bluuebunny/crossref_metadata_2025_split huggingface dataset:
DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome
Authors: Bruand Claude , Farache Michaël , McGovern Stephen , Ehrlich S. Dusko , Polard Patrice 
Published: March 2003

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single‐stranded DNA in a PriA‐independent, DnaD‐ and DnaI‐dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.
The polyphosphate kinase plays a negative role in the control of antibiotic production in Streptomyces lividans
Authors: Chouayekh Hichem , Virolle Marie‐Joelle 
Published: March 2003

The polyphosphate kinase gene (ppk) from Streptomyces lividans, which encodes a 774‐amino‐acid protein (86.4 kDa) showing extensive homology to other bacterial polyphosphate kinases, was cloned by polymerase chain reaction (PCR) using oligonucleotides derived from the putative ppk gene from the closely related species, Streptomyces coelicolor. In vitro, the purified Ppk was shown to be able to synthesize the polyphosphate [poly(P)] from ATP (forward reaction) as well as to regenerate ATP from the poly(P) in the presence of an excess of ADP (reverse reaction). In conditions of poly(P) synthesis, a phosphoenzyme intermediate was detected, indicating an autophosphorylation of the enzyme in the presence of ATP. The ppk gene was shown to be transcribed as a monocistronic mRNA from a unique promoter. Its transcription was only detectable during the late stages of growth in liquid minimal medium. A mutant strain interrupted for ppk was characterized by increased production of the antibiotic actinorhodin on rich R2YE solid medium (0.37 mM KH2PO4 added). This production was enhanced on the same medium with no KH2PO4 added but was completely abolished by the addition of 1.48 mM KH2PO4. In the ppk mutant strain, this increased production correlated with enhanced transcription of actII‐ORF4 encoding the specific activator of the actinorhodin pathway. In that strain, the transcription of redD and cdaR, encoding the specific activators of the undecylprodigiosin and calcium‐dependent antibiotic biosynthetic pathways, respectively, was also increased but to a lesser extent. The enhanced expression of these regulators did not seem to be related to increased relA‐dependent ppGpp synthesis, as no obvious increase in relA expression was observed in the ppk mutant strain. These results suggested that the negative regulatory effect exerted by Ppk on antibiotic biosynthesis was most probably caused by the repression exerted by the endogenous Pi, resulting from the hydrolysis of the poly(P) synthesized by Ppk, on the expression of the specific activators of the antibiotic biosynthetic pathways.
The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model
Authors: Stephenson Aimee E. , Wu Hui , Novak Jan , Tomana Milan , Mintz Keith , Fives‐Taylor Paula 
Published: March 2003

Summary Streptococcus parasanguis is a primary colonizer of the tooth surface and
plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis
are important in mediating adhesion to saliva‐coated hydroxylapatite (SHA), an in
vitro tooth adhesion model. The Fap1 adhesin has been identified as the major
fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide
analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated
the presence of rhamnose, glucose, galactose, N‐acetylglucosamine and N‐acetylgalactosamine.
A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by
immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide
was similar to that of the intact molecule. The functionality of the glycan moiety
was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein.
These antibodies were grouped into two categories based on their ability to block
adhesion of S. parasanguis to SHA and their corresponding specificity for
either protein or glycan epitopes of the Fap1 protein. ‘Non‐blocking’
MAb epitopes were mapped to unique protein sequences in the N‐terminus of the Fap1
protein using non‐glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed
in Escherichia coli. In contrast, the ‘blocking’ antibodies
did not bind to the recombinant Fap1 proteins, and were effectively competed by the
binding to the purified glycopeptide. These data suggest that the ‘blocking’
antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.
Regulation of the Bacillus subtilis ccpC gene by CcpA and CcpC
Authors: Kim Hyun‐Jin , Jourlin‐Castelli Cécile , Kim Sam‐In , Sonenshein Abraham L. 
Published: March 2003

Summary Bacillus subtilis CcpC, a LysR‐type transcriptional regulator, represses the transcription of genes for citrate synthase (citZ) and aconitase (citB) in response to citrate availability. Transcription of ccpC was shown to initiate at two promoters, P1, located just upstream of the ccpC gene, and P2, located within or upstream of the neighbouring ykuL gene. Expression from the ccpC‐specific promoter (P1) was negatively regulated by CcpC but independent of the carbon source in the medium. Gel shift and DNase I footprinting experiments revealed that CcpC binds to an interrupted dyad sequence that surrounds the ccpC transcriptional start point. Transcription of ccpC from the upstream promoter (P2) was repressed by glucose in a CcpA‐dependent manner. A putative CcpA binding site (cre) was identified upstream of the –35 region of the P1 promoter. Transcriptional fusion studies demonstrated that glucose repression of ccpC expression from the P2 promoter depends on this cre site. In addition, DNase I footprinting experiments showed that CcpA specifically binds to this cre site and that the introduction of mutations (cre*) into this site abolished the binding. These results suggest that CcpA may control CcpC syn‐thesis by acting as a road‐block to readthrough transcription from the P2 promoter.
Differential roles of two SARP‐encoding regulatory genes during tylosin biosynthesis
Authors: Bate Neil , Stratigopoulos George , Cundliffe Eric 
Published: March 2003

SummaryThe tylosin biosynthetic gene cluster of Streptomyces fradiae is remarkable in harbouring at least five regulatory genes, two of which (tylS and tylT) encode proteins of the Streptomyces antibiotic regulatory protein (SARP) family. The aim of the present work was to assess the respective contributions of TylS and TylT to tylosin production. A combination of targeted gene disruption, fermentation studies and gene expression analysis via reverse transcriptase–polymerase chain reaction (RT–PCR) suggests that tylS is essential for tylosin production and controls the expression of tylR (previously shown to be a global activator of the biosynthetic pathway) plus at least one other gene involved in polyketide metabolism or regulation thereof. This is the first demonstration of a SARP acting to control another regulatory gene during antibiotic biosynthesis. In contrast, tylT is not essential for tylosin production.
A novel type II secretion system in Pseudomonas aeruginosa
Authors: Ball Geneviève , Durand Éric , Lazdunski Andrée , Filloux Alain 
Published: March 2003

SummaryThe genome sequence of Pseudomonas aeruginosa strain PAO1 has been determined to facilitate post‐genomic studies aimed at understanding the capacity of adaptation of this ubiquitous opportunistic pathogen. P. aeruginosa produces toxins and hydrolytic enzymes that are secreted via the type II secretory pathway using the Xcp machinery or ‘secreton’. In this study, we characterized a novel gene cluster, called hxc for homologous to xcp. Characterization of an hxcR mutant, grown in phosphate‐limiting medium, revealed the absence of a 40 kDa protein found in the culture supernatant of wild‐type or xcp derivative mutant strains. The protein corresponded to the alkaline phosphatase L‐AP, renamed LapA, which is secreted in an xcp‐independent but hxc‐dependent manner. Finally, we showed that expression of the hxc gene cluster is under phosphate regulation. This is the first report of the exist‐ence of two functional type II secretory pathways within the same organism, which could be related to the high adaptation potential of P. aeruginosa.
OmpR‐dependent and OmpR‐independent responses of Escherichia coli to sublethal attack by the neutrophil bactericidal/permeability increasing protein
Authors: Prohinar Polonca , Forst Steve A. , Reed Deoine , Mandic‐Mulec Ines , Weiss Jerrold 
Published: March 2003

SummaryBactericidal/permeability‐increasing protein (BPI) of neutrophils is a lipopolysaccharide (LPS)‐binding antibacterial protein with specificity for Gramnegative bacteria. BPI binding to the bacterial surface rapidly triggers potentially reversible bacterial growth inhibition and alterations of the outer membrane and, later, disruption of the inner membrane and lethal injury. Initial effects include selective OmpR‐dependent changes in the synthesis of outer membrane porins (OmpF and OmpC). Because OmpR is a global transcriptional regulator, we have examined its possible role in responses of E. coli to sublethal injury caused by BPI. Early (<15 min) reversible

effects of BPI on bacterial colony‐forming ability and outer membrane permeability

were virtually identical in isogenic wild‐type (wt) and ompR−E. coli. Both strains could repair the outer membrane permeability barrier

after Mg2+‐induced displacement of bound BPI. However, OmpR was

essential for the ability of E. coli to tolerate low doses of BPI and escape

the progression of sublethal to lethal damage. Scanning electron microscopy revealed

that BPI treatment produced greater membrane perturbations in the ompR−

strain, apparent even before lethal injury. These findings suggest that the fate

of E. coli exposed to BPI depends on both OmpR‐independent mechanisms engaged in outer membrane repair and OmpR‐ dependent processes that modulate porin synthesis and retard progression of injury from the outer to the inner membrane.
Transcytosis of iota‐toxin across polarized CaCo‐2 cells
Authors: Richard Jean François , Mainguy Gael , Gibert Maryse , Marvaud Jean Christophe , Stiles Bradley G. , Popoff Michel R. 
Published: March 2003

SummaryIota‐toxin from Clostridium perfringens type E is a binary toxin consisting of two independent proteins, an enzymatic Ia and binding Ib component. Ia catalyses ADP‐ribosylation of actin monomers, thus disrupting the actin cytoskeleton. In this report, we show that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo‐2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side, slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. Interestingly, the two iota‐toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti‐Ib sera, raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, trans‐cytosed and permanently exposed on the opposite cell surface or continuously recycled between an endosomal compartment and the cell surface.
Control of chitin synthesis through Shc1p, a functional homologue of Chs4p specifically induced during sporulation
Authors: Sanz Maria , Trilla Ose Angel , Duran Ngel , Roncero Cesar 
Published: March 2003

SummaryThe Saccharomyces cerevisiae SHC1 gene encodes a protein with a high homology

to Chs4p, a positive regulator of chitin synthase III (CSIII) during vegetative growth.

SHC1 is not expressed during vegetative growth but is strongly induced during

sporulation as a mid‐late gene. shc1/shc1 mutants do not show any defect in

the total rate of sporulation and meiosis occurs normally. However, shc1/shc1

ascospores be‐come highly permeable to DAPI, much more sensitive to glusulase treatment,

and have very low levels of chitosan in their cell walls. All these observations

indicate that Shc1p is required for proper maturation of the ascospore through its

participation in the synthesis of the chitosan layer. Lack of SHC1 during

sporulation can be partially compensated by over‐expression of the CHS4 gene.

During vegetative growth, SHC1 has no apparent function but, when ectopically

overexpressed, it can substitute Chs4p as an activator of the CSIII activity; however,

Shc1p fails to localize it properly, as Chs4p does. In conclusion, S. cerevisiae

contains two functionally redundant genes in the control of CSIII activity: CHS4,

whose function is restricted to vegetative growth because Chs4p is rapidly degraded

during sporulation, and SHC1, whose function in cell wall ascospore assembly is transcriptionally restricted to the sporulation process.
Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4
Authors: Kumar Renu B. , Das Anath 
Published: March 2003

Summary Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants.

VirD4 presumably functions as a coupling factor that facilitates communication between

a substrate and the transport pore. To serve as a coupling protein, VirD4 may be

required to localize near the transport apparatus. In a previous study, we observed

that several constituents of the transport apparatus localize to the cell membranes.

In this study, we demonstrate that VirD4 has a unique cellular location. In immunofluorescence

microscopy, cells probed with anti‐VirD4 antibodies had foci of fluorescence primarily

at the cell poles, indicating that VirD4 localizes to the cell pole. Polar location

of VirD4 was not dependent on T‐DNA processing, the formation of the transport apparatus

and the presence of other Vir proteins. VirD4 is an integral membrane protein with

one periplasmic domain. The large cytoplasmic region contains a nucleotide‐binding

domain. To investigate the role of these domains in DNA transfer, we introduced mutations

in virD4 and studied the effect of a mutation on substrate transfer. A deletion

of most of the periplasmic domain as well as the alterations of glycine 151 to serine

and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that

both domains are essential for substrate transfer. Subcellular localization of the

mutant proteins indicated that both the periplasmic and the nucleotide‐binding domains

are required for polar localization of VirD4. The periplasmic domain mutant VirD4Δ36–61

was distributed throughout the cell membrane, whereas the nucleotide binding site

mutant VirD4G151S localized to sites other than the cell poles. Polar location of VirD4 suggests a role for the cell pole in DNA transfer.
The flagella of enteropathogenicEscherichia colimediate adherence to epithelial cells
Authors: Girón Jorge A. , Torres Alfredo G. , Freer Enrique , Kaper James B. 
Published: March 2003

SummaryEnteropathogenicEscherichia coli(EPEC) utilizes a type III protein secretion system to target effector molecules into the host cell leading to effacement of the intestinal mucosa. This secretion apparatus shares many structural features of the flagellar type III export system involved in flagella assembly and motility. We report here thatfliCinsertional mutants constructed in two wild‐type EPEC strains were markedly impaired in adherence and microcolony formation on cultured cells. AnE. coliK‐12 strain harbouring the EPEC H6fliCgene on a plasmid showed discrete adhering clusters on HeLa cells, albeit to less extent than the wild‐type EPEC strain. Flagella purified from EPEC bound to cultured epithelial cells and antiflagella antibodies blocked adherence of several EPEC serotypes. We determined that eukaryotic cells in culture stimulate expression of flagella by motile and non‐motile EPEC. Isogenic strains mutated inperA(a transcriptional activator),bfpA(a type IV pilin),luxS(a quorum‐sensing autoinducer gene) and in the type III secretion genes were reduced for motility in Dulbecco’s modified Eagle medium (DMEM) motility agar and produced none or few flagella when associated with epithelial cells. Growth of these mutants in preconditioned tissue culture medium restored motility and their ability to produce flagella, suggesting the influence of a signal provided by mammalian cells that triggers flagella production. This study shows for the first time that the flagella of EPEC are directly involved in the adherence of these bacteria and supports the existence of a molecular relationship between the two existing type III secretion pathways of EPEC, the EPEC adherence factor (EAF) plasmid‐encoded regulator, quorum sensing and epithelial cells.
A NarX–Tar chimera mediates repellent chemotaxis to nitrate and nitrite
Authors: Ward Scott M. , Delgado Asuncion , Gunsalus Robert P. , Manson Michael D. 
Published: March 2003

SummaryMembrane receptors communicate between the external world and the cell interior. In bacteria, these receptors include the transmembrane sensor kinases, which control gene expression via their cognate response regulators, and chemoreceptors, which control the direction of flagellar rotation via the CheA kinase and CheY response regulator. Here, we show that a chimeric protein that joins the ligand‐binding, transmembrane and linker domains of the NarX sensor kinase to the signalling and adaptation domains of the Tar chemoreceptor of Escherichia coli mediates repellent responses to nitrate and nitrite. Nitrate induces a stronger response than nitrite and is effective at lower concentrations, mirroring the relative sensitivity to these ligands exhibited by NarX itself. We conclude that the NarX–Tar hybrid functions as a bona fide chemoreceptor whose activity can be predicted from its component parts. This observation implies that ligand‐dependent activation of a sensor kinase and repellent‐initiated activation of receptor‐coupled CheA kinase involve a similar transmembrane signal.
Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction
Authors: Singh Upinder , Brewer Jeremy L. , Boothroyd John C. 
Published: March 2003

SummaryDevelopmental switching in Toxoplasma gondii, from the virulent tachyzoite

to the relatively quiescent bradyzoite stage, is responsible for disease propagation

and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd−)

mutants in T. gondii and used these in combination with a cDNA microarray

to identify developmental pathways in bradyzoite formation. Four independently generated

Tbd− mutants were analysed and had defects in bradyzoite development

in response to multiple bradyzoite‐inducing conditions, a stable phenotype after

in vivo passages and a markedly reduced brain cyst burden in a murine model

of chronic infection. Transcriptional profiles of mutant and wild‐type parasites,

growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated

genes, including many bradyzoite‐induced genes whose transcripts were reduced in

all mutants. A set of non‐developmentally regulated genes whose transcripts were

less abundant in Tbd− mutants were also identified. These may represent

genes that mediate downstream effects and/or whose expression is dependent on the

same transcription factors as the bradyzoite‐induced set. Using these data, we have

generated a model of transcription regulation during bradyzoite development in T.

gondii. Our approach shows the utility of this system as a model to study developmental biology in single‐celled eukaryotes including protozoa and fungi.
Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS‐1
Authors: Yoshida Takao , Ideno Akira , Suzuki Rintaro , Yohda Masafumi , Maruyama Tadashi 
Published: March 2003

SummaryThe thermostability of the recombinant α‐ and β‐subunit homo‐oligomers (α16mer and β16mer) and of natural chaperonins purified from cultured Thermococcus strain KS‐1 cells was measured to understand the mechanism for the thermal acclimatization of T. KS‐1. The β‐subunit content of the natural chaperonin from cells grown at 90°C was higher than that at 80°C. The optimum temperature for ATPase activity of the natural chaperonins was 80–90°C, whereas that for α16mer and β16mer was 60°C and over 90°C respectively. Judging from the ATPase activity, β16mer was more thermostable than α16mer. The thermostabilities of the natural chaperonins were intermediate between α16mer and β16mer, whereas the natural chaperonin with a higher β‐subunit content was more stable than that with a lower β‐subunit content. Native polyacrylamide gel electrophoresis (PAGE) revealed that the chaperonin oligomers thermally dissociated to their ATPase‐inactive monomers. The thermal denaturation process monitored by circular dichroism showed that the free β‐subunit was more stable than the free α‐subunit, and that the secondary structure of the chaperonin monomer in the oligomer was more stable than that in the free monomer. These results suggest that the structure of these subunits was stabilized in the oligomer, and that an increase in the β‐subunit content conferred higher thermostability to the natural hetero‐oligomeric chaperonin.
Mg2+ homeostasis and avoidance of metal toxicity
Authors: Chamnongpol Sangpen , Groisman Eduardo A. 
Published: March 2003

SummaryBecause Mg2+ is required for a wide variety of cellular functions, its intracellular levels must be tightly regulated. In the Gram‐negative bacterium Salmonella enterica, three transporters mediate Mg2+ uptake: the P‐type ATPases MgtA and MgtB, whose expression is transcriptionally induced in low Mg2+ by the Mg2+‐regulated PhoP/PhoQ two‐component system; and CorA, whose transcription is regulated neither by the levels of Mg2+ nor by the PhoP/PhoQ system. We now report that mutants defective in phoP or in both mgtA and mgtB are hypersensitive to oxidative stress‐dependent Fe (II)‐mediated killing. These mutants display increased iron accumulation and heightened Ni2+ uptake. Inactivation of the corA gene restored Fe(II) resistance to the phoP mutant and eliminated uptake of Ni2+. Neither corA transcription nor CorA protein levels were altered in the phoP mutant, suggesting that CorA alters its activity in response to the presence of PhoP‐regulated determinants. Downregulation of CorA activity in low Mg2+ environments may enable Salmonella to avoid the uncontrolled influx of toxic metals.