bluuebunny/crossref_metadata_2025_split huggingface dataset:
NovelspoIIEMutation That Causes Uncompartmentalized σFActivation inBacillus subtilis
Authors: Hilbert David W. , Piggot Patrick J. 
Published: February 2003

ABSTRACTDuring sporulation,Bacillus subtilisundergoes an asymmetric division that results in two cells with different fates, the larger mother cell and the smaller forespore. The protein phosphatase SpoIIE, which is required for activation of the forespore-specific transcription factor σF, is also required for optimal efficiency and timing of asymmetric division. We performed a genetic screen forspoIIEmutants that were impaired in sporulation but not σFactivity and isolated a strain with the mutationspoIIEV697A. The mutant exhibited a 10- to 40-fold reduction in sporulation and a sixfold reduction in asymmetric division compared to the parent. Transcription of the σF-dependentspoIIQpromoter was increased more than 10-fold and was no longer confined to the forespore. The excessive σFactivity persisted even when asymmetric division was prevented. Disruption ofspoIIGBdid not restore asymmetric division to thespoIIEV697Amutant, indicating that the deficiency is not a consequence of predivisional activation of the mother cell-specific transcription factor σE. Deletion of the gene encoding σF(spoIIAC)restored asymmetric division; however, a mutation that dramatically reduced the number of promoters responsive to σF,spoIIAC561(spoIIACV233 M), failed to do so. This result suggests that the block is due to expression of one of the small subset of σF-dependent genes expressed in this background or to unregulated interaction of σF with some other factor. Our results indicate that regulation of SpoIIE plays a critical role in coupling asymmetric division to σFactivation in order to ensure proper spatial and temporal expression of forespore-specific genes.
The Cyclic AMP-Cyclic AMP Receptor Protein Complex Regulates Activity of the
traJ
Promoter of the
Escherichia coli
Conjugative Plasmid pRK100
Authors: Starčič Marjanca , Žgur-Bertok Darja , Jordi Bart J. A. M. , Wösten Marc M. S. M. , Gaastra Wim , van Putten Jos P. M. 
Published: February 2003

ABSTRACT

The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of
traJ
expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in
traJ
transcription using a
traJ-lacZ
reporter system. A comparison of the enzyme activities in the wild-type
Escherichia coli
strain MC4100 with those in
cya
and
crp
mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the
traJ
promoter of the F-like plasmid pRK100. The defect in the
cya
mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the
traJ
promoter region. DNase I footprint experiments mapped the CRP binding site around position −67.5 upstream of the putative
traJ
promoter. Targeted mutagenesis of the
traJ
promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in
cya
or
crp
exhibited reduced conjugal transfer from pRK100.
Role of the Cytoplasmic C Terminus of the FliF Motor Protein in Flagellar Assembly and Rotation
Authors: Grünenfelder Björn , Gehrig Stefanie , Jenal Urs 
Published: February 2003

ABSTRACT

Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the
Caulobacter crescentus
FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first
fliF
mutations described so far with a Fla
+
but Mot
−
phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5′ region of the
fliG
gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.
Synthesis of a
Klebsiella pneumoniae
O-Antigen Heteropolysaccharide (O12) Requires an ABC 2 Transporter
Authors: Izquierdo Luis , Merino Susana , Regué Miguel , Rodriguez Florencia , Tomás Juan M. 
Published: February 2003

ABSTRACT

A recombinant clone encoding enzymes for
Klebsiella pneumoniae
O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of
K. pneumoniae
KT776 (O12:K80) introduced into
Escherichia coli
DH5α. A total of eight open reading frames (ORFs) (
wb
O12
gene cluster) were necessary to produce
K. pneumoniae
O12-antigen LPS in
E. coli
K-12. A complete analysis of the
K. pneumoniae wb
O12
cluster revealed an interesting coincidence with the
wb
O4
cluster of
Serratia marcescens
from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of
K. pneumoniae
strain KT776 (O12) and to study complementation between the two enterobacterial
wb
clusters using mutants of
S. marcescens
N28b (O4) obtained previously. Both
wb
gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The
wzm
-
wzt
genes and the
wbbA
or
wbbB
genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (
wzm
-
wzt
-
wbbA
or
wzm
-
wzt
-
wbbB
) into mutants unable to produce O antigen allowed production of the specific O antigen. The
K. pneumoniae
O12 WbbL protein performs the same function as WbbL from
S. marcescens
O4 in either the
S. marcescens
O4 or
E. coli
K-12 genetic background.
Rhamnogalacturonate Lyase RhiE Is Secreted by the Out System in
Erwinia chrysanthemi
Authors: Laatu Minna , Condemine Guy 
Published: February 2003

ABSTRACT

Supernatants of rhamnose-induced
Erwinia chrysanthemi
strain 3937 cultures contain a principal secreted protein named RhiE. A
rhiE
mutant has been found among a set of rhamnose-induced MudI1681
lacZ
fusions. RhiE is a 62-kDa protein that has rhamnogalacturonate lyase activity on rhamnogalacturonan I (RG-I). It does not require a divalent cation for its activity and has an optimal pH of 6.0.
rhiE
expression is strongly induced in the presence of rhamnose but is also regulated by PecT and Crp, two regulators of the transcription of pectinolytic enzyme genes. RhiE is secreted through the type II Out secretion pathway. RhiE has no disulfide bond. The absence of RhiE secretion in a
dsb
mutant indicated that disulfide bond formation is required for the biogenesis of the secretion apparatus. RhiE was searched for in several
E. chrysanthemi
strains by using antibodies, and it was found to be present in one-third of the strains tested. However, the reduced virulence of the
rhiE
mutant indicates that degradation of the RG-I region of pectin is important for full virulence of
E. chrysanthemi
.
Functional Characterization of Penicillin-Binding Protein 1b fromStreptococcus pneumoniae
Authors: Di Guilmi Anne Marie , Dessen Andréa , Dideberg Otto , Vernet Thierry 
Published: February 2003

ABSTRACTThe widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by β-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region ofStreptococcus pneumoniaePBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.
Overexpression of the
waaZ
Gene Leads to Modification of the Structure of the Inner Core Region of
Escherichia coli
Lipopolysaccharide, Truncation of the Outer Core, and Reduction of the Amount of O Polysaccharide on the Cell Surface
Authors: Frirdich Emilisa , Lindner Buko , Holst Otto , Whitfield Chris 
Published: February 2003

ABSTRACT

The
waa
gene cluster is responsible for the biosynthesis of the lipopolysaccharide (LPS) core region in
Escherichia coli
and
Salmonella.
Homologs of the
waaZ
gene product are encoded by the
waa
gene clusters of
Salmonella enterica
and
E. coli
strains with the K-12 and R2 core types. Overexpression of WaaZ in
E. coli
and
S. enterica
led to a modified LPS structure showing core truncations and (where relevant) to a reduction in the amount of O-polysaccharide side chains. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the predominant LPS structures in an
E. coli
isolate with an R1 core (
waaZ
is lacking from the type R1
waa
gene cluster) with a copy of the
waaZ
gene added on a plasmid. Novel truncated LPS structures, lacking up to 3 hexoses from the outer core, resulted from WaaZ overexpression. The truncated molecules also contained a KdoIII residue not normally found in the R1 core.
Complex Regulation of theBacillus subtilisAconitase Gene
Authors: Kim Hyun-Jin , Kim Sam-In , Ratnayake-Lecamwasam Manoja , Tachikawa Kiyoshi , Sonenshein Abraham L. , Strauch Mark 
Published: February 2003

ABSTRACTThe roles of the CcpC, CodY, and AbrB proteins in regulation of theBacillus subtilisaconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulatingcitBexpression in minimal-glucose-glutamine medium, also contributed to repression ofcitBduring exponential-phase growth in broth medium. A null mutation incodYhad no effect oncitBexpression during growth in minimal medium even when combined withccpCandabrBmutations. However, acodYmutation slightly relieved repression during exponential growth in broth medium and completely derepressedcitBexpression when combined with accpCmutation. AnabrBmutation led to decreased expression ofcitBduring stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within thecitBregulatory region. Interaction of CcpC and CodY with thecitBpromoter region was partially competitive.
Conjugative Transfer of p42a from
Rhizobium etli
CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing
Authors: Tun-Garrido Cristina , Bustos Patricia , González Víctor , Brom Susana 
Published: February 2003

ABSTRACT

Rhizobium etli
CFN42 contains six plasmids. Only one of them, p42a, is self-conjugative at high frequency. This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym). To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated. Its sequence showed that most of the
tra
genes are highly similar to genes of
Agrobacterium tumefaciens
pTiC58 and other related plasmids. Four putative regulatory genes were identified; three of these (
traI
,
traR
, and
cinR
) belong to the LuxR-LuxI family. Mutagenesis of these genes confirmed their requirement for p42a transfer. We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer. Although
R. etli
has been shown to produce different
N
-acyl-homoserine lactones, only one of them, a 3-oxo-C
8
-homoserine lactone encoded by the
traI
gene described here, was involved in transfer. Mutagenesis of the fourth regulatory gene,
traM
, had no effect on transfer. Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer. Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C
8
-homoserine lactone synthesized by TraI. The
traI
gene was autoregulated by these elements and positively regulated by CinR, while
cinR
expression required
traI
. Finally, we did not detect expression of
traM
, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a.
P15 and P3, the Tail Completion Proteins of Bacteriophage T4, Both Form Hexameric Rings
Authors: Zhao Li , Kanamaru Shuji , Chaidirek Chatree'chalerm , Arisaka Fumio 
Published: February 2003

ABSTRACT
Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.
The
Escherichia coli
Methyl-Directed Mismatch Repair System Repairs Base Pairs Containing Oxidative Lesions
Authors: Wyrzykowski Jennifer , Volkert Michael R. 
Published: February 2003

ABSTRACT

A major role of the methyl-directed mismatch repair (MMR) system of
Escherichia coli
is to repair postreplicative errors. In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis. When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically. In addition, we show that a
dam
mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by mutations that inactivate MMR. In a
dam
mutant, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted to single- and double-strand DNA breaks. Thus, base pairs containing oxidized bases will be converted to strand breaks if they are repaired by MMR. This is demonstrated by the increased peroxide sensitivity of a
dam
mutant and the finding that the sensitivity can be suppressed by mutations inactivating MMR. We demonstrate further that this repair activity results from MMR recognition of base pairs containing 8-oxoguanine (8-oxoG) based on the finding that overexpression of the MutM oxidative repair protein, which repairs 8-oxoG, can suppress the
mutH
-dependent increase in transversion mutations. These findings demonstrate that MMR has the ability to prevent oxidative mutagenesis either by removing 8-oxoG directly or by removing adenine misincorporated opposite 8-oxoG or both.
Characterization of a Cytosolic NiFe-Hydrogenase from the Hyperthermophilic Archaeon
Thermococcus kodakaraensis
KOD1
Authors: Kanai Tamotsu , Ito Sota , Imanaka Tadayuki 
Published: February 2003

ABSTRACT

We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon
Thermococcus kodakaraensis
KOD1 (
T. kodakaraensis
hydrogenase). A gene cluster encoding
T. kodakaraensis
hydrogenase was composed of four open reading frames (

hyhBGSL
Tk

), where the

hyhS
Tk

and

hyhL
Tk

gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (

hybD
Tk

) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL

Tk

were used for immunoaffinity purification of
T. kodakaraensis
hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified
T. kodakaraensis
hydrogenase was composed of four subunits (β, γ, δ, and α), corresponding to the products of

hyhBGSL
Tk

, respectively. Each αβγδ unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the
T. kodakaraensis
hydrogenase was 95°C for H
2
uptake and 90°C for H
2
production with methyl viologen as the electron carrier. We found that NADP
+
and NADPH promoted high levels of uptake and evolution of H
2
, respectively, suggesting that the molecule is the electron carrier for the
T. kodakaraensis
hydrogenase.
DNA Microarray Analysis of Redox-Responsive Genes in the Genome of the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Hihara Yukako , Sonoike Kintake , Kanehisa Minoru , Ikeuchi Masahiko 
Published: February 2003

ABSTRACT

Whole-genome DNA microarrays were used to evaluate the effect of the redox state of the photosynthetic electron transport chain on gene expression in
Synechocystis
sp. strain PCC 6803. Two specific inhibitors of electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-
p
-benzoquinone (DBMIB), were added to the cultures, and changes in accumulation of transcripts were examined. About 140 genes were highlighted as reproducibly affected by the change in the redox state of the photosynthetic electron transport chain. It was shown that some stress-responsive genes but not photosynthetic genes were under the control of the redox state of the plastoquinone pool in
Synechocystis
sp. strain PCC 6803.
tRNA
2
Thr
Complements Temperature Sensitivity Caused by Null Mutations in the
htrB
Gene in
Escherichia coli
Authors: Mohri Yoshio , Goto Simon , Nakahigashi Kenji , Inokuchi Hachiro 
Published: February 2003

ABSTRACT

According to the wobble rule, tRNA2Thr is nonessential for protein synthesis, because the codon (ACG) that is recognized by tRNA2Thr is also recognized by tRNA4Thr. In order to investigate the reason that this nonessential tRNA nevertheless exists in
Escherichia coli
, we attempted to isolate tRNA2Thr-requiring mutants. Using strain JM101F
−
, which lacks the gene for tRNA2Thr, we succeeded in isolating two temperature-sensitive mutants whose temperature sensitivity was complemented by introduction of the gene for tRNA2Thr. These mutants had a mutation in the
htrB
gene, whose product is an enzyme involved in lipid A biosynthesis. Although it is known that some null mutations in the
htrB
gene give a temperature-sensitive phenotype, our mutants exhibited tighter temperature sensitivity. We discuss a possible mechanism for the requirement for tRNA2Thr.
Mechanism of
cis-trans
Isomerization of Unsaturated Fatty Acids in
Pseudomonas putida
Authors: von Wallbrunn Angelika , Richnow Hans Hermann , Neumann Grit , Meinhardt Friedhelm , Heipieper Hermann J. 
Published: February 2003

ABSTRACT

We studied the pattern of the
cis
-
trans
isomerization of unsaturated fatty acids in cells of
Pseudomonas putida
S12 grown in a medium supplemented with oleic acid which was deuterated at both of the C atoms of its double bond. Direct evidence that isomerization does not include a transient saturation of the double bond was obtained. In addition, analysis of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome
c
type.
A Suppressor of the Menadione-Hypersensitive Phenotype of a
Xanthomonas campestris
pv. phaseoli
oxyR
Mutant Reveals a Novel Mechanism of Toxicity and the Protective Role of Alkyl Hydroperoxide Reductase
Authors: Vattanaviboon Paiboon , Whangsuk Wirongrong , Mongkolsuk Skorn 
Published: February 2003

ABSTRACT

We isolated menadione-resistant mutants of
Xanthomonas campestris
pv. phaseoli
oxyR
(

oxyR
Xp

). The
oxyR
R2

Xp

mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H
2
O
2
and
tert
-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the
oxyR
R2

Xp

mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of
ahpC
in the
oxyR
R2

Xp

mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned
ahpC
and
ahpF
in the

oxyR
Xp

mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in
X. campestris.
Hemin Binding, Functional Expression, and Complementation Analysis of Pap 31 fromBartonella henselae
Authors: Zimmermann Rainer , Kempf Volkhard A. J. , Schiltz Emile , Oberle Karin , Sander Anna 
Published: February 2003

ABSTRACTGrowth ofBartonella henselaeis strongly heme dependent, andB. henselaeis unable to synthesize heme itself. At least five outer membrane-associated proteins fromB. henselaebind hemin, including the 31-kDa protein designated Pap31. The gene of this protein was heterologously expressed inEscherichia coliM15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction. Complementation of thehemA-deficient mutantE. coliK-12 EB53 (aroB tsx malT hemA) withpap31demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis ofB. henselae.