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Microbiology and Molecular Biology Reviews, September 2001, p. 422-444, Vol. 65, No. 3
Department of Molecular and Cell Biology, The
University of Texas at Dallas, Richardson, Texas 75083-0688
1092-2172/01/$04.00+0 DOI: 10.1128/MMBR.65.3.422-444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Metabolic Context and Possible Physiological Themes
of
54-Dependent Genes in Escherichia
coli
SUMMARY
INTRODUCTION AND OVERVIEW
Scope
Sigma Subunits and Their Function in E. coli
Unique Features of
54-Dependent Transcription
Control of
54-Dependent Promoters
COMMON FEATURES OF
54-DEPENDENT PROMOTERS
COMPUTER IDENTIFICATION OF POTENTIAL
54-DEPENDENT PROMOTERS
Site Identification and the Problem of False Positives
Predictive Value of the Promoter Scores
Estimating the Number of
54-Dependent
Promoters in E. coli
54-DEPENDENT GENES OF NITROGEN METABOLISM
Nitrogen Assimilation and Its Control
Nitrogen assimilation.
Control of ammonia assimilation and GS activity: role of
glutamine.
Nitrogen-Regulated (Ntr) Response
Nitrogen sources.
Control of the Ntr response by glutamine.
-Ketoglutarate counters the effects of unmodified
PII.
NRI regulon.
Nac regulon.
Why are there two general regulators of the Ntr response?
Function of the Ntr response.
glnALG (glnA-ntrBC) Operon
glnK-amtB Operon
GlnK: a PII paralog.
Why have GlnK?
Product of amtB and ammonia transport.
nac
Catabolism of Arginine, Agmatine, Ornithine,
Putrescine, and
-Aminobutyrate
astCADBE operon and catabolism of arginine and
ornithine.
GABA and putrescine catabolism and the gabDTPC
operon.
ygjG.
54-dependent Amino Acid Transport Systems
Arginine.
(i) The three transport systems.
(ii) Repression by arginine.
(iii) Transport and activation during nitrogen limitation.
Histidine.
Glutamine.
Glutamate-aspartate.
ddpXABCDE operon.
Peptide transport and ompF.
Potential
54-Dependent Genes That Are
Induced by Nitrogen Limitation
54-DEPENDENT GENES THAT ARE NOT INVOLVED
IN NITROGEN METABOLISM
Formate Catabolic Genes and the FhlA Regulon
Formate metabolism.
The four confirmed
54-dependent operons of
formate metabolism.
Hydrogenase 4.
ato Operon and Acetoacetate Catabolism
prpBCDE Operon and Propionate Catabolism
psp Operon and Phage Shock Response
rtcBA Operon
zraSR (hydHG), zraP,
and the Response to Zn2+ and Pb2+
OTHER GENES WITH HIGHLY RANKED POTENTIAL
E
54 BINDING SITES
54-DEPENDENT ACTIVATORS
PHYSIOLOGICAL FUNCTION OF
54
Possible Relationship between the
54-Dependent Genes
Evolutionary Persistence of
54
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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54 has several features that distinguish it from other sigma factors in Escherichia coli: it is not homologous to other
subunits,
54-dependent expression absolutely requires an activator, and the activator binding sites can be far from the transcription start site. A rationale for these properties has not been readily apparent, in part because of an inability to assign a common physiological function for
54-dependent genes. Surveys of
54-dependent genes from a variety of organisms suggest that the products of these genes are often involved in nitrogen assimilation; however, many are not. Such broad surveys inevitably remove the
54-dependent genes from a potentially coherent metabolic context. To address this concern, we consider the function and metabolic context of
54-dependent genes primarily from a single organism, Escherichia coli, in which a reasonably complete list of
54-dependent genes has been identified by computer analysis combined with a DNA microarray analysis of nitrogen limitation-induced genes. E. coli appears to have approximately 30
54-dependent operons, and about half are involved in nitrogen assimilation and metabolism. A possible physiological relationship between
54-dependent genes may be based on the fact that nitrogen assimilation consumes energy and intermediates of central metabolism. The products of the
54-dependent genes that are not involved in nitrogen metabolism may prevent depletion of metabolites and energy resources in certain environments or partially neutralize adverse conditions. Such a relationship may limit the number of physiological themes of
54-dependent genes within a single organism and may partially account for the unique features of
54 and
54-dependent gene expression.
INTRODUCTION AND OVERVIEW
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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.
Sigma Subunits and Their Function in E. coli
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).
E
70 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 E
70 (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.
Unique Features of
54-Dependent Transcription
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,
E
54 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 E
54-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 E
54 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).
Control of
54-Dependent Promoters
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.
COMMON FEATURES OF
54-DEPENDENT PROMOTERS
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There are 11
54-dependent promoters for which the
transcription start site has been determined in vivo, in vitro, or
both. These promoters precede astCADBE,
fdhF, glnALG, glnHPQ,
hycABCDEFGHI, hydN-hypF,
hypABCDE-fhlA, prpBCDE, pspABCDE,
rtcBA, and ygjG. It is virtually a certainty that
atoDAEB, glnK-amtB, nac,
zraP, and zraSR (hydHG) have a
54-dependent promoter, since their expression absolutely
requires
54 and their promoter regions contain an easily
recognizable site for E
54. Also, argT-hisJQMP
has a verified
54-dependent promoter in Salmonella
enterica serovar Typhimurium, and nitrogen limitation
induces these genes in E. coli, which suggests that this
operon possesses a
54-dependent promoter.
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 E
54 in relation to the binding
sites for the
54-dependent activators (when known) and
the nearest upstream structural gene.
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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 E
54 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 E
54 and an activator. Fourth,
the distance from the 3' end of the E
54 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
E
54 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 E
54 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 E
54, which is described in the next section.
COMPUTER IDENTIFICATION OF POTENTIAL
54-DEPENDENT PROMOTERS
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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.
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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 E
54
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.
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Predictive Value of the Promoter Scores
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
E
54 binding sites, which implies that the failure to
detect such a site is a reliable indicator that such a site does not
exist.
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Estimating the Number of
54-Dependent
Promoters in E. coli
There are 17
54-dependent promoters which either
have been verified by direct evidence or whose expression requires
54; there are 7 other operons which are induced by
nitrogen limitation (often associated with
54), appear
to require a
54-dependent activator, and for which
computer analysis suggests the presence of an appropriately located
sequence for a
54-dependent promoter: b1012-b1006,
chaC, ddpXABCDE, gltIJKL,
potFGHI, yeaGH, and yhdWXYZ. Assuming that
all of these genes have a functional
54-dependent
promoter (which is unlikely) and that a few have been missed for
various reasons (e.g., misidentified open reading frames), we estimate
that E. coli contains about 30
54-dependent promoters.
54-DEPENDENT GENES OF NITROGEN METABOLISM
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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 and Its Control
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.
Ammonia can be considered the focal point of nitrogen assimilation. There are two routes of ammonia assimilation (Fig. 4). For the first pathway, glutamate dehydrogenase assimilates ammonia and synthesizes glutamate. For the second pathway, glutamine synthetase (GS) assimilates ammonia, and glutamate synthase synthesizes glutamate. The former pathway is often associated with the presence of ammonia, and the latter pathway is associated with low ammonia levels or growth with a nitrogen source other than ammonia, since the Km for ammonia for glutamate dehydrogenase is about 20-fold higher than that for GS. However, the most important difference between the two pathways appears to be that the former does not consume ATP but the latter does. Helling has shown that the glutamate dehydrogenase pathway is physiologically advantageous during carbon- and energy-limited growth while the GS-glutamate synthase pathway is used whenever energy is readily available (66, 67). (The energy difference between the ammonia assimilation pathways can be calculated, and it is significant. A 1-g amount of E. coli requires the synthesis of about 57,000 µmol of ATP and contains about 10,500 µmol of nitrogen. The ATP requirement for glutamine synthesis depends on the pathway of ammonia assimilation. If glutamate dehydrogenase assimilates ammonia, the cell requires about 2,300 µmol of glutamine and a corresponding amount of ATP for its synthesis. If the GS-glutamate synthase route assimilates ammonia, glutamine is also the precursor for glutamate and the cell must synthesize an additional 8,070 µmol of glutamine and ATP, or 14% more energy.)
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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.
-ketoglutarate to glutamine (a sensor of relative carbon-to-nitrogen sufficiency) controls UTase-UR activity and therefore GS adenylylation. This conclusion was based on the properties of partially purified UTase-UR (2), which were not confirmed with purified
UTase-UR (83). Furthermore, metabolite measurements
suggested that low intracellular glutamine levels might be sufficient
to control the response to nitrogen limitation (discussed below), which
UTase-UR also controls (79). These results suggest that
glutamine is the primary effector of UTase-UR and therefore of GS
adenylylation (Fig. 5). Low glutamine
levels stimulate UTase activity, which uridylylates
PII. PII-UMP interacts with
adenylyltransferase, which now removes adenylyl groups from GS and
activates GS activity. High glutamine (nitrogen excess) stimulates UR
activity, which results in the formation of unmodified PII,
whose interaction with adenylyltransferase stimulates adenylylation and
reduces GS specific activity. Even though
-ketoglutarate does not
affect UTase-UR, it does control the activity of unmodified
PII (discussed below).
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Nitrogen-Regulated (Ntr) Response
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.
Control of the Ntr response by glutamine. The two proteins that control GS adenylylation, UTase-UR and PII, also control the Ntr response. High glutamine levels (nitrogen sufficiency) stimulate UR activity, which prevents uridylylation of PII. Unmodified PII interacts with nitrogen regulator II (NRII, also called NtrB) and stimulates the dephosphorylation of nitrogen regulator I (NR1, also called NtrC). The net effect is low expression of the glnALG operon and failure to activate Ntr genes. Low glutamine levels (nitrogen limitation) result in the formation of PII-UMP, which is unable to interact with NRII. In this situation, NRII phosphorylates itself and transfers the activated phosphate to NRI. NRI-P then activates the expression of the glnALG operon and other Ntr genes.
-Ketoglutarate counters the effects of unmodified
PII.
Even though recent studies have suggested that
the ratio of glutamine to
-ketoglutarate does not regulate UTase-UR
activity,
-ketoglutarate does affect PII activity
(83-85).
-Ketoglutarate counteracts the effects of
unmodified PII (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 PII) 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 PII is uridylylated. It will be suggested
elsewhere in this review that such coordination might be a function of
GlnK, a PII-like protein.
NRI regulon.
NRI 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 PII 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 NRI 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, NRI represses the two minor promoters,
glnAp1 and glnLp, of the glnALG operon.
Nac regulon.
There are two majors regulators of the
Ntr response: NRI and Nac. NRI 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).
-aminobutyrate [GABA] metabolism), nupC
(nucleoside transport), ompF (outer membrane protein F),
oppABCD (oligopeptide transport), yedL (unknown
function), and yhiE (unknown function) (176).
Nac-dependent control has been directly verified for the gab
operon (S. Ruback and L. Reitzer, unpublished observation) but
not for the other genes.
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 NRI-dependent control.
NRI-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).
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Function of the Ntr response.
It is likely that most
of the
54-dependent Ntr genes have been identified using
results from a microarray analysis and the complementary computer
analysis of potential
54-dependent promoters. The Ntr
genes can be divided into a few categories: GS, regulators, transport
proteins, and catabolic enzymes.
glnALG (glnA-ntrBC) Operon
The glnALG operon codes for GS, NRII, and
NRI, respectively (127, 129). All three
products of this operon are required for nitrogen assimilation and the
Ntr response (discussed above).
54 and
NRI~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 NRI indicate that nitrogen limitation
induces NRI synthesis 14-fold (128). Minor
promoters, glnAp1 and glnLp, ensure
basal synthesis of the important products of this operon (130,
157).
glnK-amtB Operon
Nitrogen limitation and NRI 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
E
54 and NRI (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 PII paralog.
The existence of a
PII paralog was first suspected because of the rapid
deadenylylation of GS in an E. coli glnB
(PII-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 PII 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 NRI, which has been suggested to cause
inappropriate overexpression of an Ntr gene (10). An
alternate explanation is NRI-dependent overexpression of a
54-dependent gene that is not normally regulated by
NRI.
-ketoglutarate is more efficient in inhibiting the activity of GlnK than of PII (9).
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 PII 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).
nac
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 NRI~P (16, 54,
106). Nitrogen limitation induces E. coli nac
(114, 176), and computer analysis indicates a likely binding site for E
54 with a score of 85.2, which is very
high. These results suggest that NRI and E
54
are also required for E. coli nac expression. Nac negatively modulates its own synthesis in K. aerogenes by interfering
with the interaction between NRI and RNA polymerase
(55, 114), and results from the DNA microarray analysis
are consistent with such regulation in E. coli
(176).
Catabolism of Arginine, Agmatine, Ornithine,
Putrescine, and
-Aminobutyrate
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.
|
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.
54, while expression from the other promoter
requires E
S. SeqScan gives the
54-dependent promoter a score of 85.5. Some evidence
suggests that transcription from one promoter prevents transcription
from the other (60). One unusual feature of the
ast operon in E. coli is that ArgR binds to the
region between the E
54 and NRI binding sites
and has been proposed to stimulate the interaction between the two
proteins (103). ArgR is required for optimal transcription
of the E. coli ast operon but is not absolutely necessary
(Kiupakis and Reitzer, unpublished). In contrast, ArgR appears to be
required for the S. enterica serovar Typhimurium ast operon (103). The ast operon
contains the only known E. coli NRI-dependent
promoter that also requires specific induction. Microarray analysis
indicates that general nitrogen limitation (i.e., without arginine
induction) increases ast transcription 7- to 11-fold
(176), which is consistent with the results of a direct
assay of the gene products (144). Arginine induces the enzymes three- to fourfold further (144), which is
consistent with in vitro results (Kiupakis and Reitzer, unpublished).
Another unusual aspect of ast expression is that a strain
with a glnL (ntrB) deletion cannot utilize
arginine as a nitrogen source (L. Reitzer, unpublished observation) but
can still activate glnA expression, albeit not as rapidly
(130). Expression of the ast operon requires
phosphorylation of NRI by both NRII and small phosphodonors (B. L. Schneider, D. Fewell, and L. J. Reitzer, unpublished observation).
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.
-aminobutyraldehyde) is a presumed intermediate in
putrescine catabolism. Unexpectedly, a strain with a deletion of
gabDT grew normally with putrescine or agmatine as a
nitrogen source (Kasbarian et al., unpublished). Even more surprising
is the observation that an rpoN mutant (
54
deficient) grew normally with putrescine as a nitrogen source (Kiupakis
and Reitzer, unpublished). These results would suggest that nitrogen
limitation is not required for induction of putrescine catabolic genes,
but this is not the case. Microarray analysis suggests that nitrogen
limitation activates the expression of two different putrescine
transport operons: potFGHI (five- to sixfold) and b1440-1444
(five- to sevenfold) (176). In addition to these transport
systems, E. coli possesses two
54-independent
transport systems, the products of potABCD (which preferentially transport spermidine but also transport putrescine) and
of potE (78). The genes of putrescine
catabolism and the physiological function of the four transport systems
have yet to be established.
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).
54-dependent Amino Acid Transport Systems
More than half of the genes activated by nitrogen limitation in
E. coli code for transport systems. Such activation usually does not require specific induction. Such regulation combined with the
observation that mutational inactivation of the genes for
54-dependent amino acid transport system does not
prevent growth on the respective amino acid suggests a scavenging
function. In this section, the
54-dependent amino acid
transport systems are considered within the context of the multiple
transport systems for these amino acids.
Arginine.
(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.
Only one of the three E. coli arginine transport systems requires
54 for its synthesis. It contains the
periplasmic ArgT protein, also called the LAO protein, which binds
lysine, ornithine, and arginine with high affinity (136).
The argT gene and its product have been extensively studied
in S. enterica serovar Typhimurium but not in E. coli. ArgT interacts with HisP of S. enterica serovar Typhimurium (98). HisJ, the periplasmic histidine binding
protein, also interacts with HisP (5). An S. enterica serovar Typhimurium hisP mutant grows much
more slowly with arginine as the nitrogen source than an
argT mutant does, which suggests that HisP also interacts
with an arginine binding periplasmic protein other than ArgT
(98).
54-independent arginine transport systems have been
studied only in E. coli. One system contains AbpS, also
called the arginine-ornithine protein, which binds arginine and
ornithine in the periplasm with lower affinity than the LAO protein
does (34). Early reports refer to this protein as a
low-affinity, arginine-specific protein (41, 136). The
abpS gene has been approximately mapped to min 63.5 of the
most recent E. coli map (35). AbpS has been
purified, and its size and amino acid composition have been determined
(36). However, no gene near min 63 specifies a protein
with the published amino acid composition. The nearest matches to this
amino acid composition in the E. coli genome, in descending
order, are the products of artI, artJ, and
hisJ, which are located at min 19.4, 19.4, and 52.3, respectively. It is possible that the sequenced MG1655 does not contain
abpS. The second
54-independent system
consists of the artPIQM-artJ operons, which are at min 19 of
the E. coli chromosome (169). ArtJ is a
periplasmic protein that binds arginine but not ornithine
(169). ArtI is another putative periplasmic binding
protein, but it does not detectably bind any amino acid. Mutants with
mutations in the ArtJ system do not exist, but overexpression increases
arginine transport, which is consistent with a proposed function in
arginine transport (169). Promoters precede
artP and artJ, and neither appears to require
54 (169).
(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).
argT is adjacent to the hisJQMP operon, which codes for components of a histidine transport system. Transcript mapping in wild-type S. enterica serovar Typhimurium, not in an extensively studied dhuA1 mutant which appears to have a mutationally created promoter, shows a
54-dependent
promoter immediately preceding argT but not immediately preceding hisJ (6). This is consistent with
expression studies with reporter gene fusions, which failed to identify
an Ntr promoter preceding hisJ (142, 143). The
potential
54-dependent promoter preceding
argT in E. coli has a score of 72.3. There is
only one binding site for NRI, and it is in the
argT-hisJ intercistronic region and not upstream from
argT (6). The function of this site is not
clear. It does not appear to be necessary for expression, which would
imply that NRI activates the argT promoter
without binding to DNA (143). There is precedent for NRI-dependent transcription that does not require a DNA
binding site (131, 171).
Histidine. The genes and regulation of the HisJ transport system were discussed in the preceding section because of their relation to arginine transport. It is not known whether there are other histidine transport systems.
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).
Expression of glnHPQ requires nitrogen limitation but not glutamine (18, 98, 168). Nitrogen limitation increases the production of glnHPQ transcripts five- to ninefold (176). The operon contains two promoters (118). Transcription from the downstream promoter, glnHp2, requires
54 and
NR1 and is enhanced by IHF (44). The promoter
has a score of 80.7. The factors that control the upstream promoter,
glnHp1, have not been examined.
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.
Nitrogen limitation induces a periplasmic protein that binds both glutamate and aspartate in S. enterica serovar Typhimurium (98). Such a protein has been purified and characterized from E. coli (167). The closest match in the entire SWISS-PROT database with the published amino acid composition of the glutamate-aspartate binding protein is gltI (also called ybeJ) from E. coli. The gltI product was the only protein in E. coli with the correct pI, size, and number of cysteines. gltI might be part of a gltIJKL operon. Despite the annotation of gltIJKL as part of a glutamate-aspartate transport system, no published evidence supports this possibility (17). There are no potential
54-dependent promoters preceding
gltI with a score greater than 60. However, the gene
preceding gltI specifies an IS5 transposase, and
the promoter region for the transposase gene contains a possible
54-dependent promoter with a score of 88, which is very
high. Microarray analysis indicates a three- to sixfold increase of
gltI transcription during nitrogen limitation, and
NRI-dependent activation (176). These results
suggest that the
54-dependent promoter preceding the
transposase gene can initiate gltIJKL transcription.
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 NRI
(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
NRI or Nac.
54-dependent promoter with a score of
64.4. However, like oppABCDF expression, the level of
induction in mutants with constitutively active NRII is
dramatically stronger with glutamine (25-fold) than with ammonia
(4-fold) (176), and Lrp has been implicated in
ompF regulation (57). Perhaps nitrogen
limitation controls ompF indirectly, as was suggested for
oppABCDF.
Potential
54-Dependent Genes That Are
Induced by Nitrogen Limitation
Nitrogen limitation results in a 20-fold increase in the
expression of the putative b1012-1006 operon (176). The
potential E
54 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 NRI 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
NRI or Nac (176). The potential
E
54-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
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|
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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 FDHH. The
divergently transcribed polycistronic hyc and hyp
operons code for components of FDHH, 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
FDHH 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.
54-dependent activators (reference 141 and
references therein). Footprinting experiments have established the
binding sites for FhlA (73). Like other
54-dependent activators, it can activate when bound to
distant sites (104).
Several factors regulate FHL complex synthesis (22, 125).
Low pH and formate induce its synthesis, while oxygen, nitrate, and
glucose repress it (138). The formate and oxygen control are regulated via FhlA. Formate stimulates the ATPase activity of
FhlA (74) and is required for in vitro transcription,
which implies that formate binds FhlA (73). Oxygen control
may be mediated by OxyS, which is induced by oxidative stress
(3). OxyS is an abundant stable untranslated RNA that
binds to the FhlA mRNA and blocks its translation (4).
Glucose, pH, nitrate, and additional oxygen control is probably
indirect, i.e., via regulation of the synthesis of the major formate
transport system, which is part of the pfl (pyruvate
formate-lyase) operon (93, 138). HycA is a regulator that
antagonizes the activation of FhlA (unpublished results cited in
reference 24). The mechanism of this regulation has not
been characterized. Mo availability also regulates expression of the
hyc operon (146). ModE is a sensor of
intracellular Mo, and a ModE-Mo complex represses the modABCD operon, which specifies components of a Mo transport
system. ModE-Mo also stimulates transcription of the hyc
operon by binding to a site centered 190 bases from the start site of
transcription (146). (The FhlA binding site is centered
100 bases from the transcription start site [73]).
ModE-Mo is not required for expression, but its presence accounts for a
two- to threefold stimulation. In addition, the MoeA protein, a
component of Mo metabolism, stimulates hyc expression two-
to threefold by an unknown mechanism (146). The
multiplicity of regulators might strengthen the binding of
E
54 to FhlA-dependent promoters and may account for
their low scores.
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).
ato Operon and Acetoacetate Catabolism
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 E
54. 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 NRI (NtrC),
and the putative AtoS is homologous to sensor kinases, such as
NRII (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-E
54 interaction (42).
prpBCDE Operon and Propionate Catabolism
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
NRI (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).
psp Operon and Phage Shock Response
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, NRI, and perhaps
others (51). It is not known whether this inhibition of
NRI 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.
rtcBA Operon
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.
zraSR (hydHG), zraP, and the Response to Zn2+ and Pb2+
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
E
54 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.
OTHER GENES WITH HIGHLY RANKED POTENTIAL
E
54 BINDING SITES
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Computer analysis identified 25 properly oriented intergenic sites
with a score greater than 76, which is a good indicator of an
E
54-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 E
54 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 NRI 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, PII (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 E
54 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 E
54 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 E
54 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 NRI
(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.
54-DEPENDENT ACTIVATORS
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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
E
54. 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
NRI as probes for BLAST searches. Both probes identified
the same proteins: AtoC, the product of b1201, FhlA, HyfR,
NRI, 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 E
54
(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,
NRI and FhlA, are known to activate more than one operon.
|
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
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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 NRI
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 NRI, 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.
Evolutionary Persistence of
54
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 E
54, 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.
ACKNOWLEDGMENTS
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We acknowledge Juan Gonzalez and Alexandros Kiupakis for comments on the manuscript.
Grants GM47965 from the National Institute of General Medical Sciences and MCB-9723003 and MCB-0077904 from the National Science Foundation supported the work of L.R. on nitrogen metabolism.
FOOTNOTES
* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Mail Station FO 31, The University of Texas at Dallas, P.O. Box 830688, Richardson, TX 75083-0688. Phone: (972) 883-2502/2523. Fax: (972) 883-2409. E-mail: reitzer{at}utdallas.edu.
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