bluuebunny/crossref_metadata_2025 huggingface dataset:
Candida dubliniensis: phylogeny and putative virulence factors
Authors: D. Gilfillan Gregor , Derek J. Sullivan , Parkinson Tanya , Coleman David C. , Gow Neil A.R.
Published: June 2009

Candida dubliniensisis a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to,Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubliniensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubliniensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The G dubliniensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced usingN-acetylglucosamine. Oral G dubliniensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The G dubliniensis isolates were sensitive to fluconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested G albicans secretory aspartyl proteinase (SAP) genes were detected in G dubliniensis by Southern analysis.In vivovirulence assays using a systemic mouse model suggest that G dubliniensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of G dubliniensis and suggest that this species may be particularly adapted to colonization of the oral cavity.
Physiological function and regulation of flavocytochrome c3, the soluble fumarate reductase from Shewanella putrefaciens NCIMB 400
Authors: Gordon Euan H. J , Pealing Sara L. , Chapman Stephen K. , Ward F. Bruce , Reid Graeme A.
Published: June 2009

Shewanella putrefaciens produces a soluble flavocytochrome c under anaerobic growth conditions. This protein shares sequence similarity with the catalytic subunits of membrane-bound fumarate reductases from Escherichia coli and other bacteria and the purified protein has fumarate reductase activity. It is shown here that this enzyme, flavocytochrome c
3, is essential for fumarate respiration in vivo since disruption of the chromosomal fccA gene, which encodes flavocytochrome c
3, leads to a specific loss of the ability to grow with fumarate as terminal electron acceptor. Growth with nitrate, trimethylamine N-oxide (TMAO) and other acceptors was unaffected. The fccA gene is transcribed as a 2 kb monocistronic mRNA. An adjacent reading frame that bears limited sequence similarity to one of the membrane anchor subunits of E. coli fumarate reductase is not co-transcribed with fccA. Expression of the fccA gene is regulated by anaerobiosis and by the availability of alternative electron acceptors, particularly nitrate and TMAO. DNA sequences have been identified that are required for this regulation.
Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358
Authors: Venturi Vittorio , Zennaro Francesca , Degrassi Giuliano , Okeke Benedict C. , Bruschi Carlo V.
Published: June 2009

Transposon Tn5 genomic mutants of plant-growth-promoting Pseudomonas putida strain WCS358 have been isolated which no longer utilize ferulic and coumaric acids as sole sources of carbon and energy. Genetic studies confirmed previous biochemical data showing that ferulic acid is degraded via vanillic acid, and coumaric acid via hydroxybenzoic acid. The genes involved in these enzymic steps were cloned and characterized. Two proteins designated Fca (26.5 kDa) and Vdh (50.3 kDa) were identified as responsible for the conversion of ferulic acid to vanillic acid; the proteins are encoded by the fca and vdh genes which are organized in an operon structure in the chromosome. The Vdh protein is 69% identical at the amino acid level to the Vdh protein recently identified in Pseudomonas sp. strain HR199 and converts vanillin to vanillic acid. Homology studies revealed that the Vdh proteins exhibited significant identity to aldehyde dehydrogenases from different organisms whereas Fca belonged to the enoyl-CoA hydratase family of proteins. Two proteins, designated VanA (39.9 kDa) and VanB (34.3 kDa), encoded by two genes, vanA and vanB, are organized in an operon in the chromosome. They were found to be responsible for the demethylation of vanillic acid to protocatechuic acid. The VanA proteins showed no homology to any other known protein, while VanB belonged to the ferredoxin family of proteins. This two-component enzyme system demethylated another phenolic monomer, veratric acid, thus indicating broad specificity. Studies of the regulation of the vanAB operon demonstrated that the genes were induced by the substrate, vanillic acid; however, the strongest induction was observed when cells were grown in the presence of the product of the reaction, protocatechuic acid.
Analysis of lipids reveals differences between ‘Mycobacterium habana’ and Mycobacterium simiae
Authors: Mederos Lilian M. , Valdivia José A. , Sempere Marco A. , Valero-Guillén Pedro L
Published: June 2009

Fatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of ‘Mycobacterium habana’ and in two strains of Mycobacterium simiae. Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC. The strains of ‘M. habana’ were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-II and GPL-III; group B contained GPL-I, GPL-II’, GPL-II and GPL-III; group C contained GPL-II’, GPL-II and GPL-III; group B did not contain any of these compounds. Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant. Mycolic acids were also similar by TLC and HPLC, and consisted of α-, α- and ketomycolates. Partial structural analysis by MS carried out in strains ‘M. habana’ TMC 5135 and M. simiae ATCC 25275T revealed that α- and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and α-mycolates from 58 to 67 carbon atoms. The α- and ketomycolates belonged to several structural series, and minor variations were found between the two strains examined. The data obtained justified the synonymy between ‘M. habana’ and M. simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis. Most strains of ‘M. habana’ can be defined by the presence of GPL-II and GPL-III, a finding that could be useful in the quality control of potential vaccine strains.
Structure of asparagine-linked oligosaccharides of an aspartic proteinase from the zygomycete fungus Rhizomucor pusillus
Authors: Murakami Kohji , Takeuchi Kyoko , Beppu Teruhiko , Horinouch Sueharu
Published: June 2009

The zygomycete fungus Rhizomucor pusillus (previously called Mucor pusillus) secretes an aspartic proteinase containing two asparagine-linked, high-mannose type oligosaccharide chains at Asn79 and Asn188. For structural elucidation of the carbohydrate moieties, the protein was divided into two portions, an N-terminal portion containing Asn79 and a C-terminal portion containing Asn188, by a specific autocatalytic cleavage under alkaline conditions. Each of the asparagine-linked oligosaccharides was then released by peptide-N-glycosidase F digestion and pyridylaminated with a fluorescent reagent, 2-aminopyridine, at the reducing end. High-performance liquid chromatography analyses showed that the structure of the asparagine-linked oligosaccharide chain attached to residue Asn79 was Man5 GlcNAc2, and that bound to residue Asn188 was Man5 GlcNAc2 and Man5GlcNAc2. These observations suggest that the processing of mannose residues in asparagine-linked oligosaccharides in the Golgi apparatus of Rhizomucor resembles that in mammalian cells.
Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris
Authors: Hu Nien-Tai , Hung Ming-Ni , Chen David Chanhan , Tsai Rong-Tzong
Published: June 2009

XpsD is an outer-membrane protein required for extracellular protein secretion in Xanthomonas campestris pv. campestris. Cross-linking and gel-filtration chromatography analyses have suggested that it forms a multimer. To determine its structure-function relationship, linker-insertion mutants were constructed in an xpsD gene carried on a plasmid. To assay for secretion function, each mutant gene was introduced into an xpsD::Tn5 mutant strain (XC1708) and assayed for α-amylase secretion on starch plates. To test whether the mutant genes exerted a dominant-negative effect, each was introduced into the parental strain XC1701 and examined for secretion interference. Nine functional, one semi-functional and eleven non-functional mutants were obtained. All the non-functional mutants, except two for which the mutant proteins were undetectable on immunoblots, showed interference of normal secretion. The insertion sites in the different mutant proteins are randomly distributed throughout the entire sequence of the XpsD protein. All the permissive insertion sites are located where β-turn or coiled secondary structure is predicted. Over half of the non-permissive sites are located within predicted helical or β-sheet regions. By pretreating total membranes of XC1701 in SDS at 50 °C, an immunoreactive band with high molecular mass (HMM) could be detected that remained in the stacking gel during SDS-PAGE. The semi-functional and all functional mutant proteins formed HMM complexes that were as SDS-resistant as those of the wild-type, whereas all except three of the non-functional mutant proteins formed HMM structures that were less resistant to SDS than the wild-type. By analysing the appearance of SDS-resistant HMM complexes, we were able to detect conformational alterations in XpsD that are too subtle to be detected by other assays.
Multiple oligomieric forms of glucoses-6-phosphate dehydrogenase in cyanobacteria and the role of OpeA in the assembly process
Authors: Sundaram Shanthy , Karakaya Haydar , Scanlan David J. , Mann Nicholas H.
Published: June 2009

Multiple molecular forms of glucose-6-phosphate dehydrogenase (G6PDH) were detected by activity staining in non-denaturing polyacrylamide gels of cell-free extracts from a range of cyanobacteria including Anabaena sp. PCC 7120, Synechococcus sp. PCC 7942, Plectonema boryanum PCC 73110, Synechocystis sp. PCC 6803, Nostoc sp. MAC PCC 8009 and the marine strain Synechococcus sp. WH7803. In most of the species tested, the profile of G6PDH activities was modulated by the growth of the cells in the presence of exogenous 10 mM glucose. Using an antiserum raised against a fragment of G6PDH from Anabaena sp. PCC 7120, it was shown that the different molecular forms of G6PDH all contained an antigenically related subunit, suggesting that the different forms arose from different quaternary structures involving the same monomer. An insertion mutant of Synechococcus sp. PCC 7942 was constructed in which the opcA gene, adjacent to zwf (encoding G6PDH), was disrupted. Although no reduction in the amount of G6PDH monomers (Zwf) was observed in the opcA mutant, activity staining of native gels indicated that most of this protein is not assembled into one of the active oligomeric forms. The oligomerization of G6PDH in extracts of the opcA mutant was stimulated in vitro by a factor present in crude extracts of the wild-type, suggesting that the product of the opcA gene is involved in the oligomerization and activation of G6PDH.
Pleiotropic effects of potassium deficiency in a heterocystous, nitrogen-fixing cyanobacterium, Anabaena torulosa
Authors: Alahari Anuradha , Apte Shree K.
Published: June 2009

Omission of potassium from the growth medium caused multiple metabolic impairments and resulted in cessation of growth of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anahaena torulosa, during both diazotrophic and nitrogen-supplemented growth. Prominent defects observed during potassium deprivation were: (i) the loss of photosynthetic pigments, (ii) impairment of photosynthetic functions, (iii) reduced synthesis of dinitrogenase reductase (Fe-protein), (iv) inhibition of nitrogenase activity, and (v) specific qualitative modifications of protein synthesis leading to the repression of twelve polypeptides and synthesis and accumulation of nine novel polypeptides. The observed metabolic defects were reversible, and growth arrested under prolonged potassium deficiency was fully restored upon re-addition of potassium. Such pleiotropic effects of potassium deficiency demonstrate that apart from its well-known requirement for pH and turgor homeostasis, K+ plays other vital specific roles in cyanobacterial growth and metabolism.
Distribution of 14C-labelleed carbon from glucose and glutamate during anaerobic growth of Saccharomyces cerevisiae
Authors: Albers Eva , Gustafsson Lena , Niklasson Claes , Lidén Gunnar
Published: June 2009

The distribution of carbon from glucose and glutamate was studied using anaerobically grown Saccharomyces cerevisiae. The yeast was grown on glucose (20 g I-1) as the carbon/energy source and glutamic acid (3.5 g I1) as additional carbon and sole nitrogen source. The products formed were identified using labelled [U-14C]glucose or [U-14C]glutamic acid. A seldom-reported metabolite in S. cerevisiae, 2–hydroxyglutarate, was found in significant amounts. It is suggested that 2-hydroxyglutarate is formed from the reduction of 2-oxoglutarate in a reaction catalysed by a dehydrogenase. Succinate, 2-oxoglutarate and 2-hydroxyglutarate were found to be derived exclusively from glutamate. Based on radioactivity measurements, 55%, 17% and 14% of the labelled glutamate was converted to 2-oxoglutarate, succinate and 2-hydroxyglutarate, respectively, and 55%, 9% and 3% of the labelled glucose was converted to ethanol, glycerol and pyruvate, respectively. No labelled glucose was converted to 2-oxoglutarate, succinate or 2-hydroxyglutarate. Furthermore, very little of the evolved CO2 was derived from glutamate. Separation of the amino acids from biomass by paper chromatography revealed that the glutamate family of amino acids (glutamic acid, glutamine, proline, arginine and lysine) originated almost exclusively from the carbon skeleton of glutamic acid. It can be concluded that the carbon flow follows two separate paths, and that the only major reactions utilized in the tricarboxylic acid (TCA) cycle are those reactions involved in the conversion of 2-oxoglutarate to succinate.
Structural differences between two types of basidiomycete septal pore caps
Authors: Müller Wally H. , Montijn Roy C. , Humbel Bruno M. , van Aelst Adriaan C. , Boon Eline J. M. C. , van der Krift Theo P. , Boekhout Teun
Published: June 2009

The septal pore cap (SPC) of Trichosporon sporotrichoides CBS 8245 is vesicular-tubular, connected with flat-tubular endoplasmic reticulum (ER), and stains densely with zinc/iodine/osmium tetroxide, as does the ER. The SPC of Schizophyllum commune CBS 340.81 is more complex, about 600 nm in diameter, with perforations of 80--120 nm diameter, and stains less densely with zinc/iodine/osmium tetroxide than the ER. In high-pressure frozen and freeze-substituted hyphae of T. sporotrichoides the ER is present parallel to the dolipore septa, and electron-dense material occurs opposite the septal pore channel; the SPC rarely showed smooth vesicular-tubular membranes, suggesting that this is an ephemeral function of the SPC. The SPC of S. commune has a smooth outer and inner membrane, which enclose a matrix with a palisade-like substructure. A thin layer of electron-dense material covers the inner surface of the SPC of S. commune, from which beaded filamentous structures connect the SPC and the pore-occluding material. These filamentous structures may maintain the intracellular position of the SPC and possibly play a role in plugging the septal pore channel. The septal pore swellings of T. sporotrichoides contain more 1,6--glucan than the septum, and intracellular glucans are also present near the septal pore channel. This cytosolic 1,6--glucan in T. sporotrichoides may serve as a matrix to keep the tubular membranous structures of the SPC together. In contrast, 1,6--glucan is not observed in the SPC and in the pore-occluding material of S. commune, and hyphal septa of this species show less labelling of 1,6--glucan than the septal swelling. The evolutionary transition from simple to more complex types of SPCs may have resulted in a requirement for different components to maintain the morphological integrity and cell biological function.
A transposition-induced mutant of Nostoc ellipsosporum implicates an arginine-biosynthetic gene in the formation of cyanophycin granules and of functional heterocysts and akinetes
Authors: Leganés F. , Fernández-Piñas F. , Wolk C. P.
Published: June 2009

In strain NE1 of Tn5-1058-mutagenized Nostoc ellipsosporum, the transposon was found within a gene whose translation product is similar in amino acid sequence to the arginine-biosynthetic protein N-acetylglutamate semialdehyde dehydrogenase encoded by argC of Bacillus subtilis. The argC reported from Anabaena sp. strain PCC 7120 hybridized to a sequence different from the one interrupted by the transposon in NE1. The newly identified gene from N. ellipsosporum was denoted argL. The argL mutation renders certain processes in strain NE1 conditionally dependent on provision of L-arginine. Heterocysts and apparent akinetes that formed in the absence of added L-arginine failed to fix dinitrogen or to germinate, respectively, and lacked granules of cyanophycin, composed of copolymers of arginine and aspartic acid. However, apparent akinetes that differentiated upon growth of the mutant in the presence of L-arginine plus nitrate formed cyanophycin granules and could regenerate a new culture.
The lipopolysaccharide biosynthesis locus of Campylobacter jejuni 81116
Authors: Fry Ben N. , Korolik Victoria , ten Brinke Janna A , Pennings Maarten T. T , Zalm Robert , Teunis Bart J. J , Coloe Peter J. , van der Zeijst Bernard A. M
Published: June 2009

MostCampylobacter jejunistrains express lipo-oligosaccharides. Some strains also express lipopolysaccharides (LPS), with O-antigen-like carbohydrate repeats.C. jejuni81116 expresses an LPS containing both lipo-oligosaccharides and O-antigen-like repeats, but nothing is known about the structure or sugar composition of these LPS species. A cosmid library of the genome of C.jejuni81116 was constructed and probed withCampylobacter hyoileigenes involved in LPS synthesis. Five cosmids hybridized with the probe and two of these expressed C.jejuni81116 LPS inEscherichia coli. By subcloning, a 16 kb DNA region was identified which contains the genetic information required to express C.jejuniLPS. DNA sequence analysis revealed 11 ORFs homologous to genes involved in LPS synthesis of other bacteria. They consisted of three homologues of sugar biosynthesis genes, two homologues of transport genes and six homologues of sugar transferases.
16S rRNA gene sequences of ‘Candidatus Campylobacter hominis’, a novel uncultivated species, are found in the gastrointestinal tract of healthy humans
Authors: Lawson Andrew J. , Linton Dennis , Stanley John
Published: June 2009

Although some Campylobacter species are agents of gastroenteritis and periodontal disease in humans, little is known of the variety of campylobacters in the gastrointestinal tract of healthy individuals. This paper provides evidence for the existence of a previously undescribed, uncultivated Campylobacter species that may be a commensal in the healthy human gut. Saliva and faeces from 20 healthy individuals were examined by PCR assays specific for nine species of campylobacter (C. sputorum, C. concisus, C. upsaliensis, C. helveticus, C. Iari, C. fetus, C. hyointestinalis, C. jejuni and C. coli) and for the genus as a whole. Genus-specific amplicons were produced from 19 of 20 saliva samples and from 18 of 20 faecal samples. C. concisuss species-specific amplicons were produced from 19 of 20 saliva samples and 3 of 20 faecal samples. The faecal samples were all PCR-negative for other Campylobacter species. Three unidentified 16S rRNA Campylobacter genus-specific amplicons of faecal origin were sequenced. Phylogenetic analysis showed that these sequences were 99% similar, and clustered within the genus as a novel group which was termed HS (HS = healthy subject). A PCR primer pair specific for the HS group was designed from the sequence data and used to reexamine the original samples. Although it was not possible to culture the organism from faeces, specific PCR assay detected it in 10 of the 20 faecal samples, but not in any corresponding saliva samples. The authors propose that the source of the amplicons is a previously undescribed and so far uncultivated species, which they term ‘Candidatus Campylobacter hominis’.
rRNA-targeted fluorescent in situ hybridization analysis of bacterial community structure in river water
Authors: Kenzaka Takehiko , Yamaguchi Nobuyasu , Tani Katsuji , Nasu Masao
Published: June 2009

An improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2′-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Flavobacterium-Cytophaga; Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes; Vibrio-Aeromonas; Pseudomonas (rRNA I); the genus Acinetobacter] were used to investigate the bacterial community structure at two sites differing in organic carbon pollution level. At the eutrophic site, 54-68% of all cells visualized by staining with DAPI (4′,6-diamidino-2-phenylindole) could be detected with probe EUB338. In samples from the oligotrophic site, 39-45% of the total cells hybridized with EUB338. At the eutrophic site, approximately 50% of the total cells were identified with the five group-specific probes; the bacterial community structure was dominated by the Flavobacterium-Cytophaga group and Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes group. At the oligotrophic site, only 26-38% of the total cells were identified with the five group-specific probes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectable cells differed. No appreciable change was found in the community structure during the sampling period at either site. The improved HNPP-FISH technique should be a useful tool for the analysis of microbial community composition.
Cloning and heterologous expression of the gene for BLIP-II, a -lactamase-inhibitory protein from Streptomyces exfoliatus SMF19
Authors: Park Hyeon Ung , Lee Kye Joon
Published: June 2009

A -lactamase-inhibitory protein (BLIP-II) was purified from the culture filtrate of Streptomyces exfoliatus SMF19 and its N-terminal amino acid sequence was determined. A clone containing the gene encoding BLIP-II (bliB) was selected from a cosmid library by colony hybridization using an oligonucleotide probe based on the N-terminal amino acid sequence of BLIP-II. The bliB gene was isolated and sequenced. Analysis of the nucleotide sequence revealed that the gene consists of 1116 bp and encodes a mature protein of 332 amino acids preceded by a 40 amino acid signal sequence. bliB, expressed under the control of the T7 promoter in Escherichia coli, was accumulated in an inactive form in inclusion bodies, but -lactamase-inhibitory activity was recovered after refolding. In addition, bliB was heterologously expressed in Streptomyces lividans TK24 using the me/C1 promoter. The BLIP-II protein produced in recombinant strains of S. lividans was secreted into the culture supernatant in a biologically active form.
Lysogenic bacteriophage M1 from Selenomonas ruminantium: isolation, characterization and DNA sequence analysis of the integration site
Authors: Cheong Judy P. E , Brooker John D.
Published: June 2009

Bacteriophage M1 from the ruminal bacterium Selenomonas ruminantium strain ML12 comprises a 30 nm icosahedral capsid, a 25 nm tail and 48 kb of linear dsDNA with cohesive ends. A restriction map of the phage genome has been constructed. The presence of bacteriophage M1 in the rumen has been demonstrated by PCR amplification and Southern blot analysis of DNA from rumen bacterial samples obtained from ten different sheep. Lysogeny was demonstrated by hybridization of M1 DNA to host chromosomal DNA and by identification and cloning of a 2.3 kb region of the phage containing the predicted attP domain which promotes chromosomal integration. DNA sequencing of the attP region demonstrated two major ORFs surrounding the predicted attP site and structural analysis of this region revealed a motif comprising three different inverted repeats surrounding a 12 bp palindrome. Analysis of the translated amino acid sequence upstream of the attP site demonstrated the presence of conserved residues found within integrase proteins of several temperate phages of different bacterial species.
Asymmetric reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli and Proteus species
Authors: Hanlon Steven P. , Graham Daniel L. , Hogan Philip J. , Holt Robert A. , Reeve Christopher D. , Shaw Anthony L. , McEwan Alastair G.
Published: June 2009

The enantioselective reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli, Proteus mirabilis and Proteus vulgaris was investigated. Purified dimethyl sulfoxide reductase from Rhodobacter capsulatus catalysed the selective removal of (S)-methyl p-tolyl sulfoxide from a racemic mixture of methyl p-tolyl sulfoxide and resulted in an 88% recovery of enantiomerically pure (R)-methyl p-tolyl sulfoxide. Rhodobacter capsulatus was shown to be able to grow photoheterotrophically in the presence of certain chiral sulfoxides under conditions where a sulfoxide is needed as an electron sink. Whole cells of Rhodobacter capsulatus were shown to catalyse the enantioselective reduction of methyl p-tolyl sulfoxide, ethyl 2-pyridyl sulfoxide, methylthiomethyl methyl sulfoxide and methoxymethyl phenyl sulfoxide. Similarly, whole cells of Escherichia coli, Proteus mirabilis and Proteus vulgaris reduced these sulfoxides but with opposite enantioselectivity.
Nitrate reduction and the isolation of Nit− mutants in Hansenula polymorpha
Authors: Pignocchi Cristina , Berardi Enrico , Cox Brian S.
Published: June 2009

Hansenula polymorpha (syn. Pichia angusta) is able to grow on nitrate as sole nitrogen source. Nitrate reductase (NR) assays, optimized in crude extracts from nitrate-grown cells, revealed that NR preferentially used NADPH, but also used NADH, as electron donor and required FAD for maximum activity. NR activity was present in nitrate-grown and nitrite-grown cells, and was absent in cells grown in ammonium, glutamate and methylamine. Addition of reduced nitrogen compounds to nitrate-grown cells led to loss of NR activity, even if added with nitrate. Under nitrogen starvation, NR activity was not observed; however, following growth on nitrate, NR activity is maintained in the absence of nitrate. Increases but not decreases in NR activity were dependent on protein synthesis. Conditions for chlorate selection were optimized, and Nit− (nitrate−) mutants were isolated. Some of these mutants showed reduced or absent NR activity. Sixty-one NR− mutants revealed the monogenic recessive nature of their lesions and were grouped in 10 complementation classes. These mutants will be used in gene cloning experiments aimed at identifying structural and regulatory elements involved in the first step of nitrate reduction.
The Rhizopus oryzae secreted aspartic proteinase gene family: an analysis of gene expression
Authors: Farley Peter C. , Sullivan Patrick A.
Published: June 2009

Rhizopus oryzae was shown to possess a secreted aspartic proteinase gene family (sap) of at least four members (sap1-sap4). Within the family there was 77-87% identity at the nucleotide level and 76-92% identity at the amino acid level. Transcription of three members of this gene family (sap1-sap3) required an acidic medium (pH<4.5) and either nitrogen or sulphur derepression. Regulation was co-ordinate and hierarchical, with pH occupying the higher position in the hierarchy. Exogenous protein increased transcript levels, probably via the provision of metabolic intermediates rather than by direct induction of gene expression. sap4 was not expressed under these conditions. SAP1-SAP4 are predicted to have almost identical substrate-binding sites and therefore substrate specificity. It is proposed that sap1-sap3 exist to provide amplified expression of the secreted aspartic proteinase because protein, an important secondary nitrogen source for this fungus, requires extensive degradation to make its nitrogen available to the cell.
Biochemistry of methanogenesis: a tribute to Marjory Stephenson:1998 Marjory Stephenson Prize Lecture
Authors: Thauer Rudolf K.
Published: June 2009

Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043 Marburg, and Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karl-von-Frisch-Straße, D-35032 Marburg, Germany

In 1933, Stephenson & Stickland (1933a) published that they had isolated from river mud, by the single cell technique, a methanogenic organism capable of growth in an inorganic medium with formate as the sole carbon source.
Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae
Authors: Warit Saradee , Walmsley Richard M. , Stateva Lubomira I.
Published: June 2009

Two genomic fragments have been isolated from Candida albicans which strongly hybridize to SRB1/PSA1/VIG9, an essential gene which encodes GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae. A common 2.5 kb Xbal-Pstl fragment has been identified, which Southern analysis suggests is most likely unique in the C. albicans genome. The fragment contains an ORF, which is 82% identical and 90% homologous to the Srb1p/Psa1p/Vig9p from S. cerevisiae, contains one additional amino acid at position 254 and is able to functionally complement the major phenotypic characteristics of S. cerevisiae srb1 null and conditional mutations. The authors therefore conclude that they have cloned and sequenced from C. albicans the bona fide homologue of SRB1/PSA1/VIG9, named hereafter CaSRB1. Northern analysis data indicate that the gene is expressed in C. albicans under conditions of growth in the yeast and hyphal form and suggest that its expression might be regulated.
Hydrolysis of the soft amphiphilic antimicrobial agent tetradecyl betainate is retarded after binding to and killing Salmonella typhimurium
Authors: Ahlström Britta , Edebo Lars
Published: June 2009

Hydrolysis of tetradecyl betainate (B14), a fast-acting microbicidal amphiphilic quaternary ammonium compound (QAC) being an ester of betaine and tetradecanol, occurred after binding to Salmonella typhimurium, resulting in release of the water-soluble betaine portion and retention of the lipophilic tetradecanol. The rate of the hydrolysis was significant but retarded in comparison to B14 in solution. As in free solution, the hydrolysis of substance bound to S. typhimurium was increased in an alkaline environment and by heat. At pH 6.0 and 20 ° the hydrolysis of bound ester was about 10% after 180 min, whereas at pH 9.0 and 50 ° it was about 50% after 60 min. These results are consistent with a model where amphiphilic QACs are inserted into the bacterial outer membrane (OM) with the quaternary ammonium head group facing outwards and the lipophilic portion, including the ester bond, being in the membrane lipid environment enough for retarding the hydrolysis. However, calculation of the mean concentration of B14 in the bacteria at MBC99 (minimum bactericidal concentration required to kill 99% of cells) showed a 7000-8000 times greater concentration than in the medium. At this concentration, when most B14 is considered to be bound to the OM, the available surface area for each molecule was only 2 å2. This is only 6-7% of that required for close packing of the quaternary ammonium head group (30 å2), indicating that a three-dimensional, presumably continuous arrangement was formed. Since B14 is hydrolysed after its binding to bacteria with microbicidal effect, it may be used under conditions where stable QACs might be harmful to the close or the common environment.
Close proximity of the tdh, trh and ure genes on the chromosome of Vibrio parahaemolyticus
Authors: lida Tetsuya , Park Kwon-Sam , Suthienkul Orasa , Kozawa Junji , Yamaichi Yoshiharu , Yamamoto Koichiro , Honda Takeshi
Published: June 2009

The distribution and location of the virulence-factor genes of Vibrio parahaemolyticus, tdh and trh, and the structural gene of urease, ureC, were examined on the genomic DNAs of 115 clinical isolates of V. parahaemolyticus. The majority of strains (81%) had two copies of tdh on the chromosome, and no copies of trh or ure. Southern hybridization with a tdh probe, after pulsed-field gel electrophoresis of Noti-digested genomic DNA of each strain revealed only single bands, suggesting that the two copies of tdh exist on single Notl fragments in each strain. Of the 115 strains, 7% had the tdh, trh and ure genes on chromosomal DNA. The three genes were also detected on single Notl fragments in these strains. More detailed analysis revealed that the three genes were localized within 40 kb. By long and accurate polymerase chain reactions (LA-PCR), the distance between trh and ure was shown to be less than 8.5 kb. These results reveal a close proximity of the tdh, trh and ure genes on the chromosome of pathogenic V. parahaemolyticus strains.
Intracellular chitinase gene from Rhizopus oligosporus: molecular cloning and characterization
Authors: Takaya Naoki , Yamazaki Daisuke , Horiuchi Hiroyuki , Ohta Akinori , Takagi Masamichi
Published: June 2009

Multiple chitinases have been found in hyphae of filamentous fungi, which are presumed to have various functions during hyphal growth. Here it is reported, for the first time, the primary structure of one such intracellular chitinase, named chitinase III, fromRhizopus oligosporus,a zygomycete filamentous fungus. Chitinase III was purified to homogeneity from actively growing mycelia ofR. oligosporususing three steps of column chromatography. Its molecular mass was 43.5 kDa and the pH optimum was 6.0 whenp-nitrophenylN,N’,N"-β-D-triacetylchitotrioside was used as a substrate. Chitinase III also hydrolysed chromogenic derivatives of chitobiose, but had noN-acetylglucosaminidase activity. The gene encoding chitinase III(chi3)was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The deduced amino acid sequence ofchi3was similar to that of bacterial chitinases and chitinases from mycoparasitic fungi, such asAphanocladium albumandTrichoderma harzianum,but it had no potential secretory signal sequence in its amino terminus. Northern blot analysis showed thatchi3was transcribed during hyphal growth. These results suggest that chitinase III may function during morphogenesis inR. oligosporus.
Phylogenetic diversity of mesophilic and thermophilic granular sludges determined by 16S rRNA gene analysis
Authors: Sekiguchi Yuji , Kamagata Yoichi , Syutsubo Kazuaki , Ohashi Akiyoshi , Harada Hideki , Nakamura Kazunori
Published: June 2009

The microbial diversity of two types of methanogenic granular sludge, mesophilic (35 °C) and thermophilic (55 °C), which had been treating sucrose/propionate/acetate-based artificial wastewater were compared. 16S rDNA clone libraries were constructed by PCR with a prokaryote-specific primer set, and partial sequencing of the clonal 16S rDNAs was conducted for phylogenetic analysis. Of 115 mesophilic granule and 110 thermophilic granule clones sequenced, 19 and 22%, respectively, were phylogenetically affiliated with the domain Archaea, and the remainder in each case were assigned to the domain Bacteria. Within the domain Archaea, the 16S rDNA clones in both libraries showed relatively close relationships with those of methanogens. Within the Bacteria, a major group represented in the mesophilic clone library was the delta subclass of the Proteobacteria (27%), in which high degrees of relatedness were observed between the clonal 16S rDNA sequences and those of previously identified syntrophic bacteria and sulfate-reducing bacteria. In contrast, in the thermophilic clone library, the Thermodesulfovibrio group (19%), the green non-sulfur bacteria (18%) and the low G+C subclass of the Gram-positive bacteria (18%) were predominant. A significant difference between the two libraries was that no clone affiliated with the Proteobacteria was detected in the thermophilic clone library, whereas the Proteobacteria was the most predominant group in the mesophilic clones. Thirty-six and 24 different sequences were found in the mesophilic and thermophilic clones, respectively, suggesting that the microbial diversity of the thermophilic granule was lower than that of the mesophilic granule.
Effect of phytoplankton cell size on transient-state nitrate and ammonium uptake kinetics
Authors: Stolte Willem , Riegman Roel
Published: June 2009

The uptake of nitrate or ammonium (at a concentration of 10 mol I-1) by marine phytoplankton was studied in relation to cell size. Initial specific nitrate uptake rates by small (35000 m3) and large (130000 m3) cells of the diatom Ditylum brightwellii did not differ significantly. However, the larger cells maintained a high uptake rate for a longer time. Therefore, they accumulated nitrate in a higher biomass-specific pool than the smaller cells. In the dark, this effect was even more pronounced. Two smaller diatom species, Lauderia borealis (7474 m3) and Thalassiosira pseudonana (98 (m3), had lower initial specific nitrate uptake rates and lower intracellular pools. Transient-state ammonium uptake did not result in accumulation of large intracellular pools of ammonium. Theoretically, and on the basis of the presented results, we stress the dualistic functional role of the vacuole. A large vacuole is an effective way for larger algal species to possess a minimum cell nutrient quota/cell surface ratio which is in the range of smaller species. Furthermore, by functioning as a storage reservoir it reduces inhibition of the uptake rate by cytoplasmic accumulated nutrients. The effect of the latter mechanism is that larger algal species are better at nitrate uptake under fluctuating conditions. These results imply that, in nitrogen-controlled marine systems, resource competition under fluctuating nutrient concentrations can only lead to a shift towards larger phytoplankton species if nitrate rather than ammonium is the main nitrogen source. From theoretical considerations it is argued that the maximum growth rate of algae is determined by nutrient assimilation properties rather than by photosynthetic capacity.
Tactic response of zoospores of the fungus Phytophthora palmivora to solutions of different pH in relation to plant infection
Authors: Morris B. M. , Reid B. , Gow N. A. R.
Published: June 2009

Zoospores of the plant pathogen Phytophthora palmivora use a number of tactic responses to target specific infectible regions of host roots. Although the dominant one is believed to be chemotaxis, it has been shown that zoospores of oomycetes may also use the exogenous proton/electrical currents generated by plant roots for guidance. Since these proton currents also generate significant pH gradients in the rhizosphere, the tactic response of zoospores to changes in pH was examined. Using ‘swim-in’ capillary tests, zoospores of P. palmivora were found to be repelled by solutions of high pH and attracted to solutions of low pH, relative to a control at neutrality. This in vitro tactic response was generally consistent with the measured pH at sites of zoospore accumulation around intact and wounded roots. However, the endogenous pH gradient around host roots could be abolished with buffers and this treatment did not affect the extent or pattern of zoospore accumulation. Therefore, detection of root-generated pH gradients is unlikely to have a major role in the homing response of zoospores towards plant roots.
A gene from Renibacterium salmoninarum encoding a product which shows homology to bacterial zinc-metalloproteases
Authors: Grayson Thomas H. , Evenden Andrew J. , Gilpin Martyn L. , Martin Keith L. , Munn Colin B.
Published: June 2009

A genomic library constructed from Renibacterium salmoninarum isolate MT444 DNA in the plasmid vector pBR328 was screened using Escherichia coli host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 C and found to contain a 31 kb Hindlll fragment of inserted DNA. This fragment was present in seven isolates of R. salmoninarum which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of R. salmoninarum and failed to identify any additional proteins compared to control E. coli containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for R. salmoninarum. The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 557. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designated hly. Neither protease nor lecithinase activities were detectable in E. coli recombinants expressing gene hly. Haemolytic activity was observed from 6 C to 37 C for erythrocytes from a number of mammalian species and also from fish. Gene hly was expressed in E. coli as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of R. salmoninarum cultured under conditions of iron-sufficiency or iron-restriction. The results indicate that the availability of iron modulates the expression of the hly gene.
Two genes encoding proteins with similarities to rubredoxin and rubredoxin reductase are required for conversion of dodecane to lauric acid in Acinetobacter calcoaceticus ADP1
Authors: Haspel Gerhard , Ehrt Sabine , Hillen Wolfgang
Published: June 2009

Mutants of Acinetobacter calcoaceticus ADP1 unable to grow on dodecane, but retaining the ability to grow on lauric acid were isolated after ethylmethanesulphonate (EMS) treatment. This growth deficiency was complemented by a clone from a gene library constructed from chromosomal DNA of the wild-type strain. The complementing DNA mapped in a gene encoding a polypeptide with homology to rubredoxins. The deduced putative rubredoxin amino acid sequence is more similar to related proteins from Gram-positive bacteria than to the Pseudomonas oleovorans rubredoxin involved in alkane oxidation. An adjacent gene encodes a protein with similarity to rubredoxin reductase from Pseudomonas oleovorans and related NAD(P)-dependent reductases. Disruption of the rubredoxin-encoding gene by insertion of a KmR/lacZ cassette rendered the resulting strain unable to grow on dodecane or hexadecane. This demonstrates that these genes are necessary for alkane degradation. Transcriptional fusion of lacZ to the rubredoxin-encoding gene led to low level constitutive
-galactosidase expression, whereas the fusion oriented in the opposite direction was not expressed.
A Bacillus subtilis spore coat polypeptide gene, cotS
Authors: Abe Akiko , Koide Hidekatsu , Kohno Takeyuki , Watabe Kazuhito
Published: June 2009

A gene, cotS, encoding a spore coat polypeptide of Bacillus subtilis, was isolated from an EcoRI fragment (54 kb) of the chromosome by using synthetic oligonucleotide probes corresponding to the NH2-terminal amino acid sequence of Cot40-2 previously purified from the spore coat of B. subtilis. The nucleotide sequence (2603 bp) was determined and sequence analysis suggested the presence of two contiguous ORFs, ORF X and cotS, followed by the 5'-region of an additional ORF, ORF Y, downstream of cotS. The cotS gene is 1053 nucleotides long and encodes a polypeptide of 351 amino acids with a predicted molecular mass of 41083 Da. The predicted amino acid sequence was in complete agreement with the NH2-terminal amino acid sequence of Cot40-2. The orfX gene is 1131 nucleotides long and encodes a polypeptide of 377 amino acids with a predicted molecular mass of 42911 Da. The gene product of cotS was confirmed to be identical to Cot40-2 by SDS-PAGE and immunoblotting from Escherichia coli transformed with a plasmid containing the cotS region. Northern hybridization analysis indicated that a transcript of cotS and orfX appeared at about 5 h after the onset of sporulation. The transcriptional start point determined by primer extension analysis suggested that -10 and -35 regions are present upstream of orfX and are very similar to the consensus sequence for the k-dependent promoter. Terminator-like sequences were not found in the DNA fragment (2603 bp) sequenced in this paper, which suggested that the cotS locus may be part of a multicistronic operon. The cotS gene is located between dnaB and degQ at about 270-275 on the genetic map. Insertional mutagenesis of the cotS gene by introducing an integrative plasmid resulted in no alteration of growth or sporulation, and had no effect on germination or resistance to chloroform.
Paracoccus thiocyanatus sp. nov., a new species of thiocyanate-utilizing facultative chemolithotroph, and transfer of Thiobacillus versutus to the genus Paracoccus as Paracoccus versutus comb. nov. with emendation of the genus
Authors: Katayama Yoko , Hiraishi Akira , Kuraishi Hiroshi
Published: June 2009

A facultatively chemolithotrophic thiocyanate-degrading bacterium, strain THI 011T, which was previously isolated from activated sludge and tentatively named Thiobacillus sp., was studied taxonomically and phylogenetically. This bacterium utilizes thiocyanate as sole energy source and the specific growth rate for chemolithoautotrophic growth with thiocyanate was 0059 h-1. Molecular phylogenetic relationships of strain THI 011T to Thiobacillus versutus and members of the genus Paracoccus were elucidated by comparing 16S rRNA gene sequences. Binary sequence comparisons showed that strain THI 011T was most related to Paracoccus aminophilus, at a similarity level of 970%, and T. versutus was most similar to Paracoccus denitrificans, at a level of 991%. A neighbour-joining phylogenetic tree showed that strain THI 011T formed a cluster together with T. versutus and known species of the genus Paracoccus within the α-3 subclass of the Proteobacteria. DNA-DNA hybridization assays and phenotypic studies indicated that strain THI 011T differed from T. versutus and known species of the genus Paracoccus. On the basis of these results, we propose to classify strain THI 011T into a new species of the genus Paracoccus with the name Paracoccus thiocyanatus sp. nov. We also propose to transfer T. versutus to the genus Paracoccus and present an emended description of the genus.