bluuebunny/crossref_metadata_2025 huggingface dataset:
BacSim, a simulator for individual-based modelling of bacterial colony growth
Authors: Kreft Jan-Ulrich , Booth Ginger , Wimpenny Julian W. T.
Published: June 2009

Summary: The generic, quantitative, spatially explicit, individual-based model BacSim was developed to simulate growth and behaviour of bacteria. The potential of this approach is in relating the properties of microscopic entities – cells – to the properties of macroscopic, complex systems such as biofilms. Here, the growth of a single Escherichia coli cell into a colony was studied. The object-oriented program BacSim is an extension of Gecko, an ecosystem dynamics model which uses the Swarm toolkit for multi-agent simulations. The model describes bacterial properties including substrate uptake, metabolism, maintenance, cell division and death at the individual cell level. With the aim of making the model easily applicable to various bacteria under different conditions, the model uses as few as eight readily obtainable parameters which can be randomly varied. For substrate diffusion, a two-dimensional diffusion lattice is used. For growth-rate-dependent cell size variation, a conceptual model of cell division proposed by Donachie was examined. A mechanistic version of the Donachie model led to unbalanced growth at higher growth rates, whereas including a minimum period between subsequent replication initiations ensured balanced growth only if this period was unphysiologically long. Only a descriptive version of the Donachie model predicted cell sizes correctly. For maintenance, the Herbert model (constant specific rate of biomass consumption) and for substrate uptake, the Michaelis-Menten or the Best equations were implemented. The simulator output faithfully reproduced all input parameters. Growth characteristics when maintenance and uptake rates were proportional to either cell mass or surface area are compared. The authors propose a new generic measure of growth synchrony to quantify the loss of synchrony due to random variation of cell parameters or spatial heterogeneity. Variation of the maximal uptake rate completely desynchronizes the simulated culture but variation of the volume-at-division does not. A new measure for spatial heterogeneity is introduced: the standard deviation of substrate concentrations as experienced by the cells. Spatial heterogeneity desynchronizes population growth by subdividing the population into parts synchronously growing at different rates. At a high enough spatial heterogeneity, the population appears to grow completely asynchronously.
Population structure and diversity of avian isolates of Pasteurella multocida from Australia
Authors: Blackall Patrick J. , Fegan Narelle , Chew Gerard T. I. , Hampson David J.
Published: June 2009

A total of 110 isolates ofPasteurella multocidafrom Australian poultry and reference strains for the 16 somatic serovars plus the three subspecies (gallicida, multocida, septica) were analysed to examine their population structure and diversity. The 81 field isolates examined by multilocus enzyme electrophoresis (MLEE) were diverse, being divided into 56 electrophoretic types (ETs), with the 19 reference strains in another 15 ETs. The population was clonal and somatic serotyping was not particularly useful in establishing relationships between isolates. The 71 ETs formed three distinct subclusters (A, B and C) at a genetic distance of 0.36. Biovars tended to be associated with these subclusters: A with biovars 1, 3, 4, 5 and 8 and B with biovars 2, 6, 7, 9 and 10. Ribotyping, performed on all 110 isolates usingHpall, recognized 21 ribotypes forming nine clusters (R1-R9). The isolates in ribotype cluster R1 were almost identical to those in MLEE cluster B. Using both MLEE and ribotyping, the 19 non-Australian reference strains were found to be distributed over the full diversity of the Australian isolates ofP. multocida. This study has shown that a range ofP. multocidaclones are associated with fowl cholera in Australia and that many of the Australian isolates are similar to non-Australian reference strains. Both the MLEE results and the ribotyping data identified a previously unrecognized subset ofP. multocidastrains.
Regulation of chitin synthesis during dimorphic growth of Candida albicans
Authors: Munro C. A. , Schofield D. A. , Gooday G. W. , Gow N. A. R.
Published: June 2009

Candida albicans has three genes encoding chitin synthase enzymes. In wild-type strains, the expression of CHS2 and CHS3 peaked 1-2 h after the induction of hyphal growth, whilst mRNA levels in a non-germinative strain, CA2, remained low under the same conditions. CHS1 gene expression did not peak during germ tube formation but remained at low levels in both yeast and hyphal growth. The pattern of gene expression did not predict the changes in measured chitin synthase activities or changes in chitin content during dimorphic transition. Chitin synthase activity increased steadily, and did not peak shortly after germ tube induction, and activity profiles were similar in germ-tube-competent and germ-tube-negative strains. The phenotype of a Δchs2 null mutant suggested that CHS2 encoded the major enzyme activity in vitro and was largely responsible for elevated chitin synthase activities in microsomal preparations from hyphal cells compared to yeast cells. However, CaChs3p was responsible for synthesis of most chitin in both yeast and hyphae. Three independent chitin assays gave markedly different estimates of the relative chitin content of yeast and hyphae and wild-type and chs mutants. Only one of the methods gave a significantly higher chitin content for hyphal compared to yeast cell walls and a lower chitin content for hyphae of the Δchs2 null mutant compared to the parental strain.
Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’ with separate glycosyl hydrolase family 9 and 48 catalytic domains
Authors: Zverlov Vladimir , Mahr Sabine , Riedel Kathrin , Bronnenmeier Karin
Published: June 2009

A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium ‘Anaerocellum thermophilum’. Full-length CelA and a truncated enzyme species designated CelA' were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and β-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 ° (CM-cellulase) and 85 ° (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelA-encoding gene was isolated from genomic DNA of ‘A. thermophilum’ by PCR and the nucleotide sequence was determined. The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the N-terminus of CelA purified from ‘A. thermophilum’. Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family III cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of Caldicellulosiruptor saccharolyticus (sequence identities of 96 and 97% were found for the N- and C-terminal catalytic domains, respectively). Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound to microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.
High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca
Authors: Zhang Lianhui , Xu Jinling , Birch Robert G.
Published: June 2009

Albicidins are a family of phytotoxins and antibiotics which play an important role in the pathogenesis of sugarcane leaf scald disease. The albA gene from Klebsiella oxytoca encodes a protein which inactivates albicidin by heat-reversible binding. Albicidin ligand binding to a recombinant AlbA protein, purified by means of a glutathione S-transferase gene fusion system, is an almost instant and saturable reaction. Kinetic and stoichiometric analysis of the binding reaction indicated the presence of a single high affinity binding site with a dissociation constant of 6.4 x 10−8 M. The AlbA-albicidin complex is stable from 4 to 40 °, from pH 5 to 9 and in high salt solutions. Treatment with protein denaturants released all bound albicidin. These properties indicate that AlbA may be a useful affinity matrix for selective purification of albicidin antibiotics. AlbA does not bind to p-nitrophenyl butyrate or α-naphthyl butyrate, the substrates of the albicidin detoxification enzyme AlbD from Pantoea dispersa. The potential exists to pyramid genes for different mechanisms in transgenic plants to protect plastid DNA replication from inhibition by albicidins.
The recombinant antigen ASPND1r from Aspergillus nidulans is specifically recognized by sera from patients with aspergilloma
Authors: Puente Pilar , Leal Fernando
Published: June 2009

A 996 bpAspergillus nidulanscDNA encoding the ASPND1 immunodominant antigen was cloned and expressed inEscherichia colias a fusion protein with the enzyme glutathioneS-transferase (GST) fromSchistosoma japonicum. The GST-ASPND1 fusion protein was purified from isolated bacterial inclusion bodies by preparative SDS-PAGE. After cleavage with thrombin, the ASPND1 recombinant antigen (ASPND1r) and the GST protein were separated by SDS-PAGE and immunoblotted with a number of different human sera. The sera from 22 (88%) of 25 patients with an aspergilloma recognized the ASPND1r recombinant antigen on immunoblots. Forty-nine normal human sera and 14 sera from patients with other infections were unreactive. The ASPND1r expressed inE. colicould therefore be used, in combination with previously reported recombinant antigens, as a standardized antigen for serological and clinical diagnosis ofAspergillus-associated diseases.
Extensive genetic diversity among clinical isolates of Streptococcus pyogenes serotype M5
Authors: Desai Meeta , Tanna Asha , Efstratiou Androulla , George Robert , Clewley Jonathan , Stanley John
Published: June 2009

The genetic diversity of clinical isolates ofStreptococcus pyogenesserotype M5 has been characterized. Strain genotypes were defined by macrorestriction profile, 16S ribotype,emmgene subtype, insertion element IS1239profile, and exotoxin gene determinant. By these criteria, clinical isolates of M5 constituted a multiplicity of strain clusters rather than a homogeneous population as found for certain serotypes. Distance matrices and an unrooted tree were constructed from macrorestriction data with three rarely cutting endonucleases, determined by PFGE. A singleIS1239profile was common to 85% of isolates but there was great diversity of both ribotype and macrorestriction profile, and 18 differentemmgene subtypes were detected by PCR-RFLP. DNA sequence analysis of the antigen-coding 5' (hypervariable) region ofemmgene amplicons (about 240 bp) showed that 14/18 exhibited up to 6% divergence. Four amplicons had highly divergent sequences - corresponding to those previously determined foremm6,emm11,emm18 andemm77. Further serological and hybridization studies were used to analyse the discrepancy between the Lancefield serotype of these strains (W5) and theiremmgenotype. Overall, this study shows a high degree of genetic diversity in serotype M5, with implications for the Lancefield scheme itself, for the epidemiology of group A streptococci, and for recombinant DNA strategies for M protein-based vaccine development.
Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress
Authors: Hounsa Charlemagne-Gilles , Brandt E. Vincent , Thevelein Johan , Hohmann Stefan , Prior Bernard A.
Published: June 2009

Trehalose is an enigmatic compound that accumulates in Saccharomyces cerevisiae and has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate that may be mobilized during stress. In this study, specific mutants in trehalose metabolism were used to evaluate whether trehalose contributes to survival under severe osmotic stress and generates the compatible solute glycerol under moderate osmotic stress. The survival under severe osmotic stress (0.866 a
W, NaCI or sorbitol) of mutants was compared to that of the wild-type strain when cultivated to either the mid-exponential or the stationary growth phase on glucose, galactose or ethanol. Stationary-phase cells survived better than exponential-phase cells. The death rates of ethanol-grown cells were lower than those of galactose-grown cells, which in turn survived better than glucose-grown cells. There was a strong relationship between intracellular trehalose levels and resistance to osmotic stress. The mutant strains unable to produce trehalose (tps1Δ tps2Δ and tps1Δ hxk2 Δ) were more sensitive to severe osmotic stress (0.866 a
W) than the isogenic wild-type strain, confirming a role for trehalose in survival. Hyperaccumulation of trehalose found in the nth1Δ and the nth1Δ gpd1Δ mutant strains, however, did not improve survival rates compared to the wild-type strain. When wild-type, nth1Δ and nth1Δ gpd1Δ cells were exposed to moderate osmotic stress (0.98 and 0.97 a
W, NaCI), which permits growth, glycerol production did not appear to be related to the intracellular trehalose levels although glycerol levels increased more rapidly in nth1Δ cells than in wild-type cells during the initial response to osmotic stress. These data indicate that trehalose does not act as a reserve compound for glycerol synthesis under these conditions. No evidence was found for solutes other than glycerol and trehalose being significant for the survival of or growth by S. cerevisiae under osmotic stress conditions.
Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli
Authors: Kuzj Ann E. S. , Medberry Poonam S. , Schottel Janet L.
Published: June 2009

The functional stability of the chloramphenicol acetyltransferase (cat) mRNA, as well as the functional stability of the total mRNA pool, change during the course ofEscherichia coliculture growth. mRNA half-lives are long during lag phase, decrease during the exponential phase and increase again during the stationary phase of the bacterial growth cycle. The half-lives ofcatmRNA and total mRNA also increase three- to fourfold during amino acid starvation when compared to exponential culture growth. Even though the stability of thecatmessage changes about fourfold during culture growth, the amount ofcatmRNA per cell mass does not vary significantly between the culture growth phases, indicating that there are compensating changes incatgene transcription. Translation ofcatmRNA also changes during culture growth. In exponential phase, the rate ofcattranslation is about 14-fold higher than when the culture is in stationary phase. This is in contrast to the fourfold increase in stability ofcatmRNA in the stationary-phase culture compared to the exponentially growing culture and indicates that active translation is not correlated with increased mRNA stability. When a stationary-phase culture was diluted into fresh medium, there was a five- to sevenfold increase in CAT synthesis and a threefold increase in total protein synthesis in the presence or absence of rifampicin. These results suggest that while mRNA becomes generally more stable and less translated in the stationary-phase culture, the mRNA is available for immediate translation when nutrients are provided to the culture even when transcription is inhibited.
Carbapenems as inhibitors of OXA-13, a novel, integron-encoded β-lactamase in Pseudomonas aeruginosa
Authors: Mugnier P. , Podglajen I. , Goldstein F. W. , Collatz E.
Published: June 2009

A clinical Pseudomonas aeruginosa strain, PAe391, was found to be resistant to a number of antibiotics including ticarcllin, piperacillin, cefsulodin and amikacin, and a disk diffusion assay showed evidence of pronounced synergy between imipenem and various β;-lactam antibiotics. Cloning and nucleotide sequence analysis revealed the dicistronic arrangement of an aac(6’)-lb variant and a novel bla
OXA-type gene between the intl and qacEδ1 genes typical of integrons. In PAe391, this integron was apparently chromosome-borne. The β;-lactamase, named OXA-13, displayed nine amino acid changes with respect to OXA-10: I in position 10 of OXA-10 to T (I10T), G20S, D55N, N73S, T107S, Y174F, E229G, S245N and E259A. OXA-13 (plapp = 8.0) showed poor catalytic activity against penicillins as well as cephalosporins, but was efficient in hydrolysing some penicillinase-resistant β;-lactams, such as cefotaxime and aztreonam. It was efficiently inhibited by imipenem (K
iapp = 11 nM), and formed a stable complex. While the K
iapp value of meropenem was similar (16 nM), the corresponding complex was less stable.
Growth of green sulphur bacteria in experimental benthic oxygen, sulphide, pH and light gradients
Authors: Pringault Olivier , Kühl Michael , de Wit Rutger , Caumette Pierre
Published: June 2009

The green sulphur bacterium Prosthecochloris aestuarii (strain CE 2401) was cultured in a benthic gradient chamber to study its growth and photosynthetic activity in experimental gradients of oxygen, sulphide and light. An axenic biofilm was obtained within evenly inoculated artificial sediment after 5 weeks of incubation. The phototrophic biofilm was located 2.2-3.5 mm below the sediment surface, i.e. below the maximal penetration depth of oxygen, thus confirming that growth of P. aestuarii was restricted to strictly anoxic sediment layers. The activity was limited by the diffusive flux of sulphide, showing the role of molecular diffusion in growth of this benthic species. Scalar irradiance was attenuated strongly in the biofilm, with distinct attenuation maxima at 750 nm corresponding to bacteriochlorophyll c (Bchl c) absorption and at 800 nm corresponding to bacteriochlorophyll a (Bchl a) absorption. Using radiance attenuation data as a proxy for photopigment contents it was shown that the ratio Bchl a/Bchl c changed with depth. This indicates chromatic adaptation to changing light climates in the sediment. Total sulphide oxidation was estimated from the sulphide fluxes from below into the reaction zone. Measurements of sulphide oxidation as a function of scalar irradiance in the reaction zone showed that anoxygenic photosynthesis of the biofilm was saturated at a scalar irradiance (430-830 nm)>2 μmol photons m-2 s-1.
Molecular analysis of Physarum haemagglutinin l: lack of a signal sequence, sulphur amino acids and post-translational modifications
Authors: Morita Masashi
Published: June 2009

The cDNAs encoding haemagglutinin I from plasmodia of Physarum polycephalum have been cloned using PCR protocols. The composite haemagglutinin I cDNA sequence, derived from several overlapping clones from PCR fragments, spans 408 nt and the 315 bp ORF encodes a polypeptide of 104 aa without a typical signal sequence. The putative molecular mass deduced from the amino acid sequence (10760.76 Da) corresponds exactly to that determined by electrospray ionization MS (10759.86±.15 Da), suggesting that haemagglutinin I is not subject to post-translational modification. Haemagglutinin I lacks sulphur amino acids and has a β-sheet as the major secondary structure. Expression of the coding sequence in Escherichia coli yielded a product that exhibits the same sugar-binding specificity as natural haemagglutinin I. The deduced amino acid sequence shows little similarity to that of any known lectins and thus apparently represents a novel type of lectin.
Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants
Authors: Zhou Guo-Lei , Miyazaki Yasumasa , Nakagawa Tsuyoshi , Tanaka Katsunori , Shishido Kazuo , Matsuda Hideyuki , Kawamukai Makoto
Published: June 2009

The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroomLentinus edodes. TheL. edodes capgene contains eight introns and an ORF encoding a 518 amino acid protein. TheL. edodesCAP is 35.5% and 40.9% identical at the amino acid level withSaccharomyces cerevisiaeCAP andSchizosaccharomyces pombeCAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest thatL. edodes capis a sinμle-copy gene and uniformly expressed. Expression of theL. edodesCAP in bothSchiz. pombeandSacch.cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between theL. edodesCAP andSchiz. pombeactin. This result and the functional complementation test indicate that CAP fromL. edodeshas a conserved C-terminal domain function.
Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene
Authors: Peters-Wendisch Petra G. , Kreutzer Caroline , Kalinowski Jörn , Pátek Miroslav , Sahm Hermann , Eikmanns Bernhard J.
Published: June 2009

In addition to phosphoenolpyruvate carboxylase (PEPCx), pyruvate carboxylase (PCx) has recently been found as an anaplerotic enzyme in the amino-acid-producing bacterium Corynebacterium glutamicum. Using oligonucleotides designed according to conserved regions of PCx amino acid sequences from other organisms, a 200 bp fragment central to the C. glutamicum PCx gene (pyc) was amplified from genomic DNA by PCR. This fragment was then used to identify and to subclone the entire C. glutamicum pyc gene. The cloned pyc gene was expressed in C. glutamicum, as cells harbouring the gene on plasmid showed four- to fivefold higher specific PCx activities when compared to the wild-type (WT). Moreover, increased PCx protein levels in the pyc-plasmid-carrying strain were readily detected after SDS-PAGE of cell-free extracts. DNA sequence analysis of the pyc gene, including its 5’ and 3’ flanking regions, and N-terminal sequencing of the pyc gene product predicts a PCx polypeptide of 1140 amino acids with an M
r of 123070. The amino acid sequence of this polypeptide shows between 62% and 45% identity when compared to PCx enzymes from other organisms. Transcriptional analyses revealed that the pyc gene from C. glutamicum is monocistronic (3.5 kb mRNA) and that its transcription is initiated at an A residue 55 bp upstream of the translational start. Inactivation of the chromosomal pyc gene in C. glutamicum WT led to the absence of PCx activity and to negligible growth on lactate, indicating that PCx is essential for growth on this carbon source. Inactivation of both the PCx gene and the PEPCx gene in C. glutamicum led additionally to the inability to grow on glucose, indicating that no further anaplerotic enzymes for growth on carbohydrates exist in this organism.
An antigenic protein gene of a phytoplasma associated with sweet potato witches’ broom
Authors: Yu Yen-Ling , Yeh Kai-Wun , Lin Chan-Pin
Published: June 2009

A gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches’ broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma. The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78. The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes. The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus. This major antigenic protein was also present in phytoplasma associated with peanut witches’ broom (PNWB) and the two showed homology based on the results of Western blot analysis. Southern hybridization. Northern hybridization, primer extension analysis and PCR. The homologous genes of the antigenic protein of SPWB phytoplasma arid PNWB phytoplasma were not found in other phytoplasmas tested.
Structural elements of the Streptomyces oriC region and their interactions with the DnaA protein
Authors: Jakimowicz Dagmara , Majka Jerzy , Messer Walter , Speck Christian , Fernandez Marisol , Cruz Martin M , Sanchez Jesus , Schauwecker Florian , Keller Ullrich , Schrempf Hildgund , Zakrzewska-Czerwinńska Jolanta
Published: June 2009

Streptomycetes differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large, linear, GC-rich chromosome. To deduce structural features of the Streptomyces origin of chromosomal replication, the oriC sequences of three Streptomyces species (S. antibioticus, S. chrysomallus and S. lividans) were compared. In Streptomyces, the oriC region contains 19 DnaA boxes whose location, orientation and spacing are conserved. The consensus sequence of the DnaA box identified within Streptomyces oriC is (T/C)(T/C)(G/A/C)TCCACA (preferred bases underlined). The interactions of DnaA with DNA fragments containing single, two or three DnaA boxes were studied using surface plasmon resonance. The dissociation constant (K
D) for specific binding of individual DnaA boxes varied between 12 and 78 nM. Streptomyces oriC does not contain the three AT-rich 13-mer direct repeats present in the 5′ part of the Escherichia coli oriC region. However, short AT-rich sequences are distributed among the DnaA boxes of Streptomyces oriC. Repeated attempts to unwind Streptomyces oriC have been unsuccessful. It remains to be elucidated whether DnaA interacts with putative accessory proteins which help in unwinding Streptomyces oriC.
The Streptomyces reticuli α-chitin-binding protein CHB2 and its gene
Authors: Kolbe Steffi , Fischer Sabine , Becirevic Ardina , Hinz Petra , Schrempf Hildgund
Published: June 2009

When co-cultivated with chitin-containing fungi, Strepfomyces reticuli secretes the chitin-binding protein CHB2. Microscopical and immunological investigations revealed that CHBZ acts like a glue to mediate the contact between the fungal and the Streptomyces hyphae. CHBZ was purified to homogeneity, and the sequence of its N-terminal amino acids was determined and used to deduce an oligonucleotide, which was then used to probe a subgenomic library. The ch62 gene was cloned, sequenced and overexpressed. The deduced mature protein has a molecular mass of 18.6 kDa, and a large number of its amino acids are identical to those of CHBl from Streptomyces oliwaceowiridis. CHB2 effectively targets different types of α-chitin, but no other polysaccharide. The dissociation constant (Kd) for binding to purified crab shell chitin is 0 27 μM. Immunological studies suggest that homologues of CHBl and CHBZ are secreted by streptomycetes while growing in the presence of a-chitin-containing substrates.
Accumulation and effects of cadmium on sulphate-reducing bacterial biofilms
Authors: White Chris , Gadd Geoffrey M.
Published: June 2009

Biofilms comprising a pure and a mixed culture of sulphate-reducing bacteria (SRB) were grown in continuous culture. When exposed to 20 or 200 μM Cd, both cultures accumulated Cd but the mixed culture accumulated more and continued to accumulate Cd during the experiment, whereas accumulation by the pure cultures ceased after 4-6 d. Unlike the pure culture, the mixed culture also accumulated both protein and carbohydrate throughout the experiment proportionally to Cd which showed that accumulation required the production of biofilm material. Electron microscopy showed the presence of polysaccharide and particulates in both pure and mixed cultures, irrespective of the presence of Cd. However, energy-dispersive X-ray analysis (EDXA) showed that accumulation of Cd in the form of CdS occurred in biofilms exposed to Cd while back-scattered electron imaging of sections indicated that the accumulation of Cd was localized in a superficial layer of the biofilm. The mechanism of uptake, therefore, appeared to be entrapment and/or precipitation of CdS at the biofilm surface. The relatively low Cd uptake by the pure culture biofilm was attributed to its less efficient growth and polysaccharide production. These results indicate that mixed SRB cultures are more effective than pure cultures for metal removal and underlines significant differences between the biology of pure and mixed cultures.
Lipopolysaccharide expression within the genus Bordetella: influence of temperature and phase variation
Authors: van den Akker Willem M. R.
Published: June 2009

LPSs play an important role in bacterial pathogenesis. In this study, the LPS expression of the seven known Bordetella species and its dependency on growth temperature was analysed by oxidative silver staining of proteinase-K-treated whole bacteria separated by Tricine-SDS-PAGE. The bordetellae were found to have extensively variable LPS in a species-specific way. In addition, the human and ovine Bordetella parapertussis strains exhibited host-specific LPS expression. LPSs from human B. parapertussis strains grown at 37 and 25 °C were distinct. Growth temperature also affected LPS production by several Bordetella bronchiseptica strains. In some of these cases, BvgAS, the global regulator of virulence factors, was involved in this regulation of LPS biosynthesis. In contrast, no evidence was found for the involvement of the Bordetella pertussis BvgAS system in regulation of LPS synthesis. The obligate human pathogens B. pertussis and Bordetella holmesii are closely related but were shown to produce immunologically distinct LPSs. These species are isolated from the upper respiratory tract and blood, respectively. This raises several interesting questions concerning the potential role of LPS as a virulence factor in the infection processes.
Genes for D-arabinitol and ribitol catabolism from Klebsiella pneumoniae
Authors: Heuel H. , Shakeri-Garakani A. , Turgut S. , Lengeler J. W.
Published: June 2009

The enzymes for catabolism of the pentitols D-arabinitol (Dal) and ribitol (Rbt) and the corresponding genes from Klebsiella pneumoniae (dal and rbt) and Escherichia coli (atl and rtl) have been used intensively in experimental evolutionary studies. Four dal and four rbt genes from the chromosome of K. pneumoniae 1033-5P14 were cloned and sequenced. These genes are clustered in two adjacent but divergently transcribed operons and separated by two convergently transcribed repressor genes, daIR and rbtR. Each operon encodes an NAD-dependent pentose dehydrogenase (daID and rbtD), an ATP-dependent pentulose kinase (daIK and rbtK) and a pentose-specific ion symporter (dalT and rbtT). Although the biochemical reactions which they catalyse are highly similar, the enzymes showed interesting deviations. Thus, DalR (313 aa) and RbtR (270 aa) belong to different repressor families, and DalD (455 aa) and RbtD (248 aa), which are active as a monomer or as tetramers, respectively, belong to different dehydrogenase families. Of the two kinases (19.3% identity), DalK (487 aa) belongs to the subfamily of short D-xylulokinases and RbtK (D-ribulokinase; 535 aa) to the subfamily of long kinases. The repressor, dehydrogenase and kinase genes did not show extensive similarity beyond local motifs. This contrasts with the ion symporters (86.6% identity) and their genes (82.7% identity). Due to their unusually high similarity, parts of dalT and rbtT have previously been claimed erroneously to correspond to ‘inverted repeats’ and possible remnants of a ‘metabolic transposon’ comprising the dal and rbt genes. Other characteristic structures, e.g. a secondary attλ site and chi-like sites, as well as the conservation of this gene group in E. coli C are also discussed.
Actin enhances the haemolytic activity of Escherichia coli
Authors: Basaraba R. J. , Byerly A. N. , Stewart G. C. , Mosier D. A. , Fenwick B. W. , Chengappa M. M. , Laegreid W. W.
Published: June 2009

Act in is a major cytoskeletal protein of mammalian muscle and non-muscle cells. Exposure of cells to soluble factors that damage cell membranes results in the release of actin into the extracellular spaces. The α-haemolysin (HlyA) of Escherichia coli is the prototype RTX (repeat in toxin) toxin and is thought to be important in virulence because of its ability to lyse cells by formation of pores in the cell membrane. These studies were conducted to determine if actin influences growth and haemolytic activity of E. coli. Growth of E. coli in the presence of actin resulted in culture supernatant haemolytic activity that was 2.4-, 2.7- and 3.3-fold greater than that of E. coli grown in medium containing BSA, non-supplemented medium, or medium containing heat-denatured actin, respectively. The enhanced haemolytic activity occurred only when actin was present during the growth phase and there was no effect when actin was added to culture supernatants containing haemolysin. The increased haemolytic activity by actin was concentration-dependent, detectable in early-exponential-phase growth, and associated with increased concentrations of secreted HlyA by Western blotting. Actin induced a 2.9-fold increase in alkaline phosphatase activity in E. coli CC118 with a TnphoA insertion in the hlyB determinant of the recombinant haemolysin piasmid pWAM04. These results indicate that extracellular actin enhances haemolysin production by E. coli and may have implications in the pathogenesis of E. coli infections.
Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are involved in the oxidation of intracellular sulfur
Authors: Pott Andrea S. , Dahl Christiane
Published: June 2009

The sequence of the dsr gene region of the phototrophic sulfur bacterium Chromatium vinosum D (DSMZ 180T) was determined to clarify the in vivo role of ‘reverse’ sirohaem sulfite reductase. The dsrAB genes encoding dissimilatory sulfite reductase are part of a gene cluster, dsrABEFHCMK, that encodes four small, soluble proteins (DsrE, DsrF, DsrH and DsrC), a transmembrane protein (DsrM) with similarity to haem-b-binding polypeptides and a soluble protein (DsrK) resembling [4Fe---4S]-cluster-containing heterodisulfide reductase from methanogenic archaea. Northern hybridizations showed that expression of the dsr genes is increased by the presence of reduced sulfur compounds. The dsr genes are not only transcribed from a putative promoter upstream of dsrA but primary transcripts originating from (a) transcription start site(s) downstream of dsrB are also formed. Polar insertion mutations immediately upstream of dsrA, and in dsrB, dsrH and dsrM, led to an inability of the cells to oxidize intracellularly stored sulfur. The capability of the mutants to oxidize sulfide, thiosulfate and sulfite under photolithoautotrophic conditions was unaltered. Photoorganoheterotrophic growth was also unaffected. ‘Reverse’ sulfite reductase and DsrEFHCMK are, therefore, not essential for oxidation of sulfide or thiosulfate, but are obligatory for sulfur oxidation. These results, together with the finding that the sulfur globules of C. vinosum are located in the extracytoplasmic space whilst the dsr gene products appear to be either cytoplasmic or membrane-bound led to the proposal of new models for the pathway of sulfur oxidation in this phototrophic sulfur bacterium.
Isolation of mutants deficient in acetyl-CoA synthetase and a possible regulator of acetate induction in Aspergillus niger
Authors: Sealy-Lewis Heather M. , Fairhurst Valerie
Published: June 2009

Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.
Evaluation of the intranasal challenge route in mice as a mucosal model for Candida albicans infection
Authors: Londono Patricia , Gao Xiao M. , Bowe Frances , McPheat William L. , Booth George , Dougan Gordon
Published: June 2009

The intranasal route was used to studyCandida albicansinfections in mice. Mice from two different inbred strains were challenged intranasally withC. albicansand the level of local and systemic colonization was monitored. DBA/2 mice were highly susceptible to challenge and viableC. albicansdisseminated from the lungs to deeper tissues, including kidneys, liver and spleen within 48 h. In contrast, in BALB/c mice challenged in the same manner,C. albicanswere retained within the lungs and cleared. Local and systemic anti-C. albicansimmune responses were investigated. BALB/c mice exhibited higher titres of serum and mucosal anti-C. albicansIgA than DBA/2 mice. Splenocytes from BALB/c mice, but not from DBA/2 mice, produced detectable levels of interleukin-4 and -5 following stimulation withC. albicansantigens. Both DBA/2- and BALB/c-derived splenocytes produced interferon-γ and interleukin-10 in response to similar stimulation. In conclusion, the intranasal route provided a simple, non-invasive murine model for investigatingC. albicansinfection through mucosal surfaces.
A controllable gene-expression system for the pathogenic fungus Candida glabrata
Authors: Nakayama Hironobu , Izuta Miho , Nagahashi Shigehisa , Sihta Emi Y. , Sato Yasuko , Yamazaki Toshikazu , Arisawa Mikio , Kitada Kunio
Published: June 2009

A system for controlling gene expression was established in the pathogenic fungus Candida glabrata to elucidate the physiological functions of genes. To control the expression of the gene of interest, the C. glabrata cells were first transformed with the plasmid carrying the tetracycline repressor-transactivator fusion tetR::GAL4, then with the DNA fragment containing the controllable cassette, the tetracycline operator chimeric promoter (tetO::ScHOP1). The peptide elongation factor 3 (CgTEF3) and DNA topoisomerase II (CgTOP2) genes from C. glabrata were cloned and their expression assessed using this system. When the promoter of CgTEF3 or CgTOP2 was replaced with tetO::ScHOP1, doxycycline almost completely repressed the expression of both mRNAs, and impaired growth. Repression of the TOP2 or TEF3 gene by doxycycline also hampered the survival of C. glabrata cells in mice; in mouse kidneys the number of C. glabrata cells, in which the TOP2 or TEF3 promoter was replaced with the tetO::ScHOP1 controllable cassette, did not increase when the mice were given doxycycline. Thus, it appears that the gene repression mediated by doxycycline occurred not only in culture media but also in animals; therefore, this system can be used to elucidate the function of the gene in fungal infections and pathogenesis.
Bacteria in post-glacial freshwater sediments
Authors: Miskin Ian , Rhodes Glenn , Lawlor Kirsten , Saunders Jon R. , Pickup Roger W.
Published: June 2009

Prokaryote communities in post-glacial profundal freshwater sediments of Windermere, representing 10-12000 years of deposition, were examined for culturability, viability and community structure. The potential for active geochemical cycles was inferred from the presence of specific groups of bacteria. Direct count procedures revealed 1012cells (g dry wt sediment)-1in the surface sediments, which declined to approximately 109cells (g dry wt sediment)-1at 6 m depth of core (representing approximately 10000 years of deposition). The majority of the cells in the upper sediments were metabolically active when challenged with viability probes and responded to the direct viable count method. Below 250 cm, viability shown by 5-cyano-2,3-diotyl tetrazolium chloride (CTC) dye was not significantly different from the direct count; however, counts obtained with 5-carboxyfluorescein diacetate (CFDA) and the direct viable count both declined significantly from the direct count below 250 cm and 1 m, respectively. Culture was achieved from samples throughout the core, although the numbers of culturable bacteria decreased significantly with depth, from 107c.f.u. (g dry wt sediment)-1to 101-102c.f.u. (g dry wt sediment)-1below 3 m depth. Among culturable isolates, Gram-positives and Gram-negatives were found at all levels of the core, and spore-forming heterotrophs dominated. Although sulphate-reducing bacteria were not detected below 20 cm, isolates demonstrating denitrifying activity were detected at all depths. PCR performed on samples taken below 3 m (deposited more than 7000 years ago) using eubacterial and archaeal primers revealed sequences similar to those found in deep sediments of the Pacific Ocean and the presence of methanogenic archaea. These observations indicate that bacteria and archaea are capable of long-term persistence and activity in deep, aged freshwater sediments.
Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions
Authors: Moter Annette , Leist Gregor , Rudolph Roland , Schrank Kirstin , Choi Bong-Kyu , Wagner Michael , G�bel Ulf B
Published: June 2009

Fluorescencein situhybridization (FISH) was performed on sections of plastic-embedded tissue using 16S rRNA-directed oligonucleotide probes to visualize uncultured treponemes in skin biopsies of cows with digital dermatitis. Plastic as embedding material allowed sectioning of hard and soft tissue with a defined thickness, avoiding the risk of dragging bacteria into the tissue while sectioning. Furthermore, it provided a good signal-to-noise ratio. Using this method the spatial distribution of three different bacterial phylotypes was visualized simultaneously within the tissue. Whereas debris covering the ulcers contained a mixture of different micro-organisms, a layering of certain treponemal phylotypes was observed deeper in the epidermis. Confocal laser scanning microscopy and subsequent three-dimensional reconstruction of series of optical sections confirmed that the treponemes migrated intercellularly around the cells, most of them directed towards the dermis.In situhybridization on tissue embedded in plastic proved to be a useful method to study mixed bacterial infections since it combines excellent histological conservation of tissue with identification of bacterial species by simultaneous use of probes labelled with different fluorescent dyes. This technique may have implications forin situdetection, identification and localization of microorganisms in veterinary as well as in human medicine.
Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates
Authors: van der Ploeg Jan R. , Cummings Nicola J. , Leisinger Thomas , Connerton Ian F.
Published: June 2009

A 5 kb region upstream ofkatAat 82°G on theBacillus subtilischromosome contains five ORFs organized in an operon-like structure. Based on sequence similarity, three of the ORFs are likely to encode an ABC transport system(ssuBAC)and another to encode a monooxygenase(ssuD).The deduced amino acid sequence of the last ORF(ygaN)shows no similarity to any known protein.B. subtiliscan utilize a range of aliphatic sulfonates such as alkanesulfonates, taurine, isethionate and sulfoacetate as a source of sulfur, but not whenssuAandssuCare disrupted by insertion of a neomycin-resistance gene. Utilization of aliphatic sulfonates was not affected in a strain lacking 3′-phosphoadenosine 5′-phosphosulfate (PAPS) sulfotransferase, indicating that sulfate is not an intermediate in the assimilation of sulfonate-sulfur. Sulfate or cysteine prevented expression of β-galactosidase from a transcriptionalssuD::IacZfusion. It is proposed thatssuBACDencode a system for ATP-dependent transport of alkanesulfonates and an oxygenase required for their desulfonation.
F- phenocopies: characterization of expression of the F transfer region in stationary phase
Authors: Frost Laura S. , Manchak Jan
Published: June 2009

The phenomenon of ‘F- phenocopies’ in which F+ cells become transfer-deficient in stationary phase seems contradictory to the proposed role for F transfer in adaptive mutation during stationary phase induced by nutrient limitation. The expression of a range of transfer genes at the transcriptional and translational level in stationary phase has been characterized as well as the degree of nicking at the origin of transfer, oriT. Transfer efficiency rapidly decreased in mid-exponential phase, coincident with a decrease in traM transcripts. Approximately 2 h later, the transcript for traA, encoding F-pilin, also decreased to undetectable levels. The levels of TraA (pilin), TraD, TraJ and TraT remained fairly constant well into stationary phase while the levels of TraM and Tral decreased to undetectable levels in early stationary phase. A null mutation in the gene for the alternative s factor, rpoS, did not affect mating efficiency or transcript levels but did increase the stability of TraM and Tral in stationary phase. Nicking at oriT was detected at maximal levels in early stationary phase and at low levels in late stationary phase. The results suggest that the F-pilus transfer apparatus is maintained in the cell envelope after transcription of the transfer region from the main promoter, Py, has ceased with down-regulation of traM transcription being the first step detected in this process. The presence of a low level of nicking at oriT in stationary phase is consistent with a role for F in promoting adaptive mutation.
Division-inhibition capacity of penicillin in Escherichia coli is growth-rate dependent
Authors: Hadas Hilla , Einav Monica , Fishov Itzhak , Zaritsky Arieh
Published: June 2009

Growing bacteria are sensitive to various
-lactam derivatives due to their interference with peptidogly can biosynthesis. At low concentrations, penicillin G (benzylpenicillin) blocks cell division without affecting mass growth rate. The MIC for division of Escherichia coli B/r (H266) was found to depend on the growth rate, which was modified by the nutritional conditions. Our hypothesis, that division sensitivity is proportional to the rate of peptidoglycan synthesis for septum formation, as well as to cell circumference, was thus confirmed.