bluuebunny/crossref_metadata_2025 huggingface dataset:
Propionibacterium acnes, a resident of lipid-rich human skin, produces a 33 kDa extracellular lipase encoded by gehA
Authors: Miskin James E. , Farrell Angela M. , Cunliffe William J. , Holland Keith T.
Published: June 2009

Five independent clones of the Propionibacterium acnes P-37 lipase gene (gehA) were obtained in Escherichia coli, and the gene was localized to a 2.75 kb Xhol fragment by subcloning. The five clones were shown to contain the same gene by Southern blotting with a DIG-labelled probe to gehA. The nucleotide sequence of gehA was determined, and shown to contain a single ORF of 1017 kb, encoding a protein of 339 amino acids. The predicted molecular mass was 36 kDa. A 33 kDa (PAGE) radiolabeled polypeptide was detected from E. coli minicell preparations harbouring gehA, which could correspond to GehA after cleavage of the putative 26 amino acid residue signal peptide. gehA was overexpressed in E. coli under the control of the bacteriophage T7 promoter, and the corresponding polypeptide was found to be present in insoluble aggregates. Active lipase was produced when the overexpressing strain was incubated at a reduced temperature in the presence of sucrose. Purification of lipase from P. acnes culture supernatant fluids confirmed the production of a 33 kDa (PAGE) lipase.
Escherichia coli LT enterotoxin subunit A demonstrates partial toxicity independent of the nicking around Arg192
Authors: Tsuji Takao , Kato Michio , Kawase Hidetsugu , Imamura Seiji , Kamiya Hirofumi , Ichinose Yoshio , Miyama Akio
Published: June 2009

A study was conducted into whether or not nicking of the A subunit of Escherichia coli LT enterotoxin at position Arg192 or its neighbouring amino acids Arg192 to The 195 is required for its toxicity. The toxic activity of mutants created by substitution or deletion at this position, which lacked ADP-ribosyltransferase activity in vitro, was not completely obliterated and cyclic AMP was partially induced in the target cells, showing that they still displayed enzymic activity in vivo. Moreover, although the A subunit possesses three potential sites for cleavage by furin, furin was not involved in the partial toxicity and cyclic AMP induction observed. These data suggest that target cells have a nick mechanism that operates at sites other than those around Arg192 or those recognized by furin, which generates an active fragment by processing the A subunit after toxin binding to the cell membrane.
Nucleotide sequence and analysis of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome
Authors: Sadaie Yoshito , Yata Katsunori , Fujita Masaya , Sagai Hitoshi , Itaya Mitsuhiro , Kasahara Yasuhiro , Ogasawara Naotake
Published: June 2009

A 36 kb sequence of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome at around 55° has been determined. The sequenced region contains 36 ORFs including the phoB and groESL genes, and the whole rrnE operon. The phoB gene is transcribed in the direction opposite to that of chromosome replication, while most ORFs, including groESL and the rrnE operon, are transcribed in the same direction. Two newly identified tRNA genes upstream of the rrnE operon were those for Arg-tRNA and Gly-tRNA. The sequenced region contains an operon consisting of genes for degradation and uptake of mannan. The rrnE operon and its downstream ORFs are well conserved among Mycoplasma genitalium, Haemophilus influenzae, Synechocystis sp. and Methanococcus jannaschii. σH consensus sequences are present in the promoter regions of three ORFs, including groESL.
CHS2, a chitin synthase gene from the oomycete Saprolegnia monoica
Authors: Mort-Bontemps Maryline , Gay Lucien , Févre Michel
Published: June 2009

PCR was used to amplify fragments corresponding to the chitin synthase (CHS) genes from the Oomycetes Saprolegnia monoica, Phytophthora capsicum and Achlya ambisexualis, utilizing as primers, oligonucleotides designed from the conserved region of CHS genes of chitinous fungi. Chitin synthase homologues were found in the three cellulosic fungi. The chitin synthase 2 gene (CHS2) from S. monoica was cloned, sequenced and characterized. The amino acid sequence deduced from the CHS2 genomic DNA revealed several domains, corresponding to the catalytic domains and polypeptide signatures, of high identity with CHS genes from chitinous fungi. Existence of a CHS gene family in S. monoica was supported by the identification of two CHS sequences among the PCR products, the localization of CHS homologues on two chromosomes, and the detection of two transcripts in mycelia and protoplasts. Polyclonal anti-chitin synthase antibodies raised against the N-terminal and the neutral fragments of the CHS2 products revealed, respectively, two and four proteins in membrane fractions and a truncated active form in entrapped product. The overall comparison of the structure and organization of CHS genes indicates that in spite of their divergent evolution, Oomycetes and chitinous fungi have evolved with conserved chitin synthase systems.
The genetic structure of Escherichia coli populations in feral house mice
Authors: Gordon David M.
Published: June 2009

Escherichia coliwas isolated from feral house mice (Mus domesticus) during the course of a mouse plague in the state of Victoria, Australia. Two farms were sampled over a period of 7 months and a total of 447 isolates were collected. The isolates were characterized using the techniques of randomly amplified polymorphic DNA and multi-locus enzyme electrophoresis. The mean genetic diversity of thisE. colipopulation (H= 0.24) was found to be substantially lower than the diversity of anE. colipopulation reported elsewhere for a single human host. Analysis of the allozyme data revealed that there were significant differences in the relative abundance of genotypes between the two localities sampled and among sample dates. Overall, however, spatial and temporal effects accounted for less than 5% of the genotypic diversity. Allele frequencies and the relative abundance of the more common genotypes did not differ between male and female hosts. The number of genotypes and genotype diversity increased as the age of the host increased, suggesting that the mice are continuing to acquire newE. coliclones throughout their life. The frequency of some alleles changed with respect to host age, which indicates that clone acquisition may not be a random process. It is argued that the low level of genetic diversity observed in this population ofE. colireflects the boom and bust nature of mouse population density in this region of Australia.
bctA: a novel pBF4 gene necessary for conjugal transfer in Bacteroides spp.
Authors: Morgan Roderick M. , Macrina Francis L.
Published: June 2009

Summary: pBF4 is a 41 kb conjugative R-plasmid that confers MLS (macrolide-lincosamide-streptogramin B) resistance in Bacteroides spp. To identify pBF4 genes governing conjugation, recombinational mutagenesis using a suicide vector carrying fragments of the pBF4 plasmid was employed. One of six independent insertion mutants of pBF4 isolated using this method was found to be conjugation-deficient. Nucleotide sequence analysis around the insertion site on this plasmid revealed a 2.8 kb ORF that encoded a putative 110 kDa protein. A corresponding protein was observed when a 12 kb DNA fragment containing this ORF was used to program an in vitro transcription-translation system. Both the ORF and the predicted protein were novel when compared to available database sequences. This gene was designated bctA (Bacteroides conjugal transfer). Polyclonal rabbit antibodies that recognized a sub-sequence polypeptide of BctA reacted with a 55 kDa protein in Western blot analysis using a total protein extract from Bacteroides fragilis containing pBF4. The protein was not present in a B. fragilis strain containing the conjugation-deficient insertion mutant of pBF4. The 55 kDa protein was associated with the membrane fraction of B. fragilis. Although the cellular and biochemical basis of bctA-promoted conjugation remains unknown, this work demonstrates the existence of a heretofore unrecognized gene in bacterial conjugation, and the mutagenesis system used provides the means to isolate and characterize other genes involved in conjugal transfer in Bacteroides spp.
Physiology of poly-3-hydroxybutyrate (PHB) production by Alcaligenes eutrophus growing in continuous culture
Authors: Henderson Robin A. , Jones Colin W.
Published: June 2009

Summary: Alcaligenes eutrophus was grown in continuous culture (34 °C, pH 6.8) under various conditions with respect to dilution rate, nutrient limitation and carbon substrate. Poly-3-hydroxybutyrate (PHB) content, the rate of PHB production (q
PHB) and the rate of carbon substrate utilization (q
s) during growth on glucose were maximum at low dilution rate under ammonia limitation (ammonia limitation > potassium/oxygen limitation > glucose limitation). PHB content decreased in a linear manner as a function of dilution rate, from approximately 80% at D 0-025 h−1 during ammonia-limited growth to approximately 5% during growth at the maximum specific growth rate (μmax) in batch culture. PHB content, q
PHB and qs varied with the nature of the carbon substrate during ammonia-limited growth at fixed dilution rate, and were maximum during growth on lactate [lactate>pyruvate>glucose/gluconate>fructose; highest q
PHB 0.38 g PHB (g non-PHB biomass)−1 h−1]. qPHB was related in an approximately linear manner to the q
s in excess of that required solely for the production of non-PHB biomass. This surplus q
s was higher during growth on lactate than on glucose because q
s was approximately equal to the maximum rate of carbon substrate utilization (q
smax) during growth on lactate, but much lower than q
smax during growth on glucose. The relationship between q
PHB and surplus q
s was confirmed by the effect of adding formate (as an additional source of NADH and/or ATP) and the uncoupling agent carbonyl cyanide-m-chlorophenylhydrazone (CCCP) to ammonia-limited cultures. It is concluded that A. eutrophus is unable to regulate the rate at which it takes up excess carbon substrate to match that required solely for growth, particularly during growth on lactate at low dilution rate, and thus produces PHB as a means of avoiding the potentially deleterious effects of generating high concentrations of intracellular metabolites. Possible ways of further increasing PHB production are discussed.
Mycobacteriophage D29 contains an integration system similar to that of the temperate mycobacteriophage L5
Authors: Ribeiro Graça , Viveiros Miguel , David H. L. , Costa João V.
Published: June 2009

Summary: A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis, was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally held view of D29 as a virulent phage must be reviewed.
Protein F, a fibronectin-binding protein of Streptococcus pyogenes, also binds human fibrinogen: isolation of the protein and mapping of the binding region
Authors: Katerov Viacheslav , Andreev Andrej , Schalén Claes , Totolian Artem A.
Published: June 2009

Summary: During screening of a gene library of Streptococcus pyogenes type M15 for fibrinogen-binding material, a protein of approximately 100 kDa, encoded outside the vir region, was found. DNA sequencing revealed this component to be identical to protein F, a fibronectin-binding protein. Isolation of the recombinant protein, termed F15, was performed by the use of fibrinogen affinity chromatography. The affinity constant (K
a) of protein F15 for fibrinogen, 1.25 x 107 mol−1, was lower than that for fibronectin, 1.8 x 108 mol−1. The fibrinogen-binding domain was located in the N-terminal part of the molecule, while the fibronectin-binding domains, as previously determined, were in the C-terminal portion of protein F. To examine the amino acid sequence heterogeneity of protein F, the 5′ part of the prtF gene, corresponding to the N-terminal variable region of the protein, was amplified by PCR from 12 strains of S. pyogenes belonging to six different M-types. Alignment of these nucleotide sequences indicated that the 5′ portion of the prtF gene had probably undergone a number of intragenic recombination and horizontal gene transfer events, allowing a pattern of structural diversity of protein F observed earlier for some other streptococcal virulence factors. There was no strict correlation between M-type and nucleotide sequence of the variable region of the prtF gene and, compared to streptococcal M protein, the overall variation observed for protein F appeared more limited.
Isoschizomers of the restriction endonuclease Taql (T/CGA) requiring different metal ion concentrations and having a range of thermal stabilities from Thermus species from different continents
Authors: Welch Simon G. , Al-Awadhi Sameera , Williams Ralph A. D.
Published: June 2009

Summary: One-hundred-and-fifty-two isolates of the genus Thermus, collected from hot springs on four continents, were screened for evidence of the presence of the thermophilic Type II restriction endonuclease Taql (T/CGA). The presence of isoschizomers of Taql in 27 of the isolates, originating from hot springs in New Zealand, Iceland, USA, Japan, mainland Portugal and the island of Sao Miguel in the Azores, is reported. Six of the Taql-containing isolates from diverse geographical locations, identified by means of DNA/DNA homology and 16S rRNA sequence alignment as belonging to the Thermus species T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus and T. brockianus, were selected for comparative studies. The Taql isoschizomers from each of the six isolates were partially purified. They differed in their magnesium ion requirements, isoelectric points, subunit molecular masses and thermal stability.
A pheromone-independent CarR protein controls carbapenem antibiotic synthesis in the opportunistic human pathogen Serratia marcescens
Authors: Cox A. R. J. , Thomson N. R. , Bycroft B. , Stewart G. S. A. B. , Williams P. , Salmond G. P. C.
Published: June 2009

Summary: Strain ATCC 39006 of Serratia marcescens makes the same carbapenem, (5R)-carbapen-2-em-3-carboxylic acid (Car), as the Erwinia carotovora strain GS101. Unlike E. carotovora, where the onset of production occurs in the late-exponential phase of growth in response to the accumulation of the small diffusible pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), in S. marcescens carbapenem is produced throughout the growth phase and does not appear to involve any diffusible pheromone molecule. Two cosmids capable of restoring antibiotic production in E. carotovora group I carbapenem mutants were isolated from an S. marcescens gene library. These cosmids were shown to contain a homologue of the E. carotovora carR gene, encoding a CarR protein with homology to the LuxR family of transcriptional regulators. The S. marcescens carR was subcloned and shown to be capable of complementing in trans, in the absence of OHHL, an E. carotovora carR carl double mutant, releasing the heterologous E. carotovora host from pheromone dependence for carbapenem production. The apparent OHHL-independence of the S. marcescens CarR explains the constitutive nature of carbapenem production in this strain of S. marcescens.
Differential expression of glucose-regulated (grp78) and heat-shock-inducible (hsp70) genes during asexual development of Neurospora crassa
Authors: Häfker Thomas , Techel Dieter , Steier Gaby , Rensing Ludger
Published: June 2009

Summary: The expression of a glucose-regulated gene (grp78) changes significantly during the vegetative life cycle of Neurospora crassa: the amounts of grp78 mRNA are low in dormant conidia, increase during germination and exponential growth, decline in young aerial hyphae and reach a maximum in late (15-18 h) aerial hyphae. Heat shock (30 min at 45°C) elevated the mRNA level of this gene especially in early aerial hyphae, whereas no increase above the high constitutive amount was found after heat treatment of late aerial hyphae. The expression of the inducible hsp70 gene after heat shock also varied with the state of development and showed the highest inducibility in late aerial hyphae. Surface mycelium, from which aerial hyphae emerge, showed a similar increase in the amounts of both mRNA species. A developmental mutant (acon-2), which is defective in minor constriction budding of aerial hyphae, showed lower levels of con-2 mRNA as well as of grp78 and hsp70 mRNA (after heat shock) in late aerial hyphae. The acon-2 mutant did not form conidia at this stage. It is concluded that the high constitutive and inducible expression of stress genes in late aerial hyphae is due to a developmental activation of their transcription or, alternatively, to a lower degradation rate of their mRNA during this stage.
The role of autolysins during vegetative growth of Bacillus subtilis 168
Authors: Blackman Steve A. , Smith Thomas J. , Foster Simon J.
Published: June 2009

Summary: A set of isogenic mutants of Bacillus subtilis 168, insertionally inactivated in the genes encoding a number of lytic enzymes and a sigma factor (σD, which controls the expression of a number of autolysins) was constructed. Phenotypic analysis of the mutants determined the individual and combined roles of the autolysins in vegetative growth. The major vegetative autolysins of B. subtilis, LytC (50 kDa amidase) and LytD (90 kDa glucosaminidase), were shown to have roles in cell separation, cell wall turnover, antibiotic-induced lysis and motility. LytC was also shown to have a role in general cell lysis induced by sodium azide. Renaturing SDS-PAGE of cell-wall-binding protein extracts of the mutant strains revealed the presence of a novel autolysin that was previously masked by LytC. This 49 kDa enzyme was shown to be σD-controlled and was identified as a candidate cell separation and cell wall turnover enzyme. A multiple mutant strain, lacking LytC, LytD and the 49 kDa enzyme, retained at least ten bands of autolytic activity. These may correspond to individual or proteolytically processed novel autolysins, the functions of which are unknown. The multiple mutant strains facilitate the study of these, and other lytic enzymes, to determine their cellular functions.
Osmoregulatory transporter ProP influences colonization of the urinary tract by Escherichia coli
Authors: Culham Doreen E. , Dalgado Cheryl , Gyles Carlton L. , Mamelak Daniel , MacLellan Shawn , Wood Janet M.
Published: June 2009

Summary: Osmoregulatory transporters ProP and ProU mediate the use of betaines as osmoprotectants by Escherichia coli. Glycine betaine and proline betaine are present in mammalian urines. Betaine uptake may therefore facilitate the growth of E. coli in the urinary tract, an environment of fluctuating osmolality. ProP transporter activity was approximately threefold higher in a pyelonephritis isolate, E. coli HU734, than in E. coli K-12. The growth rate of E. coli HU734 in aerated minimal salts medium was reduced twofold by 0.2 M NaCl in the absence and by 0.55 M NaCI in the presence of glycine betaine. Maximal growth rate stimulation was achieved when glycine betaine was added at a concentration as low as 25 μM. Deletion of the proP locus impaired the growth rate of E. coli HU734 in human urine but not in minimal medium supplemented with NaCI (0.4 M), with or without glycine betaine (0.1 mM). The expression of pyelonephritis-associated (P) pili was reduced when E. coli HU734 was cultured in a rich culture medium (LB) of elevated salinity. The proP lesion had no influence on P pilus expression in vitro or on the recovery of bacteria from the kidneys of inoculated mice. However, it did reduce their recovery from the bladders of inoculated mice 100-fold. These data provide the first direct evidence that osmoprotective betaine accumulation and transporter ProP are pertinent to both growth in human urine and colonization of the murine urinary tract by uropathogenic E. coli.
Pleiotropic effects of the opil regulatory mutation of yeast: its effects on growth and on phospholipid and inositol metabolism
Authors: Jiranek Vladimir , Graves J. Anthony , Henry Susan A.
Published: June 2009

SUMMARY: Key factors which impact on the biosynthesis and subsequent fate of the phospholipid precursor inositol were studied as a function of growth phase in the yeast Sacchammyces cerevisiae. Both wild-type and strains disrupted for the OPI7 gene, the principal negative regulator of the phospholipid biosynthetic genes, were examined. Overexpression of the IN07 gene and overproduction of both inositol and the major inositol-containing phospholipid, phosphatidylinositol. varied as a function of growth phase. In Opi7 cells, IN07 expression was constitutive at a high level throughout growth, although the level of transcript was reduced at stationary phase when the cells were grown in defined medium. In the wild-type strain, IN07 expression was limited to a peak in the exponential phase of growth in cells grown in the absence of inositol. Interestingly, the pattern of OW7 expression in the wild- type strain resembled that of its putative target, IN07. lntracellular inositol contents of the opi7 strain were higher than those of the wild-type strain. with peak levels occurring in the stationary phase. Membrane phosphatidylinositol content paralleled intracellular inositol content, with opil strains having a higher phosphatidylinositol content in stationary phase. The proportion of the predominant phospholipid, phosphatidylcholine, exhibited a profile that was the inverse of the phosphatidylinositol content: phosphatidylcholine content was lowest in Opi7 cells in stationary phase. The Opil mutation was also found to have effects beyond phospholipid biosynthesis. opi7 cells were smaller, and Opi7 cultures achieved a cell density twice as high as comparable wild-type cultures. Opil cells were also more salt tolerant than wild-type cells: they were partly resistant to shrinking, more rapidly resumed growth, and attained a higher culture density after upshift to medium supplemented with 8% NaCl.
In situ detection of bacteria in continuous-flow cultures of seawater sediment suspensions with f luorescently labelled rRNA-directed oligonucleotide probes
Authors: Bruns Alke , Berthe-Corti Luise
Published: June 2009

SUMMARY: RNA-targeted and f luorescently labelled oligonucleotide probes were used to study the composition of natural bacterial populations in continuous-f low cultures of seawater sediment suspensions. The cultures were run as enrichment cultures with increasing dilution rates, and hexadecane as the sole carbon source. Total cell numbers were analysed by counting DAPI (4′,6- diamidino-2-phenylindole)-stained cells. To differentiate the population composition, oligonucleotide probes for eubacteria, for Cytophagd Flavobacteria, and for four subclasses of the Proteobacteria (a, b, y and 6) were used. About 4&80°/o of the DAPI-stained cells could be detected with the EUB338 probe. Moreover, it was possible to detect a shift in the composition of the natural bacterial population with increasing dilution rate of the continuous culture, from large amounts of CytophagdFlavobacteria to large numbers of members of the pProteobacteria. The cell recovery rate for bacteria labelled with specific oligonucleotide probes was analysed with defined cell numbers of Rhodospirillum nrbnrm, Cornamonas testosteroni and Desulfowibrio vulgaris subsp. vulgaris introduced into the seawater sediment suspension, and was determined to be 13*%33*5 %. The standard deviation determined for this method applied to sediment suspensions was 28.3 %. The results suggest that the application of the in situ hybridization technique allows a good insight into the structure of populations growing in sediment suspensions.
The conjugative plasmid pSG5 from Streptomyces ghanaensis DSM 2932 differs in its transfer functions from other Streptomyces rolling-circle-type plasmids
Authors: Maas Ruth-Maria , GUtz JUrg , Wohlleben Wolfgang , Muth Gunther
Published: June 2009

SUMMARY: The Streptomyces ghanaensis plasmid pSG5 is self-transmissible but does not form the growth-retardation zones (pocks) normally characteristic of the Streptomyces plasmid-transfer process. The complete nucleotide sequence of pSG5 was determined on both strands. pSG5 is 12208 bp in length and has a GC content of 68 mol0/o. Characterization’ of the open reading frames by insertion and deletion analysis revealed that only a single gene, traB, is involved in the transfer of pSG5. The deduced amino acid sequence of TraB is similar to the SpolllE protein that is responsible for chromosome translocation during prespore formation of Bacillus subtilis. In contrast to the tra genes of the other Streptomyces plasmids, the pSG5 traB does not represent a kill function. Although pSG5 transfer is not associated with pock formation, pSG5 was shown to possess putative spd genes that are responsible for the pock phenotype of other Streptomyces plasmids. However, promoter-probe experiments revealed that the spd genes of pSG5 are not transcribed, thus explaining the deficiency in pock formation.
Electrotransformation and natural transformation of Streptococcus pneumoniae:requirement of DNA processing for recombination
Authors: LefranGois Jacques , Samrakandi M. Mustapha , Sicard A. Michel
Published: June 2009

SUMMARY: Electrotransformation has been used as a tool to introduce genes carried on replicative vectors in hundreds of bacterial species. In this study, the technique was used to try to obtain recombination of markers in the chromosome of the natura IIy transformable bacterium Streptococcus pneumoniae. Recombination was not observed even using naturally competent cultures. Both chromosomal and cloned DNA, denatured or native, were without effect. These results suggest that it is not sufficient to introduce DNA into the cell to obtain recombinants in this bacterium. The integration of markers into the chromosome in naturally competent cells must require DNA processing during entry. Electrotransformation of replicating plasmids is red-independent but can be facilitated by a red-dependent process. This facilitation required the induction of the natural competence machinery, probably involving partial homologous pairing.
PhhC is an essential aminotransferase for aromatic amino acid catabolism in Pseudornonas aeruginosa
Authors: Gu Wei , Song Jian , Bonner Carol A. , Xie Gang , Jensen Roy A.
Published: June 2009

SUMMARY: The phhC gene of Pseudomonas aeruginosa encodes a protein which is a member of the Family I aminotransferases. At high expression levels in the heterologous Escherichia oli system, PhhC can compensate for the absence of AspC (which functions in L-aspartate biosynthesis) and TyrB (which functions in aromatic biosynthesis). In the native organism, PhhC is essential for catabolism of either L-tyrosine or L-phenylalanine, as demonstrated by gene inactivation. This catabolic function of PhhC is consistent with its inclusion as the distal gene in the inducible phenylalanine hydroxylase operon. The presence of PhhC for catabolism of aromatic amino acids is required in spite of an existing
multiplicity of other P. aeruginosa aminotransferases having a similar pattern of broad substrate specificity in vitm. This implies a spatial orientation of PhhC that effectively specializes it for aromatic amino acid catabolism.
Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate
Authors: Chan Maurice , Sim Tiow-Suan
Published: June 2009

SUMMARY: Malate synthase is a key enzyme of the glyoxylate cycle, which is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, encoding malate synthase from Streptomyces clavuligems NRRL 3585, was cloned using PCR and fully sequenced. The ORF obtained encodes 541 amino acids with a deduced M, of 6OOOO, consistent with the observed M, (62000-64000) of most malate synthase enzymes reported so far. The aceB gene has a high G+C content (71.5 molO/O), especially in the third codon position. A 50 bp region upstream of the malate synthase ORF was predicted to be a prokaryotic promoter region. The relationship between carbon source, antibiotic (cephalosporin) biosynthesis and malate synthase activity was investigated. Growth of S. clavuligerus on acetate as the major carbon sorce was delayed, compared to that on glycerol. Furthermore, high levels of malate synthase activity were associated with the presence of acetate in the growth medium. Growth on acetate also resulted in lower levels of cephalosporin production, compared to that on glycerol. The cloned 5. clavuligerus aceB gene was expressed in Escherichia coli BL2l (DE3).Transformants exhibited an approximately 713old increase in malate synthase activity, compared to the control, thereby demonstrating high-level expression of soluble and enzymically active malate synthase in the heterologous host.
Protein phosphorylation in Escherichia coli L. form NC-7
Authors: Freestone P. , Grant S. , Trinei M. , Onoda T. , Norris V.
Published: June 2009

Summary: Wall-less L-forms of Escherichia coli constitute an interesting, and relatively underused, model system for numerous studies of bacterial physiology including the cell cycle, intracellular structure and protein phosphorylation. Total extracts of the L-form revealed a pattern of protein phosphorylation similar to that of an enteropathogenic strain but very different from its parental K-12 strain. In particular, the L-form extract revealed phosphorylation on tyrosine of a protein important in pathogenesis, TypA, and calcium-specific phosphorylation of a 40 kDa protein. Two new phosphoproteins were identified in the L-form as the DNA-binding protein Dps, and YfiD, a protein of 14 kDa with homology to pyruvate formate-lyase and a region containing a tRNA cluster in bacteriophage T5.
A kirromycin-resistant EF-Tu species reverses streptomycin dependence of Escherichia coli strains mutated in ribosomal protein S12
Authors: Zuurmond Anne-Marie , Zeef Leo A. H. , Kraal Barend
Published: June 2009

Summary: Streptomycin dependence can be caused by mutations in ribosomal protein S12. Mutations suppressing such streptomycin dependence have been found in ribosomal proteins S4 and S5, and in 16S rRNA. Here a new suppressor mutation localized in elongation factor Tu (EF-Tu) is described, consistent with recent models of ribosome-EF-Tu-tRNA interaction at the decoding centre. The EF-Tu mutation was obtained by genetic selection for streptomycin independence; it was identified as Ala375 → Thr, previously described as EF-TuAR and known to confer a kirromycin-resistant, error-prone phenotype. Also, other streptomycin-dependent (SmD) S12 mutations could be complemented by this mutation. The streptomycin-independent (SmI) strain grows more slowly than the wild-type (wt), suggesting that not all the defects of the S12 mutation can be complemented by EF-Tu[A375T]. Moreover, this strain is more susceptible than wt to reduction in the cellular EF-Tu concentration, and disruption of tufB led to considerable growth-rate impairment. Expression of EF-Tu from tufB, not only of wt EF-Tu and EF-Tu[A375T] but, remarkably, also of EF-Tu[G222D], known as EF-TuB0 and defective in protein synthesis, equally contributed to cell growth. In vitro analysis revealed a decreased translational activity of wt EF-Tu with SmD ribosomes as compared to EF-Tu[A375T], while EF-Tu[G222D] showed no activity at all, just as with wt ribosomes. Possible mechanisms are discussed for the improved growth rate observed in such SmI strains when they include wt EF-Tu or EF-Tu[G222D].
Disruption of the sole IdhL gene in Lactobacillus sakei prevents the production of both L- and D-Iactate
Authors: Maleret Christine , Lauret R. , Ehrlich S. Dusko , Morel-Deville Françoise , Zagorec Monique
Published: June 2009

Summary: A 7 kb DNA fragment was cloned from Lactobacillus sakei which contains the IdhL gene encoding the l(+)-lactate dehydrogenase (l-LDH). Analysis of the DNA sequence, Northern experiments and primer extension experiments showed that IdhL is transcribed from a single promoter, leading to a monocistronic 1·15 kb mRNA which yields the l-LDH. A stable mutant was constructed by chromosomal integration of a chloramphenicol cassette into IdhL by a double-crossover event. Both l- and d-lactate were produced by the wild-type strain whereas only residual amounts of both isomers were produced by the mutant. This demonstrates that L. sakei possesses an l-LDH producing l-lactate and a lactate racemase able to transform it to d-lactate, but is devoid of d-LDH activity. Moreover the ability to degrade l-lactate present in the medium that was observed with the mutant strain grown aerobically suggests that an l-lactate oxidase activity is also present in L. sakei.
Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa
Authors: Michel Gérard , Bleves Sophie , Ball Geneviève , Lazdunski Andrée , Filloux Alain
Published: June 2009

Summary: Protein secretion in Gram-negative bacteria is often dependent on the general secretory pathway (GSP). In Pseudomonas aeruginosa, this system requires at least 12 Xcp (Gsp) proteins, which are proposed to constitute a multiprotein complex localized in the bacterial envelope. Hitherto, little was known about the mutual interactions between Xcp proteins. In this study, mutants affected in the xcpZ gene encoding a bitopic inner-membrane protein were analysed to investigate the role of this protein in the architecture of the secretory machinery. The absence of XcpZ resulted in a decreased amount of XcpY. Reciprocally, XcpZ was not detectable in a xcpY mutant demonstrating a mutual stabilization of these two proteins. These results strongly suggest that XcpZ and XcpY interact within the functional secretory apparatus.
Permeabilizing action of polyethyleneimine on Salmonella typhimurium involves disruption of the outer membrane and interactions with lipopolysaccharide
Authors: Helander Ilkka M. , Latva-Kala Kyösti , Lounatmaa Kari
Published: June 2009

Polyethyleneimine (PEI), a polycationic polymer substance used in various bioprocesses as a flocculating agent and to immobilize enzymes, was recently shown to make Gram-negative bacteria permeable to hydrophobic antibiotics and to detergents. Because this suggests impairment of the protective function of the outer membrane (OM), the effect of PEI on the ultrastructure of Salmonella typhimurium was investigated. Massive alterations in the OM of PEI-treated and thin-sectioned bacteria were observed by electron microscopy. Vesicular structures were seen on the surface of the OM, but no liberation of the membrane or its fragments was evident. Since a potential mechanism for the action of PEI could be its binding to anionic LPSs on the OM surface, the interaction of PEI with isolated LPSs was assayed in vitro. The solubility of smooth-type LPSs of Salmonella, regardless of the sugar composition of their O-specific chains, was not affected by PEI, nor was that of Ra-LPS (lacking O-specific chains but having a complete core oligosaccharide). PEI strongly decreased the solubility of rough-type LPSs of the chemotypes Rb2 and Re, whereas it had only a weak effect on the abnormally cationic Rb2-type pmrA mutant LPS, suggesting that the negative charge to mass ratio of LPS plays a critical role in the interaction.
Depolarized cell growth precedes filamentation during the process of ethanol-induced pseudohyphal formation in the yeast Candida tropicalis
Authors: Suzuki Takahito , Imanishi Yumi , Iwaguchi Shin-Ichi , Kamihara Teijiro
Published: June 2009

Ethanol has been reported to cause mycelial growth in Candida tropicalis Pk233, and mycelial growth has also been shown to be abolished by concomitant addition of myo-inositol. In this study, the process of ethanol-induced mycelial growth in this organism was examined in combination with cytological characterization of actin localization. Cultivation with ethanol gave biphasic growth curves. During the first growth phase (doubling time 2.4 h), there was an accumulation of swollen spherical yeast cells, instead of the oblong ones observed in the control culture, followed by the appearance of spherical daughter cells in chains. Randomly distributed actin patches were observed on these swollen yeast cells and the bud initiation sites of these cells appeared random. These observations suggested that ethanol caused depolarization of cell growth during the first phase. During the second growth phase (doubling time 7.4 h), pseudohyphal cells appeared, projecting from the swollen yeast cells. Activity of chitinase in the control culture rose during the exponential phase. In the ethanol culture the activity stayed at a low level throughout the growth phases. When pseudohyphal cells were transferred to fresh ethanol medium, yeast cells appeared from pseudohyphal filaments and changed their shape to spherical, and filamentation appeared to be inhibited during the first phase. From these observations, an initial effect of ethanol on C. tropicalis cells appeared to be depolarization of cell growth, and the resulting swollen cells grew as polar pseudohyphal cells. In the culture supplemented with both ethanol and inositol, or with both ethanol and sorbitol, the accumulation of swollen cells was not observed and single yeast cells with normal oblong shape were seen throughout the growth phases.
Organization around the dnaA gene of Streptococcus pneumoniae
Authors: Gasc Anne-Marie , Giammarinaro Philippe , Richter Stefan , Sicard Michel
Published: June 2009

The dnaA gene region of Streptococcus pneumoniae was cloned and sequenced. A tRNA gene, seven ORFs and three DnaA box clusters were identified. The order of the genes and intergene regions found was tRNAArg-orf1-DnaA box cluster 3-htrA-spo0J-DnaA box cluster 2-dnaA-DnaA box cluster 1-dnaN-orfX-orfY. Five ORFs are homologous to known bacterial genes. The tRNAArg gene and orf1, also called orfL, have already been described in pneumococci and have been reported to be preceded by the competence regulation locus comCDE. In Escherichia coli, htrA encodes a serine protease. In Bacillus subtilis, spo0J plays a role in sporulation and partition. dnaA encodes an initiator replication protein, very well conserved in several bacteria and dnaN encodes the β subunit of DNA polymerase III in E. coli. The function of orfX is unknown. The N-terminal part of another reading frame, orfY, revealed high homology with a GTP-binding protein. DnaA box clusters were found upstream and downstream from dnaA. The presence of two such clusters suggests that the chromosomal origin of S. pneumoniae is located within this region. The position of dnaA, and therefore the putative origin of replication, were localized on the physical map of S. pneumoniae.
Antiterminator protein GlpP of Bacillus subtilis binds to glpD leader mRNA
Authors: Glatz Elisabeth , Persson Martin , Rutberg Blanka
Published: June 2009

The Bacillus subtilis glpD gene encodes glycerol-3-phosphate (G3P) dehydrogenase. Expression of glpD is mainly controlled by termination/antitermination of transcription at an inverted repeat in the glpD leader. Antitermination is mediated by the antiterminator protein GlpP in the presence of G3P. In this paper, interaction between GlpP and glpD leader mRNA in vivo and in vitro is reported. In vivo, the antiterminating effect of GlpP can be titrated in a strain carrying the glpD leader on a plasmid. GlpP has been purified and gel shift experiments have shown that it binds to glpD leader mRNA in vitro. GlpP is not similar to other known antiterminator proteins, but database searches have revealed an Escherichia coli ORF which has a high degree of similarity to GlpP.
Identification of the contiguous Paracoccus denitrificans ccmF and ccmH genes: disruption of ccmF, encoding a putative transporter, results in formation of an unstable apocytochrome c and deficiency in siderophore production
Authors: Pearce David A. , Page M. Dudley , Norris Hilary A. C. , Tomlinson Esther J. , Ferguson Stuart J.
Published: June 2009

Apocytochrome c
550 was detected in the periplasm of a new mutant of Paracoccus denitrificans, HN48, that is pleiotropically lacking c type cytochromes, produces reduced levels of siderophores and carries a Tn5 insertion in the ccmF gene for which sequence data, along with that for the contiguous ccmH, are reported. A counterpart to the ccmF gene was found in an archaebacterium but could not be located in the yeast genome, whereas mitochondrial haem lyases in the latter were not present in an archaeobacterial or in eubacterial genomes. A topological analysis for CcmF is presented which indicates at least eleven transmembrane helices, suggesting a role as a transporter; evidence against the substrate being haem is presented but sequence similarity with Escherichia coli γ-aminobutyric acid transporter was identified. Analysis by pulse-chase methodology has shown that, in this and another cytochrome-c-deficient mutant, the apo form of P. denitrificans cytochrome c
550 is much less stable than the holo form, directly demonstrating the presence of a periplasmic degradation system in P. denitrificans that removes non-functional proteins. A variety of phenotypes are observed for P. denitrificans mutated in different ccm genes, thus indicating that the stability of the ccm gene products does not require assembly of a complex of all the Ccm proteins.
Floc stability and adhesion of green-fluorescent-protein-marked bacteria to flocs in activated sludge
Authors: Olofsson Ann-Cathrin , Zita Anna , Hermansson Malte
Published: June 2009

Wastewater is often treated using the activated sludge process. Flocculation and subsequent sedimentation of flocs are vital steps in this process that have direct influence on the quality of the effluent water from wastewater treatment plants. Since cells that remain free-living will decrease the quality of the effluent water it is important to understand the mechanisms of bacterial adhesion to flocs. The green fluorescent protein (GFP) was used as a cellular marker to study bacterial adhesion to activated sludge flocs in situ in sludge liquor. Cell surface hydrophobicity (CSH) was shown to be an important factor that determined the relative bacterial adhesion potential. High CSH correlated with high numbers of attached cells. However, the absolute adhesion of two test bacteria to different sludge flocs varied and could not be explained by the floc characteristics. Confocal laser scanning microscopy of GFP-marked cells showed their position in the floc matrix in situ. Hydrophobic cells attached not only on the surface but also within the floc, whereas hydrophilic cells did not. This indicates that cells may penetrate the flocs through channels and pores and increase the effective surface, which in turn makes the clarification of the wastewater effluent more efficient. The addition of polymers is common practice in wastewater treatment and was shown to increase bacterial adhesion to the flocs. A decrease in surface tension caused by addition of DMSO decreased adhesion, indicating the detrimental effect of surfactants on flocculation. An understanding of basic bacterial adhesion and aggregation mechanisms is important for the managment and control of biotechnological wastewater treatment.
The loading domain of the erythromycin polyketide synthase is not essential for erythromycin biosynthesis in Saccharopolyspora erythraea
Authors: Pereda Ana , Summers Richard G. , Stassi Diane L. , Ruan Xiaoan , Katz Leonard
Published: June 2009

6-Deoxyerythronolide B synthase (DEBS) is a large multifunctional enzyme that catalyses the biosynthesis of the erythromycin polyketide aglycone. DEBS is organized into six modules, each containing the enzymic domains required for a single condensation of carboxylic acid residues which make up the growing polyketide chain. Module 1 is preceded by loading acyltransferase (AT-L) and acyl carrier protein (ACP-L) domains, hypothesized to initiate polyketide chain growth with a propionate-derived moiety. Using recombinant DNA technology several mutant strains of Saccharopolyspora erythraea were constructed that lack the initial AT-L domain or that lack both the AT-L and ACP-L domains. These strains were still able to produce erythromycin, although at much lower levels than that produced by the wild-type strain. In addition, the AT-L domain expressed as a monofunctional enzyme was able to complement the deletion of this domain from the PKS, resulting in increased levels of erythromycin production. These findings indicate that neither the initial AT-L nor the ACP-L domains are required to initiate erythromycin biosynthesis; however, without these domains the efficiency of erythromycin biosynthesis is decreased significantly. It is proposed that in these mutants the first step in erythromycin biosynthesis is the charging of KS1 with propionate directly from propionyl-CoA.
A novel regulatory switch mediated by the FNR-like protein of Lactobacillus casei
Authors: Gostick Dominic O. , Green Jeffrey , Irvine Alistair S. , Gasson Michael J. , Guest John R.
Published: June 2009

FNR (regulator for fumarate and nitrate reduction) and CRP (cAMP receptor protein) are global regulators which regulate the transcription of overlapping modulons of target genes in response to anaerobiosis and carbon source in Escherichia coli. An ORF, designated flp because it encodes an FNR-like protein of the FNR-CRP family, has been found in Lactobacillus casei. The product of the flp coding region (FLP) was overproduced in E. coli, purified and crystallized. FLP is a homodimeric protein in which each subunit can form an intramolecular disulphide bond. The isolated protein also contains non-stoichiometric amounts of Cu and Zn. Although the DNA recognition helix of FLP resembles that of FNR, the flp gene failed to complement the anaerobic respiratory deficiency of an fnr mutant when expressed in E. coli and it neither activated nor interfered with transcription from FNR- or CRP-dependent promoters in E. coli. Site-specific DNA binding by oxidized FLP (the form containing intrasubunit disulphide bonds) was abolished by reduction. The interconversion between disulphide and dithiol forms thus provides the basis for a novel redox-mediated transcriptional switch. Two non-identical FLP-binding sites, distinct from FNR- and CRP-binding sites, were identified in the meIR region of E. coli by gel-retardation analysis. A further eight FLP-binding sites were selected from a random library. A synthetic oligonucleotide conforming to a putative FLP site consensus, CA/cTGA-N4-TCAG/TG (the most significant bases are underlined), was retarded by FLP. Functional tests showed that FLP represses the aerobic transcription of a semi-synthetic promoter in E. coli. A C5S variant of FLP lacking the ability to form intramolecular disulphide bonds was unable to bind to FLP sites and failed to repress transcription in vivo.