(Received for publication, September 7, 1994)
From the
Two rpoN-linked Tn10-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.
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) ()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
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.
, 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.
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
-initiated transcription. To date,
-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
-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
-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.
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
Tn10::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
Tn10::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
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
Tn10 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.
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
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
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).
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
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
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
8.5 cm, 10-ml bed volume) which
was developed with a salt concentration gradient (400 ml, 0-0.35 M NaCl) in TDPG.
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
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).
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 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
Tn10-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) (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
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.
Figure 3:
Average similarity plot (A) and
phylogenetic tree (B) of the 19 sequenced RpoN
() 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.
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-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.
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.
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--hydroxybutyrate metabolism (Pries et al., 1991).
This close relationship of NPr(Eco) with HPr(Aeu) may suggest a
regulatory role for NPr.
In the
wild-type strain, constructs that fused sequences upstream of rpoN to lacZ (BP125 and
BP124) resulted in the
highest levels of
-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
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
-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 Tn10-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
Tn10-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.
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::Tn10, was
also tested. In the rnc40::
Tn10 mutant, normal
RNaseIII-dependent transcriptional regulation is severed by the
placement of a
Tn10 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, (
)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.
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 -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.
-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
-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.
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
(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--glucopyranoside (
MG). 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
MG required the use of both carbon and nitrogen
from alanine. However, since growth with
MG 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 MG as
would be predicted if standard catabolite repression were involved.
Surprisingly, neither 5 mM cAMP nor 0.2%
MG 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
MG.
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.
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
, 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-
137 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).
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 -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).
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
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
[
(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
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 (rpoN-npr)::kan mutant on LB medium
suggests that the rpoN operon as a whole is not essential.
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
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
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
protein truncated by this mutation. Interestingly,
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 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
.
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,
(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
::
Tn10 (Takiff et al., 1989), suppression of Era-related
temperature sensitivity is not limited to the era
era
::
Tn10 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
-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
-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
-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
-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
-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
-glucoside. By conventional understanding methyl
-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.
This work is dedicated in memory of Eun Ei Yu (``Mio'') whose technical assistance aided the discovery of the nitrogen-limiting carbon repression phenotype.
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.