bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Characterization of a Developmentally Regulated Protein, EshA, Required for Sporogenic Hyphal Branches in
Streptomyces griseus
Authors: Kwak Jangyul , McCue Lee Ann , Trczianka Kristen , Kendrick Kathleen E. 
Published: July 2002

ABSTRACT

To identify sporulation-specific proteins that might serve as targets of developmental regulatory factors in
Streptomyces
, we examined total proteins of
Streptomyces griseus
by two-dimensional gel electrophoresis. Among five proteins that were present at high levels during sporulation but absent from vegetative cells, two of the proteins, P3 and P4, were absent from developmental mutants that undergo aberrant morphogenesis. The deduced amino acid sequence of the gene that encodes P3 (EshA) showed extensive similarity to proteins from mycobacteria and a cyanobacterium,
Synechococcus
, that are abundant during nutritional stress but whose functions are unknown. Uniquely among these proteins, EshA contains a cyclic nucleotide-binding domain, suggesting that the activity of EshA may be modulated by a cyclic nucleotide. The
eshA
gene was strongly expressed from a single transcription start site only during sporulation, and accumulation of the
eshA
transcript depended on a developmental gene,
bldA
. During submerged sporulation, a null mutant strain that produced no EshA could not extend sporogenic hyphae from new branch points but instead accelerated septation and spore maturation at the preexisting vegetative filaments. These results indicated that EshA is required for the growth of sporogenic hyphae and localization of septation and spore maturation but not for spore viability.
Flagellum-Independent Surface Migration of
Vibrio cholerae
and
Escherichia coli
Authors: Brown Igor I. , Häse Claudia C. 
Published: July 2002

ABSTRACT

Surface translocation has been described in a large variety of microorganisms, including some gram-negative enteric bacteria. Here, we describe the novel observation of the flagellum-independent migration of
Vibrio cholerae
and
Escherichia coli
on semisolid surfaces with remarkable speeds. Important aspects of this motility are the form of inoculation, the medium composition, and the use of agarose rather than agar. Mutations in several known regulatory or surface structure proteins, such as ToxR, ToxT, TCP, and PilA, did not affect migration, whereas a defect in lipopolysaccharide biosynthesis prevented translocation. We propose that the observed surface migration is an active process, since heat, protease, or chloramphenicol treatments of the cells have strong negative effects on this phenotype. Furthermore, several
V. cholerae
strains strongly expressing the hemagglutinin/protease but not their isogenic
hap
-negative mutants, lacked the ability of surface motility, and the treatment of migrating strains with culture supernatants from
hap
strains but not
hap
-null strains prevented surface translocation.
Localization of a Germinant Receptor Protein (GerBA) to the Inner Membrane of
Bacillus subtilis
Spores
Authors: Paidhungat Madan , Setlow Peter 
Published: July 2002

ABSTRACT

Dormant
Bacillus subtilis
spores germinate in response to specific nutrients called germinants, which are recognized by multisubunit receptor complexes encoded by members of the
gerA
family of operons, of which the
gerB
operon is a member. The germinant receptors are expected to be membrane associated, but there is some debate about whether they are located in the inner or outer spore membrane. In this study we have used Western blot analysis to determine the precise location of GerBA, a
gerB
-encoded receptor protein, in various spore fractions. GerBA was not extracted from spores by a decoating treatment that removes the coat and outer membrane but was present in lysates from decoated spores and in the insoluble fraction (termed P100) from such lysates that contained inner-membrane vesicles. GerBA was also solubilized from the P100 fraction with detergent but not with high salt. These findings suggest that GerBA is an integral membrane protein located in the spore's inner membrane. Consistent with this idea, GerBA was present in the cell membrane of the outgrowing spore, a membrane that is derived from the dormant spore's inner membrane. Based on these observations we propose that GerBA and probably the entire GerB germinant receptor are located in the inner membrane of the dormant spore. We also estimated that there are only 24 to 40 molecules of GerBA per spore, a number that is consistent with the previously reported low level of
gerB
operon expression and with the putative receptor function of the proteins encoded by the
gerB
operon.
Mapping ofMycobacterium tuberculosis katGPromoters and Their Differential Expression in Infected Macrophages
Authors: Master Sharon , Zahrt Thomas C. , Song Jian , Deretic Vojo 
Published: July 2002

ABSTRACTIntracellular pathogenic bacteria, includingMycobacterium tuberculosis, frequently have multitiered defense mechanisms ensuring their survival in host phagocytic cells. One such defense determinant inM. tuberculosisis thekatGgene, which encodes an enzyme with catalase, peroxidase, and peroxynitritase activities. KatG is considered to be important for protection against reactive oxygen and nitrogen intermediates produced by phagocytic cells. However, KatG also activates the front-line antituberculosis drug isoniazid, hence renderingM. tuberculosisexquisitely sensitive to this compound. In this context,katGexpression represents a double-edged sword, as it is an important virulence determinant but at the same time its activity levels determine sensitivity to INH. Thus, it is important to delineate the regulation and expression ofkatG, as this not only can aid understanding of howM. tuberculosissurvives and persists in the host but also may provide information of relevance for better management of INH therapy. Here, we report the first extensive analysis of thekatGpromoter activity examined both in vitro and in vivo. Using S1 nuclease protection analysis, we mapped thekatGmRNA 5′ ends and demonstrated that two promoters, P1furAand P1katG, control transcription ofkatG. ThefurAandkatGgenes are cotranscribed from P1furA. Both P1furAand P1katGpromoters show induction upon challenge with hydrogen peroxide and cumene hydroperoxide. Studies carried out using the transcriptional fusions P1furA-gfp, P1katG-gfp, and P1furA-P1katG-gfpconfirmed the existence of twokatGpromoters. In addition, we showed that both promoters are expressed in vivo during intracellular growth of virulentM. tuberculosisH37Rv. P1furAis induced early upon infection, and P1katGbecomes active only upon extended growth in macrophages. These studies delineate the transcriptional organization of thefurA-katGregion and indicate differential regulation in vivo of the twokatGpromoters. These phenomena most likely reflect the differing demands at sequential stages of the infection cycle and may provide information for improved understanding of host-pathogen interactions in tuberculosis and for further optimization of INH chemotherapy.
The Histone Deacetylase GenesHDA1andRPD3Play Distinct Roles in Regulation of High-Frequency Phenotypic Switching inCandida albicans
Authors: Srikantha T. , Tsai L. , Daniels K. , Klar A. J. S. , Soll D. R. 
Published: July 2002

ABSTRACTFive histone deacetylase genes (HDA1, RPD3, HOS1, HOS2,andHOS3) have been cloned fromCandida albicansand characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylasesHDA1andRPD3is down-regulated in the opaque phase of the white-opaque transition in strain WO-1.HOS3is selectively transcribed as a 2.5-kb transcript in the white phase and as a less-abundant 2.3-kb transcript in the opaque phase.HDA1andRPD3were independently deleted in strain WO-1, and both switching between the white and opaque phases and the downstream regulation of phase-specific genes were analyzed. Deletion ofHDA1resulted in an increase in the frequency of switching from the white phase to the opaque phase, but had no effect on the frequency of switching from the opaque phase to the white phase. Deletion ofRPD3resulted in an increase in the frequency of switching in both directions. Deletion ofHDA1resulted in reduced white-phase-specific expression of theEFG13.2-kb transcript,but had no significant effect on white-phase-specific expression ofWH11or opaque-phase-specific expression ofOP4, SAP1,andSAP3.Deletion ofRPD3resulted in reduced opaque-phase-specific expression ofOP4, SAP1,andSAP3and a slight reduction of white-phase-specific expression ofWH11and 3.2-kbEFG1. Deletion of neitherHDA1norRPD3affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein geneMCM1, which has been implicated in the regulation of opaque-phase-specific gene expression. In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genesHDA1andRPD3play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.
Comparison of Δ
relA
Strains of
Escherichia coli
and
Salmonella enterica
Serovar Typhimurium Suggests a Role for ppGpp in Attenuation Regulation of Branched-Chain Amino Acid Biosynthesis
Authors: Tedin K. , Norel F. 
Published: July 2002

ABSTRACT

The growth recovery of
Escherichia coli
K-12 and
Salmonella enterica
serovar Typhimurium Δ
relA
mutants were compared after nutritional downshifts requiring derepression of the branched-chain amino acid pathways. Because wild-type
E. coli
K-12 and
S. enterica
serovar Typhimurium LT2 strains are defective in the expression of the genes encoding the branch point acetohydroxy acid synthetase II (
ilvGM
) and III (
ilvIH
) isozymes, respectively, Δ
relA
derivatives corrected for these mutations were also examined. Results indicate that reduced expression of the known global regulatory factors involved in branched-chain amino acid biosynthesis cannot completely explain the observed growth recovery defects of the Δ
relA
strains. In the
E. coli
K-12 MG1655 Δ
relA
background, correction of the preexisting
rph
-1 allele which causes pyrimidine limitations resulted in complete loss of growth recovery.
S. enterica
serovar Typhimurium LT2 Δ
relA
strains were fully complemented by elevated basal ppGpp levels in an
S. enterica
serovar Typhimurium LT2 Δ
relA spoT1
mutant or in a strain harboring an RNA polymerase mutation conferring a reduced RNA chain elongation rate. The results are best explained by a dependence on the basal levels of ppGpp, which are determined by
relA
-dependent changes in tRNA synthesis resulting from amino acid starvations. Expression of the branched-chain amino acid operons is suggested to require changes in the RNA chain elongation rate of the RNA polymerase, which can be achieved either by elevation of the basal ppGpp levels or, in the case of the
E. coli
K-12 MG1655 strain, through pyrimidine limitations which partially compensate for reduced ppGpp levels. Roles for ppGpp in branched-chain amino acid biosynthesis are discussed in terms of effects on the synthesis of known global regulatory proteins and current models for the control of global RNA synthesis by ppGpp.
Assembly of an FtsZ Mutant Deficient in GTPase Activity Has Implications for FtsZ Assembly and the Role of the Z Ring in Cell Division
Authors: Mukherjee Amit , Saez Cristian , Lutkenhaus Joe 
Published: July 2002

ABSTRACT

FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the
ftsZ2
mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.
Aminopeptidases A, B, and N and Dipeptidase D Are the Four Cysteinylglycinases of
Escherichia coli
K-12
Authors: Suzuki Hideyuki , Kamatani Sachiko , Kim Eun-Soo , Kumagai Hidehiko 
Published: July 2002

ABSTRACT

Aminopeptidases A, B, and N and dipeptidase D, with broad substrate specificity, are the four cysteinylglycinases of
Escherichia coli
K-12, and there is no peptidase specific for the cleavage of cysteinylglycine.
Mobilization Function of the pBHR1 Plasmid, a Derivative of the Broad-Host-Range Plasmid pBBR1
Authors: Szpirer Cédric Y. , Faelen Michel , Couturier Martine 
Published: July 2002

ABSTRACT

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium
Bordetella bronchiseptica
(R. Antoine and C. Locht, Mol. Microbiol. 6:1785–1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the
mob
gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and
mob
gene regulation. We show that the Mob protein is a relaxase, and we located the
nic
site position in vitro. After sequence alignment, the position of the
nic
site of pBBR1 corresponds with those of the nick sites of the
Bacteroides
mobilizable transposon Tn
4555
and the streptococcal plasmid pMV158. The
oriT
of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.
Analysis of Promoters Recognized by PvdS, an Extracytoplasmic-Function Sigma Factor Protein from
Pseudomonas aeruginosa
Authors: Wilson Megan J. , McMorran Brendan J. , Lamont Iain L. 
Published: July 2002

ABSTRACT

The alternative sigma factor PvdS is required by
Pseudomonas aeruginosa
for initiation of transcription from pyoverdine (
pvd
) promoters. Two divergent PvdS-dependent promoters (
pvdE
and
pvdF
) were characterized by deletion analysis, and the minimal promoter region for each included a sequence element, the iron starvation (IS) box, that is present in other
pvd
promoters. Site-directed mutagenesis showed that the IS box elements were essential for promoter activity in vivo. Band shift assays and in vitro transcription experiments showed that a complex of PvdS and core RNA polymerase required the presence of an IS box in order to bind to and initiate transcription from
pvd
promoters. These results indicate that IS box elements participate in sequence-specific recognition by PvdS to enable initiation of transcription from
pvd
promoters and are likely to represent a −35 sequence element for this sigma factor.
Regulation of Ornibactin Biosynthesis and
N
-Acyl-
l
-Homoserine Lactone Production by CepR in
Burkholderia cepacia
Authors: Lewenza Shawn , Sokol Pamela A. 
Published: July 2002

ABSTRACT

The CepR-CepI quorum-sensing system has been shown to regulate production of the siderophore ornibactin, extracellular proteases, and
N
-octanoyl-homoserine-
l
-lactone (OHL) in
Burkholderia cepacia
strain K56-2. To examine the effect of
cepIR
on production of other siderophores,
cepR
mutants were constructed in strains that produce pyochelin in addition to salicylic acid and ornibactins. Pc715j-R1 (
cepR
::tp) hyperproduced ornibactin but produced parental levels of pyochelin and salicylic acid, suggesting that CepR is a negative regulator of ornibactin synthesis but not pyochelin or salicylic acid. Pc715j-R1 was also protease deficient and OHL negative. The effects of
cepR
on ornibactin biosynthetic genes were examined by constructing
cepR pvdA-lacZ
and
cepR pvdD-lacZ
mutants and monitoring β-galactosidase activity. There was an increase in expression of
pvdA
in the
cepR
mutant compared to the level in its parent strain in both low- and high-iron media during stationary phase. When the outer membrane protein profiles of a
cepR
mutant and the wild-type strain were compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there did not appear to be any difference in levels of expression of the ornibactin receptor. Experiments with
cepI-lacZ
and
cepR-lacZ
transcriptional fusions indicated that
cepI
was not expressed in the
cepR
mutant and that
cepR
acts as a negative regulator of its own expression. By a thin-layer chromatography assay for
N
-acyl homoserine lactones, OHL and
N
-hexanoyl-
l
-homoserine lactone (HHL) were detectable in K56-2 and Pc715j, both wild-type strains. OHL was not detectable and HHL was only weakly detectable in the
cepI
and
cepR
mutants. These results suggest that CepR is both a positive and negative transcriptional regulator and that CepR may influence the expression of ornibactin biosynthetic genes in addition to the expression of the
cepIR
quorum-sensing system.
SirA Orthologs Affect both Motility and Virulence
Authors: Goodier Robert I. , Ahmer Brian M. M. 
Published: July 2002

ABSTRACT

The
sirA
gene of
Salmonella enterica
serovar Typhimurium encodes a two-component response regulator of the FixJ family that has a positive regulatory influence on the expression of type III secretion genes involved with epithelial cell invasion and the elicitation of bovine gastroenteritis. SirA orthologs in
Pseudomonas, Vibrio
, and
Erwinia
control the expression of distinct virulence genes in these genera, but an evolutionarily conserved target of SirA regulation has never been identified. In this study we tested the hypothesis that
sirA
may be an ancient member of the flagellar regulon. We examined the effect of a
sirA
mutation on transcriptional fusions to flagellar promoters (
flhD, fliE, fliF, flgA, flgB, fliC, fliD, motA
, and
fliA
) while using fusions to the virulence gene
sopB
as a positive control. SirA had only small regulatory effects on all fusions in liquid medium (less than fivefold). However, in various types of motility agar plates,
sirA
was able to activate a
sopB
fusion by up to 63-fold while repressing flagellar fusions by values exceeding 100-fold. Mutations in the
sirA
orthologs of
Escherichia coli, Vibrio cholerae, Pseudomonas fluorescens
, and
Pseudomonas aeruginosa
result in defects in either motility or motility gene regulation, suggesting that control of flagellar regulons may be an evolutionarily conserved function of
sirA
orthologs. The implications for our understanding of virulence gene regulation in the gamma
Proteobacteria
are discussed.
Roles for Sigma Factors in Global Circadian Regulation of the Cyanobacterial Genome
Authors: Nair Usha , Ditty Jayna L. , Min Hongtao , Golden Susan S. 
Published: July 2002

ABSTRACT

The circadian clock of the unicellular cyanobacterium
Synechococcus elongatus
PCC 7942 imposes a global rhythm of transcription on promoters throughout the genome. Inactivation of any of the four known group 2 sigma factor genes (
rpoD2
,
rpoD3
,
rpoD4
, and
sigC
), singly or pairwise, altered circadian expression from the
psbAI
promoter, changing amplitude, phase angle, waveform, or period. However, only the
rpoD2
mutation and the
rpoD3 rpoD4
and
rpoD2 rpoD3
double mutations affected expression from the
kaiB
promoter. A striking differential effect was a 2-h lengthening of the circadian period of expression from the promoter of
psbAI
, but not of those of
kaiB
or
purF
, when
sigC
was inactivated. The data show that separate timing circuits with different periods can coexist in a cell. Overexpression of
rpoD2
,
rpoD3
,
rpoD4
, or
sigC
also changed the period or abolished the rhythmicity of P
psbAI
expression, consistent with a model in which sigma factors work as a consortium to convey circadian information to downstream genes.
α-Galactosidase Aga27A, an Enzymatic Component of theClostridium josuiCellulosome
Authors: Jindou Sadanari , Karita Shuichi , Fujino Emi , Fujino Tsuchiyoshi , Hayashi Hidenori , Kimura Tetsuya , Sakka Kazuo , Ohmiya Kunio 
Published: July 2002

ABSTRACTTheClostridium josui aga27Agene encodes the cellulosomal α-galactosidase Aga27A, which comprises a catalytic domain of family 27 of glycoside hydrolases and a dockerin domain responsible for cellulosome assembly. The catalytic domain is highly homologous to those of various α-galactosidases of family 27 of glycoside hydrolases from eukaryotic organisms, especially plants. The recombinant Aga27A α-galactosidase devoid of the dockerin domain preferred highly polymeric galactomannan as a substrate to small saccharides such as melibiose and raffinose.
Genetic Analysis of the RcsC Sensor Kinase from
Escherichia coli
K-12
Authors: Clarke D. J. , Joyce S. A. , Toutain C. M. , Jacq A. , Holland I. B. 
Published: July 2002

ABSTRACT

The Rcs two-component pathway is involved in the regulation of capsule production in
Escherichia coli
. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.
Tetracycline Induces Stabilization of mRNA in
Bacillus subtilis
Authors: Wei Yi , Bechhofer David H. 
Published: July 2002

ABSTRACT

The
tet
(L) gene of
Bacillus subtilis
confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of
tet
(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of
tet
(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in
tet
(L) mRNA stability. Using a plasmid-borne derivative of
tet
(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of
tet
(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a
B. subtilis
strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5" end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other
B. subtilis
mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.