bluuebunny/crossref_metadata_2025_split huggingface dataset:
NhaR and RcsB Independently Regulate the
osmCp1
Promoter of
Escherichia coli
at Overlapping Regulatory Sites
Authors: Sturny Rachel , Cam Kaymeuang , Gutierrez Claude , Conter Annie 
Published: July 2003

ABSTRACT

Transcription of the
Escherichia coli osmC
gene is induced by several stress conditions.
osmC
is expressed from two overlapping promoters,
osmCp1
and
osmCp2.
The proximal promoter,
osmCp2
, is transcribed at the entry into the stationary phase by the σ
s
sigma factor. The distal promoter,
osmCp1
, is activated by NhaR and RcsB. NhaR is a positive regulator of the LysR family and is known to be an activator of the
nhaA
gene encoding an Na
+
/H
+
antiporter. RcsB is the response regulator of the RcsCDB His-Asp phosphorelay signal transduction system. Genetic data indicated that activation of
osmCp1
by both NhaR and RcsB requires the same short sequences upstream of the −35 region of the promoter. Accordingly, DNase I footprint analysis indicated that both activators protect an overlapping region close to the −35 box of the promoter and suggested that the regulatory effect is direct. Despite the overlap of the binding sites, each activator acts independent of the other and is specific for a particular stress. NhaR can stimulate
osmCp1
in response to an osmotic signal even in the absence of RcsB. RcsB is responsible for the induction of
osmCp1
by alteration of the cell envelope, even in the absence of NhaR.
osmCp1
as an example of multiple-stress-responsive promoter is discussed in light of a comparison of the NhaR and RcsB target regions in the
Enterobacteriaceae
.
Interplay of the Czc System and Two P-Type ATPases in Conferring Metal Resistance to
Ralstonia metallidurans
Authors: Legatzki Antje , Grass Gregor , Anton Andreas , Rensing Christopher , Nies Dietrich H. 
Published: July 2003

ABSTRACT

Cadmium and zinc are removed from cells of
Ralstonia metallidurans
by the CzcCBA efflux pump and by two soft-metal-transporting P-type ATPases, CadA and ZntA. The
czcCBA
genes are located on plasmid pMOL30, and the
cadA
and
zntA
genes are on the bacterial chromosome. Expression of
zntA
from
R
.
metallidurans
in
Escherichia coli
predominantly mediated resistance to zinc, and expression of
cadA
predominantly mediated resistance to cadmium. Both transporters decreased the cellular content of zinc or cadmium in this host. In the plasmid-free
R
.
metallidurans
strain AE104, single gene deletions of
cadA
or
zntA
had only a moderate effect on cadmium and zinc resistance, but zinc resistance decreased 6-fold and cadmium resistance decreased 350-fold in double deletion strains. Neither single nor double gene deletions affected zinc resistance in the presence of
czcCBA
. In contrast, cadmium resistance of the
cadA zntA
double mutant could be elevated only partially by the presence of CzcCBA.
lacZ
reporter gene fusions indicated that expression of
cadA
was induced by cadmium but not by zinc in
R
.
metallidurans
strain AE104. In the absence of the
zntA
gene, expression of
cadA
occurred at lower cadmium concentrations and zinc now served as an inducer. In contrast, expression of
zntA
was induced by both zinc and cadmium, and the induction pattern did not change in the presence or absence of CadA. However, expression of both genes,
zntA
and
cadA
, was diminished in the presence of CzcCBA. This indicated that CzcCBA efficiently decreased cytoplasmic cadmium and zinc concentrations. It is discussed whether these data favor a model in which the cations are removed either from the cytoplasm or the periplasm by CzcCBA.
immX
Immunity Region of
Rhizobium
Phage
16
-
3
: Two Overlapping Cistrons of Repressor Function
Authors: Csiszovszki Zsolt , Buzás Zsuzsanna , Semsey Szabolcs , Ponyi Tamás , Papp Péter P. , Orosz László 
Published: July 2003

ABSTRACT

16
-
3
is a temperate phage of the symbiotic nitrogen-fixing bacterium
Rhizobium meliloti
41. Its prophage state and immunity against superinfection by homoimmune phages are governed by a complex set of controls: the
immC
and
immX
repressor systems and the
avirT
element are all located in well-separated, distinct regions which span 25 kb on the bacteriophage chromosome. The anatomy and function of the
immC
region are well documented; however, fewer analyses have addressed the
immX
and
avirT
regions. We focused in this paper on the
immX
region and dissected it into two major parts:
X

U/L

and
X

V

. The
X

U/L

part (0.6 kb) contained two overlapping cistrons,
X

U

and
X

L

, coding for proteins pXU and pXL, respectively. Inactivation of either gene inactivated the repressor function of the
immX
region. Loss-of-function mutants of
X

U

and
X

L

complemented each other in
trans
in double lysogens. The
X

V

part (1 kb) contained a target for
X

U/L

repressor action. Mutations at three sites in
X

V

led to various degree of ImmX insensitivity in a hierarchic manner. Two sites (
X

V1

and
X

V3

) exhibited the inverted-repeat structures characteristic of many repressor binding sites. However,
X

V1

could also be folded into a transcription terminator. Of the two immunity regions of
16
-
3
,
immX
seems to be unique both in its complex genetic anatomy and in its sequence. To date, no DNA or peptide sequence homologous to that of ImmX has been found in the data banks. In contrast,
immC
shares properties of a number of immunity systems commonly found in temperate phages.
Molecular Analysis of the
Enterococcus faecalis
Serotype 2 Polysaccharide Determinant
Authors: Hancock Lynn E. , Shepard Brett D. , Gilmore Michael S. 
Published: July 2003

ABSTRACT

We previously described a 15-kb genetic cluster consisting of 11 open reading frames (
cps2A
to
cps2K
) of
Enterococcus faecalis
FA2-2 that is responsible for the production of the serotype 2 capsular polysaccharide. By using transcriptional fusions to a promoterless
lac
Z gene, we identified two independent promoters related to the expression of the polysaccharide. Both transcription initiation sites were mapped by primer extension. Reverse transcription-PCR (RT-PCR) demonstrated the transcriptional linkage of genes present in both transcripts. Real-time RT-PCR quantification of transcripts revealed maximum transcription during log phase growth, an observation confirmed by promoter fusion studies. The heterologous expression of this pathway in
Escherichia coli
caused reactivity with
E. faecalis
type 2 antiserum, thus demonstrating the essential role of this pathway in the synthesis of the type-specific polysaccharide.
The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from
Streptococcus pneumoniae
Catalyzes the Polymerization of Murein Glycan Chains
Authors: Di Guilmi Anne Marie , Dessen Andréa , Dideberg Otto , Vernet Thierry 
Published: July 2003

ABSTRACT

The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked
N
-acetylglucosaminyl-
N
-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant
Streptococcus pneumoniae
PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters
K

m

and
k
cat
/
K

m

were measured at 40.6 μM (± 15.5) and 1 × 10
−3
M
−1
s
−1
, respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of
S
.
pneumoniae
PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.
CsrA Regulates Translation of the
Escherichia coli
Carbon Starvation Gene,
cstA
, by Blocking Ribosome Access to the
cstA
Transcript
Authors: Dubey Ashok K. , Baker Carol S. , Suzuki Kazushi , Jones A. Daniel , Pandit Pallavi , Romeo Tony , Babitzke Paul 
Published: July 2003

ABSTRACT

CsrA is a global regulator that binds to two sites in the
glgCAP
leader transcript, thereby blocking ribosome access to the
glgC
Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the
Escherichia coli
genomic sequence for other genes that might be regulated by CsrA.
cstA
contained an exact match that overlapped its Shine-Dalgarno sequence.
cstA
was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a
cstA
′-′
lacZ
translational fusion in wild-type and
csrA
mutant strains was examined. Expression levels in the
csrA
mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5- to 10-fold higher when LB was supplemented with glucose. It was previously shown that
cstA
is regulated by the cyclic AMP (cAMP)-cAMP receptor protein complex and transcribed by Εσ
70
. We investigated the influence of σ
S
on
cstA
expression and found that a σ
S
deficiency resulted in a threefold increase in
cstA
expression in wild-type and
csrA
mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated
cstA
regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to
cstA
RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to
cstA
transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the
cstA
Shine-Dalgarno sequence, as predicted. These results establish that CsrA regulates translation of
cstA
by sterically interfering with ribosome binding.
Transcription and Analysis of Polymorphism in a Cluster of Genes Encoding Surface-Associated Proteins ofClostridium difficile
Authors: Savariau-Lacomme Marie-Pierre , Lebarbier Carole , Karjalainen Tuomo , Collignon Anne , Janoir Claire 
Published: July 2003

ABSTRACTRecent investigations of theClostridium difficilegenome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins. Two of these genes have been proven to encode proteins involved in cell adherence:slpAencodes the precursor of the two proteins of the S-layer, P36 and P47, whereascwp66encodes the Cwp66 adhesin. To gain further insight into the function of this cluster, we further focused onslpA,cwp66, andcwp84,the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases. It displayed nonspecific proteolytic activity when expressed as a recombinant protein inEscherichia coli. Polymorphism ofcwp66andcwp84genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots. TheslpAgene is strongly transcribed during the entire growth phase as a bicistronic transcript;cwp66is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream. The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence. No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains. Thecwp84gene is strongly transcribed as a monocistronic message. This feature, together with the highly conserved sequence pattern ofcwp84, suggests a significant role in the physiopathology ofC. difficilefor the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.
IS
981
-Mediated Adaptive Evolution Recovers Lactate Production by
ldhB
Transcription Activation in a Lactate Dehydrogenase-Deficient Strain of
Lactococcus lactis
Authors: Bongers Roger S. , Hoefnagel Marcel H. N. , Starrenburg Marjo J. C. , Siemerink Marco A. J. , Arends John G. A. , Hugenholtz Jeroen , Kleerebezem Michiel 
Published: July 2003

ABSTRACT

Lactococcus lactis
NZ9010 in which the
las
operon-encoded
ldh
gene was replaced with an erythromycin resistance gene cassette displayed a stable phenotype when grown under aerobic conditions, and its main end products of fermentation under these conditions were acetate and acetoin. However, under anaerobic conditions, the growth of these cells was strongly retarded while the main end products of fermentation were acetate and ethanol. Upon prolonged subculturing of this strain under anaerobic conditions, both the growth rate and the ability to produce lactate were recovered after a variable number of generations. This recovery was shown to be due to the transcriptional activation of a silent
ldhB
gene coding for an Ldh protein (LdhB) with kinetic parameters different from those of the native
las
operon-encoded Ldh protein. Nevertheless, cells producing LdhB produced mainly lactate as the end product of fermentation. The mechanism underlying the
ldhB
gene activation was primarily studied in a single-colony isolate of the recovered culture, designated
L
.
lactis
NZ9015. Integration of IS
981
in the upstream region of
ldhB
was responsible for transcription activation of the
ldhB
gene by generating an IS
981
-derived −35 promoter region at the correct spacing with a natively present −10 region. Subsequently, analysis of 10 independently isolated lactate-producing derivatives of
L
.
lactis
NZ9010 confirmed that the
ldhB
gene is transcribed in all of them. Moreover, characterization of the upstream region of the
ldhB
gene in these derivatives indicated that site-specific and directional IS
981
insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate.
The Phosphate Starvation Stimulon ofCorynebacterium glutamicumDetermined by DNA Microarray Analyses
Authors: Ishige Takeru , Krause Malgorzata , Bott Michael , Wendisch Volker F. , Sahm Hermann 
Published: July 2003

ABSTRACTThe phosphate (Pi) starvation stimulon ofCorynebacterium glutamicumwas characterized by global gene expression analysis by using DNA microarrays. Hierarchical cluster analysis of the genes showing altered expression 10 to 180 min after a shift from Pi-sufficient to Pi-limiting conditions led to identification of five groups comprising 92 genes. Four of these groups included genes which are not directly involved in P metabolism and changed expression presumably due to the reduced growth rate observed after the shift or to the exchange of medium. One group, however, comprised 25 genes, most of which are obviously related to phosphorus (P) uptake and metabolism and exhibited 4- to >30-fold-greater expression after the shift to Pilimitation. Among these genes, the RNA levels of thepstSCAB(ABC-type Piuptake system),glpQ(glycerophosphoryldiester phosphodiesterase),ugpAEBC(ABC-typesn-glycerol 3-phosphate uptake system),phoH(unknown function),nucH(extracellular nuclease), and Cgl0328 (5′-nucleotidase or related esterase) genes were increased, andpstSCABexhibited a faster response than the other genes. Transcriptional fusion analyses revealed that elevated expression ofpstSCABandugpAEBCwas primarily due to transcriptional regulation. Several genes also involved in P uptake and metabolism were not affected by Pistarvation; these included the genes encoding a PitA-like Piuptake system and a putative Na+-dependent Pitransporter and the genes involved in the metabolism of pyrophosphate and polyphosphate. In summary, a global, time-resolved picture of the response ofC. glutamicumto Pistarvation was obtained.
Unusual Integrase Gene Expression on the
clc
Genomic Island in
Pseudomonas
sp. Strain B13
Authors: Sentchilo V. , Ravatn R. , Werlen C. , Zehnder A. J. B. , van der Meer J. R. 
Published: July 2003

ABSTRACT

An unusual type of gene expression from an integrase promoter was found in cultures of the bacterium
Pseudomonas
sp. strain B13. The promoter controls expression of the
intB13
integrase gene, which is present near the right end of a 105-kb conjugative genomic island (the
clc
element) encoding catabolism of aromatic compounds. The enzymatic activity of integrase IntB13 is essential for site-specific integration of the
clc
element into the bacterial host's chromosome. By creating transcription fusions between the
intB13
promoter and the
gfp
gene, we showed that integrase expression in strain B13 was inducible under stationary-phase conditions but, strangely, occurred in only a small proportion of individual bacterial cells rather than equally in the whole population. Integrase expression was significantly stimulated by growing cultures on 3-chlorobenzoate. High cell density, heat shock, osmotic shock, UV irradiation, and treatment with alcohol did not result in measurable integrase expression. The occurrence of the excised form of the
clc
element and an increase in the rates of
clc
element transfer in conjugation experiments correlated with the observed induction of the
intB13′-gfp
fusion in stationary phase and in the presence of 3-chlorobenzoate. This suggested that activation of the
intB13
promoter is the first step in stimulation of
clc
transfer. To our knowledge, this is the first report of a chlorinated compound's stimulating horizontal transfer of the genes encoding its very metabolism.
DNA Array-Based Transcriptional Analysis of Asporogenous, Nonsolventogenic
Clostridium acetobutylicum
Strains SKO1 and M5
Authors: Tomas Christopher A. , Alsaker Keith V. , Bonarius Hendrik P. J. , Hendriksen Wouter T. , Yang He , Beamish Jeffrey A. , Paredes Carlos J. , Papoutsakis Eleftherios T. 
Published: July 2003

ABSTRACT

The large-scale transcriptional program of two
Clostridium acetobutylicum
strains (SKO1 and M5) relative to that of the parent strain (wild type [WT]) was examined by using DNA microarrays. Glass DNA arrays containing a selected set of 1,019 genes (including all 178 pSOL1 genes) covering more than 25% of the whole genome were designed, constructed, and validated for data reliability. Strain SKO1, with an inactivated
spo0A
gene, displays an asporogenous, filamentous, and largely deficient solventogenic phenotype. SKO1 displays downregulation of all solvent formation genes,
sigF
, and carbohydrate metabolism genes (similar to genes expressed as part of the stationary-phase response in
Bacillus subtilis
) but also several electron transport genes. A major cluster of genes upregulated in SKO1 includes
abrB
, the genes from the major chemotaxis and motility operons, and glycosylation genes. Strain M5 displays an asporogenous and nonsolventogenic phenotype due to loss of the megaplasmid pSOL1, which contains all genes necessary for solvent formation. Therefore, M5 displays downregulation of all pSOL1 genes expressed in the WT. Notable among other genes expressed more highly in WT than in M5 were
sigF
, several two-component histidine kinases,
spo0A
,
cheA
,
cheC
, many stress response genes,
fts
family genes, DNA topoisomerase genes, and central-carbon metabolism genes. Genes expressed more highly in M5 include electron transport genes (but different from those downregulated in SKO1) and several motility and chemotaxis genes. Most of these expression patterns were consistent with phenotypic characteristics. Several of these expression patterns are new or different from what is known in
B. subtilis
and can be used to test a number of functional-genomic hypotheses.
Characterization of Transcriptional Regulation ofShewanella frigidimarinaFe(III)-Induced FlavocytochromecReveals a Novel Iron-Responsive Gene Regulation System
Authors: Reyes-Ramirez Francisca , Dobbin Paul , Sawers Gary , Richardson David J. 
Published: July 2003

ABSTRACTThe bacteriumShewanella frigidimarinacan grow anaerobically by utilizing Fe(III) as a respiratory electron acceptor. This results in the synthesis of a number of periplasmicc-type cytochromes, which are absent when the organism is grown in the absence of added Fe(III). One cytochrome, IfcA, is synthesized when Fe(III) is present as the sole respiratory electron acceptor or when it is present in combination with oxygen, fumarate, or nitrate. TheifcAgene was thus selected for a study of iron-responsive gene regulation of respiratory proteins inS. frigidimarina. The monocistronicifcAgene clusters with two other monocistronic genes,ifcO, encoding a putative outer membrane porin, andifcR, encoding a putative transcriptional regulator of the LysR superfamily. Analysis of transcription of all three genes under a range of growth conditions in the wild type and anifcRinsertion mutant and analysis of a strain that constitutively expressesifcRrevealed that iron regulation is exerted at the level ofifcRtranscription. In the presence of Fe(III) IfcR is synthesized and acts positively to regulate expression ofifcOandifcA. Control of Fe(III) respiration by this novel regulatory system differs markedly from Fur-mediated regulation of iron assimilation, in which Fur serves as an Fe(II)-activated repressor.
Initial Proteome Analysis of Model MicroorganismHaemophilus influenzaeStrain Rd KW20
Authors: Kolker Eugene , Purvine Samuel , Galperin Michael Y. , Stolyar Serg , Goodlett David R. , Nesvizhskii Alexey I. , Keller Andrew , Xie Tao , Eng Jimmy K. , Yi Eugene , Hood Leroy , Picone Alex F. , Cherny Tim , Tjaden Brian C. , Siegel Andrew F. , Reilly Thomas J. , Makarova Kira S. , Palsson Bernhard O. , Smith Arnold L. 
Published: July 2003

ABSTRACTThe proteome ofHaemophilus influenzaestrain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism ofH. influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) ofH. influenzaestrain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.