bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Stringent Response of
Mycobacterium tuberculosis
Is Required for Long-Term Survival
Authors: Primm Todd P. , Andersen Susan J. , Mizrahi Valerie , Avarbock David , Rubin Harvey , Barry Clifton E. 
Published: July 2002

ABSTRACT

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins.
Mycobacterium tuberculosis
contains a single homolog of these proteins (Rel
Mtb
) and responds to nutrient starvation by producing (p)ppGpp. A

rel
Mtb

knockout strain was constructed in a virulent strain of
M. tuberculosis
, H37Rv, by allelic replacement. The

rel
Mtb

mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel
Mtb
-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel
Mtb
-mediated adaptation. Long-term survival of the

rel
Mtb

mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anerobic incubation than the wild-type virulent organism. Thus, the Rel
Mtb
protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.
Lesions in
gshA
(Encoding γ-
l
-Glutamyl-
l
-Cysteine Synthetase) Prevent Aerobic Synthesis of Thiamine in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Gralnick Jeffrey , Webb Eric , Beck Brian , Downs Diana 
Published: July 2002

ABSTRACT

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in
Salmonella enterica
serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in
gshA
, encoding γ-
l
-glutamyl-
l
-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including
S. enterica
LT2. Phenotypic characterization of the
gshA
mutants indicated they were similar enough to
apbC
and
apbE
mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of
l
-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.
Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of
Corynebacterium glutamicum
Authors: Molenaar Douwe , van der Rest Michel E. , Drysch André , Yücel Raif 
Published: July 2002

ABSTRACT

Like many other bacteria,
Corynebacterium glutamicum
possesses two types of
l
-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC
1.1.99.16
) and a cytoplasmic malate dehydrogenase (MDH; EC
1.1.1.37
) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of
C. glutamicum
. A mutant with a site-directed deletion in the
mqo
gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an
mqo
mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from
C. glutamicum
catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an
mdh
deletion alone was observed, which leaves a physiological function for MDH in
C. glutamicum
obscure.
Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of
Escherichia coli
Authors: van der Rest Michel E. , Frank Christian , Molenaar Douwe 
Published: July 2002

ABSTRACT

Oxidation of malate to oxaloacetate in
Escherichia coli
can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC
1.1.1.37
) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC
1.1.99.16
), encoded by the gene
mqo
(previously called
yojH
). Expression of the
mqo
gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the β-galactosidase reporter fused to the promoter of the
mqo
gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress
mqo
expression. On the contrary, MQO and MDH are active at the same time in
E. coli
. For
Corynebacterium glutamicum
, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of
E. coli
. In this organism, a defined deletion of the
mdh
gene led to severely decreased rates of growth on several substrates. Deletion of the
mqo
gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an
mqo
mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes
sfcA
and
b2463
(coding for EC
1.1.1.38
and EC
1.1.1.40
, respectively) and of the phosphoenolpyruvate synthase (EC
2.7.9.2
) gene
pps
were created. They were introduced separately or together with the deletion of
mqo
. These studies did not reveal a significant role for MQO in malate oxidation in wild-type
E. coli
. However, comparing growth of the
mdh
single mutant to that of the double mutant containing
mdh
and
mqo
deletions did indicate that MQO partly takes over the function of MDH in an
mdh
mutant.
Subunit Interactions and Glutamine Utilization by
Escherichia coli
Imidazole Glycerol Phosphate Synthase
Authors: Klem Thomas J. , Chen Yuan , Davisson V. Jo 
Published: July 2002

ABSTRACT

A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric
Escherichia coli
imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however,
k
cat
/

K
m

for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.
Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of an
l
-Arginine/γ-Aminobutyric Acid Permease in
Mycobacterium bovis
BCG
Authors: Seth Anjali , Connell Nancy D. 
Published: July 2002

ABSTRACT

Genes encoding
l
-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter from
Mycobacterium bovis
BCG and the effects of its deletion on
l
-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to the
rocE
gene of
Bacillus subtilis
, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using
14
C-labeled substrates. Greatly reduced uptake of
l
-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),
l
-[
14
C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenous
l
-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium.
l
-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.
Molecular Basis for the Temperature Sensitivity of
Escherichia coli pth
(Ts)
Authors: Cruz-Vera L. Rogelio , Toledo Ivonne , Hernández-Sánchez Javier , Guarneros Gabriel 
Published: July 2002

ABSTRACT

The gene
pth
, encoding peptidyl-tRNA hydrolase (Pth), is essential for protein synthesis and viability of
Escherichia coli
. Two
pth
mutants have been studied in depth: a
pth
(Ts) mutant isolated as temperature sensitive and a
pth
(rap) mutant selected as nonpermissive for bacteriophage λ vegetative growth. Here we show that each mutant protein is defective in a different way. The Pth(Ts) protein was very unstable in vivo, both at 43°C and at permissive temperatures, but its specific activity was comparable to that of the wild-type enzyme, Pth(wt). Conversely, the mutant Pth(rap) protein had the same stability as Pth(wt), but its specific activity was low. The thermosensitivity of the
pth
(Ts) mutant, presumably, ensues after Pth(Ts) protein levels are reduced at 43°C. Conditions that increased the cellular Pth(Ts) concentration, a rise in gene copy number or diminished protein degradation, allowed cell growth at a nonpermissive temperature. Antibiotic-mediated inhibition of mRNA and protein synthesis, but not of peptidyl-tRNA drop-off, reduced
pth
(Ts) cell viability even at a permissive temperature. Based on these results, we suggest that Pth(Ts) protein, being unstable in vivo, supports cell viability only if its concentration is maintained above a threshold that allows general protein synthesis.
Multiple Interactions between Pullulanase Secreton Components Involved in Stabilization and Cytoplasmic Membrane Association of PulE
Authors: Possot Odile M. , Vignon Guillaume , Bomchil Natalia , Ebel Frank , Pugsley Anthony P. 
Published: July 2002

ABSTRACT

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from
Klebsiella oxytoca
encoded by a multiple-copy-number plasmid in
Escherichia coli
. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of
Erwinia chrysanthemi
and
Erwinia carotovora
, showing that they do not play a role in secretory protein recognition and secretion specificity.
Intrinsic Polymerase Activities of UmuD′
2
C and MucA′
2
B Are Responsible for Their Different Mutagenic Properties during Bypass of a T-T
cis-syn
Cyclobutane Dimer
Authors: O'Grady Paul I. , Borden Angela , Vandewiele Dominique , Ozgenc Ali , Woodgate Roger , Lawrence Christopher W. 
Published: July 2002

ABSTRACT

In wild-type
Escherichia coli
, translesion replication is largely dependent upon the UmuD′
2
C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA′
2
B. Interestingly, both the efficiency of translesion replication of a T-T
cis-syn
dimer and the spectra of mutations observed are different in Umu- and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of
dnaQ
(ɛ subunit) or
holE
(θ subunit) or carrying the
dnaQ
allele
mutD5
, which is deficient in proofreading but is competent in the structural function of ɛ, or the
dnaE
antimutator allele
spq-2
. The chromosomal copy of the
umuDC
operon was deleted in each strain, and the UmuDC, UmuD′C, MucAB, or MucA′B proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA′
2
B complex is more efficient in promoting translesion replication than the UmuD′
2
C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA′
2
B than by UmuD′
2
C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass.
Action of RNase II and Polynucleotide Phosphorylase against RNAs Containing Stem-Loops of Defined Structure
Authors: Spickler Catherine , Mackie George A. 
Published: July 2002

ABSTRACT
The 3′→5′ exoribonucleases, RNase II and polynucleotide phosphorylase (PNPase), play an essential role in degrading fragments of mRNA generated by prior cleavages by endonucleases. We have assessed the ability of small RNA substrates containing defined stem-loop structures and variable 3′ extensions to impede the exonucleolytic activity of these enzymes. We find that stem-loops containing five G-C base pairs do not block either enzyme; in contrast, more stable stem-loops of 7, 9, or 11 bp block the processive action of both enzymes. Under conditions where enzyme activity is limiting, both enzymes stall and dissociate from their substrates six to nine residues, on average, from the base of a stable stem-loop structure. Our data provide a clear mechanistic explanation for the previous observation that RNase II and PNPase behave as functionally redundant.
Characterization and Mutational Analysis of Three Allelic
lsc
Genes Encoding Levansucrase in
Pseudomonas syringae
Authors: Li Hongqiao , Ullrich Matthias S. 
Published: July 2002

ABSTRACT

In the plant pathogen
Pseudomonas syringae
pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180:
lscA
,
lscB
, and
lscC
. In this study,
lscB
and
lscC
were cloned from a genomic library of strain PG4180. Sequence analysis of the two
lsc
genes showed that they were virtually identical to each other and highly similar to the previously characterized
lscA
gene.
lscA
and
lscC
had a chromosomal location, whereas
lscB
resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual
lsc
genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the
lscB
gene product was secreted but not that of
lscA
or
lscC
. Our results indicated that
lscB
and
lscC
but not
lscA
contributed to periplasmic levan synthesis of PG4180. The
lscB lscC
double mutant was completely defective in levan formation and could be complemented by either
lscB
or
lscC.
Our data suggested a compartment-specific localization of two
lsc
gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that
lscA
was not expressed and that
lscA
was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P

lac

promoter. PCR screening in various
P. syringae
strains with primers derived from the three characterized
lsc
genes demonstrated the presence of multiple Lsc isoenzymes in other
P. syringae
pathovars.
DNA Microarray-Mediated Transcriptional Profiling of the
Escherichia coli
Response to Hydrogen Peroxide
Authors: Zheng Ming , Wang Xunde , Templeton Lori J. , Smulski Dana R. , LaRossa Robert A. , Storz Gisela 
Published: July 2002

ABSTRACT

The genome-wide transcription profile of
Escherichia coli
cells treated with hydrogen peroxide was examined with a DNA microarray composed of 4,169
E. coli
open reading frames. By measuring gene expression in isogenic wild-type and
oxyR
deletion strains, we confirmed that the peroxide response regulator OxyR activates most of the highly hydrogen peroxide-inducible genes. The DNA microarray measurements allowed the identification of several new OxyR-activated genes, including the
hemH
heme biosynthetic gene; the six-gene
suf
operon, which may participate in Fe-S cluster assembly or repair; and four genes of unknown function. We also identified several genes, including
uxuA,
encoding mannonate hydrolase, whose expression might be repressed by OxyR, since their expression was elevated in the Δ
oxyR
mutant strain. In addition, the induction of some genes was found to be OxyR independent, indicating the existence of other peroxide sensors and regulators in
E. coli
. For example, the
isc
operon, which specifies Fe-S cluster formation and repair activities, was induced by hydrogen peroxide in strains lacking either OxyR or the superoxide response regulators SoxRS. These results expand our understanding of the oxidative stress response and raise interesting questions regarding the nature of other regulators that modulate gene expression in response to hydrogen peroxide.
Three Target Genes for the Transcriptional Activator Cat8p of
Kluyveromyces lactis
: Acetyl Coenzyme A Synthetase Genes
KlACS1
and
KlACS2
and Lactate Permease Gene
KlJEN1
Authors: Lodi Tiziana , Saliola Michele , Donnini Claudia , Goffrini Paola 
Published: July 2002

ABSTRACT

The aerobic yeast
Kluyveromyces lactis
and the predominantly fermentative
Saccharomyces cerevisiae
share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the
KlCAT8
gene (a homologue of
S. cerevisiae CAT8
, encoding a DNA binding protein) as a multicopy suppressor of a
fog1
mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of
S. cerevisiae
. While
CAT8
controls the key enzymes of gluconeogenesis in
S. cerevisiae
,
KlCAT8
of
K. lactis
does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193–203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes,
KlACS1
and
KlACS2
, were specifically regulated by
KlCAT8
, but very differently from the
S. cerevisiae
counterparts.
KlACS1
was induced by acetate and lactate, while
KlACS2
was induced by ethanol, both under the control of
KlCAT8.
Also,
KlJEN1
, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of
KlCAT8
.