INTRODUCTION

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The nitrogen assimilation control protein (NAC) of Klebsiella aerogenes plays an important role in regulating the nitrogen metabolism of this enteric bacterium (4, 25, 40). NAC allows the coupling of operons transcribed by RNA polymerase carrying ⁷⁰ to the nitrogen regulatory (Ntr) system (26), which uses RNA polymerase carrying ⁵⁴. In brief, nitrogen-limited growth leads to a starvation for glutamine. Through a complex cascade of events, glutamine starvation leads to phosphorylation (and activation) of the transcriptional regulator NtrC. Phosphorylated NtrC activates RNA polymerase carrying ⁵⁴ to transcribe a number of genes, one of which is nac, which codes for NAC. NAC in turn activates RNA polymerase carrying ⁷⁰ to transcribe a number of operons whose products can supply the cell with ammonium or glutamate from alternative organic sources (4). NAC also represses operons whose function is to assimilate ammonium when ammonium is present in abundance (4). The operons activated by NAC in K. aerogenes include hutUH, putP, and ureDABCEFG, which code for enzymes required for the catabolism of histidine, proline, and urea, respectively. The operons repressed by NAC include gdhA (glutamate dehydrogenase [GDH]), gltBD (glutamate synthase), and nac itself (4, 16, 25).

Using the hutUH operon as a model system, we have learned that NAC is both necessary and sufficient to activate transcription from a ⁷⁰-dependent promoter, that no coeffector is needed for NAC's activity, and that a NAC-binding site placed at 64 relative to the start of transcription can activate RNA polymerase even at the lacZ promoter (19, 36, 40). NAC is a member of the LysR family of transcriptional regulators (41), which includes over 50 members. NAC is a typical member of this family, with two differences: NAC does not require a coeffector for its actions, and the nac gene is not divergently transcribed from an operon that it regulates.

Although NAC plays an important role in the nitrogen regulation of K. aerogenes, Salmonella typhimurium appears to lack a functional nac gene. The hutUH operon of S. typhimurium does not respond to nitrogen starvation unless it is moved to a K. aerogenes cytoplasm (34) or unless a nac⁺ plasmid from K. aerogenes is present in S. typhimurium (6).

The existence of a nac gene in Escherichia coli K-12 has remained an open question. Many of the NAC-regulated operons of K. aerogenes either are absent from E. coli (hut and ure) or do not respond to nitrogen regulation (put and gdh) (45). When the hutUH operon from K. aerogenes (or S. typhimurium) was transferred to an E. coli cytoplasm, hutUH expression was nitrogen regulated but the degree of that regulation was much less than is seen in K. aerogenes (18). In other words, the fact that hutUH shows some nitrogen regulation in E. coli suggests the presence of an active nac gene, but the weakness of that regulation and the lack of NAC-regulated targets is suggestive of the absence of an active nac gene. The data presented here show that S. typhimurium does indeed lack a nac gene and that E. coli has a nac gene that is a fully functional analog of the nac gene from K. aerogenes.

	MATERIALS AND METHODS

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Strains and plasmids. All K. aerogenes and E. coli strains used in this study are derived from W70 and K-12 respectively, and are listed in Table 1.

Media and chemicals. Strains were grown in W4 salts (W salts adjusted to an initial pH of 7.4 [25]) supplemented with carbon and nitrogen sources at 0.4 and 0.2% (wt/vol), respectively, or in rich LB medium (29). TB medium (44) was used for plasmid isolation. Media for plasmid-bearing strains were supplemented with ampicillin (100 µg/ml), kanamycin sulfate (50 µg/ml), or tetracycline (25 µg/ml) as indicated. Glutamine was always Calbiochem A grade. IPTG (isopropyl--D-thiogalactopyranoside) was from Sigma Chemical Company. Sequencing reagents (Sequenase) were from United States Biochemicals, and [-³²P]dATP at 3,000 Ci/mmol was from ICN Pharmaceuticals.

Genetic techniques. Recombinant DNA techniques were carried out essentially as described by Maniatis et al. (27). DNA was often purified by separating digested DNA fragments in agarose buffered with TAE (27) buffer and then recovering fragments by use of glass resin or by electroelution and precipitation with ethanol. Plasmid constructs were often passed through E. coli DH5 as a host, and in all cases, only secondary transformants were studied. Strains were made competent for transformation by treatment with calcium chloride (30). Transduction using phage P1vir was performed as described previously (17).

DNA sequence analysis. DNA nucleotide sequence was determined by the chain-terminating method, using modified T7 DNA polymerase (Sequenase) on double-stranded templates, with the following modifications to the protocol supplied by the manufacturer. Strand denaturation by NaOH was replaced by heat denaturation. The reactions mixture, containing 1 µg of plasmid DNA, 50 pmol of primer (usually 17- to 20-bp oligomer), and water to a final volume of 10 µl, was heated to 94°C for 3 min, followed by quick cooling on ice. Reaction buffer was added after denaturation, and subsequent steps followed the Sequenase protocol. Overlapping sequences were determined for both strands with pCB511 and pCB524 as templates. The sequencing strategy involved primer walking, in which new primers were synthesized by using sequence determined by the results of the previous sequence determination.

Enzyme assays. All assays were performed with cells that had been washed in 1% KCl and suspended at a concentration that contained 1 to 1.5 mg of protein per ml. Most assays were performed with cells permeabilized by hexadecyltrimethylammonium bromide. Toluene was used to make cells permeable for proline oxidase assays. Assays for histidase (37), GDH (7), glutamate synthase (28), urease (25), and proline oxidase (37) have been described. Histidase, glutamate synthase, and GDH activities were measured at 37°C for historical reasons; all other enzymatic activities were measured at 30°C. Specific activities are reported as nanomoles of product formed or substrate consumed per minute per milligram of total cell protein. Cell protein was measured by the method of Lowry et al. (24) with bovine serum albumin as the standard, except that whole cells suspended in 1% KCl were added directly to the test mixture without prior disruption.

Complementation of K. aerogenes nac with genomic clones from E. coli. Derivatives of several of the members of the Kohara E. coli genomic phage library (22) (miniset 345-348, generously provided by F. Neidhardt) that carried a kanamycin resistance (Km^r) gene were isolated as described by Henry and Cronan (20). To insert these derivatives, a K. aerogenes host strain required a preexisting  prophage to provide sufficient homology for recombination. Since K. aerogenes lacks the  receptor and an att site, plasmid pTROY11 was used to transform several stains to  sensitivity as described previously (12). To provide a selectable marker for lysogeny, phage pgal8 (14) (provided by K. McKenny) was used to transduce strain KC2330 to Gal⁺. The details of this lysogeny will be described elsewhere (35). The resulting pgal8-containing strains were used as recipients for transduction by derivatized (Km^r) Kohara phage  clones.

Southern hybridizations. Chromosomal DNA from E. coli, S. typhimurium, and K. aerogenes was prepared from log-phase cultures by using a Puregene DNA isolation kit (Gentra Systems Inc.). Phage DNA from the Kohara miniset members was prepared as described by Chisholm (10). Restriction digests were performed with enzymes supplied by Boehringer Mannheim. DNA fragments were separated by electrophoresis on an 0.8% agarose gel buffered with Tris-borate-EDTA (TBE) and blotted as described by Maniatis et al. (27). Hybridization with probe prepared by random-primed labeling of the 920-bp AflII fragment from pCJ5 (6) containing the K. aerogenes nac gene was performed as described previously (27), varying the hybridization and wash temperatures between 50 and 65°C.

Mutagenesis of E. coli nac. Using the cloned nac gene on plasmid pCB511, we introduced various disruptions into the coding sequence. Initially, a Km^r gene cartridge from plasmid pWW97 (31) was introduced as a BamHI fragment into the compatible BglII site internal to nac (see Fig. 2). An amber stop codon linker (SpeI; New England Biolabs) was introduced into the SmaI site present in the cartridge's multiple cloning site (just 3' to the now destroyed BglII site) to create plasmid pCB529. This plasmid produces a truncated NAC of 169 amino acids, 165 amino acids from NAC and 4 amino acids provided by the multiple cloning site from the BglII site to the stop codon. This construct was used to make the nac-10 allele. A further and more complete disruption was made by deleting the region between the SacI and PvuII sites of this plasmid to delete the promoter and 5' end (N terminus) of the nac gene. This construct, on plasmid pCB547, was nac-28. Replacement of the chromosomal nac gene with the disrupted gene was achieved following electroporation of linearized plasmid DNA into a recD strain of E. coli (32). These Km^r-tagged nac alleles were immediately transferred into stable host backgrounds by P1vir-mediated transduction of various E. coli host strains.

Mobility shift retardation assays. Mobility shift assays were performed with purified K. aerogenes NAC protein. Pure NAC protein was isolated as described by Goss and Bender (19). DNA targets were prepared by digestion and precipitation followed by resuspension in Tris-EDTA (TE). Radioactively labeled targets were prepared by filling in the overhanging 5' ends with [-³²P]dATP (ICN Pharmaceuticals) and the Klenow fragment of DNA polymerase I. Reaction mixtures contained 1 µl of DNA, 1 µl of poly(dI-dC) (50 ng/µl), 4 µl of double-distilled H₂O, and 1 µl NAC dilution in dilution buffer (50 mM NaPO₄ [pH 7], 125 mM NaCl, 0.5 mM MgCl₂, 0.1 mM -mercaptoethanol, 50% glycerol, 1 mg of bovine serum albumin/µl). Reaction mixtures contained dilutions of NAC with 1 to 200 ng of protein/µl and were allowed to incubate with the DNA for 30 min before 1.5 µl of loading buffer (40 mM Tris [pH 8.4], 4 mM EDTA, 0.2% bromothymol blue, 0.2% xylene cyanol, 15% Ficoll) was added and the mixture was loaded for electrophoresis onto 4% acrylamide gels buffered with TE or 2% agarose gels with TBE.

Primer extension assays. Primer extension analysis was carried out as described by Ausubel et al. (1), with the following modifications: RNA was purified from log-phase cultures grown in rich broth (LB) or in W4 salts medium supplemented with 0.4% glucose and 0.2% L-arginine as the sole nitrogen source (ammonia limiting) or 0.2% ammonium sulfate plus 0.2% glutamine (ammonia excess). A template of 25 µg of total RNA was used in each reaction. A modified reverse transcriptase (Superscript II) was used in the extension reaction at 52°C. Reactions were loaded alongside DNA sequencing reactions on a 0.8% sequencing gel. The same primer was used for primer extension and for sequencing.

Expression of the E. coli NAC protein. Initial expression of the E. coli NAC protein was carried out by putting the nac gene under the control of a temperature-inducible promoter in the expression vector pJLA503 (38). This construct, pCB552, was made by PCR amplification of nac from pCB511. Plasmid pCB554 was made by cloning the -galactosidase gene from pRS415 into the EcoRI and SalI sites of pCB552 such that lacZ would be transcriptionally fused to nac and thus coexpressed when the promoter was induced by raising the temperature above 35°C.

SDS-PAGE analysis of expressed protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole-cell extracts was carried out as described by Ausubel et al. (1), using a polyacrylamide concentration of either 10, 12.5, or 15%.

Nucleotide sequence accession number. The DNA sequence has been deposited in GenBank with accession no. U56736.

	RESULTS

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E. coli carries a functional nac gene. Interspecies complementation has suggested that E. coli expresses a functional NAC but S. typhimurium does not (6, 18). To test whether E. coli and S. typhimurium possess a homolog of the K. aerogenes nac gene, we looked for hybridization between the K. aerogenes nac gene and DNA from these organisms. DNA from K. aerogenes W70 (strain KC1043), E. coli K-12 (strain W3110), and S. typhimurium 15-59 (strain NE7) was digested with EcoRI or with BamHI. After separation of the resulting fragments by agarose gel electrophoresis, a hybridization analysis by the method of Southern was carried out with an AflII fragment from within the K. aerogenes nac gene as probe. Under stringent conditions (hybridization and washes at 65°C), this 920-bp probe hybridized to a single fragment of K. aerogenes DNA but did not hybridize to any DNA fragment from E. coli or S. typhimurium. When the stringency of the hybridization and washes was reduced (58°C), we detected a single fragment from E. coli DNA but no hybridization with S. typhimurium DNA (Fig. 1). The positions of the bands suggested that the EcoRI and BamHI fragments from E. coli were about 11.6 and 5.1 kb in size. When the stringency of the hybridization and washes was reduced still further (50°C), hybridization to S. typhimurium DNA was detected but there were many fragments showing hybridization in the lanes containing DNA from each of the three organisms under these conditions (not shown). Thus, E. coli appears to have a locus with strong sequence similarity to nac from K. aerogenes; S. typhimurium appears to lack such a sequence.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Southern blot of digested chromosomal DNA probed with a 920-bp AflII fragment (carrying the K. aerogenes nac gene). Lanes 1 to 3 and 4 to 6, K. aerogenes, E. coli, and S. typhimurium, respectively, digested with EcoRI (lanes 1 to 3) and BamHI (lanes 4 to 6); lanes 7 and 8, K. aerogenes and E. coli plasmid clones (pCJ5 and pCB577, respectively) digested with BamHI.

Isolation and characterization of the E. coli nac gene. The E. coli nac gene was isolated as a 5.4-kb BamHI fragment from the Kohara miniset clone 347 in both a low-copy-number vector (pGB2) and a high-copy-number vector (pBGS9). High-copy-number plasmids carrying nac cause the cells that carry them to grow slowly, perhaps because of the high-level expression of the adjacent asnV gene (tRNA^Asn). A restriction map of this fragment is shown in Fig. 2.

View larger version (8K): [in this window] [in a new window]   FIG. 2.   E. coli nac region and clones.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   Comparison of the promoter regions from the nac genes of E. coli (top) and K. aerogenes (bottom). |, identity; , space added for better alignment; *, start of transcription as determined by primer extension.

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Primer extension analysis of the E. coli nac promoter. RNA was extracted from YMC10 cells grown in nitrogen-excess medium containing ammonium sulfate (0.2%) and glutamine (0.2%) as nitrogen sources (lane 1) and from YMC10 cells grown in nitrogen-limiting medium containing arginine (0.2%) as the nitrogen source (lane 2). A sequencing ladder was generated by using the nac containing plasmid pCB524 as template. The same oligonucleotide (5' GCTGGTTGTGCGATATGCA 3') was used as a primer for primer extension and for the sequencing reactions; thus, the sequencing ladder represents the complement of the sequence shown. The sequence at the bottom is the E. coli nac promoter, with the start of transcription indicated by the bold A.

Isolation of nac mutations. Two nac mutations were isolated by reverse genetics, taking advantage of a BglII site at the 165th codon of the nac coding sequence and a PvuII site upstream of the nac promoter. The nac-10 mutation is an insertion of a Km^r cassette from plasmid pWW97 into the BglII site of nac. Such a mutation leads to a truncated NAC with only the N-terminal 165 amino acids of NAC plus 4 amino acids encoded in the linker used to provide a stop codon. The nac-28 mutation was derived from nac-10 by deletion of the DNA from the PvuII site upstream of the nac promoter to a SacI site just inside the Km^r cassette. Thus, nac-28 is a null allele for nac that deletes the nac promoter and the entire N-terminal half of the nac coding sequence. The nac-28 mutation was crossed onto the E. coli chromosome as described in Materials and Methods, and replacement of the wild-type nac gene was confirmed by Southern blot analysis (not shown). The resulting mutant strains were tested for regulation of the K. aerogenes hutUH operon in these E. coli strains (Table 3).

Effect of nac mutations on growth rate of E. coli. In K. aerogenes, nac mutations have no phenotype except slower growth on substrates like histidine, proline, or urea that are catabolized by products of NAC-dependent operons (3). This was also seen in E. coli in that nac mutants grew significantly more slowly in glucose minimal medium with cytosine as the sole nitrogen source, with doubling times of 180 ± 5 and 235 ± 10 min for the nac⁺ and nac strains, respectively. The nac mutant also grew somewhat more slowly when either serine or threonine was the sole nitrogen source. This was readily seen as a difference in colony size, but the doubling times of nac mutants on serine or threonine were only about 5 min (about 3%) longer than the nac⁺ parent.

Effect of NAC on nac expression in E. coli. Transcription of the K. aerogenes nac gene is strongly regulated by the Ntr system in response to the nitrogen supply (25). It is also repressed by the binding of NAC to a site centered about 77 bp upstream from the start of transcription (6, 16). When the K. aerogenes nac promoter was cloned on a high-copy-number plasmid driving -galactosidase expression, the E. coli Ntr system was fully functional in regulating transcription from the K. aerogenes nac promoter (Table 5, lines 1 and 2 or 3 and 4). The chromosomally encoded NAC exerted a weak repression on nac-driven -galactosidase expression from this multicopy plasmid (Table 5, lines 2 and 4). When the E. coli nac promoter replaced that from K. aerogenes, a similar regulation in response to the nitrogen supply (Table 5, lines 5 and 6 or 7 and 8), as well as a similar repression by NAC (Table 5, lines 6 and 8), was seen. The E. coli nac promoter contains a sequence resembling the NAC-binding site from the K. aerogenes nac promoter, and this site is also in a very similar position (centered at 76). A gel mobility shift assay (Fig. 5) confirms that NAC binds to the E. coli nac promoter region, consistent with the argument that the E. coli nac gene is autogenously regulated, just like that from K. aerogenes.

View larger version (56K): [in this window] [in a new window]   FIG. 5.   Gel mobility shift assay showing the interaction of NAC with the E. coli nac region. Lanes: 1 to 4, 330-bp EcoRI-HindIII fragment containing the K. aerogenes ureD promoter incubated with 0, 0.35, 0.7, 1.7 pmol of purified K. aerogenes NAC, respectively; 5 to 8, 378-bp EcoRI-to-BamHI fragment containing the E. coli nac promoter incubated with 0, 0.35, 0.7, and 1.7 pmol of K. aerogenes NAC, respectively.

Expression of E. coli nac coding sequence. The coding sequence of E. coli nac was expressed from a plasmid expression vector with a temperature-inducible phage  p_L promoter (38). SDS-PAGE of expressed NAC showed a protein of 33 kDa as expected from the sequence. Curiously, no NAC protein was seen when strains bearing this construct were grown at 42°C. To visualize protein, the strains had to be grown at 46°C. Control experiments using this vector (38) suggested that an ample amount of protein should have been made at 42°C. To reconcile this discrepancy, a transcriptional fusion was made by placing the lacZ coding sequence immediately downstream of the nac coding sequence, creating an artificial operon with nac and lacZ cotranscribed. Expressed -galactosidase was readily detected at the lower temperatures (37 and 42°C), suggesting that both genes were being adequately transcribed. It seemed likely that either the NAC transcript was being inefficiently translated or the NAC protein was being degraded. Therefore, we compared the stabilities of the NAC and -galactosidase polypeptides. NAC and -galactosidase were produced by growing strains at 46°C for 1 h and then shifting the temperature to 30°C along with the addition of chloramphenicol and rifampin to prevent further translation and transcription. The disappearance of the NAC and -galactosidase polypeptides was monitored by SDS-PAGE (Fig. 6). The NAC bands exhibited a half-life of about 15 min following overexpression and growth at 30°C, in contrast to the more stable -galactosidase protein (half-life of >35 min).

View larger version (53K): [in this window] [in a new window]   FIG. 6.   Relative stabilities of E. coli NAC and -galactosidase. E. coli NAC and -galactosidase were cotranscribed from the temperature-inducible phage pL promoter in plasmid pCB554. Cells were grown to a density of approximately 50 Klett units, and expression was induced by growth at 45°C for 40 min. At time zero, the culture was shifted to 30°C and chloramphenicol and rifampin were added. Samples (0.2 ml) were removed at 5-min intervals and concentrated 10-fold, and 10 µl was applied to an SDS-12.5% polyacrylamide gel. Lanes: 1, 5 µg of K. aerogenes NAC as a standard; 2, sample at time zero; 3 to 9, samples at 5-min intervals; 10 and 11, E. coli NAC expressed from plasmid pCB552 (which transcribes NAC alone).

	DISCUSSION

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The data presented here show clearly that E. coli has a functional homolog of the K. aerogenes nac gene. DNA sequence analysis of E. coli nac (hereafter referred to as nac_E) predicts a protein with about 80% identity to the K. aerogenes NAC (hereafter referred to as NAC_K). Moreover, nac_E is fully able to complement a K. aerogenes nac mutant for activation of hut, put, and ure operon expression as well as for repression of gdhA expression and probably for autogenous repression of nac_K expression. On one hand, the existence of nac_E was not surprising given the close evolutionary relationship between E. coli and K. aerogenes. On the other hand, we have long known that the operons that NAC regulates in K. aerogenes are absent from E. coli (hut and ure) or are either unregulated or only slightly regulated by nitrogen in E. coli (gdhA and put). That raised the question of whether E. coli had a nac gene and, if so, what that gene did. We continue to find operons in K. aerogenes with a NAC-dependent regulation, most recently the dadAB operon which is involved in alanine catabolism (21). Again, dad is NAC regulated in K. aerogenes but not in E. coli. Nevertheless, it seems likely that as more NAC-regulated operons are discovered, some of them will be NAC regulated in E. coli as well.

Although NAC_E is a functional homolog of NAC_K, the degree of sequence divergence between NAC_E and NAC_K is surprising. Most regulatory proteins are highly conserved between these two bacteria. NtrC and Lrp are identical in the two strains, and catabolite gene activator protein and the  subunit of RNA polymerase differ by only one and two amino acids in these bacteria. Homologous LysR family members also tend to be highly conserved within the enteric bacteria. For example, the CysB proteins from E. coli and S. typhimurium are 95% identical in amino acid sequence (39). In fact, the entire LysR family shows surprising sequence conservation. NAC from K. aerogenes is 50% identical to OxyR (a regulator that senses oxidative stress) from E. coli (41). Thus, the >20% nonidentity between NAC_E and NAC_K is striking. It should be noted that there is strong sequence conservation in the amino-terminal one-third of the protein and that most of the sequence divergence is found in the carboxy-terminal two-thirds. The carboxy-terminal portion is generally thought to be important for interaction of LysR family proteins with their regulatory coeffectors (39). NAC_K appears to lack any coeffectors and to be a constitutively active transcriptional regulator (25). This may explain the lack of sequence conservation in this region. However, it must be noted that NAC_E and NAC_K both have exactly 305 amino acids and that their length is quite typical of all other LysR family members. Therefore, the carboxy-terminal domain(s) may play some role (such as stability) other than response to a coeffector signal. In support of this argument, we noted that the hydropathy plots of NAC_E and NAC_K are remarkably similar, despite the 25% divergence of the amino acid sequence in the carboxy-terminal region.

The physiological role for NAC_E remains unproved, though the data presented here suggest that NAC_E, like NAC_K, is involved in regulating the nitrogen metabolism of the cell. Several lines of evidence support this hypothesis. First, the nac-28 mutation affects the growth rate of E. coli on a variety of nitrogen sources, including arginine (faster), cytosine (slower), and serine (allowing growth of Ntr-constitutive strains). Second, even though the strong repression of gdhA by NAC seen in K. aerogenes does not occur in E. coli, a small but significant effect is detectable. In the nac-28 mutant, GDH expression increases about twofold in response to nitrogen limitation. In wild-type strains, this increase does not occur. In a gltB mutant, where glutamate formation from ammonia is completely dependent on GDH, a strain carrying nac-28 grows faster with ammonium as nitrogen source than does a nac⁺ strain, suggesting that this twofold effect on GDH expression is important. Third, the key regulatory sites in the nac_K promoter region (15, 16) are well conserved, both in sequence and in position, in the nac_E promoter region. These include the ⁵⁴-dependent promoter and the NtrC-binding enhancer sequence. Thus, it is likely that nac_E, like nac_K, is transcribed as part of the Ntr response during nitrogen limitation.

The absence of any nac-like sequences in S. typhimurium is consistent with older observations that NAC-dependent operons cannot be expressed in response to nitrogen limitation (6, 8). The absence of a nac gene in S. typhimurium also raises questions about the evolutionary plasticity of this chromosomal region in the Enterobacteriaceae. The nac_K gene is preceded by asnV, the gene that codes for tRNA^Asn, and it is followed by an apparent operon composed of cblA (orf2), itself a member of the LysR family, and orf3. The function of this operon is unknown but may be related to sulfur metabolism. In E. coli, nac is again preceded by asnV and followed by cblA, but orf3 is located upstream from nac, on the other side of asnV. In S. typhimurium, nac is not present. There is evidence that the asnV region may be where the large cluster of cob genes, encoding the enzymes of cobalamine synthesis, lies in S. typhimurium (23). (E. coli lacks this cob cluster.) Similarly, the large cluster of nif genes (encoding the enzymes of nitrogen fixation) in K. pneumoniae appear to be linked to nac. (The nif genes are not present in K. aerogenes.) In short, this region has been subject to considerable insertion or deletion during the evolutionary divergence of the enteric bacteria. It is tempting to note that asnV and asnU flank nac_E and present an 86-bp direct repeat. If no essential genes lie between these (as is seen for E. coli), simple deletion of the intervening region by asnV-asnU recombination could lead to loss of nac, but such a speculation remains untested.

In summary, E. coli has a nac gene that is a functional homolog of the nac gene from K. aerogenes, and this nac gene is probably involved in the nitrogen regulation of E. coli. The strong sequence conservation within the first 100 or so amino acids suggests an important role for the domain(s) in this region, whereas the large number of differences in the last 200 or so amino acids suggests that the domain(s) in this region are quite plastic and can withstand considerable amino acid substitution without loss of function.