bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of
Escherichia coli dnaE
(
polC
) Mutants with Altered Sensitivity to 2′,3′-Dideoxyadenosine
Authors: Hiratsuka Koji , Reha-Krantz Linda J. 
Published: July 2002

ABSTRACT

Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S. A. Rashbaum and N. R. Cozzarelli, Nature 264:679–680, 1976). We have used this observation as the basis of a genetic strategy to identify mutations in the
dnaE
(
polC
) gene of
Escherichia coli
that alter sensitivity to 2′,3′-dideoxyadenosine (ddA). Two
dnaE
(
polC
) mutant strains with increased sensitivity to ddA and one strain with increased resistance were isolated and characterized. The mutant phenotypes are due to single amino acid substitutions in the α subunit, the protein product of the
dnaE
(
polC
) gene. Increased sensitivity to ddA is produced by the L329F and H417Y substitutions, and increased resistance is produced by the G365S substitution. The L329F and H417Y substitutions also reduce the accuracy of DNA replication (the mutator phenotype), while the G365S substitution increases accuracy (the antimutator phenotype). All of the amino acid substitutions are in conserved regions near essential aspartate residues. These results prove the effectiveness of the genetic strategy in identifying informative
dnaE
(
polC
) mutations that can be used to elucidate the molecular basis of nucleotide interactions in the α subunit of the DNA polymerase III holoenzyme.
Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by
Streptomyces clavuligerus
Authors: Li Rongfeng , Khaleeli Nusrat , Townsend Craig A. 
Published: July 2002

ABSTRACT

Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain
Streptomyces clavuligerus
were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236,
In
V. P. Gullo et al., ed.,
Development in industrial microbiology series
, 1993). In contrast, we report the identification of three new genes,
orf10
(
cyp
),
orf11
(
fd
), and
orf12
, that are required for clavulanic acid biosynthesis as indicated by gene replacement and
trans
-complementation analysis in
S. clavuligerus
. These genes are contained within a 3.4-kb DNA fragment located directly downstream of
orf9
(
cad
) in the clavulanic acid cluster. While the
orf10
(
cyp
) and
orf11
(
fd
) proteins show homologies to other known
CYP-150
cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by
orf12
shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.
Construction and Use of Derivatives of Transposon Tn
4001
That Function in
Mycoplasma pulmonis
and
Mycoplasma arthritidis
Authors: Dybvig Kevin , French C. Todd , Voelker LeRoy L. 
Published: July 2002

ABSTRACT

Previous attempts to introduce transposon Tn
4001
into
Mycoplasma pulmonis
and
Mycoplasma arthritidis
have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn
4001C
and Tn
4001T
were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the
tetM
tetracycline resistance determinant into Tn
4001
. Both Tn
4001C
and Tn
4001T
transposed in
M. pulmonis
, and Tn
4001T
transposed in
M. arthritidis
. The incorporation of a Tn
4001T
derivative that contained
lacZ
into either
Mycoplasma
species resulted in transformants with readily detectable LacZ activity. Tn
4001T
may be of general utility for use as a mycoplasma cloning vehicle because
tetM
functions in all species of
Mycoplasma
examined thus far.
Regulation of Quorum Sensing by RpoS in
Pseudomonas aeruginosa
Authors: Whiteley Marvin , Parsek Matthew R. , Greenberg E. P. 
Published: July 2002

ABSTRACT

The LasR-LasI and RhlR-RhlI quorum-sensing systems are global regulators of gene expression in the opportunistic pathogen
Pseudomonas aeruginosa
. Previous studies suggest that the RhlR-RhlI system activates expression of
rpoS
. We constructed merodiploid strains of
P. aeruginosa
containing the native
rpoS
gene and an
rpoS-lacZ
fusion. Studies of
lacZ
transcription in these strains indicated that
rpoS
was not regulated by RhlR-RhlI. We also generated an
rpoS
null mutant. This
rpoS
mutant showed elevated levels of
rhlI
(but not
rhlR
) transcription, elevated levels of the RhlI-generated acylhomoserine lactone quorum-sensing signal, and elevated levels of RhlR-RhlI-regulated gene transcription. These findings indicate that there is a relationship between RpoS and quorum sensing, but rather than the RhlR-RhlI system influencing the expression of
rpoS
, it appears that RpoS regulates
rhlI
.
Phosphate Starvation-Inducible Proteins of
Bacillus subtilis
: Proteomics and Transcriptional Analysis
Authors: Antelmann Haike , Scharf Christian , Hecker Michael 
Published: July 2002

ABSTRACT

The phosphate starvation response in
Bacillus subtilis
was analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of ς
B
, the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a
sigB
mutant and a
phoR
mutant. By matrix-assisted laser desorption ionization–time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of
glpQ
and
ydhF
confirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that
glpQ
is transcribed monocistronically from a ς
A
promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore,
ydhF
might be cotranscribed with
phoB
initiating from the
phoB
promoter. Only a small group of proteins remained phosphate starvation inducible in both
phoR
and
sigB
mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by ς
B
-dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a ς
B
-dependent manner and in the
sigB
mutant probably via ς
H
. YxiE was induced by phosphate starvation independently of ς
B
and PhoPR.
Stationary-Phase Variation Due to Transposition of Novel Insertion Elements in
Xanthomonas oryzae
pv. oryzae
Authors: Rajeshwari R. , Sonti R. V. 
Published: July 2002

ABSTRACT

Xanthomonas oryzae
pv. oryzae causes bacterial leaf blight, a serious disease of rice. Spontaneous mutants which are deficient for virulence and extracellular polysaccharide (Eps) production accumulate in large numbers in stationary-phase cultures of this bacterium, a phenomenon which we have called stationary-phase variation. A clone (pSD1) carrying the Eps biosynthetic gene (
gum
) cluster of
X. oryzae
pv. oryzae restored Eps production and virulence to several
spv
(for stationary-phase variation) mutants. Data from localized recombination analysis, Southern hybridization, PCR amplification, and sequence analysis showed that the mutations are due to insertion of either one of two novel endogenous insertion sequence (IS) elements, namely, IS
Xo1
and IS
Xo2
, into
gumM
, the last gene of the
gum
gene cluster. The results of Southern analysis indicate the presence of multiple copies of both IS elements in the genome of
X. oryzae
pv. oryzae. These results demonstrate the role of IS elements in stationary-phase variation in
X. oryzae
pv. oryzae.
Changes in
cspL
,
cspP
, and
cspC
mRNA Abundance as a Function of Cold Shock and Growth Phase in
Lactobacillus plantarum
Authors: Derzelle Sylviane , Hallet Bernard , Francis Kevin P. , Ferain Thierry , Delcour Jean , Hols Pascal 
Published: July 2002

ABSTRACT

An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in
Lactobacillus plantarum
. In addition to the two previously reported
cspL
and
cspP
genes, a third gene,
cspC
, has been cloned and characterized. All three genes encode small 66-amino-acid proteins with between 73 and 88% identity. Comparative Northern blot analyses showed that the level of
cspL
mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of
cspC
and
cspP
remain unchanged or increase only slightly (about two- to threefold). Cold induction of
cspL
mRNA is transient and delayed in time as a function of the severity of the temperature downshift. The cold shock behavior of the three
csp
mRNAs contrasts with that observed for four unrelated non-
csp
genes, which all showed a sharp decrease in mRNA level, followed in one case (
bglH
) by a progressive recovery of the transcript during prolonged cold exposure. Abundance of the three
csp
mRNAs was also found to vary during growth at optimal temperature (28°C).
cspC
and
cspP
mRNA levels are maximal during the lag period, whereas the abundance of the
cspL
transcript is highest during late-exponential-phase growth. The differential expression of the three
L. plantarum csp
genes can be related to sequence and structural differences in their untranslated regions. It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.
Binding of Ferric Enterobactin by theEscherichia coliPeriplasmic Protein FepB
Authors: Sprencel Cathy , Cao Zhenghua , Qi Zengbiao , Scott Daniel C. , Montague Marjorie A. , Ivanoff Nora , Xu Jide , Raymond Kenneth M. , Newton Salete M. C. , Klebba Phillip E. 
Published: July 2002

ABSTRACTThe periplasmic protein FepB ofEscherichia coliis a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay theKdof the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (Kd= 30 nM) and ferric enantioenterobactin (Kd= 15 nM), the left-handed stereoisomer of the naturalE. colisiderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (Kd= 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.
Spermidine Acetyltransferase Is Required To Prevent Spermidine Toxicity at Low Temperatures in
Escherichia coli
Authors: Limsuwun Kornvika , Jones Pamela G. 
Published: July 2002

ABSTRACT

Polyamines are required for optimal growth in most cells; however, polyamine accumulation leads to inhibition of cellular growth. To reduce intracellular polyamine levels, spermidine is monoacetylated in both prokaryotes and eukaryotes. In
Escherichia coli
, the
speG
gene encodes the spermidine acetyltransferase, which transfers the acetyl group to either the N-1 or N-8 position. In addition to polyamine accumulation, stress conditions, such as cold shock, cause an increase in the level of spermidine acetylation, suggesting an adaptive role for reduced polyamine levels under stressful growth conditions. The effect of spermidine accumulation on the growth of
E. coli
at low temperature was examined using a
speG
mutant. At 37°C, growth of the
speG
mutant was normal in the presence of 0.5 or 1 mM spermidine. However, following a shift to 7°C, the addition of 0.5 or 1 mM spermidine resulted in inhibition of cellular growth or cell lysis, respectively. Furthermore, at 7°C, spermidine accumulation resulted in a decrease in total protein synthesis accompanied by an increase in the synthesis of the major cold shock proteins CspA, CspB, and CspG. However, the addition of 50 mM Mg
2+
restored growth and protein synthesis in the presence of 0.5 mM spermidine. The results indicate that the level of spermidine acetylation increases at low temperature to prevent spermidine toxicity. The data suggest that the excess spermidine replaces the ribosome-bound Mg
2+
, resulting in ribosome inactivation at low temperatures.
Genes Involved in Anaerobic Metabolism of Phenol in the Bacterium
Thauera aromatica
Authors: Breinig Sabine , Schiltz Emile , Fuchs Georg 
Published: July 2002

ABSTRACT

Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium
Thauera aromatica
have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor (“phenylphosphate synthase”) and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate (“phenylphosphate carboxylase”). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the
ubiD
gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the
ubiX
gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different
Thauera
and
Azoarcus
strains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the
mutT
gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of
Pseudomonas putida
. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator.
On the Architecture of the Gram-Negative Bacterial Murein Sacculus
Authors: Pink David , Moeller Jeremy , Quinn Bonnie , Jericho Manfred , Beveridge Terry 
Published: July 2002

ABSTRACT

The peptidoglycan network of the murein sacculus must be porous so that nutrients, waste products, and secreted proteins can pass through. Using
Escherichia coli
and
Pseudomonas aeruginosa
as a baseline for gram-negative sacculi, the hole size distribution in the peptidoglycan network has been modeled by computer simulation to deduce the network's properties. By requiring that the distribution of glycan chain lengths predicted by the model be in accord with the distribution observed, we conclude that the holes are slits running essentially perpendicular to the local axis of the glycan chains (i.e., the slits run along the long axis of the cell). This result is in accord with previous permeability measurements of Beveridge and Jack and Demchik and Koch. We outline possible advantages that might accrue to the bacterium via this architecture and suggest ways in which such defect structures might be detected. Certainly, large molecules do penetrate the peptidoglycan layer of gram-negative bacteria, and the small slits that we suggest might be made larger by the bacterium.
Characterization of the DNA- and Metal-Binding Properties of
Vibrio anguillarum
Fur Reveals Conservation of a Structural Zn
2+
Ion
Authors: Zheleznova Ekaterina E. , Crosa Jorge H. , Brennan Richard G. 
Published: July 2002

ABSTRACT

The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under iron-replete conditions. Here we report in vitro solution studies on
Vibrio anguillarum
Fur binding to the consensus 19-bp
Escherichia coli
iron box in the presence of several divalent metals. We found that
V. anguillarum
Fur binds the iron box in the presence of Mn
2+
, Co
2+
, Cd
2+
, and to a lesser extent Ni
2+
but, unlike
E. coli
Fur, not in the presence of Zn
2+
. We also found that
V. anguillarum
Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding.
N
-Acetyl-1-
d
-
myo
-Inosityl-2-Amino-2-Deoxy-α-
d
-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis
Authors: Newton Gerald L. , Av-gay Yossef , Fahey Robert C. 
Published: July 2002

ABSTRACT

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria. Mycothiol was previously shown to be synthesized from 1-
d
-
myo
-inosityl-2-amino-2-deoxy-α-
d
-glucopyranoside by ligation with cysteine followed by acetylation. A novel mycothiol-dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in
Mycobacterium smegmatis
and shown to have a homolog, Rv1082, in
Mycobacterium tuberculosis
. In the present study we found that a protein encoded by the
M. tuberculosis
open reading frame Rv1170, a homolog of Rv1082, possesses weak mycothiol conjugate amidase activity but shows substantial deacetylation activity with 1-
d
-
myo
-inosityl-2-acetamido-2-deoxy-α-
d
-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic precursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in
M. smegmatis
at a level about twice that of mycothiol. It was shown that GlcNAc-Ins is absent in mycothiol-deficient mutant strain 49 of
M. smegmatis
and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol. This demonstrates that GlcNAc-Ins is a key intermediate in the pathway of mycothiol biosynthesis. Assignment of Rv1170 as the gene coding the deacetylase in the
M. tuberculosis
genome represents the first identification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.
Proteome Analysis of the Effect of Mucoid Conversion on Global Protein Expression in
Pseudomonas aeruginosa
Strain PAO1 Shows Induction of the Disulfide Bond Isomerase, DsbA
Authors: Malhotra Sonal , Silo-Suh Laura A. , Mathee Kalai , Ohman Dennis E. 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion. The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT [also called AlgU]), which is required for the activation of genes for alginate biosynthesis. In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis. To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its
mucA22
derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion. Six proteins that were clearly more abundant in the mucoid strain were observed. The amino termini of such proteins were determined and used to identify the gene products in the genomic database. Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified. This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by
mucA
mutation. Two other protein spots were also clearly upregulated in the
mucA22
background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase). Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The
oprF-lacZ
fusion showed little difference in levels of expression in the two strains. However, the
dsbA-lacZ
fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity. A
dsbA
-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase activity, increased sensitivity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase. Although efficient secretion of elastase in the
dsbA
mutant was still demonstrable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds. Disruption of
dsbA
in the mucoid PDO300 background did not affect alginate production. Thus, even though
dsbA
is coregulated with mucoid conversion, it was not required for alginate production. This suggests that
mucA
mutation, which deregulates sigma-22, results in a global response that includes other factors in addition to increasing the production of alginate.