bluuebunny/crossref_metadata_2025 huggingface dataset:
A spiroplasma associated with tremor disease in the Chinese mitten crab (Eriocheir sinensis)
Authors: Wang Wen , Wen Bohai , Gasparich Gail E. , Zhu Ningning , Rong Liwen , Chen Jianxiu , Xu Zaikuan
Published: September 2004

An epidemic of tremor disease has been a serious problem in Chinese mitten crabs,Eriocheir sinensis, in China in recent years. The disease-causing agent was previously considered to be a rickettsia-like organism. Here, analysis of the 16S rRNA gene sequence, light and electron microscopy and cultivationin vitrowere used to identify the agent. Sequence analysis of the 16S rRNA gene found it to have 98 % identity with that ofSpiroplasma mirum. The agent was able to be passed through membrane filters with pores 220 nm in diameter and could be cultivated by inoculating the yolk sac of embryonated chicken eggs and M1D medium. Rotary motion and flexional movement were seen by light microscopy, and electron microscopy showed that the organism had a helical morphology and lacked a cell wall. The organism produced small colonies with a diameter of 40–50 μm after 17–25 days of incubation on solid M1D medium. The agent was found in blood cells, muscles, nerves and connective tissues of crabs inoculated with a filtrate of yolk sacs or with cultures grown in M1D medium, and it was similar in structure to those grown in eggs and cultivation broth. Disease was reproduced by experimental infection with the cultivated organisms. This study has demonstrated that the causative agent of tremor disease in the Chinese mitten crab is a member of the genusSpiroplasma. This is believed to be the first time a spiroplasma has been found in a crustacean. These findings are not only significant for studies on pathogenic spiroplasmas, but also have implications for studies of freshwater ecology.
Use of community genome arrays (CGAs) to assess the effects of Acacia angustissima on rumen ecology
Authors: Krause Denis O. , Smith Wendy J. M. , McSweeney Christopher S.
Published: September 2004

This research developed a community genome array (CGA) to assess the effects of Acacia angustissima on rumen microbiology. A. angustissima produces non-protein amino acids as well as tannins, which may be toxic to animals, and CGA was used to assess the effects of this plant on the ecology of the rumen. CGAs were developed using a 7·5 cm×2·5 cm nylon membrane format that included up to 96 bacterial genomes. It was possible to separately hybridize large numbers of membranes at once using this mini-membrane format. Pair-wise cross-hybridization experiments were conducted to determine the degree of cross-hybridization between strains; cross-hybridization occurred between strains of the same species, but little cross-reactivity was observed among different species. CGAs were successfully used to survey the microbial communities of animals consuming an A. angustissima containing diet but quantification was not precise. To properly quantify and validate the CGA, Fibrobacter and Ruminococcus populations were independently assessed using 16S rDNA probes to extracted rRNA. The CGA detected an increase in these populations as acacia increased in the diet, which was confirmed by rRNA analysis. There was a great deal of variation among strains of the same species in how they responded to A. angustissima. However, in general Selenomonas strains tended to be resistant to the tannins in the acacia while Butyrivibrio fibrisolvens was sensitive. On the other hand some species, like streptococci, varied. Streptococcus bovis-like strains were sensitive to an increase in acacia in the diet while Streptococcus gallolyticus-like strains were resistant. Strep. gallolyticus has independently been shown to be resistant to tannins. It is concluded that there is significant variation in tannin resistance between strains of the same species. This implies that there are specific molecular mechanisms at play that are independent of the phylogenetic position of the organism.
Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis
Authors: Kimura Keitarou , Tran Lam-Son Phan , Itoh Yoshifumi
Published: September 2004

Many bacteria, including Escherichia coli, have a unique gene that encodes glutamate racemase. This enzyme catalyses the formation of d-glutamate, which is necessary for cell wall peptidoglycan synthesis. However, Bacillus subtilis has two glutamate racemase genes, named racE and yrpC. Since racE appears to be indispensable for growth in rich medium, the role of yrpC in d-amino acid synthesis is vague. Experiments with racE- and yrpC-knockout mutants confirmed that racE is essential for growth in rich medium but showed that this gene was dispensable for growth in minimal medium, where yrpC executes the anaplerotic role of racE. LacZ fusion assays demonstrated that racE was expressed in both types of media but yrpC was expressed only in minimal medium, which accounted for the absence of yrpC function in rich medium. Neither racE nor yrpC was required for B. subtilis cells to synthesize poly-γ-dl-glutamate (γ-PGA), a capsule polypeptide of d- and l-glutamate linked through a γ-carboxylamide bond. Wild-type cells degraded the capsule during the late stationary phase without accumulating the degradation products, d-glutamate and l-glutamate, in the medium. In contrast, racE or yrpC mutant cells accumulated significant amounts of d- but not l-glutamate. Exogenous d-glutamate utilization was somewhat defective in the mutants and the double mutation of race and yrpc severely impaired d-amino acid utilization. Thus, both racemase genes appear necessary to complete the catabolism of exogenous d-glutamate generated from γ-PGA.
Cell-surface alterations in class IIa bacteriocin-resistant Listeria monocytogenes strains
Authors: Vadyvaloo Viveka , Arous Safia , Gravesen Anne , Héchard Yann , Chauhan-Haubrock Ramola , Hastings John W. , Rautenbach Marina
Published: September 2004

Strains of the food-borne pathogenListeria monocytogenes, showing either intermediate or high-level resistance to class IIa bacteriocins, were investigated to determine characteristics that correlated with their sensitivity levels. Two intermediate and one highly resistant spontaneous mutant ofL. monocytogenesB73, a highly resistant mutant ofL. monocytogenes412, and a highly resistant, defined (mptA) mutant ofL. monocytogenesEGDe were compared with their respective wild-type strains in order to investigate the contribution of different factors to resistance. Decreased mannose-specific phosphotransferase system gene expression (mptA, EIIABMancomponent) was implicated in all levels of resistance, confirming previous studies by the authors' group. However, a clear correlation betweend-alanine content in teichoic acid (TA), in particular the alanine : phosphorus ratio, and a more positive cell surface, as determined by cytochromecbinding, were found for the highly resistant strains. Furthermore, two of the three highly resistant strains showed a significant increase in sensitivity towardsd-cycloserine (DCS). However, real-time PCR of thedltA(d-alanine esterification), anddalandddlAgenes (peptidoglycan biosynthesis) showed no change in transcriptional levels. The link between DCS sensitivity and increasedd-alanine esterification of TA may be that DCS competes with alanine for transport via the alanine transporter. A possible tendency towards increased lysinylation of membrane phospholipid in the highly resistant strains was also found. A previous study reported that cell membranes of all the resistant strains, including the intermediate resistant strains, contained more unsaturated phosphatidylglycerol, which is an indication of a more fluid cell membrane. The results of that study correlate with the possible lysinylation, decreasedmptAexpression,d-alanine esterification of TA and more positive cell surface charge found in this study for resistant strains. The authors' findings strongly indicate that all these factors could contribute to class IIa bacteriocin resistance and that the combination and contribution of each of these factors determine the level of bacteriocin resistance.
Calnexin, calreticulin and cytoskeleton-associated proteins modulate uptake and growth of Legionella pneumophila in Dictyostelium discoideum
Authors: Fajardo Marcela , Schleicher Michael , Noegel Angelika , Bozzaro Salvatore , Killinger Silke , Heuner Klaus , Hacker Jörg , Steinert Michael
Published: September 2004

The haploid amoebaDictyostelium discoideumis a versatile host system for studying cellular aspects ofLegionellapathogenicity. Previous studies have shown that the internalization ofL. pneumophilaleads to an endoplasmic reticulum (ER)-derived organelle that supports intracellular replication of the bacteria. In this study a roadmap of host-cell factors involved in this process was developed. Phagocytosis assays with specific cellular inhibitors and the effects of well defined host-cell mutants revealed that cytoplasmic calcium levels, cytoskeleton-associated proteins and the calcium-binding proteins of the ER, calreticulin and calnexin, specifically influence the uptake and intracellular growth ofL. pneumophila. Confocal microscopic time series with green fluorescent protein (GFP)-tagged calnexin and calreticulin demonstrated the accumulation of both proteins in the phagocytic cup ofL. pneumophila-infected host cells. In contrast to the control experiment withEscherichia coli-containing phagosomes, both proteins decorated the replicative vacuole ofL. pneumophiladuring the entire growth phase of the bacteria. The cumulative effects of cytosolic calcium levels, the spatial distribution of calnexin and calreticulin, and the defective invasion and replication ofL. pneumophilain calnexin- and calreticulin-minus cells suggest that these factors are part of a regulatory system that leads to the specific vacuole ofL. pneumophila.
Identification of the dialysable serum inducer of germ-tube formation in Candida albicans
Authors: Hudson Debbie A. , Sciascia Quentin L. , Sanders Rebecca J. , Norris Gillian E. , Edwards Pat J. B. , Sullivan Patrick A. , Farley Peter C.
Published: September 2004

Yeast cells of Candida albicans are induced by serum at 37 °C to produce germ tubes, the first step in a transition from yeast to hyphal growth. Previously, it has been shown that the active component is not serum albumin but is present in the dialysable fraction of serum. In this study, serum induction of germ-tube formation is shown to occur even in the presence of added exogenous nitrogen sources and is therefore not signalled by nitrogen derepression. The active component in serum was purified by ion-exchange, reverse-phase and size-exclusion chromatography from the dialysable fraction of serum and was identified by NMR to be d-glucose. Enzymic destruction of glucose, using glucose oxidase, demonstrated that d-glucose was the only active component in these fractions. Induction of germ-tube formation by d-glucose required a temperature of 37 °C and the pH optimum was between pH 7·0 and 8·0. d-Glucose induced germ-tube formation in a panel of clinical isolates of C. albicans. Although d-glucose is the major inducer in serum, a second non-dialysable, trichloroacetic acid precipitable inducer is also present. However, whereas either 1·4 % (v/v) serum or an equivalent concentration of d-glucose induced 50 % germ-tube formation, the non-dialysable component required a 10-fold higher concentration to induce 50 % germ-tube formation. Serum is, therefore, the most effective induction medium for germ-tube formation because it is buffered at about pH 8·5 and contains two distinct inducers (glucose and a non-dialysable component), both active at this pH.
Complex regulation of the synthesis of the compatible solute ectoine in the halophilic bacterium Chromohalobacter salexigens DSM 3043T
Authors: Calderón M. Isabel , Vargas Carmen , Rojo Fernando , Iglesias-Guerra Fernando , Csonka Laszlo N. , Ventosa Antonio , Nieto Joaquín J.
Published: September 2004

The synthesis of the compatible solute ectoine, mediated by the ectABC gene products, is the main mechanism used by the halophilic bacterium Chromohalobacter salexigens to cope with osmotic stress. Evidence was found that this process is regulated at the transcriptional level. S1 protection analyses performed with RNA extracted from cells grown in minimal medium at low (0·75 M NaCl) or high (2·5 M NaCl) osmolarity suggested the existence of four promoters upstream of ectA. Two of these (PectA1 and PectA2) might be recognized by the main vegetative sigma factor σ
70, and one (PectA3) might be dependent on the general stress sigma factor σ
S. The S1 protection assays suggest that PectA1 and PectA3 may be osmoregulated promoters. In addition, an internal promoter showing sequences homologous to promoters dependent on the heat-shock sigma factor σ
32 was found upstream of ectB. Transcription from PectA in C. salexigens followed a pattern typical of σ
S-dependent promoters, and was reduced by 50 % in an E. coli rpoS background. These data strongly suggest the involvement of the general stress sigma factor σ
S in ectABC transcription in C. salexigens. Expression of PectA–lacZ and PectB–lacZ trancriptional fusions was very high at low salinity, suggesting that ectABC may be a partially constitutive system. Both transcriptional fusions were induced during continuous growth at high temperature and their expression was reduced in cells grown in the presence of osmoprotectants (ectoine or glycine betaine) or the DNA gyrase inhibitor nalidixic acid. Moreover, PectA–lacZ expression was negatively modulated in cells grown with an excess of iron (FeCl3). Measurement of ectoine levels in the presence of glycine betaine at different NaCl concentrations suggests that an additional post-transcriptional control may occur as well.
A differential effect of σ
S on the expression of the PHO regulon genes of Escherichia coli
Authors: Taschner Natalia Pasternak , Yagil Ezra , Spira Beny
Published: September 2004

The RNA polymerase core associated with σ
S transcribes many genes related to stress or to the stationary phase. When cells enter a phase of phosphate starvation, the transcription of several genes and operons, collectively known as the PHO regulon, is strongly induced. The promoters of the PHO genes hitherto analysed are recognized by σ
D-associated RNA polymerase. A mutation in the gene that encodes σ
S, rpoS, significantly increases the level of alkaline phosphatase activity and the overproduction of σ
S inhibits it. Other PHO genes such as phoE and ugpB are likewise affected by σ
S. In contrast, pstS, which encodes a periplasmic phosphate-binding protein and is a negative regulator of PHO, is stimulated by σ
S. The effect of σ
S on the PHO genes is at the transcriptional level. It is shown that a cytosine residue at position −13 is important for the positive effect of σ
S on pst. The interpretation of these observations is based on the competition between σ
S and σ
D for the binding to the core RNA polymerase.
New roles for CDC25 in growth control, galactose regulation and cellular differentiation in Saccharomyces cerevisiae
Authors: Folch-Mallol Jorge Luis , Martínez Luz María , Casas Sergio J. , Yang Runying , Martínez-Anaya Claudia , López Lorena , Hernández Alejandra , Nieto-Sotelo Jorge
Published: September 2004

Living organisms display large differences in stress resistance throughout their life cycles. To study the coordinated regulation of development and stress responses in exponentially growing yeast, mutants that displayed elevated heat-shock resistance at this stage were screened for. Here, two new mutant alleles ofCDC25inSaccharomyces cerevisiae,cdc25-21andcdc25-22, are described. During exponential growth in glucose at 25 °C, these mutants are resistant to heat, oxidative, osmotic and ionic shock, accumulate stress-protein transcripts, show slow growth rates, thick cell walls and glycogen hyperaccumulation and lack cAMP signalling in response to glucose. Genetic and cellular analyses revealed that the stationary-phase phenotypes ofcdc25-21andcdc25-22mutants are not due to entrance to a G0state during exponential growth, but are the result of a prolonged G1phase. It was found that, in the W303 background,CDC25is dispensable for growth in glucose media. However,CDC25is essential for growth in galactose, in non-fermentable carbon sources and under continuous incubation at 38 °C. In conclusion, the function of the catalytic, C-terminal domain of Cdc25p is not only important for fermentative growth, but also for growth in non-fermentable carbon sources and to trigger galactose derepression.
Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme
Authors: Kiiskinen Laura-Leena , Kruus Kristiina , Bailey Michael , Ylösmäki Erkko , Siika-aho Matti , Saloheimo Markku
Published: September 2004

Previous studies onMelanocarpus albomyceslaccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungusTrichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin–laccase fusion protein. Both proteins were expressed inT. reeseiunder thecbh1promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l−1). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin–laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l−1. The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.
Polymorphonuclear leukocytes (PMNs) induce protective Th1-type cytokine epithelial responses in an in vitro model of oral candidosis
Authors: Schaller Martin , Boeld Ursula , Oberbauer Sylvia , Hamm Gerald , Hube Bernhard , Korting Hans C.
Published: September 2004

The immune response and the anticandidal activity of keratinocytes and polymorphonuclear leukocytes (PMNs) play a key role in host defence against localizedCandida albicansinfection. An established model of oral candidosis based on reconstituted human oral epithelium (RHE) was supplemented with PMNs to study the effect of these immune cells during experimental oral candidosis. Infection of RHE withC. albicansinduced a strong expression of the chemokine interleukin-8 (IL-8) and the cytokine granulocyte-macrophages colony-stimulating factor (GM-CSF), and a moderate stimulation of interleukin-1α(IL-1α), interleukin-1β(IL-1β), interleukin-6 (IL-6), interferonγ(IFN-γ) and tumour necrosis factorα(TNF-α) by keratinocytes. This immune response was associated with chemoattraction of PMNs to the site of infection, whereas uninfected RHE failed to induce cytokine expression or to attract PMNs. Growth of the pathogen and tissue damage ofC. albicans-infected RHE were significantly reduced when PMNs were applied to the apical epithelial surface or when PMNs migrated through a perforated basal polycarbonate filter of the model. Notably, protection against epithelial tissue damage was also observed when PMNs were placed on the basal side of non-perforated filters, which prevented PMN migration into the RHE. Addition of PMNs enhanced a Th1-type immune response (IFN-γ, TNF-α), down-regulated the expression of the Th2-type cytokine interleukin-10 (IL-10), and was associated with protection againstCandida-induced tissue damage. This PMN-supplemented model of oral candidosis mimics thein vivosituation, and provides a promising tool for studying the immunological interactions between keratinocytes andC. albicans, as well as the influence of PMNs onC. albicanspathogenesis.
Involvement of genes of genome maintenance in the regulation of phase variation frequencies in Neisseria meningitidis
Authors: Martin Patricia , Sun Li , Hood Derek W. , Moxon E. Richard
Published: September 2004

InNeisseria meningitidis, the reversible expression of surface antigens, i.e. phase variation, results from changes within repeated simple sequence motifs located in coding or promoter regions of the genes involved in their biosynthesis. The mutation rates of these simple sequences, which have a major influence on the generation of phenotypic diversity, can affect the fitness of the population. The aim of the present study was to investigate the involvement of genetic factors involved (mutSanddam) and not yet analysed (drganddinB) in the regulation of phase variation frequencies of genes associated with a variety of repeat tracts. The frequency of frameshifts occurring in the polycytidine (polyC) tracts associated withsiaD,sprandlgtGand in the tetranucleotide (TAAA) repeat tract associated withnadAwas determined by colony immunoblotting or using thelacZgene as a reporter. Inactivation ofmutSincreased the frequency of phase variation of genes presenting homopolymeric tracts of diverse length. Overexpression ofdinBenhanced the instability of the homopolymeric tract associated withsiaD. Investigation of thedamlocus in a population of genetically distinctN. meningitidisstrains revealed that 27 % of strains associated with invasive disease contained thedamgene. In all strains where a Dam function was absent, thedrggene had been inserted into thedamlocus. Disruption ofdamanddrgin strains representative of each genotype, i.e.dam+/drganddam/drg+, did not modify phase variation frequencies. In contrast to the effects of certain genes on homopolymeric tracts, none of the genetic factors investigated affected the stability of tetranucleotide repeat tracts.
Diversity of stx
2 converting bacteriophages induced from Shiga-toxin-producing Escherichia coli strains isolated from cattle
Authors: Muniesa Maite , Blanco Jesus E. , de Simón Merce , Serra-Moreno Ruth , Blanch Anicet R. , Jofre Juan
Published: September 2004

The presence of bacteriophages encoding Shiga toxin 2 (stx
2 phages) was analysed in 168 strains of Shiga-toxin-producing Escherichia coli (STEC) isolated from cattle. Following mitomycin C induction, strains carrying stx
2 phages were screened by plaque blot and hybridization with an stx
2
A-probe. In the stx
2-phage-carrying strains, the amounts of phage production, phage DNA extracted and Stx2 produced after induction were assessed. The induced stx
2 phages were characterized morphologically and genetically. Assays to obtain lysogens from different strains were also carried out and phages induced from the lysogens were compared with those induced from the STEC isolates. Results indicated that 18 % of the strains carried an inducible stx
2 phage. Most of them showed a direct relationship between phage induction and toxin production. Each strain carried only one inducible stx
2 phage, although a few strains had two copies of the stx
2 in the chromosome. The stx
2 phages showed diverse morphology and a wide variability in their genome. Assays to obtain lysogens showed that not all the phages were transduced with the same frequency and only six lysogens were obtained. Phages in the lysogens were the same as those induced from their respective initial STEC host strains, although the induction and relative toxin production of the lysogens varied. Most phages carried the stx
2 gene, while a few carried stx
2 variants. Infectivity of the phages depended on the different hosts, although O157 : H7 was preferentially infected by phages induced from O157 strains. The results show that inducible stx
2 phages are common among STEC of animal origin and that they may enhance the spread of stx
2.
Metabolic properties of Eubacterium pyruvativorans, a ruminal ‘hyper-ammonia-producing’ anaerobe with metabolic properties analogous to those of Clostridium kluyveri
Authors: Wallace R. John , Chaudhary Lal C. , Miyagawa Eiichi , McKain N. , Walker Nicola D.
Published: September 2004

Eubacterium pyruvativoransI-6Tis a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source. This study investigated its metabolic properties and its likely ecological niche. Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate. No single amino acid supported growth, and peptides were required for growth on amino acids. Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation. Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate.l-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated. Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively. Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate. The metabolic strategy ofE. pyruvativoranstherefore resembles that ofClostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization. The fermentative niche ofE. pyruvativoransappears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with C2units derived from other substrates to dispose of reducing equivalents.
Characterization of the Aspergillus parasiticus Δ12-desaturase gene: a role for lipid metabolism in the Aspergillus–seed interaction
Authors: Wilson Richard A. , Calvo Ana M. , Chang Perng-Kuang , Keller Nancy P.
Published: September 2004

In the mycotoxigenic oilseed pathogens Aspergillus flavus and Aspergillus parasiticus and the model filamentous fungus Aspergillus nidulans, unsaturated fatty acids and their derivatives act as important developmental signals that affect asexual conidiospore, sexual ascospore and/or sclerotial development. To dissect the relationship between lipid metabolism and fungal development, an A. parasiticus Δ12-desaturase mutant that was unable to convert oleic acid to linoleic acid and was thus impaired in polyunsaturated fatty acid biosynthesis was generated. The Δ12-desaturase mutant demonstrates delayed spore germination, a twofold reduction in growth, a reduced level of conidiation and complete loss of sclerotial development, compared to the wild-type. Host colonization is impaired, as reflected by a decrease in conidial production on live peanut and corn seed by the mutant compared to the wild-type. Similarly, the previously isolated A. nidulans Δ12-desaturase mutant has reduced colonization capabilities compared to the wild-type. Therefore, desaturation mutants display a key requisite that affords a genetic solution to oilseed crop contamination by mycotoxigenic Aspergillus species: a reduction in the production of conidia, the infectious particle of the pathogenic aspergilli.
Functional diversity of three different DsbA proteins from Neisseria meningitidis
Authors: Sinha Sunita , Langford Paul R. , Kroll J. Simon
Published: September 2004

The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes – nmb0278, nmb0294 and nmb0407 – encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.
A linear megaplasmid, p1CP, carrying the genes for chlorocatechol catabolism of Rhodococcus opacus 1CP
Authors: König Christina , Eulberg Dirk , Gröning Janosch , Lakner Silvia , Seibert Volker , Kaschabek Stefan R. , Schlömann Michael
Published: September 2004

The Gram-positive actinobacterium Rhodococcus opacus 1CP is able to utilize several (chloro)aromatic compounds as sole carbon sources, and gene clusters for various catabolic enzymes and pathways have previously been identified. Pulsed-field gel electrophoresis indicates the occurrence of a 740 kb megaplasmid, designated p1CP. Linear topology and the presence of covalently bound proteins were shown by the unchanged electrophoretic mobility after S1 nuclease treatment and by the immobility of the native plasmid during non-denaturing agarose gel electrophoresis, respectively. Sequence comparisons of both termini revealed a perfect 13 bp terminal inverted repeat (TIR) as part of an imperfect 583/587 bp TIR, as well as two copies of the highly conserved centre (GCTXCGC) of a palindromic motif. An initial restriction analysis of p1CP was performed. By means of PCR and hybridization techniques, p1CP was screened for several genes encoding enzymes of (chloro)aromatic degradation. A single maleylacetate reductase gene macA, the clc gene cluster for 4-chloro-/3,5-dichlorocatechol degradation, and the clc2 gene cluster for 3-chlorocatechol degradation were found on p1CP whereas the cat and pca gene clusters for the catechol and the protocatechuate pathways, respectively, were not. Prolonged cultivation of the wild-type strain 1CP under non-selective conditions led to the isolation of the clc- and clc2-deficient mutants 1CP.01 and 1CP.02 harbouring the shortened plasmid variants p1CP.01 (500 kb) and p1CP.02 (400 kb).
Balamuthia mandrillaris from soil samples
Authors: Dunnebacke Thelma H. , Schuster Frederick L. , Yagi Shigeo , Booton Gregory C.
Published: September 2004

Balamuthia mandrillarisamoebas are recognized as a causative agent of granulomatous amoebic encephalitis, a disease that is usually fatal. They were first recognized when isolated from the brain of a mandrill baboon that died in the San Diego Zoo Wild Life Animal Park. Subsequently, the amoebas have been found in a variety of animals, including humans (young and old, immunocompromised and immunocompetent persons), in countries around the world. Until recently, the amoebas had not been recovered from the environment and their free-living status was in question. The recovery of aBalamuthiaamoeba from a soil sample taken from a plant at the home of a child from California, USA, who died ofBalamuthiaamoebic encephalitis, was reported previously. In a continued investigation, a second amoeba was isolated from soil that was obtained from an outdoor potted plant in a spatially unrelated location. A comparison of these two environmental amoebas that were isolated from different soils with the amoeba that was obtained from the child's clinical specimen is reported here. Included are the isolation procedure for the amoebas, their growth requirements, their immunological response to anti-Balamuthiaserum, their sensitivity to a selection of antimicrobials and sequence analysis of their 16S rRNA gene. The evidence is consistent that the amoebas isolated from both soil samples and the clinical isolate obtained from the Californian child areB. mandrillaris.
Dynamic localization of membrane proteins in Bacillus subtilis
Authors: Johnson A. S. , van Horck S. , Lewis P. J.
Published: September 2004

The subcellular localization of membrane proteins in Bacillus subtilis was examined by using fluorescent protein fusions. ATP synthase and succinate dehydrogenase were found to localize within discrete domains on the membrane rather than being homogeneously distributed around the cell periphery as expected. Dual labelling of cells indicated partial colocalization of ATP synthase and succinate dehydrogenase. Further analysis using an ectopically expressed phage protein gave the same localization patterns as ATP synthase and succinate dehydrogenase, implying that membrane proteins are restricted to domains within the membrane. 3D reconstruction of images of the localization of ATP synthase showed that domains were not regular and there was no bias for localization to cell poles or any other positions. Further analysis revealed that this localization was highly dynamic, but random, implying that integral membrane proteins are free to diffuse two-dimensionally around the cytoplasmic membrane.
Hypermutable Haemophilus influenzae with mutations in mutS are found in cystic fibrosis sputum
Authors: Watson Michael E. , Burns Jane L. , Smith Arnold L.
Published: September 2004

Hypermutable bacterial pathogens exist at surprisingly high prevalence and benefit bacterial populations by promoting adaptation to selective environments, including resistance to antibiotics. Five hundredHaemophilus influenzaeisolates were screened for an increased frequency of mutation to resistance to rifampicin, nalidixic acid and spectinomycin: of the 14 hypermutable isolates identified, 12 were isolated from cystic fibrosis (CF) sputum. Analysis by enterobacterial repetitive intergenic consensus (ERIC)-PCR and ribotyping identified eight distinct genetic fingerprints. The hypermutable phenotype of seven of the eight unique isolates was associated with polymorphisms in conserved sites ofmutS. Four of the mutantmutSalleles were cloned and failed to complement the mutator phenotype of amutS : : TSTE mutant ofH. influenzaestrain Rd KW20. Antibiotic susceptibility testing of the hypermutators identified oneβ-lactamase-negative ampicillin-resistant (BLNAR) isolate with two isolates producingβ-lactamase. Six isolates from the same patient with CF, with the same genetic fingerprint, were clonal by multilocus sequence typing (MLST). In this clone, there was an evolution to higher MIC values for the antibiotics administered to the patient during the period in which the strains were isolated. HypermutableH. influenzaewith mutations inmutSare prevalent, particularly in the CF lung environment, and may be selected for and maintained by antibiotic pressure.
Evidence for a copper-dependent iron transport system in the marine, magnetotactic bacterium strain MV-1
Authors: Dubbels Bradley L. , DiSpirito Alan A. , Morton John D. , Semrau Jeremy D. , Neto J. N. E. , Bazylinski Dennis A.
Published: September 2004

Cells of the magnetotactic marine vibrio, strain MV-1, produce magnetite-containing magnetosomes when grown anaerobically or microaerobically. Stable, spontaneous, non-magnetotactic mutants were regularly observed when cells of MV-1 were cultured on solid media incubated under anaerobic or microaerobic conditions. Randomly amplified polymorphic DNA analysis showed that these mutants are not all genetically identical. Cellular iron content of one non-magnetotactic mutant strain, designated MV-1nm1, grown anaerobically, was ∼20- to 80-fold less than the iron content of wild-type (wt) MV-1 for the same iron concentrations, indicating that MV-1nm1 is deficient in some form of iron uptake. Comparative protein profiles of the two strains showed that MV-1nm1 did not produce several proteins produced by wt MV-1. To understand the potential roles of these proteins in iron transport better, one of these proteins was purified and characterized. This protein, a homodimer with an apparent subunit mass of about 19 kDa, was an iron-regulated, periplasmic protein (p19). Two potential ‘copper-handling’ motifs (MXM/MX2M) are present in the amino acid sequence of p19, and the native protein binds copper in a 1 : 1 ratio. The structural gene for p19,chpA(copperhandlingprotein) and two other putative genes upstream ofchpAwere cloned and sequenced. These putative genes encode a protein similar to the iron permease, Ftr1, from the yeastSaccharomyces cerevisiae, and a ferredoxin-like protein of unknown function. A periplasmic, copper-containing, iron(II) oxidase was also purified from wt MV-1 and MV-1nm1. This enzyme, like p19, was regulated by media iron concentration and contained four copper atoms per molecule of enzyme. It is hypothesized that ChpA, the iron permease and the iron(II) oxidase might have analogous functions for the three components of theS. cerevisiaecopper-dependent high-affinity iron uptake system (Ctr1, Ftr1 and Fet3, respectively), and that strain MV-1 may have a similar iron uptake system. However, iron(II) oxidase purified from both wt MV-1 and MV-1nm1 displayed comparable iron oxidase activities using O2as the electron acceptor, indicating that ChpA does not supply the multi-copper iron(II) oxidase with copper.
Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence
Authors: Pascon Renata C. , Ganous Tonya M. , Kingsbury Joanne M. , Cox Gary M. , McCusker John H.
Published: September 2004

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.
Functional and phylogenetic analysis of a plant-inducible oligoribonuclease (orn) gene from an indigenous Pseudomonas plasmid
Authors: Zhang Xue-Xian , Lilley Andrew K. , Bailey Mark J. , Rainey Paul B.
Published: September 2004

Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenousPseudomonasplasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3′ to 5′ exoribonuclease specific for small oligoribonucleotides. The deduced amino acid sequence of the plasmid-derivedorn(ornpl) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes. Deletion ofornplgenerated no observable phenotype, but inactivation of the chromosomal copy caused slow growth inPseudomonas putidaKT2440. This defect was fully restored by complementation withornfromEscherichia coli(ornE.coli). Plasmid-derivedornplwas capable of partially complementing theP. putida ornmutant, demonstrating functionality ofornpl. Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria. A survey ofornplfrom relatedPseudomonasplasmids showed a sporadic distribution but no sequence diversity. These data suggest that theornplwas acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of theProteobacteria.
Genomic subtraction for the identification of putative new virulence factors of an avian pathogenic Escherichia coli strain of O2 serogroup
Authors: Schouler Catherine , Koffmann Frédérique , Amory Cécile , Leroy-Sétrin Sabine , Moulin-Schouleur Maryvonne
Published: September 2004

To identify putative new virulence factors of avian pathogenicEscherichia coli(APEC) strains, a genomic subtraction was performed between the APEC strain MT512 and the non-pathogenicE. colistrain of avian origin EC79. Seventeen DNA fragments were cloned that were specific for the APEC strain. Among them, nine were identified that were more frequent among pathogenic than non-pathogenic isolates in a collection of 67 avianE. coli. Chromosome or plasmid location, and the nucleotide sequence of these nine fragments were characterized. Four fragments were plasmid-located. The nucleotide sequence of two of them exhibited identity with the sequence of the RepF1B replicon ofE. coliplasmids, and the amino-acid deduced sequences from the two other fragments exhibited similarity to the products of genessitAofSalmonellaTyphimurium andiroDofE. coli, which are involved in iron metabolism. Of the five chromosome-located fragments, three were predicted to encode parts of proteins that were significantly homologous to previously described proteins: TktA (transketolase) ofHaemophilus influenzae, a FruA (fructokinase) homologue ofListeria innocuaand Gp2 (large terminal subunit) of phage 21. The putative products of the two other chromosome-located fragments were homologous to proteins with unknown functions: Z0255 ofE. colistrain EDL933 (EHEC) and RatA ofSalmonellaTyphimurium strain LT2. Both these chromosomal fragments, whose presence is correlated with serogroups O1 and O2 and to the virulence of APEC strains belonging to these serogroups, are good candidates for being part of novel virulence determinants of APEC. Moreover, several fragments were shown to be located close to tRNAselC,asnTorthrW, which suggests they could be part of pathogenicity islands. Six fragments that were shown to be part of whole ORFs present in the APEC strain MT 512 were also present in extra-intestinal pathogenicE. coli(ExPEC) strains of human and animal origin. Thus, the putative novel virulence factors identified in this study could be shared by ExPEC strains of different origins.
Transcription of Proteus mirabilis flaAB
Authors: Manos Jim , Belas Robert
Published: September 2004

Proteus mirabilis, a Gram-negative urinary tract pathogen, has two highly homologous, tandemly arranged flagellin-encoding genes, flaA and flaB. flaA is transcribed from a σ
28 promoter, while flaB is a silent allele. Previous studies have demonstrated the presence of a family of hybrid flagellin genes, referred to as flaAB. These genes are composed of the 5′ end of flaA and the 3′ end of flaB, and are produced through excision of the intervening DNA between the two genes. Although the existence of flaAB DNA has been documented, it was not known if transcription of flaAB occurs in wild-type P. mirabilis. In this study, proof of flaAB transcription was obtained from a combination of RNA dot-blots and RT-PCR assays using specific primers and probes for flaAB and flaA. The RNA data were further supported by the demonstration of phenotypic switching of the locus using a FlaAB-detector strain. The results show that flaAB mRNA is transcribed and is 1/64 as abundant as flaA in the population of wild-type cells, suggesting that flaAB constitutes 1·0–1·5 % of the total flagellin message. Nucleotide sequence analysis of flaAB products produced by RT-PCR from the wild-type confirms previous reports of a variable fusion site between flaA and flaB resulting in a hybrid flagellin transcript. These data support the hypothesis that the production of FlaAB is integral to the physiology of P. mirabilis.
Ciliostasis is a key early event during colonization of canine tracheal tissue by Bordetella bronchiseptica
Authors: Anderton Tracy L. , Maskell Duncan J. , Preston Andrew
Published: September 2004

The primary site of infection for Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis is the ciliated respiratory epithelium. Previous studies have implicated adherence of bacteria to cilia, induction of mucus production, induction of ciliostasis and damage to the ciliated epithelium in Bordetella pathogenesis. This paper describes the use of an air-interface organ culture system using canine tracheal tissue infected with B. bronchiseptica to assess the temporal relationship between these pathologies. Ciliostasis occurs very early during the host tissue–pathogen interaction, before mucus production and obvious signs of epithelial damage occur. A B. bronchiseptica bvg mutant does not colonize the organ culture model, induce ciliostasis or cause damage to the epithelial cell layer, but it does induce similar amounts of mucus release as does infection by wild-type bacteria. The authors propose that ciliostasis is a key early event during the B. bronchiseptica–host tissue interaction that abrogates the muco-ciliary defences of the host tissue, renders it susceptible to colonization by the bacteria and allows subsequent damage to the epithelium. The organ culture model described offers a physiologically relevant tool with which to characterize the molecular basis for interactions between Bordetella and its primary site of infection, the ciliated respiratory epithelium.
Choosing materials to solve corrosion problems in the pulp and paper industry
Authors: Delblanc Bauer Anna , Lundberg Mats
Published: September 2004

Several process stages in the pulp and paper industry are undergoing changes. This is done partly for optimization, and partly to discontinue the use of substances hazardous to the environment. A side effect of these measures is that the problem of corrosion has increased. Explains why certain environments in the pulp and paper industry represent corrosion risks, with some examples given of corrosion failures, and suggests appropriate materials for these process stages.
Chamber music and coaching managers
Authors: Megginson David
Published: September 2004

Argues that, in the fields of team working and coaching, the metaphor of sporting excellence is overused. Considers the downside of the sporting analogy and proposes instead a musical analogy. Examines the value of the symphony orchestra, the jazz combo and the chamber music ensemble as alternatives. Describes a public masterclass where an established and successful musician coached a young quartet and a subsequent session that discussed the masterclass and it
Timing the adoption of a new technology: an option‐based approach
Authors: Scarso Enrico
Published: September 2004

Deals with the problem of timing the adoption of a new technology. This is a very important question of strategy which directly affects the firm’s competitive capability and whose analysis cannot be adequately faced by conventional capital budgeting techniques. Aims to provide a procedure for deciding on the appropriate adoption time, which is based on the option pricing approach. In particular, since innovative technologies generally disclose a set of strategic opportunities, makes the assumption that adoption occurs when it is possible to profit better from these opportunities. The decision procedure introduced also provides the starting point for developing a practical, option‐based framework to formulate a technology strategy. In this perspective, a proactive attitude of firms towards the planning and looking for future technology opportunities is proposed.