bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pneumococcal HtrA Protease Mediates Inhibition of Competence by the CiaRH Two-Component Signaling System
Authors: Sebert M. E. , Patel K. P. , Plotnick M. , Weiser J. N. 
Published: June 2005

ABSTRACTActivation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation inStreptococcus pneumoniaethrough a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression ofhtrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of aciaRH+strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.
Regulation of Bacteriocin Production in
Streptococcus mutans
by the Quorum-Sensing System Required for Development of Genetic Competence
Authors: van der Ploeg Jan R. 
Published: June 2005

ABSTRACT

In
Streptococcus mutans
, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by
comC
). Here, it is shown that the same system is also required for expression of the
nlmAB
genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional
nlmAB
′-
lacZ
fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the
comDE
genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the
nlmAB
promoter region led to a decrease in CSP-dependent expression of
nlmAB
′
-lacZ
. In agreement with these results, a
comDE
mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci.
Membrane-Associated Maturation of the Heterotetrameric Nitrate Reductase ofThermus thermophilus
Authors: Zafra Olga , Cava Felipe , Blasco Francis , Magalon Axel , Berenguer Jose 
Published: June 2005

ABSTRACTThenaroperon, coding for the respiratory nitrate reductase ofThermus thermophilus(NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants ofT. thermophiluslacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized bynarJmutants, but in contrast to itsEscherichia colihomologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that ofE. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.
Characterization of the Small Untranslated RNA RyhB and Its Regulon inVibrio cholerae
Authors: Davis Brigid M. , Quinones Mariam , Pratt Jason , Ding Yanpeng , Waldor Matthew K. 
Published: June 2005

ABSTRACTNumerous small untranslated RNAs (sRNAs) have been identified inEscherichia coliin recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved inVibrio cholerae, and both identification and characterization of sRNAs inV. choleraeremain at a preliminary stage. We have characterized one of the few sRNAs conserved betweenE. coliandV. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB inV. choleraeis significantly larger than inE. coli. As inE. coli,V. choleraeRyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB inV. choleraeandE. coliappear to differ, although some overlap is evident. Analysis of gene expression inV. choleraein the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host byV. cholerae.
A VicRK Signal Transduction System in
Streptococcus mutans
Affects
gtfBCD
,
gbpB
, and
ftf
Expression, Biofilm Formation, and Genetic Competence Development
Authors: Senadheera M. Dilani , Guggenheim Bernard , Spatafora Grace A. , Huang Yi-Chen Cathy , Choi Jison , Hung David C. I. , Treglown Jennifer S. , Goodman Steven D. , Ellen Richard P. , Cvitkovitch Dennis G. 
Published: June 2005

ABSTRACT

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the
vicRK
signal transduction system in
Streptococcus mutans
. A
vicK
(putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a
vicR
(putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the
vicRKX
operon (Smuvic
+
). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the
vicK
mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic
+
. Based on real-time PCR, Smuvic
+
showed increased
gtfBCD
,
gbpB
, and
ftf
expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the
gtfB
,
gtfC
, and
ftf
genes. Also, transformation efficiency in the presence or absence of the
S. mutans
competence-stimulating peptide was altered for the
vic
mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of
vicK
did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the
vicRK
signal transduction system in regulating several important physiological processes in
S. mutans
.
ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in EnterohemorrhagicEscherichia coli
Authors: Iyoda Sunao , Watanabe Haruo 
Published: June 2005

ABSTRACTExpression of the type III protein secretion system (TTSS), encoded in the locus of enterocyte effacement (LEE) of enterohemorrhagicEscherichia coli(EHEC), has been shown to be controlled by various regulators. In a search for additional regulatory genes, we identified a DNA fragment containingclpXandclpPthat has a positive regulatory effect on LEE expression in EHEC O157. The expression of LEE-encoded Esp proteins was significantly reduced in aclpXPdeletion mutant. Deletion ofgrlR, a negative regulatory gene within LEE, markedly increased LEE expression even in theclpXPmutant. To verify the regulatory mechanism of GrlR expression, a chromosomal epitope-tagged allele ofgrlR(grlR-FLAG) was constructed. GrlR-FLAG expression was increased significantly in theclpXPdeletion mutant, suggesting that the GrlR level is under the control of ClpXP, and this regulation is critical for the ClpXP-dependent expression of LEE in EHEC. Deletion ofrpoS, the gene encoding a stationary-phase-inducing sigma factor that is a substrate for ClpXP protease, partially restored LEE expression in theclpXPmutant. A multicopy plasmid carryingrpoSstrongly repressed expression of Esp proteins, suggesting that positive regulation by ClpXP is partially mediated through a negative effect of RpoS on LEE expression. We also found thatrpoSdeletion induces transcription ofpchA, which encodes one of the positive regulators for LEE expression in EHEC. These results suggest that ClpXP controls expression of LEE through the regulation of RpoS and GrlR levels in EHEC.
Genomic Sequence and Receptor for the
Vibrio cholerae
Phage KSF-1Φ: Evolutionary Divergence among Filamentous Vibriophages Mediating Lateral Gene Transfer
Authors: Faruque Shah M. , Bin Naser Iftekhar , Fujihara Kazutaka , Diraphat Pornphan , Chowdhury Nityananda , Kamruzzaman M. , Qadri Firdausi , Yamasaki Shinji , Ghosh A. N. , Mekalanos John J. 
Published: June 2005

ABSTRACT

KSF-1Φ, a novel filamentous phage of
Vibrio cholerae
, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1Φ infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the
mshA
gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1Φ infection, naturally occurring MSHA-negative strains and an
mshA
deletion mutant were resistant. Furthermore,
d
-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1Φ. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1Φ, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXΦ, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of
V. cholerae
strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to
V. cholerae
evolution.
Characterization of Gla
KP
, a UDP-Galacturonic Acid C4-Epimerase from
Klebsiella pneumoniae
with Extended Substrate Specificity
Authors: Frirdich Emilisa , Whitfield Chris 
Published: June 2005

ABSTRACT

In
Escherichia coli
and
Salmonella enterica
, the core oligosaccharide backbone of the lipopolysaccharide is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. In contrast,
Klebsiella pneumoniae
lacks phosphoryl groups in its core oligosaccharide but instead contains galacturonic acid residues that are proposed to serve a similar function in outer membrane stability. Gla
KP
is a UDP-galacturonic acid C4-epimerase that provides UDP-galacturonic acid for core synthesis, and the enzyme was biochemically characterized because of its potentially important role in outer membrane stability. High-performance anion-exchange chromatography was used to demonstrate the UDP-galacturonic acid C4-epimerase activity of Gla
KP
, and capillary electrophoresis was used for activity assays. The reaction equilibrium favors UDP-galacturonic acid over UDP-glucuronic acid in a ratio of 1.4:1, with the

K
m

for UDP-glucuronic acid of 13.0 μM. Gla
KP
exists as a dimer in its native form. NAD
+
/NADH is tightly bound by the enzyme and addition of supplementary NAD
+
is not required for activity of the purified enzyme. Divalent cations have an unexpected inhibitory effect on enzyme activity. Gla
KP
was found to have a broad substrate specificity in vitro; it is capable of interconverting UDP-glucose/UDP-galactose and UDP-
N
-acetylglucosamine/UDP-
N
-acetylgalactosamine, albeit at much lower activity. The epimerase GalE interconverts UDP-glucose/UDP-galactose. Multicopy plasmid-encoded
gla
KP
partially complemented a
galE
mutation in
S. enterica
and in
K. pneumoniae
; however, chromosomal
gla
KP
could not substitute for
galE
in a
K. pneumoniae galE
mutant in vivo.
Two Thimet Oligopeptidase-Like Pz Peptidases Produced by a Collagen- Degrading Thermophile,
Geobacillus collagenovorans
MO-1
Authors: Miyake Ryoma , Shigeri Yasushi , Tatsu Yoshiro , Yumoto Noboru , Umekawa Midori , Tsujimoto Yoshiyuki , Matsui Hiroshi , Watanabe Kunihiko 
Published: June 2005

ABSTRACT

A collagen-degrading thermophile,
Geobacillus collagenovorans
MO-1, was found to produce two metallopeptidases that hydrolyze the synthetic substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-
d
-Arg (Pz-PLGPR), containing the collagen-specific sequence -Gly-Pro-X-. The peptidases, named Pz peptidases A and B, were purified to homogeneity and confirmed to hydrolyze collagen-derived oligopeptides but not collagen itself, indicating that Pz peptidases A and B contribute to collagen degradation in collaboration with a collagenolytic protease in
G. collagenovorans
MO-1. There were many similarities between Pz peptidases A and B in their catalytic properties; however, they had different molecular masses and shared no antigenic groups against the respective antibodies. Their primary structures clarified from the cloned genes showed lower identity (22%). From homology analysis for proteolytic enzymes in the database, the two Pz peptidases belong to the M3B family. In addition, Pz peptidases A and B shared high identities of over 70% with unassigned peptidases and oligopeptidase F-like peptidases of the M3B family, respectively. Those homologue proteins are putative in the genome database but form two distinct segments, including Pz peptidases A and B, in the phylogenic tree. Mammalian thimet oligopeptidases, which were previously thought to participate in collagen degradation and share catalytic identities with Pz peptidases, were found to have lower identities in the overall primary sequence with Pz peptidases A and B but a significant resemblance in the vicinity of the catalytic site.
Identification of the Ω4406 Regulatory Region, a Developmental Promoter of
Myxococcus xanthus
, and a DNA Segment Responsible for Chromosomal Position-Dependent Inhibition of Gene Expression
Authors: Loconto Jennifer , Viswanathan Poorna , Nowak Scott J. , Gloudemans Monica , Kroos Lee 
Published: June 2005

ABSTRACT

When starved,
Myxococcus xanthus
cells send signals to each other that coordinate their movements, gene expression, and differentiation. C-signaling requires cell-cell contact, and increasing contact brought about by cell alignment in aggregates is thought to increase C-signaling, which induces expression of many genes, causing rod-shaped cells to differentiate into spherical spores. C-signaling involves the product of the
csgA
gene. A
csgA
mutant fails to express many genes that are normally induced after about 6 h into the developmental process. One such gene was identified by insertion of Tn
5 lac
at site Ω4406 in the
M. xanthus
chromosome. Tn
5 lac
fused transcription of
lacZ
to the upstream Ω4406 promoter. In this study, the Ω4406 promoter region was identified by analyzing mRNA and by testing different upstream DNA segments for the ability to drive developmental
lacZ
expression in
M. xanthus
. The 5′ end of Ω4406 mRNA mapped to approximately 1.3 kb upstream of the Tn
5 lac
insertion. A 1.0-kb DNA segment from 0.8 to 1.8 kb upstream of the Tn
5 lac
insertion, when fused to
lacZ
and integrated at a phage attachment site in the
M. xanthus
chromosome, showed a similar pattern of developmental expression as Tn
5 lac
Ω4406. The DNA sequence upstream of the putative transcriptional start site was strikingly similar to promoter regions of other C-signal-dependent genes. Developmental
lacZ
expression from the 1.0-kb segment was abolished in a
csgA
mutant but was restored upon codevelopment of the
csgA
mutant with wild-type cells, which supply C-signal, demonstrating that the Ω4406 promoter responds to extracellular C-signaling. Interestingly, the 0.8-kb DNA segment immediately upstream of Tn
5 lac
Ω4406 inhibited expression of a downstream
lacZ
reporter in transcriptional fusions integrated at a phage attachment site in the chromosome but not at the normal Ω4406 location. To our knowledge, this is the first example in
M. xanthus
of a chromosomal position-dependent effect on gene expression attributable to a DNA segment outside the promoter region.
Evolution of Transcription Regulatory Genes Is Linked to Niche Specialization in the Bacterial Pathogen
Streptococcus pyogenes
Authors: Bessen Debra E. , Manoharan Anand , Luo Feng , Wertz John E. , Robinson D. Ashley 
Published: June 2005

ABSTRACT

Streptococcus pyogenes
is a highly prevalent bacterial pathogen, most often giving rise to superficial infections at the throat or skin of its human host. Three genotype-defined subpopulations of strains exhibiting strong tropisms for either the throat or skin (specialists) or having no obvious tissue site preference (generalists) are recognized. Since the microenvironments at the throat and skin are distinct, the signal transduction pathways leading to the control of gene expression may also differ for throat versus skin strains of
S. pyogenes
. Two loci (
mga
and
rofA/nra
) encoding global regulators of virulence gene expression are positioned 300 kb apart on the genome; each contains alleles forming two major sequence clusters of ∼25 to 30% divergence that are under balancing selection. Strong linkage disequilibrium is observed between sequence clusters of the transcription regulatory loci and the subpopulations of throat and skin specialists, against a background of high recombination rates among housekeeping genes. A taxonomically distinct commensal species (
Streptococcus dysgalactiae
subspecies
equisimilus
) shares highly homologous
rof
alleles. The findings provide strong support for a mechanism underlying niche specialization that involves orthologous replacement of regulatory genes following interspecies horizontal transfer, although the directionality of gene exchange remains unknown.
Requirement of themymAOperon for Appropriate Cell Wall Ultrastructure and Persistence ofMycobacterium tuberculosisin the Spleens of Guinea Pigs
Authors: Singh Amit , Gupta Radhika , Vishwakarma R. A. , Narayanan P. R. , Paramasivan C. N. , Ramanathan V. D. , Tyagi Anil K. 
Published: June 2005

ABSTRACTWe had recently reported that themymAoperon (Rv3083 to Rv3089) ofMycobacterium tuberculosisis regulated by AraC/XylS transcriptional regulator VirS (Rv3082c) and is important for the cell envelope ofM. tuberculosis. In this study, we further show that avirSmutant (MtbΔvirS) and amymAmutant (Mtbmym::hyg) ofM. tuberculosisexhibit reduced contents and altered composition of mycolic acids along with the accumulation of saturated C24and C26fatty acids compared to the parental strain. These mutants were markedly more susceptible to major antitubercular drugs at acidic pH and also showed increased sensitivity to detergent (sodium dodecyl sulfate) and to acidic stress than the parental strain. We show that disruption ofvirSandmymAgenes impairs the ability ofM. tuberculosisto survive in activated macrophages, but not in resting macrophages, suggesting the importance of themymAoperon in protecting the bacterium against harsher conditions. Infection of guinea pigs with MtbΔvirS, Mtbmym::hyg, and the parental strain resulted in an ∼800-fold-reduced bacillary load of the mutant strains compared with the parental strain in spleens, but not in the lungs, of animals at 20 weeks postinfection. Phenotypic traits were fully complemented upon reintroduction of thevirSgene into MtbΔvirS. These observations show the important role of themymAoperon in the pathogenesis ofM. tuberculosisat later stages of the disease.