bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-β-glucosyltransferase
Authors: Sarthy Aparna V. , McGonigal Thomas , Coen Michael , Frost David J. , Meulbroek Jonathan A. , Goldman Robert C.
Published: June 2009

The BGL2 gene encodes a unique 1,3-β-glucosyltransferase (BgI2p) present in the cell wall of Candida albicans and other fungi. Although believed to be involved in cell wall assembly, disruption of the gene in Saccharomyces cerevisiae showed no apparent phenotype. We performed sequential disruptions of the BGL2 loci in a homozygous ura3 clinical isolate of C. albicans using the URA3 blaster method, in order to investigate the role of BgI2p in this dimorphic, pathogenic fungus. Strain CACW-1 contained disruptions of both homologues of the BGL2 gene and lacked BgI2p, as assessed by protein extraction, SDS-PAGE and Western blot analysis, and enzyme assay; however, residual non-BgI2p transferase activity was detected. CACW-1 was attenuated in virulence for mice when compared to an isogenic parent strain, and fewer organisms were recovered from the kidneys of infected animals. Additional phenotypic changes included: (1) a dramatic increase in the sensitivity to the chitin synthesis inhibitor nikkomycin Z when CACW-1 cells were incubated at 37 or 42 °; (2) an 8.7+1.6% slower growth rate at 37 ° for CACW-1 when compared to its isogenic parent; and (3) aggregation of CACW-1 cells during stationary phase and/or incubation of stationary phase cells in phosphate buffer. Characterization of SDS-extracted cell walls did not reveal any significant differences in the levels of 1,3-β- or 1,6-β-glucan. These data reveal that loss of BgI2p does have a phenotype in C. albicans, and indicate that (1) loss of BgI2p function renders cells more dependent on chitin for wall integrity, and attenuates virulence (probably due to subtle changes in wall structure), and (2) that additional 1,3-β-glucosyltransferases are present in the C. albicans BGL2 disruptant.
Biochemical and genetic characterization of Rbf1p, a putative transcription factor of Candida albicans
Authors: Ishii Nobuya , Yamamoto Mutumi , Yoshihara Fumie , Arisawa Mikio , Aoki Yuhko
Published: June 2009

A Candida albicans gene encoding a novel DNA-binding protein that bound to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence o C albicans was previously cloned and designated RBF1 (RPG-box-binding factor). In this report, determination of the functional domains of the protein is described. The DNA-binding domain was 140 aa in length, was centrally located between two glutamine-rich regions, and correlated with transcriptional activation in S. cerevisiae. The results, together with the previous finding that showed its predominant localization in the nucleus, suggest that this DNA-binding protein could be a transcription factor. Disruption of the functional RBF1 gene of C. albicans strains caused an alteration in cell morphology to the filamentous form on all solid and liquid media tested. Thus, we speculate that Rbf1p may be involved in the regulation of the transition between yeast and filamentous forms at the level of transcription.
Metabolism of inositol 1,4,5-trisphosphate in Candida albicans: significance as a precursor of inositol polyphosphates and in signal transduction during the dimorphic transition from yeast cells to germ tubes
Authors: Gadd Geoffrey M. , Foster Sally A.
Published: June 2009

The metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was examined in yeast cells and germ tubes ofCandida albicans.Methods have been developed for analysis of the two key metabolic enzymes, Ins(1,4,5)P3kinase and phosphatase. ATP-dependent Ins(1,4,5)P3kinase activity was detected predominantly in the soluble fraction of cell extracts and exhibited aKmof approximately 9 μM. The apparentKmof Ins(1,4,5)P3phosphatase for Ins(1,4,5)P3was approximately 480 μM. The slow rate of dephosphorylation of Ins(1,4,5)P3to inositol bisphosphate suggests a lower importance of the phosphatase within cells compared to the kinase. Since both yeast cells and germ tubes ofC. albicansrapidly phosphorylated Ins(1,4,5)P3to inositol tetrakisphosphate and inositol penta/hexakisphosphate, it is suggested that Ins(1,4,5)P3has an important role as a precursor for production of these compounds. A sustained increase in cellular Ins(1,4,5)P3levels was observed during germ tube formation and, prior to the onset of germination between 1 and 2 h incubation, the Ins(1,4,5)P3content increased up to eightfold. Transien increases in the level of Ins(1,4,5)P3were also observed during yeast-like growth ofC. albicans.The possible role and relative importance of Ins(1,4,5)P3as a precursor for inositol polyphosphates and in signal transduction involving Ca2+release from internal stores is discussed.
The Paracoccus denitrificans ccmA, B and C genes: cloning and sequencing, and analysis of the potential of their products to form a haem or apo- c-type cytochrome transporter
Authors: Page M. Dudley , Pearce David A. , Norris Hilary A. C. , Ferguson Stuart J.
Published: June 2009

Two c-type cytochrome deficient mutants of Paracoccus denitrificans, HN49 and HN53, were isolated by Tn5 mutagenesis and screening for failure to oxidize dimethylphenylenediamine (the Nadi test). Both were completely deficient in c-type cytochromes. Genomic DNA flanking the site of Tn5 insertion in HN53 was cloned by marker rescue and a 3.1 kb region sequenced. Three of the genes, designated ccmA, ccmB and ccmC, present in this region are proposed to encode the components of a membrane transporter of the ABC (ATP-binding cassette) super-family, which is similar to a group of transporters postulated to translocate either haem or apocytochromes c. The Tn5 elements in HN49 and HN53 were shown to be inserted in ccmB and ccmA, respectively. Sequence analysis suggested that both CcmB and CcmC have the potential to interact with CcmA and thus that the three gene products probably associate to form a complex with (CcmA)2-CcmB-CcmC stoichiometry; it also indicated a lack of similarity between CcmB and CcmC and the membrane-integral components of transporters mediating uptake of haem or other iron complexes. Supplementation of growth media with haem did not stimulate c-type cytochrome formation in HN49 or HN53, although it elevated levels of soluble haemoproteins and membrane-bound cytochromes b, suggesting that exogenous haem can traverse both outer and inner membranes of P. denitrificans. HN49 and HN53 accumulated apocytochrome c
550 to much lower levels than other c-type cytochrome deficient mutants of P. denitrificans but expression and translocation of an apocytochrome c
550-alkaline phosphatase fusion protein and apocytochrome cd
1 were unaffected in HN53. The results suggest that the substrate for the putative CcmABC-transporter is probably neither haem nor c-type apocytochromes.
The nucleoside-specific Tsx channel from the outer membrane of Salmonella typhimurium, Klebsiella pneumoniae and Enterobacter aerogenes: functional characterization and DNA sequence analysis of the tsx genes
Authors: Nieweg Annette , Bremer Erhard
Published: June 2009

The Escherichia coli tsx gene encodes an integral outer-membrane protein (Tsx) that functions as a substrate-specific channel for deoxynucleosides and the antibiotic albicidin, and also serves as a receptor for bacteriophages and colicins. We cloned the structural genes of the Tsx proteins from Salmonella typhimurium, Klebsiella pneumoniae and Enterobacter aerogenes and expressed them in an E. coli tsx mutant. The heterologous Tsx proteins fully substituted the E. coli Tsx protein with respect to its function in deoxynucleoside and albicidin uptake, and as receptor for colicin K. The Tsx proteins from K. pneumoniae and Ent. aerogenes were also proficient as receptors for several Tsx-specific bacteriophages, whereas the corresponding protein from S. typhimurium did not confer sensitivity against these phages. The nucleotide sequence of the tsx genes from S. typhimurium, K. pneumoniae and Ent. aerogenes was established. Each of the Tsx proteins is initially synthesized with typical bacterial signal sequence peptides and the predicted mature forms of the Tsx proteins have a calculated Mr of 30567 (265 residues), 31412 (272 residues) and 31477 (272 residues), respectively. Multiple sequence alignments between the Tsx proteins showed a high degree of sequence identity and revealed the presence of four hypervariable regions, which are thought to constitute segments of the polypeptide chain exposed at the cell surface. Most notable was a deletion of 8 amino acids in one of these hypervariable domains in the S. typhimurium Tsx protein. When this deletion was introduced by site-directed mutagenesis into the corresponding region of the E. coli tsx gene, the mutant Tsx-515 protein lost its phage receptor function but still served as a colicin K receptor and as a substrate-specific channel, indicating that the region between residues 198 and 207 might be part of the bacteriophage receptor area. Multiple sequence alignments, structural predictions and the properties of previously characterized Tsx missense mutants were taken into account to develop a two-dimensional model for the topological organization of the Tsx protein within the outer membrane.
Product formation and phosphoglucomutase activities in Lactococcus lactis: cloning and characterization of a novel phosphoglucomutase gene
Authors: Qian Ny , Stanley Grant A. , Bunte Annicka , Rdstrm Peter
Published: June 2009

Maltose metabolism in Lactococcus lactis involves the conversion of -glucose 1-phosphate to glucose 6-phosphate, a reaction which is reversibly catalysed by a maltose-inducible and glucose-repressible -phosphoglucomutase (-PGM). The gene encoding -PGM (pgmB) was cloned from a genomic library of L. lactis using antibodies. The nucleotide sequence of a 5695 bp fragment was determined and six ORFs, including the pgmB gene, were found. The gene expressed a polypeptide with a calculated molecular mass of 24210 Da, which is in agreement with the molecular mass of the purified -PGM (25 kDa). A short sequence at the N-terminus was found to be similar to known metal-binding domains. The expression of -PGM in L. lactis was found to be induced also by trehalose and sucrose, and repressed by lactose in the growth medium. This indicates that -PGM does not serve solely to degrade maltose, but that it is also involved in the metabolism of other carbohydrates. The specific activity of a-PGM during fermentation was dependent on the maltose concentration in the medium. The maximum specific activity of -PGM increased by a factor of 4.6, and the specific growth rate by a factor of 7, when the maltose concentration was raised from 0.8 to 11.0 g I-1. Furthermore, a higher amount of lactate produced relative to formate, acetate and ethanol was observed when the initial maltose concentration in the medium was increased. The specific activity of -PGM responded similarly to -PGM, but the magnitude of the response was lower. Preferential sugar utilization and a- and -PGM suppression was observed when L. lactis was grown on the substrate combinations glucose and maltose, or lactose and maltose; maltose was the least-preferred sugar. In contrast, galactose and maltose were utilized concurrently and both PGM activities were high throughout the fermentation.
Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-β-semialdehyde dehydrogenase
Authors: Hoang Tung T. , Williams Scott , Schweizer Herbert P. , Lam Joseph S.
Published: June 2009

asdmutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains ofSalmonella typhimuriumand cloning vectors containingasdas anin vivoselectable marker. As a first step for development of such systems forPseudomonas aeruginosa, theasdgene from wild-type strain PAO1 was cloned by a combined approach of PCR amplification from chromosomal DNA, construction of mini-libraries and by complementation of anEscherichia coli δasdmutant. The nucleotide sequence of a 2433 bpSmal-Nsilfragment was determined. This fragment contained the C-terminal 47 nucleotides ofleuB, encoding 3-isopropylmalate dehydrogenase;asd, encoding aspartate-β-semialdehyde dehydrogenase (Asd); andorfA, whose product showed similarity to the Asd proteins fromVibriospp. By subcloning,asdwas localized to a 1.24 kb DNA fragment which in anE. coliT7 expression system strongly expressed a 40000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins fromP. aeruginosa, E. coliandHaemophilus influenzaerevealed greater than 63% identity, demonstrating the conserved nature of Asd in Gram-negative bacteria, and defined the active-site-containing consensus sequence GGNCTVXMLMXXXLGLF as a possible signature motif. Chromosomal δasdmutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated thatorfAprobably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, theasdgene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 MbpP. aeruginosachromosome.
Intraclonal mating in Trypanosoma brucei is associated with out-crossing
Authors: Gibson Wendy , Winters Kathleen , Mizen Ginny , Kearns Julia , Bailey Mick
Published: June 2009

To examine whether mating can occur within as well as between clones ofTrypanosoma brucei, we transformed threeT. bruceisubspecies stocks with heterologous genes conferring resistance to either hygromycin or Geneticin and carried out a series of inter- and intraclone matings in all possible double drug combinations. Double drug-resistant hybrids were recovered from three of the six out-crosses, but not from any of the three intraclone matings. However, further analysis of cloned progeny trypanosomes from one of the out-crosses using RFLP markers, molecular karyotyping and RAPD (random amplification of polymorphic DNA) produced unequivocal evidence that intraas well as interclone mating had occurred. The progeny of interclone mating were double drug-resistant and heterozygous at 9 of 13 loci examined. In contrast, the progeny of intraclone mating had no demonstrable input of genetic material from the hygromycin-resistant parent and were similar to the Geneticin-resistant parent for most markers, except for five loci which were heterozygous in the Geneticin-resistant parent but homozygous in these clones (aldolase, THT1 glucose transporter, procyclin, tubulin and cDNA 23). In addition, PFGE showed considerable karyotypic rearrangements in these clones and loss of genetic material was evident from RAPD and VSG (variant surface glycoprotein) gene fingerprint analysis. We conclude that intraclone mating can occur in trypanosomes, but only during out-crossing, suggesting that meiosis and/or fusion are triggered by a diffusible factor.
Expression analysis of the ssgA gene product, associated with sporulation and cell division in Streptomyces griseus
Authors: Kawamoto Shinichi , Watanabe Hajime , Hesketh Andrew , Ensign Jerald C. , Ochi Kozo
Published: June 2009

The ssgA gene of Streptomyces griseus B2682, when present in high copy number, results in both suppression of sporulation and fragmented growth of mycelia. Western analysis with polyclonal antibodies against the gene product (SsgA) revealed a close correlation between SsgA accumulation and the onset of sporulation in wild-type cells. The protein was only detected in the cytoplasm. Certain developmental mutants of S. griseus (afs, relC and brgA) which are defective in aerial mycelium formation in solid culture and submerged spore formation in liquid culture failed to accumulate SsgA. The SsgA protein appeared shortly (1 h) after nutritional shift-down of strain B2682 cells, afs mutant cells sporulated and expressed SsgA only when A-factor was present both before and after nutritional shift-down. Introduction of the ssgA gene in a low-copy-number vector into strain B2682 resulted in fivefold overexpression of SsgA, and was accompanied by fragmented growth of mycelia and suppression of submerged spore formation (in liquid culture) and aerial mycelium formation (in solid culture). Streptomycin production was not inhibited. In a control experiment, a nonfunctional ssgA gene possessing a frameshift mutation near its N-terminus had no effect on either growth or sporulation. It is proposed that the ssgA gene product plays a role in promoting the developmental process of S. griseus.
A carboxy-terminal processing protease gene is located immediately upstream of the invasion-associated locus from Bartonella bacilliformis
Authors: Mitchell Samuel J. , Minnick Michael F.
Published: June 2009

A gene with homology to those encoding an unusual class of C-terminal processing proteases that flanks the invasion-associated locusiaIABofBartonella bacilliformishas been identified. The 1302 bp gene, termedctpA,is located immediately upstream of theialAgene and encodes a predicted nascent product of 434 amino acids, producing a mature protein of 411 amino acid residues. TheBartonellaCtpA appears to undergo autolysisin vitro,producing multiple products of 43-46 kDa, and a second group of products of 36-37 kDa. Production of CtpAin vivogives a single product of 41.8 kDa. In addition to a computer-predicted N-terminal secretory signal sequence, the molecular mass differencein vivoversusin vitroindicates that CtpA is likely to be secreted and post-translationally modified. The full-length CtpA protein shows 30% identity to the CtpA protein ofSynechocystissp. 6803 (69% overall sequence similarity). The mature CtpA protein also has significant homology to the tail-specific protease (Tsp) ofEscherichia coli,with 22% identity and 62% similarity to an internal region of the 660 amino acid Tsp. The CtpA protein does not appear to exhibit haemolysin, collagenase, or caseinase activity. ThectpAgene is conserved in allBartonellaspecies examined, as determined by hybridization analyses, but it was not found inBrucella abortusorE. coli.ThectpAgene does not directly affect the erythrocyte-invasion phenotype conferred byiaIAB,but its homology to other stress-response processing proteases implies an important role in survival of this intracellular pathogen.
Molecular characterization of the bet genes encoding glycine betaine synthesis in Sinorhizobium meliloti 102F34
Authors: Pocard Jean-Alain , Vincent Nadine , Boncompagni Eric , Smith Linda Tombras , Poggi Marie-Christine , Rudulier Daniel Le
Published: June 2009

As a first step towards the elucidation of the molecular mechanisms responsible for the utilization of choline and glycine betaine (betaine) either as carbon and nitrogen sources or as osmoprotectants inSinorhizobium meliloti,we selected a Tn5 mutant, LTS23-1020, which failed to grow on choline but grew on betaine. The mutant was deficient in choline dehydrogenase (CDH) activity, failed to oxidize [methyl-14C]choline to [methyl-14C]betaine, and did not use choline, but still used betaine, as an osmoprotectant. The Tn5 mutation in LTS23-1020 was complemented by plasmid pCH034, isolated from a genomic bank ofS. meliloti102F34. Subcloning and DNA sequencing showed that pCH034 harbours two ORFs which showed 60% and 57% identity with theEscherichia coli betBgene encoding betaine-aldehyde dehydrogenase (BADH) andbetAgene encoding CDH, respectively. In addition to the homology withE. coligenes, the deduced sequence of the sinorhizobial BADH protein displays consensus sequences also found in plant BADHs. The deduced sequence of the sinorhizobial CDH protein shares only 21% identical residues with choline oxidase fromArthrobacter globiformis.The structural organization of thebetBAgenes inS. melilotidiffers from that described inE. coli: (i) the two ORFs are separated by a 210 bp sequence containing inverted repeats ressembling a putative rho-independent transcription terminator, and (ii) no sequence homologous tobetT(high-affinity choline transport system) orbetI(regulator) was found in the vicinity of the sinorhizobialbetBAgenes. Evidence is also presented that theS. meliloti betBAgenes are not located on the megaplasmids.
Use of multilocus enzyme electrophoresis to examine genetic relationships amongst isolates of Mycobacterium intracellulare and related species
Authors: Feizabadi Mohammad M. , Robertson Ian D. , Cousins Debby V. , Dawson David J. , Hampson David J.
Published: June 2009

As part of a larger study investigating diversity and distribution of Mycobacterium spp. in Australia, multilocus enzyme electrophoresis was used to assess genetic relationships at 17 enzyme loci amongst a collection of reference strains and isolates initially identified on biochemical and other grounds as M. intracellulare (70), ‘X’ mycobacteria (10), M. scrofulaceum (7), M. avium (8) and M. avium subsp. paratuberculosis (2). Two of the isolates initially identified as M. intracellulare were shown to be quite distinct from the others. Both gave negative results in a species-specific DNA probe test, whilst one was positive by PCR. These results emphasize the uncertainties involved in identifying members of this group. The other M. intracellulare isolates formed a cohesive but diverse group, being divided into 48 electrophoretic types (ETs), with a mean genetic diversity of 0∙38. Forty-three of these ETs contained only single isolates. There was no clear relationship between the serovar and ET designation. The index of association calculated for M. intracellulare was significantly different from zero, suggesting that it is a clonal species. PFGE was also applied to selected isolates from the ETs containing multiple isolates, and some of these could be differentiated further. The strains of M. scrofulaceum and ‘X’ mycobacteria were distinct from M. intracellulare, but themselves were highly heterogeneous, with mean genetic diversities of 0∙66 and 0∙65, respectively. Each of these groups may represent more than one species. M. avium strains were distinct from the two M. avium subsp. paratuberculosis strains, as well as from the other mycobacteria studied.
Escherichia coli flavohaemoglobin (Hmp) reduces cytochrome c and Fe(III)-hydroxamate K by electron transfer from NADH via FAD: sensitivity of oxidoreductase activity to haem-bound dioxygen
Authors: Poole Robert K. , Rogers Nicola J. , D'mello Rita A. M. , Hughes Martin N. , Orii Yutaka
Published: June 2009

Escherichia coli flavohaemoglobin (Hmp) reduced purified mitochondrial cytochrome c aerobically in a reaction that was not substantially inhibited by superoxide dismutase, demonstrating that superoxide anion, the product of O2 reduction by Hmp, did not contribute markedly to cytochrome c reduction. Cytochrome c was reduced by Hmp even in the presence of 0⋅ 5 mM CO, when the haem B was locked in the ferrous, low-spin state, demonstrating that electron transfer to cytochrome c from NADH was via FAD, not haem. Hmp also reduced the ferrisiderophore complex Fe(III)-hydroxamate K from Rhizobium leguminosarum bv. viciae anaerobically in a CO-insensitive manner, but at low rates and with low affinity for this substrate. The NADH-cytochrome c oxidoreductase activity of Hmp was slightly sensitive to the binding and reduction of O2 at the haem. The V
max of cytochrome c reduction fell from 7.1 s-1in the presence of 0⋅5 mM CO to 5⋅0 s-1in the presence of 100 μM O2with no significant change in Km for cytochrome c (6⋅8 to 7⋅3 μM, respectively). O2 at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 μM O2 Thus, Hmp acts as a reductase of broad specificity, apparently without involvement of electron transfer via the globin-like haem. These data are consistent with the hypothesis that Hmp could act as an intracellular sensor of O2 since, in the absence of O2 electron flux from FAD to other electron acceptors increases. However, the nature of such acceptors in vivo is not known and alternative models for O2 sensing are also considered.
Targeted gene-replacement mutagenesis of dcrA, encoding an oxygen sensor of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough
Authors: Fud Rongdian , Voordouw Gerrit
Published: June 2009

A gene-replacement mutagenesis method has been developed for the anaerobic, sulfate-reducing bacteriumDesulfovibrio vulgarisHildenborough and used to deletedcrA,encoding a potential oxygen or redox sensor with homology to the methyl-accepting chemotaxis proteins. A suicide plasmid, containing acat-markeddcrAallele and a counter-selectablesacBmarker was transferred fromEscherichia coliS17-1 toD. vulgarisby conjugation. Following plasmid integration the desireddcrAdeletion mutant (D. vulgarisF100) was obtained in media containing sucrose and chloramphenicol. Southern blot screening was required to distinguishD. vulgarisF100 from strains in which thesacBmarker was inactivated by transposition of an endogenous IS element. No anaerotactic deficiency has so far been detected inD. vulgarisF100, which was found to be more resistant to inactivation by oxygen than the wild-type. Increased transcription of therbo-ruboperon, located immediately downstream fromdcrA,was demonstrated by Northern blotting and may be the cause of this unusual phenotype, in view of the recent discovery that Rbo can complement the deleterious effects of superoxide dismutase deficiency inE. coli.
Plasmid diversity in Chlamydia
Authors: Thomas N. S. , Lusher M. , Storey C. C. , Clarke I. N.
Published: June 2009

Chlamydiae exhibit low interspecies DNA homology and plasmids from different chlamydial species can be readily distinguished by Southern blot analysis and restriction enzyme profiling. In contrast, available plasmid sequence data from within the speciesChlamydia trachomatisindicate that plasmids from human isolates are highly conserved. To evaluate the nature and extent of plasmid variation, the complete nucleotide sequences were determined for novel plasmids from three diverse non-human chlamydial isolates: pCpA1 from avianChlamydia psittaci(N352); pCpnE1 from equineChlamydia pneumoniae(N16); and pMoPn fromC. trachomatismouse pneumonitis. Comparison of the sequence data did not identify an overall biological function for the plasmid but did reveal considerable sequence conservation (> 60%) and a remarkably consistent genomic arrangement comprising eight major ORFs and four 22 bp tandem repeats. The plasmid sequences were close to 7500 nucleotides in length (pCpA1, 7553 bp; pMoPn, 7502 bp) however the equineC. pneumoniaeplasmid was smaller (7362 bp) than all other chlamydial plasmids. The reduced size of this plasmid was due to a single large deletion occurring within ORF 1; this potentially generates two smaller ORFs. The disruption of ORF 1 is the only significant variation identified amongst the chlamydial plasmids and could prove important for future vector development studies.
Effects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evidence for a stress-induced recycling of glycogen and trehalose
Authors: Parrou Jean Luc , Teste Marie-Ange , François Jean
Published: June 2009

It is well known that glycogen and trehalose accumulate in yeast under nutrient starvation or entering into the stationary phase of growth, and that high levels of trehalose are found in heat-shocked cells. However, effects of various types of stress on trehalose, and especially on glycogen, are poorly documented. Taking into account that almost all genes encoding the enzymes involved in the metabolism of these two reserve carbohydrates contain between one and several copies of the stress-responsive element (STRE), an investigation was made of the possibility of a link between the potential transcriptional induction of these genes and the accumulation of glycogen and trehalose under different stress conditions. Using transcriptional fusions, it was found that all these genes were induced in a similar fashion, although to various extents, by temperature, osmotic and oxidative stresses. Experiments performed with an msn2/msn4 double mutant proved that the transcriptional induction of the genes encoding glycogen synthase (GSY2) and trehalose-6-phosphate synthase (TPS1) was needed for the small increase in glycogen and trehalose upon exposure to a mild heat stress and salt shock. However, the extent of transcriptional activation of these genes upon stresses in wild-type strains was not correlated with a proportional rise in either glycogen or trehalose. The major explanation for this lack of correlation comes from the fact that genes encoding the enzymes of the biosynthetic and of the biodegradative pathways were almost equally induced. Hence, trehalose and glycogen accumulated to much higher levels in cells lacking neutral trehalase or glycogen phosphorylase exposed to stress conditions, which suggested that one of the major effects of stress in yeast is to induce a wasteful expenditure of energy by increasing the recycling of these molecules. We also found that transcriptional induction of STRE-controlled genes was abolished at temperatures above 40 °C, while induction was still observed for a heat-shock-element-regulated gene. Remarkably, trehalose accumulated to very high levels under this condition. This can be explained by a stimulation of trehalose synthase and inhibition of trehalase by high temperature.
Xanthobacter flavus employs a single triosephosphate isomerase for heterotrophic and autotrophic metabolism
Authors: Meijer Wim G. , de Boer Paulo , van Keulen Geertje
Published: June 2009

The expression of the cbb and gap–pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ. This mutant was unable to grow autotrophically and had a reduced growth rate of medium supplemented with gluconate or succinate. The regulation of the gap–pgk operon in the mutant was indistinguishable from the wild-type strain, but induction of the cbb operon upon transition to autotrophic growth conditions was delayed. Complementation of the mutant with a genomic library of X. flavus resulted in the isolation of a 1∙1 kb ApaI fragment which restored autotrophic growth of the mutant. One open reading frame (ORF) was present on the ApaI fragment, which could encode a protein highly similar to triosephosphate isomerase proteins from other bacteria. Cell extracts of the mutant grown under glycolytic or gluconeogenic conditions had severely reduced triosephosphate isomerase activities. The ORF was therefore identified as tpi, encoding triosephosphate isomerase. The tpi gene is not linked to the previously identified operons encoding Calvin cycle enzymes and therefore represents a third transcriptional unit required for autotrophic metabolism.
Interruption of the Streptococcus gordonii M5 sspA/sspB intergenic region by an insertion sequence related to IS1167 of Streptococcus pneumoniae
Authors: Demuth Donald R. , Duan Yang , Jenkinson Howard F. , McNab Rod , Gil Sucheol , Lamont Richard J.
Published: June 2009

Streptococcus gordonii M5 and DL1 each express two related adhesin polypeptides, SspA and SspB, which are members of the antigen I/II family of streptococcal surface proteins. The sspA and sspB genes are tandemly arranged in both strains, with sspA residing upstream of sspB. The genes are separated by approximately 400 nucleotides in S. gordonii DL1 and 1300 nucleotides in S. gordonii M5. The nucleotide sequence of the sspA/sspB intergenic region of strain M5 is reported and the difference in length compared to S. gordonii DL1 shown to arise from the presence of an insertion sequence, designated ISSg1, consisting of 1197 bp. The nucleotide sequence of ISSg1 is highly homologous to IS1167 of Streptococcus pneumoniae and is related to a lesser extent to other members of the IS1096 family of bacterial insertion sequences. It contains a single ORF of 1026 bp, encoding a putative transposase polypeptide of 342 amino acids. The deduced transposase sequence exhibits 93% identity with the transposase polypeptides encoded by IS1167. However, the S. gordonii protein lacks a 90 residue central domain that is present in the IS1167 transposase and in the transposase polypeptides encoded by the related IS elements. In addition, the organization of the inverted repeats flanking the transposase gene in S. gordonii differs from IS1167. Extension products generated from a sspB-specific primer indicated that transcription initiates within the intergenic region in both S. gordonii strains, suggesting that sspA and sspB are independently transcribed. Transcription appears to initiate 42 bases upstream of sspB in S. gordonii DL1 In contrast, sspB transcription in M5 initiates at least 125 bases upstream of sspB, in close proximity to the terminal inverted repeat of ISSg1. These results indicate that the sspB promoters of S. gordonii M5 and DL1 are not conserved and suggest that ISSg1 sequences may play a role in directing the expression of sspB in S. gordonii M5.
Conjugative transfer of tet(S) between strains of Enterococcus faecalis is associated with the exchange of large fragments of chromosomal DIMA
Authors: Francois Bénédicte , Charles Murielle , Courvalin Patrice
Published: June 2009

Summary: The tetracycline resistance determinant tet(S) was first detected in antibiotic multiresistant Listeria monocytogenes BM4210 and subsequently in strains of Enterococcus faecalis. Transfer of tet(S) from clinical isolate E. faecalis BM4242 to E. faecalis strains JH2-2 and OG1RF was found to require the presence in the donor strain of the 55 kb conjugative plasmid pIP825. Comparison of restriction endonuclease generated maps of the donor, the two recipients, and of four transconjugants indicated that transfer of tet(S) (i) was from chromosome to chromosome, (ii) resulted in the acquisition of an approximately 40 kb element in the same chromosomal region and (iii) was associated with the exchange of large chromosomal fragments. Similar observations were made following conjugal transfer of tet(S) from four other E. faecalis clinical isolates.
The role of sulfoacetaldehyde sulfo-lyase in the mineralization of isethionate by an environmental Acinetobacter isolate
Authors: King Janice E. , Jaouhari Rabih , Quinn John P.
Published: June 2009

Summary: An environmentalAcinetobacterisolate, strain ICD, utilized isethionate at concentrations up to at least 20 mM as carbon and energy source, with essentially quantitative sulfate accumulation. The initial step in isethionate metabolism is likely to be its oxidation to sulfoacetaldehyde since inducible sulfoacetaldehyde sulfo-lyase activity was demonstrated in isethionate-grown cells byin vitroassay and gel zymography; sulfoacetaldehyde itself did not induce the enzyme. Isethionate-grown cells ofAcinetobactersp. ICD, unlike those of most other C-S bond-cleaving strains described, also contained an inducible sulfite-oxidizing activity. The results provide further evidence that sulfoacetaldehyde sulfo-lyase plays a central role in the mineralization of biogenic sulfonates.
The Staphylococcus aureus allelic genetic loci for serotype 5 and 8 capsule expression contain the type-specific genes flanked by common genes
Authors: Sau Subrata , Bhasin Navneet , Wann Elisabeth R. , Lee Jean C. , Foster Timothy J. , Lee Chia Y.
Published: June 2009

Summary: The nucleotide sequences of two gene clusters, cap5 and cap8, involved in the synthesis of Staphylococcus aureus type 5 and type 8 capsular polysaccharides (CPs), respectively, were determined. Each gene cluster contained 16 ORFs, which were named cap5A through cap5P for type 5 CP and cap8A through cap8P for type 8 CP. The cap5 and cap8 loci were allelic and were mapped to the Smal-G fragment in the standard Smal map of Staph, aureus strain NCTC 8325. The predicted gene products of cap5A through cap5G and cap5L through cap5P are essentially identical to those of cap8A through cap8G and cap8L through cap8P, respectively, with very few amino acid substitutions. Four ORFs located in the central region of each locus are type-specific. A comparison of the predicted amino acid sequences of cap5 and cap8 with sequences found in the databases allowed tentative assignment of functions to 15 of the 16 ORFs. The majority of the capsule genes are likely to be involved in amino sugar synthesis; the remainder are likely to be involved in sugar transfer, capsule chain-length regulation, polymerization and transport.
Medium- and long-chain fatty acid uptake and utilization by Streptomyces coelicolor A3(2): first characterization of a Gram-positive bacterial system
Authors: Banchio Claudia , Gramajo Hugo C.
Published: June 2009

Summary: The first characterization of fatty acid uptake in a Gram-positive bacterium is reported. Streptomyces coelicolor A3(2) utilizes fatty acids of different chain length (C4-C18) as sole carbon and energy sources. In vivo β-oxidation studies and the assay of two enzymes of the β-oxidation cycle proved that fatty acid degradation is constitutive in this micro-organism. Uptake of the medium-chain fatty acid octanoate showed the characteristics of simple diffusion, whereas the uptake of palmitate, a long-chain fatty acid, occurred by both simple diffusion and active transport. After correcting for non-mediated transport, palmitate uptake measured over a wide range of concentrations followed Michaelis-Menten kinetics. The apparent K
m for palmitate was 97.8 μM and the V
max was 19.3 nmol min−1 (mg protein)−1. Competition experiments showed specificity of the mediated transport component for long-chain fatty acids (> C10). Metabolic inhibitors such as oligomycin, NaF and vanadate, and the ionophores gramicidin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited palmitate uptake to different degrees, consistent with the existence of an active transport mechanism. Uptake rates measured at different pH values indicated that both the ionized and the unionized forms of octanoate crossed the cytoplasmic membrane by simple diffusion. Palmitate in its ionized form appears to be transported by an active mechanism, whereas the unionized molecule diffuses through the membrane. When present in the medium, glucose stimulated the degradation of long-chain fatty acids by increasing the rate of uptake and the level of acyl-CoA synthetase.
Reduction of ATPase activity accompanied by photodecomposition of ergosterol by near-UV irradiation in plasma membranes prepared from Saccharomyces cerevisiae
Authors: Arami Shin-ichiro , Hada Megumi , Tada Mikiro
Published: June 2009

Summary: When plasma membranes prepared from the yeastSaccharomyces cerevisiaewere exposed to near-UV radiation, photodecomposition of ergosterol and reduction of ATPase activity occurred simultaneously. TheVmaxfor ATPase activity decreased markedly with increasing near-UV dosage while theKmvalue remained constant. When ATPase solubilized from the plasma membrane was exposed to near-UV, the activity remained constant irrespective of dosage, indicating that the ATPase molecule itself was not damaged by near-UV irradiation. The relationship between content of ergosterol and ATPase activity was examined using liposomes constructed with lipids extracted from the membrane. Maximum activity of ATPase was seen at 5% ergosterol in liposomes; this activity was 2.5 times greater than that in liposomes without ergosterol. Activity of ATPase bound to liposomes with 5% ergosterol was reduced after near-UV irradiation, while the activity remained unchanged in the case of the liposomes without ergosterol. Fluidity of the liposomes with 5% ergosterol also decreased with increasing near-UV dosage. Dosage-response curves for reduction of ATPase activity and for decrease in fluidity were similar to that for photodecomposition of ergosterol. These results suggested that the reduction of ATPase activity in the membrane by near-UV irradiation was not caused by photochemical degradation of the primary structure of the ATPase molecule, but was attributable to conformational change resulting from an alteration in the higher-order structure of the membrane due to photochemical decomposition of ergosterol.
Diversity and differential distribution of IS231, IS232 and IS240 among Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides
Authors: Léonard Catherine , Chen Yahua , Mahillon Jacques
Published: June 2009

Summary: Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides are very closely related bacteria, generally considered as subspecies of B. cereus sensu lato. Different transposable elements have been isolated from B. thuringiensis, including IS231, IS232 and IS240 and their variants. The distribution of these three insertion sequences (IS) within the B. cereus group has been investigated in 90 strains of B. thuringiensis (representing 61 serovars), in 30 reference strains of B. cereus and in 33 strains of B. mycoides. Since these IS elements art delimited by well-conserved and specific inverted repeats, the use of primers corresponding to these ends allowed their amplification by PCR. The results showed that IS231 is the most abundant element in the three taxa, whereas IS232 is apparently exclusively associated with B. thuringiensis. Hybridization and Dral RFLP analysis of the PCR products confirmed and extended knowledge of the heterogeneity previously observed among iso-IS231 elements. Moreover, a similar diversity was observed among iso-IS240 elements. This contrasted with the relative homogeneity displayed by iso-IS232 elements. No specific association appeared to exist between any particular iso-element and a specific strain or serotype.
Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases
Authors: Dalrymple Brian P. , Cybinski Daisy H. , Layton Ingrid , McSweeney Christopher S. , Xue Gang-Ping , Swadling Yolande J. , Lowry J. Brian
Published: June 2009

Summary: Acetylesterase and cinnamoyl ester hydrolase activities were demonstrated in culture supernatant of the anaerobic ruminal fungusNeocallimastix patriciarum.A cDNA expression library fromN. patriciarumwas screened for esterases using β-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydrolase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity.bnaA, bnaBandbnaCencode proteins with several distinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologous to protein-docking domains in other enzymes fromNeocallimastixspecies and another anaerobic fungue, aPiromycessp. The catalytic domains of BnaB and BnaC are members of a recently described family of Ser/His active site hydrolases [Upton, C. & Buckley, J. T. (1995).Trends Biochem Sci20, 178-179]. BnaB exhibits 40% amino acid identity to a domain of unknown function in the CeIE cellulase fromClostridium thermocellumand BnaC exhibits 52% amino acid identity to a domain of unknown function in the XynB xylanase fromRuminococcus flavefaciens.BnaA, whilst exhibiting less than 10% overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the same family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturonan acetylesterase fromAspergillus aculeatus.However, none of the other previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.
A reassessment of the genetic determinants, the effect of growth conditions and the availability of an electron donor on the nitrosating activity of Escherichia coli K-12
Authors: Metheringham Rachael , Cole Jeff A.
Published: June 2009

Summary: Anaerobic, but not aerobic, cultures of Escherichia coli K-12 catalysed the rapid nitrosation of the model substrate 2,3-diaminonaphthalene when incubated with nitrite. Formate and lactate were effective electron donors for the nitrosation reaction, which was inhibited by nitrate. Optimal growth conditions for the expression of nitrosation activity by various strains and mutants were determined. Highest activities were found with bacteria that had been grown anaerobically in a minimal medium rather than in Lennox broth, with glycerol and fumarate rather than glucose as the main carbon and energy source, and in the presence of a low concentration of nitrate. Bacteria harvested in the early exponential phase were more active than those harvested in later stages of growth. Well-characterized mutants defective in the synthesis of one or more anaerobically induced electron transfer chains were screened for nitrosation activity under these optimal growth conditions: only the respiratory nitrate reductase encoded by the narGHJI operon was implicated as a major contributor to nitrosation activity. Due to the limited sensitivity of the assays currently available, a minor contribution from the two alternative nitrate reductases or even other molybdoproteins could not be excluded. The role of formate in nitrosation was complex and was clearly not limited simply to that of an electron donor in the bacterial reduction of nitrite to nitric oxide: at least two further, chemical roles were inferred. This extensive study of more than 400 independent cultures of E. coli K-12 and its derivatives resolved some, but not all, of the apparently conflicting data in the literature concerning nitrosation catalysed by enteric bacteria.
The Staphylococcus aureus scdA gene: a novel locus that affects cell division and morphogenesis
Authors: Brunskill Eric W. , de Jonge Boudewijn L. M. , Bayles Kenneth W.
Published: June 2009

Summary: A new Staphylococcus aureus gene termed scdA was found upstream of the autolysis regulatory genes, lytS and lytR, and was shown to potentially encode a hydrophilic 25 kDa protein. Analysis of scdA transcription revealed that it is transcribed as a monocistronic message and is lytSR-independent. A role in cell wall metabolism was indicated by examination of the scdA mutant S. aureus KB323, which had a grossly aberrant cellular morphology and formed large cell clusters when grown in liquid culture medium. Furthermore, KB323 exhibited a reduced rate of autolysis and had increased peptidoglycan cross-linking compared to the parental strain, NCTC 8325-4. These data suggest that scdA plays an important role in staphylococcal cell division.
An RNA polymerase preparation from Methylobacterium extorquens AM1 capable of transcribing from a methylotrophic promoter
Authors: Davagnino Juan , Springer Amy L. , Lidstrom Mary E.
Published: June 2009

Summary: RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM1 cells grown on methanol or on succinate. The β, β', α and ω subunits were approximately the same size as those of Escherichia coli, and the identity of the ω subunit was confirmed by N-terminal sequence analysis. N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be σ factors, a 40 kDa polypeptide that shared identity with σ32 homologues, and a 97 kDa polypeptide that shared identity with σ70 homologues in other bacteria. The 97 kDa polypeptide did not cross-react with antibody to E. coli σ70. The same complement of putative σ factors was found in RNAP purified from M. extorquens AM1 grown on succinate and those grown on methanol, indicating that no major methanol-inducible σ factor is present in this strain. Run-off assays showed that the purified RNAP was capable of initiating transcription specifically at the transcriptional start site of a methylotrophic gene, mxaF, which encodes the large subunit of methanol dehydrogenase and is found only in methylotrophic bacteria.