bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo studies disprove an obligatory role of azurin in denitrification in Pseudomonas aeruginosa and show that azu expression is under control of RpoS and ANR
Authors: Vijgenboom Erik , Busch Julie E. , Canters Gerard W.
Published: June 2009

Summary: The role of the blue copper protein azurin and cytochrome C551 as the possible electron donors to nitrite reductase in the dissimilatory nitrate reduction pathway in Pseudomonas aeruginosa have been investigated. It was shown by an in vivo approach with mutant strains of P. aeruginosa deficient in one or both of these electron-transfer proteins that cytochrome C551, but not azurin, is functional in this pathway. Expression studies demonstrated the presence of azurin in both aerobic and anaerobic cultures. A sharp increase in azurin expression was observed when cultures were shifted from exponential to stationary phase. The stationary-phase sigma factor, σs, was shown to be responsible for this induction. In addition, one of the two promoters transcribing the azu gene was regulated by the anaerobic transcriptional regulator ANR. An azurin-deficient mutant was more sensitive to hydrogen peroxide and paraquat than the wild-type P. aeruginosa. These results suggest a physiological role of azurin in stress situations like those encountered in the transition to the stationary phase.
Identification of residues in the putative TolA box which are essential for the toxicity of the endonuclease toxin colicin E9
Authors: -Schneider Carole Garinot , Penfold Christopher N. , Moore Geoffrey R. , Kleanthous Colin , James Richard
Published: June 2009

Summary: E colicins are plasmid-coded, protein antibiotics which bind to the BtuB outer membrane receptor of Escherichia coli cells and are then translocated either to the outer surface of the cytoplasmic membrane in the case of the pore-forming colicin E1, or to the cytoplasm in the case of the enzymic colicins E2-E9. Translocation has been proposed to be dependent on a putative TolA box; a pentapeptide (DGSGW) located in the N-terminal 39 residues of several Toldependent colicins. In this study, site-directed mutagenesis was used to change each of the residues of the putative TolA box of colicin E9 to alanines. In the case of the two glycine residues, the resulting mutant proteins were indistinguishable from the native colicin E9 protein in a biological assay; whereas the other three residues when mutated to alanines resulted in a complete loss of biological activity. Mutagenesis of two serine residues flanking the putative TolA box, Ser34 and Ser40, to alanines did not abolish the biological activity of the mutant colicin E9, although the zones of growth inhibition were hazy and slow to form. The size of the zone of inhibition was significantly smaller than the control in the case of the Ser40Ala mutant. The ColE9/lm9 complex was isolated from the three biologically inactive TolA box alanine mutants. In competition assays all three mutant protein complexes were capable of protecting sensitive E. coli cells against killing by the native ColE9/lm9 complex. On removal of the Im9 protein from the three mutant ColE9/lm9 complexes, all three mutant colicins exhibited DNase activity. These results confirm the importance of the putative TolA box in the biological activity of colicin E9, and suggest that the TolA box has a role in the translocation of colicin E9.
A Rhodococcus species that thrives on medium saturated with liquid benzene
Authors: Paje M. Luz F. , Neilan Brett A. , Couperwhite lain
Published: June 2009

Summary: A bacterium isolated from a contaminated site in Sydney, Australia, utilized benzene in the liquid phase as a sole carbon source at levels toxic to other micro-organisms. The organism was a short Gram-positive rod which grew at 6% NaCI, 0-37 ° and pH 2-10. Biochemical tests, fatty acid analysis, and 16S rDNA sequencing identified the organism as a member of the genus Rhodococcus. Vapour-phase addition of benzene to the medium in batch and continuous systems resulted in initial concentrations averaging 200 p.p.m. Under these conditions, 95% of the benzene was degraded. In separate experiments, medium spiked with liquid benzene resulted in concentrations of up to 2789 p.p.m. and supported good growth of the organism. To confirm utilization of benzene at levels known to be toxic to other micro-organisms, continuous cultures were used; benzene added at 2% (v/v) per day resulted in growth and 89% degradation, which was maintained for more than 30 d. Rhodococcus sp. strain 33 appears to be the only organism known that can grow at these levels of benzene.
Phenotypic characterization of a Candida albicans strain deficient in its major exoglucanase
Authors: del Mar González María , Díez-Orejas Rosalía , Molero Gloria , álvarez Alberto M. , Pla Jesús , Pla Jesús , Nombela CéAsar , Sánchez-PéArez Miguel
Published: June 2009

Summary: Both alleles of theXOG1gene ofCandida albicans, which encodes a protein with exoglucanase activity, were sequentially disrupted. Enzymic analysis of either cell extracts or culture supernatants of disrupted strains revealed that this gene is responsible for the major exoglucanase activity inC. albicans, although residual exoglucanase activity could still be detected.xog1null mutants showed similar growth rates in both rich and minimal liquid medium as compared to the wild-type strain, indicating that the enzyme is not essential forC. albicansgrowth. In addition, no differences were observed between wild-type andxog1null mutants with respect to their ability to undergo dimorphic transition. However, small but repeatable differences were found between the wild-type and the null mutant with respect to susceptibility to chitin and glucan synthesis inhibitors. Using a murine model of experimental infection, no significant differences in virulence were observed. Thexog1null strain is thus a suitable recipient for studyingCandidagene expression using the exoglucanase as a reporter gene.
Haemolysin production by strains of Verocytotoxin-producing Escherichia coli
Authors: Chart Henrik , Jenkins Claire , Smith Henry R. , Hedges Dawn , Rowe Bernard
Published: June 2009

Summary: Twenty-one strains of Verocytotoxin-producing Escherichia coli (VTEC) that hybridized with DNA probe CVD419 were examined for the ability to produce haemolysin. With solid media, all strains produced most haemolysin when grown in blood agar tubes and least when grown on blood agar plates incubated in air. Haemolysin production was increased considerably by incubating blood agar plates in an atmosphere comprising 8% carbon dioxide, 40% hydrogen and 52% nitrogen at 37 °C for 16 h, followed by 6 h at 21 °C in air. Haemolysin production was also increased when strains were grown on L-agar containing the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) prior to subculture on blood agar. Intracellular haemolysin was detected in five out of the 21 strains of E. coli grown on L-agar in the atmosphere described above, but haemolysin was not detected in L-broth culture supernatants. The haemolysins lysed guinea pig, mouse and ferret erythrocytes, but not human, rabbit, rat, turkey or chicken erythrocytes. Also, the addition of calcium ions to culture media was not required for haemolytic activity. It was concluded that haemolysins produced by VTEC appear to be quite distinct from E. coli α-haemolysin and resemble a form of β-haemolysin.
Recombinational reassortment among opa genes from ET-37 complex Neisseria meningitidis isolates of diverse geographical origins
Authors: Hobbs Marcia M. , Malorny Burkhard , Prasad Parachuri , Morelli Giovanna , Kusecek Barica , Heckels John E. , Cannon Janne G. , Achtman Mark
Published: June 2009

Summary: Opacity (Opa) proteins are a family of antigenically variable outer-membrane proteins of Neisseria meningitidis. ET-37 complex meningococci, defined by multilocus enzyme electrophoresis, have been isolated on different continents. Twenty-six different Opa proteins have been observed within strains of the ET-37 complex isolated between the 1960s and the 1980s, although individual strains have only four opa genes per chromosome. In this work the opa genes of four closely related ET-37 complex N. meningitidis strains recently isolated from Mali, West Africa were characterized and compared with the opa genes of strain FAM18, an ET-37 complex isolate from the USA. DNA sequence analysis and Southern blot experiments indicated that recombinational reassortment, including gene duplication and import by horizontal genetic exchange, has occurred in the opa genes within the ET-37 complex, resulting in two partially different Opa repertoires being present in FAM18 and the Mali isolates. Using synthetic peptides derived from the hypervariable (HV) regions of opa genes, the epitopes for nine mAbs were mapped. These bacteria, isolated on different continents, contain both shared and unique opa HV regions encoding epitopes recognized by mAbs and show evidence of recombinational reassortment of the HV regions.
pqqA is not required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1
Authors: Toyama Hirohide , Lidstrom Mary E.
Published: June 2009

Summary: Methylobacterium extorquens AM1 is a facultative methylotroph that oxidizes methanol via the pyrroloquinoline quinone (PQQ)-linked enzyme methanol dehydrogenase. In M. extorquens AM1 and other PQQ-synthesizing bacteria, several genes are involved in the synthesis of PQQ and one of these, pqqA, has been proposed to encode a peptide precursor of PQQ. In other PQQ-synthesizing bacteria, pqqA is required for PQQ production. In this study, it is shown that both deletion and insertion mutants of pqqA in M. extorquens AM1 grow normally on methanol and produce PQQ. The level of PQQ production is reduced in the insertion mutant, but it is sufficient to allow normal growth on methanol. These results suggest either that a different peptide in M. extorquens AM1 can substitute for PqqA in pqqA mutants, or that PqqA-like peptides may not be obligatory precursors of PQQ. In addition, it is shown that the methanol oxidation transcriptional regulator gene, mxbM, is required for normal methanol induction of PQQ synthesis.
Identification of a region responsible for binding to the cell wall within the S-layer protein of Clostridium thermocellum
Authors: Lemaire Marc , Miras Isabelle , Gounon Pierre , Béguin Pierre
Published: June 2009

Summary: The protomer forming the S-layer of Clostridium thermocellum was identified as a 140 kDa protein which was non-covalently bound to the cell wall. Cloning and sequencing of the corresponding gene revealed an open reading frame of 3108 nucleotides encoding a polypeptide of 1036 amino acids, termed SIpA. The amino acid composition of SIpA matches the composition of a previously described exocellular glycoprotein. SIpA shared extensive similarity with the S-layer protein of Bacillus sphaericus and with the outer wall protein of Bacillus brevis. In addition, the amino-terminal region of SIpA contained a segment presenting similarities with segments termed SLH (S-layer homologous), which are found in several bacterial exoproteins. A polypeptide of 209 residues comprising this segment was shown to bind to cell walls extracted from C. thermocellum cells.
Metabolism is required for chemotaxis to sugars in Rhodobacter sphaeroides
Authors: Jeziore-Sassoon Yehudit , Hamblin Paul A. , Bootle-Wilbraham Carolyn A. , Poole Philip S. , Armitage Judith P.
Published: June 2009

Summary: Chemotaxis towards carbohydrates is mediated, in enteric bacteria, either by the transport-independent, methylation-dependent chemotaxis pathway or by transport and phosphorylation via the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). This study shows that Rhodobacter sphaeroides is chemotactic to a range of carbohydrates but the response involves neither the classical methyl-accepting chemotaxis protein (MCP) pathway nor the PTS transport pathway. The chemoattractant fructose was transported by a fructose-specific PTS system, but transport through this system did not appear to cause a chemotactic signal. Chemotaxis to sugars was inducible and occurred with the induction of carbohydrate transport systems and with substrate incorporation. A mutation of the glucose-6-phosphate dehydrogenase gene (zwf) inhibited chemotaxis towards substrates metabolized by this pathway although transport was unaffected. Chemotaxis to other, unrelated, chemoattractants (e.g. succinate) was unaffected. These data, in conjunction with the fact that mannitol and fructose (which utilize different transport pathways) compete in chemotaxis assays, suggest that in R. sphaeroides the chemotactic signal is likely to be generated by metabolic intermediates or the activities of the electron-transport chain and not by a cell-surface receptor or the rate or mode of substrate transport.
The Nhal antiporter of Saccharomyces cerevisiae mediates sodium and potassium eft: lux
Authors: BaAueIos Maria A. , Sychrová Hana , Bleykasten-Grosshans Claudine , Souciet Jean-Luc , Potier Serge
Published: June 2009

SUMMARY: The NHAl gene of Saccharomyces cerevisiae, transcribed into a 3-5 kb mRNA, encodes a protein mediating Na+ and K+ efflux through the plasma membrane that is required for alkali cation tolerance at acidic pH. Deletion of the gene in a wild-type strain resulted in higher sensitivity to both K+ and Na+ at acidic pH. Measurements of cation loss in strains carrying deleted or overexpressed alleles of NHAl demonstrated its role in K+ and Na+ efflux. In addition, high K+ and Na+ efflux observed upon alkalinization of the cytoplasm implies a role of Nhalp in the regulation of intracellular pH. Moreover, the overexpression of ENA1 and NHAl genes in an enal-46-nhalb strain showed that the Nhal alkalication antiporter is responsible for growth on high concentrations of KCI and NaCl at acidic pH, and Ena alkali-cation ATPases are necessary at higher pH values. Both systems have a complementary action to maintain the intracellular steadystate concentration of K+ and Na+.
Transmembrane topology of the two FhuB domains representing the hydrophobic components of bacterial ABC transporters involved in the uptake of siderophores, haem and vitamin B
Authors: Groeger Wolfram , KOstert Wolfgang
Published: June 2009

SUMMARY: Transport of siderophores of the hydroxamate type across the Escherichia coli cytoplasmic membrane depends on a periplasmic binding-protein-dependent (PBT) system. This uptake system consists of the binding protein FhUD, the membrane-associated putative ATP-hydrolase FhuC and the integral membrane protein FhuB. The two halves of FhuB [FhuB(N) and FhuB(C)], both essential for transport, are similar with respect to structure and function. Regions were identified in FhuB(N) and FhuB(C) which are presumably involved in the interaction of the two FhuB halves with each other or with other components of the uptake system, or with the different substrates. To determine the topology of the membrane-embedded polypeptide chain, FhuB‘-’/?-lactamase protein fusions were analysed. The experimental data suggest that each half of the FhuB is able to fold autonomously into the lipid bilayer, which is a prerequisite for the assembly of both halves into a transport-competent formation. The hydrophobic components from PBT systems involved in the uptake of siderophores, haem and vitamin B, define a subclass of polytopic integral membrane proteins. The topology of these ‘siderophore family’ proteins differs from that of the equivalent components of other PBT systems in that each polypeptide (and each half of FhuB) consists of 10 membrane- spanning regions, with the N- and C-termini located in the cytoplasm. The conserved region at a distance of about 90 amino acids from the C-terminus, typical of all hydrophobic PBT proteins, is also oriented to the cytoplasm. However, in the siderophore family’ proteins this putative ATPase interaction loop is followed by four instead of two transmembrane spans.
Identification of two linear plasmids in the actinomycete Planobispora rosea
Authors: Polo Simona , Guerini Oscar , Sosio Margherita , Dehb Gianni
Published: June 2009

SUMMARY: Two linear plasmids (pPR1, 27.5 kb, and pPR2, 16 kb) were identified in Planobispora msea, an actinomycete that produces the antibiotic GE2270, an inhibitor of the elongation factor Tu. Strains lacking both plasmids still produce and are resistant to GE2270. The two plasmids share an internal region of high similarity, but no cross-hybridization was detected between their telomeric regions or between plasmid and chromosomal DNA. The 5‵ ends of the plasmids appear to be linked to terminal proteins. The telomeric regions of pPR2 were cloned after 3‵-end homopolymer tailing and PCR amplification. The approximately 650 nt telomeric DNA sequences of pPR2 are repeated in inverted orientation and are rich in direct and inverted repeats; the 350 bp terminal region is less G+C-rich than the rest of the plasmid. The structural organization of these plasmids appears to be similar to Streptomyces linear rep1icons.
Two-component signal-transducing systems involved in stress responses and vancomycin susceptibility in Lactobacillus sakei
Authors: Morel-Deville Franqoise , Fauvel Franck , Morel Patrice
Published: June 2009

SUMMARY: Fragments of five rrp genes encoding response regulators (RRs) in LactobscMus sakei were amplified by PCR using degenerate oligonucleotide primers. The five rrp genes were part of distinct loci that also comprised hpk genes encoding histidine protein kinases (HPKs). The putative RRs belonged tom the OmpR-PhoB subclass of response regulators that consist of N-terminal receiver and C-terminal DNA-binding domains. The putative HPKs were members of the EnvZ-NarX family of orthodox histidine protein kinases which possess two transmembrane segments in a non-conserved N-terminal domain and a C-terminal cytoplasmic kinase domain. Insertional inactivation of the rrp genes indicated that the RRs are implicated in susceptibility to the glycopeptide antibiotic vancomycin, and to extreme pH, temperature and oxidative conditions.
Clustered organization and transcriptional analysis of a family of five csp genes of Lactococcus /actis MGl363
Authors: Woufers Jeroen A. , sander Jan-Willem , Kok Jan , de Vos Willem M. , Kuipers Oscar P. , Abee Tjakko
Published: June 2009

SUMMARY: A family of genes encoding cold-shock proteins, named cspA, cspB, cspC, cspD and cspE, was cloned and sequenced from Lactococcus lactis MG1363. The genes cspA and cspB and the genes cspC and cspD are located in tandem repeats, an organization of csp genes that has never been encountered before. The five genes encode small (7.1-706 kDa) proteins with high mutual sequence identities (up to 85 O/O) and high identities (about 45-65 %) with the major cold- shock proteins from Escherichia coli (CspA) and Bacillus subtilis (CspB)., Northern-blot analysis revealed single transcripts of about 300 nucleotides for each csp gene and showed that cspA, cspB, cspC and cspD mRNA levels were strongly increased upon cold shock to 10 "C (about lo-, 40-, 10- and 30-fold compared to 30 "C, respectively), whereas the cspE mRNA level was not increased. The expression of the cold-induced csp genes was highest in the. 6-8 h lag phase after cold shock. A differential expression in time, in which cspA and cspC were maximally expressed at 2 h and cspB and cspD at 4 h after cold shock, was observed. The -35 and -10 regions of the five promoters were identified and transcriptional start sites were mapped in each case by primer extension at different temperatures which confirmed that regulation takes place at the transcriptional level. Significant differences were observed between the 5′-untranslated leader regions of the four cold-induced csp genes and the corresponding region of the non-cold-induced cspE gene.
A functional myo-inositol catabolism pathway is essential for rhizopine utilization by Sinorhizobium meliloti
Authors: Galbraith Mark P. , Feng Szi Fei , Borneman James , Triplett Eric W. , de Bruijn Frans J. , Rossbachl Silvia
Published: June 2009

SUMMARY: Rhizopine (~-3-O-methyl-scy//o-inosamine) is a symbiosis-specif ic compound found in alfalfa nodules induced by specific Sinorhilobium meliloti strains. It has been postulated that rhizobial strains able to synthesize and catabolize rhizopine gain a competitive advantage in the rhizosphere. The pathway of rhizopine degradation is analysed here. Since rhizopine is an inositol derivative, it was tested whether inositol catabolism is involved in rhizopine utilization. A genetic locus required for the catabolism of inositol as sole carbon source was cloned from S. meliloti. This locus was delimited by transposon Tn5 mutagenesis and its DNA sequence was determined. Based on DNA similarity studies and enzyme assays, this genetic region was shown to encode an S, meliloti myo-inositol dehydrogenase. Strains that harboured a mutation in the myo-inositol dehydrogenase gene (idhA) did not display myo-inositol dehydrogenase activity, were unable to utilize myo-inositol as sole carbodenergy source, and were unable to catabolize rhizopine. Thus, myo-inositol dehydrogenase activity is essential for rhizopine utilization in s. meliloti.
ldentif ication of three differentially expressed hydrophobins in Pleurotus ostreatus (oyster mushroom)
Authors: Asgeirsddttir Sigridur A. , de Vries Onno M. H , Wessels Joseph G. H
Published: June 2009

SUMMARY: Three proteins with characteristic features of class I hydrophobins, designated POH1, POH2 and POH3, were isolated from the basidiomycete PIeurotus ostreatus. Based on N-terminal sequence analyses, their cDNAs were isolated using RT-PCR; the cDNAs and corresponding genes were sequenced and their regulation studied. POHI is expressed in the fruiting bodies but not in vegetative mycelium. The regulation of fWf2 and poH3 is tightly correlated. Both genes are switched off in the fruiting bodies but abundantly expressed in the vegetative mycelium of both monokaryon and dikaryon. POH2 and POH3 were isolated from the culture medium and from aerial hyphae. Co-purified POH2 and POH3 assembledin witro into a protein membrane with a typical rodlet pattern as found previously withother hydrophobins. Similar structures were detected on the surface of aerial hyphae.
Internalization and cytotoxicity are important virulence mechanisms in vibrio-f ishepithelial cell interactions
Authors: Wang X. H. , Oon H. L. , Ho G. W. P. , Wong W. S. F. , Lim T. M. , Leung K. Y.
Published: June 2009

SUMMARY: Vibrio anguillarum and Vibrio damselee are Gram-negative bacteria that cause systemic infections called vibriosis in fish. They can enter fish cells and survive as intracellular parasites. The host-pathogen interactions between these Vibrio species and the fish epithelial cell lines epithelioma papillosum of carp (EPC) and grunt-f in tissue (GF) cells, were examined using phase-contrast, scanning electron and confocal microscopy. In addition, potential signal transduction pathways that precede bacterial internalization were studied by using signal transduction inhibitors. Some Vibrio species induced morphological changes in fish cells and this allowed classification into a cytopathic group and a noncytopathic group. The cytopathic group could be subdivided into two invasive groups (I and II) and a cytotoxic group. Of the invasive strains V. anguillarum 811218-5W (group I) and GNirus/5(3) (group II), genistein, a tyrosine kinase inhibitor, only inhibited internalization of V. anguillarum GNirus/5(3) into EPC cells, whereas staurosporine, a protein kinase C inhibitor, accelerated internalization of both strains. Cytochalasin D, an inhibitor of microfilament polymerization, prevented internalization of both strains, whilst vincristin, a microtubule inhibitor, only inhibited internalization of V. anguillarum GNirus/5(3). For the cytotoxic strain V. damselae ATCC 33539, extracellular products (ECP) alone caused morphological changes in EPC and GF. Bacterial internalization may not be important in the pathogenesis of this group. The non-cytopathic strain V. anguillarum SU5/93(2) did not enter cells or induce any changes in EPC and GF monolayen. This study has identified some major differences between Vibrio species in their interactions with fish cells in vitm and will thus facilitate future studies of the molecular basis of pathogenesis of vibriosis.
Construction of a physical and preliminary genetic map of Aerornonas hydrophila IMP636
Authors: Dodd Helen N. , Pemberton John M.
Published: June 2009

SUMMARY: A physical and preliminary genetic map of the Aeromonas hydrophila JMP636 chromosome has been constructed. The topology of the genome was predicted to be circular as chromosomal DNA did not migrate from the origin during PFGE unless linearized by 51 nuclease. Cleavage of the chromosome with Pacl and PmeI produced 23 and 14 fragments, respectively, and enabled calculation of the genome size at 4.5 Mb. Digestion of the chromosome with I-Ceul produced 10 fragments, indicating that 10 rrl(23S) genes were likely t o be present. Hybridizations between DNA fragments generated with Pad, PmeI and I-Ceul were used t o initially determine the relationship between these segments. To accurately map genes previously characterized from JMP636, the suicide vector pJP5603 was modified to introduce restriction sites for Pad and PmeI, producing pJP9540. Following cloning of genes into this vector and recombinational insertion into the JMP636 chromosome, Pad and Pmel cleavage determined the location of genes within macrorestriction fragments
with the additional bands produced forming hybridization probes. From the data generated, it was possible t o form a physical map comprising all the fragments produced by Pacl and Pmel, and assign the contig of I-Ceul fragments on this map. The preliminary genetic map defines the location of six loci for degradative enzymes previously characterized from JMP636, while the locations of the 10 sets of ribosomal genes were assigned with less accuracy from hybridization data.
A vector for systematic gene inactivation in Bacillus subtilis
Authors: Vagner Valerie , Dervyn Etienne , Ehrlich S. Dusko
Published: June 2009

SUMMARY: To study the functions of the uncharacterized open reading frames identified in the Bacillus subtih genome, several vectors were constructed t o perform insertional mutagenesis in the chromosome. All the pMUTlN plasmids carry a lac2 reporter gene and an inducible Pspac promoter, which is tightly regulated and tan be induced about 1000-fold. The integration of a pMUTlN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lac2 becomes transcriptionally fused t o the gene, allowing its expression pattern to be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion. This last feature is important because B. subti/is genes are often organized in operons. The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG. Also, conditional mutants of essential genes can be obtained by integrating pMUTlN vectors upstream of the target gene. The vectors are currently being used for systematic inactivation of genes without known function within the B. subtilis European consortium. pMUTlN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.
The product of the yvoC(geri)gene of Bacillus subtillis is required for spore germination
Authors: Robinson Carl , Rivolta Carlo , Karamata Dimitri , Moir Anne
Published: June 2009

SUMMARY: All known gerF mutations affecting Bacillus subtilis spore germination have been mapped, by a combination of recombination and complementation analysis, to yvoC (/gt)# a gene belonging to the yvoB CptsK) yvoC (/gt) yvoDEF operon. Examination of the properties of null mutants confirmed that the only gene in the operon that affects germination is poC, which encodes a homologue of known prelipoprotein diacylglyceryl transferases. As several germination proteins (GerAC, GerBC, GerKC, GerD) are predicted lipoproteins, it is not unreasonable to assume that a defect in prelipoprotein processing will affect spore germination. Two other null mutants in this chromosomal region showed a clear phenotype: the nagA gene is required for growth on N-acetylglucosamine, whereas a null mutation in yvoB (ptsK) affects colony formation from single cells.
Streptococcus pyogenes protein F promotes invasion of HeLa cells
Authors: Okada Nobuhiko , Tatsuno lchiro , Hanski Emanuel , Caparon Michael , Sasakawa Chihiro
Published: June 2009

SUMMARY: Although the Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) has been considered an extracellular pathogen which adheres to human mucosal epithelium, the streptococcus possesses invasive capacity for cultured human epithelial cells. This study provides genetic and functional evidence supporting the conclusion that protein F is capable of mediating entry of S. pyogenes into HeLa cells. Using 111916 insertion mutagenesis or an isogenic 5. pyogenes strain with a defined mutation in the gene encoding protein F (prtF), it was observed that the invasive capacity was affected by the
levels of surface-exposed protein F, but not by those of M protein. In addition, heterologous expression of protein F on Enterococcus faecalis conferred upon the bacteria an efficient invasive phenotype. Several assays demonstrated that both the fibronectin-binding domains of protein F, UR and RD2, were involved in host-cell invasion. In addition, coinfection experiments of HeLa cells with 5. pyogenes and an Escherichia coli K-12 strain expressing an afimbrial adhesin AFA-I showed that the uptake of S. pyogenes did notpermit internalization of the E. coli cells.
Growth polarity transitions in a dimorphic fission yeast
Authors: Sipiczki M. , Takeo K. , Grallert A.
Published: June 2009

Summary: Fission yeast cells grow by extension at the ends (poles) and divide by transverse fission. It has previously been reported thatSchizosaccharomyces japonicusvar.japonicuscan switch to unipolar, filamentous growth. Here it is shown that the yeast-to-mycelium transition is a gradual process involving a changeover to unipolar growth associated with asymmetric divisions, the development of large polarly located vacuoles, the modifications of the actin and microtubular cytoskeleton and the repression of cell separation after division. High concentrations of glucose in the medium or supplementation of the medium with caffeine or cAMP support the bipolar yeast phase, inhibit the transition to the mycelial phase and induce the conversion of hyphae to yeasts. These effects suggest that cAMP may be involved in the regulation of dimorphism. Temperatures below 18 °C or over 35 °C are restrictive for the mycelial phase and provoke a return to yeast phase.
Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model
Authors: Wong Christopher Y. F. , Heuzenroeder Michael W. , Flower Robert L. P.
Published: June 2009

The contribution of two unrelated Aeromonas hydrophila β-haemolytic toxins to virulence was assessed in a suckling mouse model. The first haemolysin gene, isolated from an A. hydrophila A6 cosmid bank, encoded a potential gene product of 621 amino acids and a predicted molecular size of 69.0 kDa. The inferred amino acid sequence showed 89% identity to the AHH1 haemolysin of A. hydrophila ATCC 7966, and 51% identity to the HlyA haemolysin of Vibrio cholerae El Tor strain O17. The second haemolysin gene (designated aerA), which encodes aerolysin, a pore-forming toxin, was partially cloned by PCR for the purpose of mutant construction. This PCR product was a 1040 bp fragment from the C-terminal region of aerA. It is proposed that the 69.0 kDa V. cholerae-HlyA-like haemolysin gene be termed hlyA to contrast with the aerA terminology for the aerolysin. A suicide vector was used to inactivate both the hlyA and aerA genes in A. hydrophila A6. When assessed in the suckling mouse model, only the hlyA aerA double mutant showed a statistically significant reduction in virulence - a 20-fold change in LD50 (Scheffe test, P<0.05). Cytotoxicity to buffalo green monkey kidney cell monolayers and haemolysis on horse blood agar were eliminated only in the hlyA aerA double mutants. This is the first report of cloning and mutagenesis of two unrelated haemolytic toxin genes in the same strain of a mesophilic aeromonad. For A. hydrophila, a two-toxin model provides a more complete explanation of virulence.
Streptococcus suis serotype 2 mutants deficient in capsular expression
Authors: Charland Nathalie , Harel Josée , Kobisch Marylène , Lacasse Serge , Gottschalk Marcelo
Published: June 2009

Streptococcus suis serotype 2 is responsible for a wide variety of porcine infections. In addition, it is considered a zoonotic agent. Knowledge about the virulence factors for this bacterium is limited but its polysaccharide capsule is thought to be one of the most important. Transposon mutagenesis with the self-conjugative transposon Tn916 was used to obtain acapsular mutants from the virulent S. suis type 2 reference strain S735. Clones were screened by colony-dot ELISA with a monoclonal antibody specific for a type 2 capsular epitope and clones that failed to react with the antibody were characterized. Two mutants, 2A and 79, having one and two Tn916 insertions respectively, were chosen for further characterization. Absence of capsule was confirmed by coagglutination, capillary precipitation and capsular reaction tests and by transmission electron microscopy. Absence of capsular polysaccharides correlated with increased hydrophobicity and phagocytosis by both murine macrophages and porcine monocytes compared to the wild-type strain. Furthermore, both mutants were shown to be avirulent in murine and pig models of infection. Finally, mutant 2A was readily eliminated from circulation in mice compared to the wild-type strain, which persisted more than 48 h in blood. Thus, isogenic mutants defective in capsule production demonstrate the importance of capsular polysaccharides as a virulence factor for S. suis type 2.
Metabolism of cellobiose by Clostridium cellulolyticum growing in continuous culture: evidence for decreased NADH reoxidation as a factor limiting growth
Authors: Payot S. , Guedon E. , Cailliez C. , Gelhaye E. , Petitdemange H.
Published: June 2009

Previous results indicated that molar growth yields are reduced when Clostridium cellulolyticum is cultured in media containing cellobiose concentrations greater than 1 g I−1. Continuous cultures were examined to determine the physiological basis of these poor growth yields. Acetate was the main product of C. cellulolyticum metabolism, whereas the production of reduced compounds such as ethanol or lactate was low. Such patterns of product formation were accompanied by a 12-fold increase in intracellular NADH concentration when the cellobiose flow was increased. Catabolic enzymic activities were measured in vitro. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acetate kinase and phosphoroclastic activities were found at similar levels as in cells metabolizing higher substrate concentrations. In contrast, lactate dehydrogenase activity was low and correlated with the rate of lactate production. Furthermore, an inhibition of GAPDH activity by high NADH/NAD+ ratios was established. These results suggested that a decreased NADH reoxidation could be responsible for limiting C. cellulolyticum growth. Lactate and ethanol production were not sufficient to balance out the NADH produced in the GAPDH step of glycolysis. One consequence of poor NADH reoxidation would be an increase in intracellular concentration of NADH, which in turn could inhibit GAPDH activity.
Localization of denitrification genes on the chromosomal map of Pseudomonas aeruginosa
Authors: Vollack Kai-Uwe , Xie Jun , Härtig Elisabeth , Römling Ute , Zumft Walter G.
Published: June 2009

Cleavage of chromosomal DNA from Pseudomonas aeruginosa PAO by SpeI and DpnI has been used together with PFGE and Southern hybridization to establish the map location of the following principal denitrification genes: narGH (encoding the large and small subunits of respiratory nitrate reductase), nirS (cytochrome-cd
1 nitrite reductase), nirE (uroporphyrinogen-III methyltransferase for haem d
1 biosynthesis), norCB (nitric-oxide reductase complex), nosZ (nitrous-oxide reductase) and nosA (an outer-membrane protein and OprC homologue). The study also included several genes related to anaerobic or microaerophilic metabolism: napA (encoding the catalytic subunit of the periplasmic nitrate reductase), ccoN (catalytic subunit of the cytochrome-cbb
3 oxidase), hemN (oxygen-independent coproporphyrinogen-III oxidase), an fnr-like regulatory gene, and azu and fdxA (electron carriers azurin and ferredoxin, respectively). Genes necessary for denitrification are concentrated at 20 to 36 min on the P. aeruginosa chromosome, where they form three separate loci, the nir-nor, nar and nos gene clusters. Genomic DNA of Pseudomonas stutzeri ZoBell was also subjected to SpeI restriction and Southern analysis to assign denitrification genes to individual fragments. A homologue of nosA encoding a putative component of the Cu-processing apparatus for nitrous-oxide reductase was identified. In both P. aeruginosa and P. stutzeri there is evidence for the linkage of anr (fnrA) with hemN and ccoN; and for the presence of a napA gene.
Physical map of the chromosome of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola
Authors: De Ita M. Esther , Marsch-Moreno Rodolfo , Guzmán Plinio , Alvarez-Morales Ariel
Published: June 2009

Pseudomonas syringae pv. phaseolicola (P.s. phaseolicola) is one of about 45 recognized pathovars within the P. syringae group and is the causal agent of halo-blight disease of beans. DNA from this bacterium digested to completion with two different restriction enzymes, PacI and PmeI, yielded 15 and 16 fragments, respectively. These were separated using PFGE and sized by comparison to known molecular mass markers. The P.s. phaseolicola chromosome was determined to be approximately 5.64 Mb in size. To link the different fragments obtained into a circular chromosome map for both enzymes, 150 random Tn5 mutants of P.s. phaseolicola were used as a source of DNA and the identification of the band carrying the transposon ‘tag’ in each mutant was done after PFGE and Southern hybridization of a complete chromosomal digestion using a Tn5 probe. Partial digestions of DNA from different Tn5 mutants ‘tagging’ specific bands were then generated and the complete and partial products of the digestion separated by PFGE and identified with a Tn5 probe. By calculating the size of the partial products, it was then possible to link different bands into a physical map. This is the first report on the construction of a physical map of a member of the P. syringae group and should be invaluable for molecular genetic analysis in this species and in evolutionary or taxonomic studies when compared to similar data obtained for any of the other recognized pathovars.
Horizontal spread of mer operons among Gram-positive bacteria in natural environments
Authors: Bogdanova E. S. , Bass I. A. , Minakhin L. S. , Petrova M. A. , Mindlin S. Z. , Volodin A. A. , Kalyaeva E. S. , Tiedje J. M. , Hobman J. L. , Brown N. L. , Nikiforov V. G.
Published: June 2009

Horizontal dissemination of the genes responsible for resistance to toxic pollutants may play a key role in the adaptation of bacterial populations to environmental contaminants. However, the frequency and extent of gene dissemination in natural environments is not known. A natural horizontal spread of two distinct mercury resistance (mer) operon variants, which occurred amongst diverse Bacillus and related species over wide geographical areas, is reported. One mer variant encodes a mercuric reductase with a single N-terminal domain, whilst the other encodes a reductase with a duplicated N-terminal domain. The strains containing the former mer operon types are sensitive to organomercurials, and are most common in the terrestrial mercury-resistant Bacillus populations studied in this work. The strains containing the latter operon types are resistant to organomercurials, and dominate in a Minamata Bay mercury-resistant Bacillus population, previously described in the literature. At least three distinct transposons (related to a class II vancomycin-resistance transposon, Tn 1546, from a clinical Enterococcus strain) and conjugative plasmids are implicated as mediators of the spread of these mer operons.
Thigmotropism and stretch-activated channels in the pathogenic fungus Candida albicans
Authors: Watts H. J. , Veacute;ry A.-A. , Perera T. H. S. , Davies J. M. , Gow N. A. R.
Published: June 2009

The direction of growth of hyphae of the pathogenic fungus Candida albicans responds thigmotropically to surface contours by following scratches, ridges and grooves and by penetrating pores. Here it is shown that the thigmotropic response to ridges is attenuated by GdCl3 and verapamil [blockers of stretch-activated (SA) ion channels and L-type calcium channels, respectively]. At low concentrations, both compounds reduced the percentage of hyphae reorienting on contact with a ridge without markedly affecting hyphal extension rate, suggesting a possible role for SA or other calcium channels in the transduction of the thigmotropic response. In addition, patch-clamp recordings demonstrated SA channel activity in the plasma membrane of both yeast and hyphal cells of C. albicans. Two distinct SA channels with conductances of 54 pS and 20-25 pS in 200 mM KCI were observed in protoplasts from yeast cells and one channel of 51 pS was found in protoplasts from hyphal cells.
Distribution of repC plasmid-replication sequences among plasmids and isolates of Rhizobium leguminosarum bv. viciae from field populations
Authors: Rigottier-Gois Lionel , Turner Sarah L. , Young J. Peter W. , Amarger Noëlle
Published: June 2009

The distribution of four classes of related plasmid replication genes (repC) within three field populations of Rhizobium leguminosarum in France, Germany and the UK was investigated using RFLP, PCR-RFLP and plasmid profile analysis. The results suggest that the four repC classes are compatible: when two or more different repC sequences are present in a strain they are usually associated with different plasmids. Furthermore, classical incompatibility studies in which a Tn5-labelled plasmid with a group IV repC sequence was transferred into field isolates by conjugation demonstrated that group IV sequences are incompatible with each other, but compatible with the other repC groups. This supports the idea that the different repC groups represent different incompatibility groups. The same field isolates were also screened for chromosomal (plac12) and symbiotic gene (nodD-F region) variation. Comparison of these and the plasmid data suggest that plasmid transfer does occur within field populations of R. leguminosarum but that certain plasmid-chromosome combinations are favoured.
The cadmium-stress stimulon of Escherichia coli K-12
Authors: Ferianc Peter , Farewell Anne , Nyström Thomas
Published: June 2009

The influence of cadmium on stress protein production in Escherichia coli K-12 (strain MG1655) was analysed using two-dimensional polyacrylamide gel electrophoresis and the gene-protein database of E. coli K-12. Cadmium (273 μM) caused complete but transient inhibition of growth accompanied by the synthesis of cadmium-induced proteins (CDPs). It was found that some CDPs induced during the growth-arrested phase belong to the heat-shock, oxidation stress, SOS and stringent response regulons, while others are general stress inducible proteins (e.g. H-NS, UspA). In addition, trigger factor, adenylate kinase, W-protein, the cold shock protein G041.2, and seven unknown proteins whose synthesis is not known to be controlled by a global regulator, were identified as immediate responders to cadmium exposure. The rate of synthesis of most of the immediate responders to cadmium exposure decreased when the growth of the cells resumed. However, seven CDPs, including those encoded by argl, tyrA and xthA, maintained a high production rate during growth in the presence of cadmium. Two of the unidentified proteins were N-terminally sequenced by Edman degradation. The N-terminal amino acid sequence of one of these proteins (designated F023.3) matches the E. coli open reading frame o216. This ORF is similar to the N-terminal third of the copper-binding protein amine oxidases (encoded by maoA) of both E. coli and Klebsiella pneumoniae (K. aerogenes). The other N-terminally sequenced protein (designated C044.6) matches perfectly the product of the metK gene, S-adenosylmethionine synthetase I. In comparison to untreated cells, cadmium-stressed cells were found to recover more rapidly during subsequent stress conditions, such as ethanol, osmotic, heat shock, and nalidixic acid treatment. The role of the CDPs is discussed in view of their physiological assignments in the cell.
Fructose and mannose metabolism in Aeromonas hydrophila: identification transport systems and catabolic pathways
Authors: Binet Marie R. B , Rager Marie-Noelle , Bouvet Odile M. M
Published: June 2009

Aeromonas hydrophila was examined for fructose and mannose transport systems. A. hydrophila was shown to possess a phosphoenolpyruvate (PEP): fructose phosphotransferase system (fructose-PTS) and a mannose-specific PTS, both induced by fructose and mannose. The mannose-PTS of A. hydrophila exhibited cross-reactivity with Escherichia coli mannose-PTS proteins. The fructose-PTS proteins exhibited cross-reactivities with E. coli and Xanthomonas campestris fructose-PTS proteins. In A. hydrophila grown on mannose as well as on fructose, the phosphorylated derivative accumulated from fructose was fructose 1-phosphate. Identification of fructose 1-phosphate was confirmed by 13C-NMR spectroscopy. 1-Phosphofructokinase (1-PFK), which converts the product of the PTS reaction to fructose 1,6-diphosphate, was present in A. hydrophila grown with fructose but not on mannose. An inducible phosphofructomutase (PFM) activity, an unusual enzyme converting fructose 1-phosphate to fructose 6-phosphate, was detected in extracts induced by mannose or fructose. These results suggest that in cells grown on fructose, fructose 1-phosphate could be converted to fructose 1,6-diphosphate either directly by the 1-PFK activity or via fructose 6-phosphate by the PFM and 6-phosphofructokinase activities. In cells grown on mannose, the degradation of fructose 1-phosphate via PFM and the Embden-Meyerhof pathway appeared to be a unique route.