bluuebunny/crossref_metadata_2025 huggingface dataset:
A mutation in the Rho1-GAP-encoding gene BEM2 of Saccharomyces cerevisiae affects morphogenesis and cell wall functionality
Authors: Cid Víctor J. , Cenamor Rosa , Sánchez Miguel , Nombela César
Published: June 2009

Summary: Saccharomyces cerevisiae strain V918 was previously isolated in a search for thermosensitive autolytic mutants and found to bear a recessive mutation that caused the development of multinucleate swollen cells undergoing cell lysis. The BEM2 gene has been isolated by complementation of the phenotype of a V918 segregant. BEM2 encodes a Rho-GTPase-activating protein (GAP) which is thought to act as a modulator of the Rho1 small GTPase. It is shown that the mutation causing the morphogenetic and autolytic phenotype in strain V918 and its segregants lies in the BEM2 gene, defining a new mutant allele, bem2-21. Mutants in the BEM2 gene have been reported to display loss of cell polarity and depolarization of the actin cytoskeleton, causing a bud-emergence defect. Low resistance to sonication and to hydrolytic enzymes proved that the cell wall is less protective in bem2-21 mutants than in wild-type strains. Moreover, bem2-21 mutants are more sensitive than the wild-type to several antifungal drugs. Transmission electron microscopy revealed the development of abnormally thick and wide septa and the existence of thin areas in the cell wall which probably account for cell lysis. The depolarization of actin in bem2-21 mutants did not preclude morphogenetic events such as cell elongation in homozygous diploid strains during nitrogen starvation in solid media, hyperpolarization of growth in a background bearing a mutated septin, or sporulation. Multinucleate cells from bem2-21 homozygous diploids underwent sporulation giving rise to multispored asci (‘polyads’), containing up to 36 spores. This phenomenon occurred only under osmotically stabilized conditions, suggesting that the integrity of the ascus wall is impaired in cells expressing the bem2-21 mutation. It is concluded that the function of the BEM2 gene product is essential for the maintenance of a functional cell wall.
Antisense RNA to ahpC, an oxidative stress defence gene involved in isoniazid resistance, indicates that AhpC of Mycobacterium bovis has virulence properties
Authors: Wilson Theresa , de Lisle Geoffrey W. , Marcinkeviciene Jovita A. , Blanchardand John S. , Collins Desmond M.
Published: June 2009

SUMMARY: Antisense RNA is a versatile tool for reducing gene expression. It was used to determine if ahpC, a gene that is involved in defence against oxidative stress and isoniazid (INH) resistance, is important for virulence of Mycobacterium bovis, a member of the Mycobecterium tuberculosis complex. Antisense RNA constructs of ahpC were made using different strength promoters in front of a reversed coding sequence of ahpC. These constructs were electroporated into a virulent wild-type M. bowis strain and a moderately virulent INH-resistant M. bo wis strain that was cata lase/peroxi dase-negat ive. Down- regulation of protein synthesis occurred and this was visualized by immunoblotting. All strains containing antisense RNA were markedly less virulent than their parent strains in guinea pigs. M. bowis with an up-regulated ahpC gene was more resistant to cumene hydroperoxide than its parent strain, which had a wild- type ahpC promoter. These results agree with a model of INH resistance in which overexpression of AhpC compensates in some INH-resistant strains for loss of catalase/peroxidase by maintaining the ability to defend against oxidative stress mediated through organic peroxides. In addition, normal expression of AhpC is crucial for maintaining the virulence of wild-type M. bovis, which has normal catalase/peroxidase levels.
The mabA gene from the inhA operon of Mycobacterium tuberculosis encodes a 3lketoacyl reductase that fails to confer isoniazid resistance
Authors: Banerjee Asesh , Sugantino Michele , Sacchettini James C. , Jacobs William R.
Published: June 2009

SUMMARY: A target of the anti-tuberculosis drugs isoniazid (INH) and ethionamide (ETH) has been shown to be an enoyl reductase, encoded by the inhA gene. The mabA (mycolic acid biosynthesis A) gene is located immediately upstream of inhA in Mycobacterium tuberculosis, Mycobacterium bo wis and Mycobacterium smegmatis. The MabA protein from M. tuberculosis was expressed in Escherichia coli and shown to have 3-ketoacyl reductase activity, consistent with a role in mycolic acid biosynthesis. In M. smegmatis, inhA and mabA are independently transcribed, but in M. tuberculosis and M. bowis BCG, mabA and inhA constitute a single operon. Several INH-ETH-resistant M. tuberculosis clinical isolates contain point mutations in the ribosome-binding site of mabA in the mabA-inhA operon. However, genetic dissection of this operon reveals that the INH-ETH-resistance phenotype is encoded only by hhA, and not by mabA.
Differential regulation of SAP8 and SAPS, which encode two new members of the secreted aspartic proteinase family in Candida albicans
Authors: Monod Michel , Hube Bernhard , Hess Daniela , Sanglard Dominique
Published: June 2009

SUMMARY: Secreted aspartic proteinases (Saps) contribute to the virulence of Candida albicans in systemic animal models of infection. Seven genes encoding Saps (SAM-SAP7) have been identified to date but evidence suggested the existence of additional SAP genes. The screening of a C. albicans iZEMBL3 genomic library for the presence of other SAP genes was undertaken. Two new genes, SAP8 and SAPS, were isolated. The N-terminal amino acid sequence deduced from SAP8 downstream of a Kex2plike cleavage site corresponds to the N-terminal amino acid sequence of the 41 kDa Sap isolated and characterized previously. SAP8 mRNA was expressed preferentially in yeasts at 25 "C after 6 and 9 h growth in BSA-containing medium. SAPS encodes an aspartic proteinase with a Kex2pllike cleavage site and contains a putative glycophosphatidylinositol-anchor signal at the C-terminus. Although the SAPS gene product has not yet been isolated from cultures of C. albicans, transcripts of SAPS were observed preferentially in later growth phases when SAP8 expression had decreased.
Roles of calcium gradients in hyphal tip growth: a mathematical model
Authors: Regaladot Carlos M.
Published: June 2009

SUMMARY: A tip-high Ca2+ gradient is observed in growing fungal hyphae, but so far its role remains unknown. A mathematical model is presented, which provides evidence for the functions of such a Ca2+ gradient, in terms of its non-linear effect on the visco-elastic properties of the hyphal cytoskeleton. The model explains how the Ca2+ status at the tip may be responsible for the apical accumulation of vesicles and for an increase in the cytogel osmotic pressure, accompanied by the contraction of the cytoskeleton. The experimentally observed retraction of the spitzenkarper preceding the initiation of a branch is also reproduced, by simulating a subapical transient release of Cat+ from internal stores.
A PCR technique based on the Hipl interspersed repetitive sequence distinguishes cyanobacterial species and strains
Authors: Smith J. K. , Parry J. D. , Day J. G. , Smith R. J.
Published: June 2009

SUMMARY: The use of primers based on the Hipl sequence as a typing technique for cyanobacteria has been investigated. The discovery of short repetitive sequence structures in bacterial DNA during the last decade has led t o the development of PCR-based methods for typing, i.e. distinguishing and identifying, bacterial species and strains. An octameric palindromic sequence known as Hipl has been shown t o be present in the chromosomal DNA of many species of cyanobacteria as a highly repetitious interspersed sequence. PCR primers were constructed that extended the Hipl sequence at the 3` end by two bases. Five of the 16 possible extended primers were tested. Each of the five primers produced a different set of products when used t o prime PCR from cyanobacterial genomic DNA. Each primer produced a distinct set of products for each of the 15 cyanobacterial species tested. The ability of Hipl-based PCR to resolve taxonomic differences was assessed by analysis of independent isolates of Anabaena flos-aquae and Nostoc ellipsosporum obtained from the CCAP (Culture Collection of Algae and Protozoa, IFE, Cumbria, UK). A PCR-based RFLP analysis of products amplified from the 235-165 rDNA intergenic region was used to characterize the isolates and to compare with the Hipl typing data. The RFLP and Hipl typing yielded similar results and both techniques were able to distinguish different strains. On the basis of these results it is suggested that the Hipl PCR technique may assist in distinguishing cyanobacterial species and strains.
pBLA8, from Brevibacterium linens, belongs to a Gram-positive subfamily of ColE2-related plasmids
Authors: Leret Veronique , Trautwetter Annie , Rind Alain , Blanco Carlos
Published: June 2009

SUMMARY: A 3.1 kb DNA fragment from pBLA8, a Brevibacterium linens cryptic plasmid, containing all the information required for autonomous replication was cloned and sequenced. Using deletion analysis, the fragment essential and sufficient for autonomous replication was delimited to 1.5 kb. This fragment is characterized by the presence of an ori site located upstream of an operon encoding two proteins, RepA and RepB, both essential for replication. Based on structural similarities and a strong conservation of ori, RepA and RepB, pBLA8 was assigned to a new subfamily of the ColE2 plasmid family. This subfamily is distinguished by the requirement for two Rep proteins and the location of an ori site upstream of the mpAB operon. RepA is thought to encode primase activity, whereas RepB could be a DNA-binding protein. An Escherichia coli-B. linens shuttle vector, derived from pBLA8, was constructed. Its host spectrum was extended to Arthrobacter species.
Heterologous expression of the bacteriocin mesentericin Y105 using the dedicated transport system and the general secretion pathway
Authors: Biet Franck , Berjeaud Jean Marc , Worobo Randy W. , Cenatiempo Yves , Fremaux Christophe
Published: June 2009

SUMMARY: Two different N-terminal extensions have been identified within class II bacteriocin precursors. The first one is a two-glycine-type leader peptide associated with a dedicated ATP-binding cassette transporter. The second is a signal peptide which directs the bacteriocin precursor to the general secretion machinery. Mesentericin Y105 is a class II anti-Listeria bacteriocin produced by Leuconostoc mesentemides Y105 via a dedicated transport system (DTS). To investigate heterologous expression systems capable of producing mesentericin Y105 in various hosts, two different secretion vectors were constructed. One of them, containing the mesentericin Y105 structural gene fused to the segment encoding the divergicin A signal peptide, was introduced into Escherichia coli, Leuconostoc subsp. and Lactococcus subsp. In E. coli, mesentericin Y105 production was linked to a putative periplasmic toxicity. To take advantage of this secretion system, the mesentericin Y105 precursor was also produced in E. coli. It was demonstrated that this pre-bacteriocin exhibited some antagonistic activity against Listeria. To allow for comparison between the two different transport systems, mesentericin Y105 production using the vector containing the mesentericin Y105 structural genem and i t s DTS transporter operon was examined. The production of mesentericin Y105 was monitored by a new fast purification method followed by MS analysis. It was shown that, in Leuconostoc, the production of mesentericin Y105 is enhanced via the DTS compared to the general secretion pathway.
Roles for GcvA-binding sites 3 and 2 and the Lrp-binding region in gcvT:: lacZ expression in Escherichia coli
Authors: Stauffer Lorraine T. , Stauffer George V.
Published: June 2009

SUMMARY: GcvA and Lrp are both necessary for activation of the gcv operon. The upstream GcvA-binding sites 3 and 2 were separated from the Lrplbinding region and the rest of the gcv control region. Moving these sites by 1 or 2 helical turns of DNA further from the gcv promoter reduces, but does not eliminate, either GcvA-mediated activation or repression of a gcvT:: lac2 gene fusion. However, moving these sites by 1-5 or 2.5 helical turns of DNA results inm a GcvA-mediated super-repression of the operon. This repression is dependent on Lrp and is partially dependent on GcvR. Lrp bound to the gcv control region induces a bend in the DNA. Based on these results, a model for gcw regulation is presented in which Lrp plays a primarily structural role, by bending the DNA and GcvA functions as the activator protein.
Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF, are induced by extracellular pyochelin in Pseudornonas aeruginosa
Authors: Reimmann Cornelia , Serino Laura , Beyeler Markus , Haa Dieter
Published: June 2009

SUMMARY: The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes, pChEF8 encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha), the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE(156 kDa) and PchF (197 kDa) proteins had adenylation, thiolation and condensationkyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon, which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchf-'/acZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin, the end product of the pathway. Iron replete conditions led t o Fur (ferric uptake regulator)-dependent repression of the pchE -laciZ fusion. A translational pchD-lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus, autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa.
Cloning and transcriptional analysis of the nifUHDK genes of Trichodesmium sp. IMS101 reveals stable nifD, nifDK and nifK transcripts
Authors: Dominic Benny , Chen Yi-Bu , Zehr Jonathan P.
Published: June 2009

Summary: Trichodesmium spp. are marine filamentous, non-heterocystous cyanobacteria capable of aerobic nitrogen fixation. In this study, the nitrogenase structural genes (nifHDK) and nifU gene of Trichodesmium sp. IMS101 were cloned and sequenced. The Trichodesmium sp. IMS101 nifH, nifD and nifK amino acid sequences showed only 79%, 66% and 68% identity, respectively, to those of Anabaena sp. strain PCC 7120. A potential transcription start site for nifH was found 212 bases upstream of the nifH start codon. Promoter-like nucleotide sequences upstream of the transcription start site were identified that were very similar to those identified for the nitrogenase genes of Anabaena spp. Sequence analysis revealed regions of DNA that may form stem-loop structures in the intercistronic regions downstream of nifH and nifD. RNA analysis by Northern hybridization revealed the presence of transcripts corresponding to nifH, nifHD and nifHDK. Surprisingly, Northern hybridization also revealed the presence of transcripts that corresponded to nifD, nifDK and nifK, which have not been previously reported as transcripts in contiguous nifHDK genes of cyanobacteria. Transcription of the nifHDK genes was not significantly repressed in the presence of nitrate at a final concentration of 20 mM or at oxygen concentrations of up to 40%, whereas ammonium and urea inhibited nifHDK transcription. The transcription of the nifHDK genes was not affected by darkness, which suggests that transcription of these genes in Trichodesmium is not directly regulated by light.
Siderophore-mediated iron uptake in Saccharomyces cerevisiae: the SIT1 gene encodes a ferrioxamine B permease that belongs to the major facilitator superfamily
Authors: Lesuisse Emmanuel , Simon-Casteras Monique , Labbe Pierre
Published: June 2009

Summary: Uptake of iron from various siderophores by a Δfet3Δfet4 strain of Saccharomyces cerevisiae was investigated. The catecholate enterobactin and the hydroxamate coprogen were taken up by the cells by passive diffusion, whereas the hydroxamates ferrioxamine B (FOB) and ferricrocin (FC) were taken up via a high-affinity energy-dependent mechanism. The kinetics of FOB and FC uptake showed reciprocal competitive inhibition. The transport was regulated by iron availability, but was independent of the Aft1p and Mac1p transcriptional activators. Mutants affected in the transport of FOB were isolated. The transport of FC was not impaired in these mutants. Functional complementation of one mutant allowed the identification of the SIT1 gene (Siderophore iron Transport) encoding a putative permease belonging to the major facilitator superfamily. The Sit1 protein is probably a permease specific for the transport of ferrioxamine-type siderophores. The evidence suggests that the uptake of ferrichrome-type siderophores like FC involves other specific permease(s), although there seems to be a common handling of FOB and FC following their internalization by the cell.
Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis
Authors: Raynaud Catherine , Etienne Gilles , Peyron Pascale , Lanéelle Marie-Antoinette , Daffé Mamadou
Published: June 2009

To evaluate the potential contribution of extracellular enzymes to the pathogenicity of mycobacteria, the presence of selected enzyme activities was investigated in the culture filtrates of the obligate human pathogen Mycobacterium tuberculosis, M. bovis BCG, the opportunistic pathogens M. kansasii and M. fortuitum, and the non-pathogenic species M. phlei and M. smegmatis. For M. tuberculosis and M. bovis, 22 enzyme activities were detected in the culture filtrates and/or cell surfaces, of which eight were absent from the culture fluids of non-pathogens: alanine dehydrogenase, glutamine synthetase, nicotinamidase, isonicotinamidase, superoxide dismutase, catalase, peroxidase and alcohol dehydrogenase. These activities, which correspond to secreted enzymes, formed a significant part (up to 92%) of the total enzyme activities of the bacteria and were absent from the culture fluids and the cell surfaces of the non-pathogenic species M. smegmatis and M. phlei. The extracellular location of superoxide dismutase and glutamine synthetase seemed to be restricted to the obligate pathogens examined. The difference in the enzyme profiles was not attributable to the growth rates of the two groups of bacteria. The presence of the eight enzyme activities in the outermost compartments of obligate pathogens and their absence in those of non-pathogens provides further evidence that these enzymes may be involved in the pathogenicity of mycobacteria. In addition, the eight enzyme activities were demonstrated in the cell extract of M. smegmatis. Stepwise erosion of the cell surface of M. smegmatis to expose internal capsular constituents showed that the various enzyme activities, with the possible exception of superoxide dismutase, were located more deeply in the cell envelope of this bacterium. This suggests that the molecular architecture of the mycobacterial envelopes may play an important role in the pathogenicity of these organisms.
Inteins in mycobacterial GyrA are a taxonomic character
Authors: Sander Peter , Alcaide Fernando , Richter Ingeborg , Frischkorn Klaus , Tortoli Enrico , Springer Burkhard , Telenti Amalio , Böttger Erik C.
Published: June 2009

The A subunit of DNA gyrase in mycobacteria is frequently subjected to splicing events as its gene, gyrA, harbours an insertion encoding an intein. Investigation of a number of different isolates of Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium ulcerans and Mycobacterium xenopi demonstrated that the presence of GyrA inteins is not random but a taxonomic character specific for a given taxon at a species or subspecies level.
Mechanisms underlying the transport and intracellular metabolism of acetic acid in the presence of glucose in the yeast Zygosaccharomyces bailii
Authors: Sousa Maria João , Rodrigues Fernando , Coôrte-Real Manuela , Leão Cecília
Published: June 2009

Zygosaccharomyces bailii ISA 1307 displays biphasic growth in a medium containing a mixture of glucose (0.5%, w/v) and acetic acid (0.5%, w/v), pH 5.0 and 3.0. In cells harvested during the first growth phase, no activity of a mediated acetic acid transport system was found. Incubation of these cells in phosphate buffer with cycloheximide for 1 h restored activity of an acetic acid carrier which behaved as the one present in glucose-grown cells. These results indicated that the acetic acid carrier is probably present in cells from the first growth phase of the mixed medium but its activity was affected by the presence of acetic acid in the culture medium. In glucose-grown cells, after incubation in phosphate buffer with glucose and acetic acid, the activity of the acetic acid carrier decreased significantly with increased acid concentration in the incubation buffer. At acid concentrations above 16.7 mM, no significant carrier activity was detectable. Furthermore, the intracellular acid concentration increased with the extracellular one and was inversely correlated with the activity of the acetic acid carrier, suggesting the involvement of a feedback inhibition mechanism in the regulation of the carrier. During biphasic growth, the first phase corresponded to a simultaneous consumption of glucose and acetic acid, and the second to the utilization of the remaining acid. The enzyme acetyl-CoA synthetase was active in both growth phases, even in the presence of glucose. Activity of isocitrate lyase and phosphoenolpyruvate carboxykinase was found only in acetic-acid-grown cells. Thus it appears that both membrane transport and acetyl-CoA synthetase and their regulation are important for Z. bailii to metabolize acetic acid in the presence of glucose. This fact correlates with the high resistance of this yeast to environments with mixtures of sugars and acetic acid such as those often present during wine fermentation.
Expression of the cold-shock gene cspB in Salmonella typhimurium occurs below a threshold temperature
Authors: Craig Jane E. , Boyle David , Francis Kevin P. , Gallagher Maurice P.
Published: June 2009

Previous studies have shown that several bacterial species exhibit a multigenic response following temperature downshift (cold shock). Evidence for such a response in Salmonella typhimurium is reported, based on the isolation of a range of low-induction-temperature gene fusions containing Mudlux insertions. The fusions exhibited different levels of basal light at 30 °C, and were induced at different rates and to different degrees over several hours following a reduction in temperature to 10 °C. Of the Mudlux gene fusions isolated, one was found which produced essentially no light when grown at 30 °C but exhibited rapid and high-level induction when the temperature was reduced to 10 °C. The target of this gene fusion (which was named cspB) was shown to lie adjacent to the umuDC operon and to encode a homologue of the major cold-shock protein of Escherichia coli, CspA. Luminescence studies revealed that substantial light production occurred from the cspB::Mudlux fusion at or below 22 °C but not at higher temperatures, even following a temperature drop from 30 °C. Moreover, cspB mRNA levels were found to mimic this pattern of luminescence, suggesting that cspB expression occurs below a defined temperature threshold. The cspB mRNA was also found to be very stable at 10 °C but to become highly unstable when the temperature was raised towards the threshold temperature, even in the presence of rifampicin. Existing cellular RNases therefore appear to mediate the decay of cspB mRNA at high temperatures, but are incapable of this at low temperatures.
The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression
Authors: Callegari M. Luisa , Riboli Barbara , Sanders J. Willem , Cocconcelli P. Sandro , Kok Jan , Venema Gerard , Morelli Lorenzo
Published: June 2009

Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (sIpH) has been cloned in Escherichia coli and sequenced. sIpH consists of 440 codons and is preceded by a ribosome-binding site (RBS) and followed by a putative ρ-independent terminator. Indeed, Northern analysis revealed that sIpH is a monocistronic gene. The gene is preceded by a possible promoter of which the -35 and -10 hexanucleotides are separated by 17 nt. By primer extension analysis the transcription start site was mapped at 7 nt downstream of the -10 sequence while the deduced amino acid sequence of SIpH shows a leader peptide of 30 aa. The sIpH gene has been amplified by PCR and the fragment, carrying the complete gene from the RBS to the stop codon, has been cloned in a lactococcal gene expression vector downstream of promoter P32. Lactococcus lactis MG1363 carrying the resulting plasmid produced and secreted an S-layer monomer with the same molecular mass as the authentic L. helveticus CNRZ 892 SIpH, as judged by SDS-PAGE. Immunoelectron microscopy revealed that SIpH was bound to the lactococcal cell walls in small clumps and accumulated in the growth medium as small sheets.
Fast purification of thioredoxin reductases and of thioredoxins with an unusual redox-active centre from anaerobic, amino-acid-utilizing bacteria
Authors: Harms Claudia , Meyer Manfred A. , Andreesen Jan R.
Published: June 2009

Thioredoxin reductase and thioredoxin are primarily involved in catabolic metabolism as important electron carriers in anaerobic, amino-acid-degrading bacteria. A general and fast procedure was developed for the purification of thioredoxin reductase and thioredoxin from Eubacterium acidaminophilum, Clostridium litorale, C. sticklandii, C. sporogenes, C. cylindrosporum and ‘Tissierella creatinophila’ based upon their properties: the binding to 2′,5′-AMP-Sepharose by thioredoxin reductase and the inability of thioredoxins to bind to a DEAE-Sephacel column. The consensus sequence at the active site of thioredoxins (-WCGPC-) was found to be modified in all of these anaerobes: Trp-31 (Escherichia coli nomenclature) was replaced by Gly or Ser, Gly-33 by Val or Glu. None of these thioredoxins reacted with thioredoxin reductase of E. coli or vice versa, but they did interact with the thioredoxin reductases obtained from the other anaerobes studied. Based upon their distinguishing features it is suggested that these thioredoxins might form an evolutionarily separate group.
Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in Saccharomyces cerevisiae
Authors: Alexandre Hervé , Plourde Lucile , Charpentier Claudine , François Jean
Published: June 2009

Apma1-1mutant ofSaccharomyces cerevisiaewith reduced H+-ATPase activity and the isogenic wild-type strain accumulated high levels of trehalose in response to a temperature upshift to 40 éC and after addition of 10% ethanol, but only modest levels in response to a rapid drop in external pH and after addition of decanoic acid. There was, however, no correlation between the absolute levels of trehalose in the stressed cells and their viability. All these treatments induced a significant decrease in intracellular pH, and surprisingly, this decrease was very similar in both strains, indicating that intracellular acidification could not be the triggering mechanism for trehalose accumulation in response to stress. A careful investigation of metabolic parameters was carried out to explain how trehalose accumulated under the four different stress conditions tested. No single and common mechanism for trehalose accumulation could be put forward and the transcriptional activation ofTPS1was not unequivocally related to trehalose accumulation. Another finding was that apma1-1mutant exhibited a two- to threefold greater capacity to accumulate trehalose than the isogenic wild-type. This enhanced disaccharide synthesis could be attributed to a twofold higher trehalose-6-phosphate synthase activity, together with a fourfold higher content of intracellular UDP-Glc. In addition, this mutant showed 1.5-fold higher levels of ATP compared to the wild-type. The various stress treatments studied showed that a drop in intracellular pH does not correlate with trehalose accumulation. It is suggested that plasma membrane alteration could be the physiological trigger inducing trehalose accumulation in yeast.
A second IgG-binding protein in Staphylococcus aureus
Authors: Zhang Lihong , Jacobsson Karin , Vasi Jozsef , Lindberg Martin , Frykberg Lars
Published: June 2009

Most strains ofStaphylococcus aureusexpress IgG-binding activity and this binding has been considered to be solely mediated by protein A. However, the existence of a second gene inS. aureusstrain 8325-4 encoding an IgG-binding polypeptide was recently reported. This novel IgG-binding polypeptide was found after panning a shotgun phage display library, made from chromosomal DNA, against immobilized human IgG. The complete gene (sbi) encoding this novel IgG-binding protein, denoted protein Sbi, has now been cloned and sequenced. Analysis of otherS. aureusstrains showed that this gene is not unique for strain 8325-4. The protein consists of 436 amino acids and exhibits an immunoglobulin-binding specificity similar to protein A. Furthermore, it is shown that Sbi is highly expressed in strain Newman 4, which shows that IgG-binding activity inS. aureuscan be mediated by proteins other than protein A.
Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi
Authors: Meehan Mary , Nowlan Peter , Owen Peter
Published: June 2009

Cell-wall-associated proteins fromStreptococcus equisubsp.equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-Mrprotein species (apparentMr220000 and 550000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-Mrprotein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge fromS. equisubsp.equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (Mr54597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified fromEscherichia coliand was shown to behave in an analogous fashion to the wild-type product in terms ofMrestimation, fibrinogen binding and seroreactivity.
Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae
Authors: Lan Ruiting , Reeves Peter R.
Published: June 2009

Individualrrnoperons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone ofVibrio cholerae.The genome of an early isolate of the seventh pandemic clone had ninerrnoperons of which two were in tandem with otherrrnoperons. The site forBgll, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate. This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation. Additionalrrnrecombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon. Spacers of 431, 509, 607 and 711 bp were found. A total of at least eightrrnrecombination events were detected. Threerrnloci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G. In addition there was variation due to deletion of tandem operons. The frequency of recombination betweenrrnoperons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study. This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study. Recombination betweenrrnoperons may be a factor in ribotype variation in all systems. The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved.
Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly-isolated strain of Pseudomonas fluorescens
Authors: Narbad Arjan , Gasson Michael J.
Published: June 2009

A soil bacterium, designated Pseudomonas fluorescens AN103, was isolated based on its ability to grow on ferulic acid as a sole source of carbon and energy. In addition, this strain was found to metabolize a number of related phenolic substrates which contained a hydroxyl group at the para position of the aromatic ring. During growth on ferulic acid, transient accumulation of vanillic acid and trace amounts of protocatechuic acid were detected in the culture medium. Washed cells grown on ferulic acid readily oxidized vanillin, vanillic acid and protocatechuic acid, the three putative intermediates of the metabolic pathway. The side-chain cleavage of ferulic acid to produce vanillin was demonstrated in vitro for the first time and this enzyme-catalysed reaction was shown to have an essential requirement for CoASH, ATP and MgCl2. This conversion involved a two-step process involving a CoA ligase followed by the side-chain cleavage. The addition of NAD increased the oxidation of vanillin to vanillic acid and had an overall effect of increasing the rate of ferulic acid cleavage. The application of 13C-NMR studies in vitro revealed acetyl-CoA as the C2 side-chain cleavage product. High levels of inducible ferulate-CoA ligase and NAD-linked vanillin dehydrogenase were detected and a novel pathway for ferulic acid metabolism in this organism is proposed.
Characterization of the glnB gene product of Nostoc punctiforme strain ATCC 29133: glnB or the PII protein may be essential
Authors: Hanson Thomas E. , Forchhammer Karl , de Marsac Nicole Tandeau , Meeks John C.
Published: June 2009

Bacterial PII proteins, encoded by glnB genes, are central signalling molecules in nitrogen regulatory pathways and are modulated by post-translational modification in response to the cellular nitrogen status. The glnB gene was cloned from the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme strain ATCC 29133 (PCC 73102) by heterologous hybridization to a Synechococcus sp. strain PCC 7942 gene fragment. Expression of the cloned gene was verified by hybridization to N. punctiforme total RNA and a single cross-reactive polypeptide was observed in immunoblots of N. punctiforme extracts probed with anti-Synechococcus 7942 PII antiserum. Modification of the purified N. punctiforme PII protein by a Synechococcus 7942 PII kinase was observed, but modified forms of PII were not detected in extracts of N. punctiforme from a variety of incubation conditions. The N. punctiforme glnB gene could not be disrupted by targeted gene replacement unless a second copy of glnB was provided in trans, suggesting that the gene or gene product is essential for growth under the conditions tested.
PAGE analysis of the heteroduplexes formed between PCR-amplified 16S rRNA genes: estimation of sequence similarity and rDNA complexity
Authors: Espejo Romilio T. , Feijóo Carmen Gloria , Romero Jaime , Vásquez Mónica
Published: June 2009

Analysis of the 16S rRNA genes retrieved directiy from different environments has proven to be a powerful tool that has greatly expanded our knowledge of microbial diversity and phylogeny. It is shown here that sequence similarity between 80 and 100% among 16S rDNAs can be estimated by the electrophoretic migration of their heteroduplexes. This was measured by hybridization and electrophoresis in polyacrylamide gels of the product obtained after PCR amplification of almost the entire 16S rRNA gene from different bacterial species. These heteroduplexes were also observed after amplification of samples containing DNA from two or more bacterial species and a procedure was applied to identify reliably heteroduplexes among the amplification products. The electrophoretic migration of the heteroduplexes observed after PCR was used to detect the presence of 16S rDNAs with different sequences in DNA extracted from both a mixture of two bacterial species and samples containing a natural bacterial community.
Molecular analysis of the DNA gyrB gene from Myxococcus xanthus
Authors: Paitan Yossi , Boulton Neil , Ron Eliora Z. , Rosenberg Eugene , Orr Elisha
Published: June 2009

DNA gyrase, an essential type II topoisomerase, mediates negative supercoiling of the bacterial chromosome, thereby affecting the processes of DNA replication, transcription, recombination and repair. The gyrB gene from the Gram-negative soil bacterium Myxococcus xanthus was sequenced. The sequence predicts a protein of 815 amino acid residues displaying significant homology to all known GyrB proteins. A 6-His-GyrB fusion protein was overexpressed in Escherichia coli and purified to near homogeneity using affinity chromatography on Ni-nitrilotriacetic acid-agarose and novobiocin-Sepharose columns. The fusion protein bound novobiocin and cross-reacted with anti-E. coli GyrB antibodies, indicating structural and functional similarities to the E. coli DNA GyrB. The gene was mapped to the region of the origin of replication (oriC of M. xanthus.
Phenotypic variation of lipid composition in Burkholderia cepacia: a response to increased growth temperature is a greater content of 2-hydroxy acids in phosphatidylethanolamine and ornithine amide lipid
Authors: Taylor Catherine J. , Anderson Alistair J. , Wilkinson Stephen G.
Published: June 2009

Burkholderia cepacia produces an unusual range of polar lipids, which includes two forms each of phosphatidylethanolamine (PE) and ornithine amide lipid (OL), differing in the presence or absence of 2-hydroxy fatty acids. By using chemostat cultures in chemically defined media, variations in the lipid content and the proportions of individual lipids have been studied as a function of (a) growth temperature, (b) growth rate and (c) growth-limiting nutrient (carbon, magnesium, phosphorus or oxygen). Total cellular lipid in carbon-limited cultures was lowest at high growth temperatures and low growth rates. Increases in growth temperature over the range 25--40 ° led to increases in the proportions of molecular species of PE and OL containing 2-hydroxy acids, without changing the PE: OL ratio. Growth temperature did not alter the balance between neutral and acidic lipids, but the contribution of phosphatidylglycerol to the latter increased with rising growth temperature and growth rate. Pigmentation of cells and the presence of flagella were also temperature-dependent. Change in growth rate also affected the PE: OL ratio and the extent to which monoenoic acids were replaced by their cyclopropane derivatives. Whereas similar lipid profiles were found for carbon-, magnesium-and oxygen-limited cultures, ornithine amides were the only polar lipids detected in phosphorus-limited cells.
SepA, the 110 kDa protein secreted by Shigella flexneri: two-domain structure and proteolytic activity
Authors: Benjelloun-Touimi Zineb , Si Tahar Mustapha , Montecucco Cesare , Sansonetti Philippe J. , Parsot Claude
Published: June 2009

Shigellosis is characterized by a strong inflammatory response which is induced by bacteria invading the colonic mucosa. Characterization of a sepA mutant indicated that SepA, the major protein secreted by Shigella flexneri growing in laboratory media, might be involved in invasion and destruction of the host intestinal epithelium. The sequence of the first 500 residues of mature SepA (110 kDa) is homologous to that of the N-terminal region of lgA1 proteases. To investigate the potential proteolytic activity of SepA, the activity of the purified protein on a wide range of synthetic peptides was tested. SepA hydrolysed several of these substrates and the activity was inhibited by PMSF. Several peptides which were hydrolysed by SepA have been described as specific substrates for cathepsin G, a serine protease produced by polymorphonuclear leukocytes that was proposed to play a role in inflammation. However, unlike cathepsin G, SepA degraded neither fibronectin nor angiotensin I and had no effect on aggregation of human platelets. In addition, analysis of SepA hydrolysis by proteinase K suggested that the protein is composed of two domains of about 450 residues separated by a hinge region of 100 residues. The 47 kDa N-terminal domain was stable and endowed with proteolytic activity.
Probing the receptor recognition site of the FimH adhesin by fimbriae-displayed FimH---FocH hybrids
Authors: Knudsen Thomas B. , Klemm Per
Published: June 2009

Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces. This binding is conferred by the minor fimbrial component FimH. The binding domain of the FimH adhesin has been studied by constructing hybrids of FimH and a homologous protein, FocH, originating from F1C fimbriae. F1C fimbriae do not bind to D-mannosides or confer agglutination of any known types of erythrocytes or yeast. It was previously shown that the FocH protein can be readily substituted by the FimH adhesin, resulting in hybrid fimbriae with the same binding characteristics as type 1 fimbriae. The receptor binding of fimbriae-presented chimeric FimH--FocH hybrids was studied. FimH--FocH fusions encompassing 72% of the N-terminus of FimH fused to the complementary sector of FocH conferred agglutination of erythrocytes and yeast cells at a comparable level to FimH. Surprisingly, it was also found that similar fusions containing between 56 and 66% FimH still conferred binding to yeast cells, D-mannose--BSA and D-mannose--beads but did not give rise to agglutination. The receptor binding capacity of fusions containing 50% or less of the FimH N-terminal region was virtually abolished. The results point to the presence of a D-mannose-receptor-binding core domain in FimH, the affinity of which is modulated by other sectors of the protein to enable binding to extended mannose-containing targets.
Aggregation in Azospirillum brasilense: effects of chemical and physical factors and involvement of extracellular components
Authors: Burdman Saul , Jurkevitch Edouard , Schwartsburd Boris , Hampel Michal , Okon Yaacov
Published: June 2009

A medium for consistent induction of aggregation of Azospirillum brasilense cells was developed and used to study the effects of chemical and physical factors as well as extracellular components involved in this phenomenon. Growth of A. brasilense strain Cd in a high C:N medium using fructose and ammonium chloride as C and N sources, respectively, resulted in flocculation visible to the naked eye after 24 h. No cell aggregates were formed after 72 h growth in low C:N medium. Aggregating cells, but not cells grown under low C:N, accumulated high amounts of poly--hydroxybutyrate and the cell envelope contained a well-defined electron-dense layer outside the outer membrane. Suspending the aggregates in 0.2 or 0.5 M urea was the only treatment effective for disrupting aggregates. The concentration of exopolysaccharide produced by four different strains of A. brasilense, differing in their capacity to aggregate, strongly correlated with the extent of aggregation. Electrophoretic protein profiles from different fractions of aggregating and non-aggregating cells were compared. Differences were observed in the pattern of low-molecular-mass proteins and in the polar flagellin that has previously been proposed to be involved in adhesion processes. However, a mutant lacking both lateral and polar flagella showed the strongest aggregation. The involvement of polysaccharides and/or proteins in aggregation of A. brasilense is discussed.
Rickettsia Rickettsii Infection of the Ea.Hy 926 Endothelial Cell Line: Morphological Response To Infection and Evidence for Oxidative Injury
Authors: Eremeeva Marina E. , Silverman David J.
Published: June 2009

EA.hy 926 is a permanent human cell line that expresses highly differentiated functions characteristic of human vascular endothelium.Rickettsia rickettsiican efficiently infect and cause a cytopathic effect in EA.hy 926 cells.R. rickettsiiproduced visible lytic plaques in EA.hy 926 cells at 10 d postinfection (p.i.) following application of a secondary agarose overlay containing 2 μg emetine ml-1and 40 μg NaF ml-1on day 2. Rickettsial growth in EA.hy 926 cells had a similar profile to that occurring in human umbilical vein endothelial cells (HUVEC) and rickettsiae catalysed polymerization of actin tails. Intracellular multiplication ofR. rickettsiiresulted in significant changes in the internal morphology of EA.hy 926 cells, most notably extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope by 72 h p.i. These events correlated with significant alterations in the host-cell antioxidant system, including decreased levels of intracellular reduced glutathione and glutathione peroxidase activity and increased amounts of intracellular peroxide through to 96 h of infection. These findings are similar to the changes described previously forR. rickettsii-infected HUVEC and suggest that common mechanisms associated with rickettsia-induced oxidative injury occur in the two cell lines. EA.hy 926 cells were also used to investigate the influence of the antioxidant α-lipoic acid on rickettsial infection. Overnight pretreatment with 1-500 μM α-lipoic acid did not prevent cells from being destroyed following infection with rickettsiae. Supplementation of the culture medium with 1 and 10 μM α-lipoic acid 2 h after rickettsial inoculation also did not provide any protective effect. However, 100, 200 and 500 μM α-lipoic acid increased the viability of infected cells at 96 h to 45, 51 and 70%, respectively compared with 26% for untreated, infected samples. Thiol levels and glutathione peroxidase activity in treated, infected cells increased and peroxide content decreased proportionally to increasing α-lipoic acid concentrations. Furthermore, treatment with 500 μM α-lipoic acid for 72 h p.i. completely prevented ultrastructural changes in infected cells. In conclusion, the permanent endothelial cell line EA.hy 926 is susceptible to injury induced byR. rickettsiiinfection. Although the cellular changes resulting from infection are not identical in all aspects to that demonstrated previously in HUVEC, the increased reproducibility and convenience of EA.hy 926 cells make them suitable for biochemical and morphological studies.