bluuebunny/crossref_metadata_2025 huggingface dataset:
A bacteriophage of Rhodopseudomonas blastica
Authors: Gorham H. , Dow C. S.
Published: June 2009

A bacteriophage, øBHG1, was isolated from a small eutrophic pond from which its host,Rhodopseudomonas blastica, was originally obtained. It is a lytic bacteriophage specific forR. blasticawhich also causes non-specific lysis ofRhodobacter sphaeroides8253. øBHG1 has an icosahedral head of 62 nm diameter and a short 39 nm tail. Caesium chloride density gradient centrifugation of infected cell lysates gave a single bacteriophage band at a density of 1.385 g cm-3, but also occasionally a second band was observed at a lower density. No differences were apparent between bacteriophage taken from either of the two bands. øBHG1 contained double-stranded DNA with a size of 48 kb and a G + C content of 50.6 mol%. The bacteriophage adsorbed to both photosynthetically and chemoheterotrophically grownR. blasticaat an identical rate of 1.39 × 10-9ml−1min−1. One-step growth curves and kinetic studies of the bacteriophage under these physiological regimes showed no differences in the latent and rise periods and only slight changes in the burst size. Adsorption of this bacteriophage is cell-surface specific and attachment only occurs to the ‘older’ pole of the budding reproductive cell.
The Plasmid-Located Haloalkane Dehalogenase Gene from Rhodococcus Rhodochrous NCIMB 13064
Authors: Kulakova Anna N. , Larkin Michael J. , Kulakov Leonid A.
Published: June 2009

The haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB 13064 was cloned and sequenced. Its comparison with the previously studied dhIA gene from Xanthobacter autotrophicus GJ10 did not show homology. However, the amino acid sequences of the products of these genes showed approximately 30% identity and several of the catalytic amino acid residues were conserved in the NCIMB 13064 dehalogenase. A high level of dhaA expression was demonstrated in Escherichia coli cells and this gene was shown to encode a dehalogenase with the activity against chloroalkanes of chain length C3-C10. Also, some dehalogenase activity against 1,2-dichloroethane encoded by the cloned dhaA gene was detected. The analysis of NCIMB 13064 derivatives lacking dehalogenase activity showed that the dhaA gene was located on the 100 kbp pRTL1 plasmid. It was also found that reversible rearrangements of DNA in the dhaA region may be responsible for the control of expression of haloalkane dehalogenase in R. rhodochrous NCIMB 13064. A number of repeated and inverted sequences which may cause genetic instability at the locus were found in the haloalkane dehalogenase gene region.
ScCypB is a Novel Second Cytosolic Cyclophilin from Streptomyces Chrysomallus which is Phylogenetically Distant from ScCypA
Authors: Pahl Andreas , Gewies Andreas , Keller Ullrich
Published: June 2009

A novel second streptomycete cyclophilin gene - designated sccypB - was isolated from a cosmid gene library of Streptomyces chrysomallus by using as gene probe a fragment of the previously isolated cyclophilin gene sccypA of the same organism. From its sequence the gene sccypB should encode a protein of Mr
18868. Expression of sccypB in Escherichia coli as a hexaHis-tagged fusion protein (H6ScCypB) and enzymic characterization of the purified protein showed that, like ScCypA, ScCypB is a peptidyl-prolyl cis-trans isomerase (PPIase). The specific activity and substrate specificity of the enzyme were comparable to that of ScCypA, but it was threefold less sensitive to inhibition by cyclosporin A (CsA). In contrast to ScCypA, which is abundant and exists in free and liganded form, ScCypB was 50- to 100-fold less abundant in cytosol-derived protein fractions of S. chrysomallus or Streptomyces lividans, as revealed by Western blot analyses, suggesting a specialized function for this enzyme in the streptomycete cell. Both sccypB and sccypA were found to be present as single copies in the genome of S. chrysomallus and hybridized to a single band in chromosomal DNAs of other streptomycetes. High-level expression of sccypB as well as of sccypA cloned into the expression vector pIJ702 did not produce detectable changes in growth and morphology of S. chrysomallus and S. lividans. Calculations of similarities to known cyclophilin sequences and construction of phylogenetic trees indicated that ScCypB and ScCypA are phylogenetically distant from each other. While ScCypA is clearly related to the eukaryotic cyclophilins, the analyses show the sequence of ScCypB to be the most divergent of all cyclophilin sequences, indicating that it possibly constitutes a cluster by itself.
Malic Enzyme is a Major Source of NADPH for Lipid Accumulation by Aspergillus Nidulans
Authors: Wynn James P. , Ratledge Colin
Published: June 2009

Malic enzyme was shown to be a major source of NADPH for the synthesis of storage lipid by Aspergillus nidulans. A previously produced mutant lacking malic enzyme activity (acuK248) accumulated only half the lipid (12%, w/w, of cell dry weight) accumulated by strains of A. nidulans possessing malic enzyme. When cultivated under conditions designed not to promote the production of storage lipid all three strains of A. nidulans studied contained equivalent amounts of lipid (5%, w/w, of cell dry weight). Thus malic enzyme did not appear to be limiting the synthesis of metabolically active lipid (e.g. membrane lipids). No evidence of a role for malic enzyme in the desaturation of fatty acids was found.
Fungicides and sterol-deficient mutants of Ustilago maydis: plasma membrane physico-chemical characteristics do not explain growth inhibition
Authors: Hernández AgustÍn , Cooke David T. , Lewis Mervyn , Clarkson David T.
Published: June 2009

Plasma membrane vesicles from erg11 and erg2 sterol-deficient mutants and from wild-type Ustilago maydis sporidia treated with and without inhibitors of sterol 14α-demethylase or sterol ∆8–∆7 isomerase (triadimenol and fenpropimorph fungicides, respectively) were purified by aqueous two-phase partitioning. Changes in plasma membrane lipid composition were mostly restricted to sterols and complex lipid-bound fatty acids (CLB fatty acids). There was a greater accumulation of abnormal sterols (14α-methyl-or ∆ 8-unsaturated sterols) in plasma membranes from sterol-deficient mutants than from those treated with their fungicide counterparts. However, greater growth inhibition was observed on fungicide-treated wild-type than on mutants. Changes in CLB fatty acids were restricted to alterations in the relative proportion of linoleic acid (18:2) with respect to oleic acid (18:1). The 18:2 to 18:1 ratio found in CLB fatty acids in plasma membranes could be correlated to rates of sporidial growth but not to accumulation of a particular abnormal sterol or to the extent of sterol replacement. Plasma membrane permeability to protons was increased moderately in the mutants only. No changes were observed in plasma membrane fluidity. Plasma membrane H+-ATPase activity was increased up to twofold in those cases with lower growth rate. It was concluded that fungicide-induced growth inhibition in U. maydis was not due to accumulation of abnormal sterols in plasma membranes but probably due to intracellular ATP depletion by the H+-ATPase and that changes in 18:2 to 18:1 ratio in CLB fatty acids were not directly dependent on the plasma membrane physical state or lipid composition but were possibly part of a stress adaptation mechanism.
Polyethyleneimine is an effective permeabilizer of Gram-negative bacteria
Authors: Helander Ilkka M. , Alakomi Hanna-Leena , Latva-Kala KyÖsti , Koski Pertti
Published: June 2009

The effect of the polycation polyethyleneimine (PEI) on the permeability properties of the Gram-negative bacterial outer membrane was investigated using Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium as target organisms. At concentrations of less than 20 µg ml−1, PEI increased the bacterial uptake of 1-N-phenylnaphthylamine, which is a hydrophobic probe whose quantum yield is greatly increased in a lipid environment, indicating increased hydrophobic permeation of the outer membrane by PEI. The effect of PEI was comparable to that brought about by the well-known permeabilizer EDTA. Permeabilization by PEI was retarded but not completely inhibited by millimolar concentrations of MgCl2. PEI also increased the susceptibility of the test species to the hydrophobic antibiotics clindamycin, erythromycin, fucidin, novobiocin and rifampicin, without being directly bactericidal. PEI sensitized the bacteria to the lytic action of the detergent SDS in assays where the bacteria were pretreated with PEI. In assays where PEI and SDS were simultaneously present, no sensitization was observed, indicating that PEI and SDS were inactivating each other. In addition, a sensitizing effect to the nonionic detergent Triton X-100 was observed for P. aeruginosa. In conclusion, PEI was shown to be a potent permeabilizer of the outer membrane of Gram-negative bacteria.
Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cerous
Authors: Lund Terje , Granum Per Einar
Published: June 2009

The cytotoxicity of the two different enterotoxin complexes ofBacillus cereuswas compared after isolation from three different strains. Protein components of non-haemolytic enterotoxin (NHE) of 39 kDa, 45 kDa and 105 kDa were isolated from all of the three strains, whilst proteins B, L1and L2of haemolysin BL (HBL) were isolated from supernatants of two strains (F837-76 and 1230-88). These proteins were not detected in strain 0075-95. Inhibition of protein synthesis in Vero cells was used as a measure of cytotoxicity. The HBL complex from strain F837-76 was highly toxic. This strain also produced the NHE complex. However, when purified, at least two of the components of NHE had to be present in higher amounts than those of the components of HBL to cause the same degree of toxicity. Both complexes purified from strain 1230-88 were cytotoxic. The amount required to cause the same degree of cytotoxicity was approximately equal for the components of the two complexes, except that higher amounts of the 105 kDa protein of NHE had to be present than for the other components. None of the purified complexes from strain 1230-88 was toxic in amounts comparable to those of the HBL complex of strain F837-76 and NHE of strain 0075-95. These results indicate that when measuring cytotoxic enterotoxins fromB. cereusat least two different complexes and six different proteins have to be taken into consideration.
Targets for pSAM2 integrase-mediated site-specific integration in the Mycobacterium smegmatis chromosome
Authors: Seoane Asunción , Navas Jesús , García Lobo Juan M.
Published: June 2009

An improved integrative cassette from plasmid pSAM2 has been constructed containing plasmid int and attP genes but excluding the xis gene, which should result in increased stability by suppression of the excision reaction. This cassette was included in both suicide and thermosensitive plasmids and used for integration in Mycobacterium smegmatis. Suicide plasmids containing this cassette integrated at a single site (attB1) in the M. smegmatis chromosome. The sequence of the attB1 site has been determined and was identified as a putative tRNAPro gene. Thermosensitive plasmids containing the cassette integrated both at the same attB1 site and at other different sites, often giving rise to simultaneous integration at two sites. A second integration site (attB2) has been sequenced, which was located in the region encoding 16S rRNA of one of the two rrn operons of M. smegmatis.
Pseudomonas aeruginosa in cystic fibrosis: role of mucC in the regulation of alginate production and stress sensitivity
Authors: Boucher J. C. , Schurr M. J. , Yu H. , Rowen D. W. , Deretic V.
Published: June 2009

Alginate production inPseudomonas aeruginosaand the associated mucoid phenotype of isolates from cystic fibrosis patients are under the control of thealgU mucABCDcluster. This group of genes encodes AlgU, theP. aeruginosaequivalent of the extreme heat shock σ factor σEin Gram-negative bacteria, the AlgU-cognate anti-σ factor MucA, the periplasmic protein MucB and a serine protease homologue, MucD. WhilemucA, mucBormucDact as negative regulators of AlgU, the function ofmucCis not known. In this study the role ofmucCinP. aeruginosaphysiology and alginate production has been addressed. Insertional inactivation ofmucCin the wild-typeP. aeruginosastrain PAO1 did not cause any overt effects on alginate synthesis. However, it affected growth ofP. aeruginosaunder conditions of combined elevated temperature and increased ionic strength or osmolarity. inactivation ofmucCinmucAormucBmutant backgrounds resulted in a mucoid phenotype when the cells were grown under combined stress conditions of elevated temperature and osmolarity. Each of the stress factors tested separately did not cause comparable effects. The combined stress factors were not sufficient to cause phenotypically appreciable enhancement of alginate production inmucAormucBmutants unlessmucCwas also inactivated. These findings support a negative regulatory role ofmucCin alginate production byP. aeruginosa,indicate additive effects ofmucgenes in the regulation of mucoidy in this organism and suggest that multiple stress signals and recognition systems participate in the regulation ofalgu-dependent functions.
Manipulation of the physiology of clavulanic acid production in Streptomyces clavuligerus
Authors: Ives Paul R. , Bushell Michael E.
Published: June 2009

This paper reports a novel use of cluster analysis for the identification of intermediary metabolites that are produced at rates closely correlated with those of antibiotic biosynthesis. This information was used to devise culture feeds resulting in enhanced production of clavulanic acid, an antibiotic of current worldwide commercial interest. The feeding strategies apparently alleviated a rate-limiting supply of the C3 precursor of clavulanic acid. C3 limitation may be a consequence of unusual nitrogen and carbon metabolism in Streptomyces clavuligerus. This approach has potential as a generic method for influencing biosynthetic pathway fluxes using feeds without knowledge of the biosynthetic pathway.
Differential HSC70 expression during asexual development of Neurospora crassa
Authors: Fracella Franco , Scholle Carl , Kallies Andreas , Häfker Thomas , Schröder Torsten , Rensing Ludger
Published: June 2009

The constitutive and the heat-shock-induced expression of members of heat-shock protein families changed during vegetative development and conidiation of Neurospora crassa as determined by two-dimensional gel electrophoresis. Western blot, and ELISA analyses revealed the highest amounts of the constitutive heat-shock protein 70 (HSC70) in conidiating aerial hyphae and dormant conidia. During conidial germination the amount of HSC70 decreased and subsequently increased during vegetative growth. Stationary mycelia and young aerial hyphae exhibited the lowest HSC70 level. The stationary-phase-dependent decrease in HSC70 was accompanied by a concomitant increase in its nuclear localization, whereas no significant changes in the amount of nuclear HSC70 were found during aerial hyphae development. The cAMP content during aerial hyphae development was inversely correlated with that of HSC70. To examine possible causal relations between HSC70 expression and cAMP content, the adenylate-cyclase-deficient mutant crisp (cr-1) was analysed, which exhibits low concentrations of endogenous cAMP. This mutant, however, showed a lower constitutive HSC70 level, compared to the bdA strain. Treatment of the bd strain and cr-1 mutant with 20 μM 8-bromo-cAMP did not result in significant changes of the constitutive HSC70 level, but in the level of heat-induced HSC/HSP70. In a developmental mutant (acon-2) which is defective in a differentiation step toward conidiation, the expression of HSC70 in aerial hyphae was delayed until the first proconidial chains were observed. It is concluded that the differential expression of HSC/HSP70 does not depend on different nuclear levels of HSC70 or on changes in cAMP concentrations, but rather on developmental genes controlling conidiation.
Development and verification of fingerprinting probes for Candida glabrata
Authors: Lockhart Shawn R. , Joly Sophie , Pujol Claude , Sobel Jack D. , Pfaller Michael A. , Sol David R.
Published: June 2009

Since Candida glabrata has emerged as one of the most common Candida pathogens, DNA probes have been developed which fulfil the requirements for effective fingerprinting. Using a screen for complex genomic fragments containing moderately repetitive sequences, seven DNA probes were cloned which generate complex Southern blot hybridization patterns with EcoRI-digested C. glabrata DNA. All of the probes are species-specific and the majority cross-hybridize to varying degrees. The capacity of two of the probes, Cg6 and Cg12, to measure genetic distance between independent isolates is verified by comparing clustering in dendrograms based on similarity coefficients computed between all pairs of 39 independent isolates fingerprinted with Cg6, Cg12 and randomly amplified polymorphic DNA. The capacity of the probes Cg6 and Cg12 to assess microevolution in clonal populations of infecting C. glabrata over time is also demonstrated. These probes can now be used in large computer-assisted epidemiological studies.
A neutral trehalase gene from Candida albicans: molecular cloning, characterization and disruption
Authors: Eck Raimund , Bergmann Clemens , Ziegelbauer Karl , Schönfeld Wolfgang , Künkel Waldemer
Published: June 2009

A neutral trehalase gene, NTC1, from the human pathogenic yeast Candida albicans was isolated and characterized. An ORF of 2724 bp was identified encoding a predicted protein of 907 amino acids and a molecular mass of 104 kDa. A single transcript of approxymately 3·2 kb was detected by Northern blot analysis. Comparison of the deduced amino acid sequence of the C. albicans NTC1 gene product with that of the Saccharomyces cerevisiae NTH1 gene product revealed 57% identity. The NTC1 gene was localized on chromosome 1 or R. A null mutant (Δntc1/Δntc1) was constructed by sequential gene disruption. Extracts from mutants homozygous for neutral trehalase deletion had only marginal neutral trehalase activity. Extracts from heterozygous mutants showed intermediate activities between extracts from the wild-type strain and from the homozygous mutants. The null mutant showed no significant differences in pathogenicity as compared to the wild-type strain in a mouse model of systemic candidiasis. This result indicates that the neutral trehalase of C. albicans is not a potential target for antifungal drugs.
The molecular basis for the differential regulation of the hlyE-encoded haemolysin of Escherichia coli by FNR and HlyX lies in the improved Activating Region 1 contact of HlyX
Authors: Green Jeffrey , Baldwin Mandy L.
Published: June 2009

The regulator of fumarate and nitrate reduction (FNR) protein of Escherichia coli is an oxygen-responsive transcription regulator that acts mainly to activate the transcription of genes associated with anaerobic energy generation during periods of oxygen starvation. The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae encodes an FNR homologue, HlyX, which can complement the anaerobic respiratory deficiencies of an fnr mutant. However, FNR and HlyX have distinct but overlapping regulons because during anaerobic incubation, hlyX-expressing E. coli K-12 strains produce an otherwise latent haemolysin. The gene encoding the ‘latent’ haemolysin has been designated hlyE and analysis of the promoter region by DNase I footprinting reveals the presence of an FNR- (HlyX-) binding site. Anaerobic expression of an hlyE::lacZ reporter was 6.5-fold higher in hlyX compared to fnr-expressing cells. Both FNR and HlyX recruited RNA polymerase to the hlyE promoter but formed different ternary complexes. One major transcript (tsp1) initiating at 78.5 bp downstream of the FMR-binding site and four minor transcripts initiating at 73.5 (tsp2), 71.5 (tsp3), 63.5 (tsp4) and 62.5 (tsp5) bp from the FNR site were detected. From the position of the FNR box relative to the transcript starts, hlyE is expressed from a Class I FNR-regulated promoter. Substitution of selected FNR amino acids with the residues found in the equivalent positions in HlyX indicated that Activating Region 1 (AR1) of FNR forms a surface encompassing β9 to β11 and that the AR1 contact at Class I promoters is different to that at Class II promoters, although the same surface is involved. The FNR variant, FNR-A225T, combined the properties of FNR (good activation from Class II promoters) and HlyX (good activation of Class I promoters) and conferred the haemolytic phenotype.
RpoN of the fish pathogen Vibrio (Listonella) anguillarum is essential for flagellum production and virulence by the water-borne but not intraperitoneal route of inoculation
Authors: O'Toole Ronan , Milton Debra L. , Hörstedt Per , Wolf-Watz Hans
Published: June 2009

To investigate the involvement of RpoN in flagellum production and pathogenicity of Vibrio (Listonella) anguillarum, the rpoN gene was cloned and sequenced. The deduced product of the rpoN gene displayed strong homology to the alternative σ54 factor (RpoN) of numerous species of bacteria. In addition, partial sequencing of rpoN-linked ORFs revealed a marked resemblance to similarly located ORFs in other bacterial species. A polar insertion or an in-frame deletion in the coding region of rpoN abolished expression of the flagellin subunits and resulted in loss of motility. Introduction of the rpoN gene of V. anguillarum or Pseudomonas putida into the rpoN mutants restored flagellation and motility. The rpoN mutants were proficient in the expression of other proposed virulence determinants of V. anguillarum, such as ability to grow under low available iron conditions, and expression of the LPS O-antigen and of haemolytic and proteolytic extracellular products. The infectivity of the rpoN mutants with respect to the wild-type strain was unaffected following intraperitoneal injection of fish but was reduced significantly when fish were immersed in bacteria-containing water. Thus, RpoN does not appear to regulate any factors required for virulence subsequent to penetration of the fish epithelium, but is important in the infection of fish by water-borne V. anguillarum.
The highly immunogenic enolase and Hsp70p are adventitious Candida albicans cell wall proteins
Authors: Eroles Pilar , Sentandreu Maria , Elorza M. Victoria , Sentandreu Rafael
Published: June 2009

Screening cDNA libraries with polyclonal antibody preparations against Candida albicans yeast or mycelial cell walls resulted in isolation of several positive clones. Some of them encoded enolase; others encoded a protein of the 70 kDa heat-shock protein family (Hsp70p), etc. The presence of these cytosolic proteins in the cell wall of actively growing C. albicans was discovered by analytical (SDS-PAGE and Western blot) and cytological (indirect immunofluorescence) experiments. Supplementation of cell cultures with papulacandin B, an antibiotic that inhibits formation of the -glucan skeleton, resulted in the release of enolase to the supernatant fluids; this release was prevented when 0.6 M KCI was present as an osmotic stabilizer. The cell wall of C. albicans incorporated exogenously added proteins (enolase and Escherichia coli and C albicans cytosolic proteins). The presence in the C. albicans cell wall of enolase, Hsp70p, and probably other intracellular proteins that are highly immunogenic might help the fungal cells to evade the host defences, and consequently could represent a survival mechanism for C. albicans in vivo.
Functional reconstitution of a purified proline permease from Candida albicans: interaction with the antifungal cispentacin
Authors: Jethwaney Deepa , H�fer Milan , Khaware Raj K. , Prasad Rajendra
Published: June 2009

We have purified proline permease to homogeneity from Candida albicans using an L-proline-linked agarose matrix as an affinity column. The eluted protein produced two bands of 64 and 67 kDa by SDS-PAGE, whereas it produced a single band of 67 kDa by native PAGE and Western blotting. The apparent K
m for L-proline binding to the purified protein was 153 �M. The purified permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. The main features of L-proline transport in reconstituted systems, viz. specificity and sensitivity to N-ethylmaIeimide, were very similar to those of intact cells. The antifungal cispentacin, which enters C. albicans cells via an inducible proline permease, competitively inhibited the L-proline binding and translocation in reconstituted proteoliposomes. However, the uptake of L-proline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent K
m of 40 �M.
IS900 targets translation initiation signals in Mycobacterium avium subsp. paratuberculosis to facilitate expression of its hed gene
Authors: Doran Tim , Tizard Mark , Millar Douglas , Ford Jon , Sumar Nazira , Loughlin Mark , Hermon-Taylor John
Published: June 2009

The Mycobacterium avium subsp. paratuberculosis (formerly Mycobacterium paratuberculosis) atypical insertion sequence, IS900, encodes a novel gene on the complementary strand to the putative transposase, p43. This gene requires a promoter, ribosome binding site (RBS) and termination codon to be acquired upon insertion into the M. avium subsp. paratuberculosis genome and hence is designated the hed (host expression-dependent) gene of IS900. Analysis of IS900 insertion sites suggests that this element targets translation initiation signals in M. avium subsp. paratuberculosis, specifically inserting between the RBS and start codon of a putative gene sequence. This aligns the hed initiation codon adjacent to a functional RBS and possibly downstream of an active promoter, driving expression of Hed protein. We have confirmed this unique targeting process by detecting expression of hed in M. avium subsp. paratuberculosis at the level of transcription by reverse transcription-PCR. Further, two Hed-specific antibodies detected Hed translation products in Western blots of protein extracts from M. avium subsp. paratuberculosis. A recombinant form of Hed expressed and purified from Escherichia coli will facilitate studies of IS900 transposition and will also be assessed as a diagnostic antigen for M. avium subsp. paratuberculosis disease. Implications of IS900 insertion in M. avium subsp. paratuberculosis pathogenicity are discussed.
Analysis of sequences flanking the vap regions of Dichelobacter nodosus: evidence for multiple integration events, a killer system, and a new genetic element
Authors: Bloomfield Garry A. , Whittle Gabrielle , McDonagh Matthew B. , Katz Margaret E. , Cheetham Brian F.
Published: June 2009

Dichelobacter nodosus is the causative agent of ovine footrot. The vap regions of the D. nodosus genome may have arisen by the integration of a genetic element and may have a role in virulence. The virulent D. nodosus strain A198 has multiple copies of the vap regions. In the present study, sequences to the left and right of vap regions 1, 2 and 3 of strain A198 were analysed by Southern blotting and DMA sequencing. The results suggest that vap regions 1 and 2 arose by independent integration events into different tRNA genes. The discovery of a second integrase gene (intB), a gene with similarity to bacteriophage repressor proteins (regA), and a gene similar to an ORF from a conjugative transposon (gepA), suggests that a second genetic element, either a bacteriophage or a conjugative transposon, is integrated next to vap region 3 in the D. nodosus genome. The arrangement of intB and the vap regions in three other virulent strains and one benign strain was determined using using Southern blotting and PCR. One strain, H1215, contained vapE’ and not vapE, and thus resembles vap region 3, suggesting that vap region 3 also may have arisen by an independent integration event. In all strains, a copy of intB was found next to the vap regions. The vap regions contain two genes, vapA and toxA, with similarity to the hig genes of the killer plasmid Rts1. Evidence is presented that vapA and toxA have a similar function in D. nodosus.
Sequence analysis of pqq genes required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1 and the purification of a biosynthetic intermediate
Authors: Toyama Hirohide , Chistoserdova Ludrnila , Lidstrom Mary E.
Published: June 2009

Methylobacterium extorquens AM1 produces pyrroloquinoline quinone (PQQ), the prosthetic group of methanol dehydrogenase. Two gene clusters have been shown to be required for PQQ biosynthesis in this micro-organism and complementation analysis has identified seven pqq genes, pqqDGCBA and pqqEF. The DNA sequence of pqqDGC’ was reported previously. This paper reports the sequence of the genomic region corresponding to pqqC'BA. For consistency, the nomenclature of pqq genes in Klebsiella pneumoniae will be followed. The new nomenclature for pqq genes of M. extorquens AM1 is pqqABCDE and pqqFG. In the genomic region sequenced in this study, two open reading frames were found. One of these encodes PqqE, which showed high identity to analogous pqq genes in other bacteria. PqqE also showed identity to MoaA and NifB in the N-terminal region, where a conserved CxxxCxYC sequence was identified. The sequence of the second open reading frame covered both the pqqC and pqqD regions, suggesting that both functions were encoded by this gene. It is proposed to designate this gene pqqC/D. The deduced amino acid sequence of the pqqC/D product showed identity to PqqC of K. pneumoniae and Pqql of Acinetobacter calcoaceticus in the N-terminal region, and to PqqD of K. pneumoniae and Pqqll of A. calcoaceticus in the C-terminal region. A fragment of M. extorquens AM1 DNA containing only pqqC/D produced a protein of 42 kDa in Escherichia coli, which corresponds to the size of the deduced amino acid sequence of PqqC/D, confirming the absence of a separate pqqD. This genomic region complemented the growth of pqqC mutants of M. extorquens AM1 and Methylobacterium organophilum DSM 760 on methanol. As previously reported for pqq genes of K. pneumoniae, a pqqC mutant of M. extorquens AM1 produced an intermediate of PQQ biosynthesis, which was converted to PQQ by incubation with a crude extract from E. coli cells expressing PqqC/D. The intermediate was found in both crude extract and culture supernatant, and it was purified from the crude extract. The PqqC/D enzyme reaction appeared to require molecular oxygen and reduced nicotinamide adenine dinucleotides.
Identification of novel species-specific antigens of Mycoplasma hyopneumoniae by preparative SDS-PAGE ELISA profiling
Authors: Scarman Anthony L. , Chin James C. , Earmens Graeme J. , Delaney Stephen F. , Djordjevic Steven P.
Published: June 2009

Mycoplasma hyopneumoniae, M. hyorhinisandM. flocculareare commonly isolated from the respiratory tract of pigs and are phylogenetically related. The identification and characterization of antigens specific forM. hyopneumoniaeis crucial for the development of serological reagents and for understanding the mechanisms of pathogenicity of this pathogen. Protein and antigen profiles of six strains ofM. hyopneumoniae,four strains ofM. hyorhinisand a type strain ofM. floccularewere compared using SDS-PAGE and immunoblotting. Five strains ofM. hyopneumoniaeoriginally isolated from diverse geographical regions produced similar protein and antigen profiles. One strain, C1735/2, produced a unique protein profile and was poorly immunoreactive, suggesting that some strains ofM. hyopneumoniaemay possess a structurally modified repertoire of antigens. MajorM. hyopneumoniaeantigens with molecular masses of approximately 36, 43, 48, 52, 76, 78, 80, 82, 94, 106, 114 and 200 kDa were identified by immunoblotting using hyperimmune pig sera raised against both high and low passage strains ofM. hyopneumoniae.Porcine hyperimmune sera raised against the GDL type strain ofM. hyorhinisreacted strongly with allM. hyorhinisstrains although the profiles displayed considerable variation. Major antigens of molecular mass 42, 49, 52, 78, 80 and 82 kDa were identified in type strains GDL and BTS-7 and field strain 2; however, field strain 1 produced a unique profile. A preparative SDS-PAGE profiling (PPP) technique was developed which enabled quantification of the immiunoreactivity of denatured antigens with porcine serum by ELISA. PPP facilitated the rapid identification of species-specific and cross-reactive antigens among the three mycoplasma species. PPP studies revealed several strongly immunoreactiveM. hyopneumoniae-specific antigens of 43, 76, 94, 114 and 200 kDa as well as antigens of molecular mass between 52 and 62 kDa which were not apparent in immunoblotting studies. Rabbit monospecific anti-43 kDa serum reacted specifically with a 43 kDa antigen in whole cell lysates of geographically diverse strains ofM. hyopneumoniaeand failed to cross-react withM. flocculareorM. hyorhiniswhole cell lysates. This study has identified a number of M. hyopneumoniae-specific antigens which warrant further investigation to determine their potential as diagnostic reagents and the role they play, if any, in pathogenicity.
Growth temperature dependence of channel size of the major outer-membrane protein (OprF) in psychrotrophic Pseudomonas fluorescens strains
Authors: D Emmanuelle , Orange Nicole , Saint Nathalie , Gurillon Josette , De Mot Ren , Molle Grard
Published: June 2009

The outer-membrane (OM) permeability of the psychrotrophic bacteriumPseudomonas fluorescensstrain MFO for the -lactam mezlocillin is increased at the optimum growth temperature (28 C) compared to low growth temperatures (8 C). In an attempt to explain this phenomenon, OM protein content was studied in cultures grown at both temperatures. No significant difference in proportion or composition was found, suggesting that a change in the structure and function of porins could be responsible for the differential permeability. The major OM protein OprF of two psychrotrophicP. fluorescensstrains, MFO and OE 28.3, was purified from cultures grown at 8 C and 28 C in order to reincorporate them in solvent-free lipid bilayers. From cultures grown at the same temperature, OprF displayed very similar channel-forming properties for both strains. Decreasing the growth temperature induced a threefold reduction of the major conductance values (250270 pS in 1 M NaCl for 28 C cultures and 8090 pS in 1 M NaCl for 8 C cultures). The trypsin digestion kinetics showed a very different reactivity for these porins between cultures grown at 8 C and 28 C. This may indicate that the pore structure of OprF is modified depending on the growth temperature, as suggested by its functional behaviour.
High resolution X-ray micrography of live Candida albicans using laser plasma pulsed point X-ray sources
Authors: Rajyaguru Jayshree M. , Kado Masataka , Nekula Kerry , Richardson Martin C. , Muszynski Michael J.
Published: June 2009

Electron microscopy is still the most frequently used method for visualization of subcellular structures in spite of limitations due to the preparation required to visualize the specimen. High resolution X-ray microscopy is a relatively new technique, still under development and restricted to a few large synchrotron X-ray sources. We utilized a single-shot laser (nanosecond) plasma to generate X-rays similar to synchrotron facilities to image live cells of Candida albicans. The emission spectrum was tuned for optimal absorption by carbon-rich material. The photoresist was then scanned by an atomic force microscope to give a differential X-ray absorption pattern. Using this technique, with a sample image time of 90 min, we have visualized a distinct 152.24 nm thick consistent ring structure around cells of C. albicans representing the cell wall, and distinct ‘craters’ inside, one of 570.90 nm diameter and three smaller ones, each 400 nm in diameter. This technique deserves further exploration concerning its application in the ultrastructural study of live, hydrated microbiological samples and of macromolecules.
Pirellulosomes: a new type of membrane-bounded cell compartment in planctomycete bacteria of the genus Pirellula
Authors: Lindsay Margaret R. , Webb Richard I. , Fuerst John A.
Published: June 2009

A distinct type of cellular organization was found in two species of the planctomycete genus Pirellula, Pirellula marina and Pirellula staleyi. Both species possess two distinct regions within the cell which are separated by a single membrane. The major region of the cell, the pirellulosome, contains the fibrillar condensed nucleoid. The other area, the polar cap region, forms a continuous layer surrounding the entire pirellulosome and displays a cap of asymmetrically distributed material at one cell pole. Immuno- and cytochemical-labelling of P. marina demonstrated that DNA is located exclusively within the pirellulosome; cell RNA is concentrated in the pirellulosome, with some RNA also located in the polar cap region.
Identification of a 95 kDa putative adhesin from Actinomyces serovar WVA963 strain PK1259 that is distinct from type 2 fimbrial subunits
Authors: Klier Christiane M. , Kolenbrander Paul E. , Roble Arlene G. , Marco Maria L. , Cross Sharon , Handley Pauline S.
Published: June 2009

The species Actinomyces serovar WVA963 is among the 20 bacteria most frequently isolated from human subgingival plaque. The interactions of this species with streptococci are inhibited by lactose, a function associated with type 2 fimbrial surface structures in Actinomyces naeslundii. Type 1 fimbriae mediate binding of cells to salivary proline-rich proteins. Specific polyclonal antisera against type 1 and type 2 fimbriae of A. naeslundii T14V revealed both types of fimbriae on Actinomyces serovar WVA963 strain PK1259. To investigate the role of type 2 fimbriae of strain PK1259 in Actinomyces-Streptococcus lactose-inhibitable coaggregations, spontaneous coaggregation-defective (Cog-) mutants that failed to coaggregate with streptococci were isolated; three were chosen for study. All three mutant strains synthesized type 1 fimbriae and a 59 kDa protein; mutant strains PK2415 and PK3092 synthesized type 2 fimbriae and a 57 kDa protein. In contrast, the Cog-strain PK2407 did not agglutinate with anti-type 2 antibodies or show the 57 kDa band, suggesting that the 57 kDa protein was the type 2 fimbrial subunit. Polyclonal antiserum raised against the Actinomyces serovar WVA963 strain PK2399, an antibiotic-resistant derivative of wild-type PK1259, blocked coaggregation between this strain and streptococci. Anti-PK2399 serum absorbed with mutant strain PK3092 bearing type 2 fimbriae retained its blocking ability. Surface sonicates of the parent and mutant strains were adsorbed to streptococcal cells and to lactose-agarose beads. Lactose eluates from both the streptococcal cells and the affinity beads were characterized by SDS-PAGE and corresponding immunoblots using anti-PK2399 serum absorbed with Cog-mutant PK3092. These blots revealed a 95 kDa putative adhesin in the parent strain PK2399 that was absent in the Cog-mutant strain PK3092. These results suggest the presence of a putative 95 kDa actinomyces adhesin distinct from the 57 kDa type 2 fimbrial subunit and that this adhesin mediates lactose-inhibitable coaggregation with streptococci.
A novel fusidic acid resistance gene from Streptomyces lividans 66 encodes a highly specific esterase
Authors: von der Haar Beate , Walter Stefan , Schwäpenheer Susanne , Schrempf Hildgund
Published: June 2009

Resistance to fusidic acid in Streptomyces lividans is due to secretion of an extracellular enzyme (FusH) that converts the steroid antibiotic into an inactive derivative. NH2-terminal and several internal amino acid sequences were prepared from the purified enzyme. Using one of the deduced oligonucleotides to probe a subgenomic DNA library, the fusH gene was cloned and sequenced. Sequence analysis located an ORF which, owing to the presence of two putative start codons, indicates a predicted protein with 520 or 509 amino acids. A signal peptide was identified by aligning the deduced amino acids with the N-terminal sequence determined for the mature enzyme. The C-terminal part of the deduced FusH contains a region of three tandemly repeated stretches of 50 amino acids, which is preceded and followed by amino acids showing high homology with the repeats. FusH was found to share a GDS motif with some deduced esterases. S. lividans transformants carrying fusH on a multicopy vector synthesized high levels of FusH. Purified FusH cleaved equally well an acetyl, a thioacetyl or a formyl group from the 16β-position of fusidic acid and its derivatives. However, a propionyl group at the 16β-position was attacked with difficulty and a 16β-acetyl group was not hydrolysed at all. These data indicate that FusH is a highly specific esterase. The fusH gene is widely distributed among streptomycetes that modify fusidic acid to its inactive lactone derivative.
Membrane topology analysis of the Bacillus subtilis BofA protein involved in pro-σ K processing
Authors: Varcamonti Mario , Marasco Rosangela , Maurilio De Felice , Sacco Margherita
Published: June 2009

The Bacillus subtilis BofA protein is involved in regulation of pro-σ K processing in the mother cell during the late stages of sporulation. A computer analysis of the BofA amino acid sequence indicates that it is an integral membrane protein. To determine the membrane topology of the protein, a series of gene fusions of bofA with lacZ or phoA reporter genes in Escherichia coli were analysed. A BofA topological model with two membrane-spanning segments, and with the N- and the C-terminal domains located in the region between the inner and outer membranes surrounding the forespore is presented. The analysis of different modifications of the last five amino acid residues of the BofA protein, obtained by PCR site-directed mutagenesis, suggests a possible role of the C-terminal domain in the regulation of pro-σ K processing.
Contact with a solid substratum induces cysts in axenic cultures ofPhysarum polycephalum amoebae: mannitol-induced detergent-resistant cells are not true cysts
Authors: Dee Jennifer , Foxon Jennifer L. , Hill Wendy , Roberts Evaline M. , Walker Muriel H.
Published: June 2009

Previous workers reported thatPhysarum polycephalum amoebae cultured in liquid axenic medium were induced to form cysts by the addition of mannitol. Their criterion for encystment was the formation of detergent (Triton)-resistant cells (TRC). In this study the frequencies of TRC in suspensions of amoebae from various treatments were compared with counts of cell types identified by transmission electron microscopy. Amoebae treated with mannitol in axenic liquid culture formed 50% TRC after 17 h but no walled cysts were found. It was concluded that TRC induced by mannitol were dense, rounded cells without walls. In contrast, TRC formed after growth to stationary phase on bacterial lawns were walled cells. When resuspended in growth medium, most mannitol-induced TRC reverted to active amoebae within a few minutes, whereas TRC formed on bacteria remained Triton resistant for many hours. It was concluded that delayed reversion of TRC was a more reliable indication of wall formation than Triton resistance alone. Transfer of amoebae from liquid culture to the surface of diluted axenic agar medium resulted in the formation of walled cysts identical in appearance with those formed on bacterial lawns. The results indicated that efficient encystment requires a solid substratum as well as nutrient deprivation.
Effects of alternative methyl group acceptors on the growth energetics of the O-demethylating anaerobe Holophaga foetida
Authors: Kappler Oliver , Janssen Peter H. , Kreft Jan-U. , Schink Bernhard
Published: June 2009

The anaerobicbacterium Holophaga foetidacan metabolize the methyl groups of methoxylated aromatic compounds either to acetate or to dimethyl sulphide. The effects of this metabolic flexibility were investigated under conditions of excess; substrate (batch culture) and substrate limitation (chemostat culture). Growth yield data suggest that transfer of the methyl groups to sulphide, in contrast to the homoacetogenic transfer to CO2, was not coupled to energy conservation. Under conditions of excess substrate, methyl groups were quantitatively transferred to sulphide. Growth yields decreased but growth rates increased upon the addition of sulphide during exponential growth in pH- and sulphide-regulated batch cultures. From the measured growth yields, the Gibbs free energy dissipation of catabolism plus anabolism () was calculated using stoichiometric equations incorporating biomass formation (macrochemical equations). The observed increase in growth rate correlated well with an increase in, suggesting a relationship between growth kinetics and growth energetics. During steady-state growth in pH- and sulphide-regulated chemostat culture, a considerable fraction of the methyl groups was converted to acetate, despite the presence of sulphide. This resulted in similar growth yields and correspondingly similarvalues in the presence and absence of sulphide. Apparently,H. foetidauncouples catabolism and anabolism in batch culture under conditions of excess substrate to a greater extent than in the chemostat under substrate limitation, by transferring the methyl groups quantitatively to sulphide and thereby dissipating the Gibbs free energy change of the methyl transfer. The physiological significance of these findings could be thatH. foetidaadjusts the energetics of its metabolism to the growth conditions (i) to maximize the growth rate if substrate is available in excess or, (ii) to maximize the growth yield if substrate is limiting.
Comparative physiology of salt tolerance in Candida tropicalis and Saccharomyces cerevisiae
Authors: García María J. , Ríos Gabino , Ali Rashid , Bellés Jose M. , Serrano Ramón
Published: June 2009

The salt tolerance of the respiratory yeast Candida tropicalis and the fermentative yeast Saccharomyces cerevisiae have been compared in glucose media. C. tropicalis showed a better adaptation to Na+ and Li+ and maintained higher intracellular K+:Na+ and K+:Li+ ratios than S. cerevisiae However, C. tropicalis showed a poorer adaptation to osmotic stress (produced by KCI and sorbitol) and exhibited reduced glycerol production as compared to S. cerevisiae In media with the non-repressing sugar galactose as carbon source, S. cerevisiae exhibited reduced glycerol production and increased sensitivity to osmotic stress. Under these conditions, S. cerevisiae, but not C. tropicalis, utilized trehalose as a more important osmolyte than glycerol. These results suggest that the relative tolerance of yeast to the osmotic and cation toxicities of NaCl, and the underlying relative capabilities for osmolyte synthesis and cation transport, are modulated by the general catabolite control exerted by glucose.