INTRODUCTION AND OVERVIEW

Top Previous Next References

Scope

The dissociable sigma subunits of RNA polymerase are responsible for specific binding to DNA and are therefore important determinants of differential gene expression (68, 170). ⁵⁴ was discovered during an analysis of glutamine synthetase and nitrogen assimilation in enteric bacteria (71, 77). Subsequent studies have confirmed its role in nitrogen assimilation but have also shown that it is involved in a variety of seemingly unrelated functions, such as carbon source utilization, certain fermentation pathways, flagellar synthesis, and bacterial virulence (97, 151). There have been several reviews about ⁵⁴ and ⁵⁴-dependent activation, including recent ones (14, 28, 97, 110, 113, 148, 151). However, discussions of ⁵⁴ function usually consider genes from several organisms and therefore remove ⁵⁴ from the metabolic or physiological context of a single organism. The primary purpose of this review is to redress this imbalance and to discuss the functions of the known ⁵⁴-dependent genes from a single organism, Escherichia coli. The most meaningful discussion of ⁵⁴ function requires a complete set of ⁵⁴-dependent genes. Two sources provide information on likely ⁵⁴-dependent promoters: a recent DNA microarray analysis of transcripts present during nitrogen-limited growth (176) and computer analysis of potential ⁵⁴-dependent promoters, which are readily identified from the completed nucleotide sequence of the E. coli genome (23). Results of these analyses are integrated into the discussion of the function of the known ⁵⁴-dependent genes. The central thesis of this review is that the ⁵⁴-dependent genes of E. coli have only a few metabolic themes and that these themes may be related.

E. coli has seven  subunits, and each has a distinct function. ⁷⁰ is considered the primary sigma factor. Core RNA polymerase (E) associated with ⁷⁰ initiates transcription of housekeeping genes (68). E⁷⁰ also initiates the transcription of nonessential genes that are induced in specialized environments. ^S has been called either an alternative sigma factor or a second primary sigma factor (69). Although E^S binds the same sequences as E⁷⁰ (unpublished results cited in reference 69), ^S is considered a general stress factor since it is associated with a variety of growth-impairing stresses: nutrient depletion, oxidative stress, high temperature, high osmolarity, acidic pH, or exposure to ethanol (69). ³² and ^E are also associated with stress. ³² is required for the response to damage of cytoplasmic proteins, which is most commonly associated with heat shock, and ^E controls the response to extracytoplasmic or extreme heat stress (174). ^FecI and ²⁸ (FliA) are required for synthesis of the ferric citrate transporter and flagella, respectively (8, 115). As mentioned above, ⁵⁴ is usually associated with nitrogen assimilation.

The  factors in E. coli are homologous to ⁷⁰, except for ⁵⁴ (110). Not surprisingly, ⁵⁴-dependent transcription has several distinctive features (reviewed in reference 28). Core RNA polymerase (E) complexed to a ⁷⁰-like factor can be sufficient for open promoter complex formation. In contrast, E⁵⁴ catalyzes strand separation only with the help of a distinct class of transcriptional activators. A consequence of this property is that transcription can be completely turned off. Such absolute control may account for the evolutionary persistence of ⁵⁴ (see "Physiological function of ⁵⁴" below).

The activators of E⁵⁴-dependent genes are unusual. (The individual E. coli activators are discussed in a separate section.) Unlike most eubacterial transcriptional activators, the ⁵⁴-dependent activators bind to sites that are effective regardless of distance and orientation (28, 29, 131). In this respect, the activator binding sites are analogous to eukaryotic enhancers, and the activators are often called enhancer binding proteins. However, the analogy to eukaryotic enhancer binding proteins is not precise, since the bacterial proteins do not enhance basal transcription but, instead, are absolutely required for any transcription. The activators interact with E⁵⁴ from these binding sites. This interaction sometimes requires a DNA bending protein. The DNA bending proteins that participate in ⁵⁴-dependent gene expression in E. coli are integration host factor (IHF) and ArgR, the arginine repressor (72, 103). When transcription from a ⁵⁴-dependent promoter does not require a DNA bending protein, DNA curvature facilitates the interaction between the activator and RNA polymerase (32). Another distinctive feature of ⁵⁴-dependent activators is an essential ATPase activity (166).

The most important control of ⁵⁴-dependent genes is through modulation of the activator's ATPase activity. The ⁵⁴-dependent activators usually contain a regulatory domain that controls ATPase activity. Several mechanisms control the interaction of the regulatory domain with the ATP binding domain: phosphorylation, interaction with a low-molecular-weight ligand, or interaction with one or several regulatory proteins (148). Some control mechanisms are extremely complex, such as that for PspF (discussed below). Variations in ⁵⁴ activity, either by ligand binding or by covalent modification, are not known in E. coli. Furthermore, the intracellular level of ⁵⁴ is apparently constant (87), and expression of rpoN, which specifies ⁵⁴, appears to be constitutive (33). Strain W3110 contains about 700 molecules of ⁷⁰ per cell and 110 molecules of ⁵⁴, whereas strain MC4100 may contain only about 285 molecules of ⁷⁰ and as few as 13 to 22 molecules of ⁵⁴ (87). The low level of ⁵⁴ could have regulatory implications. For example, it is possible that different ⁵⁴-dependent operons compete for limiting ⁵⁴. This possibility is even more plausible if there is some physiological relationship between the ⁵⁴-dependent genes. One purpose of this review is to explore this issue.

We will use the promoters for these 17 operons to characterize the ⁵⁴-dependent promoters in E. coli (Table 1). These promoters suggest an apparent consensus of aaN₃TGGCAcN₆TGCNNt, where small letters indicate two to five mismatches, capital letters denote zero or one mismatch, and underlined bases have no mismatches. By similar criteria, the consensus derived from 186 promoters from a variety of organisms is N₅tGGcacN₅ttGC (14), which is similar but not identical to the apparent E. coli consensus. Figure 1 shows the locations of the ⁵⁴-dependent operons within the context of neighboring genes, the size of the transcripts, and the direction of expression. Figure 2 shows the binding sites for E⁵⁴ in relation to the binding sites for the ⁵⁴-dependent activators (when known) and the nearest upstream structural gene.

View this table: [in this window] [in a new window]   TABLE 1.   E. coli operons with confirmed 54-dependent promoters

View larger version (33K): [in this window] [in a new window]   FIG. 1.   54-dependent genes in E. coli. The known 54-dependent operons and their transcripts are shown in the context of neighboring genes. The open and solid boxes indicate counterclockwise and clockwise transcription, respectively. If the gene has been assigned a function, this is indicated by the gene name underneath. Boxes without gene names indicate that the gene has an unknown function. The sizes of the genes and their intergenic regions are to scale.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Regulatory regions of the characterized 54-dependent genes. The following features are shown for each of the 54-dependent promoters: binding sites for activators, DNA bending proteins (when required), and RNA polymerase. Solid boxes indicate that the binding site has been demonstrated. A hatched box indicates a confirmed binding site, but binding is weak. An open box signifies a proposed binding site. The relative location and orientation of the nearest upstream gene are also shown. All diagrams are to the same scale, except for the hyp operon.

Several generalizations can be made concerning these promoters. First, all the known ⁵⁴-dependent promoters are located outside the structural genes. This does not necessarily mean that an authentic binding site for E⁵⁴ will not be found within a gene, but it does mean that such binding sites will be the exception. Second, the activator binding sites are also outside the structural genes. The most spectacular example is the FhlA binding site for the hyp operon in the hyp-hyc regulatory region. The entire hycA gene is located between the binding sites for FhlA and RNA polymerase (Fig. 2). Even though the FhlA binding site for the hyp operon is within the hyc operon, it is not within a structural gene. Instead, it is completely located within the 130-base intergenic region between hycA and hycB. Third, the activator binding sites are almost always a significant distance from the adjacent upstream gene and therefore do not apparently interfere with expression of these genes (Fig. 2). The only exception is the binding site for PspF, which activates the pspABCDE operon. The PspF sites for activation overlap with the promoter for the pspF gene (Fig. 2), and it has been proposed that this has physiological significance (see "psp operon and phage shock response" below). The average size of the intergenic region that contains a known ⁵⁴-dependent promoter is 267 ± 106 bases, with a range from 148 to 507 bases. This distance is apparently large enough for binding sites for both E⁵⁴ and an activator. Fourth, the distance from the 3' end of the E⁵⁴ binding site to the nucleotides coding for the initiation codon is, on average, 50 bases. Such a short distance reduces the potential for RNA secondary structures or protein binding regions near the translational start site and therefore reduces the potential for translational control. Finally, the average A+T content of the 50 bases just upstream from the E⁵⁴ binding site is 70%, and no upstream region has an AT content less than 50%. Two rationales for such a bias can be suggested. Some ⁵⁴-dependent promoters require a DNA bending protein. Both of the DNA bending proteins that facilitate the activation of ⁵⁴-dependent genes in E. coli, IHF and ArgR, have an AT-rich consensus sequence. Alternately, ⁵⁴-dependent promoters that do not require a DNA bending protein often have an intrinsic bend between the activator and RNA polymerase binding sites (32), and this may favor AT-rich regions. In either case, the requirement for long-range protein-protein interactions appears to bias the base composition of DNA just upstream from the E⁵⁴ binding site.

Despite the reasonably uniform properties of the known ⁵⁴-dependent promoters, there is little useful information for promoter prediction. For uncharacterized promoters, the activator binding sites will probably not be known, and there may be uncertainty in the size of intergenic regions if the protein coding regions have been misidentified (which is not uncommon). A more useful predictor of potential promoters is based on computer identification of binding sites for E⁵⁴, which is described in the next section.

COMPUTER IDENTIFICATION OF POTENTIAL ⁵⁴-DEPENDENT PROMOTERS

Top Previous Next References

Site Identification and the Problem of False Positives

The most meaningful analysis of the physiological function of ⁵⁴ requires a comprehensive set of ⁵⁴-dependent genes. One method to help determine the complete set of such promoters is a computer analysis of potential ⁵⁴-dependent promoters from the completed E. coli genome sequence. We used the SeqScan program (B. T. Nixon, Department of Biochemistry and Molecular Biology, Pennsylvania State University [http://www.bmb.psu.edu/seqscan]) for this analysis. This program uses 86 ⁵⁴-dependent promoters from several organisms to define a consensus sequence that does not differ substantially from that derived from the 17 known E. coli promoters (Table 2). The program uses a weighted matrix to give sites a score from 0 to 100, and then reports sites with a score higher than 60. 

View this table: [in this window] [in a new window]   TABLE 2.   Comparison of consensus sequence for 54-dependent promoters from known E. coli promoters and the SeqScan programa

The SeqScan program identified approximately 8,000 potential ⁵⁴-dependent promoters from the E. coli genome, or about 1 for every 600 bases. Clearly, there are a number of false positives. We can use the properties of the known ⁵⁴-dependent promoters to eliminate many of these false positives. We eliminated all the sites within structural genes, which reduced the number of potential sites over 30-fold. The 17 known ⁵⁴-dependent promoters were in the set of 213 intergenic sites in which the potential promoter transcribed a gene in the correct direction. We found that 121 potential intergenic E⁵⁴ binding sites transcribe genes in the wrong orientation. Table 3 and its footnotes list all of the intergenic sites that could potentially transcribe a gene in the correct direction.

View this table: [in this window] [in a new window]   TABLE 3.   Computer ranking of intergenic 54 sitesa

The distribution of scores for properly and improperly oriented sites is shown in Fig. 3. There are only 2 incorrectly oriented sites (1.6%), and there are 25 correctly oriented sites (11.7%) with a score of at least 76. Of the 25 high-scoring correctly oriented sites, 18 (72%) are known ⁵⁴-dependent promoters or are induced by nitrogen limitation. Together, these results imply that properly oriented high-scoring sites are not common and are likely to contain an authentic ⁵⁴-dependent promoter. In contrast, only 7 (3%) of 213 sites with a score lower than 76 are known or likely (i.e., nitrogen limitation-induced) ⁵⁴-dependent promoters. Therefore, scores below 76 are a reasonably good, but not infallible, indicator that the site is not a promoter. Despite the correlation with promoter scores, the promoter ranking is not an exact indicator of promoter strength. For example, the nac promoter has a higher score than the ⁵⁴-dependent promoter of the glnALG operon, even though the latter is stronger (54). Nonetheless, and this point cannot be emphasized too much, the computer program recognizes all known E⁵⁴ binding sites, which implies that the failure to detect such a site is a reliable indicator that such a site does not exist.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Distribution of scores with properly and improperly oriented sites. The open bars indicate sites that are oriented toward the 3' end of a gene; the solid bars indicate sites that are oriented toward the 5' end.

Of the 17 known ⁵⁴-dependent promoters, 7 are involved in nitrogen metabolism. In addition, microarray analysis has identified 7 other genes that are induced by nitrogen limitation (176), and these have an appropriately placed potential ⁵⁴-dependent promoter. This section discusses the 14 operons that are involved in nitrogen metabolism and the functions of their products. To understand the metabolic context of these proteins, it is first necessary to discuss nitrogen assimilation and the response to nitrogen deprivation. Because there have recently been some major changes in our understanding of these topics, the next sections summarize our current knowledge.

Nitrogen assimilation. Glutamate and glutamine are the major intracellular nitrogen donors, and they provide about 75 and 25% of the cell's nitrogen, respectively (calculated from numbers presented in reference 116). Nitrogen assimilation must therefore result in the synthesis of these two nitrogen donors.

View larger version (8K): [in this window] [in a new window]   FIG. 4.   Pathways of ammonia assimilation. GDH, glutamate dehydrogenase.

Control of ammonia assimilation and GS activity: role of glutamine. Ammonia assimilation involves the regulation of three enzymes. Little is known about the regulation of glutamate dehydrogenase activity or synthesis in E. coli (127). The leucine-responsive regulatory protein (Lrp) controls the synthesis of glutamate synthase (53). A discussion of the function of Lrp and Lrp-dependent regulation, which does not require ⁵⁴, is beyond the scope of the review. Instead, we will focus on the control of GS activity.

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Regulation of GS activity and the Ntr response. The pathways are shown for conditions of nitrogen excess (high glutamine) (top) and nitrogen limitation (low glutamine) with partial GlnK uridylylation (bottom). The open arrow in the bottom panel is meant to indicate that only partial uridylylation occurs. It is assumed that partial uridylylation occurs either during nitrogen limitation or during the transition to steady-state nitrogen-limited growth. The T-like symbol indicates an inhibition.

Nitrogen sources. Ammonia is considered the preferred nitrogen source for E. coli grown in a minimal medium because ammonia supports the fastest growth and its presence prevents the synthesis of several proteins of nitrogen metabolism (reviewed in references 127 and 129). In place of ammonia, E. coli and related organisms can utilize a small number of nitrogen sources, usually amino acids, nucleosides, nucleobases, and a few inorganic nitrogen sources, e.g., nitrite and nitrate, which are reduced to ammonia. Steady-state growth on alternate nitrogen sources is slower and is said to be nitrogen limited. Catabolism of the alternate nitrogen sources must produce ammonia for the synthesis of glutamine, one of the intracellular nitrogen donors. For growth with a nitrogen source that cannot transfer its nitrogen to glutamate by transamination (e.g., adenosine), ammonia becomes an obligatory intermediate for all cellular nitrogen. In these situations, GS is the primary enzyme of ammonia and nitrogen assimilation (Fig. 4). Nitrogen-limited growth results in maximal synthesis of GS and also induces proteins that transport and catabolize several nitrogen sources. The coordinated response to nitrogen limitation is called the nitrogen-regulated (Ntr) response.

-Ketoglutarate counters the effects of unmodified P_II. Even though recent studies have suggested that the ratio of glutamine to -ketoglutarate does not regulate UTase-UR activity, -ketoglutarate does affect P_II activity (83-85). -Ketoglutarate counteracts the effects of unmodified P_II (present when glutamine levels are high and nitrogen is in excess) and therefore stimulates glnALG expression and increases GS activity. In other words, the ratio of glutamine (via UTase-UR) to -ketoglutarate (via P_II) appears to control nitrogen assimilation during relative nitrogen sufficiency. This leaves the question whether there is a mechanism to coordinate carbon and nitrogen metabolism during nitrogen-limited growth when P_II is uridylylated. It will be suggested elsewhere in this review that such coordination might be a function of GlnK, a P_II-like protein.

NR_I regulon. NR_I directly or indirectly controls the vast majority of Ntr genes. It is known to activate the expression of glnALG (GS and Ntr regulators), astCADBE (arginine catabolism), glnK-amtB (an alternate P_II and an ammonia transporter), nac (a ⁷⁰-dependent transcriptional activator), and glnHPQ (glutamine transport) in E. coli. Several lines of evidence also suggest that it controls the expression of argT-hisJMPQ (arginine and histidine transport) and gltIJKL (glutamate-aspartate transport). In addition to these genes, microarray analysis suggests that NR_I might also activate b1012-b1006 (possibly for pyrimidine catabolism), chaC (calcium transport), ddpXABCDE (D-alanine-D-alanine metabolism), potFGHI (putrescine transport), yeaGH (unknown function), ygjG (a transaminase), and yhdWXYZ (amino acid transport) (176). In addition to activation, NR_I represses the two minor promoters, glnAp₁ and glnLp, of the glnALG operon.

Nac regulon. There are two majors regulators of the Ntr response: NR_I and Nac. NR_I activates ⁵⁴-dependent promoters, while Nac activates ⁷⁰-dependent promoters. Nac is homologous to LysR (114). Unlike LysR, Nac apparently does not bind a ligand, which implies that it is constitutively active (16, 63).

Why are there two general regulators of the Ntr response? The main question concerning Nac is why there is a second Ntr regulator. Clearly, it is not necessary, since S. enterica serovar Typhimurium lacks it (114). We suggest that Nac-dependent control is important physiologically and serves a different function from NR_I-dependent control. NR_I-dependent genes respond to general nitrogen limitation, i.e., to intracellular glutamine, and not to specific induction mechanisms. The only known exception is the ast operon, which requires arginine-specific induction. In contrast, many Nac-dependent genes require both general and specific regulation: pyrimidines control the codBA operon by a complex process called reiterative transcription (126); GABA controls gab operon expression via the GabC repressor (Ruback and Reitzer, unpublished); and histidine and HutC control the hut operons in K. aerogenes (16). Nac may permit specific regulation, which may be difficult for ⁵⁴-dependent promoters. This is illustrated in Fig. 6, which shows the regulatory sites for the single hutUH promoter of K. aerogenes. Other rationales for Nac have been proposed (16).

View larger version (10K): [in this window] [in a new window]   FIG. 6.   Binding sites for regulatory proteins at the hutUH promoter.

The glnALG operon codes for GS, NR_II, and NR_I, respectively (127, 129). All three products of this operon are required for nitrogen assimilation and the Ntr response (discussed above). ⁵⁴ and NR_I~P are required for transcription from the major promoter, glnAp₂, which has a score of 83.3. Nitrogen limitation activates glnA expression 11- to 24-fold, but appears to have little effect on glnL or glnG transcription (176). However, direct measurements of NR_I indicate that nitrogen limitation induces NR_I synthesis 14-fold (128). Minor promoters, glnAp₁ and glnLp, ensure basal synthesis of the important products of this operon (130, 157).

Nitrogen limitation and NR_I are required for expression of the glnK-amtB operon (10, 160). Although the transcription start site has not been directly determined, the promoter has a score of 90.2 and potential binding sites for both E⁵⁴ and NR_I (Fig. 2) (160). In addition, nitrogen limitation increases the glnK transcript at least 10-fold (176). Therefore, this operon undoubtedly contains an authentic ⁵⁴-dependent promoter. Both products of the operon contribute to the response to nitrogen limitation.

GlnK: a P_II paralog. The existence of a P_II paralog was first suspected because of the rapid deadenylylation of GS in an E. coli glnB (P_II-encoding) mutant (160). The gene coding for this protein was cloned and called glnK (160). A glnK mutant has only a subtle phenotype (10). It has higher basal expression of an Ntr gene (glnK itself) in an ammonia-containing medium (even though the GlnK concentration should be low) and lower expression of an Ntr gene (again glnK) in a nitrogen-limiting medium. The mutant also has less of a lag during the transition to growth with arginine as a nitrogen source. This might result from higher basal expression of the ast operon, whose products degrade arginine. The phenotype of a glnB glnK double mutant, which lacks both P_II and GlnK, is more dramatic. It fails to grow in a nitrogen-rich minimal medium. The reason for this lethality is not known with certainty, but it may be related to uncontrolled phosphorylation of NR_I, which has been suggested to cause inappropriate overexpression of an Ntr gene (10). An alternate explanation is NR_I-dependent overexpression of a ⁵⁴-dependent gene that is not normally regulated by NR_I.

Why have GlnK? The lower induction of an Ntr gene and higher basal expression in a glnK mutant suggest that GlnK sharpens the response to nitrogen availability. Perhaps the responsiveness of GlnK to -ketoglutarate partially explains this effect. In this case, GlnK essentially restores the coordination of carbon and nitrogen metabolism (the responsiveness to the ratio of -ketoglutarate to glutamine) that is lost when P_II is completely uridylylated, i.e., during nitrogen-limited growth, and is no longer responsive to -ketoglutarate. A second function for GlnK has been found, but not in E. coli. GlnK is required for control of NifL, which inhibits the activity of NifA in Klebsiella species (65). NifA is the transcriptional activator required for expression of the nitrogenase gene cluster in Klebsiella species. Uridylylated and nonuridylylated GlnK can relieve repression. It is not known how GlnK mediates shutoff of the nif genes when ammonia is added, but it may interact with other proteins.

Product of amtB and ammonia transport. The second gene of the operon, amtB, codes for an ammonia transporter. None of the phenotypes of the glnK mutant could be attributed to polar effects on amtB expression (10). An amtB mutant of S. enterica serovar Typhimurium has only a subtle phenotype (149). It is unable to utilize a low concentration of ammonia if the pH is less than 7. This phenotype implied that uncharged NH₃, not NH₄⁺, is transported. It was proposed that AmtB did not concentrate ammonia but only facilitated equilibrium across the membrane. This mechanism of transport might have significant physiological implications, which are discussed in "Physiological Function of ⁵⁴" (below).

Although E. coli, S. enterica serovar Typhimurium, and K. aerogenes are closely related, they differ in their ability to utilize certain nitrogen sources and in the regulation of some genes of nitrogen metabolism. For example, nitrogen limitation strongly represses glutamate dehydrogenase in K. aerogenes but not in E. coli (16, 127). The transcriptional regulator Nac accounts for many of these differences in regulation. Nac has been most extensively studied from K. aerogenes but has also been studied from E. coli. In contrast, S. enterica serovar Typhimurium lacks Nac (114).

The nac operon is monocistronic (114, 145). Transcription initiated from the K. aerogenes nac promoter requires ⁵⁴ and NR_I~P (16, 54, 106). Nitrogen limitation induces E. coli nac (114, 176), and computer analysis indicates a likely binding site for E⁵⁴ with a score of 85.2, which is very high. These results suggest that NR_I and E⁵⁴ are also required for E. coli nac expression. Nac negatively modulates its own synthesis in K. aerogenes by interfering with the interaction between NR_I and RNA polymerase (55, 114), and results from the DNA microarray analysis are consistent with such regulation in E. coli (176).

Arginine (via agmatine) and ornithine are both precursors for putrescine, which can be metabolized to GABA, and then to succinate (Fig. 7). Nitrogen limitation induces enzymes of GABA catabolism (175). Therefore, it was reasonable to propose that Ntr regulators affect the catabolism of all of these compounds, and some evidence is consistent with this regulation (147). However, recent studies with mutants containing targeted gene disruptions have indicated unsuspected pathways and a surprising complexity and redundancy of pathways and regulators. Only the enzymes of arginine and GABA catabolism require ⁵⁴, while the enzymes of putrescine catabolism may not.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   Metabolic relationships between ornithine, arginine, putrescine, and GABA. A thick black arrow indicates that nitrogen limitation induces the enzyme indicated. A reaction catalyzed by two (or more) enzymes is indicated by two arrows. The genes that specify the enzymes are shown when they are known. A dashed arrow indicates that the gene has yet to be identified.

astCADBE operon and catabolism of arginine and ornithine. The five-step arginine succinyltransferase pathway catabolizes arginine (144). The pathway is named after the first reaction, which is the succinylation of the -amino group of arginine. The astCADBE operon codes for the proteins of the pathway (144). Disruption of the operon in E. coli prevents growth with arginine as a nitrogen source and impairs but does not eliminate growth with ornithine (60, 144). It has been proposed that AstC (which catalyzes the deamination of succinylornithine) is one of at least two transaminases that can deaminate ornithine, which generates an intermediate of proline catabolism (144). The identity of the second transaminase is unknown.

GABA and putrescine catabolism and the gabDTPC operon. E. coli can utilize GABA as a nitrogen source (H. Kasbarian, S. Ruback, and L. Reitzer, unpublished results), although earlier studies indicated otherwise (50). A mutant with a gabDT deletion cannot utilize GABA as a nitrogen source (Ruback and Reitzer, unpublished). The distance between genes suggests the existence of a gabDTPC operon. Furthermore, transcript analysis indicates the presence of only one promoter for these genes, a promoter just upstream from gabD. Each gene of the putative operon has been implicated in GABA catabolism. GabT is a transaminase that deaminates GABA to succinic semialdehyde. GabD is an NADP-specific succinic semialdehyde dehydrogenase that oxidizes succinic semialdehyde to succinate. (An NAD-dependent succinic semialdehyde dehydrogenase is specified by the sad gene, and GABA or a product of GABA metabolism induces its synthesis [49].) GabP specifies a GABA permease (92). GabC appears to be a specific repressor since a deletion of gabC stimulates growth with GABA as a nitrogen source (Kasbarian et al., unpublished). Either nitrogen limitation or entry into stationary phase activates gab operon expression (12, 175; Kasbarian et al., unpublished). Expression of the gab operon requires Nac during nitrogen-limited growth, and Nac is required to reconstitute transcription in vitro (H. Kasbarian, A. Kiupakis, S. Ruback, and L. Reitzer, unpublished results). ^S is required for expression during stationary phase.

ygjG. Nitrogen limitation results in a three- to fivefold increase in the levels of steady-state ygjG transcripts (176). Expression of this gene requires ⁵⁴, and the transcription start site has been determined (A. Kiupakis, R. Ye, and L. Reitzer, unpublished result). The score for this promoter is 70.7, which is low for an authentic ⁵⁴-dependent promoter. The gene specifies a putative -transaminase which either removes the amino group from compounds with terminal primary amines (e.g., putrescine or ornithine), or adds amino groups to compounds with an aldehyde group (e.g., N-acetylglutamic semialdehyde, an intermediate in ornithine formation). Putrescine and compounds metabolized to putrescine activate ygiG expression, which suggests a possible role in putrescine catabolism. However, a mutant with a disruption of ygjG grows normally with putrescine as a nitrogen source, which suggests that YgjG is a redundant transaminase (C. Pybus and L. Reitzer, unpublished observation).

(i) The three transport systems. E. coli has three characterized arginine transport systems (described below), while S. enterica serovar Typhimurium has at least two (98). An early study suggested the presence of three periplasmic arginine binding proteins in E. coli (136). No strain has been constructed with mutations in all the characterized systems. Therefore, it is conceivable that there are other transport systems.

(ii) Repression by arginine. Arginine represses all three E. coli transport systems (41, 137, 169). A possible mechanism of repression would involve ArgR, which mediates arginine repression for the enzymes of arginine synthesis. A computer analysis of ArgR sites in E. coli identified two sites in the art operon: one preceding artP and one preceding artJ (111). However, a missense mutation in argR had no effect on the kinetically detectable arginine transport systems (39). Instead of ArgR, ArgP and ArgK have been proposed to mediate arginine repression. ArgP is a transcriptional regulator required for synthesis of ArgK, which is required for arginine transport. Mutations in argP and argK affect both the ArgT and AbpS systems (40, 41, 137). Only one gene separates argP and argK, but they appear to be independently expressed. (argK is currently not listed in either GenBank or the latest E. coli genetic map. However, argK is ygfD, also called b2918 in GenBank.) ArgP is a LysR-type regulator that activates argK expression in the absence of arginine (37). ArgP complexed with arginine fails to activate argK expression and represses its own synthesis. ArgK has an ATPase activity that is apparently required for transport activity (158). ArgK also phosphorylates the periplasmic ArgT and AbpS (36), although this phosphorylation is not required for transport (38). ArgP is the previously characterized IciA, an inhibitor of the initiation of DNA replication (37, 154). An iciA mutant has no obvious phenotype, except for difficulty during dilution into fresh growth medium (154). It is conceivable that ArgP/IciA is a sensor of amino acid sufficiency that couples DNA synthesis with metabolism in some environments.

(iii) Transport and activation during nitrogen limitation. Nitrogen limitation induces ArgT in E. coli and S. enterica serovar Typhimurium, and this induction does not require arginine (98, 176). The genetics and regulation of arginine transport have been studied in S. enterica serovar Typhimurium, and it is assumed that they will be similar in E. coli. In S. enterica serovar Typhimurium, loss of ArgT reduced but did not eliminate the binding of arginine to periplasmic proteins and an argT mutant grew normally with arginine as a nitrogen source, which implies a second transport system in S. enterica serovar Typhimurium during nitrogen-limited growth (98).

Glutamine. Glutamine transport has been studied in both E. coli and S. enterica serovar Typhimurium. The kinetically dominant system requires GlnH, a high-affinity glutamine-specific binding protein in the periplasm (162). The glnHPQ operon specifies GlnH and two membrane proteins, which presumably interact with GlnH (119). Loss of GlnH unmasks a low-affinity glutamate-inhibitable glutamine transport system in E. coli (162) but has no effect on growth in S. enterica serovar Typhimurium (98). These results suggest a second glutamine transport system. Kinetic assays of glutamine transport also suggest two transport systems (11, 168). It is possible that the glutamate-inhibitable system requires the periplasmic glutamate-aspartate binding protein (see the next section), which also binds glutamine (167).

Glutamate-aspartate. Schellenberg and Furlong defined five transport systems for glutamate and aspartate in E. coli by a combination of genetic and biochemical experiments (140). There are no studies with gene fusions; therefore, the regulation and functions of the individual systems are poorly understood.

ddpXABCDE operon. Microarray analysis shows that general nitrogen limitation (i.e., no specific induction) induces the ddpXABCDE operon 52- to 60-fold, which is more than any other operon. Expression appears to require NR_I (176). A potential ⁵⁴-dependent promoter with a score of 76.6 precedes the operon. DdpX is a zinc-containing D-alanyl-D-alanine dipeptidase, while the other products of the operon appear to code for components of a dipeptide permease (100). There are two sources of D-alanyl-D-alanine: it is an intermediate in peptidoglycan synthesis, and it may be released during cross-linking of two diaminopimelic acids. Peptidoglycan cross-linking occurs in the stationary phase (156). Entry into the stationary phase induces the ddpXABCDE operon, and this induction requires ^S (100). It is not known whether peptidoglycan remodeling also occurs in nitrogen-limited cultures. It has been proposed that the function of the ddpXABCDE products is to scavenge D-alanyl-D-alanine (176).

Peptide transport and ompF. E. coli, S. enterica serovar Typhimurium, and other gram-negative bacteria digest peptides intracellularly after their passage through the outer membrane and transport via periplasmic binding protein-dependent transport systems. Nitrogen limitation activates the expression of dppABCDF and oppABCDF (176). The products of these operons are the major peptide transporters in E. coli and S. enterica serovar Typhimurium (120), and the periplasmic components of these systems, DppA and OppA, are among the most abundant proteins in the periplasm (1, 70, 121). In addition to its function as a transport protein, DppA is required for chemotaxis to peptides (108). Neither peptide transport operon contains a potential ⁵⁴-dependent promoter. The apparent 10-fold dppABCDF induction appears to require Nac (176), which is consistent with the absence of a ⁵⁴-dependent promoter. In contrast, microarray analysis provides no evidence for Nac-dependent regulation for the four- to sixfold oppABCDF induction and equivocal evidence for NR₁-dependent regulation (176). Induction by nitrogen limitation is most evident in medium with glutamine as the nitrogen source but not in medium with ammonia as the nitrogen source for strains with constitutively active Ntr regulatory proteins. These results suggest that induction may require specific nitrogen-containing compounds. This is consistent with the known regulators of oppABCDF expression, Lrp and the gcvB transcript (a regulatory RNA), which respond to leucine (or alanine) and glycine, respectively (31, 117, 159). (The gcvB transcript also controls dppABCDF expression.) Based on these considerations and the absence of a potential ⁵⁴-dependent promoter, the effect of nitrogen limitation on oppABCDF may be indirect, i.e., independent of NR_I or Nac.

Nitrogen limitation results in a 20-fold increase in the expression of the putative b1012-1006 operon (176). The potential E⁵⁴ binding site preceding b1012 has a score of 76.8. The last gene of the putative operon codes for a possible uracil permease, which may suggest that this operon codes for enzymes of pyrimidine catabolism. This is consistent with the observation that [¹⁴C]uracil or [¹⁴C]thymine catabolism yields ¹⁴CO₂, even though E. coli cannot degrade these compounds as sole nitrogen sources (13).

Nitrogen limitation induces b2875-76 and b2882-85. Some of the genes in this region have been studied, and homology searches have suggested that they might participate in purine catabolism (172). The xdhA gene (b2866), which codes for one subunit of a recently discovered xanthine dehydrogenase, appears to have two promoters, and one of them might be ⁵⁴ dependent (172). E. coli can utilize intermediates of purine catabolism, such as allantoin, as nitrogen sources anaerobically but not aerobically (46). The potential ⁵⁴-dependent promoters preceding xdhA (b2866), ygeW (b2870), and b2878 have low scores (66.0, 75.2, and 71.5, respectively), but they might contribute to purine catabolism during anaerobic growth.

Nitrogen limitation activates the putative yhdWXYZ operon two- to ninefold, and appears to be NR_I dependent (176). The potential promoter for this operon has a score of 88.6. The products of this operon have homology to transport proteins for polar amino acids.

Nitrogen limitation activates the yeaGH operon two- to fourfold, but it is not clear whether regulation requires NR_I or Nac (176). The potential E⁵⁴-binding site has a score of 83.8. However, homology searches provide no clue to the function of the products.

⁵⁴-DEPENDENT GENES THAT ARE NOT INVOLVED IN NITROGEN METABOLISM

Top Previous Next References

Formate Catabolic Genes and the FhlA Regulon

Formate metabolism. The products of several ⁵⁴-dependent operons contribute to formate metabolism during glucose fermentation. The products of glucose fermentation in E. coli in terms of total carbon from glucose are CO₂ (14.7%), ethanol (16.6%), acetic acid (12.2%), lactic acid (40%), formic acid (0.4%), succinic acid (7.2%), and cell constituents (~10%) (reviewed in reference 24). Formate is a major intermediate even though it does not accumulate. Formate formation is linked to pyruvate metabolism because pyruvate formate lyase cleaves pyruvate to formate and acetyl coenzyme A (acetyl-CoA) during anaerobic growth. The production of fermentation products that require acetyl-CoA as a precursor, acetate and ethanol, necessarily generates a stoichiometric amount of formate. Therefore, about 14% of glucose carbon (8.3% concomitant with ethanol formation plus 6.1% with acetic acid formation) is converted to formate during fermentation.

The four confirmed ⁵⁴-dependent operons of formate metabolism. The requirement for ⁵⁴ has been established by examination of mutant phenotypes, lacZ fusions, transcript analysis from mutant and wild-type strains, and transcription with purified components (20, 21, 73, 104, 107). The four confirmed ⁵⁴-dependent operons code for components required for the FHL complex. The monocistronic fdhF specifies one subunit of FDH_H. The divergently transcribed polycistronic hyc and hyp operons code for components of FDH_H, hydrogenase 3, proteins required for processing of hydrogenase 3 and other hydrogenases, and two transcriptional regulators (24). The hydN-hypF operon specifies a protein required for FDH_H activity (possibly a component of electron transport) and a second protein required for processing of several hydrogenases (107). The promoters for the hyp, hyc, fdhF, and hydN-hypF operons have scores of 88.0, 79.1, 72.6, and 64.5, respectively. The last two are among the lowest scores for confirmed ⁵⁴-dependent promoters.

Hydrogenase 4. A 12-gene hyf operon at min 56 contains a possible ⁵⁴-dependent promoter with a score of 68.7, and upstream sequences suggest the presence of FhlA binding sites. The expression of this operon has not been characterized or established. Homology analysis suggests that the products of the operon code for a putative hydrogenase 4, which catalyzes the same reactions as the FHL complex, and for proteins of respiration-linked proton translocation. Therefore, it was proposed that the products of the hyf operon specify an energy-conserving hydrogenase 4. The putative product of one gene of the operon, hyfR, is homologous to FhlA and may bind the same sites as FhlA because of conservation of the DNA binding residues (7).

Loss of the ato operon results in failure to utilize acetoacetate as a carbon and energy source (reviewed in reference 43). The transcription start site has not been examined. Nonetheless, it is likely that this operon requires ⁵⁴ for several reasons. First, an rpoN mutant (⁵⁴ deficient) cannot utilize acetoacetate as a carbon source (C. Pybus and L. Reitzer, unpublished). Second, expression of the ato operon requires AtoC, which is homologous to other activators of ⁵⁴-dependent promoters (42, 43). Proteins homologous to AtoC usually activate ⁵⁴-dependent promoters, although two activate ⁷⁰-dependent promoters (59, 113, 173). The latter activators lack an essential region of the domain that interacts with E⁵⁴. The domain of AtoC that interacts with RNA polymerase is homologous throughout its length to the corresponding region of other ⁵⁴-dependent activators (113). Therefore, it is likely that AtoC activates from a ⁵⁴-dependent promoter. Finally, the score of the putative ⁵⁴-dependent ato promoter is 85.9, which is very high.

The genes of the atoDAEB operon code for proteins of acetoacetate catabolism (42, 81). It has been proposed that atoE codes for an acetoacetate-specific transport system (42). The atoD and atoA genes specify the subunits of acetyl-CoA:acetoacetyl-CoA transferase, which catalyzes the transfer of CoA from acetyl-CoA to acetoacetate. AtoB specifies thiolase II, which catalyzes the formation of two molecules of acetyl-CoA from CoA and acetoacetyl-CoA (43). (Thiolase I is an enzyme in fatty acid -oxidation.)

E. coli can utilize short-chain fatty acids (C₄ to C₆) such as butyrate (C₄) and valerate (C₅) as a carbon source and such catabolism requires the ato operon (43, 124). Butyrate catabolism requires the formation of butyryl-CoA by acetyl-CoA:acetoacetyl-CoA transferase, followed by dehydrogenation of the saturated fatty acid, hydration, and oxidation, which results in the formation of acetoacetyl-CoA. These reactions require enzymes of fatty acid degradation, products of the fadR regulon. Acetoacetyl-CoA is then degraded as described above. Butyrate does not induce either the ato or the fad genes. Therefore, growth with butyrate as the sole carbon source requires constitutive expression of both sets of genes (43).

AtoC is required for expression of the atoDAEB operon (82, 124). The nucleotide sequence of this region suggests that atoC is the second of two genes in an atoSC operon that is just upstream from the atoDAEB operon. AtoC is homologous to response regulators such as NR_I (NtrC), and the putative AtoS is homologous to sensor kinases, such as NR_II (NtrB). Acetoacetate or a product of acetoacetate metabolism probably binds AtoS and stimulates AtoC phosphorylation. Other aspects of regulation have not been examined. The possibility of IHF sites upstream from the atoD promoter has been suggested (42). Glucose blocks expression of the ato operon, and possible cyclic AMP sites that have been identified upstream from the atoD promoter may interfere with the AtoC-E⁵⁴ interaction (42).

Environmental propionate is the end product of several different fermentation pathways and can also result from -oxidation of odd-chain fatty acids. Propionate is a membrane-permeable anion that can alter the internal pH of bacteria. The products of the prp operon degrade propionate. Most of the genetics of propionate catabolism and analysis of gene expression has been studied with S. enterica serovar Typhimurium. Expression of this operon requires ⁵⁴ in S. enterica serovar Typhimurium, although the transcription start site has not been identified (122). In addition to ⁵⁴, expression requires IHF and PrpR, which is homologous to NR_I (122). 2-Methylcitrate or a product of its metabolism has been proposed to bind PrpR and induce the operon (155). It is assumed that regulation in E. coli is similar. The putative E. coli promoter has the third highest score, 89.6, of known ⁵⁴-dependent promoters.

The propionate catabolic pathway is called the methylcitric acid cycle (76, 153). The first reaction is the addition of CoA to propionate by PrpE, propionyl-CoA synthetase (75). In addition to PrpE, acetyl-CoA synthetase and possibly an enzyme of acetoacetate catabolism can catalyze this reaction (75, 133). The second reaction is catalyzed by methylcitrate synthase, the product of prpC, which generates the presumed inducer methylcitrate. Methylcitrate synthase also reacts with acetyl-CoA, although propionyl-CoA is the preferred substrate (76). The next reactions are a dehydration and hydration to form methylisocitrate with methylaconitate as an intermediate. An aconitase-like activity could conceivably catalyze these reactions, and PrpD might catalyze one or both of these reactions, but PrpD is not homologous to known aconitases (76). The last reaction is cleavage of methylisocitrate to succinate and pyruvate, which is catalyzed by PrpB, methylisocitrate lyase (76). The methylcitric acid cycle requires regeneration of oxaloacetate. The most obvious source of oxaloacetate is succinate. However, some strains of E. coli require the glyoxylate shunt for this oxaloacetate formation (153), and it has been proposed that S. enterica serovar Typhimurium generates oxaloacetate from pyruvate by the combined actions of phosphoenolpyruvate synthetase and phosphoenolpyruvate carboxylase (56). Several aspects of propionate catabolism are unusual. Strains lacking glutathione cannot utilize propionate as a carbon source (135). Strains lacking DNA polymerase I also fail to utilize propionate, which suggests that propionate or a product of propionate catabolism damages DNA (134). Finally, propionate is toxic in the absence of enzymes of the methylcitric acid cycle (64).

The pspABCDE operon is unusual, and its regulation is perhaps the most complicated of the ⁵⁴-dependent operons (see reference 112 for a review). This system was first discovered and studied by Peter Model and colleagues, who noticed that overexpression of a filamentous phage protein resulted in massive PspA synthesis in E. coli (25). It was subsequently shown that several different stresses also induce PspA synthesis: filamentous-phage infection, overexpression of some filamentous-phage proteins, overexpression of some outer membrane proteins (especially mutant forms), heat shock, ethanol, hyperosmotic shock, nutritional downshifts (passage into the late stationary phase of the growth cycle), proton ionophores and other uncouplers of oxidative phosphorylation (free fatty acids), and hydrophobic organic solvents (95, 112, 165). Various proteins are required to sense these stresses, and it is unlikely that there is a single inducing effector.

Mutants lacking PspA, PspB, or PspC have no dramatic phenotype during exponential growth. However, these mutants survive poorly in stationary phase in an alkaline environment (165). These mutants also have greater motility and slower protein translocation (112). PspA appears to maintain the proton motive force in stressed cells, and it has been proposed that this is the major function of PspA (94).

All the inducing stresses result in transcription from a single promoter that requires ⁵⁴ (163). The promoter has a score of 80.0 (Table 3). Activation from the psp promoter requires PspF, which is specified by a gene adjacent to, but divergently transcribed from, the pspABCDE operon (91). PspF and IHF bind cooperatively to the psp promoter (89). One function of IHF is to increase the specificity of activation by preventing an interaction with RNA polymerase by other activators (52, 91). However, PspF (and possibly other ⁵⁴-dependent activators) can activate the psp operon without the PspF binding site (the enhancer) during hyperosmotic shock (90). PspF lacks an amino-terminal regulatory domain, and it activates transcription without phosphorylation or an activating ligand (91). Instead, as described below, PspA controls PspF activity.

An unusual aspect of the psp operon is that four of the five genes specify regulators that control psp expression. PspA binds to PspF, which blocks its ability to activate transcription (51). PspA also inhibits at least one other ⁵⁴-dependent activator, NR_I, and perhaps others (51). It is not known whether this inhibition of NR_I is important in vivo. A region of PspA has homology to the RNA polymerase binding region of ⁵⁴-dependent activators, which suggests that the mechanism of inhibition may involve a nonproductive interaction with RNA polymerase (88). PspA is peripherally associated with the inner membrane. PspB and PspC are also components of the inner membrane, and they cooperate in activating transcription, probably by antagonizing the effects of PspA (112, 163). PspD may similarly antagonize PspA (unpublished results cited in reference 112). No function is apparent for PspE (112). The induction mechanism is stimulus specific. Induction by the gene IV product of the filamentous phage f1 requires PspB, PspC, and PspD, whereas induction by heat shock requires none of these proteins. Other stimuli may require one or more of these regulatory proteins (112). The IHF dependence also varies with the stimulus (164).

PspF negatively autoregulates its own synthesis by binding to the same sites that activate psp operon expression (88). PspA, PspB, or PspC does not affect this autoregulation, which implies that these proteins do not affect the binding of PspF to DNA (88).

Proteins of the heat shock response also affect psp induction. Many but not all of the stimuli that induce the psp operon also induce the heat shock response. However, loss of ³², the heat shock sigma factor, results in higher and longer expression of the psp operon (25, 26, 112). The mechanism of this regulation is not known.

The promoter for the rtcBA operon has the highest score (95.4) of known ⁵⁴-dependent promoters. The existence of the promoter was shown by primer extension in wild-type E. coli and by failure to observe the transcript in an rpoN mutant (62). The ⁵⁴-dependent transcript was the only detectable transcript in primer extension experiments. Possible IHF binding sites were identified between 46 and 68 bases upstream from the start site of transcription. Expression of the rtcBA operon appears to require the divergently transcribed rtcR, which has a deduced product that is homologous to other ⁵⁴-dependent activators. Detectable expression of the operon requires artificial overproduction of RtcR. Deletion of its amino-terminal domain also increases expression, which suggests that this domain inhibits the activity of RtcR. The stimulus that controls the activity of RtcR is not known.

RtcA catalyzes the ATP-dependent formation of 2',3'-cyclic phosphodiester from an RNA with a 3' phosphate at its 3' end. The function of this activity is unknown, although such cyclic intermediates may be required for RNA ligation reactions (19). This enzyme is found from E. coli to HeLa cell extracts. An E. coli strain with 90% of rtcA deleted had no phenotype when grown in Luria-Bertani or minimal M9 medium (62). The activity of RtcB is not known.

The products of zraSR (previously called hydHG) are a membrane-associated sensor kinase and a response regulator, respectively. They were initially implicated in the control of hydrogenase 3 synthesis (150), but this control was observed only in an fhlA mutant and was subsequently shown to be nonspecific (99). The gene divergently transcribed from zraSR, zraP, had been implicated in tolerance to high Zn²⁺; therefore, the effect of Zn²⁺ on gene expression was examined (99). In response to high Zn²⁺ or Pb²⁺ concentrations, ZraR and ZraS specifically activated zraP, which is divergently transcribed from zraSR, and also autogenously activated zraSR expression. Purified ZraR bound in the zraP-zraSR intergenic region. Metal-induced expression required ⁵⁴ in vivo, and potential binding sites for E⁵⁴ were readily identifiable for zraP and zraSR, with scores of 88.6 and 76.2, respectively. In addition to these promoters, zraSR appeared to have a weak constitutive promoter, which ensures basal synthesis of the sensor and response regulator (99).

The most important system for Zn²⁺ tolerance is the zntA-zntR system, which codes for a Zn²⁺ efflux protein and a Zn²⁺ binding MerR-like transcriptional activator, respectively (15, 27, 132). Its loss results in Zn²⁺ hypersensitivity (132). In contrast, loss of the zraP-zraSR system is observable only in a longer lag during the transition to medium with Zn²⁺ (99). The precise physiological function of the zraP-zraSR system has yet to be determined. However, it is possible that it acts as a sensor of extracellular Zn²⁺ while the ZntR system responds to intracellular Zn²⁺ (99). It is not known whether the regulatory circuits of the ZntR and ZraR systems overlap.

Computer analysis identified 25 properly oriented intergenic sites with a score greater than 76, which is a good indicator of an E⁵⁴-dependent promoter (discussed above). This section briefly describes the seven sites and the operons they potentially control, if they have not been discussed already. Nitrogen limitation does not affect the expression of any of these operons (176).

The site preceding rpoH, which codes for the heat shock sigma factor, has a score of 92.8, which is the second highest computer score for a E⁵⁴ binding site. It is normally spaced relative to the rpoH structural gene and the adjacent upstream gene. Furthermore, it is conserved among bacteria (123). There is an obvious rationale for having a ⁵⁴-dependent promoter for rpoH. ^H activates the synthesis of several proteases, which could transiently supply amino acids. However, we have been unable to demonstrate the existence of a transcript from the putative ⁵⁴-dependent promoter from nitrogen-limited cells, which assumes, perhaps erroneously, that NR_I is the activator (A. Kiupakis and L. Reitzer, unpublished).

A possible b2710-ygbD operon has a potential ⁵⁴-dependent promoter with a score of 86. BLAST analysis suggests that b2710 codes for a flavodoxin or a rubredoxin, a redox protein, and that YgbD has homology to oxidoreductases, such as rubredoxin reductase. The gene divergently transcribed from the b2710-ygbD operon is ygaA, and it specifies a potential ⁵⁴-dependent activator that might regulate b2710-ygbD expression. Nothing else is known about this operon and its expression.

The site preceding the potential yfhKGA operon has a score of 83.9. The yfhKGA operon codes for a potential sensor kinase, a protein of unknown function, and a potential ⁵⁴-dependent activator, respectively. The putative yfhKGA operon is upstream from and transcribed in the same direction as glnB, which specifies an important regulator of the Ntr response, P_II (102). glnB is not part of an operon containing yfhKGA, since the major glnB promoter precedes the glnB structural gene (102). Furthermore, glnB on a plasmid complemented the altered glnALG regulation in a glnB mutant, which implies that defects in the putative yfhKGA operon do not contribute to the altered regulation of the glnB mutant (102). Nothing else is known about the putative yfhKGA operon.

The potential E⁵⁴ binding sites preceding kch, topA, and yaiS have scores of 85.4,76.5, and 77.3, respectively. kch codes for a potassium channel, topA codes for topoisomerase I, and yaiS specifies a protein of unknown function. The distance between the putative E⁵⁴ binding site and the translational start site for kch (264 bases) or topA (201 bases) is larger than that for any known ⁵⁴-dependent promoter (the range is from 34 to 136 bases). Furthermore, only 20 bases separate the 5' end of putative E⁵⁴ binding site for kch from the adjacent upstream gene, which probably excludes the possibility of an activator binding site. The yaiS intergenic region (706 bases) is larger than that for any intergenic region containing an authentic ⁵⁴-dependent promoter (the range is from 148 to 507 bases). The large intergenic regions for these three genes suggest that these sites are false positives.

The site preceding ybhK has a score of 78.8. Its product is homologous to RocR, a regulator of arginine catabolism in Bacillus subtilis, which is homologous to NR_I (30, 61). However, the homology does not extend to the ⁵⁴ binding domain, which implies that YbhK is not an activator of ⁵⁴-dependent genes.

A discussion of the physiological function of ⁵⁴ will account for all of the functional ⁵⁴-dependent activators. The signature of these activators is a highly conserved activation domain that binds and hydrolyzes ATP and interacts with E⁵⁴. Almost all ⁵⁴-dependent activators also have an amino-terminal regulatory domain and a small carboxy-terminal DNA binding domain. To identify ⁵⁴-dependent activators, we used PspF (which lacks an amino-terminal regulatory domain) and the central activation domain of NR_I as probes for BLAST searches. Both probes identified the same proteins: AtoC, the product of b1201, FhlA, HyfR, NR_I, PrpR, PspF, RtcR, YfhA, YgaA, YgeV, and ZraR (HydH). The search also identified TyrR, an activator of ⁷⁰-dependent genes of aromatic amino acid synthesis (45). It lacks a region of the activation domain that has been implicated in the interaction with E⁵⁴ (113). All the other proteins are homologous throughout the activation domain, which suggests that they activate ⁵⁴-dependent promoters and not ⁷⁰-dependent promoters. The functions of 10 of these proteins have already been discussed. The genetic contexts for the remaining two, the product of b1201 and YgeV, provide clues to their possible function and are discussed in this section. Table 4 summarizes the functions of these proteins and the genes that they activate. Curiously, if autogenous regulation is excluded (for ZraR), then only two of these regulators, NR_I and FhlA, are known to activate more than one operon.

View this table: [in this window] [in a new window]   TABLE 4.   Activators of 54-dependent promoters in E. coli

The b1201 gene specifies an apparent ⁵⁴-dependent activator. Genes flanking b1201 are transcribed in the opposite direction, which implies that b1201 is monocistronic. The b1201 gene is divergently transcribed from three genes, b1200-b1199-ycgC, which might form an operon. BLAST analysis suggests that b1200 and b1199 are homologous to a dihydroxyacetone kinase whereas YcgC is homologous to components of the phosphoenolpyrurate-dependent phosphotransferase system. Dihydroxyacetone kinase is an enzyme in the oxidative branch of glycerol fermentation (24). Klebsiella pneumoniae can ferment glycerol, but E. coli cannot. Nonetheless, a triply mutated E. coli can convert glycerol to dihydroxyacetone, which is initially excreted and subsequently metabolized (152). Furthermore, wild-type E. coli can use dihydroxyacetone as the sole carbon source (as long as the phosphate concentration is kept low) (86). Dihydroxyacetone kinase has yet to be assayed from E. coli. In Streptococcus faecalis, phosphotransferase system-dependent phosphorylation stimulates dihydroxyacetone kinase activity (48). Similarly, YcgC may be required for kinase activity in E. coli. In Citrobacter freundii, dihydroxyacetone kinase synthesis requires ⁵⁴ (47). Therefore, it would not be surprising if similar control was found for the E. coli genes. Unfortunately, computer analysis does not identify a potential ⁵⁴ promoter in the vicinity, perhaps because the ⁵⁴-dependent promoter has been lost.

YgeV is a potential ⁵⁴-dependent activator. Several genes in the vicinity of ygeV appear to code for enzymes of purine metabolism (172). Strains with a disruption of YgeV grow faster with aspartate as the sole nitrogen source without exogenous purines (H. Xi and L. Reitzer, unpublished observation). A strain with a disruption of the xdhABC operon, which codes for subunits of a xanthine dehydrogenase, has a similar phenotype (172). xdhABC appears to have a ⁵⁴-dependent promoter and a ⁵⁴-independent promoter (172). Since strains with a disruption of ygeV or xdhA have similar phenotypes, it is possible that YgeV is required for transcription from the ⁵⁴-dependent xdhA promoter.

PHYSIOLOGICAL FUNCTION OF ⁵⁴

Top Previous Next References

Possible Relationship between the ⁵⁴-Dependent Genes

With a nearly complete set of ⁵⁴-dependent genes (verified or potential), it is appropriate to ask whether the physiological themes of these genes are related. Many ⁵⁴-dependent genes are involved in nitrogen assimilation. These genes specify GS, the regulators NR_I and Nac, several transport systems, and a few catabolic operons. Is there a relationship between the ⁵⁴-dependent genes of nitrogen metabolism and the other ⁵⁴-dependent genes? One possibility is that there is no relation between them. This possibility makes one interesting prediction: ⁵⁴ is present in excess, and expression of one ⁵⁴-dependent gene will not affect any other. Considering the low level of ⁵⁴ found in some strains (87), it is reasonable to question whether this is the case.

Another explanation for the apparent diversity of the ⁵⁴-dependent genes is that certain conditions might make nitrogen assimilation very difficult, and several products of ⁵⁴-dependent genes might remedy the problem. It has been suggested that genes of the FhlA regulon may have coevolved with the genes of nitrogen assimilation (101). pH homeostasis provides a rationale for such coevolution. The function of the FhlA regulon is to increase the pH in an acidic environment. This could help nitrogen assimilation, because of the mechanism of ammonia transport. The AmtB protein catalyzes facilitated diffusion of NH₃ but not NH₄⁺ (149). pH determines the extent of ionization. If the external environment is acidic relative to the cytoplasm, NH₃ will leak out of the more basic cytoplasm (either with the AmtB carrier or without the carrier, because NH₃ is membrane permeable). Subsequent protonation of the external NH₃ will make it difficult to bring NH₄⁺ back into the cell. The FhlA regulon, which increases the external pH, perhaps just locally, might alleviate this problem and facilitate nitrogen assimilation.

The psp operon may also alleviate a situation that impairs nitrogen assimilation. Since the psp operon responds to energy or nutrient limitation, it has been proposed that the ATP concentration controls pspABCDE expression (112, 165). The conditions that induce the psp operon may also modulate the expression of genes that require NR_I, which itself hydrolyzes ATP (166) and is severely inhibited by ADP (D. Fewell and L. Reitzer, unpublished observation). Another mechanism by which psp expression can affect nitrogen assimilation is based on the proposal that PspA maintains the proton gradient, whose collapse would impair energy generation. Under such conditions, energy-consuming nitrogen assimilation might be inappropriate. (It has been estimated that 1 g of E. coli requires about 57,000 µmol of ATP and that if all nitrogen is assimilated via GS, then the GS reaction itself consumes about 10,500 µmol of ATP. This is obviously a major strain on cellular resources.) In this context, it should be noted that the expression of the psp, ato, and prp operons are probably linked. Fatty acids can collapse the proton gradient and presumably induce the psp operon, and the subsequent products of fatty acid catabolism will induce the ato and prp operons.

If certain conditions make nitrogen assimilation difficult, then not only might the more active ⁵⁴-dependent genes that are not directly involved in nitrogen assimilation alleviate these conditions, but also their expression might downwardly modulate the expression of the ⁵⁴-dependent genes of nitrogen assimilation by competing for ⁵⁴. This is especially plausible for strains with a low level of ⁵⁴, such as strain MC4100, which may contain as few as 20 molecules of ⁵⁴ per cell (87). Furthermore, PspA may have the ability to inactivate all ⁵⁴-dependent transcription. If this is the case, expression of the psp operon might lower the availability of ⁵⁴ and also inactivate the activators of ⁵⁴-dependent genes. The inhibition of the activators may be important even if ⁵⁴ is present in excess.

In summary, if the genes of the FhlA regulon and the psp, ato, and prp operons alleviate conditions that are detrimental to nitrogen assimilation, the vast majority of ⁵⁴-dependent genes in E. coli have a function that is related to nitrogen assimilation.

The mechanism of ⁵⁴-dependent transcription is complex and requires a large regulatory region. This raises the question why such cumbersome transcriptional control has been evolutionarily maintained. One advantage of ⁵⁴-dependent control is the wide range of activity. PspA and GS (both products of ⁵⁴-dependent operons) can become a few percent of the proteins of E. coli. Furthermore, expression of these operons can be completely suppressed. This could be important for enzymes of nitrogen assimilation, which consume energy and withdraw intermediates from central metabolic pathways, especially the citric acid cycle. The advantage of such absolute control may be to prevent the rapid and catastrophic depletion of resources. The potential for such a loss has been demonstrated by removal of just one layer of nitrogen assimilation control, the adenylylation system for GS, which can result in glutamate depletion (96).

The size constraints of ⁵⁴-dependent promoters (the need for binding sites for E⁵⁴, distant activators, and perhaps a DNA bending protein) may counterbalance the potential advantages of ⁵⁴-dependent promoters and also minimize the number of such promoters in a single organism. Such reasoning could account for the seemingly limited number of ⁵⁴-dependent promoters in E. coli, which we estimate to be about 30 (discussed above). ⁵⁴ is widespread among bacteria, and the ⁵⁴-dependent operons code for proteins with a variety of functions (151). The sheer diversity of these functions suggests that these genes are not always associated with nitrogen assimilation. Nonetheless, the possible evolutionary pressure to maintain few ⁵⁴-dependent promoters within a single organism may limit the function of ⁵⁴-dependent proteins to a few physiologically related themes.