bluuebunny/crossref_metadata_2025 huggingface dataset:
Periplasmic cyclic 1,2-β-glucan in Brucella spp. is not osmoregulated
Authors: Briones Gabriel , de lannino Nora Iñón , Steinberg Marcelo , Ugalde Rodolfo A.
Published: June 2009

Biosynthesis of periplasmic cyclic 1,2-β-glucans inBrucella ovisstrain REO198 andB. abortusstrain S19 was found to be carried out by membrane-bound enzymes that use UDP-glucose (UDP-Glc) as donor substrate. Contrary to what happens in species of the generaAgrobacteriumandRhizobium, the accumulation of the reaction products inBrucellaappeared not to be osmoticaliy regulated. Incubation of permeabilized cells with UDP-[14C]Glc led to the formation of soluble neutral cyclic 1,2-β-glucans and [14C]glucose-labelled glucoproteins. PAGE of pulse–chase experiments carried out with permeabilized cells showed that the molecular mass of the labelled protein was indistinguishable fromAgrobacterium tumefaciensA348 andRhizobium frediiUSDA191 glucoproteins known to be intermediates in the synthesis of cyclic glucans.Brucellatotal membrane preparations were less efficient than permeabilized cells in the formation of cyclic glucan; this was attributed to defective cyclization. Accumulation of protein intermediates having oligosaccharides of high molecular mass that were not released from the protein was observed after chase with 2 mM UDP-Glc. This defect was not observed when permeabilized cells were used as enzyme preparation, thus suggesting that inBrucellaa factor(s) that was lost or inactivated upon the preparation of membranes was required for the effective regulation between elongation and cyclization reactions.
Isolation and characterization of the gene encoding single-stranded-DNA-binding protein (SSB) from four marine Shewanella strains that differ in their temperature and pressure optima for growth
Authors: Chilukuri Lakshmi N. , Bartlett Douglas H.
Published: June 2009

The ssb gene, coding for single-stranded-DNA-binding protein (SSB), was cloned from four marine Shewanella strains that differed in their temperature and pressure optima and ranges of growth. All four
Shewanella
ssb
genes complemented Escherichia coli ssb
point and deletion mutants, with efficiencies that varied with temperature and ssb gene source. The Shewanella SSBs are the largest bacterial SSBs identified to date (24.9-26.3 kDa) and may be divided into conserved amino- and carboxy-terminal regions and a highly variable central region. Greater amino acid sequence homology was observed between the Shewanella SSBs as a group (72-87%) than with other bacterial SSBs (52-69%). Analysis of the amino acid composition of the Shewanella SSBs revealed several features that could correlate with pressure or temperature adaptation. SSBs from the three low-temperature-adapted Shewanella strains were an order of magnitude more hydrophilic than that from the mesophilic strain, and differences in the distribution of eight amino acids were identified which could contribute to either the temperature or pressure adaptation of the proteins. The SSBs from all four Shewanella strains were overproduced and partially purified based upon their ability to bind single-stranded DNA. The differences found among the Shewanella SSBs suggest that these proteins will provide a useful system for exploring the adaptation of protein-protein and protein-DNA interactions at low temperature and high pressure.
Copper-inducible transcriptional regulation at two promoters in the Escherichia coli copper resistance determinant pco
Authors: Rouch D. A. , Brown N. L.
Published: June 2009

The pco determinant of Escherichia coli plasmid pRJ1004 encodes inducible resistance to the trace element copper. The identification of two copper-dependent transcriptional initiation regions within pco that each contain a similar upstream hyphenated dyad motif is described. Deletion constructs showed that this “copper box” motif was essential for copper-inducible activity at both pco promoters, PpcoA and PpcoE. The placement of the motif differs in the two promoters, and PpcoA contains an extended -10 nonamer typical of promoters for which RNA polymerase does not bind specifically to -35 sequences. PpcoE does not contain this motif and is the more strongly expressed promoter. The transcript from PpcoA contains the pcoABCDRS genes, while PpcoE expresses only pcoE. The induction profiles for PpcoA- and PpcoE-lacZ fusions were flattened sigmoidal curves with a gradual response to increasing copper concentration. On high-copy-number plasmids, zinc was found also to induce transcription from both promoters in vivo. Both promoters showed inducible activity in the absence of pcoRS, the plasmid-borne two-component regulatory system, indicating that a second trans-acting regulatory system is present on the chromosome. The pcoR product showed repressor action in the absence of pcoS, while still allowing induction, suggesting the chromosome encoded a similar two-component system to pco. TnphoA insertion mutagenesis identified chromosomal genes which affected promoter expression, including ptsH, ptsl (sugar phosphotransferase system) and cya (adenylate cyclase). The results support that idea that pco-encoded copper resistance is an auxiliary mechanism for handling copper, the regulation of which is integrated with the chromosomal regulation of cellular copper metabolism.
Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11
Authors: Grzeszik Claudia , Lübbers Meike , Reh Michael , Schlegel Hans G.
Published: June 2009

The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus MR11 into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7.4 kbp AsnI fragment of the linear plasmid pHG201 via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hox F, -U, -Y, -H, -W and ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hox F, -U, -Y and -H encode the subunits α, γ, δ and ß, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the ß subunit. Finally, ORF7 encodes a protein which has similarities to cAMP- and cGMP-binding protein kinases, but its function is not known. 0RF1, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus MR11 indicated that the hydrogenase genes are under control of a α70-like promoter located at the right end of ORF1 and are even transcribed under heterotrophic conditions at a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHY1 harbouring the 7.4 kbp AsnI fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.
A 23.4 kb segment at the 69°-70° region of the Bacillus subtilis genome
Authors: Yamamoto Hiroki , Uchiyama Shigeki , Nugroho Fajar Aji , Sekiguchi Junichi
Published: June 2009

Within the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 23 911 bp chromosome segment has been cloned and sequenced. This region (23433 bp; 69°-70° of the genetic map) contains 17 complete ORFs and a partial one. A homology search for the products deduced from the 18 ORFs revealed that twelve of them had significant similarity to known proteins, including the quinolone-resistance protein, ABC transporter, aldehyde dehydrogenase, amino acid transporter, fosmidomycin-resistance protein, CDP-glucose 4,6-dehydratase, glucose-1-phosphate cytidyltransferase and cytochrome P450/NADPH-cytochrome P450 reductase.
Identification of genes in Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins
Authors: Król Jaroslaw , Skorupska Anna
Published: June 2009

The specific interaction between rhizobia and their hosts requires many genes that influence both early and late steps in symbiosis. Three new genes, designated prsD, prsE (protein secretion) and orf3, were identified adjacent to the exo133 mutation in a cosmid carrying the genomic DNA of Rhizobium leguminosarum bv. trifolii TA1. The prsDE genes share significant homology to the genes encoding ABC transporter proteins PrtDE from Erwinia chrysanthemi and AprDE from Pseudomonas aeruginosa which export the proteases in these bacteria. PrsD shows at least five potential transmembrane hydrophobic regions and a large hydrophilic domain containing an ATP/GTP binding cassette. PrsE has only one potential transmembrane hydrophobic domain in the N-terminal part and is proposed to function as an accessory factor in the transport system. ORF3, like PrtF and AprF, has a typical N-terminal signal sequence but has no homology to these proteins. The insertion of a kanamycin resistance cassette into the prsD gene of the R. leguminosarum bv. trifolii TA1 wild-type strain created a mutant which produced a normal amount of exopolysaccharide but was not effective in the nodulation of clover plants.
Hydrogenosomes of Metopus contortus physiologically resemble mitochondria
Authors: Biagini Giancarlo A. , Hayes Anthony J. , Suller Marc T. E. , Winters Carole , Finlay Bland J. , Lloyd David
Published: June 2009

The anaerobic free-living ciliated protozoonMetopus contortusis a grazer in anoxic marine sediments. It does not possess mitochondria, but it does have specialized organelles termed hydrogenosomes which release hydrogen gas. The cationic lipophilic cyanine dye DiOC7(3) is an indicator of transmembrane electrochemical potential. With the aid of confocal laser scanning microscopy (CLSM), the association of this dye with hydrogenosomesin situwas followed. Flow cytometric measurements showed that fluorescence of the membrane potential dye decreased in response to an elevatedpH2in the cell. CLSM also revealed localization of fluorescence of the calcium probe Fluo 3-AM, and of the transmembrane pH gradient probe BCECF-AM, within the lumen of the hydrogenosomes. In addition, hydrogenosomal inclusions were detected. X-ray microanalysis of these electron-dense granules revealed high levels of calcium, phosphate and magnesium. It is concluded thatM. contortushydrogenosomes are calcium stores, have a membrane potential, and an alkaline lumen. These physiological features resemble those of mitochondria in aerobic protozoa.
Highly thermostable endo-1,3-β -glucanase (laminarinase) Lam A from Thermotoga neapolitana: nucleotide sequence of the gene and characterization of the recombinant gene product
Authors: Zverlov Vladimir V. , Volkov Ilia Y. , Velikodvorskaya Tatjana V. , Schwarz Wolfgang H.
Published: June 2009

The nucleotide sequence of clone pTT26 (3786 bp), containing the gene for 1,3-β -glucanase lamA (laminarinase) from Thermotoga neapolitana, was determined. It contains an ORF encoding a protein of 646 aa (73 328 Da). The central part of the protein is homologous to the complete catalytic domain of bacterial and some eukaryotic endo-1,3-β -D-glucanases and belongs to family 16 of glycosyl hydrolases. This domain is flanked on both sides by one copy on each side of a substrate binding domain homologue (family II). The recombinant laminarinase protein was purified from Escherichia coli host cells in two forms, a 73 kDa and a processed 52 kDa protein, both having high specific activity towards laminarin (3100 and 2600 U mg-1, respectively) and K
m values of 2.8 and 2.2 mg ml-1, respectively. Limited activity on 1,3-1,4-β -glucan (lichenan) was detected (90 U mg-1). Laminarin was degraded in an endoglucanase modus, yielding glucose, laminaribiose and -triose as end products. Thus lamA classifies as an endo-1,3(4)-β -glucanase (EC 3.2.1.6). The optimum temperature of the enzymes was 95° (73 kDa) and 85° (52 kDa) at an optimum pH of 6.2. The superior thermostability of the 73 kDa enzyme is demonstrated by incubation without substrate at 100°, where 57% of the initial activity remained after 30 min (82% at 95°). Thus, lamA is the most thermostable 1,3-β -glucanase described to date.
Variation in assimilating functions occurs in spontaneous Candida albicans mutants having chromosomal alterations
Authors: Rustchenko E. P. , Howard D. H. , Sherman F.
Published: June 2009

In this study, four clinical isolates and over 100 colony morphology mutants, previously derived spontaneously from strain 3153A during growth on glucose medium, were examined for their utilization of 21 carbon and 3 nitrogen sources at various growth temperatures. The results demonstrated extensive variability in the pattern of assimilation among the mutants and strains, including both the gain and loss of assimilating functions. The persistent alterations in assimilation patterns observed in sequentially produced subclones illustrated an extensive ability of C. albicans populations to constantly produce new combinations of assimilating functions. The variability among spontaneous mutants derived from a single strain explains the well documented variability among natural isolates. From these results we established a relationship between the previously documented broad spectrum of spontaneous chromosomal aberrations in these mutants to the expression of genes controlling the utilization of alternative carbon and nitrogen sources. The existence of cryptic genes, responsible for growth on alternative substrates, was previously deduced from the analysis of other mutants obtained as a response to the restrictive condition on media containing non-assimilating carbon sources. Thus, mutants with altered assimilation functions can arise either on glucose medium or by selection on restricted media. Extensive differences between the patterns of chromosomal aberrations and the distribution of correlated phenotypes in the two groups of mutants indicated that the same phenotypes may be produced by two different mechanisms involving the same or different genes.
Glucose-dependent, cAMP-mediated ATP efflux from Saccharomyces cerevisiae
Authors: Boyum Rodney , Guidotti Guido
Published: June 2009

Extracellular ATP plays an important role in the physiology of multicellular organisms; however, it is unknown whether unicellular organisms such as yeast also release ATP extracellularly. Experiments are described here which show thatSaccharomyces cerevisiaereleases ATP to the extracellular fluid. This efflux required glucose and the rate was increased dramatically by the proton ionophores nigericin, monensin, carbonyl cyanidem-chlorophenylhydrazone and carbonyl cyanidep-(trifluoromethoxy)-phenylhydrazone; ATP efflux was also increased by the plasma membrane proton pump inhibitor diethylstilbestrol. The increase in the concentration of extracellular ATP was not due to cell lysis or general disruption of plasma membrane integrity as measured by colony-forming and methylene-blue-staining assays. ATP efflux was strictly correlated with a rise in intracellular cAMP; therefore, the cAMP pathway is likely to be involved in triggering ATP efflux. These results demonstrate that yeast cells release ATP in a regulated manner.
The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase
Authors: Notley-McRobb Lucinda , Death Alison , Ferenci Thomas
Published: June 2009

The concentration of glucose in the medium influences the regulation of cAMP levels in Escherichia coli. Growth in minimal medium with micromolar glucose results in 8- to 10-fold higher intracellular cAMP concentrations than observed during growth with excess glucose. Current models would suggest that the difference in cAMP levels between glucose-rich and glucose-limited states is due to altered transport flux through the phosphoenolpyruvate : glucose phosphotransferase system (PTS), which in turn controls adenylate cyclase. A consequence of this model is that cAMP levels should be inversely related to the saturation of the PTS transporter. To test this hypothesis, the relationship between external glucose concentration and cAMP levels inside E. coli were investigated in detail, both through direct cAMP assay and indirectly through measurement of expression of cAMP-regulated genes. Responses were followed in batch, dialysis and glucose-limited continuous culture. A sharp rise in intracellular cAMP occurred when the nutrient concentration in minimal medium dropped to approximately 0∙3 mM glucose. Likewise, addition of >0∙3 mM glucose, but not <0∙3 mM glucose, sharply reduced the intracellular cAMP level of starving bacteria. There was no striking shift in growth rate or [14C]glucose assimilation in bacteria passing through the 0∙5 to 0∙3 mM concentration threshold influencing cAMP levels, suggesting that neither metabolic flux nor transporter saturation influenced the sensing of nutrient levels. The (IIA/IIBC)GIc PTS is 96–97% saturated at 0∙3 mM glucose so these results are not easily reconcilable with current models of cAMP regulation. Aside from the transition in cAMP levels initiated above 0∙3 mM, a second shift occurred below 1 μM glucose. Approaching starvation, well below saturation of the PTS, cAMP levels either increased or decreased depending on unknown factors that differ between common E. coli K-12 strains.
Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR
Authors: Reeve Wayne G. , Dilworth Michael J. , Tiwari Ravi P. , Glenn Andrew R.
Published: June 2009

A mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRI rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced. There was extensive similarity between the ORF disrupted by Tn5 in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rm1021 (71-3% identity over 892 bp). At the protein level there was 70% identity and 93-3% similarity over 267 amino acids with the ExoR protein of R. meliloti Rm1021. Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable. This gene in R. leguminosarum bv. viciae was thus designated exoR. The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R. leguminosarum bv. viciae WSM710. Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R. leguminosarum bv. viciae or R. meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar.
The site-specific recombinase encoded by pinD in Shigella dysenteriae is due to the presence of a defective Mu prophage
Authors: Tominaga Akira
Published: June 2009

The DNA inversion systems are made up of an invertible DNA segment and a site-specific recombinase gene. Five systems are known in prokaryotes: the Salmonella typhimurium H segment and hin gene (H-hin), phage Mu G-gin, phage P1 C-cin, Escherichia coli e14 P-pin, and Shigella sonnei B-pinB systems. In this report a site-specific recombinase (pinD) gene of Shigella dysenteriae was cloned and sequenced. pinD mediated inversion of five known segments at the same extent in E. coll. Although one inv sequence was identified, no invertible region was detected in a cloned fragment. The predicted amino acid sequences of PinD and three ORFs showed high homology to those of Gin and its flanking gene products. An ORF homologous to Mom of Mu conserved a functional activity to modify intracellular plasmid DNA. Southern analysis showed that the cloned fragment contains two homologous regions corresponding to the left and right ends of the Mu genome. Together these results indicated that the pinD gene in S. dysenteriae is derived from a Mu-like prophage.
Modulation of gene expression through chromosomal positioning in Escherichia coli
Authors: Sousa Carolina , de Lorenzo Victor , Cebolla Angel
Published: June 2009

Variations in expression of the nah genes of the NAH7 (naphthalene biodegradation) plasmid of Pseudomonas putida when placed in different chromosomal locations in Escherichia coli have been studied by employing a collection of hybrid mini-Tn5 transposons bearing lacZ fusions to the Psal promoter, along with the cognate regulatory gene nahR. Insertions of Psal-lacZ reporters in the proximity of the chromosomal origin of replication, oriC, increased accumulation of β-galactosidase in vivo. Position-dependent changes in expression of the reporter product could not be associated with local variations of the supercoiling in the DNA region, as revealed by probing the chromosome with mobile gyrB-lacZ elements. Such variations in β-galactosidase activity (and, therefore, the expression of catabolic genes) seemed, instead, to be linked to the increase in gene dosage associated with regions close to oriC, and not to local variations in chromosome structure. The tolerance of strains to the selection markers borne by the transposons also varied in parallel with the changes in LacZ levels. The role of chromosomal positioning as a mechanism for the outcome of adaptation phenotypes is discussed.
Cloning and disruption of the ornithine decarboxylase gene of Ustilago maydis: evidence for a role of polyamines in its dimorphic transition
Authors: Guevara-Olvera Lorenzo , Xoconostle-Cázares Beatriz , Ruiz-Herrera José
Published: June 2009

Summary: The gene encoding ornithine decarboxylase (ODC) from Ustilago maydis was cloned. A conserved PCR product amplified from U. maydis DNA was synthesized and used to screen a genomic library of the fungus. Alignment of its deduced protein sequence with those of other cloned ODCs showed a high degree of homology. Gene replacement was obtained by removal of a central part of the gene and insertion of the hygromycin resistance cassette. The null mutant thus obtained displayed no ODC activity and behaved as a polyamine auxotroph. This result is evidence that a single ODC gene exists in the fungus, and that U. maydis utilizes the ODC pathway as the only mechanism for polyamine biosynthesis. When grown in polyamine-containing media, the null mutant accumulated a polyamine pool which further sustained its normal rate of growth in polyamine-free media for approximately 12-16 h. When putrescine concentrations lower than 0.5 mM were employed, the mutant grew at a normal rate but was unable to engage in the dimorphic transition. Under conditions favourable for mycelial growth, the mutant grew with a yeast-like morphology in liquid media, and formed smooth colonies consisting of yeast cells on solid media. Reversion to normal dimorphic phenotype required high concentrations of putrescine or spermidine. These results are evidence that concentrations of polyamines higher than those necessary to sustain vegetative growth are required for the dimorphic transition in U. maydis.
Monoclonal antibodies as probes for fungal wall structure during morphogenesis
Authors: Marshall Margaret , Gull Keith , Jeffries Peter
Published: June 2009

Summary: Three monoclonal antibodies (mAbs), S4D1, S3B3 and S1E5, were produced from hybridoma cell limes raised from mice immunized with hyphal walls ofNeurospora crassaand one (Pax-1) from mice immunized with hyphal walls ofPaxillus involutus.In immunofluorescence studies, the threeN. crassamAbs recognized epitopes with different patterns of distribution at the hyphal surface ofN. crassa.S4D1 recognized an epitope which was present on the surface of both conidia and hyphae; S3B3 recognized an epitope seen only at the ends of conidia or in the septal region of hyphae and conidial chains; and S1E5 recognized an epitope present on the surface of hyphae, but not on mature conidia. mAb Pax-1 reacted with hyphal wall fragments ofPax. involutusand withN. crassaconidia in a similar way to S3B3. S4D1 reacted with an epitope found in 1,3-α-glucan preparations from hyphal walls of different fungi. The surface distribution of this epitope varied: it was found of the surface of both conidia and hyphae ofN. crassaandAspergillus nidulans,on the basidiospore surface only ofAmanita muscaria,and on the hyphae but not the conidia ofPenicillium chrysogenum.Immunogold studies revealed tha the epitope was present throughout the wall of conidia and hyphae ofN. crassa.mAbs S3B3, S1E5 and Pax-1 also reacted with other fungi: for example Pax-1 cross-reacted with all fungi tested except for a member of the Zygomycota. Immunogold studies revealed that epitopes of these three mAbs were present within the inner layers of the walls of conidia and hyphae ofN. crassa.
Novel thermophilic bacteria producing nitrile-degrading enzymes
Authors: Cramp Rebecca , Gilmour Martin , Cowan Don A.
Published: June 2009

Summary: The first known report of the isolation of thermophilic bacteria which produce nitrile-degrading enzymes is presented. One of the strains isolated was studied in detail. Strain Dac521, classified asBacillus pallidus,was capable of growth on acetonitrile, benzonitrile, propionitrile, acetamide, benzamide and propionamide as the sole carbon and nitrogen source in minimal nutrient media. The strain produced separate aliphatic-nitrile (e.g. acetonitrile)- and aromatic-nitrile (e.g. benzonitrile)-degrading activities. Acetonitrile-degrading activity was produced constitutively and enzyme production was not enhanced by the addition of substrate. Under conditions where benzonitrile was the sole carbon and nitrogen source in minimal nutrient media, acetonitrile-degrading enzyme activity was completely inhibited and benzonitrile-degrading activity was induced. Growth on substrates as sole carbon and nitrogen sources, together with the substrate specificity of cell-free extracts, suggested that acetonitrile and benzonitrile degradation may have occurred via nitrile hydratase and nitrilase pathways, respectively. Both the acetonitrile- and benzonitrile-degrading enzyme systems were significantly more thermostable in whole-cell preparations and cell-free extracts compared to their mesophilic counterparts.
Association of newly discovered IS elements with the dichloromethane utilization genes of methylotrophic bacteria
Authors: Schmid-Appert M. , Zoller K. , Traber H. , Vuilleumier S. , Leisinger T.
Published: June 2009

Summary: Dichloromethane (DCM) dehalogenases enable facultative methylotrophic bacteria to utilize DCM as sole carbon and energy source. DCM-degrading aerobic methylotrophic bacteria expressing a type A DCM dehalogenase were previously shown to share a conserved 4.2 kb BamHI DNA fragment containing the dehalogenase structural gene, dcmA, and dcmR, the gene encoding a putative regulatory protein. Sequence analysis of a 10 kb DNA fragment including this region led to the identification of three types of insertion sequences identified as IS 1354, IS1355 and IS1357, and also two ORFs, orf353 and orf192, of unknown function. Two identical copies of element IS 1354 flank the conserved 4.2 kb fragment as a direct repeat. The occurrence of these newly identified IS elements was shown to be limited to DCM-utilizing methylotrophs containing a type A DCM dehalogenase. The organization of the corresponding dcm regions in 12 DCM-utilizing strains was examined by hybridization analysis using IS-specific probes. Six different groups could be defined on the basis of the occurrence, position and copy number of IS sequences. All groups shared a conserved 5.6 kb core region with dcmA, dcmR, orf353 and orf192 as well as IS1357. One group of strains including Pseudomonas sp. DM1 contained two copies of this conserved core region. The high degree of sequence conservation observed within the genomic region responsible for DCM utilization and the occurrence of clusters of insertion sequences in the vicinity of the dcm genes suggest that a transposon is involved in the horizontal transfer of the DCM-utilization character among methylotrophic bacteria.
The form of folate affects the mechanisms of methotrexate resistance in Enterococcus faecium
Authors: Tamura Tsunenobu , Baggott Joseph E. , Johnston Kelley E. , Li Qing-Jun , Antony Asok C.
Published: June 2009

Summary: Several mechanisms have been described to explain the resistance of cells to methotrexate (MTX); however, the basis for the heterogeneity of mechanisms has been obscure. It was hypothesized that the type of MTX resistance in a single species can be influenced by the form of extracellular folate supplied during the development of resistance. Two strains of MTX-resistant Enterococcus faecium were developed by transferring the bacteria to media containing increasing concentrations of MTX in the presence of constant concentrations of either 5-formyl-5,6,7,8-tetrahydropteroylglutamic acid (5-HCO-H4PteGlu) or pteroylglutamic acid (PteGlu). These resistant strains were designated E. faecium/MTX/5-HCO-H4PteGlu and E. faecium/MTX/PteGlu, respectively. The mechanisms of MTX resistance included: (1) increased folic acid reductase (FAR) activity in both resistant strains but increased dihydrofolate reductase (DHFR) activity only in E. faecium/MTX/PteGlu; (2) decreased synthesis and intracellular retention of MTX containing two glutamyl residues; (3) decreased uptake of MTX accompanied by decreased uptake of folates; and (4) reduction of folate-binding capacity. Among these, the form of folate present in the media during the development of resistance affected DHFR and FAR activities and the transport of folates. These findings, together with data from other laboratories, suggest that it may be important to use a reduced form of folate, a more physiological form than oxidized PteGlu, in the media during the development of resistance for the study of the mechanisms of MTX resistance in cultured cells.
The Klebsiella pneumoniae cytochrome bd’ terminal oxidase complex and its role in microaerobic nitrogen fixation
Authors: Juty Navtej S. , Moshiri Farhad , Merrick Mike , Anthony Christopher , Hill Susan
Published: June 2009

Summary: Cytochrome bd’ has been implicated in having an important role in microaerobic nitrogen fixation in the enteric bacterium Klebsiella pneumoniae, where it is expressed under all conditions that permit diazotrophy. In this paper the sequence of the genes encoding this terminal oxidase (cydAB) of Klebsiella pneumoniae and the characterization of a cyd mutant are reported. The deduced amino acid sequences support the proposal that His 19, His 186 and Met 393 provide three of the four axial ligands to the Fe of the three haems in the oxidase complex. The nitrogen-fixing ability of the mutant was severely impaired in the presence of low concentrations of oxygen compared with the wild-type bacterium. Only the wild-type organism was capable of microaerobic nitrogenase activity supported by fermentation products. It is proposed that formate dehydrogenase-O may be involved in supplying electrons to a respiratory chain terminated by the bd-type oxidase, which would remove inhibitory oxygen and supply ATP for nitrogenase activity.
Molecular characterization of the Serratia marcescens OmpF porin, and analysis of S. marcescens OmpF and OmpC osmoregulation
Authors: Hutsul Jo-Anne , Worobec Elizabeth
Published: June 2009

Summary: Serratia marcescens is a nosocomial pathogen with a high incidence of β-lactam resistance. Reduced amounts of outer-membrane porins have been correlated with increased resistance to β-lactams but only one porin, OmpC, has been characterized at the molecular level. In this study we present the molecular characterization of a second porin, OmpF, and an analysis of the expression of S. marcescens porins in response to various environmental changes. Two porins were isolated from the outer membrane using urea-SDS-PAGE and the relative amounts were shown to be influenced by the osmolarity of the medium and the presence of salicylate. From a S. marcescens genomic DNA library an 8 kb EcoRI fragment was isolated that hybridized with an oligonucleotide encoding the published N-terminal amino acid sequence of the S. marcescens 41 kDa porin. A 41 kDa protein was detected in the outer membrane of Escherichia coli NM522 carrying the cloned S. marcescens DNA. The cloned gene was sequenced and shown to code for a protein that shared 60-70% identity with other known OmpF and OmpC sequences. The upstream DNA sequence of the S. marcescens gene was similar to the corresponding E. coli ompF sequence; however, a regulatory element important in repression of E. coli ompF at high osmolarity was absent. The cloned S. marcescens OmpF in E. coli increased in expression in conditions of high osmolarity. The potential involvement of micF in the observed osmoregulation of S. marcescens porins is discussed.
Towards the phylogeny of APS reductases and sirohaem sulfite reductases in sulfate-reducing and sulfur-oxidizing prokaryotes
Authors: Hipp Wolfgang M. , Pott Andrea S. , Thum-Schmitz Natalie , Faath Ilka , Dahl Christiane , Trüper Hans G.
Published: June 2009

Summary: The genes for adenosine-5′-phosphosulfate (APS) reductase, aprBA, and sirohaem sulfite reductase, dsrAB, from the sulfur-oxidizing phototrophic bacterium Chromatium vinosum strain D (DSMZ 180T) were cloned and sequenced. Statistically significant sequence similarities and similar physicochemical properties suggest that the aprBA and dsrAB gene products from Chr. vinosum are true homologues of their counterparts from the sulfate-reducing chemotrophic archaeon Archaeoglobus fulgidus and the sulfate-reducing chemotrophic bacterium Desulfovibrio vulgaris. Evidence for the proposed duplication of a common ancestor of the dsrAB genes is provided. Phylogenetic analyses revealed a greater evolutionary distance between the enzymes from Chr. vinosum and D. vulgaris than between those from A. fulgidus and D. vulgaris. The data reported in this study are most consistent with the concept of common ancestral protogenotic genes both for dissimilatory sirohaem sulfite reductases and for APS reductases. The aprA gene was demonstrated to be a suitable DNA probe for the identification of apr genes from organisms of different phylogenetic positions. PCR primers and conditions for the amplification of apr homologous regions are described.
A cryptic proline permease in Salmonella typhimurium
Authors: Liao Min-Ken , Gort Steve , Maloy Stanley
Published: June 2009

Summary: Wild-type Salmonella typhimurium expresses three proline transport systems: a high-affinity proline transport system encoded by the putP gene, and two glycine betaine transport systems with a lower affinity for proline encoded by the proP and proU genes. Although proline uptake by the ProP and ProU transport systems is sufficient to supplement a proline auxotroph, it is not efficient enough to allow proline utilization as a sole source of carbon or nitrogen. Thus, the PutP transport system is required for utilization of proline as a carbon or nitrogen source. In this study, an overexpression suppressor, designated proY, which allows proline utilization in a putP genetic background and does not require the function of any of the known proline transport systems, was cloned and characterized. The suppressor gene, designated proY, maps at 8 min on the S. typhimurium linkage map, distant from any of the other characterized proline transport genes. The DNA sequence of the proY gene predicts that it encodes a hydrophobic integral membrane protein, with strong similarity to a family of amino acid transporters. The suppressor phenotype requires either a multicopy clone of the proY
+ gene or both a single copy of the proY
+ gene and a proZ mutation. These results indicate that the proY gene is the structural gene for a cryptic proline transporter that is silent unless overexpressed on a multicopy plasmid or activated by a proZ mutation.
Lectin reactivity and virulence among strains of Listeria monocytogenes determined in vitro using the enterocyte-like cell line Caco-2
Authors: Facinelli Bruna , Giovanetti Eleonora , Magi Gloria , Biavasco Francesca , Varaldo Pietro E.
Published: June 2009

Summary: Forty-six cultures of Listeria monocytogenes (including clinical, food and collection strains) were serotyped, characterized for motility, haemolysis and phospholipase activities and tested for lectin agglutination using a four-lectin set. Lectin reactivity (i.e. agglutination by one or more of the four lectins) was observed in all 12 clinical isolates, 16 of the 23 food isolates and eight of the 11 collection strains. Virulence was evaluated in vitro based on strains' ability to invade the human enterocyte-like cell line Caco-2. In gentamicin survival experiments, recovery of viable intracellular bacteria among lectin-unreactive strains was usually 100-1000-fold lower than among lectin-reactive haemolytic strains, and lower than among nonhaemolytic strains. Considerable cytopathogenic effects were produced by lectin-reactive haemolytic strains in trypan-blue-stained cell monolayers, whereas lectin-unreactive and nonhaemolytic strains produced no detectable cytopathogenic effect. Among lectin-reactive strains, the number of listerial cells associated with Caco-2 monolayers was more than tenfold greater than among lectin-unreactive strains. Cell invasion was inhibited by pretreatment of Caco-2 cells with sugars recognized by the lectins or of listeriae with enzymes which removed the same sugars from the bacterial surface. The results suggest that the study of lectin interactions could be helpful in understanding the pathogenicity potential of isolates of food and environmental origin.
Divercin V41, a new bacteriocin with two disulphide bonds produced by Carnobacterium divergens V41: primary structure and genomic organization
Authors: Metivier Anita , Pilet Marie-France , Dousset Xavier , Sorokine Odile , Anglade Patricia , Zagorec Monique , Piard Jean-Christophe , Marlon Didier , Cenatiempo Yves , Fremaux Christophe
Published: June 2009

SUMMARY: Divercin V41 is a new bacteriocin produced by Carnobacterium divergens V41, a lactic acid bacterium isolated from fish viscera. The amino acid sequence of divercin V41 showed high homologies with pediocin PA-1 and enterocin A. Two disulphide bonds were present in the hydrophilic N-terminal domain and in the highly variable hydrophobic C-terminal domain, respectively. A DNA probe designed from the N-terminal sequence of the purified peptide was used to locate the structural gene of divercin V41. A 6 kb chromosomal fragment containing the divercin V41 structural gene (dvnA) was cloned and sequenced. The results indicate that divercin V41 is synthesized as a pre-bacteriocin of 66 amino acids. The 23-residue N-terminal extension is cleaved off t o yield the mature 43-amino-acid divercin V41. In addition, the fragment encodes putative proteins commonly found within bacteriocin operons, including an ATP- dependent transporter, two immunity-like proteins and the two components of a lantibiotic-type signal-transducing system. The genetic organization of the fragment suggested important gene rearrangements.
The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex
Authors: Rigg Gordon P. , Barrett Brendan , Roberts Ian s.
Published: June 2009

SUMMARY: Biosynthesis of the Escherichis coli K5 polysaccharide requires the Kf iA, Kf iB, KfiC and KfiD proteins. The subsequent transport of the polysaccharide onto the cell surface requires the KpsC, KpsD, KpsE, KpsM, KpsS and KpsT proteins, which are conserved between different group II capsular polysaccharides. The KfiA and KfiC, together with the KpsC, KpsS and KpsT proteins, were purified and polyclonal antisera to each protein generated. These antisera, together with one previously generated (by others) against the purified KfiD protein, were used in Western blot analysis to locate the corresponding proteins within the cell. Analysis of membrane fractions revealed that KfiA (involved in initiation of polysaccharide synthesis), Kf iC (K5 glycosyl transferase) and the Kf iD protein (UDP-glucose dehydrogenase) were associated with the inner membrane. The KpsC, KpsS and KpsT proteins involved in polysaccharide transport were associated with the inner membrane and this membrane association occurred in the absence of any other capsule-related proteins. The effect of mutations in individual kps genes on the localization of each protein was determined. Mutations in the kpC# kpsM, kpsS and kpsT genes resulted in a loss of membrane targeting for KfiA and KfiC, suggesting some form of hetero-oligomeric membrane-bound biosynthetic complex. Osmotic shock caused the release of KfiA, KfiC, KpsC and KpsS from the inner membrane into the periplasm, suggesting that the polysaccharide biosynthetic complex may be associated with sites of adhesion between the inner and outer membrane.
Molecular and biochemical analysis of a 105 kDa Mycoplasma gallisepticum cytadhesin(GapA)
Authors: Goh M. S. , Gorton T. S. , Forsyth M. H. , Troy K. E. , Geary S.J.
Published: June 2009

SUMMARY: The identification of a gene (g8pA) from Mycoplasma gallisepticum with homology to the P I cytadherence gene of Mycoplasma pneumoniae is reported. The gapA gene is a 28 pORF encoding a protein with a molecular mass of 105 kDa. Nucleotide sequence analysis of the gapA gene revealed 45% homology t o the M. pneumoniae P I gene, 46% homology t o the Mycoplasma genitalium MgPa gene and 47% homology to the Mycoplasma pirum PI-like
protein gene. It has a 64 mol% A+T content compared to 46,60 and 72 mol%respectively for the PI, MgPa and the Pl-like protein genes. As with the PI and MgPa genes, gapA is a central gene in a multi-gene operon, but unlike the P1 and MgPa genes, there is only a single copy of gapA in the genome. GapA is a trypsin-sensitive surface-exposed protein. Chicken tracheal-ring inhibition-of-attachment assays, using anti-GapA Fab fragments, resulted in 64% inhibition of attachment. These results indicated that GapA plays a rolein cytadherence of M. gallisepticum to host cells.
Immunization with recombinant class I outermembrane protein from Neisseria meningitidis: influence of liposomes and adjuvants on antibody avidity, recognition of native protein and the induction of a bactericidal immune response against meningococci
Authors: Christodoulides Myron , Brooks Joy L. , Rattue Elizabeth , Heckels John E.
Published: June 2009

SUMMARY: The porA gene from Neisseria meningitidis was cloned into the pRSETA vector and recombinant class 1 outer-membrane protein expressed at high levels in Escherichia coli. The protein was readily purified by affinity chromatography on a Ni2+ matrix and used for immunization of mice with conventional AI(OH), adjuvant, with experimental adjuvants which have the potential for human use, and with liposomes. The resulting sera were analysed for the magnitude, subclass distribution and antigenic specificity of the immune response. In addition, surface plasmon resonance (SPR) was used to quantify antibody avidity by analysis of the kinetics of binding to native class 1 protein. Immunization withconventional and experimental adjuvants induced antibodies of low avidity that did not recognize native class 1 protein. In contrast, immunization with recombinant protein inliposomes induced antibodies of high avidity which recognized native class 1 protein, as measured by their ability t o label meningococcal cells in immunofluorescence assays and to inhibit the binding of a protective mAb. These properties were associated with the presence in sera of high levels of antibodies with the ability t o induce complement-mediated killing of meningococci. These data show that liposomes containing recombinant class 1 protein represent a potential basis of future vaccines, of defined composition, designed for the prevention of group B meningococcal infections.
Siderotyping of fluorescent pseudomonads:characterization of pyoverdines of Pseudornonas fluorescens and Pseudornonas putida strains from Antarctica
Authors: Meyer Jean-Marie , Stintzi Alain , Coulanges Valie , Shivaji Sisinthy , Voss Jessica A. , Taraz Kambiz , Budzikiewic Herbert
Published: June 2009

SUMMARY: Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems. A pyoverdine-related siderotyping, which involved pyoverdine-induced growth stimulation, pyoverdine-mediated iron uptake, pyoverdine analysis by electrophoresis and isoelectric focusing, revealed three different pyoverdine-related siderotypes among the five isolates. One siderotype, including Pseudomonas fluorescens 1W and P. fluorescens lOcW, was identical to that of P. fluorescens ATCC 13525. Two other strains, P. fluorescens 9AW and Pseudomonas putida 9BW, showed identical pyoverdine-related behaviour t o each other, whereas the fifth strain, P. fluorescens 5lW, had unique features compared to the other strains or to a set of 12 fluorescent Pseudomonas strains used as comparison material. Elucidation of the structure of the pyoverdines produced by the Antarctic strains supported the accuracy of the siderotyping methodology by confirming that pyoverdines from strains 1W and 1OcW had the same structures as the P. fluorescens ATCC 13525 pyoverdine, whereas the 9AW and 9BW pyoverdines are probably identical with the pyoverdine of P. fluorescens strain 244. Pyoverdine from strain 51W appeared t o be a novel pyoverdine since its structure was different from all previously established pyoverdine structures. Together with the conclusion that the Antarctic Pseudomonas strains have no special features at the level of their pyoverdines and
pyoverdine-mediated iron metabolism compared to worldwide strains, the present work demonstrates that siderotyping provides a rapid means of screening for novel pyoverdines.