bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a phenolic 3-O-methyltransferase in the lignin-degrading fungus Phanerochaete chrysosporium
Authors: Jeffers Maurice R. , McRoberts W. Colin , Harper David B.
Published: June 2009

A methyl transferase enzyme catalysing the 3-O-methylation of isovanillic acid (3-hydroxy-4-methoxy!benzoic acid) by S-adenosylmethionine (SAM) was identified in Phanerochaete chrysosporium and purified. Gel filtration indicated an M
r of 71000 and SDS-PAGE showed that the enzyme was composed of two sublimits of Mr approximately 36000. Substrate utilization studies demonstrated that the enzyme was highly specific, displaying an exclusive preference for the methylation of the 3-hydroxyl group of several substituted benzoic acids. 3-Hydroxybenzoic acids with a methoxyl or hydroxyl substituent in the 2 or 4 position were the best substrates with isovanillic and 3,4-dihydroxybenzoic acids showing the highest rates of methylation. The 3-O-methyltransferase enzyme was induced later in the growth cycle than the 4-O-methyltransferase previously isolated from this fungus, which is believed to have a role in the 4-O-methylation of lignin degradation products. However the function of this meta-specific enzyme, the first phenolic 3-O-methyltransferase isolated from a fungus, remains unclear. The combined activities of the 3- and 4-O-methyltransferase enzymes satisfactorily account for the pattern of SAM-dependent methylating activity shown by whole mycelia to phenolic substrates.
The MtrD protein of Neisseria gonorrhoeae is a member of the resistance/nodulation/division protein family constituting part of an efflux system
Authors: Hagman Kayla E. , Lucas Claressa E. , Balthazar Jacqueline T. , Snyder Lori , Nilles Matthew , Judd Ralph C. , Shafer William M.
Published: June 2009

Summary: The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae mediates resistance of gonococci to structurally diverse hydrophobic agents (HAs) through an energy-dependent efflux process. Recently, complete or partial ORFs that encode membrane proteins (MtrC, MtrD, MtrE) forming an efflux pump responsible for removal of HAs from gonococci were identified and appeared to constitute a single transcriptional unit. In this study, the complete nucleotide sequence of the mtrD gene was determined, permitting the characterization of the MtrD protein. The full-length MtrD protein has a predicted molecular mass of nearly 114 kDa, putatively containing a 56 amino acid signal peptide. MtrD displays significant amino acid sequence similarity to a family of cytoplasmic, membrane proteins, termed resistance/nodulation/division (RND) proteins, which function as energy-dependent transporters of antibacterial agents and secrete bacterial products to the extracellular fluid. The predicted topology of the MtrD transporter protein revealed 12 potential membrane-spanning domains, which were clustered within the central and C-terminal regions of the primary sequence. Loss of MtrD due to insertional inactivation of the mtrD gene rendered gonococci hypersusceptible to several structurally diverse HAs, including two fatty acids (capric acid and palmitic acid) and a bile salt (cholic acid), but not hydrophilic antibiotics such as ciprofloxacin and streptomycin. Since gonococci often infect mucosal sites rich in toxic fatty acids and bile salts, the expression of the mtr efflux system may promote growth of gonococci under hostile conditions encountered in vivo.
Alanyl-tRNA synthetase gene of the extreme acidophilic chernolithoautotrophic Thiobacillus ferrooxidans is highly homologous to alaS genes from all living kingdoms but cannot be transcribed from its promoter in Escherichia coli
Authors: Guiliani Nicolas , Bengrine Abderrahmane , Borne Francoise , Chippaux Marc , Bonnefoy Violaine
Published: June 2009

Summary: The alaS gene of Thiobacillus ferrooxidans has been cloned and sequenced and its expression in Escherichia coli and T. ferrooxidans analysed. The same genomic organization to that in E. coli (recA-recX-alaS) has been found in T. ferrooxidans. The recA and alaS genes cannot be transcribed from their own promoters in E. coli. In addition to the well-known homology at the protein level between AlaS proteins from various organisms, a strong homology was found between all the known alaS genes from bacteria, archaea and eucarya. Two regions, one of which corresponds to the catalytic core, are particularly well-conserved at the nucleotide sequence level, a possible indication of strong constraints during evolution on these parts of the genes.
The Aspergillus fumigatus mepB gene encodes an 82 kDa intracellular metalloproteinase structurally related to mammalian thimet oligopeptidases
Authors: Ibrahim-Granet Oumaïma , D’Enfert Christophe
Published: June 2009

Summary: Aspergillus fumigatus produces an 82 kDa intracellular metalloproteinase that hydrolyses the Pz-peptide, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg, a typical substrate of members of the thimet oligopeptidase family which is ubiquitously distributed across animal species. The A. fumigatus mepB gene encoding this 82 kDa metalloproteinase was cloned and sequenced. Analysis of the deduced amino acid sequence of mepB showed that the MepB protein is a cytosolic zinc metalloproteinase of the thimet oligopeptidase family (M3) and as such is probably involved in the intracellular degradation of small peptides. An A. fumigatus mutant that lacks the MepB Pz-peptidolytic activity was constructed by gene disruption at the mepB locus. Analysis of this mutant did not reveal any detectable phenotype.
Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A
Authors: Casaus Pilar , Nilsen Trine , Cintas Luis M. , Nes Ingolf F. , Hernández Pablo E. , Holo Helge
Published: June 2009

Summary: The strain Enterococcus faecium T136 produces two bacteriocins, enterocin A, a member of the pediocin family of bacteriocins, and a new bacteriocin termed enterocin B. The N-terminal amino acid sequences of enterocins A and B were determined, and the gene encoding enterocin B was sequenced. The primary translation product was a 71 aa peptide containing a leader peptide of the double-glycine type which is cleaved off to give mature enterocin B of 53 aa. Enterocin B does not belong to the pediocin family of bacteriocins and shows strong homology to carnobacteriocin A. However, sequence similarities in their leader peptides and C-termini suggest that enterocin B and carnobacteriocin A are related to bacteriocins of the pediocin family. Enterocins A and B had only slightly different inhibitory spectra, and both were active against a wide range of Gram-positive bacteria, including listeriae, staphylococci and most lactic acid bacteria tested. Both had bactericidal activities, but survival at a frequency of 10−44-10−2 was observed when sensitive cultures were exposed to either bacteriocin. The number of survivors was drastically reduced when a mixture of the two bacteriocins was added to the cells.
Metabolic pathway of anaerobic ammonium oxidation on the basis of 15N studies in a fluidized bed reactor
Authors: van de Graaf Astrid A. , de Bruijn Peter , Robertson Lesley A. , Jetten Mike S. M. , Kuenen J. Gijs
Published: June 2009

Summary: A novel metabolic pathway for anaerobic ammonium oxidation with nitrite as the electron acceptor has been elucidated using 15N-Iabelled nitrogen compounds. These experiments showed that ammonium was biologically oxidized with hydroxylamine as the most probable electron acceptor. The hydroxylamine itself is most likely derived from nitrite. Batch experiments in which ammonium was oxidized with hydroxylamine transiently accumulated hydrazine. The conversion of hydrazine to dinitrogen gas is postulated as the reaction generating electron equivalents for the reduction of nitrite to hydroxylamine. During the conversion of ammonium, a small amount of nitrate was formed from some of the nitrite. The addition of NH2OH to an operating fluidized bed system caused a stoichiometric increase in the ammonium conversion rate (1 mmol I−1 h−1) and a decrease in the nitrate production rate (0.5 mmol I−1 h−1). Addition of hydrazine also caused a decrease in nitrate production. On the basis of these findings, it is postulated that the oxidation of nitrite to nitrate could provide the anaerobic ammonium-oxidizing bacteria with the reducing equivalents necessary for CO2 fixation.
A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins
Authors: Bhogal Peter S. , Slakeski Nada , Reynolds Eric C.
Published: June 2009

Summary: Porphyromonas gingivalis has been associated with the development of adult periodontitis and cysteine proteinases with trypsin-like specificity have been implicated as major virulence factors. We have extracted the major cell-associated trypsin-like proteolytic activity of P. gingivalis W50 using mild sonication. Anion-exchange and gel-filtration FPLC of the sonicate revealed that Arg- and Lys-specific proteinase activity was associated with a 300 kDa complex which could be dissociated into seven bands (48, 45, 44, 39, 27, 17 and 15 kDa) by SDS-PAGE with the 44 kDa band containing two different proteins as shown by N-terminal sequence analysis. On further chromatography of the 300 kDa complex on Arg-Sepharose the majority of the complex eluted from the affinity column as an undissociated complex. However, a small amount dissociated such that the Lys- and Arg-specific activities could be separated by eluting first with lysine then arginine, respectively. The 45 kDa protein of the complex was purified by further anion-exchange FPLC in the presence of octyl–D-glucopyranoside and was shown to be an Arg-specific, thiol-activated, calcium-stabilized cysteine proteinase. The 48 kDa protein was also further purified in a similar fashion and shown to be a Lys-specific cysteine proteinase that was not inhibited by EDTA. The two 44 kDa and the 39, 27, 17 and 15 kDa proteins of the complex exhibit amino acid sequence homology and are proposed to be haemagglutinins/adhesins. The 45 kDa Arg-specific proteinase and one of the 44 kDa adhesins as well as the 15, 17 and 27 kDa adhesins are processed from the single polyprotein encoded by the gene designated prtK, with all proteins preceded by an Arg or Lys residue within the polyprotein. Similarly, the 48 kDa Lys-specific proteinase, the 39 and 15 kDa adhesins as well as the other 44 kDa adhesin of the 300 kDa complex are encoded by a single gene designated prtK, with all proteins preceded by an Arg or Lys residue within the polyprotein. The 39, 15 and 44 kDa adhesins of PrtK all exhibit high homology with the 44, 15, 17 and 27 kDa adhesins encoded by prtR, particularly the 15 kDa proteins which are identical. The cell-associated proteinase-adhesin complex, designated PrtR-PrtK, is therefore composed of the two gene products, the mature PrtR (160 kDa) and mature PrtK (163 kDa) that are further proteolytically processed (most likely autolytically) to release proteinase and adhesin domains that remain non-covalently associated. The fully processed PrtR-PrtK complex comprises the cysteine proteinases PrtR45 and PrtK48 and seven sequence-related adhesin molecules, PrtR44, PrtRIS, PrtR17, PrtR27 and PrtK39, PrtK15 and PrtK44. We propose that this proteinase-adhesin complex is a major virulence factor for P. gingivalis
Mutational analysis of the early forespore/mother-cell signalling pathway in Bacillus subtilis
Authors: Londoño-Vallejo J. Arturo
Published: June 2009

Summary: Intercellular communication is a crucial phenomenon during spore development in Bacillus subtilis. It couples the establishment of a compartment-specific genetic program to the transcriptional activity of a σ factor in the other compartment. It also keeps σ factor activation in register with the morphological process. This study used directed mutagenesis to analyse the pathway that couples σE activation in the mother-cell to activation of σF in the forespore following asymmetric septation. Targets for mutagenesis in SpollGA (the receptor) were chosen based on the predicted topology of the protein when associated with the cell membrane. The results showed that a residue near the N terminus (D6), predicted to be exposed outside the cell, is required for receptor activity, whereas the major extracellular loop (between membrane domains IV and V) is dispensable for function. In contrast, mutations in SpollR (the signal) that partially blocked protein release (but not membrane translocation) had no effect on signal transduction. These results do not rule out the possibility that uncharacterized molecules intervene in the signalling pathway that establishes the mother-cell-specific developmental program during the early stage of sporulation.
The absence of D-alanine from lipoteichoic acid and wall teichoic acid alters surface charge, enhances autolysis and increases susceptibility to methicillin in Bacillus subtilis
Authors: Wecke Jörg , Madela Kazimierz , Fischer Werner
Published: June 2009

Summary: In Bacillus subtilis the physiological consequences of depriving lipoteichoic acid and wall teichoic acid of D-alanine ester were analysed using insertional inactivation of the genes of the dlt operon. Mutant strains which lacked positively charged D-alanine ester in teichoic acids bound more positively charged cytochrome C than other strains. These mutant strains also showed enhanced autolysis and a higher susceptibility to methicillin, which was expressed as accelerated wall lysis, a faster loss of viability and a slower recovery in the postantibiotic phase. The effects of methicillin could be suppressed by simultaneous addition of magnesium ions at low concentrations. The degradation of whole bacteria by bone-marrow-derived macrophages was not influenced by the surface charge and alanylation of the teichoic acids had no protective effect.
Isolation of Aspergillus niger creA mutants and effects of the mutations on expression of arabinases and L-arabinose catabolic enzymes
Authors: Ruijter George J. G. , Vanhanen Sipo A. , Gielkens Marco M. C. , van de Vondervoort Peter J. I. , Visser Jaap
Published: June 2009

Summary: Aspergillus niger mutants relieved of carbon repression were isolated from an areA parental strain by selection of colonies that exhibited improved growth on a combination of 4-aminobutanoic acid (GABA) and D-glucose. In addition to derepression of the utilization of GABA as a nitrogen source in the presence of D-glucose, three of the four mutants also showed derepression of L-alanine and L-proline utilization. Transformation of the mutants with the A. niger creA gene, encoding the repressor protein CREA, re-established the areA phenotype on GABA/D-glucose, identifying the mutations as creA
d. The creA gene mapped on chromosome IV by linkage analysis and contour-clamped homogeneous electric field hybridization. The creA mutants obtained were used to study the involvement of CREA in repression by D-glucose of arabinases and L-arabinose catabolism in A. niger. In wild-type A. niger, α-L-arabinofuranosidase A, α-L-arabinofuranosidase B, endo-arabinase, L-arabinose reductase and L-arabitol dehydrogenase were induced on L-arabinose, but addition of D-glucose prevented this induction. Repression was relieved to varying degrees in the creA mutants, showing that biosynthesis of arabinases and L-arabinose catabolic enzymes is under control of CREA.
Enzymic activity of the mycoparasite Verticillium fungicola on Agaricus bisporus fruit body cell walls
Authors: Calonje M. , García Mendoza C. , Galán B. , Novaes-Ledieu M.
Published: June 2009

Summary: The in vitro production of different hydrolytic enzyme activities by Verticillium fungicola, a mycoparasite of Agaricus bisporus fruit bodies, was examined in cultures grown with various carbon sources (glucose, fructose, sucrose and A. bisporus cell walls). Several of the identified enzymes were influenced by the carbon source. The results of A. bisporus cell wall digestion by these enzymes in vitro have been compared with V. fungicola infection of A. bisporus fruit bodies in vivo. Evidence supporting mycoparasite enzymic action on the host cell walls is presented.
Altered adherence properties of a Streptococcus gordonii hppA (oligopeptide permease) mutant result from transcriptional effects on cshA adhesin gene expression
Authors: McNab Roderick , Jenkinson Howard F.
Published: June 2009

Summary: Cell-surface polypeptide CshA (259 kDa) mediates multiple adherence interactions of Streptococcus gordonii. By generating a chromosomal cshA promoter (p-cshA)-cat gene fusion and measuring both CAT enzyme activity and cat mRNA levels, it was shown that cshA is expressed maximally in cells in the late exponential phase of growth in batch culture. The expression of CAT enzyme activity from the p-cshA-cat promoter fusion was 28% decreased in early stationary phase cell extracts of mutant strain OB528 in which the hppA (oligopeptide-binding lipoprotein) gene was insertionally inactivated. This effect was correlated with proportionally reduced cell-surface expression of CshA protein and with impaired adherence of hppA mutant cells to cells of an oral Actinomyces naeslundii strain. cshA promoter activity was enhanced in streptococcal cells that were incubated in conditioned culture medium as opposed to fresh medium, but this did not occur in an hppA genetic background. It is suggested that HppA is necessary for the response of cells to an extracellular factor that modulates cshA transcription, and hence affects cell-surface CshA expression and streptococcal cell adherence properties.
Phylogenetic mapping of bacterial morphology
Authors: Siefert't Janet L. , Fox George E.
Published: June 2009

SUMMARY: The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.
A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease
Authors: Enright Mark C. , Spratt Brian G.
Published: June 2009

SUMMARY: The population biology of Streptococcus pneumoniae is poorly understood. Most of the important issues could be addressed by the molecular characterization of large, well sampled populations from carriage and from the different manifestations of pneumococcal disease. The authors have therefore developed a pneumococcal multilocus sequence typing scheme and database by sequencing - 450 bp fragments of seven housekeeping loci from 295 isolates. The combination of alleles at the seven loci provided an allelic profile, or sequence type (ST), and the relatedness between isolates was obtained by constructing a dendrogram from the matrix of paiwvise differences between STs. The typing scheme was validated using pneumococci of known genetic relatedness and could resolve 6 billion STs. Among 274 isolates from recent cases of invasive pneumococcal disease in eight countries,143 STs were resolved, but 12 STs contained at least five isolates (range 5-21 isolates). The repeated recovery of indistinguishable isolates from invasive disease in different countries implies that these STs define strains with an increased capacity to cause invasive disease. The relationship between STs and serotypes suggested that, in the longer term, capsular genes have been distributed horizontally within the pneumococcal population, but in the short term, expansion of clones occurs with only occasional changes of serotype. The multilocus sequence typing scheme provides a powerful new approach to the characterization of pneumococci, since it provides molecular typing datathat are electronically portable between laboratories, and which can be used to probe aspects of the population and evolutionary biology of these organisms. A Web sitefor themolecular characterization of pneumococci by MLST is available (http://mlst.zoo.ox.ac.uk).
The kdgRKAT operon of Bacillus subtilis:detection of the transcript and regulation by the kdgR and ccpA genes
Authors: Pujic Petar , Dervyn Rozenn , Sorokin Alexei , Ehrlich S. Dusko
Published: June 2009

SUMMARY: Transcription of a new catabolic operon in Bacillus subtilis, involved in the late stages of galacturonic acid utilization, has been studied. The operon consists of four genes: kdgR, encoding the putative regulator protein; kdgK, encoding 2-keto-3-deoxyg I uconate kinase; kdgA, encoding 2-keto-3-deoxyg luconate-6-phosphate aldolase; and kdg, encoding a transporter. These four genes are organized in one transcriptional unit and map at 198" of the B. subtik chromosome. Primer extension experiments and Northern blot analysis show that an active σ-dependent promoter precedes kdgR and transcription is terminated at the putative pindependent terminator downstream of kdgr. The operon is negatively regulated by the kdgR and ccpA gene products, which belong t o the Lac1 family of transcription regulators. The expression of the genes in this operon can be induced by galacturonate and strongly repressed when glucose is present in the growth medium. Knockout mutations in genes kdgR and ccpA remove, respectively, the effects of galacturonate and glucose on the transcription of this operon.
Natural genetic transformation of Pseudomonas stutzeri in a non-sterile soil
Authors: Sikorski Johannes , Graupner Stefan , Lorenz Michael G. , Wackernagel Wilfried
Published: June 2009

Natural transformation of the soil bacterium Pseudomonas stutzeri JM300 in a non-sterile brown earth microcosm was studied. For this purpose, the microcosm was loaded with purified DNA (plasmid or chromosomal DNA, both containing a high-frequency-transformation marker, his
+, of the P. stutzeri genome), the non-adsorbed DNA was washed out with soil extract and then the soil was charged with competent cells (his-1). Both chromosomal and plasmid transformants were found among the P. stutzeri cells recovered from the soil. The number of plasmid transformants increased in a linear fashion with the amount of DNA added [10-600 ng (0.7 g soil)−1]. The observed efficiency of transformation, the time course of transformant formation and the complete inhibition of transformation by DNase I, when added to the soil, were similar to that seen in optimized transformations in nutrient broth. Addition of cells as late as 3 d after loading the soil with plasmid DNA still yielded 3% of the initial transforming activity. This suggests that nucleases indigenous to the soil destroyed the transforming DNA, but at a rate allowing considerable DNA persistence. Transformants were also obtained when intact P. stutzeri cells were introduced into the soil to serve as plasmid DNA donors. Apparently, DNA was released from the cells, adsorbed to the soil material and subsequently taken up by recipient cells. The results indicate that competent cells of P. stutzeri were able to find access to and take up DNA bound on soil particles in the presence of micro-organisms and DNases indigenous to the soil.
Loss-of-function mutations in the mtr efflux system of Neisseria gonorrhoeae
Authors: Veal Wendy L. , Yellen Ansley , Balthazar Jacqueline T. , Pan Wubin , Spratt Brian G. , Shafer William M.
Published: June 2009

Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents (HAs) has been ascribed to the mtr (multiple transferable resistance) operon. This operon is composed of the mtrR gene, which encodes a transcriptional repressor (MtrR), and a three-gene complex (mtrCDE), which encodes cell envelope proteins (MtrC-MtrD-MtrE) that form an energy-dependent efflux pump. HA-hypersusceptible strains are often isolated from patients, but the genetic basis for such hypersusceptibility was heretofore unknown. The genetic basis of HA hypersusceptibility in laboratory-derived strains BR54 and BR87 was studied to learn if this trait could be linked to mutations in the mtr operon. Mutations in the mtrR gene of these strains that could be phenotypically suppressed by mutations in their mtrC or mtrD genes were identified. Thus, small deletions (4-10 bp) in the mtrC or mtrD genes of strains BR87 and BR54 that would result in the production of truncated efflux pump proteins that serve as a membrane fusion protein (MtrC) or transporter of HAs (MtrD) were found to be responsible for their HA-hypersusceptible property.
Re-evaluation of the serotypes of Serratia marcescens and separation into two schemes based on lipopolysaccharide (O) and capsular polysaccharide (K) antigens
Authors: Aucken Hazel M. , Wilkinson Stephen G. , Pitt Tyrone L.
Published: June 2009

Chemical and serological analysis has revealed that many of the 29 O serotype reference strains of Serratia marcescens contain both neutral and acidic polysaccharides which correspond to LPS O antigens and capsular K antigens, respectively. New O and K antigen typing schemes have therefore been devised, based on the known chemical structures of the surface polysaccharides of the organism. These schemes were designed to allow the specific detection of these antigens on unknown strains using ELISAs. O antigens were detected using whole cells cultured in broth then autoclaved to remove capsular material, while K antigens were detected using formolized whole cells which had been cultured on glycerol agar to enhance capsule production. After testing with the 29 reference strains as well as 423 distinct clinical strains, it was apparent that different aspects of chemical structure were associated with different degrees of serological reactivity and the typing schemes were modified further to accommodate this. In general, the O antigen repeating unit structures were chemically simple with di-or trisaccharide backbones. Serological specificity was often provided solely by the presence or absence of anO-acetyl substituent, or a change in the linkage between two sugar residues. Five of the O serotypes in the new scheme were represented by 12 of the 29 reference strains, while three reference strains lacked O antigens altogether, resulting in the elimination of 10 of the original O types. In contrast, the K antigen repeating unit structures were more complex and chemically diverse, having at least four sugar residues. Three K types were each seen in two reference strains while 12 of the 29 reference strains were acapsular. Thus, the resulting schemes contain 19 O types and 14 K types and allow the definitive serotype identification ofS. marcescens.
The 172 kb prkA-addAB region from 83° to 97° of the Bacillus subtilis chromosome contains several dysfunctional genes, the glyB marker, many genes encoding transporter proteins, and the ubiquitous hit gene
Authors: Noback Michiel A. , Holsappel Siger , Kiewiet Rense , Terpstra Peter , Wambutt Rolf , Wedler Holger , Venema Gerard , Bron Sierd
Published: June 2009

A 171 812 bp nucleotide sequence betweenprkA andaddAB(83° to 97°) on the genetic map of theBacillus subtilis168 chromosome was determined and analysed. An accurate physical/genetic map of this previously poorly described chromosomal region was constructed. One hundred and seventy open reading frames (ORFs) were identified on this DNA fragment. These include the previously described genes cspB, glpPFKD, spoVR, phoAIV, papQ, citRA, sspB, prsA, hpr, pbpF, hemEHY, aprE, comKandaddAB. ORF yhaFin this region corresponds to the glyB marker. Among the striking features of this region are: an abundance of genes encoding (putative) transporter proteins, several dysfunctional genes, the ubiquitous hit gene, and five multidrug-resistance-like genes. These analyses have also revealed the existence of numerous paralogues of ORFs in this region: about two-thirds of the putative genes seem to have at least one paralogue in theB. subtilis genome
The N-acetylmuramoyl-L-alanine amidase encoded by the Bacillus subtilis 168 prophage SPβ
Authors: Regamey Alexandre , Karamata Dimitri
Published: June 2009

Heat shock ofBacillus subtilisCU1147, a strain lysogenic for SPβc2, a prophage with a thermosensitive repressor, results in phage induction and subsequent cell lysis. Cloning inEscherichia coliand sequencing of a DNA fragment of prophage SPβ led to the identification ofblyA, the gene encoding a 367 amino acid polypetide with a molecular mass of 39.6 kDa. Purified BlyA obtained from theE. coliclone exhibited anN-acetylmuramoyl-L-alanine amidase activity. Insertional mutagenesis confirmed that the latter enzyme was associated with SPβ-phage-mediated ceil lysis. Analysis of the neighbouring sequence suggested that the two ORFs immediately downstream ofblyAand belonging to the same operon encode polypeptides which may be involved in the release of the endolysin. The presence on the chromosomes of B. subtilis or relatedBacillus spp. of other, similar genes, and their possible relationship, is discussed.
An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase
Authors: Branny Pavel , de la Torre Françoise , Garel Jean-Renaud
Published: June 2009

The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3′-end of the shorter transcript and the location of this 3′-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.
Cryptic carbapenem antibiotic production genes are widespread in Erwinia carotovora: facile trans activation by the carR transcriptional regulator
Authors: Holden Matthew T. G. , McGowan Simon J. , Bycroft Barrie W. , Stewart Gordon S. A. B. , Williams Paul , Salmond George P. C.
Published: June 2009

Few strains of Erwinia carotovora subsp. carotovora (Ecc) make carbapenem antibiotics. Strain GS101 makes the basic carbapenem molecule, 1-carbapen-2-em-3-carboxylic acid (Car). The production of this antibiotic has been shown to be cell density dependent, requiring the accumulation of the small diffusible molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) in the growth medium. When the concentration of this inducer rises above a threshold level, OHHL is proposed to interact with the transcriptional activator of the carbapenem cluster (CarR) and induce carbapenem biosynthesis. The introduction of the GS101 carR gene into an Ecc strain (SCRI 193) which is naturally carbapenem-negative resulted in the production of Car. This suggested that strain SCRI 193 contained functional cryptic carbapenem biosynthetic genes, but lacked a functional carR homologue. The distribution of trans-activatable antibiotic genes was assayed in Erwinia strains from a culture collection and was found to be common in a large proportion of fee strains. Significantly, amongst the Ecc strains identified, a larger proportion contained trans-activatable cryptic genes than produced antibiotics constitutively. Southern hybridization of the chromosomal DNA of cryptic Ecc strains confirmed the presence of both the car biosynthetic cluster and the regulatory genes. Identification of homologues of the transcriptional activator carR suggests that the cause of the silencing of the carbapenem biosynthetic cluster in these strains is not the deletion of carR. In an attempt to identify the cause of the silencing in the Ecc strain SCRI 193 the carR homologue from this strain was cloned and sequenced. The SCRI 193 CarR homologue was 94% identical to the GS101 CarR and contained 14 amino acid substitutions. Both homologues could be expressed from their native promoters and ribosome-binding sites using an in vitro prokaryotic transcription and translation assay, and when the SCRI 193 carR homologue was cloned in multicopy plasmids and reintroduced into SCRI 193, antibiotic production was observed. This suggested that the mutation causing the silencing of the biosynthetic cluster in SCRI 193 was leaky and the cryptic Car phenotype could be suppressed by multiple copies of the apparently mutant transcriptional activator.
Chromosomal marker exchange in the thermophilic archaeon Sulfolobus acidocaldarius: physiological and cellular aspects
Authors: Ghané Farnaz , Grogan Dennis W.
Published: June 2009

Exchange and recombination of chromosomal markers is an intrinsic genetic property of the thermoacidophilic archaeon Sulfolobus acidocaldarius that has not been thoroughly characterized. To clarify the mechanism and experimental usefulness of this process, the frequency of S. acidocaldarius prototrophs produced from mixtures of two pyrimidine auxotrophs under a variety of conditions was determined. The apparent efficiency of genetic exchange was essentially independent of the density of cells deposited on the surface of solid media. Furthermore, recombinant formation could initiate in liquid suspensions, as indicated by high recombinant frequencies resulting from mixtures plated at low cell densities, and the formation of recombinants at equal or higher frequencies in liquid suspensions that were never plated. Apparent initiation of genetic exchange in liquid at 22 °C was not prevented by DNase, prior digestion of parental cells with protease from Streptomyces griseus, or any other non-lethal chemical agent tested. The results support prior indications that chromosomal marker exchange in S. acidocaldarius proceeds via conjugation, and further indicate that this conjugation can initiate quickly in dilute liquid suspension. The mating system of S. acidocaldarius thus appears physiologically distinct from that of Haloferax volcanii but perhaps similar to conjugational transfer of Sulfolobus plasmid pNOB8. The frequency of recombinants formed in these assays (10-4-10-5 per c.f.u.) greatly exceeds the number of spontaneous forward mutational events per generation for biosynthetic genes in S. acidocaldarius. This suggests that chromosomal exchange has the potential to influence the genetic dynamics of natural Sulfolobus populations.
Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudomonas stutzeri AK61, by cyanidase
Authors: Watanabe Atsushi , Yano Kazuyoshi , Ikebukuro Kazunori , Karube Isao
Published: June 2009

The cyanide-degrading bacterial strain AK61 was isolated from waste water at a metal-plating plant. The isolated strain was characterized by Gram-staining, quinone analysis, fatty acid profile and the API 20NE identification system, and identified as Pseudomonas stutzeri. Whole cells were able to degrade cyanide rapidly in a 1 mM solution containing no organic substances, and produced ammonia as a product. The induction of the cyanide-degrading activity of P. stutzeri AK61 did not depend on the presence of cyanide in the culture medium during growth. The cyanide-degrading enzyme was purified approximately 49-fold from a cell extract of P. stutzeri AK61. The enzyme had a Km of 1.7 mM for cyanide and a specific activity of 54.6 μmol ammonia produced min-1. The activity of the enzyme was optimal at 30 °C and pH 7.5. The results of SDS-PAGE, gel-filtration chromatography and NH2-terminal amino acid sequence analysis of the enzyme indicated that the functional enzyme was an aggregated protein consisting of a 38 kDa polypeptide. Like cyanidase (cyanide dihydratase), it was shown that the enzyme catalysed the hydrolysis of cyanide to ammonia and formate.
Starvation recovery of Staphylococcus aureus 8325-4
Authors: Clements Mark O. , Foster Simon J.
Published: June 2009

Nutrient limitation of Staphylococcus aureus induces a starvation-survival state which enables it to survive until sufficient nutrients become available to support growth. The response of starved S. aureus cells to nutritional upshift was analysed to characterize the recovery mechanism which results in the resumption of rapid growth. S. aureus 8325-4 starved for 7 d in a chemically defined medium limited for glucose was able to resume growth upon the addition of complex medium (brain heart infusion broth) or a mixture of amino acids and glucose. The addition of either glucose or amino acids alone did not lead to recovery of cells. Prior to the first cell division event, a lag period of about 120--150 min was observed, the duration of which was independent of the length of starvation survival. During this lag period, RNA synthesis increased immediately upon the addition of nutrients whilst protein synthesis was delayed by approximately 5 min. Cells rapidly enlarged within 30 min of recovery, and initiation of chromosome replication could be detected after 90 min. Changes in the profile of proteins expressed during the recovery period revealed that several starvation-specific proteins were down-regulated within 30 min, whilst other proteins were common to both starvation and recovery. Two proteins were identified which were only transiently expressed during the first 60 min of recovery. Protein synthesis could be detected during recovery even if the cells had been treated with the RNA synthesis inhibitor rifampicin for 30 min prior to the addition of recovery nutrients, demonstrating that several proteins are translated from long-lived mRNA transcripts present in starved cells.
Identification and sequencing of the groE operon and flanking genes of Lawsonia intracellularis: use in phylogeny
Authors: Dale C. Jane H , Moses Eric K. , Ong Chin-Chui , Morrow Chris J. , Reed Michael B. , Hasse Dete , Strugnell Richard A.
Published: June 2009

Proliferative enteropathy (PE) is a complex of diseases of commercial importance to the pig industry. The obligate intracellular bacterium Lawsonia intracellularis is consistently associated with PE and pure cultures of this bacterium have been used to reproduce PE in pigs. In this study L. intracellularis bacteria were purified directly from PE-affected tissue. DNA extracted from purified bacteria was used to construct a partial genomic library which was screened using sera from L. intracellularis-immunized rabbits. Two seroreactive recombinant clones were identified, one of which expressed proteins of 10 and 60 kDa. The sequence of the insert from this clone, plSI-2, revealed ORFs with sequence similarity to the groES/EL operon of Escherichia coli, the 50S ribosomal proteins L21 and L27 of E. coli, a GTP-binding protein of Bacillus subtilis and a possible protoporphyrinogen oxidase, HemK, of E. coli. Primers designed from unique sequences from the plSI-2 insert amplified DNA from infected, but not non-infected, porcine ilea; the amplicon sequence obtained from tissue-cultured L. intracellularis was identical to the corresponding sequence in plSI-2, confirming the origin of the clone. The sequence of L. intracellularis GroEL and other GroEL sequences in the databases were used to construct a partial phylogenetic tree. Analysis of the GroEL sequence relationship suggested that L. intracellularis is not significantly related to other organisms whose GroEL sequences are held in the databases and supports previous data from 16S sequence analyses suggesting that L. intracellularis is a member of a novel group of enteric pathogens.
Transcription and transcript processing in the sdh CDAB-sucABCD operon of Escherichia coli
Authors: Cunningham Louise , Guest John R.
Published: June 2009

The genes encoding succinate dehydrogenase (sdhCDAB), the specific components of the 2-oxoglutarate dehydrogenase complex (ODH, E1o and E2o; sucAB) and succinyl-CoA synthetase (sucCD) form a cluster containing two promoters at 16 · 3 min in the chromosome of Escherichia coli: Psdh sdhCDAB-Psuc sucAB-sucCD. The gene encoding the lipoamide dehydrogenase component of both the 2-oxoglutarate and pyruvate dehydrogenase complexes (E3; IpdA) is the distal gene of another cluster containing two promoters located at 2 · 7 min: Ppdh pdhR-aceEF-PIpd IpdA. The responses of the suc and Ipd promoters to different environmental conditions and to regulator defects were investigated with appropriate IacZ fusions, in order to understand how expression of the sucAB genes is co-regulated with other genes in the sdhCDAB-sucABCD cluster and with IpdA expression. Expression from the suc promoter was repressed by IHF and partially activated by s38 but it was not regulated by ArcA, FNR, CRP, FruR or Fis, and not repressed by glucose or anaerobiosis, indicating that the well-established catabolite and anaerobic repression of ODH synthesis is imposed elsewhere. In contrast, the Ipd promoter was repressed by both glucose (via a CRP-independent mechanism) and anaerobiosis (mediated by ArcA), and activated by Fis, but it was not regulated by FNR, FruR, IHF or s38. These observations support the view that transcription of the sucABCD genes is primarily initiated and regulated at the upstream sdh promoter, and that the Ipd promoter is independently co-regulated with Psdh
(primarily by ArcA-mediated repression) rather than with Psuc

suc Direct evidence for co-transcription of the entire sdhCDAB-sucABCD region from Psdh
was obtained by detecting a 10 kb transcript in rnc and rne mutants, but not in the parental strains. Three RNaseIII-specific processing sites, which contribute to the extreme instability of the readthrough transcript, were identified in the sdhCDAB-sucABCD intergenic region. Other sites of endonuclease processing were located by interpreting the patterns of transcript subfragments observed in Northern blotting.
Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects
Authors: Sampson Mark N. , Gooday Graham W.
Published: June 2009

Bacillus thuringiensis subsp. israelensis IPS78 and B. thuringiensis subsp. aizawai HD133 both secreted exochitinase activity when grown in a medium containing chitin. Allosamidin, a specific chitinase inhibitor, inhibited activity from both strains, with IC50 values of about 50 μM with colloidal chitin as substrate and between 1 and 10 μM with 4-methylumbelliferyl-diacetylchitobioside and 4-methylumbelliferyl-triacetylchitotrioside as substrates. The involvement of these chitinolytic activities during pathogenesis in insects has been investigated with B. thuringiensis subsp. israelensis IPS78 against larvae of the midge Culicoides nubeculosus, and with B. thuringiensis subsp. aizawai HD133 against caterpillars of the cotton leafworm Spodoptera littoralis. Presence of 100 μM allosamidin increased the LD50 by factors of 1.3 and 1.4, respectively, demonstrating a role for bacterial chitinases in the attack on the insects. Presence of chitinase A from Serratia marcescens considerably decreased the values for LD50' confirming previous observations with different systems of the potentiation of entomopathogenesis of B. thuringiensis by exogenous chitinases. The most likely action of the endogenous chitinases of B. thuringiensis is to weaken the insects' peritrophic membranes, allowing more ready access of the bacterial toxins to the gut epithelia. Addition of exogenous chitinases will then increase this effect. Complementary cross-infection experiments, strain HD133 against midge larvae and strain IPS78 against caterpillars, were performed to investigate the pathogen/host specificities of the effects. Results showed that much higher concentrations of bacteria were required to achieve even low mortalities, and addition of chitinase A gave no increase in death rate.
Temporal transcription of the lactococcal temperate phage TP901-1 and DNA sequence of the early promoter region
Authors: Madsen Peter L. , Hammer Karin
Published: June 2009

Transcriptional analysis by Northern blotting identified clusters of early, middle and late transcribed regions of the temperate lactococcal bacteriophage TP901-1 during one-step growth experiments. The latent period was found to be 65 min and the burst size 40.10. The eight early transcripts, all mapping in a 13 kb region adjacent to the attachment site of TP901-1, were present at maximal levels 10 min after infection. The four middle transcripts, observed at maximal levels 30 min after infection, are all located within a 2 kb region at the distal end of the early transcripts. The late class of transcripts were detected 40 min after infection and the amounts of these transcripts increased with time. The late transcripts were localized to the 13 kb region adjacent to the 2 kb middle transcribed region. The sequence of almost 4 kb of the early region was determined, allowing a detailed transcriptional map for the early region of which in total 6.4 kb was sequenced. Sequence analysis of the early region revealed two closely positioned but divergently orientated promoters, PL and PR, in accordance with the orientation of the ORFs and the transcriptional map. Nine ORFs were found, and similarities to a phage repressor, a single-stranded DNA-binding protein, a topoisomerase, a Cro-like protein and two other phage proteins of unknown function were detected. The gene arrangement in the early transcribed region of TP901-1 thus consists of two transcriptional units: one from PR containing four genes, of which at least two (the integrase gene and putative repressor) are needed for lysogeny, and the divergent and longer transcriptional unit from PL, presumably encoding functions required for the lytic life cycle. ORFs with homology to proteins involved in DNA replication were identified on the latter transcriptional unit.