bluuebunny/crossref_metadata_2025 huggingface dataset:
The ability of Escherichia coli O157:H7 to decrease its intracellular pH and resist the toxicity of acetic acid
Authors: Diez-Gonzalez Francisco , Russell James B.
Published: June 2009

Batch cultures of Escherichia coli K-12 grew well in an anaerobic glucose medium at pH 5.9, but even small amounts of acetate (20 mM) inhibited growth and fermentation. E. coli O157:H7 was at least fourfold more resistant to acetate than K-12. Continuous cultures of E. coli K-12 (pH 5.9, dilution rate 0.085 h-1) did not wash out until the sodium acetate concentration in the input medium was 80 mM, whereas E. coli O157:H7 persisted until the sodium acetate concentration was 160 mM. E. coli K-12 cells accumulated as much as 500 mM acetate, but the intracellular acetate concentration of O157:H7 was never greater than 300 mM. Differences in acetate accumulation could be explained by intracellular pH and the transmembrane pH gradient (δpH). E. coli K-12 maintained a more or less constant δpH (intracellular pH 6.8), but E. coli O157:H7 let its δpH decrease from 0.9 to 0.2 units as sodium acetate was added to the medium. Sodium acetate increased the rate of glucose consumption, but there was little evidence to support the idea that acetate was creating a futile cycle of protons. Increases in glucose consumption rate could be explained by increases in D-lactate production and decreases in ATP production. Intracellular acetate was initially lower than the amount predicted by ∆pH, but intracellular acetate and δpH were in equilibrium when the external acetate concentrations were high. Based on these results, the acetate tolerance of O157:H7 can be explained by fundamental differences in metabolism and intracellular pH regulation. By decreasing the intracellular pH and producing large amounts of D-lactate, O157:H7 is able to decrease δpH and prevent toxic accumulations of intracellular acetate anion.
Chemical characterization and spectroscopic analysis of the solubilization products from wheat straw produced by Streptomyces strains grown in solid-state fermentation
Authors: Hernández-Coronado M. J. , Hernández M. , Centenera F. , Pérez-Leblic M. I. , Ball A. S. , Arias M. E.
Published: June 2009

The effects of two extraction procedures on the yield and properties of APPL (acid-preciμltable polymeric lignin, or solubilized lignocellulose) produced by four streptomycetes during growth in solid-state fermentation were examined. When APPL was extracted with NaOH (0.1 M) rather than distilled water, yields increased threefold, with Streptomyces chattanoogensis exhibiting maximum solubilization levels [163 mg product (g straw)-1]. Alterations in the characteristics of APPL obtained during extraction with NaOH were detected using cross-polarization and magic-angle sμlnning (CPMAS) 13C NMR and IR spectroscopy and by GC-MS analysis after CuO oxidation, with the most significant changes detected in the cinnamic acid and lignin moieties. When APPL was extracted with NaOH, ester links between hemicellulose and lignin and between hemicellulose and cinnamic acid were cleaved, resulting in a decrease in the alkyl and carbonyl groups attached to lignin, enabling greater solubilization. Yields of APPL extracted with water were lower, but spectral characterization of this APPL suggested a possible role for actinomycete peroxidases and phenolic acid esterases in lignin solubilization. For industrial solubilization of lignocellulose, a possible role for the application of streptomycetes, or their enzymes, in alkali extraction is suggested as a means of increasing solubilization levels.
Genomic relationships between selected phage types of Salmonella enterica subsp. enterica serotype typhimurium defined by ribotyping, IS200 typing and PFGE
Authors: Olsen John E. , Skov Marianne N. , Angen Øystein , Threlfall E. John , Bisgaard M.
Published: June 2009

The genomic relationship between isolates representing 17 definitive phage types (DTs) of Salmonella enterica subsp. enterica serotype typhimurium (S. typhimurium) were analysed using three different typing methods: IS200 typing using the restriction enzymes EcoRI and Pvull, ribotyping using Smal and EcoRI, and PFGE using Xbal. These methods were used to study four DTs in greater detail; in all 18 (DT 49), 10 (DT 110), five (DT 120) and seven (DT 135) isolates were studied. The combined data generated two large clusters, which could be divided into five groups. Within the first cluster, a close similarity was indicated between isolates of the following phage types: group A – DTs 44, 49, 135 and 204c, with DT 9 distantly related; group B – DTs 95 and 99; and group C – DTs 104a, 110 and 120. The other large cluster contained group D – DTs 10, 20 and 146, with DT 12 distantly related, and group E – DTs 69, 103 and 153. The same grouping was observed by principal component analysis, but a minimum spanning tree linked DT 12 to group E and not group D in this analysis. Among the typing methods used, IS200 gave the best representation of the overall similarity between the S. typhimurium isolates. Five different IS200 profiles were obtained among isolates belonging to DT 49. Only one profile was observed within each of the phage types DT 110, 120 and 135. All isolates within each of these four phage types were of one ribotype. Isolates of DT 49 showed four PFGE patterns, while one pattern was present within isolates of the three other phage types. Members of these four phage types were found to be clonally related as they formed tight subclusters separated from isolates of other phage types.
Molecular analysis of mxbD and mxbM, a putative sensor-regulator pair required for oxidation of methanol in Methylobacterium extorquens AM1
Authors: Springer Amy L. , Morris Christina J. , Lidstrom Mary E.
Published: June 2009

Five genes are thought to be required for transcription of methanol oxidation genes in Methylobacterium strains. These putative regulatory genes include mxcQE, which encode a putative sensor-regulator pair, and mxbDM and mxaB, whose functions are less well-understood. In this study, mxbDM in Methylobacterium extorquens AM1 were shown to be required for expression of a xyIE transcriptional fusion to the structural gene for the large subunit of methanol dehydrogenase (mxaF), confirming the role of these genes in transcriptional regulation of mxaF. The nucleotide sequence suggests that mxbD encodes a histidiine protein kinase with two transmembrane domains and that mxbM encodes a DNA-binding response regulator. A xyIE transcriptional fusion to the putative mxbD promoter showed low-level expression in wild-type cells grown on one-carbon (C1) compounds and no detectable expression in cells grown on succinate. Deletion analysis of this promoter construct showed that the region 229-129 bp upstream of the start of mxbD is required for expression. The expression of the mxbD-xylE fusion was examined in each of the five known regulatory mutant classes. xyIE expression was reduced to non-detectable levels in MxcQ and MxcE mutants, but was not affected in the other regulatory mutants or in non-regulatory mutants defective in methanol oxidation. These results suggest a regulatory hierarchy in which the sensor-regulator pair MxcQE control expression of the sensor-regulator pair MxbDM, and MxbDM in turn control expression of a number of genes involved in methanol oxidation.
VlpA of Vibrio cholerae O1: the first bacterial member of the α2-microglobulin lipocalin superfamily
Authors: Barker Andrew , Manning Paul A.
Published: June 2009

We have identified a gene, vlpA, which is closely linked to the mfrA,B locus associated with mannose-fucose-resistant haemagglutination. VlpA is an outer-membrane protein which can be labelled with [3H]palmitate and whose processing is globomycin-sensitive, suggesting that it is a lipoprotein. Homology searches revealed that VlpA belongs to the group of lipocalins of the α2-microglobulin superfamily which function as small hydrophobic molecule transporters, and is the first identified bacterial member of this group. Multiple copies of this gene are present in Vibrio cholerae O1 and O139 and Southern hybridization reveals a biotype-specific pattern of fragment sizes. Construction of strains capable of hyperproducing VlpA suggested that it is able to bind haemin with low affinity but this may be due to a simple hydrophobic interaction. Attempts to construct specific mutants in vlpA have been unsuccessful, presumably because of the multiple copies of vlpA genes and their linkage to the VCR element.
Organization of methylamine utilization genes (mau) in ‘Methylobacillus flagellatum’ KT and analysis of mau mutants
Authors: Gak Evgeny R. , Tsygankov Yuriy D. , Chistoserdov Andrei Y.
Published: June 2009

The organization of genes involved in utilization of methylamine (maugenes) was studied in the obligate methylotroph‘Methylobacillus flagellatum’KT. Nine open reading frames were identified as corresponding to the genesmauFBEDAGLMN.In addition, an open reading frame (orf-1) encoding a polypeptide with unknown function was identified upstream of themaugene cluster. Subclones of the‘M. flagellatum’KT gene cluster were used for complementation of a series of chemically induced mau mutants of‘M. flagellatum’KT. Mutants inmauF, mauB, mauEID, mauA, mauG, mauLandmauMwere identified. Two mutants (mau-18andmau-19) were not complemented by the knownmaugenes. Since none of the chemically induced mutants studied had a defect inorf-1 ormauN,insertion mutants in these genes were constructed. Phenotypically the mutants fell into three groups. ThemauF, mauB, mauE/D, mauA, mauG, mauLandmauMmutants do not grow on methylamine as a source of carbon and lack methylamine dehydrogenase activity, but they synthesize both the large and the small subunit polypeptides albeit at different ratios. Themau-18andmau-19mutants do not grow on methylamine as a source of carbon, and lack both methylamine dehydrogenase activity and the methylamine dehydrogenase subunits. Theorf-1andmauNmutants grow on methylamine as a source of carbon and synthesize wild-type levels of methylamine dehydrogenase. It has been shown earlier that the product of themauMgene is not required for synthesis of active methylamine dehydrogenase inMethylobacterium extorquensAM1 andParacoccus denitrificans.However, MauM is required for synthesis of functional methylamine dehydrogenase in ‘M. flagellatum’.
The aldA gene of Escherichia coli is under the control of at least three transcriptional regulators
Authors: Limón Ana , Hidalgo Elena , Aguilar Juan
Published: June 2009

Expression studies on the aldA gene encoding aldehyde dehydrogenase in Escherichia coli showed induction by two types of molecule (hydroxyaldehydes and 2-oxoglutarate), carbon catabolite repression and respiration dependence. Promoter deletion analysis showed that the proximal operator, which includes inducer-regulator complex and catabolite repression protein (Crp) recognition sites, was necessary for induction by either type of inducer, and that full induction by aldehydes required the cooperation of distal operator sequences beyond position -119. Interactions of the regulator protein with the -59 to -6 fragment were shown by DNA mobility shift assays. Fusions of different deletions of the aldA promoter to lacZ indicated that a Crp site proximal to the transcriptional start point (tsp) was functional in the cAMP-dependent catabolite repression of this system, whereas a distal control site was likely to operate in a cAMP-independent catabolite repression. DNA mobility shift and footprint analyses showed that only the tsp proximal site was bound by pure Crp with a K
d of 5.4 x 10-7 M. As shown by an Arc-defective strain, the aldA gene seems to be repressed by the Arc system under anaerobiosis, displaying its physiological full induction and activity in the presence of oxygen.
Challenging food microbiology from a molecular perspective
Authors: Stewart Gordon S. A. B.
Published: June 2009

Summary: Two key themes within food microbiology are bacterial detection and control. There is a raft of sub-headings under each of these themes, but in the last decade molecular approaches within each have made a significant contribution to the field. This is a personal review of the author’s past and present contributions and future ideas for challenging food microbiology from a molecular perspective.
Regulation of the inducible acetamidase gene of Mycobacterium smegmatis
Authors: Parish Tanya , Mahenthiralingam Eshwar , Draper Philip , Davis Elaine O. , Colston Elaine O.
Published: June 2009

Summary: The inducible acetamidase ofMycobacterium smegmatisNCTC 8159 is expressed at high levels in the presence of a suitable inducer, such as acetamide. The gene and 1.5 kb of upstream sequence had previously been sequenced. A further 1.4 kb of upstream sequence has now been determined, containing an additional ORF on the opposite strand to the acetamidase gene. This ORF has significant homologies to genes encoding regulatory proteins involved in amidase expression in other organisms. Restriction fragments from the 4 kb region were subcloned into a promoter-probe shuttle vector to locate the approximate region of the acetamidase promoter and investigate the mechanism of regulation. An inducible promoter was found to lie in the 1.4 kb region situated 1.5 kb upstream from the acetamidase coding region. Expression of the acetamidase was studied at the protein and mRNA levels. Using immunoblotting, induction of the enzyme was demonstrated in minimal medium containing succinate plus acetamide, but not in a richer medium (Lemco broth) plus acetamide, confirming that regulation of acetamidase expression is mediated by both positive and negative control elements. After induction by acetamide, an increase above basal level could be detected after 1 h for both protein levels (using ELISA) and mRNA levels (using Northern blot analysis), indicating that control of expression is at the mRNA level. The size of the mRNA transcript detected was approximately 1.2 kb, the size of the acetamidase coding region. Since no promoter was identified immediately upstream of the coding region, this raises the possibility that a larger, primary transcript (possibly polycistronic) is cleaved to produce a stable form encoding the acetamidase protein.
Tn5-induced lipopolysaccharide mutations in Bordetella pertussis that affect outer membrane function
Authors: Turcotte M. L. , Martin Denis , Brodeur Bernard R. , Peppler Mark S.
Published: June 2009

Summary: An LPSB-specific mAb was used to screen for ten Tn5insertion mutants ofBordetella pertussiswhich have LPS which is phenotypically distinct from either wild-type LPSAB or LPSB. Silver-stained SDS-PAGE gels showed nine different LPS phenotypes, six of which contain two clinically undocumented LPS bands, designated IntA and IntB based on their proximity to the LPSA and LPSB bands, respectively. Binding assays with LPSA- and LPSB-specific mAbs established changes in epitope exposure for the various mutant LPS, both in cell-free form and as presented on the surface of whole cells. The possible involvement of a number of genes, both structural and regulatory, was indicated in production of the altered phenotypes. PFGE and Southern blotting showed that the Tn5inserts of seven mutants mapped to a region of theB. pertussischromosome shown previously to encode thebplgene products of LPS biosynthesis. Mutants MLT3, MLT5 and MLT8, however, mapped to distinctly different parts of the chromosome. In addition, mutants MLT2 and MLT3 contributed to an accelerated frequency in the appearance of avirulent phase organisms despite their Tn5inserts being over 1000 bp from thebvglASR locus. The alterations in LPS structure in the mutants changed their reactivity to strain-specific mAbs and their sensitivity to hydrophobic and hydrophilic antibiotics.
Cadmium-specific formation of metal sulfide ‘Q-particles’ by Klebsiella pneumoniae
Authors: Holmes Justin D. , Richardson David J. , Saed Shaheen , Evans-Gowing Richard , Russell David A. , Sodeau John R.
Published: June 2009

Summary:Klebsiella pneumoniaeovercomes cadmium toxicity through the ‘biotransformation’ of cadmium ions into photoactive, nanometre-sized CdS particles deposited on the cell surface. The kinetics of particle formation during batch culture growth was monitored by electron microscopy (EM), energy-dispersive X-ray analysis and electronic absorption spectroscopy (EAS). During the deceleration phase of bacterial growth, the presence of CdS particles on the outer cell wall of K. pneumoniae (≥ 5 nm in diameter) was detected by EM. The size of these electron-dense particles continued to increase throughout the stationary phase of growth, with some of the particles reaching a diameter >200 nm. The formation of the extracellular CdS particles contributed to around 3-4% of the total cell biomass. EAS undertaken on these extracellular ‘bio-CdS’ particles suggested that the large ‘superparticles’ observed by EM, e.g. 200 nm, were aggregates of smaller particles termed ‘Q-particles’, ~ 4 nm in diameter. Metal sulfide particles were not formed in batch cultures of K. pneumoniae grown in the presence of lead, zinc, mercury, copper or silver ions. Growth in the presence of lead ions resulted in the formation of extracellular electron-dense particles containing lead but not sulfide or phosphate. Intracellular phosphorus-containing electron-opaque particles were formed during growth in the presence of copper and mercury. Intracellular electron-dense particles were formed in the presence of zinc ions but these did not contain phosphorus. From these results it was thought that metal sulfide formation inK. pneumoniaeshowed some cadmium-specificity. When cadmium and zinc ions were both added to the growth medium, metal sulfide particles were formed that were predominantly composed of cadmium, e.g. 48.6% cadmium and 0.04% zinc. Similarly, when cadmium and lead ions were both present during growth only CdS particles formed. In both cases analysis of the cells by EAS confirmed the presence of CdS only. These observations suggest that the mechanism of CdS formation is unlikely to occur simply through a cadmium-induced release of hydrogen sulfide by the cells into the external environment. If hydrogen sulfide production was the mechanism of sulfide formation then metal sulfide particles containing lead and zinc ions in addition to cadmium ions should have been produced.
Suppression of Escherichia coli formate hydrogenlyase activity by trimethylamine N-oxide is due to drainage of the inducer formate
Authors: Abaibou Hafid , Giordano Gérard , Mandrand-Berthelot Marie-Andrée
Published: June 2009

Summary: The effect of the addition of trimethylamine N-oxide (TMAO) in the growth medium on Escherichia coli anaerobic fermentative and respiratory pathways was examined. Formate dehydrogenase H (FDH-H) activity was totally repressed by the addition of 40 mM TMAO, whereas the overall hydrogenase (HYD) activity was reduced by 25%. Accordingly, expression of lacZ operon fusions with the fdhF and hycB structural genes specifying FDH-H and HYD3 was reduced sevenfold and eightfold, respectively, leading to suppression of an active formate hydrogenlyase system. In contrast, global respiratory formate-dependent phenazine methosulphate reductase (FDH-PMS) activity, which consists of both the major anaerobic FDH-N enzyme and the aerobic FDH-Z isoenzyme, was increased approximately twofold. This was corroborated by a 2.5-fold stimulation of the sole fdoG-uidA transcriptional fusion which reflects the synthesis of the respiratory aerobic FDH-Z enzyme. In fdhD, fdhE or torA mutants lacking either FDH-PMS activity or TMAO reductase (TOR) activity, the formate hydrogenlyase pathway was no longer inhibited by TMAO. In addition, introduction of 30 mM formate in the growth medium was found to relieve the repressive effect of TMAO in the wild-type strain. When TMAO was added as terminal electron acceptor a significant enhancement of anaerobic growth was observed with the wild-type strain and the fdoG mutant. It was associated with the concomitant suppression of the formate hydrogenlyase enzymes. This was in contrast to the fdnG and torA mutants whose growth pattern and fermentative enzymes remained unaffected. Taken together, these results strongly suggest that formate-dependent reduction of TMAO via FDH-N and TOR reduces the amount of formate available for induction of the formate hydrogenlyase pathway.
A 32 kb nucleotide sequence from the region of the lincomycin-resistance gene (22°-25°) of the Bacillus subtilis chromosome and identification of the site of the lin-2 mutation
Authors: Kumano Miyuki , Tamakoshi Atsuo , Yamane Kunio
Published: June 2009

Summary: A 32 kb nucleotide sequence in the region of the lincomycin-resistance gene, located from 22° to 25° on the Bacillus subtilis chromosome, was determined. Among 32 putative ORFs identified, four [lipA for lipase, natA, natB and yzaE (renamed yccK)] have already been reported, although the functions of NatA, NatB and YccK remain to be characterized. Six putative products were found to exhibit significant similarity to known proteins in the databases, namely L-asparaginase precursor, protein aspartate phosphatase, x-glucosidase, two tellurite-resistance proteins and a hypothetical protein from B. subtilis. The region of the tellurite-resistance gene, consisting of seven ORFs, seems to correspond to an operon. The products of 14 ORFs exhibited considerable or limited similarity to known proteins. The sequenced region seems to be rich in membrane proteins, since at least 16 gene products appeared to contain membrane-spanning domains. The site of the lin-2 mutation (two nucleotide replacements) was mapped and identified by sequencing. This site is located between a putative promoter and the SD sequence of ImrA (yccB)[a putative repressor of the Imr operon, which consists of ImrA and ImrB (yccA)]. LmrB is a homologue of proteins involved in drug-export systems and seems likely to be the protein responsible for resistance to lincomycin.
Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis
Authors: Yeo Chew Chieng , Tham Jill Maelan , Yap Melvyn Wee-Ching , Poh Chit Laa
Published: June 2009

Summary: Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xylenol and harbours the plasmid RP4, was mated with a plasmid-free derivative of Pseudomonas putida NCIB 9869, strain RA713, which cannot grow on 2,5-xylenol. Some RA713 transconjugants, initially selected on 2,5-xylenol, were found to carry RP4 plasmids that had acquired additional fragments (designated XIn) which ranged in size from 2 kb to approximately 26 kb. Instability of DNA inserts in RP4::XIn hybrid plasmids was observed. The smallest insert present in a stable RP4::XIn6 hybrid plasmid, termed XIn6, yielded multiple bands when it was used as a probe with digested P25X chromosomal DNA. Sequence analysis of XIn6 led to the discovery of an open reading frame with homology to the maturases of group II introns. The XIn6 insert also exhibited several features characteristic of a group II intron. These included the presence of the consensus sequence GUGYG at the 5′ end and RAY at the 3′ end of the intron. RNA secondary structure modelling of XIn6 also revealed the presence of perfectly conserved domains V and VI. Differences were detected in the XIn6 hybridization profiles of several P25X catabolic mutants that have lost the ability to grow on 2,5-xylenol. In these mutants the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the XIn6 group II intron. This is the first reported group II intron isolated from Pseudomonas spp. and the first time that the mobility of a bacterial group II intron has been demonstrated.
Molecular and functional analysis of pTAV320, a repABC-type replicon of the Paracoccus versutus composite plasmid pTAV1
Authors: Bartosik Dariusz , Baj Jadwiga , Wlodarczyk Miroslawa
Published: June 2009

SUMMARY: The second replicator region of the native plasmid pTAVl of Paracoccus versutus has been identified thus proving the composite nature of this replicon. The minimal replicon designated pTAV320 (43 kb) was cloned and sequenced. pTAV320 encodes three putative proteins - RepA, RepB and RepC. This replicator region shows strong structural and functional similarity to mpABC-type rep1 icons found in several Agrobacterium and Rhizobium
plasmids. The origin of replication appears to be localized within the coding sequence of the repC gene. RepC was shown t o be essential for replication. RepA and RepB were necessary for stable maintenance of the plasmid, which implies a role in active partitioning. The presence of the complete sequence of pTAV320 (in its non-replicative form) could stabilize in cis pTAV202, a rninireplicon derived from the other pTAVl replicator region. Deletions introduced into the mpC gene abolished the ‘stabilizing’ activity of pTAV320, suggesting that the centromere-like sequence, necessary for partitioning, might be localized within this gene. The two replicator regions of pTAV1 (pTAV320 and pTAV202) expressed incompatibility towards the parental plasmid but were compatible in trans in P. wersutus cells. The pTAV320 replicon can be maintained in several Paracoccus, Agrobacterium, Rhizobium and Rhodohcter strains in addition t o P. versutus.
Isolation and characterization of Staphylococcus aureus starvat ion4 nduced, stationary-phase mutants defective in survival or recovery
Authors: Watson Sean P. , Antonio Martin , Foster Simon J.
Published: June 2009

SUMMARY: Ten Staphylococcus aureus mutants, defective in the starvation-induced stationary phase of growth were isolated from two independent Tn917-LTVI transposon insertion libraries and were designated suv as they had apparent-suryival defects. Seven of these mutants were defective under amino-acid-limiting conditions alone. Two mutants (suv-3 and suw-20) demonstrated lower plating efficiency when starved for glucose, phosphate or amino acids and one mutant (suv-11) had reduced plating efficiency after amino acid or glucose starvation. All of the mutants tested were as resistant to hydrogen peroxide assault as the parent, but six were more sensitive to low pH conditions. All the mutants were physically mapped on the 5. aureus chromosome using PFGE. Chromosomal DNA flanking the Tn917-LNI insertion sites was rescued by cloning into Escherichia coli. DNA sequence analysis resulted in theidentification of a number of transposon-disrupted ORFs encoding putative components such as superoxide dismutase (suv-I), haem A synthase (suv-3)# a component of the 505 response (suv-9) and hypoxanthine-guanine phosphoribosyltransferase (suv-20). The Tn917-LTVI insertion created lac2 transcriptional fusions for some of the stationary-phase loci. Expression analysis indicated that suv-4 was induced at mid-exponential phase, whereas suv-3 and suv-II were induced at the onset of stationary phase. The possible roles of these suv components in stationary-phase survival or recovery is discussed.
Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector
Authors: Van Mellaert Lieve , Mei Lijuan , Lammertyn Elke , Schacht Sabine , Ann Jozef
Published: June 2009

Summary: The temperate bacteriophage VWB integrates into the chromosome of Streptomyces venezuelae ETH14630 via site-specific integration. Following recombination of the VWB attP region with the chromosomal attB sequence, the host-phage junctions attL and attR are formed. Nucleotide sequence analysis of attP, attB, attL and attR revealed a 45 bp common core sequence. In attB this 45 bp sequence consists of the 3' end of a putative tRNAArg(AGG) gene with a 3'-terminal CCA sequence which is typical for prokaryotic tRNAs. Phage DNA integration restores the putative tRNAArg(AGG) gene in attL. However, following recombination the CCA sequence is missing as is the case for most Streptomyces tRNA genes described so far. Adjacent to VWB attP, an ORF encoding a 427 aa protein was detected. The C-terminal region of this protein shows high similarity to the conserved C-terminal domain of site-specific recombinases belonging to the integrase family. To prove the functionality of this putative integrase gene (int), an integrative vector pKT02 was constructed. This vector consists of a 23 kb HindIII-Sphl restriction fragment of VWB DNA containing attP and int cloned in a non-replicative Escherichia coli vector carrying a thiostrepton-resistance (tsr) gene. Integration of pKT02 was obtained after transformation of Streptomyces venezuelae ETH14630 and Streptomyces lividans TK24 protoplasts. This vector will thus be useful for a number of additional Streptomyces species in which a suitable tRNA gene can be functional as integration site.
The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site OB3 but other KorB-binding sites form destabilizing complexes in the absence of Ob3
Authors: Williams D. Ross , Macartney Donia P. , Thomas Christopher M.
Published: June 2009

Summary: The sector of the genome of broad-host-range IncP plasmid RK2 from kb coordinate 54·0 to 60·0 confers an active partitioning phenotype, increasing the segregational stability of low-copy-number unstable plasmids. This Par region encodes the central control operon (korA, incC, korB, korF and korG) and the associated genes kfrA, upf54.8 and upf54.4. Each ORF in this region was knocked out in turn and it was shown that only incC and korB are needed for the stability phenotype. incC encodes two polypeptides from alternative translational starts. A deletion of the start of the operon showed that only IncC2, the shorter product is essential for partitioning. Directed mutation or deletion was used to inactivate in turn each of the three KorB-binding sites (Obs) which were candidate cis-acting sequences needed for stability. Only inactivation of OB3, which lies between upf54.4 and upf54.8, resulted in an increased rate of segregational loss. However, the rate of loss was significantly higher than the rate of loss of the test plasmid carrying none of this RK2 Par region. Either inactivation of korB or deletion of Ob1 from this Ob3 mutant resulted in restoration of the loss rate to that expected for the unstable test plasmid alone. Thus KorB can act on Ob1 to create a complex that either inhibits replication or reduces the effective plasmid copy number, perhaps by promoting pairing between plasmid molecules. This implies that RK2 goes through a cycle of pairing and separation, akin to the mitotic cycle of eukaryotic chromosomes.
The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue
Authors: Kirby S. D. , Lainson F. A. , Donachie W. , Okabe A. , Tokuda M. , Hatase O. , Schryvers A. B.
Published: June 2009

Summary: In a previous investigation, a 35 kDa iron-regulated protein was identified from total cellular proteins of Pasteurella haemolytica grown under iron-depleted conditions. This study reports identification of the gene (fbpA) encoding the 35 kDa protein based on complementation of an entA Escherichia coli strain transformed with a plasmid derived from a P. haemolytica lambda ZAP II library. Cross-reactivity was demonstrated between an anti-35 kDa mAb and a 35 kDa protein expressed in this strain. Furthermore, a translated ORF identified on the recombinant plasmid corresponded with the N-terminal amino acid sequence of the intact and a CNBr-cleaved fragment of the 35 kDa iron-regulated protein. Nucleotide sequence analysis of the gene encoding the 35 kDa protein demonstrated homology with the cluster 1 group of extracellular solute-binding proteins, especially to the iron-binding proteins of this family. Complete sequence analysis of the recombinant plasmid insert identified three other predominant ORFs, two of which appeared to be in an operonic organization with fbpA. These latter components (fbpB and fbpC) showed homology to the transmembrane and ATPase components of ATP-binding cassette (ABC)-type uptake systems, respectively. Based on amino acid/DNA sequencing, citrate competition assay of iron affinity and visible wavelength spectra, it was concluded that the P. haemolytica 35 kDa protein functions as an FbpA homologue (referred to as PFbpA) and that the gene encoding this protein is part of an operon comprising a member of the FbpABC family of iron uptake systems. Primary sequence analysis revealed rather surprisingly that PFbpA is more closely related to the intracellular Mn/Febinding protein IdiA found in cyanobacteria than to any of the homologous FbpA proteins currently known in commensal or pathogenic members of the Pasteurellaceae or Neisseriaceae.
Internalization of Aeromonas hydrophila by fish epithelial cells can be inhibited with a tyrosine kinase inhibitor
Authors: Tan E. , Low K. W. , Wong W. S. F. , Leung K. Y.
Published: June 2009

Aeromonas hydrophila is a Gram-negative bacterium that is pathogenic in fish, causing motile aeromonad septicaemia. It can enter (invade) fish cells, and survive as an intracellular parasite. The host-pathogen interaction and signal transduction pathway were studied by screening signal transduction inhibitors using carp epithelial cells and a virulent strain of the bacterium, PPD134/91. Genistein, a tyrosine kinase inhibitor, postponed internalization of A. hydrophila into host cells, suggesting that tyrosine phosphorylation plays a role in internalization. In contrast, staurosporine, a protein kinase C inhibitor, and sodium orthovanadate, a protein tyrosine phosphatase inhibitor, accelerated internalization of PPD134/91. Other virulent strains of A. hydrophila were also examined and it is likely that all strains, irrespective of serogroup, use the same signalling pathway to facilitate bacterial uptake.
Escherichia coli ColV plasmid pRK100: genetic organization, stability and conjugal transfer
Authors: AmbrožKičK Jerneja , OstroveršKnik Alenka , StarčKičK Marjanca , Kuhar Irena , Grabnar MiklavžK , Žgur-Bertok Darja
Published: June 2009

UropathogenicEscherichia colistrains express chromosomal and plasmid-encoded virulence-associated factors such as specific adhesins, toxins and iron-uptake systems. A ColV plasmid (pRK100) of a uropathogenic strain and its host KS533 were studied. The host strain encodes the K1 capsule, and P and S fimbriae, but neither haemolysin nor the cytotoxic-necrotic factor CNF1, indicating that this strain does not harbour a larger pathogenicity island. A restriction map of pRK100 was constructed on the basis of hybridization experiments and nucleotide sequencing. pRK100 harbours ColV, the conserved replication region RepFIB, the aerobactin-uptake system, a RepFIC replicon and additionally Colla as well as transposon Tn5431. The location of the RepFIC replicon was similar to that in plasmid F. ColV plasmids and F thus share a region spanning more than half the length of plasmid F. Even though their replication and transfer regions are homologous, ColV plasmids are found only inE. colistrains. Among the four other species tested, conjugal transfer of pRK100 was demonstrated, with low frequency, only toKlebsiella pneumoniae, suggesting that a natural barrier effectively bars transfer.In vitrostability of the plasmid with integration into the chromosome to ensure maintenance in the presence of an incompatible plasmid was demonstrated.
Genes and enzymes of the acetyl cycle of arginine biosynthesis in the extreme thermophilic bacterium Thermus thermophilus HB27
Authors: Baetens Margot , Legrain Christianne , Boyen Anne , Glansdorff Nicolas
Published: June 2009

An arginine biosynthetic gene cluster, argC-argJ, of the extreme thermophilic bacterium Thermus thermophilus HB27 was isolated by heterologous complementation of an Escherichia coli acetylornithinase mutant. The recombinant plasmid (pTHM1) conferred ornithine acetyltransferase activity to the E. coli host, implying that T. thermophilus uses the energetically more economic pathway for the deacetylation of acetylornithine. pTHM1 was, however, unable to complement an E. coli argA mutant and no acetylglutamate synthase activity could be detected in E. coli argA cells containing pTHM1. The T. thermophilus argJ-encoded enzyme is thus monofunctional and is unable to use acetyl-CoA to acetylate glutamate (contrary to the Bacillus stearothermophilus homologue). Alignment of several ornithine acetyltransferase amino acid sequences showed no obvious pattern that could account for this difference; however, the monofunctional enzymes proved to have shorter N-termini. Sequence analysis of the pTHM1 3.2 kb insert revealed the presence of the argC gene (encoding N-acetylglutamate-5-semialdehyde dehydrogenase) upstream of the argJ gene. Alignment of several N-acetylglutamate-5-semialdehyde dehydrogenase amino acid sequences allowed identification of two strongly conserved putative motifs for cofactor binding: a putative FAD-binding site and a motif reminiscent of the NADPH-binding fingerprint. The relationship between the amino acid content of both enzymes and thermostability is discussed and an effect of the GC content bias is indicated. Transcription of both the argC and argJ genes appeared to be vector-dependent. The argJ-encoded enzyme activity was twofold repressed by arginine in the native host and was inhibited by ornithine. Both upstream of the argC gene and downstream of the argJ gene an ORF with unknown function was found, indicating that the organization of the arginine biosynthetic genes in T. thermophilus is new.
Bioremediation: towards a credible technology
Authors: Head Ian M.
Published: June 2009

Bioremediation is the technological process whereby biological systems are harnessed to effect the clean-up of environmental pollutants. Currently, microbial systems are most widely employed in bioremediation programmes, generally in the treatment of soils and waters contaminated with organic pollutants. Micro-organisms have a huge metabolic repertoire that enables them to degrade a panoply of organic pollutants and in many cases the complex biochemistry and molecular biology of the catabolic pathways involved have been unravelled (e.g. Gibson, 1984; Frantz et al., 1987; Evans & Fuchs, 1988; Burlage et al., 1989; Abramowicz, 1990; Assinder & Williams, 1990; Chaudhry & Chapalamadugu, 1991; Cerniglia, 1992; Knackmuss, 1996). Despite valuable basic knowledge on the mechanisms of pollutant bio-degradation, bioremediation has yet to be accepted as a routine treatment technology and the environmental industry is wary of applying bioremediation for the treatment of contaminated sites.
Mycoplasma hominis expresses two variants of a cell-surface protein, one a lipoprotein, and one not
Authors: Ladefoged Soren A. , Christiansen Gunna
Published: June 2009

A protein similar to the previously characterized variable surface-exposed membrane protein P120 was identified (P120′), establishing that Mycoplasma hominis PG21 possesses a novel gene family. The gene, p120', was sequenced and found to have some distinctive properties including a putative start codon of GTG, rather than the common ATG codon, and a coding region with a high G+C content, characteristic of essential housekeeping genes in mycoplasmas. No sequence homology was found to known proteins. The genomic locations of the p120 and p120' genes were determined on the restriction map of five M. hominis strains by PFGE. The genes were localized in two separate regions separated by more than 6 kb. Genes as well as proteins corresponding to P120′ were identified in 24/24 M. hominis isolates tested and no size variation was detected. P120′ had a molecular mass of 98 kDa, 20 kDa smaller than P120 as estimated by SDS-PAGE. The protein was surface-exposed and associated with the mycoplasma membrane, but had predominantly hydrophilic characteristics upon Triton X-114 extraction. The N-terminal part of P120′ had a hydrophobic leader sequence without the characteristics of a prolipoprotein. This might explain the membrane association of the protein. Unlike P120, which is frequently recognized by sera of patients seropositive for M. hominis, P120′ was only rarely recognized. The conserved nature of the P120 gene family indicates that it has an essential, although currently unknown, function.
The rrs (16S)-rrl (23S) ribosomal intergenic spacer region as a target for the detection of Haemophilus ducreyi by a heminested-PCR assay
Authors: Gu X. X. , Rossau R. , Jannes G. , Ballard R. , Laga M. , Van Dyck E
Published: June 2009

The intergenic spacer region between the rrs and rrl ribosomal RNA genes of Haemophilus ducreyi was analysed and the DNA sequence was used for the selection of specific PCR primers. A highly sensitive and specific heminested-PCR assay for the identification of H. ducreyi was developed. The assay showed a sensitivity of 96% on genital ulcer specimens from patients with clinically diagnosed chancroid, compared with a sensitivity of 56% for culture methods. These results indicate that this PCR assay has the potential to become an accurate and easy reference method for the detection of H. ducreyi.
Fused nucleoids resegregate faster than cell elongation in Escherichia coli pbpB(Ts) filaments after release from chloramphenicol inhibition
Authors: Van Helvoort J. M. L. M , Huls P. G. , Vischer N. O. E. , Woldringh C. L.
Published: June 2009

The course of nucleoid movement during and upon release from protein synthesis inhibition by chloramphenicol in filaments of Escherichia coli pbpB(Ts) was analysed. Cells were grown at 42 °C in glucose minimal medium for two mass doublings and were treated with chloramphenicol to generate fusion (coalescence) of the nucleoids. Upon release from protein synthesis inhibition, the large distance between the border of the fused nucleoids and the cell poles immediately decreased, before full recovery of the rates of mass growth and length increase at 30 °C. This indicates that nucleoids can reoccupy the DNA-free cell ends independently of cell elongation. During filamentation at 42 °C, the pbpB cells established initial constrictions at midcell and at one-quarter and three-quarter positions. Nevertheless, divisions only started 75 min after chloramphenicol removal at 30 °C, when most nucleoids had moved back into the vacated cell ends. No ‘guillotine-like’ constrictions at the site of the nucleoids occurred.This suggests that segregating nucleoids postpone division recovery at previously established sites. The results are discussed in the light of a working model for transcription/translation-mediated chromosome segregation and nucleoid occlusion of cell division.
Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates
Authors: Hummerjohann Jörg , Küttel Erika , Quadroni Manfredo , Ragaller Jürgen , Leisinger Thomas , Kertes Michael A.
Published: June 2009

Pseudomonas aeruginosaPAO1 grew in defined synthetic medium with any of a broad variety of single sulfur sources, including sulfate, cysteine, thiocyanate, alkanesulfonates, organosulfate esters and methionine, but not with aromatic sulfonates, thiophenols or organothiocyanates or isothiocyanates. During growth with any of these compounds except sulfate, cysteine or thiocyanate, a set of 10 sulfate starvation-induced (SSI) proteins was strongly up-regulated, as observed by two-dimensional protein electrophoresis of total cell extracts. A comparable level of up-regulation was found for the hydrolytic enzyme arylsulfatase, which has previously been used as a marker enzyme for the sulfate starvation response. One of the SSI proteins was identified by N-terminal sequencing as a high-affinity periplasmic sulfate-binding protein, and another was related to thiol-specific antioxidants, but the N-terminal sequences of the other SSI proteins revealed no similarity to N-termini of proteins of known function, and they probably represent uncharacterized enzymes involved in sulfur scavenging when preferred sulfur sources are absent. To study the role that cysteine biosynthetic intermediates play in the synthesis of these proteinsin vivo, we isolated mini-Tn5transposon mutants ofP. aeruginosawith insertions in thecysNandcyslgenes, which encode subunits of ATP-sulfurylase and sulfite reductase, respectively. These two genes were cloned and sequenced.cyslshowed high similarity to the cognate gene inEscherichia coli, whereascysNencoded a 69.3 kDa protein with two domains corresponding to theE. coliCysN and CysC proteins. Sulfate no longer repressed synthesis of the SSI proteins incysNmutants, but repression was restored by sulfite; in thecyslmutant, sulfate, sulfite and sulfide all led to repression of SSI protein synthesis. This suggests that there are at least two independent corepressors of the sulfate starvation response in this species.
Implications of the xylQ gene of TOL plasmid pWW102 for the evolution of aromatic catabolic pathways
Authors: Aemprapa Sirinun , Williams Peter A.
Published: June 2009

Pseudomonas putida strain O2C2 is able to grow on toluene, m-xylene and p-xylene through benzoate and the corresponding methylbenzoates (toluates). The catabolic genes are encoded on a large TOL plasmid, pWW102, of >220 kb. The complete catabolic genes were cloned on four large overlapping restriction fragments covering a total of 28 kb of the plasmid, which was carefully mapped by restriction enzyme analysis. The presence of the xyl genes on the cloned DNA was confirmed by assay of representative enzymes of both operons. Virtually all the genes were located on the cloned DNA by hybridization of Southern blots with gene-specific probes from related pathways of other catabolic plasmids. Within the limitations of available restriction sites, the analysis showed that the genes are in two blocks. The major block carries the meta pathway operon xylXYZLTEGFJQKIH with the two regulatory genes xylSR immediately downstream. The upper pathway operon xylUWCMAB(N) is about 2-3 kb downstream of the regulatory genes and transcribed in the same direction as the meta pathway operon. Within each operon the gene order appears to be identical to that found in other TOL plasmids, but the relative location of the operons most closely resembles that found on plasmid pWW53, although there is no evidence of any xyl duplications on pWW102. The nucleotide sequence of the xylQ gene for the acetaldehyde dehydrogenase (acylating; ADA), together with the 3-end of the upstream xylJ (for 2-oxopent-4-enoate hydratase) and the 5-end of the downstream xylJ (for 4-hydroxy-2-oxovalerate aldolase), was determined. The xylQ gene was ligated into expression vector pTrc99a and high levels of XylQ protein were detected by enzyme assay and by SDS-PAGE. All three genes xylJQK showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas meta pathways, the highest being with the naphthalene catabolic genes nahLOM from the plasmid of Pseudomonas sp. NCIB 9816. The implications of the sequence homologies to the evolution of these pathways are discussed.