bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of CEL2 and CEL4, Two Proteins from Agaricus Bisporus with Similarity to Fungal Cellobiohydrolase I and -mannanase, Respectively, is Regulated by the Carbon Source
Authors: Yage Ernesto , Mehak-Zunic Milena , Morgan Louis , Wood David A. , Thurston Christopher F.
Published: June 2009

Two new cellulose-growth specific (cel) cDNAs, cel2 and cel4, have been isolated from an Agaricus bisporus cDNA expression library by immunoscreening with an A. bisporus anti-endoglucanase antibody. The deduced amino acid sequences showed that both CEL2 and CEL4 proteins have a modular structure consisting of a fungal-type cellulose-binding domain (CBD) and a catalytic domain separated by a linker region rich in Pro, Ser and Thr. The CEL2 and CEL4 catalytic domains were homologous to fungal cellobiohydrolases (CBH) in family 7 and to fungal mannanases in family 5 of the glycosyl hydrolases, respectively. A previously isolated cDNA derived from a constitutive gene was also sequenced. The deduced amino acid sequence corresponded to 5-aminolaevulinic acid synthase (ALA), the first enzyme in the haem biosynthetic pathway, and was most similar to other fungal ALAs. RNA analysis showed that the expression of cel2 and cel4 genes was induced by cellulose and repressed by glucose, fructose and lactose. The soluble cellulose derivative CM-cellulose induced mRNA accumulation for cel1 but not cel2, cel3 or cel4. Mannitol, maltose, sorbitol and glycerol decreased cel2 and cel4 mRNA levels to different extents, cel1, cel2, cel3 and cel4 mRNAs all disappeared after the addition of glucose with apparent half-lives of less than 20 min. Whether cel mRNAs have short half-lives or glucose affects the stability of cel transcripts remains to be investigated.
The Phytase Subfamily of Histidine Acid Phosphatases: Isolation of Genes for Two Novel Phytases from the Fungi Aspergillus Terreus and Myceliophthora Thermophila
Authors: Mitchell David B. , Vogel Kurt , Weimann Bernd J. , Pasamontes Luis , van Loon Adolphus P. G. M.
Published: June 2009

Phytases catalyse the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. In this study genes encoding novel phytases from two different filamentous fungi, Aspergillus terreus strain 9A-1 and Myceliophthora thermophila were isolated. The encoded PhyA phytase proteins show 60% (A. terreus) and 48% (M. thermophila) identity, respectively, to the PhyA of Aspergillus niger and have 21-29% identity compared to other histidine acid phosphatases. All three PhyA proteins, in contrast to the A. niger pH 2.5-optimum acid phosphatase, prefer phytic acid as substrate and show enzyme activity at a broad range of acidic pH values. Based on their enzyme characteristics and protein sequence homology, the phytases form a novel subclass of the histidine acid phosphatase family.
An unusual illegitimate recombination occurs in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii
Authors: Wang Jianhui , Masuzawa Toshiyuki , Li Muqing , Yanagihara Yasutake
Published: June 2009

In this study, we describe an unusual illegitimate recombination in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii. A 96 bp DNA segment was deleted from the ospA structural gene of B. afzelii strain R9. The nature of the rearrangement suggested that it arose by a strand slippage mechanism, which was stimulated by a 18-mer palindromic sequence and 5-mer short direct repeats at both termini of the deleted DNA. The deleted sequence could form a complex hairpin structure suggesting that it may have played important roles in pausing of replication and slippaging of the nascent strand across the replication fork. In addition, the mutant strain was isolated from a chronic Lyme disease patient, implying that the variation mechanism may have been used by the borrelial strain to avoid host immune elimination.
Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors
Authors: Fish Steven A. , Cundliffe Eric
Published: June 2009

Three glycosyltransferases are involved in tylosin biosynthesis in Streptomyces fradiae. The first sugar to be added to the polyketide aglycone (tylactone) is mycaminose and the gene encoding mycaminosyltransferase is orf2
* (tylM2). However, targeted disruption of orf2
* did not lead to the accumulation of tylactone under conditions that normally favour tylosin production; instead, the synthesis of tylactone was virtually abolished. This may, in part, have resulted from a polar effect on the expression of genes downstream of orf2
*. particularly orf4
* (ccr) which encodes crotonyl-CoA reductase, an enzyme that supplies 4-carbon extender units for polyketide metabolism. However, that cannot be the entire explanation, since tylosin production was restored at about 10% of the wild-type level when orf2
* was re-introduced into the disrupted strain. When glycosylated precursors of tylosin were fed to the disrupted strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis were still intact. Interestingly, however, tylactone also accumulated under such conditions and, to a much lesser extent, when tylosin was added to similar fermentations. It is concluded that glycosylated macrolides exert a pronounced positive effect on polyketide metabolism in S. fradiae.
Analysis of secreted aspartic proteinases from Candida albicans: purification and characterization of individual Sap1, Sap2 and Sap3 isoenzymes
Authors: Smolenski G. , Sullivan P. A. , Cutfield S. M. , Cutfield J. F.
Published: June 2009

The recently discovered secreted aspartic proteinase multi-gene (SAP) family in Candida albicans has complicated assessment of proteolytic activity as a factor in the onset and development of Candida infections. Differential expression of the SAP genes under various conditions, as well as possible variation in the properties of the individual isoenzymes, have consequences for immunological detection, for targeted drug design and possibly for pathogenicity. It is therefore important to be able to monitor Sap isoenzyme profiles in different strains of C. albicans cultures, and to know the biochemical properties of each isoenzyme. We have employed a simple purification protocol based on strong anion exchange chromatography for the direct analysis of C. albicans Sap isoenzymes from culture filtrates, as well as recovery of individual Sap1, Sap2 and Sap3 products. In the case of Sap1, this involved development of an overexpression system using the pEMBLyex4 vector transformed into Saccharomyces cerevisiae. The C albicans strains ATCC 10231 and 10261 were shown to produce different ratios of Sap2 and Sap3 under the same conditions. Analysis of all three purified proteins by gel electrophoresis, immunoblotting and proteinase assays which were designed to evaluate pH dependence, thermal stability and substrate specificity revealed similar but distinct properties for each isoenzyme. Although Sap3 was shown to be antigenically more similar to Sap2 than was Sap1, it was less similar in terms of thermal stability and activity at low pH, being more stable and more active.
A physiological model for the control of erythromycin production in batch and cyclic fed batch culture
Authors: Bushell M. E. , Smith J. , Lynch H. C.
Published: June 2009

Reported differences in antibiotic production dynamics resulting from altering the growth-limiting nutrient (growth-dissociated production in carbon-limited culture and apparent growth-associated production in nitrogen-limited culture) are due to the different effects on growth kinetics. The substrate affinity for nitrate is significantly lower than that for glucose, resulting in nitrogen limitation effectively occurring throughout the culture. Glucose limitation occurs later in the culture, coinciding with the induction of antibiotic production. Induction occurs at the start of nitrogen-limited culture so that production appears to be growth-associated. Evidence that this hypothesis is consistent with production kinetics in cyclic fed batch culture was also obtained.
Distribution of amine oxidases and amine dehydrogenases in bacteria grown on primary amines and characterization of the amine oxidase from Klebsiella oxytoca
Authors: Hacisalihoglu Ayse , Jongejan Jaap A. , Duine Johannis A.
Published: June 2009

The bacteria Klebsiella oxytoca LMD 72.65 (ATCC 8724), Arthrobacter P1 LMD 81.60 (NCIB 11625), Paracoccus versutus LMD 80.62 (ATCC 25364), Escherichia coli W LMD 50.28 (ATCC 9637), E. coli K12 LMD 93.68, Pseudomonas aeruginosa PAO1 LMD 89.1 (ATCC 17933) and Pseudomonas putida LMD 68.20 (ATCC 12633) utilized primary amines as a carbon and energy source, although the range of amines accepted varied from organism to organism. The Gram-negative bacteria K. oxytoca and E. coli as well as the Gram-positive methylotroph Arthrobacter P1 used an oxidase whereas the pseudomonads and the Gram-negative methylotroph Paracoccus versutus used a dehydrogenase for amine oxidation. K. oxytoca utilized several primary amines but showed a preference for those containing a phenyl group moiety. Only a single oxidase was used for oxidation of the amines. After purification, the following characteristics of the enzyme indicated that it belonged to the group of copper-quinoprotein amine oxidases (EC 1.4.3.6): the molecular mass (172000 Da) of the homodimeric protein; the UV/visible and EPR spectra of isolated and p-nitrophenylhydrazine-inhibited enzyme; the presence and the content of copper and topaquinone (TPQ). The amine oxidase appeared to be soluble and localized in the periplasm, but catalase and NAD-dependent aromatic aldehyde dehydrogenase, enzymes catalysing the conversion of its reaction products, were found in the cytoplasm. From the amino acid sequence of the N-terminal part as well as that of a purified peptide, it appears that K. oxytoca produces a copper-quinoprotein oxidase which is very similar to that found in other Enterobacteriaceae.
The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF
Authors: Thomas Joanna D. , Reeves Philip J. , Salmond George P. C.
Published: June 2009

The out gene cluster of Erwinia carotovora subsp. carotovora (Ecc) encodes the proteins of the type II or general secretory pathway (GSP) apparatus which is required for secretion of pectinase and cellulase. In this study, fusions between Ecc out genes and the topology probe blaM were constructed. The ability of Out protein domains to export BlaM across the cytoplasmic membrane in both Escherichia coli and the cognate host was utilized to confirm the computer-predicted cytoplasmic membrane topology of OutC and OutF. When outC was fused to blaM, the resulting phenotype suggested that the majority of OutC is targeted to the periplasm, typical of a type II bitopic conformation in the cytoplasmic membrane. In contrast, for the outF gene product, three transmembrane regions were identified which connect a large N-terminal cytoplasmic domain, a smaller periplasmic domain, and a large cytoplasmic loop. Fusions between blaM and outD and outE were used to further substantiate the locations of these gene products in the outer membrane and the cytoplasm respectively. The data derived suggest that a number of the Out apparatus components possess domains in the cytoplasm and/or the periplasm with potential for protein-protein interactions which facilitate the secretion of periplasmic enzyme intermediates across the outer membrane to the external milieu.
Classification of Enterobacteriaceae by minimization of stochastic complexity
Authors: Gyllenberg H. G. , Gyllenberg M. , Koski T. , Lund T. , Schindler J. , Verlaan M.
Published: June 2009

A new method for classifying bacteria is presented and applied to a large set of biochemical data for the Enterobacteriaceae. The method minimizes the bits needed to encode the classes and the items or, equivalently, maximizes the information content of the classification. The resulting taxonomy of Enterobacteriaceae corresponds well to the general structure of earlier classifications. Minimization of stochastic complexity can be considered as a useful tool to create bacterial classifications that are optimal from the point of view of information theory.
Assay and properties of acetone carboxylase, a novel enzyme involved in acetone-dependent growth and CO2 fixation in Rhodobacter capsulatus and other photosynthetic and denitrifying bacteria
Authors: Birks Stephen J. , Kelly David J.
Published: June 2009

The photosynthetic bacterium Rhodobacter capsulatus is able to grow, in the presence of carbon dioxide, under anaerobic (photosynthetic) conditions with the solvents acetone or butanone as carbon source. The carboxylation of acetone to form acetoacetate is the most likely initial step in acetone metabolism. This paper describes an assay for acetone carboxylation, in which fixation of radiolabeled carbon dioxide from NaH14CO3 is measured in the presence of acetone, ATP, magnesium ions and acetyl-CoA. Acetone carboxylase activity was specifically induced by growth of R. capsulatus on acetone or butanone and was associated with a high-molecular-mass protein complex containing two major polypeptides, of 70 and 85 kDa. Partial purification of the activity was achieved by FPLC ion-exchange chromatography, which confirmed that the 70 and 85 kDa proteins were subunits of the enzyme and suggested that at least one additional protein (60 kDa) may be associated with carboxylase activity. N-terminal sequences of the two major subunits were not significantly similar to any other carboxylases in the databases and neither contained covalently bound biotin, indicating that the enzyme represents a novel type of carboxylase. Acetone carboxylase activity was also demonstrated in cell-free extracts of acetone-grown Rhodomicrobium vannielii and the denitrifying bacterium Thiosphaera pantotropha.
FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites
Authors: Meng Wenmao , Green Jeffrey , Guest John R.
Published: June 2009

The ndh gene of Escherichia coli encodes a non-proton-translocating NADH dehydrogenase (NdhII) that is anaerobically repressed by the global transcription regulator, FNR. FNR binds at two sites (centred at −50.5 and −94.5) in the ndh promoter but the mechanism of FNR-mediated repression appears not to be due to promoter occlusion. This mechanism has been investigated using an aerobically active derivative of FNR, FNR*(FNR-D154A), with ndh promoters containing altered FNR-binding sites. FNR*repressed ndh gene expression both aerobically and anaerobically in vivo. Gel retardation analysis and DNase I footprinting with purified FNR*protein confirmed that FNR interacts at two sites in the ndh promoter, and that FNR and RNA polymerase (RNAP) can bind simultaneously. Studies with three altered ndh promoters, each containing an impaired or improved FNR-site, indicated that both FNR-sites are needed for efficient repression in vivo. The α-subunit of RNAP interacted with two regions (centred at −105 and −46), each overlapping one of the FNR-sites in the ndh promoter. Footprints of the FNR*-RNAP-ndh ternary complex indicated that FNR*-binding at −50.5 prevents the α-subunit of RNAP from docking with the DNA just upstream of the −35 element. Binding of a second FNR*molecule at the −105 site likewise prevents binding of the α-subunit at its alternative site, thus providing a plausible mechanism for FNR-mediated repression based on displacement of the α-subunit of RNAP.
Fluorescent oligonucleotide rDNA probes that specifically bind to a common nanoflagellate,
Paraphysomonas vestita
Authors: Rice Jason , O'Connor C. David , Sleigh Michael A. , Burkill Peter H. , Giles Ian G. , Zubkov Mikhail V.
Published: June 2009

Nanoflagellates are ecologically important, but morphological identification requires techniques which are not practicable for use in quantitative studies of populations; alternative methods of accurate recognition of nanoflagellate species in mixed populations are therefore desirable. Fluorescent oligonucleotide probes which hybridize with unique sequences of the small subunit (SSU) rRNA have been exploited as ‘phylogenetic stains’ in the identification of bacteria. In this paper we describe the preparation and application of probes which specifically hybridize with a common nanoflagellate species, Paraphysomonas vestita. The sequence of nucleotides in the SSU rRNA gene of this flagellate was determined and compared with those of related species to select unique P. vestita sequences 18-21 nucleotides in length. Five sequences in different parts of the SSU rRNA gene were used to design 5 -fluorescently labelled oligonucleotide probes. Published sequences were used to make probes that hybridized with all eukaryotes (EUK) or any cellular organism (UNI), and probes were designed not to hybridize with rRNA (CON). Optimum conditions for hybridization were determined. In all cases, UNI probes hybridized with the cells, but CON probes were only bound to a limited extent. All five probes targeted to P. vestita proved to be species-specific; they hybridized well with this species, but not with three other species of the same genus, nor with three more distantly related flagellate species, nor with a ciliate, nor with bacteria. These probes provide a means of quantitatively measuring the proportion of P. vestita cells in samples of mixed protists.
Mutations to antibiotic resistance occur during the stationary phase in Lactobacillus plantarum ATCC 8014
Authors: Thompson J. K. , McConville K. J. , McReynolds Christine , Moorhouse S. D. , Collins M. A.
Published: June 2009

When Lactobacillus plantarum ATCC 8014 was maintained in LCM broth (which consists of buffered tryptone and is sufficient to support the growth of some species of Lactobacillus ) for long periods (120 d), viable bacteria persisted. Rifampicin-, streptomycin- and sodium-fusidate-resistant mutants were recovered from parallel LCM broth cultures following a stochastic pattern. Individual cultures appeared to yield mutants intermittently. One culture in particular yielded rifampicin-resistant colonies at a frequency of 1 in 100 viable bacteria after 20 d incubation and these persisted until the experiment was terminated at 115 d. In a separate experiment two parallel cultures yielded mutants resistant to low concentrations of streptomycin at a similar frequency. Using a chemostat it was shown that in continuous culture in LCM at slow growth rates the highest frequency of recovery of antibiotic-resistant mutants was achieved when the bacteria exhibited doubling times of 90 h or greater. The frequency of recovery of mutants was as high as 1 in 1000 viable bacteria. Thus, mutations to antibiotic resistance in L. plantarum ATCC 8014 can take place in the absence of measurable cell division. The data are consistent with the notion that populations of starved bacteria in stationary phase can be genetically dynamic.
The cydR gene product, required for regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii, is an Fnr-like protein
Authors: Wu Guanghui , Hill Susan , Kelly Mark J. S. , Sawers Gary , Poole Robert K.
Published: June 2009

Summary: The cytochrome bd complex in the obligately aerobic diazotroph Azotobacter vinelandii is an oxidase, which, in vivo, has a low affinity for oxygen and is required for respiratory protection of nitrogenase. Mutations caused by insertion of Tn5-B20 upstream of the structural genes (cydAB) for cytochrome bd result in over-expression of this oxidase and, for unexplained reasons, inability of the organism to grow microaerobically. Cloning and sequencing of this upstream region revealed a gene, cydR. The deduced amino acid sequence of CydR indicates that it is a new member of the Fnr class of regulators and that it represses cydAB expression. Refined mapping data for three insertions in cydR are presented. The cloned cydR gene complemented anaerobic growth of Escherichia coli fnr mutants and strongly enhanced expression of a narG-lacZ fusion in an E. coli fnr mutant.
The Sch9 protein kinase in the yeast Saccharomyces cerevisiae controls cAPK activity and is required for nitrogen activation of the fermentable-growth-medium-induced (FGM) pathway
Authors: Crauwels Marion , Donaton Monica C. V. , Pernambuco Maria Beatriz , Winderickx Joris , de Winde Johannes H. , Thevelein Johan M.
Published: June 2009

Summary: In cells of the yeast Saccharomyces cerevisiae, trehalase activation, repression of CTT1 (catalase), SSA3 (Hsp70) and other STRE-controlled genes, feedback inhibition of cAMP synthesis and to some extent induction of ribosomal protein genes is controlled by the Ras-adenylate cyclase pathway and by the fermentable-growth-medium-induced pathway (FGM pathway). When derepressed cells are shifted from a non-fermentable carbon source to glucose, the Ras-adenylate cyclase pathway is transiently activated while the FGM pathway triggers a more lasting activation of the same targets when the cells become glucose-repressed. Activation of the FGM pathway is not mediated by cAMP but requires catalytic activity of cAMP-dependent protein kinase (cAPK; Tpk1, 2 or 3). This study shows that elimination of Sch9, a protein kinase with homology to the catalytic subunits of cAPK, affects all target systems in derepressed cells in a way consistent with higher activity of cAPK in vivo. In vitro measurements with trehalase and kemptide as substrates confirmed that elimination of Sch9 enhances cAPK activity about two- to threefold, in both the absence and presence of cAMP. In vivo it similarly affected the basal and final level but not the extent of the glucose-induced responses in derepressed cells. The reduction in growth rate caused by delation of SCH9 is unlikely to be responsible for the increase in cAPK activity since reduction of growth rate generally leads to lower cAPK activity in yeast. On the other hand, deletion of SCH9 abolished the responses of the protein kinase A targets in glucose-repressed cells. Re-addition of nitrogen to cells starved for nitrogen in the presence of glucose failed to trigger activation of trehalase, caused strongly reduced and aberrant repression of CTT1 and SSA3, and failed to induce the upshift in RPL25 expression. From these results three conclusions can be drawn: (1) Sch9 either directly or indirectly reduces the activity of protein kinase A; (2) Sch9 is not required for glucose-induced activation of the Rasadenylate cyclase pathway; and (3) Sch9 is required for nitrogen-induced activation of the FGM pathway. The latter indicates that Sch9 might be the target of the FGM pathway rather than cAPK itself.
Sub-specific differentiation of intestinal spirochaete isolates by macrorestriction fragment profiling
Authors: Rayment Sarah J. , Livesley M. Anne , Barrett Stephen P.
Published: June 2009

Summary: Macrorestriction fragment profile analysis by PFGE was used to distinguish intestinal spirochaetes, some of which were isolated from cases of swine dysentery and intestinal spirochaetosis in humans, pigs, mice, chickens and dogs. Macrorestriction fragment profiles using Smal and Sacll restriction enzymes were produced and used in statistical analysis. This permitted the division of the isolates into two major clusters. One cluster contained isolates which were identified as Serpulina pilosicoli and the second cluster contained isolates identified as Serpulina hyodysenteriae by immunoblotting with species-specific mAbs. Both species contained sub-specific groups, although these rarely correlated with the source of the isolates. We conclude that PFGE is capable of sub-specific differentiation of intestinal spirochaetes, but that the current species contain a large variety of genotypes among which cross-species transmission may be feasible.
Carbon and energy flux constraints in continuous cultures of Alcaligenes eutrophus grown on phenol
Authors: Leonard David , Lindley Nicholas D.
Published: June 2009

Summary: The growth behaviour of Alcaligenes eutrophus on phenol was investigated in continuous cultures to identify the phenomena limiting both growth efficiency and substrate degradation rates. It was shown that the fixed stoichiometry of the meta pathway of phenol degradation, leading to equimolar quantities of pyruvate and acetate, and the structure of the central pathways, which do not allow gluconeogenesis of acetate during growth on phenol, provoke the accumulation of polyhydroxybutyrate (PHB) under certain growth conditions. Acetate is predominantly used as an energy source and PHB accumulates when the cells are carbon-limited rather than energy-limited. The maximum rates of phenol degradation can be attributed to the expression of the enzymes of the catabolic pathway. This is particularly true of phenol hydroxylase and 2-hydroxymuconate semialdehyde (2-hms) dehydrogenase, whose substrates accumulated to physiologically significant concentrations at high growth rates. Indeed the concentration of 2-hms that accumulated in the medium indicated that this enzyme was substrate-saturated at maximum growth rates. However, the specific activity profiles of other catabolic enzymes associated with phenol degradation were close to the estimated flux through the pathway. This suggests a complex control structure in which several enzymes contribute to the control of pathway flux, as would be expected in a catabolic pathway.
Candida albicans SSDl can suppress multiple mutations in Saccharomyces cere visiae
Authors: Chen Chung-Yung , Rosamondt John
Published: June 2009

SUMMARY: The SSD1 gene of Saccharomyces encodes a 160 kDa cytoplasmic protein that can suppress mutations in a number of other genes. A functional homologue of SSD1 from the humanpathogen Candida albicans was isolated on the basis of its ability to restore viabilityat the restrictive temperature in a Saccharomyces cerevisiae swi4 ssdl-d strain. The C. albicans gene, designated CaSSD1, encodes a 1262 aa protein which has 47% identity overall to S. cerevisiae SSDI as well as significant identity to Schizosaccharomyces pombe dis3 and sts5 products. It is shown that CaSSD1 expression is constitutive through the mitotic cell cycle, which is consistent with a role for the protein in cell growth. CaSSD1 rescues the swiP defect in an ssd1-d background when expressed from its own promoteron a single-copy plasmid and under the same conditions can rescue mutations in genes encoding protein phosphatase type 2A catalytic subunits. These data suggest that CaSSD1, like its S. cerevisiae homologue, can limit the effect of mutations on a variety of cellular processes.
Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes
Authors: Neef Alexander , Amann Rudolf , Schlesner Heinz , Schleifer Karl-Heinz
Published: June 2009

Summary: The group of planctomycetes represents a separate line of descent within the domain Bacteria. Two phylum-specific 16S rRNA-targeted oligonucleotide probes for planctomycetes have been designed, optimized for in situ hybridization and used in different habitats to detect members of the group in situ. The probes, named PLA46 and PLA886, are targeting all or nearly all members of the planctomycete line of descent. Planctomycetes could be detected in almost all samples examined, e.g. a brackish water lagoon, activated sludge, and other wastewater habitats. In situ probing revealed quite uniform morphology and spatial arrangement of the detected cells but profound differences in abundance ranging from less than 01% to several percentage of the total cells. Single coccoid cells with diameters between 1 and 25 m were dominating in most samples with the exception of the lagoon, in which rosettes of pear-shaped cells were abundant. The planctomycetes showed generally no hybridization signals with the bacterial probe EUB338, which is in accordance with base changes in their 16S rRNA sequences. A discrete ultrastructure of planctomycete cells was suggested by double staining with rRNA-targeted probes and the DNA-binding dye 4',6-diamidino-2-phenylindole (DAPI). The probe-conferred fluorescence was distributed in a ring-shaped manner around a central DAPI spot. The two probes developed extend the existing set of group-specific rRNA-targeted probes and help to elucidate the basic composition of bacterial communities in a first step of differential analysis. In situ hybridization of environmental samples indicated widespread presence of planctomycetes in different ecosystems.
Phenotypic and molecular characterization of a Brucella strain isolated from a minke whale (Balaenoptera acutorostrata)
Authors: Clavareau Chantal , Wellemans Vincent , Walravens Karl , Tryland Morten , Verger Jean-Michel , Grayon Maggy , Cloeckaert Axel , Letesson Jean-Jacques , Godfroid Jacques
Published: June 2009

Summary: Isolation of Brucella spp. in marine mammals has been reported during the past several years. A Brucella strain from the spleen and liver of a minke whale (Balaenoptera acutorostrata) was isolated. Conventional typing methods indicated that this isolate was related to the genus Brucella but did not match the profiles of any known Brucella species or biovar. Successful PCR amplification of the Brucella rrs-rrl spacer sequence and of the insertion sequence IS6501 also indicated that the minke whale strain was related to the genus Brucella. In addition, the rrs gene of this strain shared a very high degree of nucleotide identity (>98%) with published Brucella spp. rrs sequences. However, RFLP studies using an IS6501-specific probe showed a unique profile for this strain in comparison with the profiles of the six known Brucella species. Moreover, analysis of the omp2 locus by PCR-RFLP, by Southern hybridization using omp2a- and omp2b-specific probes, and by DNA sequencing showed that the minke whale isolate possesses two copies of the omp2b gene instead of one omp2a and one omp2b gene copy or two copies of the omp2a gene described in the six known Brucella species. Thus, molecular typing methods showed that this isolate is clearly distinct from all other known Brucella species and strains. The specific molecular features of this minke whale Brucella isolate raise questions about the lineage between the Brucella strains isolated from marine mammals and the Brucella species isolated from terrestrial mammals.
IS990, a new species-specific insertion-sequence-related element of the Mycobacterium tuberculosis complex
Authors: Dziadek J. , Sajduda A. , Dale J. W. , McFadden J. J.
Published: June 2009

Summary: The structure and distribution of IS990, a new Mycobacterium tuberculosis DNA sequence with homology to characterized insertion sequences (ISs), were investigated. IS990 was related to IS elements of the IS3 family and was present as a single copy in all 21 investigated M. tuberculosis strains, two Mycobacterium bovis strains and two M. bovis BCG strains. The sequence appears to be specific for the M. tuberculosis complex. The element carries two frameshift mutations and appears to be defective.
Mycoplasma penetrans infection of Molt-3 lymphocytes induces changes in the lipid composition of host cells
Authors: Salman Michael , Borovsky Zipi , Rottem Shlomo
Published: June 2009

Summary: The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. The ability of M. penetrans to both adhere to and invade Molt-3 lymphocytes was markedly increased in the presence of polyethylene glycol 8000 (PEG). The effect of PEG was more pronounced in the more alkaline pH range, where the binding kinetics were much faster and almost unaffected by temperature (4-37 C). Incubation of [14C]oleic-acid-labelled Molt-3 cells with viable M. penetrans resulted in a substantial release of radioactive fatty acids, whereas treating the host cells with heat-inactivated mycoplasmas, isolated M. penetrans membrane preparations, or M. penetrans growth medium, had no effect. Total lipid analysis of Molt-3 lymphocytes infected by M. penetrans revealed an augmented level of the neutral lipid fraction that was associated with a decrease in the relative amounts of polar lipids, mainly a decrease in the amount of phosphatidylserine and diphosphatidylglycerol. Analysis of the neutral lipid fraction in the infected Molt-3 cells revealed a fivefold increase in the relative amount of diacylglycerol and a marked increase in the free fatty acid (FFA) fraction. The profile of the FFAs released was dominated by a relatively high concentration of the polyunsaturated fatty acid docosahexaenoic acid. The release of lipid intermediates suggests that the degradation of Molt-3 cell phospholipids induced by M. penetrans may initiate a signal transmission cascade in the host cell.
Isolation of CaSLN1 and CaNIK1, the genes for osmosensing histidine kinase homologues, from the pathogenic fungus Candida albicans
Authors: Nagahashi Shigehisa , Mio Toshiyuki , Ono Naomi , Yamada-Okabe Toshiko , Arisawa Mikio , Bussey Howard , Yamada-Okabe Hisafumi
Published: June 2009

Recent studies have revealed that fungi possess a mechanism similar to bacterial two-component systems to respond to extracellular changes in osmolarity. In Saccharomyces cerevisiae, SIn1p contains both histidine kinase and receiver (response regulator) domains and acts as an osmosensor protein that regulates the downstream HOG1 MAP kinase cascade. SLN1 of Candida albicans was functionally cloned using an S. cerevisiae strain in which SLN1 expression was conditionally suppressed. Deletion analysis of the cloned gene demonstrated that the receiver domain of C. albicans SIn1p was not necessary to rescue SLN1-deficient S. cerevisiae strains. Unlike S. cerevisiae, a null mutation of C. albicans SLN1 was viable under regular and high osmotic conditions, but it caused a slight growth retardation at high osmolarity. Southern blotting with C. albicans SLN1 revealed the presence of related genes, one of which is highly homologous to the NIK1 gene of Neurospora crassa. Thus, C. albicans harbours both SLN1 and NIK1 type histidine kinases.
Physical and genetic map of the genome of Staphylococcus carnosus TM300
Authors: Wagner Elke , Doskar Jirí , Götz Friedrich
Published: June 2009

A genome map of Staphylococcus carnosus TM300, an important micro-organism in the food industry and long used as a starter culture, was constructed by pulsed-field gel electrophoresis of DNA fragments obtained after digestion with NotI, SfiI and ApaI. The size of the chromosome was estimated to be 2590 kb. The fragments were assembled into a physical map using a combination of complementary methods including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. Fifteen genes or gene clusters were positioned on the physical map by Southern hybridization analysis. The map provides a basis for further analysis of the S. carnosus chromosome.
A dissimilatory sirohaem-sulfite-reductase-type protein from the hyperthermophilic archaeon Pyrobaculum islandicum
Authors: Molitor Michael , Dahl Christiane , Molitor Ilka , Schäfer Ulrike , Speich Norbert , Huber Robert , Deutzmann Rainer , Trüper Hans G.
Published: June 2009

A sulfite-reductase-type protein was purified from the hyperthermophilic crenarchaeote Pyrobaculum islandicum grown chemoorganoheterotrophically with thiosulfate as terminal electron acceptor. In common with dissimilatory sulfite reductases the protein has an α α β structure and contains high-spin sirohaem, non-haem iron and acid-labile sulfide. The oxidized protein exhibits absorption maxima at 280, 392, 578 and 710 nm with shoulders at 430 and 610 nm. The isoelectric point of pH 8.4 sets the protein apart from all dissimilatory sulfite reductases characterized thus far. The genes for the α- and β-subunits (dsrA and dsrB) are contiguous in the order dsrAdsrB and most probably comprise an operon with the directly following dsrG and dsrC genes. dsrG and dsrC encode products which are homologous to eukaryotic glutathione S-transferases and the proposed α-subunit of Desulfovibrio vulgaris sulfite reductase, respectively. dsrA and dsrB encode 44.2 kDa and 41.2 kDa peptides which show significant similarity to the two homologous subunits DsrA and DsrB of dissimilatory sulfite reductases. Phylogenetic analyses indicate a common protogenotic origin of the P. islandicum protein and the dissimilatory sulfite reductases from sulfate-reducing and sulfide-oxidizing prokaryotes. However, the protein from P. islandicum and the sulfite reductases from sulfate-reducers and from sulfur-oxidizers most probably evolved into three independent lineages prior to divergence of archaea and bacteria.
Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains
Authors: Kulakov Leonid A. , Delcroix Valerie A. , Larkin Michael J. , Ksenzenko Vladimir N. , Kulakova Anna N.
Published: June 2009

Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases. The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. I1 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.
Comparative analysis of Pseudomonas aeruginosa penicillin-binding protein 7 in the context of its membership in the family of low-molecular-mass PBPs
Authors: Song Jian , Xie Gary , Elf Pamela K. , Young Kevin D. , Jensen Roy A.
Published: June 2009

The Pseudomonas aeruginosa pbpG gene encoding penicillin-binding protein 7, a homologue of the Escherichia coli gene encoding a DD-endopeptidase, was cloned and sequenced. pbpG was located immediately downstream of the phenylalanine hydroxylase (phh) operon. DNA sequencing revealed an open reading frame of 936 bp (starting with a GTG codon) which encodes a protein of 34115 Da. N-terminal amino acid sequencing confirmed the presence of a cleavable N-terminal signal peptide of 23 amino acids. Verification that the protein is a penicillin-binding protein was directly demonstrated by labelling with 125l-labelled penicillin X. Inactivation of P. aeruginosa pbpG by interposon mutagenesis resulted in no obvious phenotypic changes, but when P. aeruginosa PbpG was overexpressed in E. coli using a T7 expression system, cell lysis resulted. P. aeruginosa PbpG resembled E. coli PbpG in being associated with the membrane fraction. Two additional members of the PbpG subfamily were identified in the database. P. aeruginosa PbpG shows 63% identity with E. coli penicillin-binding protein 7 (PbpG) and 60% identity with Vibrio cholerae PbpG, but only 23% identity with Haemophilus influenzae PbpG. The PbpG subfamily and three other subfamilies constituting the low-molecular-mass PBP protein family were analysed by multiple alignment of 26 sequences. PbpG exhibited the consensus motifs of other penicillin-binding proteins. Ten anchor residues were identified that are conserved at the family level within the superfamily of serine-active-site penicillin-interacting proteins.
Physical map of the genome of Oenococcus oeni PSU-1 and localization of genetic markers
Authors: Zé-Zé Líbia , Tenreiro Rogério , Brito Luisa , Santos Mário A. , Paveia Helena
Published: June 2009

A physical map of the chromosome of Oenocccus oeni PSU-1 was constructed. This represents the first map for a strain of this species. A total of 37 restriction sites for the rare-cutting endonucleases Ascl, Fsel, Notl and Sfil were mapped on the chromosome, which was found to be circular with an estimated size of 1857 kb. Fragment order was determined using several approaches: analysis of partial and double digestions, two-dimensional pulsed-field gel electrophoresis, isolation of linking clones, and Southern hybridization with labelled restriction fragments both from PSU-1 and from O. oeni strain GM. Oenococcal genes alsS/alsD, mleA and mir, two phage attachment sites and recurrent sequences such as IS 1165-like elements and rrn loci were located on the physical map. Specific fragments hybridizing with gene probes from Lactococcus lactis, Leuconostoc mesenteroides and Bacillus subtilis were also identified. The two ribosomal operons have been precisely located and their transcription direction determined.
Identification and analysis of a gene (abpA) encoding a major amylase-binding protein in Streptococcus gordonii
Authors: Rogers Jeffrey D. , Haase Elaine M. , Brown Alan E. , Douglas Charles W. I , Gwynn Justin P. , Scannapieco Frank A.
Published: June 2009

Oral streptococci such as Streptococcus gordonii bind the abundant salivary enzyme x-amylase. This interaction may be important in dental plaque formation and metabolism, thus contributing to the initiation and progression of dental caries and periodontal disease, the two most common plaque-mediated diseases. The conjugative transposon Tn916 was used to insertionally inactivate gene(s) essential to the expression of amylase-binding components of S. gordonii Challis, and a mutant deficient in amylase-binding (Challis Tn1) was identified. While wild-type strains of S. gordonii released both 20 kDa and 82 kDa amylase-binding proteins into culture supernatants, Challis Tn1 expressed the 82 kDa but not the 20 kDa protein. The 20 kDa amylase-binding protein was isolated from culture supernatants of S. gordonii Challis by hydroxyapatite chromatography. A partially purified, functionally active 20 kDa protein was sequenced from blots, and the N-terminal sequence obtained was found to be DEP (A) TDAAT(R)NND. A novel strategy, based on the single-specific-primer polymerase chain reaction technique, enabled the gene inactivated by Tn916 to be cloned. Analysis of the resultant nucleotide sequence revealed an open reading frame of 585 bp, designated amylase-binding protein A (abpA), encoding a protein of 20 kDa (AbpA), immediately downstream from the insertion site of Tn916. This protein possessed a potential signal peptide followed by a region having identity with the N-terminal sequence of the 20 kDa amylase-binding protein. These results demonstrate the role of the 20 kDa protein in the binding of amylase to S. gordonii. Knowledge of the nature of amylase-binding proteins may provide a better understanding of the role of these proteins in the colonization of S. gordonii in the oral cavity.
Psychroflexus torquis gen. nov., sp. nov. a psychrophilic species from Antarctic sea ice, and reclassification of Flavobacterium gondwanense (Dobson et al. 1993) as Psychroflexus gondwanense gen. nov., comb. nov.
Authors: Bowman John P. , McCammon Sharee A. , Lewis Tom , Skerratt Jennifer H. , Brown Janelle L. , Nichols David S. , McMeekin Tom A.
Published: June 2009

A group of sea-ice-derived psychrophilic bacterial strains possessing the unusual ability to synthesize the polyunsaturated fatty acids eicosapentaenoic acid (20:5.3) and arachidonic acid (20:4.6) belong to the Family Flavobacteriaceae (Flexibacter-Bacteroides-Flavobacterium phylum), according to 16S rRNA sequence analysis. Surprisingly, the isolates were also found to cluster closely to the moderately halophilic and psychrotrophic species [Flavobacterium] gondwanense (sequence similarity 97-8-98-1 %). The whole-cell fatty acid profiles of this group and [Flavobacterium] gondwanense were very similar and distinct from other related flavobacteria. The sea ice strains and [Flavobacterium] gondwanense differed substantially in terms of ecophysiology, possibly representing divergent adaptations to sympagic and planktonic marine habitats, respectively. Evidence based on phylogeny and fatty acid profiles supports the conclusion that the taxa are close relatives distinct from other bacterial groups. It is thus proposed that the sea ice strains represent a novel taxon designated Psychroflexus torquis gen. nov., sp., nov. (type strain ACAM 623T) while [Flavobacterium] gondwanense becomes Psychroflexus gondwanense gen. nov., comb. nov.
The NADH-dependent glutamate dehydrogenase enzyme of Bacteroides fragilis Bf1 is induced by peptides in the growth medium
Authors: Abrahams Garth L. , Abratt Valerie R.
Published: June 2009

Bacteroides fragilis Bf1 possesses two enzymes having glutamate dehydrogenase (GDH) activity. One is dual cofactor NAD(P)H-dependent, while the other has NADH-specific activity. The gene encoding the NADH-GDH (gdhB) was cloned by complementation of the glutamate auxotrophic mutant Escherichia coli MX3004 and the recombinant protein was characterized with respect to the GDH activities present in the parental organism grown under different nitrogen conditions. The NAD(P)H-dependent GDH of B. fragilis was confirmed to be most active under high ammonia conditions, but the NADH-specific GDH levels were increased by high peptide concentrations in the growth medium and not regulated by the levels of ammonia. Northern blotting analysis showed that gdhB regulation was at the transcription level, with a single transcript of ∼ 1.6 kb being produced. GDH activity was demonstrated by zymography of the parental and recombinant enzymes. The recombinant GDH was NADH-specific and co-migrated with the equivalent enzyme band from B. fragilis cell extracts. The gdhB structural gene comprises 1335 bp and encodes a protein of 445 aa (49 kDa). Comparisons of the derived protein sequence with that of GDH from other bacteria indicated that significant sequence homology and conservation of functional domains exists with enzymes of Family I-type hexameric GDH proteins.
A novel quantitative mating assay for the fungal pathogen Cryptococcus neoformans provides insight into signalling pathways responding to nutrients and temperature
Authors: Dong Hui , Courchesne William
Published: June 2009

Cryptococcus neoformans is a fungal pathogen that causes a lethal meningitis in immunocompromised individuals. Several factors are associated with virulence of this fungus, including its mating type; however, the mechanism by which mating type affects virulence is unknown. C. neoformans is a basidiomycete that exists in two mating types called a and a α that can fuse to form an a/α dikaryon. A mating assay was developed that allowed a quantitative analysis of cryptococcal mating physiology. Interestingly, the efficiency of mating appeared to be dependent on temperature, being highest at 30 °C and almost completely absent at 37 °C. Thus, while mating type itself may be associated with virulence (which must occur at 37 °C), the ability to mate is probably not a virulence factor. Mating efficiency was increased by altering the carbon or nitrogen sources to give so-called starvation media. The addition of various drugs also seemed to alter the frequency of mating, depending on the composition of mating medium. The data suggested that cAMP, 8-bromo-cAMP and caffeine increased mating on starvation medium but only cAMP and 8-bromo-cAMP stimulated mating on rich medium; caffeine was unable to stimulate mating on rich medium. Aluminium fluoride, an activator of heterotrimeric GTP-binding proteins (G-proteins), was also found to stimulate mating, suggesting the involvement of a G-protein that may regulate the level of cAMP.