bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation of Pseudomonas aeruginosa into the alginate‐producing form: inactivation of mucB causes conversion to mucoidy
Authors: Martin D. W. , Schurr M. J. , Mudd M. H. , Deretic V.
Published: October 2006

Mucoidy in Pseudomonas aeruginosa is a critical virulence factor associated with chronic respiratory infections in cystic fibrosis. A cluster of three tightly linked genes. algU, mucA and mucB located at 67.5 min, controls development of mucoid phenotype. This locus is allelic with a group of mutations (muc) associated with conversion into constitutively mucoid forms. One of the genes previously characterized in this region, algU, is absolutely required for the transcriptional activation of algD, a critical event in the establishment of mucoidy. AlgU is homologous to the alternative sigma factor σ;H (Spo0H) controlling sporulation and competence in Bacillus. Two genes downstream of algU, mucA and mucB were further characterized in this study. Previous complementation studies have demonstrated that mucA is required for suppression of mucoidy in the muc‐2 strain PAO568. In this work, complementation analysis indicated that, in addition. mucB was required for suppression of mucoidy in the muc‐25 strain PAO581, and for enhanced complementation of the muc‐2 mutation in PAO568. The complete nucleotide sequence of mucA and mucH was determined. Insertional inactivation of mucB on the chromosome of the standard genetic strain PAO resulted in mucoid phenotype, and in a strong transcriptional activation of algD. Thus, a loss of mucB function is sufficient to cause conversion of P. aeruginosa into the mucoid phenotype. Since the algU‐mucA‐mucB region is a general site where muc mutations have been mapped, it is likely that mucB participates in the emergence of mucoid forms. Both mucA and mucB play a regulatory role in concert with the sigma‐like factor AlgU; all three genes, along with signal transduction and histone‐like elements, control differentiation of P. aeruginosa into the mucoid phenotype.
The Myf fibrillae of Yersinia enterocolitica
Authors: Iriarte Maite , Vanooteghem Jean‐Claude , Delor Isabelle , Diaz Ramon , Knutton Stuart , Cornelis Guy R.
Published: October 2006

The Myf antigen produced by Yersinia enterocolitica appeared as a proteic polymer composed of 21 kDa subunits. By transposon mutagenesis we isolated Myf‐defective mutants. Those allowed us to clone and sequence a 4.4 kb chromosomal locus involved in Myf production. This region was found to contain three genes that we called myfA, myfB and myfC. Genes myfB and myfC encode an assembly machine related to those involved in the synthesis of many fimbriae; MyfB, the putative chaperone, possesses the consensus residues of the PapD family and MyfC encodes a putative outer‐membrane protein. MyfA, the major subunit, was found to be 44% identical to the pH 6 antigen of Y.pestis. Myf is thus the K enterocolitica counterpart of this antigen, but it is by far not so well conserved as the other virulence determinants such as the Yops, suggesting that Myf and pH 6 antigen do not necessarily play the same role in Y. enterocolitica and Y. pestis. The study of the prevalence of myfA in various species of Yersinia reveaied that, like the yst enterotoxin gene, its presence is restricted to the pathogenic serotypes of Y. enterocolitica. By immuno‐gold labelling, Myf appeared as a layer of extracellular material extending locally 2μm from the bacterial surface, indicative of a fibrillar structure.
Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem‐loops in the mRNA leader
Authors: Oliveira Caria C. , van den Heuvel Joop J. , McCarthy John E. G.
Published: October 2006

A new modular gene‐expression system for use in studies of translational control in Saccharomyces cerevisiae was constructed. A GAL::PGK fusion promoter (GPF) directed the inducible synthesis of mRNAs initiated at a single major site. A series of leader sequences were tested in combination with each of two reporter genes (encoding chloramphenicol acetyl transferase (cat) and luciferase (luc)). Stem‐loop structures of three different sizes and predicted stabilities were inserted into each of two different unique restriction sites in the leader. After correction for relative mRNA abundance, a stem‐loop of predicted stability equivalent to approximately −18kcal mol−1 inhibited translation by up to 89%. The degree of inhibition exerted by the other stem‐loops correlated positively with their predicted stabilities. Combinations of two stem‐l oops at different sites yielded an inhibitory effect greater than that of either individual stem‐loop alone. Similar inhibitory effects were observed with both reporter genes. However, inhibition of translation, particularly of the cat gene, was more effective when the stem‐loop was positioned close to the start codon rather than at the 5′ end of the leader. The observed results reflect an important form of post‐transcriptional control that is expected to act on a large number of genes in yeast.
NAD+ binding to the Escherichia coli K+‐uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport
Authors: Schlosser Andreas , Hamann Angela , Bossemeyer Dirk , Schneider Erwin , Bakker Evert P.
Published: October 2006

The nucleotide sequence of trkA, a gene encoding a surface component of the constitutive K+‐uptake systems TrkG and TrkH from Escherichia coli, was determined. The structure of the TrkA protein deduced from the nucleotide sequence accords with the view that TrkA is peripherally bound to the inner side of the cytoplasmic membrane. Analysis by a dot matrix revealed that TrkA is composed of similar halves. The M‐terminal part of each TrkA half (residues 1–130 and 234–355, respectively) is similar to the complete NAD+‐binding domain of NAD+‐dependent dehydrogenases. The C‐terminal part of each TrkA half (residues 131–233 and 357–458, respectively) aligns with the first 100 residues of the catalytic domain of glyceraldehyde‐3‐phosphate dehydrogenase. Strong u.v. illumination at 252 nm led to cross‐linking of NAD+ or NADH, but not of ATP to the isolated TrkA protein.
Identification of a new nuclear gene (CEM1) encoding a protein homologous to a β‐keto‐acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae
Authors: Harington A. , Herbert C. J. , Tung B. , Getz G. S. , Slonimski P. P.
Published: October 2006

We have analysed a new gene, CEM1, from Saccharomyces cerevisiae. Inactivation of this gene leads to a respiratory‐deficient phenotype. The deduced protein sequence shows strong similarities with β‐keto‐acyl synthases or condensing enzymes. Typically, enzymes of this class are involved in the synthesis of fatty acids or similar molecules. An analysis of the mitochondrial lipids and fatty acids shows no major difference between the wild type and deleted strains. Implying that the CBM1 gene product is not involved in the synthesis of the bulk fatty acids. Thus it Is possible that the CEM1 protein is involved in the synthesis of a specialized molecule, probably related to a fatty acid, which is essential for mitochondrial respiration.
Escherichia coli endoribonuclease RNase E: autoregulation of expression and site‐specific cleavage of mRNA
Authors: Mudd Elisabeth A. , Higgins Christopher F.
Published: October 2006

Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo. Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180kDa. However, proteolytic fragments as small as 70kDa, which can arise during purification, also exhibit RNase E activity, in vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites. RNase E cleaves rne mRNA and autoregulates the expression of the rne gene. In addition we demonstrate RNase E‐dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate‐determining for degradation and reported to be cieaved by RNase K. Our data are consistent with RNase K being a proteolytic fragment of RNase E.
An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase
Authors: Frenken Leon G.J. , Bos J. Wil , Visser Chris , Müller Wally , Tommassen Jan , Verrips C. Theo
Published: October 2006

Pseudomonas glumae PG1 is able to secrete lipase into the extracellular medium. The lipase is produced as a precursor protein, with an N‐terminal signal sequence. A second open reading frame (ORF) was found immediately downstream of the lipase structural gene, lip A, a situation found for the lipases of some other Pseudomonas species. Inactivation of this ORF resulted in a lipase‐negative phenotype, indicating its importance in the production of active extracellular lipase. The ORF, lipB, potentially encodes a protein of 353‐amtno‐acid residues, having a hydrophobic N‐terminal (amino acids 1 to 90) and a hydrophilic C‐terminal part. As a first step in determining the role of LipB, its subcellular location was determined. The protein was found to fractionate with the inner membranes. The expression of fusions of lipB fragments with phoA revealed an Nin–Cout topology for the LipB protein, which was confirmed by protease accessibility studies on EDTA‐permeabilized cells and on inverted inner membrane vesicles. These and other results indicate that most of the LipB polypeptide is located in the periplasm and anchored to the inner membrane by an an N‐terminal transmembrane helix, located between amino acids 19 and 40.
Role of the lipB gene product in the folding of the secreted lipase of Pseudomonas glumae
Authors: Frenken Leon G. J. , de Groot Arjan , Tommassen Jan , Verrips C. Theo
Published: October 2006

The LipB protein of Pseudomonas glumae is essential for the production of active extracellular lipase encoded by the lipA gene. When lipase is overproduced in P. glumae in the absence of a functional lipB gene, the enzyme accumulates intracellularly in an inactive conformation. Heterologous expression of the lipase in Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli indicated that LipB is not directly involved in the trans location of the lipase across the inner or outer membrane. However, the presence of LipB was essential for obtaining active lipase and had a profound influence on the stability of the protein to proteolytic degradation. Inactive iipase, produced in the absence of LipB could be activated in vitro by unfolding and refolding, which demonstrates that LipB activity is not responsible for an essential covalent modification of the enzyme. We propose that LipB is a lipase‐specific foldase. Furthermore, proper folding of the lipase in the periplasm appears to be essential for Xcp‐mediated translocation across the outer membrane.
Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites
Authors: Mojica F. J. M. , Juez G. , Rodriguez‐Valera F.
Published: October 2006

Two genomic sequences from the halophilic archaeon Haloferax mediterranei, where we had found PstI restriction‐pattern modifications depending on the salinity of the growth medium, have been studied. A markedly salt‐dependent differential expression has been detected in the nearby regions. Two of the open reading frames characterized correspond to two of the differentially expressed transcripts. In both cases the PstI sites were included in purine–pyrimidine alternancies suggestive of Z‐DNA structures and located in non–coding regions with frequent repetitive motifs. A long alternating adenine‐thymine tract also appears in the upstream regions of one of these open reading frames. A possible role of local DNA configuration in osmoregulation in this organism is discussed.
Isolation of a putative fimbrial adhesin from Bordetella pertussis and the identification of its gene
Authors: Willems Rob J. L. , Geuijen Cecile , van der Heide Han G. J. , Matheson Mary , Robinson Andrew , Versluis Leone F. , Ebberink Rob , Theelen Joop , Mooi Frits R.
Published: October 2006

We report the purification of a minor Bordetella pertussis fimbrial subunit, designated FimD, and the identification of its gene (fimD.) FimD could be purified from the bulk of major fimbrial subunits by exploiting the fact that major subunit‐subunit interactions are more stable in the presence of SDS than minor‐major subunit interactions. To locate the gene for FimD, internal peptides of FimD were generated, purified and sequenced. Subsequently, an oligonucleotide probe, based on the primary sequence of one peptide, was used to clone fimD. The primary structure of FimD, derived from the DNA sequence of its gene, showed homology with a number of fimbrial adhesins. Most pronounced homology was observed with MrkD, a fimbrial adhesin derived from Klebsieila pneumoniae. These observations suggest that FimD may represent a B. pertussis fimbrial adhesin. With a fimD‐specific probe we detected the presence of a fimD homologue in Bordetella parapertussis and Bordetella bron‐chiseptica but not in Bordetella avium. Cloning and sequencing revealed that the B. parapertussis and B. bronchiseptica fimD product differed from the B. pertussis fimD product in 20 and 1 amino acid residues, respectively. Since B. bronchiseptica is normally not a human pathogen, but causes respiratory disease in a wide range of non‐human mammalian species, this may suggest that FimD recognizes a receptor that is well conserved in mammalian species. An in‐frame deletion in fimD completely abolished FimD expression and also affected the expression of the major subunits Fim2 and Fim3 suggesting that, in contrast to other adhesins that are minor components of fimbriae, FimD is required for formation of the fimbrial structure.
Solution of the ribosome riddle: how the ribosome selects the correct aminoacyl‐tRNA out of 41 similar contestants
Authors: Nierhaus Knud H.
Published: October 2006

Three tRNA binding sites, the A, P and E sites, have been demonstrated on ribosomes of bacterial, archaebacterial and eukaryotic origin. In all these cases the first and the third site, the A and the E site, are allosterically coupled in the sense of a negative co‐operativity. Therefore, the allosteric three‐site model seems to be a generally valid description of the ribosomal elongation phase, where in a cycle of reactions the nascent peptide chain is prolonged by one amino acid. The molecular concept of the allosteric three‐site model explains the astonishing ability of the ribosome to select the correct substrate out of a large number of very similar substrates, and it provides a framework within which the mechanisms of the elongation factors could be understood.

Molecular recognition: the special case of the ribosomal selection of the correct aminoacyl‐tRNA
Regulation of the Escherichia coli heat‐shock response
Authors: Bukau Bernd
Published: October 2006

Steady‐state‐ and stress‐induced expression of Escherichia coli heat‐shock genes is regulated at the transcriptional level through controls of concentration and activity of the positive regulator, the heat‐shock promoter‐specific subunit of RNA polymerase, σ;32. Central to these controls are functions of the DnaK, DnaJ, GrpE heat‐shock proteins as negative modulators that mediate degradation as well as repression of activity and, in some conditions, of synthesis of σ32. DnaJ has a key role in modulation since it binds σ32 and, jointly with DnaK and GrpE, represses its activity. Furthermore, DnaJ is capable of binding heat‐damaged proteins, targeting DnaK and GrpE to these substrates, and thereby mediating DnaK‐, DnaJ‐, GrpE‐dependent repair. It is proposed that one important signal transduction pathway that converts stress to a heat‐shock response relies on the sequestering of DnaJ through binding to damaged proteins which derepresses and stabilizes σ32. Damage repair ameliorates the inducing signal and frees DnaJ, DnaK, GrpE to shut off the heat‐shock response.
The interaction between coumarin drugs and DNA gyrase
Authors: Maxwell Anthony
Published: October 2006

The coumarin group of antibiotics have as their target the bacterial enzyme DNA gyrase. The drugs bind to the B subunit of gyrase and inhibit DNA supercoiling by blocking the ATPase activity. Recent data show that the binding site for the drugs lies within the N‐terminal part of the B protein, and individual amino acids involved in coumarin interaction are being identified. The mode of inhibition of the gyrase ATPase reaction by coumarins is unlikely to be simple competitive inhibition, and the drugs may act by stabilizing a conformation of the enzyme with low affinity for ATP.
Bacterial proteins binding to the mammalian extracellular matrix
Authors: Westerlund B. , Korhonen T. K.
Published: October 2006

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best‐characterized bacterial proteins active in these interactions are the mycobacterial fibronectin‐binding proteins, the fibronectin‐ and the collagen‐binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A‐protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein‐protein or on a protein‐carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.
AmpG, a signal transducer in chromosomal β‐lactamase induction
Authors: Lindquist Susanne , Weston‐Hafer Kathleen , Schmidt Herbert , Pul Christian , Korfmann Gisela , Erickson Jay , Sanders Christine , Martin Hans H. , Normark Staffan
Published: October 2006

The chromosomal ampCβ‐lactamase in Citrobacter freundii and Enterobacter cloacae is inducible by β‐lactam antibiotics. When an inducible ampC gene is introduced on a plasmid into Escherichia coli together with its transcriptional regulator ampR, the plasmid‐borne β‐lactamase is still inducible. We have isolated mutants, containing alterations in a novel E. coli gene, ampG, in which a cloned C. freundii ampC gene is unable to respond to β‐lactam inducers. The ampG gene was cloned, sequenced and mapped to minute 9.6 on the E. coli chromosome. The deduced amino acid sequence predicted AmpG to be a 53kDa, trans‐membrane protein, which we propose acts as a signal transducer or permease in the β‐lactamase induction system. Immediately upstream of ampG there is another 579‐base‐pair‐long open reading frame (ORF) encoding a putative lipoprotein shown to be non‐essential for β‐lactamase induction. We have found that ampG and this ORF form an operon, whose promoter is located in front of the ORF. Located closely upstream of the putative promoter is the morphogene bolA, which is transcribed in the opposite orientation. However, using transcription fusions, we have found that the ampG transcription is not regulated by bolA. In addition, we show that transcription is probably not regulated by either the starvation specific sigma factor RpoS, which controls bolA, or by AmpD the negative regulator for ampC transcription.
Formation of several bacterial c‐type cytochromes requires a novel membrane‐anchored protein that faces the periplasm
Authors: Ritz D. , Bott M. , Hennecke H.
Published: October 2006

We report here the discovery of a novel bacterial gene (cycH) whose product is involved in the biogenesis of most of the cellular cytochromes c. The cycH gene was detected in the course of characterizing a cytochrome oxidase‐deficient Bradyrhizobium japonicum Tn5 mutant (strain CO×3) in which the transposon insertion disrupted cycH. Ali of the c‐type cytochromes detectable in aerobically grown B. Japonicum wild‐type cells were absent in the C0X3 mutant, with the exception of cytochrome c1. A secondary phenotypic effect was the spectroscopic absence of the aa3‐type cytochrome c oxidase. The nucleotide sequence of the cloned wild‐type cycH gene predicted a membrane‐bound 369‐amino‐acid protein with an Mr of 39727. Results from studies on its membrane topology suggested that approximately 110 N‐terminal amino acids are involved in anchoring the protein in the membrane, whereas the remaining two‐thirds of the protein are exposed to the periplasm. We postulate that the CycH protein plays an essential role in an as yet unidentified periplasmic step in the biogenesis of holocytochromes c, except that of cytochrome c1.
Phosphorylation of Spo0A activates its stimulation of in vitro transcription from the Bacillus subtilis spollG operon
Authors: Bird T. H. , Grimsiey J. K. , Hoch J. A. , Spiegelman G. B.
Published: October 2006

The spollG operon of Bacillus subtilis codes for a sporulation‐specjfic sigma factor, σ;E, In vivo expression of the spollG promoter is activated shortly after the onset of sporulation and is dependent on kinA, spo0F, spo0B and spo0A genes. The products of these genes have been shown to participate in a phosphorelay reaction in vitro, culminating in phosphorylation of the transcription factor, Spo0A. The effect of Spo0A phosphorylation on in vitro transcription from the spollG promoter was determined. Aliquots from phos‐phorelay reactions enhanced spollG promoter activity 10‐fold in transcription assays and stimulation of transcription was dependent on Spo0A phosphorylation. Our results provide biochemical evidence that Spo0A and the phosphoreiay form a signal transduction pathway which activates spoil gene expression in development.
SyrD is required for syringomycin production by Pseudomonas syringae pathovar syringae and is related to a family of ATP‐binding secretion proteins
Authors: Quigley Neil B. , Mo Yin‐Yuan , Gross Dennis C.
Published: October 2006

The syrD gene of Pseudomonas syringae pathovar syringae strain B301D‐R was characterized and sequenced. The syrD open reading frame is 1695bp long and encodes a predicted protein, SyrD, of =63kDa. Database searches revealed that SyrD shares a high degree of similarity with the ATP‐binding cassette (ABC) superfamily of transporter proteins which are responsible for specific nutrient uptake and for secretion of certain cellular products in prokaryotes, and for multiple drug resistance in mammals. The amino acid sequence homology between SyrD and the ABC proteins was greatest at the conserved residues which constitute the ATP‐binding cassette of these proteins; these residues lie in the hydrophilic C‐terminal half of SyrD. The N‐terminus of SyrD is predicted to be hydrophobic and to contain six membrane‐spanning α‐helices. syrD mutants of strain B301D‐R were significantly less virulent than other syr mutants, were deficient in four large polypeptides thought to be components of a syringomycin synthetase complex, and showed reduced expression of a syrB‐lacZ reporter gene fusion in trans. It is proposed that SyrD is a cyto‐plasmic membrane protein that functions as an ATP‐driven efflux pump for the secretion of syringomycin.
The virD4 gene is required for virulence while virD3 and orf5 are not required for virulence of Agrobacterium tumefaciens
Authors: Lin Tzu‐Se , Kado Clarence I.
Published: October 2006

The virD operon of the resident Ti plasmid of Agrobacterium tumefaciens contains loci involved in T‐DNA processing and undefined virulence functions. Nucleotide sequence of the entire virD operon of pTiC58 revealed similarities to the virD operon of the root‐inducing plasmid pRiA4b and to that of the octopine‐type plasmid pTiA6NC. However, comparative sequence data show that virD of pTiC58 is more akin to that of the pRiA4b than to that of the pTiA6NC. T7f10::virD gene fusions were used to generate polypeptides that confirm the presence of four open reading frames virD1, virD2, virD3, and virD4 within virD which have a coding capacity for proteins of 16.1, 49.5, 72.6, and 73.5 kDa, respectively. virD3 therefore encodes a polypeptide 3.4 times larger (72.6 versus 21.3 kDa) than that encoded by virD3 of octopine Ti plasmids. Non‐polar virD4 mutants could not be complemented by a distant homologue, TraG protein of plasmid RP4. An independently regulated fifth ORF (orf5) is located immediately downstream of 3′ end of virD4 and encodes a polypeptide of 97.4 kDa. The expression of orf5 is dependent on its own promoter and is independent of acetosyringone induction in A. tumefaciens. Recently, it has been shown that virD3 of octopine Ri or Ti plasmids is not required for virulence. In this report, we confirm and extend these findings on a nopaline Ti plasmid by using several virD non‐polar mutants that were tested for virulence. virD3 and orf5 non‐polar mutants showed no effect on tumorigenicity on 14 different plant species, while virD4 mutants lost their tumorigenicity completely on all these test plants. These data suggest that virD3 and orfS are not essential for virulence whereas virD4 is absolutely required on a wide range of host plants.
An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtllis
Authors: Meens Jochen , Frings EIke , Klose Michael , Freudl Roland
Published: October 2006

The translocation of secretory proteins derived from a Gram‐positive (Staphylococcus hyicus prolipase) or a Gram‐negative (Escherichia coli pre‐OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. in both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre‐prolipase signal peptide (38 amino acids in length) or the pre‐OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre‐OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram‐positive and Gram‐negative bacteria.
The maturation pathway of microcin B17, a peptide inhibitor of DNA gyrase
Authors: Yorgey Peter , Davagnino Juan , Kolter Roberto
Published: October 2006

The maturation pathway of microcin B17 (MccB17), a ribosomally synthesized peptide antibiotic which inhibits DNA gyrase, has been characterized. Synthesis of MccB17 involves several steps beginning with the translation of the MccB17 structural gene, mcbA, to yield a 69 amino acid precursor, preMccB17. PreMccB17 is then modified and folded by the action of three gene products, McbBCD, to yield proMccB17. Mutations in mcbA were isolated that permit modifications of the resulting mutant peptides, but prevent folding, suggesting that modification and folding are sequential steps. ProMccB17 is subsequently converted to MccB17 by removal of the W‐terminal 26‐amino‐acid leader by a chromosomally encoded protease. Removal of the leader resulted in aggregation of the peptide, suggesting that the leader may function to maintain peptide solubility during synthesis in the cell. Finally, polyclonal antibodies raised against MccB17 recognize both MccB17 and proMccB17, but do not recognize preMccB17. This demonstrates the dramatic structural changes that result from the modifications and has been used to distinguish intermediates in the steps of maturation.
Osmostress response of the yeast Saccharomyces
Authors: Mager Willem H. , Varela João C. S.
Published: October 2006

Exposure of yeast cells to high osmolarities leads to dehydration, collapse of ion gradients over the plasma membrane and decrease in cell viability. The response of yeast cells to high external osmolarities is designated osmostress response. It is likely that both osmoregulatory and general stress reactions are involved in this so far poorly understood process. Part of the response aims at raising the internal osmotic potential, i.e. the production of osmolytes such as glycerol, and exclusion of toxic solutes. In addition, heat‐shock proteins and trehalose are synthesized, probably to protect cellular components and to facilitate repair and recovery. Recent analyses of osmosensitive yeast mutants strongly suggest the involvement of protein kinase‐mediated signal‐transduction pathways in the maintenance of the osmotic integrity of the cell. This has stimulated interesting hypotheses as to the actual osmosensing mechanism.
Expression of the gene encoding the major bacterial nucleoid protein H‐NS is subject to transcriptional auto‐repression
Authors: Falconi Maurizio , Higgins N. Patrick , Spurio Roberto , Pon Cynthia L. , Gualerzi Claudio O.
Published: October 2006

Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at –313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild‐type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid‐log cultures and negligible in cells entering the stationary phase. The level of repression in early‐log phase was lower than in mid‐log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H‐NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H‐NS and Fis protein to transcriptional systems programmed with the same hns caf fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation get analyses revealed that H‐NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around –150. When these sites are filled by H‐NS, an additional site between approximately –20 and –65, which partly overlaps the promoter, is also occupied. Binding of H‐NS to this site is probably the ultimate cause of transcriptional auto‐repression.
Inhibition of bacteriophage Mu transposition by Mu repressor and Fis
Authors: van Drunen C. M. , van Zuylen C. , Mientjes E. J. , Goosen N. , van de Putte P.
Published: October 2006

In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini‐Mu 3–80‐fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site leads to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4bp insertion in the IAS, which changes the spacing of the repressor‐binding site, abolishes the enhancing function of the IAS but leaves the repressor‐Fis effect intact. A likely target for Fis in this regulation is a strong Fis‐binding site, which is located adjacent to the L2 transposase‐binding site. However, when this Fis‐binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non‐specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.
Deletions of Tn916‐like transposons are implicated in tetM‐mediated resistance in pathogenic Neisseria
Authors: Swartley J. S. , McAllister C. F. , Hajjeh R. A. , Heinrich D. W. , Stephens D. S.
Published: October 2006

Using the tetM‐containing conjugative transposon Tn916 as a mutagenesis tool, we identified two distinct classes of transposon insertions in the meningococcal chromosome. Class I Insertions have an intact copy of Tn916 that appears to have transposed by a novel recombinational mechanism, similar to the transposition of conjugative transposons in Gram‐positive bacteria. Class II insertions were characterized by deletions of Tn916 but preservation of the tetM determinant. In addition, we identified Class II Tn916‐like insertions in the naturally occurring 25.2 MDa tetM‐containing plasmids of both Neisseria meningitidis and Neisseria gonorrhoeae. The truncated Tn916 like insertions appeared to be present in the same site in these two plasmids; however, the deletions of the transposon were different. Plasmid sequence adjacent to the truncated transposon in the 25.2 MDa plasmids was found in a tetracycline‐sensitive N. gonorrhoeae 24.5 MDa conjugative plasmid. These data suggest that the 25.2 MDa plasmids are the result of one or a series of Class II Tn916‐like insertions into 24.5 MDa conjugative plasmids. Class II insertions of Tn916‐like transposons are implicated in the dissemination of tetM resistance in pathogenic Neisseria.
Theta replication of the lactococcal plasmid pWVO2
Authors: Kiewiet Rense , Bron Sierd , de Jonge Karel , Venema Gerard , Seegers Jos F. M. L.
Published: October 2006

pWVO2 is a 3.8 kb narrow‐host‐range plasmid from Lactococcus lactis ssp. cremoris Wg2, which does not replicate in Bacillus subtilis or Escherichia coli. Single‐stranded pWVO2 DNA was not observed in lactococcal cells, indicating that this plasmid does not replicate via a rolling‐circle mechanism. The sequence of pWVO2 neither showed the structural organization typical for rolling‐circle plasmids, nor were sequence similarities with known rolling‐circle plasmids present. By 2‐D agarose gel electrophoresis of replication intermediates, it was shown that pWVO2 replicates via a theta mechanism. This is the first proof for the existence of theta‐replicating plasmids in lactococci. The pWVO2 minimal replicon is strongly related to that of several other lactococcal plasmid replicons. It contains one open reading frame encoding the replication protein, which is preceded by a 22 bp sequence tandemly repeated three and a half times. Further upstream is another 10bp direct repeat present in an A/T‐rich sequence. This structural organization resembles that of several iteroncontaining theta‐type plasmids from E. coli. Derivatives of pWVO2 were stably maintained in L. lactis and are good candidates for the development of stable food‐grade cloning vectors for this organism.
Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg;
Authors: Glaser P. , Kunst F. , Arnaud M. , Coudart M.‐P. , Gonzales W. , Hullo M.‐F. , Ionescu M. , Lubochinsicy B. , Marcelino L. , Moszer I. , Presecan E. , Santana M. , Schneider E. , Schwelzer J. , Vertes A. , Rapoport G. , Danchin A.
Published: October 2006

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety‐two open reading frames were predicted to encode putative proteins among which only forty‐two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.
Homologous pairing proteins encoded by the Escherichia coii recE and recT genes
Authors: Kolodner Richard , Hall Sharynn D. , Luisi‐DeLuca Cynthia
Published: October 2006

Early genetic analysis of alternate recombination pathways in Escherichia coli identified the RecE recombination pathway and the required exonuclease VIII encoded by the recE gene. Observations that not ail recombination events promoted by the RecE pathway require recA suggest the existence of an additional homologous pairing protein besides RecA in E. coli. Genetic and biochemical analysis of the recE gene region indicates there are two partially overlapping genes, recE and recT, encoding at least two proteins: exoVIII and the RecT protein. Biochemical analysis has shown that the RecT protein, in combination with exoVIII, promotes homologous pairing and strand exchange in reactions containing linear duplex DNA and homologous, circular, single‐stranded DNA as substrates. This reaction occurs in the absence of any high‐energy cofactor. These two proteins, RecT and exoVIII, appear to be members of a second class of homologous pairing proteins that are required in genetic recombination and differ from the class of homologous pairing proteins that includes RecA. Members of this second class of proteins appear to include both bacteriophage‐encoded proteins and proteins from eukaryotes and their viruses.
Structure and expression of the alternative sigma factor, RpoN, in Rhodobacter capsulatus; physiological relevance of an autoactivated nifU2‐rpoN superoperon
Authors: Cullen Paul J. , Foster‐Hartnett Dawn , Gabbert Karen K. , Kranz Robert G.
Published: October 2006

The alternative sigma factor, RpoN (σ54) is responsible for recruiting core RNA polymerase to the promoters of genes required for diverse physiological functions In a variety of eubacterial species. The RpoN protein In Rhodobacter capsulatus is a putative sigma factor specific for nitrogen fixation (nif) genes. Insertional mutagenesis was used to define regions important for the function of the R. capsulatus RpoN protein. Insertions of four amino acids in the predicted helix‐turn‐helix or in the highly conserved C‐terminal eight amino acid residues (previously termed the RpoN box), and an in‐frame deletion of the glutamine‐rich M‐terminus completely inactivated the R. capsulatus RpoN protein. Two separate insertions in the second hydrophobic heptad repeat, a putative leucine zipper, resulted in a partially functional RpoN protein. Eight other linkers in the rpoN open reading frame (ORF) resulted in a completeiy or partially functional RpoN protein. The rpoN gene in R capsulatus is downstream from the nifHDKU2 genes, in a nifU2‐rpoN operon. Results of genetic experiments on the nifU2‐rpoN locus show that the rpoN gene is organized in a nifU2‐rpoN superoperon. A primary promoter directly upstream of the rpoN ORF is responsible for the initial expression of rpoN. Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predicted rpoN translational start site. This primary promoter is expressed constitutively with respect to nitrogen, and it is necessary and sufficient for growth under nitrogen‐limiting conditions typically used in the laboratory. A secondary promoter upstream of nifU2 is autoactivated by RpoN and NifA to increase the expression of rpoN, which ultimately results in higher expression of RpoN dependent genes. Moreover. rpoN expression from this secondary promoter is physiologically beneficial under certain stressful conditions, such as nitrogen‐limiting environments that contain high salt (>50mM NaCl) or low iron (<400nM FeS04).
Molecular analysis of the Erwinia chrysanthemi region containing the kdgA and zwf genes
Authors: Hugouvieux‐Cotte‐Pattat Nicole , Robert‐Baudouy Janine
Published: October 2006

The pathways of pectin and galacturonate catabolism In Erwinia chrysantheml converge to form a common intermediate, 2‐keto‐3‐deoxygluconate, which is phosphorylated to form 2‐keto‐3‐deoxy‐6‐phospho‐giuconate (KDGP) and then cleaved by the aldolase encoded by the kdgA gene. We cloned the kdgA gene of the E. chrysanthemi strain 3937 by complementing an Escherichia coli kdgA mutation, using an RP4‐derivative plasm id. Restriction mapping of the kdgA region and isolation of kdgA‐lac fusions allowed the more precise localization of the kdgA gene and determination of its transcriptional direction. The nucleotide sequence of the kdgA region indicated that the kdgA reading frame is 639 bases long, corresponding to a protein of 213 amino acids with a molecular mass of 22187 Da. Comparison of the deduced primary amino acid sequences of the E. chrysanthemi KDGP‐aldolase to the E. coli, Zymomonas mobilis and Pseudomonas putida enzymes showed that they are highly conserved. The E. chrysanthemi kdgA structural gene begins 153 bases downstream of an open reading frame that has a high homology with the zwf E. coli gene encoding giucose‐6‐phosphate dehydrogenase. The zwf gene is also linked to eda (kdgA) in E. coli and P. putida but genetic organization is different. Regulation of zwf and kdgA expression in E. chrysanthemi was analysed using lacZ fusions. The expression of zwf is independent of the growth rate, but is repressed in the presence of glucose. Induction of kdgA by pectin‐degradation products is mediated in vivo by the negative regulatory gene kdgR, which also controls all the steps of pectin degradation. However, the KdgR repressor is unable to bind in vitro to the 5′‐untranslated end of the kdgA gene.
Evidence that residues −15 to −46 of the haemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator
Authors: Kenny Brendan , Chervaux Christian , Holland I. Barry
Published: October 2006

We previously identified three well‐dispersed mutations, E978‐K, F989‐L and D1009‐R within the haemolysin A signal region, located at positions –46, –35 and –15, with respect to the C‐terminus, respectively. Each mutation reduces the efficiency of secretion two‐ to threefold leaving 30–45% of the wild‐type activity. We have constructed by in vitro manipulations double mutants of HlyA carrying all combinations of these mutations and a triple mutant carrying all three mutations. The effects on secretion were determined and the results, including residual levels of secretion with the triple mutant of only 0.6%, compared with the wild type, indicated that these residues may interact to form a single function in the wild‐type signal. To test this further, we developed a secretion competition assay in order to classify signal mutations. We demonstrated that a CIZ‐HlyA fusion protein, containing the C‐terminal 81 kDa of HlyA fused to virtually the whole LacZ protein, strongly inhibits the secretion of the wild‐type HlyA co‐expressed In the same cell. The properties of the fusion indicate that it blocks the translocator. The three mutations singly and in combinations were recombined in vitro into the 3′‐end of the hybrid gene. In every case, the presence of a mutation in the secretion signal of the hybrid protein alleviated the inhibition of secretion of the co‐expressed HiyA. All the mutations are therefore essentially recessive and we propose that they all affect an early function, probably recognition of the translocator, rather than a subsequent step involved in translocation or final release of the toxin to the medium. This would indicate that residues involved in recognition for steps
The relative contributions of transcription and translation to plasmid DNA supercoiling in Salmonella typhimurium
Authors: Spirito Flavia , Figueroa‐Bossi Nara , Bossl Lionello
Published: October 2006

Mutations affecting DNA topoisomerase I (topA) in Saimonella typhimurium were isolated and graded on the basis of their ability to reverse the effects of gyrB mutations on his operon expression. Different topA and gyrB alleles (in otherwise isogenic strains) were used to gather insights into the transcription‐dependent variability of plasmid DNA‐linking deficit in growing bacteria. This study showed that modulation of DNA supercoiling by transcription results from the action of two components: one is highly dependent on the coupling of translation to RNA‐chain elongation; and the other is unrelated to protein synthesis and entirely dependent on promoter determinants. The former greatly predominates In DNA topoisomerase I mutants (topA and topA gyrB) while the latter is the sole contributor to plasmid DNA‐linking deficit in wild‐type cells. Altogether, these data suggest that whereas translation acts by enhancing the formation of twin supercoiled domains during elongation, the promoter‐dependent effects bear no relation to the twin‐supercoiled‐domain model and are latter explained by a mechanism which responds to the binding/unwinding of template DNA by RNA polymerase.
Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosynthesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus
Authors: Darzins Aldis
Published: October 2006

The type 4 pill of Pseudomonas aeruginosa are important cell‐associated virulence factors that play a crucial role in mediating (i) bacterial adherence to, and colonization of, mucosal surfaces, (ii) a novel mode of fiagetia‐independent surface translocation known as‘twitching motility, and (iii) the initial stages of the infection process for a number of bacteriophages. A new set of loci involved in pilus biogenesis and twitching motility was identified based on the ability of DNA sequences downstream of the pilG gene to complement the non‐piliated (pil) strain, PAO6609. Sequence analysis of a 3.2 kb region directly downstream of pilG revealed the presence of three genes, which have been designated pilH, pill, and pilJ. The predicted translation product of the pilH gene (13 272 Da), like PilG, exhibits significant amino acid identity with the enteric single‐domain response regulator CheY. The putative Pili protein (19933 Da) is 28% identical to the FrzA protein, a CheW homologue of the gliding bacterium Myxococcus xanthus, and the PMJ protein (72 523 Da) is 26% identical to the enteric methyl‐accepting chemotaxis protein (MCP) Tsr. Mutants containing insertions in pill and pilJ were severely impaired in their ability to produce pili and did not translocate across solid surfaces. The pilH mutant remained capable of pilus production and twitching motility, but displayed an altered motility pattern characterized by the presence of many doughnut‐shaped swirls. Each of these pil mutants, however, produced zones that were at least as large as the parent in flagellar‐mediated swarm assays. The sequence similarities between the putative pilG, H, I and J gene products and several established chemotaxis proteins, therefore, lend strong support to the hypothesis that these proteins are part of a signal‐transduction network that controls P. aeruginosa pilus biosynthesis and twitching motility.