The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: John R. Guest. Tel: +44 114 222 4406. Fax:. +44 114 272 8697 e-mail: j.r.guest{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: CRPFNR protein superfamily, ironsulphur proteins, transcriptional regulation, nitrogen starvation, stationary phase
Abbreviations: CRP, cAMP receptor protein; FNR, fumarate and nitrate reduction regulator; MALDI-TOF, matrix-associated laser desorption ionization-time of flight; MBP, maltose-binding protein
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INTRODUCTION |
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The complete genome of Escherichia coli (Blattner et al., 1997 ) was recently shown to encode a third member of the CRPFNR family (YeiL) when searched with a sequence profile derived from a multiple alignment of five members of the family: CRP and FNR from E. coli; FLP from L. casei; and FlpA and FlpB from Lactococcus lactis (Gostick et al., 1999
). The YeiL protein (SWISS-PROT accession no. P33023) is the 219-residue product of the 657 bp yeiL open reading frame (GenBank accession no. U00007). A BLAST analysis showed that YeiL is more closely related to members of the CRPFNR family (1622% identical, 4245% similar) than to other known proteins. A phylogenetic tree derived from 38 members of the CRPFNR family indicated that YeiL is a distant relative of the CRP group (Gostick et al., 1999
).
The yeiL gene is located in the nfofruA region of the E. coli linkage map at 48·57 min (co-ordinates 2253·38 to 2254·03 kb; Blattner et al., 1997 ), where it is flanked by a cluster of unidentified reading frames, URFs (Fig. 1
). Putative functions have here been assigned to the products of these URFs by similarity searches. They include two potential guanosine kinases, YeiC (P30235) and YeiI (P33020), which are 38% identical (61% similar), and two potential pyrimidine nucleoside transport proteins, YeiJ (P33021) and YeiM (P33024), which are 88% identical (95% similar). There is also a potential nucleoside hydrolase, YeiK (P33022), which is 40% identical (66% similar) both to YbeK (P41409; an unlinked E. coli URF) and to the inosine-uridine nucleoside hydrolase of Crithidia fasciculata (Degano et al., 1996
). At least 26 of the 27 residues surrounding the catalytic site of this trypanosomal purine salvage pathway enzyme are conserved in YeiK and YbeK. Thus it would appear that several genes involved in nucleoside metabolism are located in the nfofruA region of the E. coli chromosome together with a novel paralogue of CRP and FNR.
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METHODS |
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For carbon, phosphorus and nitrogen starvation, M9 medium was used with 0·02% glucose (normally 0·4%), 0·25 mM phosphate with 0·1 M Tris/HCl buffer (pH 7·6) and 0·2 mM NH4Cl, respectively (Davis et al., 1986 ), and the cultures (600 ml) were maintained at pH 7·4, 90% air saturation (stirring speed, 200 r.p.m.) and 37 °C in chemostat vessels for 7 d. The inocula (1%) were derived from single colonies from freshly streaked minimal plates, grown to stationary phase in M9 medium with 0·4% glucose. Serial dilutions of daily samples, plated in triplicate on pre-warmed LB agar and scored after 18 h at 37 °C, were used to determine viable counts (c.f.u. ml-1), which were then expressed as percentages relative to the 24 h values. All of the findings were reproducible in two or three independent experiments.
Protein purification.
The yeiL coding region was PCR-amplified from W3110 chromosomal DNA using forward primer S556 (5'-TTAAGGATCCCATATGAGTGAATCCGCGTTTAAGG-3') and reverse primer S559 (5'-TATGTCGACTTACGTCATCATCCCGGAGAATTTAT-3'), each incorporating flanking restriction sites. The 682 bp product was cloned initially in pGEM-T Easy (Promega) and then between the BamHISalI sites of pMal-c2 (NEB) to place it downstream and in frame with the maltose-binding protein (MBP) coding region and Factor Xa sensitive linker, to generate pGS1226. After confirmatory resequencing, pGS1226 transformants of E. coli CAG626 were used for overproduction and purification of the MBPYeiL fusion protein. Soluble MBPYeiL was purified from aerobic 2-litre cultures grown initially at 30 °C in LB plus 0·2% glucose and ampicillin to OD600 0·2, and then at 37 °C for 1 h after adding IPTG (0·3 mM). The bacteria harvested from one culture were washed and resuspended in 10 ml buffer containing 20 mM Tris/HCl (pH 7·4), 200 mM NaCl and 1 mM DTT, for ultrasonic disruption. The clarified cell-free extract was applied to an amylose column (10 ml; NEB), washed with 20 vols of the same buffer, and eluted with buffer containing 10 mM maltose.
Reconstitution of ironsulphur clusters, and biochemical assays.
The fusion protein was reconstituted using NifS from Azotobacter vinelandii according to Green et al. (1996) . Typically, 1 ml of the purified protein at a concentration of 1 mg ml-1 in a quartz cuvette was equilibrated in an anaerobic glove-box for 34 h on ice before adding DTT (2·5 mM), L-cysteine (1 mM), Fe2+ [40 mol (NH4)2Fe(SO4)2 per mol MBPYeiL monomer] and NifS (0·7 µM) to the stated final concentrations, and then incubated in the stoppered cuvette for up to 16 h at 4 °C. Spectra were obtained with a Unicam UV-2 spectrophotometer (Pye-Unicam). The reconstituted protein was desalted anaerobically, using a Sephadex G25 column to remove excess reagent. Iron and sulphur contents were determined by the methods of Woodland & Dalton (1984)
and Beinert (1983)
, respectively. Reactive thiol groups and the disulphide contents of the native protein were assayed according to Thelander (1973)
and Thannhauser et al. (1987)
, respectively.
Protein analysis, gel filtration and mass spectrometry.
Protein was assayed by the Bio-Rad procedure with bovine serum albumin as standard, and the Laemmli method (Sambrook et al., 1989 ) was used for SDS-PAGE. Native molecular masses were determined by gel filtration with a Protein-Pak 300SW column (7·8x300 mm) equilibrated with 0·2 M NaH2PO4 (pH 6·7) containing 0·2 M Na2SO4: DTT (1 mM) was added to the buffer when reducing conditions were required. Thyroglobulin, catalase, lactate dehydrogenase, bovine serum albumin, ovalbumin, chymotrypsin, ribonuclease, aprotonin and myoglobin were the standards used for calibration. N-terminal amino acid sequences were determined by Edman degradation using an Applied Biosystems protein sequencer. Accurate molecular mass measurements were made by electrospray and MALDI-TOF mass spectrometries.
Construction of a yeiLlacZ transcriptional fusion and ß-galactosidase assay.
A yeiLlacZ transcriptional fusion was constructed by PCR-amplification of the yeiL promoter region (1017 bp of upstream non-coding region and 25 bp of yeiL coding region) with two primers containing embedded EcoRI and BamHI restriction sites: S470 (5'-TTCCGGAATTCTTGCCAAGAACGCCCAG-3') and S471 (5'-TAGGCGGATCCAATCCTTAAACGCGGAT-3'). The product was cloned into pGEM-T Easy (Promega), generating pGS1145, from which the 1046 bp EcoRIBamHI promoter fragment was transferred to pRS415 (Simons et al., 1987 ) to generate pGS1224. The yeiLlacZ promoter fusion was then transferred to
RZ5 by homologous recombination to yield
G271, and monolysogenic derivatives of various hosts were selected and screened with
h80del9c,
cI90c17 and
vir. ß-Galactosidase specific activities were assayed according to Miller (1972)
using at least two independent cultures.
RNA extraction and primer extension analysis.
The hot acid phenol procedure (Aiba et al., 1981 ) with rapid cooling was used to extract total RNA from aerobic stationary-phase cultures grown in peptone broth plus 0·2% glucose. Primer extension analysis was performed with 100 µg RNA, 10 pmol primer and AMV reverse transcriptase (50 U; NEB) by a procedure that allowed continuous incorporation of [
-32P]dCTP (Cunningham et al., 1997
). The primers were: S557 (5'-TCACTCATCAATTGCTGCTT-3') for yeiL; and both S499 (5'-TCGCCGCCATCATTATAG-3') and S558 (5'-ATCCAGAATAATTTTTCTCTTTTCCA-3') for yeiK. The extension products were fractionated on 6% acrylamide/7 M urea gels alongside sequence ladders derived from pGS1109 and the corresponding primers (Cunningham et al., 1997
).
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RESULTS |
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A multiple sequence alignment of YeiL, CRP and FNR (Fig. 2) confirmed their close relationships and indicated that most of the secondary structural elements of CRP are retained by YeiL. The alignment shows that the glycine and proline residues flanking some of the ß-strands of the ß-roll in CRP are conserved in YeiL. The regions of greatest sequence similarity (P
0·001) identified by pairwise DIAGON comparisons (Staden, 1982
) between YeiL and either CRP or FNR are also indicated in Fig. 2
. These are different in each case but they represent major structural features such as the sensory domain (ß-roll) and dimer interface (
C) of CRP and the DNA-recognition helix (
F) of FNR. In addition, the presence of a characteristic helixturnhelix structure (
E
F) was strongly predicted by the GCG program HELIXTURNHELIX. However, the motifs associated with the DNA-binding specificities of FNR (E--SR) and CRP (RE--G) in
F (Guest et al., 1996
) are not conserved in YeiL, suggesting that YeiL recognizes different sites to those recognized by both FNR and CRP.
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The cysteine residues in YeiL were modelled at the corresponding positions in the X-ray structure of the cAMPCRPDNA complex (Schultz et al., 1991 ) as shown in Fig. 3(a)
. This hypothetical structure suggested that a disulphide bond could be formed between the cysteine residues (C116) at the dimer interfaces of two subunits. The measured distance between the ß carbon atoms (Cß) of C116A and C116B in the model was 4·76
, which is close to the values found in typical protein disulphides, 3·7 to 4·3
(1
=0·1 nm). The model also suggested that three of the cysteine residues (C68, C91 and C93) could potentially serve as ligands for the assembly of an ironsulphur cluster in each monomer (Fig. 3a
). The inter-molecular distances between their Cßs were measured as 10·6
(C68C91), 5
(C91C93) and 10·2
(C93C68). Then, by using the three-dimensional-database search program ASSAM (Artymiuk et al., 1994
), it was found that this arrangement of three cysteine residues is most closely matched by that associated with the [4Fe4S] cluster of porcine mitochondrial aconitase (Robbins & Stout, 1989
), where the corresponding inter-molecular distances are 8
(C358C421), 4·76
(C421C424) and 8
(C424C358). The relevant cysteine residues of YeiL and aconitase are superimposed in Fig. 3(b)
and it is predicted that such a [4Fe4S] cluster could be accommodated by YeiL. The fourth iron ligand could be provided by one of the remaining cysteine residues (C9 and C116, from the same or an adjacent subunit), by a hydrogen-bonded water molecule (as found in aconitase), or by some other residue. From these structural predictions it is conceivable that the proposed transcription regulatory activity of YeiL might be modulated by a sensory ironsulphur cluster (as in FNR), by reversible disulphide formation, or by a combination of both mechanisms. Intra-molecular disulphide bridges could be formed between the cysteine residue near the N-terminus and a centrally located cysteine residue (as in FLP) or between another pair(s) of cysteine residues. Alternatively, inter-molecular disulphide bonds could be formed between independent subunits at the dimer interface, e.g. between C116a and C116b (Fig. 3a
), or at other sites.
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As isolated, MBPYeiL contained approximately three free thiol groups and one disulphide bond per monomer (Table 2). This is consistent with its being a multimeric aggregate linked by one inter-molecular disulphide bond per monomer. However, because three or four thiols might be used for assembling the ironsulphur cluster, and because disulphide-bond formation could occur between thiols within and between monomers (including iron-liganding thiols after cluster disassembly), it is impossible to deduce the precise disposition of the five cysteine residues in YeiL. Nevertheless, YeiL clearly has the potential to assemble an ironsulphur cluster and to form intra- and inter-molecular disulphide bonds, as predicted. By analogy with FNR and FLP the DNA-binding form of YeiL might be dimeric and its activity might likewise be modulated by a sensory ironsulphur cluster, by reversible dithioldisulphide formation, or by both mechanisms.
Construction and phenotypic characterization of a yeiL mutant
The yeiL gene was disrupted by replacing a segment of the coding region by a kanR cassette (in vitro), then transferred to the E. coli chromosome (Oden et al., 1990 ), and ultimately to W3110 to generate JRG3827 (yeiL::kanR). The growth patterns of the yeiL mutant (JRG3827) and its isogenic parent (W3110) were compared under aerobic, microaerobic and anaerobic (±nitrate) conditions at 37 °C in LB broth (±glucose) and in minimal medium E containing different carbon sources. This showed that the growth yields (OD600) achieved by the parental strain were consistently 1015% higher than those of the mutant during aerobic growth in rich media and minimal media containing glucose, maltose or succinate. No differences in growth rates or other features of the growth cycle were observed between the mutant and parental strains. No complementation of the growth defect was observed with pGS1109 because this high-copy yeiL+ plasmid adversely affected the growth of both mutant and wild-type transformants, particularly in minimal media. In contrast, the growth yields (OD600 after fourfold dilution) observed in LB medium for mutant and wild-type transformants containing a low-copy plasmid (pGS1376) increased by 1014% and 2530% (respectively) relative to the untransformed host strains. However, the deleterious effects again became apparent in minimal media, where complementation varied with substrate from very weak (succinate) to insignificant (glucose) and detrimental (maltose).
Studies with a yeiLlacZ transcriptional fusion
Insights into the temporal expression and regulation of the yeiL gene were sought by constructing monolysogens containing G271 prophages in which the yeiL promoter region is fused to a lacZ reporter (see Methods). During aerobic growth in rich media yeiL expression increased 6·5-fold in stationary phase relative to late exponential phase (Fig. 6a
) and the degree of induction relative to early exponential phase may have been even greater if exponential phase inocula had been used. The reason for the reproducible two-stage increase is not known. Similar high levels of expression were observed in late-stationary-phase cultures relative to exponential-phase cultures grown in glucose (0·2%), glycerol (40 mM) and succinate (40 mM) minimal media (data not shown). However, the growth-phase-dependent response was not observed under anaerobic fermentative conditions, where the stationary-phase activities of the wild-type monlysogen (JRG3885) were about fourfold lower than those of the comparable aerobic cultures shown in Fig. 6(a)
(data not shown).
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Identification of transcription start sites for the yeiL and yeiK genes
Primer extension analysis with RNA from cultures of two fnr mutants, RK5279(pGS1109) and RK5279(G271) (also designated JRG3886), chosen for their enhanced yeiL expression and sampled at different times in early stationary phase (610 h), revealed a single extension product, which was most abundant in the 8 h sample (Fig. 7a
). This corresponded to a potential yeiL transcription start site located 65 bp upstream of the translation initiator codon and 7 bp downstream of a potential
70 or
S promoter containing plausible -35 (TTGAAT) and -10 (TATGCT) hexamers (Fig. 7b
). The -35 hexamer is overlapped by a potential FNR site, which closely matches the FNR-site consensus (TTGAT-N4-ATCAA) and its location is consistent with the observed FNR-mediated repression of yeiL (Fig. 7c
). There is a potential Lrp site (YAGHAWATTWTDCTR; Y=T or C; H=A, C or T; W=A or T; D=G, A or T; R=A or G; Cui et al., 1995
) centred at -120 bp just inside the yeiK coding region, and there are two sequences matching the IHF-site consensus (WATCAANNNNTTR; Freundlich et al., 1992
), which are consistent with the observed effects of Lrp and IHF on yeiL gene expression (Fig. 7c
). There are two overlapping regions of partial dyad symmetry centred at -62 (TTTTCTacagcaaAGAAAA) and -64·5 (TTaTtTTTtCTacAGcAAAgAaAA), one of which could conceivably represent a YeiL-binding site associated with its positive autoregulatory effect on yeiL gene expression.
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Starvation response of the yeiL mutant
A role for YeiL in starvation survival was sought because the yeiL gene was more highly expressed in the post-exponential phase. The viabilities of the yeiL::kanR mutant (JRG3827) and parent (W3110) were accordingly compared in M9 minimal medium under non-limiting and under carbon-, phosphorus- and nitrogen-limiting conditions. Under carbon- and phosphate-limiting conditions the viabilities of both strains declined in parallel over 7 d (data not shown). However, when medium containing 0·2 mM NH4Cl (1% of the normal concentration) was inoculated with bacteria that had been pre-grown for 16 h in nitrogen-sufficient medium, modest but significant and reproducible differences in viability were observed (Fig. 8). After starting at comparable values, mutant viability fell to 10% during the second day compared to 75% for the parental strain. After 3 d, mutant viability stabilized between 2% and 1% while parental viability continued to fall from 21% to 2% (Fig. 8
). In comparable studies with a JRG3827 transformant containing the low-copy yeiL+ plasmid (pGS1376), viability was partially restored from 10% to 63% (day 2) and 2% to 12% (day 3). It was therefore concluded that YeiL performs a direct or indirect role in maintaining mid-term, but not long-term, stationary-phase viability under N-starvation.
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DISCUSSION |
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The problems experienced in amplifying YeiL as a soluble protein or fusion protein, the inherent instability of isolated MBPYeiL, and the presence of GroEL as the main contaminant, may be functionally significant. They might indicate that considerable chaperone activity is needed to promote correct assembly of YeiL when overproduced. On the other hand they could mean that YeiL resembles the heat-shock sigma factor (32) in being associated with chaperones (DnaJ, DnaK, GrpE) which promote instability in unstressed bacteria, or in becoming resistant to proteolysis and hence functional when dissociated from chaperones in a stressed host (Gamer et al., 1996
; Straus et al., 1990
).
32 is also responsible for the induction of heat-shock proteins including GroEL under starvation conditions (VanBogelen & Neidhardt, 1990
), so the association of MBPYeiL with GroEL may serve as a post-translational mechanism for controlling the concentration of functional YeiL during starvation. Alternatively, YeiL might resemble the temperature-sensing TlpA protein of Salmonella typhimurium, which is a DNA-binding regulator that can shift between unfolded monomeric and folded oligomeric states in response to changes in temperature by a process that involves inter-molecular disulphide formation (Hurme et al., 1997
). However, the occurrence of the oligomeric form of TlpA at normal physiological concentrations is uncertain and the same might apply to the multimeric form of YeiL.
Clues about the role of YeiL came from studying the consequences of inactivating the yeiL gene and the use of a yeiLlacZ reporter fusion to investigate yeiL gene expression under different physiological conditions and in different hosts. Studies with the yeiL::kanR mutant (JRG3287) revealed a small but significant and reproducible deficiency in aerobic growth yield in several media and a remarkable enhancement of growth yield in rich medium by low-copy (but not high-copy) yeiL+ plasmids, especially in the wild-type host. Although the apparent increase in growth efficiency was not observed under all growth conditions it deserves further investigation as a mechanism for enhancing biotechnological productivity. The mutant also exhibited a specific deficiency in nitrogen-starvation survival, which was partially complemented by supplying yeiL+ in trans. It is not clear how the two features of the mutant phenotype are related. A role in the aerobic stationary phase was inferred from the very significant post-exponential increase in yeiL gene expression in rich (Fig. 6a) and minimal media. This view was strengthened by finding that yeiL expression is significantly dependent on RpoS (
S), the stationary-phase sigma factor (Kolter et al., 1993
), which is also essential for starvation survival (Lange & Hengge-Aronis, 1991
). The requirements for Lrp and IHF (especially in the absence of FNR) are consistent with their greater abundance in slow-growing or stationary-phase organisms (Landgraf et al., 1996
; Goosen & van de Putte, 1995
). No rationale can be offered at present for the positive autoregulatory effect of YeiL on yeiL gene expression, nor for the aerobic repression by FNR, be it direct or indirect. However, the fact that expression of the rpoS, lrp and ihf genes is stimulated by ppGpp (Aviv et al., 1994
; Lange et al., 1995
) and that many genes induced by carbon, phosphorus and nitrogen starvation are regulated by the stringent response (Nyström, 1995
; Rao et al., 1998
), is consistent with YeiL being involved in the response to nutrient starvation.
Although the nitrogen-starvation-survival defect may not be a direct consequence of yeiL inactivation, it may be significant that yeiL is located within a cluster of genes concerned with nucleoside metabolism. Indeed, yeiL and the adjacent yeiK gene, encoding a potential purine salvage pathway enzyme, are divergently transcribed from overlapping promoters, suggesting that YeiL might regulate yeiK expression as well as autoregulate its own synthesis. Salvage pathways recycle nucleosides and nucleobases for nucleic acid synthesis but these intermediates can also serve as carbon and nitrogen sources. It is therefore conceivable that YeiL might control expression of the salvage pathways or in some other way repress the recycling of nucleobases to nucleic acids and enhance their use as general nitrogen sources, during nitrogen-limited growth. Lack of YeiL might exacerbate the nitrogen deficiency and thus be responsible for the modest nitrogen-starvation-survival defect of the yeiL mutant. The toxic effects of excess YeiL might likewise be due (at least in part) to repression of the salvage pathways. There is no clear connection between the presence of potential ironsulphur or disulphide-dithiol redox sensors in YeiL, and post-exponential growth and nitrogen metabolism. Regulator defects often generate pleiotropic phenotypes and it is hoped that further studies will reveal the basis of the relatively diverse and subtle changes observed here. The curious and as yet unexplained fourfold stationary-phase activation/deactivation of overproduced apo-aconitase A observed by Prodromou et al. (1991) may be related insofar as it indicates that ironsulphur cluster assembly/disassembly is occurring when YeiL is being expressed and/or activated.
This work has laid a foundation for future investigations aimed at defining the precise role of the third member of the CRP family in E. coli. Further studies will be needed to elucidate exactly how extra YeiL enhances growth yield whereas excess YeiL is toxic, and also how YeiL regulates yeiL and yeiK gene expression. It will also be important to identify other potential members of the YeiL regulon and to find ways of stabilizing the free protein in order to define the nature of the DNA-binding form, the nucleotide sequence to which it binds, and the physiological signal that is recognized, as well as to establish whether the ironsulphur cluster and disulphidedithiol formation participate in DNA-binding or signal recognition.
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ACKNOWLEDGEMENTS |
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Received 22 June 2000;
revised 17 August 2000;
accepted 22 August 2000.