::kan]. The former mutation did not suppress era, thus negating the possibility that suppression can be caused by a loss of NPr activity. The latter mutation did suppress, supporting the contention that ptsN is involved irregardless of involvement by rpoN. The conclusion that ptsN is largely responsible for suppression agrees with our observation that the rpoN::kan disruption (ersB2) is less active for suppression of era than is the ptsN::kan disruption (ersB1). Although possible roles for either rpoN or orf95 immediately downstream cannot be discounted with certainty, our data do not support a significant contribution by either ^N or ORF95 to era temperature sensitivity.

All seven gene products encoded within the rpoN region have been analyzed using computer-aided approaches. The upstream gene orf185 (genes are diagramed in Fig. 1) is newly defined in this report but predicts a protein that lacks significant similarity to other proteins currently included in the databases. ORF241 is an ATP-binding protein homologous to hundreds of its type which function in several capacities. While orf241 was shown to be essential in R. meliloti (Albright et al., 1989) the essentialities of orf241, or orf185 just upstream, have not yet been established for E. coli. In this respect, the viabilty of our Delta(rpoN-npr)::kan mutant on LB medium suggests that the rpoN operon as a whole is not essential.

^N is well characterized functionally, and 19 genes encoding this protein in various bacterial species have been sequenced. Analyses have revealed their regions of relative conservation as well as their phylogenies. All homologous members of this family apparently serve a single function, namely to direct RNA polymerase to a specific class of promoters. The amino acid sequence predicted by us for ^N differs slightly from two previous predictions (Sasse-Dwight and Gralla, 1990; Imaishi et al., 1993), but agrees with a recent submission by Jones et al.(1994) (see ``Results'' for details). With regard to the known role of ^N in nitrogen gene expression, our rpoN disruption mutant ersB2::kan differs slightly from the classical mutation glnF208::Tn10 by being leaky with respect to the requirement of glutamine on glucose minimal plates (Table 4). One possible explanation is that Gln leakiness reflects residual activity of the ^N protein truncated by this mutation. Interestingly, ^N protein truncated by the ersB2 insertion would not contain the conserved ``RpoN box'' motif.

Protein ORF95, encoded by orf95 just downstream of rpoN, is homologous to a set of proteins, all but one of which are encoded by genes immediately downstream of rpoN in various Gram-negative bacteria. The one exception is a protein encoded by a gene, orf113 of E. coli, found immediately upstream of the pheA gene (Hudson and Davidson, 1984). The homologous B. japonicum protein is considerably larger (203 residues) than other protein members of this family (95-130 residues) suggesting the presence of an additional functional domain in this protein. Since this organism has two unlinked rpoN operons of different structure exhibiting different regulatory properties, and only one of these operons (the rpoN(2) operon) is known to contain the orf95 homologue (Kullick et al., 1991), B. japonicum may provide a good subject for further investigation into the biological significance of the ORF95 protein and its possible relation to ^N.

The protein encoded by ptsN (orf163) is homologous to the fructose- and mannitol-specific IIA domains. The greatest degree of sequence similarity among these proteins is found within the region surrounding the histidyl phosphorylation site. This fact led to the suggestion that the product of this gene may be phosphorylated by Enzyme I and HPr using PEP as the phosphoryl donor (Reizer et al., 1992). In fact, following its overproduction and purification, facile phosphorylation of this protein was demonstrated in a process that depends on PEP, Enzyme I, and HPr ( Fig. 7and Fig. 8). The further demonstration of reversible phosphoryl transfer between ORF163 and other PTS proteins has allowed us to designate this protein as Enzyme IIA and its gene as ptsN.

The phylogenetic tree for the family of proteins that includes IIA of E. coli revealed that IIA exhibits comparable degrees of similarity to IIA and IIA of the same organism. It is, however, more divergent from all previously characterized sugar-specific IIA proteins than from other ptsN homologs encoded within bacterial rpoN operons (Fig. 5). This fact correlates with our observations that IIA cannot substitute under in vitro conditions for either of these two homologous proteins in the phosphoryl transfer reactions catalyzed by them. These findings strongly suggest that the function of IIA in vivo does not concern the phosphorylation of fructose or mannitol. Genetic tests further supported this conclusion.

ORF284, encoded by the penultimate gene of the rpoN operon, contains a canonical ATP binding motif. This motif provides the only available clue as to the function of ORF284. A function dependent on ATP is therefore proposed, but what this function is has yet to be determined.

The protein encoded by the last gene of the rpoN operon, npr (orf90), encodes a protein we have designated NPr because of its HPr-like structure and activity. NPr clusters together with the A. eutrophus HPr-like protein (Fig. 6B) that is believed to function exclusively in a regulatory capacity. This observation suggests that NPr may similarly function in a regulatory capacity. The fact that the E. coli Enzyme I phosphorylates NPr much less efficiently than it does HPr or the HPr-like domain of DTP (FPr) may suggest that NPr phosphorylation requires a distinct Enzyme I not yet identified. Additionally, efficient transfer of phosphate to NPr from IIA, IIA, or IIA (Fig. 10) indicates that one of these phosphoryl transfer pathways may be of physiological significance. We consider it possible that NPr may not be directly targeted by Enzyme I in the sequential relay of phosphate through the PTS, and that instead, the main function of NPr may be to control the state of phosphorylation of IIA (see Fig. 11).

We focused our investigation on IIA for two reasons: first, because of its involvement in the essential and unknown function regulated by Era, and second, because of its predicted roles in regulating nitrogen or carbon metabolism (Reizer et al., 1992). The data presented here (Table 3) establish that the normal occurrence of IIA exacerbates the high temperature growth defect of an era mutant. In fact, high level expression of ptsN causes cells with an era chromosomal background to acquire temperature sensitivity. All mutations preventing expression of ptsN (e.g.ersB1::kan, ersB2::kan, glnF208::Tn10, Delta(rpoN-npr)::kan) relieve the era defect. The suppression of era by loss of ptsN expression may have represented a special allelic relationship between these genes, thereby implying a narrow scope of functional interaction. However, since the ptsN null allele suppresses high temperature lethality of the tetracycline dependent era mutant rnc::DeltaTn10 (Takiff et al., 1989), suppression of Era-related temperature sensitivity is not limited to the era era::DeltaTn10 allele. On the other hand, the ersB mutations do not suppress a null mutation of era at any temperature, and therefore do not bypass the Era requirement. Altogether these results suggest that the IIA mutations compensate for effects associated with reduced Era activity at elevated temperatures. The available evidence suggests that IIA operates on Era independently of ^N-dependent glutamine gene expression, of cAMP-dependent control, and of Enzyme IIA-dependent catabolite control (see ``Results''). Era is reported to possess an autophosphorylation activity (Sood et al., 1994), and consequently the possibility arises that phosphotransfer activity of IIA may affect the phosphorylation state of Era. Additionally, since both Era (March et al., 1988) and IIA are likely to function in association with the inner membrane, this co-localization may assist possible signaling between the two proteins.

We also investigated the effect of the ptsN mutation in the wild-type (era) cell. In looking specifically for a functional association between rpoN and ptsN, we found no convincing connection between ptsN and ^N-dependent induction of the glnAp2 promoter in E. coli ( Table 4and Table 5). Two other investigations have revealed the effects of ptsN null mutations on ^N-dependent gene expression and nitrogen metabolism in Klebsiella pneumoniae and P. aeruginosa and have drawn opposite conclusions with respect to glutamine regulation. Merrick and Coppard(1989) suggest that the normal presence of ptsN has a mild negative effect on the induction of glutamine synthetase and a stronger negative role in the regulation of nitrogen fixation genes. Jin et al.(1994) show that ptsN has a positive role for glutamine metabolism and does not affect some other ^N-regulated genes such as those for pilin synthesis. We propose that a common function for ptsN can be identified in experiments that simultaneously control for both nitrogen and carbon sources. It is also conceivable that the ptsN gene may encode a multifunctional protein or have diverse functions among different bacterial species.

The genetic and biochemical controls that regulate nitrogen and carbon assimilation must coordinate nitrogen with carbon utilization so that the global mechanisms of carbon repression do not block the uptake and use of organic nitrogen sources. The data presented in this paper indicate that nitrogen-carbon coordination in E. coli is affected by the newly characterized PTS constituent, Enzyme IIA. Our data strongly indicate that IIA does not affect expression of glutamine synthetase, but the possibility remains that IIA may affect other modes of ^N-dependent nitrogen regulation in E. coli. By testing for an effect of IIA on nitrogen related functions, we discovered that IIA allows maximal use of alanine or adenosine when one of these compounds represents the sole source of nitrogen. The need for IIA is more evident in the presence of an aditional carbon source such as a sugar. Surprisingly, the function of IIA becomes essential when the supplement is a ``poorer'' carbon source such as a tricarboxylic acid cycle intermediate ( Table 4and Table 6). It appears, in fact, that E. coli growth varied directly with the level of IIA under these conditions, i.e. two copies of ptsN are better than one (Table 6). Interestingly, another study has shown that depletion of Era is associated with elevated oxidation of some tricarboxylic acid cycle substrates (Lerner and Inouye, 1991). Thus, a reciprocal relationship bewteen Era and IIA appears to be operating. Era reduces the capacity for utilization of the citric acid while IIA enhances this capacity. On complex media such as LB, in which nitrogen is available as amino acids and peptides, such coordination may be important for growth.

Finally, we propose that the known mechanisms of IIA-dependent catabolite repression do not operate in the IIA-mediated control. First, cyclic AMP did not restore growth on alanine or adenosine in the presence of the inhibitory carbon sources. This indicates a direct effect by IIA rather than an indirect effect by the PTS on adenylate cyclase. Second, growth inhibiton by glucose was mild, and moreover, similar to that by the nonmetabolizable glucose analog, methyl alpha-glucoside. By conventional understanding methyl alpha-glucoside should affect regulation through dephosphorylating the PTS proteins. Possibly, replacement of ammonium salts with organic sources of nitrogen such as alanine can circumvent the standard hierarchy of carbon repression so that glucose is no longer dominant. Alternatively, high level production of D-alanine dehydrogenase in succinate containing medium may deplete cells of their natural pool of D-alanine thereby preventing synthesis of cell wall material and consequently inhibiting growth.

The work presented in this report demonstrates that Enzyme IIA of the phosphotransferase system affects the unknown but essential activity governed by Era, and therefore suggests that functions regulated by these two proteins converge on a common pathway. Future investigations of other suppressors of era that are unlinked to rpoN may reveal whether the physiological state of nitrogen and carbon metabolism influences Era or if the interactive role of IIA involves some other cellular process.


FOOTNOTES

*
This work was supported by grants from the Ministry of Education, Science and Culture, Japan (to Y. N.), the Human Frontier Science Program (to Y. N.), the Invitation Fellowship/Grant Program of the Ministry of Education, Science and Culture, Japan (to B. S. P.), United States Public Health Service Grants 5RO1AI 21702 and 2RO1AI 14176 from the National Institute of Allergy and Infectious Diseases (to M. H. S.) from the National Cancer Institute, Department of Health and Human Services, under Contract No. NO1-CO74101 with ABL. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is dedicated in memory of Eun Ei Yu (``Mio'') whose technical assistance aided the discovery of the nitrogen-limiting carbon repression phenotype.

§
To whom correspondence should be addressed.

Present address: Dept. of Molecular Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya 464-01 Japan.

(^1)
The abbreviations used are: PTS, phosphoenolpyruvate:sugar phosphotransferase system; PEP, phosphoenolpyruvate; kb, kilobase(s); PCR, polymerase chain reaction; DTP; diphosphoryl transfer protein; ORF, open reading frame; alphaMG, methyl-alpha-glucopyranoside.

(^2)
J. Reizer, unpublished data.

(^3)
H. Takiff and D. Court, unpublished results.

(^4)
B. Powell and D. Court, unpublished results.


ACKNOWLEDGEMENTS

We thank B. Bender, S. Brown, S. Kustu, B. Magasanik, A. Ninfa, L. Reitzer, and R. Simons for insightful discussions and for gifts of strains, phages and plasmids. We are grateful to T. Sugimoto, S. Sugano, H. Ikeda, and other members of the Institute of Medical Science for their many accommodations. Mio's contributions to this work are deeply appreciated. We are grateful to Mary Beth Hiller for her assistance in the preparation of this manuscript.

Note Added in Proof-The entire sequence of the gene encoding the previously designated 13.6-kDa hypothetical protein of B. subtilis (ORF117; SWISSPROT identifier P28368) was recently published (Chen, L., and Helmann, J. D.(1994) J. Bacteriol.176, 3093-3101). Its protein product (ORF189; 21.98 kDa) now proves to be homologous to ORF121 of Staphylococcus carnosus (GENEBANK accession no. X79725) since the two proteins exhibit 50% identity in 117 overlapping residues (comparison score 33 S.D.). ORF189 and ORF121 are encoded by genes located immediately upstream of the secA genes of B. subtilis and S. carnosus, respectively. The calculated comparison scores of ORF189 with the 11 members of the ORF95 family (9-24 S.D.) and with ORF121 of S. carnosus establish that all of these proteins arose from a common ancestor.


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