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NUTRIENT CONTROL OF BACTERIAL GENE EXPRESSION
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Michael
Cashel, MD, PhD, Head, Section on
Molecular Regulation Rajendran
Harinarayanan, PhD, Visiting Fellow Katarzyna
Potrykus, PhD, Visiting Fellow Daniel
Vinella, PhD, Courtesy Associate Helen
Murphy, MS, Microbiologist |
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Our
goal is to understand how global patterns of bacterial gene expression are coordinated
with nutrient availability. We continue to focus on the roles of (p)ppGpp,
which are two regulatory nucleotide analogs similar to GTP and GDP but with a
pyrophosphate esterified on the ribose 3´ hydroxyl. Nutrient limitation
elevates (p)ppGpp while nutrient sufficiency restores low basal levels. The
mechanism operates during starvation for amino acids, phosphate, nitrogen, or
energy sources. Regulatory roles are assigned to (p)ppGpp because eliminating
(p)ppGpp can abolish regulation during starvation, and artificially elevating
(p)ppGpp without starvation mimics many regulatory effects of starvation.
Responses to (p)ppGpp are a key element in cellular adaptive responses. We
wish to understand in molecular detail how nutrient limitation governs (p)ppGpp
metabolism and how regulation by (p)ppGpp works, i.e., the roles of (p)ppGpp
in the coordination of cellular gene expression. Our work also has
potentially practical applications as an abundant literature links (p)ppGpp
with bacterial pathogenicity in Gram-negative bacteria, synthesis of toxins
and antibiotics in Gram positives, and persistence of chronic infections in Mycobacteria
tuberculosis. Structural
determinants for reciprocal control of (p)ppGpp synthesis and degradation Harinarayanan, Murphy,
Cashel The
balance of rates of (p)ppGpp synthesis and hydrolysis determines the level of
(p)ppGpp. Regulation of the balance of opposing activities is coordinated
without simultaneously increasing synthesis and/or degradation. We reported
earlier that proteins encoded by the rsh gene family (rel spo
homolog) contain three domains. The N-terminal (NTD) half contains
hydrolase and synthetase catalytic domains while the C-terminal half (CTD) is
needed to regulate the balance of activities. Synthesis of (p)ppGpp is
activated by sensing amino acid starvation through uncharged tRNA binding to
unoccupied ribosomal acceptor sites. Hydrolysis of (p)ppGpp is controlled by
sensing starvation for other nutrients through undefined mechanisms. We
previously reported that the two catalytic domains of Rsh protein from
Streptococcus could exist in two activity states: hydrolase
OFF–synthetase ON or hydrolase ON–synthetase OFF. Switching
between these activity states is determined by the C-terminal domain. Additional
conformational control elements exist within the two catalytic domains. Many
missense mutants in the solitary synthesis catalytic domain were activated by
the added presence of the wild-type hydrolase domain to the catalytic half
protein. Screening for hydrolase domain mutants unable to activate synthetase
revealed two instances in which a special set of point mutants were found to
be capable of reversing one synthetase but not another. These examples of
intragenic allele-specific suppression together with an additional regulatory
role of the CTD suggested that the switch leading to reciprocal regulation of
opposing catalytic activities is an intrinsic feature of the protein that
could be changed by special NTD mutants, by NTD-CTD interactions, or by
signals operating on the CTD as for activation by uncharged tRNA. A
collaboration leading to structural resolution of the catalytic NTD fragment
supported these deductions because two conformations were found that could
represent each of the two reciprocal activity states. We set forth a
hypothesis to explain how an intrinsic conformational antagonism between
opposing active sites could be triggered by ligand binding and thereby
coordinate switching between activity states. A promising observation
supporting the ligand-binding hypothesis was that the presence of a
nonhydrolyzable substrate analogue for synthesis (alpha-beta methylenyl ATP)
inhibits hydrolase even though the ATP analog is not known to interact
directly with the hydrolysis site. We have constructed a series of mutants
designed to abolish one or the other of the two opposing activities yet
retain conformational changes allowing regulation of the unaltered activity.
We assume that measurements of each single activity unobscured by the
opposing activity will provide clues as to how environmental signals of
starvation provoke the transitions between the two activity states. Regulation
of (p)ppGpp mediated by the SpoT protein Harinarayanan,
Vinella, Murphy, Cashel; in collaboration with D'Ari, Schneider We
are searching for mechanisms that lead to (p)ppGpp regulation during
starvation for nutrients other than amino acids. In contrast to amino acid
starvation, little is known about such mechanisms. Inhibitory effects of
chelators of the essential manganese ion cofactor for the hydrolase are known
but are not thought to be physiologically relevant. We suspect instead that
signals of nutritional stress interact with the bifunctional protein (either
on the CTD or the NTD). If we can learn the sites of action of the signals,
we can attempt to identify the signals either through ligand fishing or
through genetic selections. Our
approach exploits mutants predicted from the protein structure just
mentioned. Mutants defective in either hydrolase or synthetase have been constructed,
tested, and inserted in single copy in the E. coli chromosome under
control of the native spoT gene promoter. We have also made a parallel
series of constructs deriving the rsh gene from M. tuberculosis,
swapping its open reading frame (orf) with the spoT orf. We imposed
stress conditions on such cells and then measured individual hydrolase or
synthetase activities. The rationale for comparisons between the E. coli and
M. tuberculosis genes is that we expect different responses because of
differences in copy number, in specialization, and the complexity of
cytoplasmic lipids. A
genetic approach to learning about new stress conditions culminating in
elevation of (p)ppGpp scored as mecillinam resistance in a relA gene–deleted
host led to isolation at NIH of an insertion allele in the fes gene,
encoding enterochelin esterase. Later systematic genetic studies in the
Jacques Monod Institute (by Vinella and D’Ari) led to the prediction
that iron starvation would provoke (p)ppGpp accumulation. Direct measurement
at NIH verified the prediction. We
are continuing to characterize spontaneous CTD-region spoT mutants
found repeatedly among populations of E. coli B strains evolving under
repeated glucose starvation growth conditions. The spoT alleles appear
to arise as a consequence of earlier spontaneous topA mutations, which
also confer a growth advantage. Measurements of (p)ppGpp reveal only minor
effects on synthesis and degradation. The lack of a convincing explanation
for the growth advantage indirectly suggests the possibility of a new
function of the SpoT protein. Transcription
regulation by (p)ppGpp Potrykus, Vinella,
Murphy, Cashel Recent
work in other laboratories has revealed several important features of the
interactions between RNA polymerase (RNAP) and (p)ppGpp. First,
co-crystallization experiments reveal that (p)ppGpp can be localized in two
orientations within RNAP at the end of the secondary (NTP entry) channel near
the active site (Artisimovitch et al., Cell 2004;117:299). Second,
crystallization of DksA has shown it to be a structural homolog of GreA or
GreB proteins, with a long coil-coil hairpin protruding deep into the
secondary channel and with an RNase activity toward RNA chains backtracked
into the secondary channel when RNAP is paused during elongation. Binding of
DksA to RNAP is thought to stabilize (p)ppGpp binding by coordinating Mg2+
bound to the pyrophosphate residues of (p)ppGpp. DksA is also thought to lack
the RNase activity of its GreA and GreB homologues (without actual
measurements), although it also contains two acidic residues at the tip of
its hairpin that are implicated in RNase activity for TFIIB, the eukaryotic
homologue of GreA and GreB (Perederina et al., Cell 2004;118:297).
Third, DksA has been shown to be a necessary regulatory component that
synergistically potentiates ppGpp inhibition of ribosomal RNA promoter
activity to levels that are equivalent to those seen physiologically in whole
cells (Paul et al., Cell 2004;118:311). We have shown that positive
regulation of stationary-phase sigma factor function by (p)ppGpp is dependent
on DksA and that the phenotype of a DksA deletion is a subset of the
broader pleiotropic phenotype of a (p)ppGpp-deficiency (Brown et al., J
Bacteriol 2002;184:4455). We
have been using genetic approaches to explore the possibility that DksA might
compete with GreA or GreB to alter its regulatory effects. Promising early
observations suggest the existence of a competition that influences gene
expression in a manner dependent on (p)ppGpp. We
have also begun to characterize the initial biochemical stages of early
transcription from a promoter. We focus on the transition between unstable
binary DNA-RNAP open complexes at a ribosomal P1 promoter and the formation
of stable ternary elongation complexes accompanied by the loss of the
sigma-70 subunit, called promoter clearance. For most promoters, clearance is
complete after the formation of eight to 12 phosphodiester bonds. By
selective addition of dinucleotide primers, an incomplete array of
template-specific NTP substrates, and appropriately modified templates,
progress of the enzyme can be halted at progressive stages during initial
transcription. Using biotinylated templates for immobilization, complexes at
these stages are purified free of enzyme and substrates by washing. These
stages in promoter clearance are being analyzed with respect to (p)ppGpp
effects on changes in stability and subunit structure (sigma-70 or DksA
release). With this new procedure, we hope to document both promoter-specific
and regulation-specific differences in conformational changes of initiating
complexes during promoter clearance. Brown L, Gentry D, Elliott T, Cashel M. DksA affects ppGpp
induction of RpoS at a translational level. J Bacteriol 2002;184:4455-4465. Cashel M, Hsu LM, Hernandez VJ. Changes in conserved region 3 of
Escherichia coli sigma-70 reduce abortive transcription and enhance
promoter escape. J Biol Chem 2003;278:5539-5547. Cashel M, Murphy H. Isolation of RNA polymerase suppressors of a
(p)ppGpp deficiency. Methods Enzymol 2003;371:596-601. Hogg T, Mechold U, Malke H, Cashel M, Hilgenfeld R.
Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT
homolog modulates (p)ppGpp metabolism during the stringent response. Cell
2004;117:57-68. COLLABORATORS Richard D’Ari, PhD, Institut Jacques
Monod, Centre National de la Recherche Scientifique, Université Paris 7,
France Dominique Schneider, PhD, CERMO, Université
Joseph Fourier,
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