bluuebunny/crossref_metadata_2025_split huggingface dataset:
The esg locus of Myxococcus xanthus encodes the E1α and E1β subunits of a branched‐chain keto acid dehydrogenase
Authors: Toal Douglas R. , Clifton Sandra W. , Roe Bruce A. , Downard John 
Published: October 2006

SummaryThe esg locus of Myxococcus xanthus appears to control the production of a signal that must be transmitted between cells for the completion of multicellular development DNA sequence analysis suggested that the esg locus encodes the E1 decarboxylase (composed of E1α and E1β subunits) of a branched‐chain keto acid dehydrogenase (BCKAD) that is involved in branched‐chain amino acid (BCAA) metabolism. The properties of an esg ::Tn5 insertion mutant supported this conclusion. These properties include: (i) the growth yield of the mutant was reduced with increasing concentrations of the BCAAs in the medium while the growth yield of wild‐type cells increased, (ii) mutant extracts were deficient in BCKAD activity, and (iii) growth of the mutant in media with short branched‐chain fatty acids related to the expected products of the BCKAD helped to correct the mutant defects in growth, pigmentation and development. The esg BCKAD appears to be involved in the synthesis of long branched‐chain fatty acids since the mutant contained reduced levels of this class of compounds. Our results are consistent with a model in which the esg‐encoded enzyme is involved in the synthesis of branched‐chain fatty acids during vegetative growth, and these compounds are used later in cell‐cell signalling during development.
A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export
Authors: Havarstein Leiv Sigve , Diep Dzung Bao , Nes Ingolf F. 
Published: October 2006

SummaryLantibiotic and non‐lantibiotic bacteriocins are synthesized as precursor peptides containing N‐terminal extensions (leader peptides) which are cleaved off during maturation. Most non‐lantibiotics and also some lantibiotics have leader peptides of the so‐ called double‐glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues In positions ‐1 and 2. The double‐glycine‐type leader peptides are unrelated to the N‐terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP‐binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N‐terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.
Entry of Listeria monocytogenes into hepatocytes requires expression of InIB, a surface protein of the internalin multigene family
Authors: Dramsi Shaynoor , Biswas Indranil , Maguin Emmanuelle , Braun Laurence , Mastroeni Pietro , Cossart Pascale 
Published: October 2006

SummaryThe intracellular bacterium Listeria monocytogenes can invade several types of normally non‐phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800‐amino‐acid protein, and inIB, which encodes a 630‐amino‐acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB In invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.
Activation of a temporally regulated Caulobacter promoter by upstream and downstream sequence elements
Authors: Marques Marills V. , Gober James W. 
Published: October 2006

SummaryThe flagellar genes of Caulobacter crescentus are expressed under cell‐cycle control. Expression is regulated by both flagellar assembly cues and cell‐cycle events. In this paper we define the sequences required for the expression of the fIgF operon, a new class of σ54 flagellar promoter. This promoter type is expressed in the middle portion of the cell cycle and regulates the expression of basal‐body genes. DNase I footprinting and mutagenesis demonstrates that an integration host factor (IHF)‐binding site is required for maximal levels of transcription of the fIgF promoter. In addition to containing a conventional upstream enhancer element (RE‐1), this promoter is unusual in that it also requires sequences (element RE‐2) immediately downstream of the transcriptional start site for maximal levels of gene expression. Cell‐cycle experiments indicate that RE‐1 and RE‐2 contribute equally to the regulation of temporal transcription. The presence of two intact elements in the promoter results in a fourfold increase in promoter activity compared with a promoter containing only one intact element, suggesting that these two elements may function synergistically to activate transcription.
A second ABC transporter is involved in oleandomycin resistance and its secretion by Streptomyces antibioticus
Authors: Olano Carlos , Rodriguez Ana Maria , Méndez Carmen , Salas José A. 
Published: October 2006

SummaryA 3.2 kb Sstl‐Sphl DNA fragment of Streptomyces antibioticus, an oleandomycin producer, conferring resistance to oleandomycin was sequenced and found to contain an open reading frame of 1710 bp (oleB). Its deduced gene product (OleB) showed a high degree of similarity with other proteins belonging to the ABC‐transporter superfamily including the gene product of another oleandomycin‐resistance gene (OleC). The OleB protein contains two ATP‐binding domains, each of approximately 200 amino acids in length, and no hydrophobic transmembrane regions. Functional analysis of the oleB gene was carried out by deleting specific regions of the gene and assaying for oleandomycin resistance. These experiments showed that either the first or the second half of the gene containing only one ATP‐binding domain was sufficient to confer resistance to oleandomycin. The gene oleB was expressed in Escherichia coli fused to a maltose‐binding protein (MBP) using the pMal‐c2 vector. The MBP‐OleB hybrid protein was purified by affinity chromatography on an amylose resin and polyclonal antibodies were raised against the fusion protein. These were used to monitor the biosynthesis and physical location of OleB during growth. By Western analysis, the OleB protein was detected both in the soluble and in the membrane fraction and its synthesis paralleled oleandomycin biosynthesis. It was also shown that a Streptomyces albus strain, containing both a glycosyltransferase (OleD) able to inactivate oleandomycin and the OleB protein, was capable of glycosylating oleandomycin and secreting the inactive glycosylated molecule. It is proposed that OleB constitutes the secretion system by which oleandomycin or its inactive glycosylated form could be secreted by S. antibioticus.
Effects of upstream deletions on light‐ and oxygen‐regulated bacterio‐opsin gene expression in Halobacterium halobium
Authors: Gropp Felix , Gropp Roswitha , Betlach Mary C. 
Published: October 2006

SummaryThe bacterio‐opsin gene (bop) of Halobacterium halobium is located within a cluster with three other genes. Growth conditions of high light intensity and low oxygen tension induce bop gene cluster expression. To identify putative regulatory factor binding sites upstream of the bop gene, we have compared sequences upstream of the bop gene with the corresponding sequences from two other genes in the bop gene cluster. Conserved sequence motifs were observed which may mediate the effect of high light intensity and/or low oxygen tension on bop gene expression. Based on these motifs, a set of mutants was constructed which contained deletions upstream of the bop gene. These constructs were tested in a host strain where bop gene expression is independent of oxygen regulation and in another strain where it is regulated by oxygen and light. The minimal upstream sequence required for both light‐ and oxygen‐regulated bop gene expression was determined to be 54 bp.
Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase‐induced strand separation of double helical DNA
Authors: DeHaseth Pieter L. , Helmann John D. 
Published: October 2006

Summary Escherichia coli RNA polymerase is able to site‐specifically melt 12 bp of promoter DNA at temperatures far below those normally associated with DNA melting. Here we consider several models to explain how RNA polymerase destabilizes duplex DNA. One popular model proposes that upon binding to the promoter, RNA polymerase untwists the spacer DNA between the –10 and –35 regions, which results in a destabilization of the –10 region at a TA base step where melting initiates. Promoter untwisting may result, in part, from extensive wrapping of the DNA around RNA polymerase. Formation of the strand‐separated open complex appears to be facilitated by specific protein‐DNA interactions which occur predominantly on the non‐template strand. Recent evidence suggests that these include important contacts with Sigma factor region 2.3, which we propose binds the displaced single strand of DNA.
The PTR family: a new group of peptide transporters
Authors: Steiner Henry‐York , Naider Fred , Becker Jeffrey M. 
Published: October 2006

SummaryThe transport of peptides into cells is a well‐documented biological phenomenon which is accomplished by specific, energy‐dependent transporters found in a number of organisms as diverse as bacteria and humans. Until recently, the majority of peptide transporters cloned and characterized were found to be proteins of the ATP‐binding cassette (ABC) family. We report the identification of a new family of peptide transporters, which we call the PTR family. This group of proteins, distinct from the ABC‐type peptide transporters, was uncovered by sequence analyses of a number of recently discovered peptide transport proteins. Alignment of these proteins demonstrated a high number of identical and similar residues and identified conserved glycosylation and phosphorylation sites, as well as a structural motif unique to this group of proteins. Cluster analysis among the proteins indicated these sequences were indeed related and could be further divided into two subfamilies. A phylogenetic analysis of these new peptide transport sequences, compared to over 50 other peptide and membrane‐bound transporters, showed that these proteins comprise a distinct, separate group of proteins.
Molecular mechanisms and implications for infection of lipopolysaccharide variation in Neisseria
Authors: van Putten Jos p. M. , Robertson Brian D. 
Published: October 2006

SummaryThe lipopolysaccharides of the pathogenic Neisseria species are subject to structural variation owing to a combination of intrinsic changes in lipopolysaccharide (LPS) biosynthesis and external modification of the LPS molecule with sialic acid. This variation appears to control bacterial behaviour by altering their ability to interact with human cells and to evade host Immune defences. This interconversion of LPS phenotypes, which is also observed during the natural infection, is probably due to environmental regulation of LPS biosynthesis superimposed on spontaneous changes in the DNA of distinct LPS loci. LPS variation may be a common strategy of mucosal pathogens to colonize and persist within the human host.
Negative control of fae (K88) expression by the‘global’regulator Lrp is modulated by the‘local’regulator FaeA and affected by DNA methylation
Authors: Huisman Tako T. , de Graaf Frits K. 
Published: October 2006

SummaryExpression of the K88 (fae) operon is negatively controlled by the co‐operative binding of Lrp and FaeA to the fae regulatory region and is dependent on the methylation status of three GATC sites present in this region. In this paper, we describe the binding of Lrp to a T‐rich DNA helix between GATC site I and site II. FaeA stabilized and modified the Lrp binding, thereby extending the Lrp footprint over GATC site I and site III. Methylation of GATC site I prevented the binding of Lrp/FaeA at this site and appeared to be essential for the cells, since mutation of this site into GTTC resulted in a lethal overproduction of K88 fimbriae. Methylation of GATC site II and site III reduced the stability of Lrp/FaeA binding. Moreover, methylation of GATC site III stimulated faeB promoter activity. The plasmid population in cells harbouring multiple copies of a K88 plasmid consisted of two differentially methylated forms. Form A plasmids with a methylated GATC site I and site III and a non‐methylated site II (+,‐,+) represented 20% of the population and were responsible for high‐level expression. Form B plasmids with a methylated GATC site I and a non‐methylated site II and site III (+,‐,‐) represented 80% of the population and were responsible for low‐level expression. Apparently, K88 fimbriae expression in vivo is balanced at its maximal possible level by modulation of the methylation status of GATC site III. The ratio (1:4) between these populations is stabilized by a constitutive synthesis of FaeA resulting from the presence of an IS1 insertion upstream of faeA. This IS1 insertion separates the faeA promoter from the FaeB‐binding sites, thereby neutralizing the control by FaeB activity on expression of FaeA. Instead, faeA transcription is stimulated by binding of FaeA to the faeA promoter region.
Bacillus licheniformis bacitracin‐resistance ABC transporter: relationship to mammalian multidrug resistance
Authors: Podlesek Zdravko , Comino Aleksandra , Herzog‐Velikonja Blagajana , Žgur‐Bertok Darja , Komel Radovan , Grabnar Miklavž 
Published: October 2006

SummaryThe nucleotide sequence of the Bacillus licheniformis bacitracin‐resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP‐binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro‐phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram‐negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide‐binding sites, BcrA protein and mammalian multidrug transporter or P‐glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide‐binding protein to determine the Importance of various regions in transport.
rpsL+: a dominant selectable marker for gene replacement in mycobacteria
Authors: Sander Peter , Meier Albrecht , Böttger Erik C. 
Published: October 2006

SummaryMolecular genetic manipulations in mycobacteria would benefit from procedures which efficiently select for double‐crossover events by homologous recombination. Here we describe a vector‐host system for gene replacement in mycobacteria, the utility of which was investigated using functional inactivation of the pyrF gene in Mycobacterium smegmatis as a model. This system is based on the expression of the wild‐type rpsL gene coding for ribosomal protein S12 in a streptomycin‐resistant host. Owing to the absence of a mycobacterial origin the plasmids are unable to replicate autonomously in mycobacteria. The first selection for maintenance of cloned sequences is conferred by the kanamycin‐resistance gene. The second simultaneous selection by streptomycin is against maintenance of cloned sequences which contain the gene encoding the streptomycin‐sensitive allele of the rpsL gene. By placing the gene for positive selection and that used for negative selection within and outside the target gene of interest, respectively, gene replacement is obtained. A one‐step double selection procedure provides a means to distinguish strictly between gene replacement by double crossover versus homologous recombination by single crossover events. The system should have considerable potential for genera or species where single‐crossover events or even illegitimate recombination are the predominant recombination mechanisms. It should also be of wide use for the construction of mutants without a selectable phenotype.
Molecular dissection of PapD interaction with PapG reveals two chaperone‐binding sites
Authors: Xu Zheng , Jones C. Hal , Haslam David , Pinkner Jerome S. , Dodson Karen , Kihlberg Jan , Hultgren Scott J. 
Published: October 2006

SummaryP pili are composite adhesive fibres that allow uropathogenic Escherichia coli to gain a foothold in the host by binding to receptors present on the uroepithalium via the adhesin PapG. The assembly of P pili requires a periplasmic chaperone, PapD, that has an immunoglobulin‐like three‐dimensional structure. PapD‐subunit complex formation involves a conserved anchoring mechanism in the chaperone cleft and a‘molecular zippering’to the extreme C‐terminus of pilus subunits. A chaperone‐binding assay was developed using fusions of the C‐terminus of PapG to maltose‐binding protein (MBP/G fusions) to investigate whether chaperone‐subunit complex formation requires additional interactions. PapD bound strongly to an MBP/G fusion containing the C‐terminal 140 amino acids of PapG (MBP/G175‐314) but only weakly to the MBP/G234‐314 fusion containing 81 C‐terminal residues, arguing that the region between residues 175‐234 contains additional information that is required for strong PapD‐PapG interactions. PapD was shown to interact with a PapG C‐terminal truncate containing residues 1‐198 but not a truncate containing residues 1‐145, suggesting the presence of a second, independent PapD interactive site. Four peptides overlapping the second site region were tested for binding to PapD in vitro to further delineate this motif. Only one of the peptides synthesized was recognized by PapD. The MBP/G fusion containing both binding sites formed a tight complex with PapD in vivo and inhibited pilus assembly by preventing chaperone‐subunit complex formation.
Treponema pallidum and the quest for outer membrane proteins
Authors: Radolf Justin D. 
Published: October 2006

Summary Treponema pallidum, the syphilis spirochaete, has a remarkable ability to evade the humoral and cellular responses it elicits in infected hosts. Although formerly attributed to the presence of an outer coat comprised of serum proteins and/or mucopolysaccharides, current evidence indicates that the immuno‐evasiveness of this bacterium is largely the result of its unusual molecular architecture. Based upon a combination of molecular, biochemical, and ultrastructural data, it is now believed that the T. pallidum outer membrane (OM) contains a paucity of poorly immunogenic transmembrane proteins (‘rare outer membrane proteins’) and that its highly immunogentc proteins are lipoproteins anchored predominantly to the periplasmic leaflet of the cytoplasmic membrane. The presence in the T. pallidum OM of a limited number of transmembrane proteins has profound implications for understanding syphilis pathogenesis as well as treponemal physiology. Two major strategies for molecular characterization of rare outer membrane proteins have evolved. The first involves the identification of candidate OM proteins as fusions with Escherichia coli alkaline phosphatase. The second involves the characterization of candidate OM proteins identified in outer membranes isolated from virulent T. pallidum. Criteria to define candidate OM proteins and for definitive identification of rare OM proteins are proposed as a guide for future studies.
The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli
Authors: Ramseier Tom M. , Bledig Stefan , Michotey Valerie , Feghali Rita , Jr Saier Milton H. 
Published: October 2006

SummaryThe Escherichia coli fructose repressor, FruR, is known to regulate expression of several genes concerned with carbon utilization. Using a previously derived consensus sequence for FruR binding, additional potential operators were identified and tested for FruR binding in DNA band migration retardation assays. Operators in the control regions of operons concerned with carbon metabolism bound FruR, while those in operons not concerned with carbon metabolism did not. In vivo assays with transcriptional lacZ fusions showed that FruR controls the expression of FruR operator‐containing genes encoding key enzymes of virtually every major pathway of carbon metabolism. Moreover, a fruR null mutation altered the rates of utilization of at least 36 carbon sources. In general, oxidation rates for glycolytic substances were enhanced while those for gluconeogenic substances were depressed. Alignment of FruR operators revealed that the consensus sequence for FruR binding is the same for operons that are activated and repressed by FruR and permitted formulation of a revised FruR‐binding consensus sequence. The reported observations indicate that FruR modulates the direction of carbon flow by transcriptional activation of genes encoding enzymes concerned with oxidative and gluconeogenic carbon flow and by repression of those concerned with fermentative carbon flow.
The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence
Authors: Dai Zhihao , Sirard Jean‐Claude , Mock Michèle , Koehler Theresa M. 
Published: October 2006

Summary Bacillus anthracis plasmid pXO1 carries the structural genes for the three anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag (protective antigen). Expression of the toxin genes by B. anthracis is enhanced during growth under elevated levels of CO2. This CO2 effect is observed only in the presence of another pXO1 gene, atxA, which encodes a trans‐activator of anthrax toxin synthesis. Here we show that transcription of atx A does not appear to differ in cells grown in 5% CO2 compared with cells grown in air. Using a new efficient method for gene replacement in B. anthracis, we constructed an atx A‐null mutant in which the atx A‐coding sequence on pXO1 is replaced with an ωkm‐2 cassette. Transcription of all three toxin genes is decreased in the absence of atx A. The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5′ ends mapping to P1 are decreased in the atx A‐null mutant. Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the Pi site is sufficient for atx A‐mediated activation of this transcript. The cya and lef genes each have one apparent start site for transcription. Transcripts with 5′ ends mapping to these sites are not detected in the afxA‐null mutant. The atx A‐null mutant is avirulent in mice. Moreover, the antibody response to all three toxin proteins is decreased significantly in atx A‐null mutant‐infected mice. These data suggest that the atx A gene product also regulates toxin gene expression during infection.