bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnesium transport in Salmonella typhimurium: biphasic magnesium and time dependence of the transcription of the mgtA and mgtCB loci
Authors: Tao Tao , Grulich Paul F. , Kucharski Lisa M. , Smith Ronald L. , Maguire Michael E.
Published: June 2009

Salmonella typhimurium has three distinct Mg2+ transport systems, the constitutive high-capacity CorA transporter and two P-type ATPases, MgtA and MgtB, whose transcription is repressed by normal concentrations of Mg2+ in the growth medium. The latter Mg2+-transporting ATPase is part of a two-gene operon, mgtCB, with mgtC encoding a 23 kDa protein of unknown function. Transcriptional regulation using fusions of the promoter regions of mgtA and mgtCB to luxAB showed a biphasic time and Mg2+ concentration dependence. Between 1 and 6 h after transfer to nitrogen minimal medium containing defined concentrations of Mg2+, transcription increased about 200-fold for mgtCB and up to 400-fold for mgtA, each with a half-maximal dependence on Mg2+ of 0.5 mM. Continued incubation revealed a second phase of increased transcription, up to 2000-fold for mgtCB and up to 10000-fold for mgtA. This secondary increase occurred between 6 and 9 h after transfer to defined medium for mgtCB but between 12 and 24 h for mgtA and had a distinct half-maximal dependence for Mg2+ of 0.01 mM. A concomitant increase of at least 1000-fold in uptake of cation was seen between 8 and 24 h incubation with either system, showing that the transcriptional increase was followed by functional incorporation of large amounts of the newly synthesized transporter into the membrane. Regulation of transcription by Mg2+ was not dependent on a functional stationary-phase sigma factor encoded by rpoS, but it was dependent on the presence of a functional phoPQ two-component regulatory system. Whereas mgtCB was completely dependent on regulation via phoPQ, the secondary Sate Mg2+-dependent phase of mgtA transcription was still evident in strains carrying a mutation in either phoP or phoQ, albeit substantially diminished. Several divalent cations blocked the early phase of the increase in transcription elicited by the decrease in Mg2+ concentration, including cations that inhibit Mg2+ uptake (Co2+, Ni2+ and Mn2+) and those which do not (Ca2+ and Zn2+). In contrast, the second later phase of the transcriptional increase was not well blocked by any cation except those which inhibit uptake. Overall, the data suggest that at least two distinct mechanisms for transcriptional regulation of the mgtA and mgtCB loci exist.
A 28 kbp segment from the spoVM region of the Bacillus subtilis 168 genome
Authors: Foulger David , Errington Jeffery
Published: June 2009

The sequence of a 28 kbp segment of DNA surrounding the spoVM gene of Bacillus subtilis 168 (lying at approximately 145 on the standard genetic map) has been determined. The region contains 27 ORFs, a number of which have predicted products significantly similar to proteins in sequence databases, particularly to proteins involved in macromolecular synthesis of nucleic acids, proteins and phospholipids. A pair of closely linked genes encode a likely serine protein phosphatase and a serine protein kinase, respectively. Such proteins play important regulatory roles in eukaryotic cells but are rare in prokaryotes.
Efficient conversion of 5-substituted hydantoins to D-α-amino acids using recombinant Escherichia coli strains
Authors: Grifantini Renata , Galli Giuliano , Carpani Giovanna , Pratesi Claudio , Frascotti Gianni , Grandi Guido
Published: June 2009

D-Amino acids, important intermediates in the production of semisynthetic penicillins and cephalosporins, are currently prepared from the corresponding hydantoins using bacterial biomass containing two enzymes, hydantoinase and carbamylase. These enzymes convert the hydantoins first into carbamyl derivatives and then into the corresponding D-amino acids. In an attempt to select more efficient biocatalysts, the hydantoinase and carbamylase genes from Agrobacterium tumefaciens (formerly A. radiobacter) were cloned in Escherichia coli. The genes were assembled to give two operon-type structures one having the carbamylase gene preceding the hydantoinase gene and the other with the carbamylase gene following the hydantoinase gene. The recombinant strains stably and constitutively produced the two enzymes and efficiently converted the corresponding hydantoins into p-hydroxyphenyl-glycine and phenylglycine. The order of the genes within the operon and the growth temperature of the strains turned out to be important for both enzyme and D-amino acid production. The configuration with the carbamylase gene preceding the hydantoinase gene was the most efficient one when the biomass was grown at 25°C rather than 37°C. This biomass produced D-amino acid twice as efficiently as the industrial strain of A. tumefaciens. The efficiency was found to be correlated with the level of carbamylase produced, indicating that the concentration of this enzyme is the rate-limiting factor in D-amino acid production under the conditions used on an industrial scale.
A chitin-binding domain in a marine bacterial chitinase and other microbial chitinases: implications for the ecology and evolution of 1,4-β-glycanases
Authors: Svitil Amy L. , Kirchman David L.
Published: June 2009

To examine the ecology and evolution of microbial chitinases, especially the chitin-binding domain, one of the chitinase genes (chiA) from the marine bacterium Vibrio harveyi was analysed. The deduced amino acid sequence of ChiA is not very similar overall to other proteins, except for two regions, the putative catalytic and chitin-binding domains. Among all bacterial chitinases sequenced to date, there is no relationship between percentage similarity of catalytic domains and chitin-binding domains in pairwise comparisons, suggesting that these two domains have evolved separately. The chitin-binding domain appears to be evolutionarily conserved among many bacterial chitinases and is also somewhat similar to cellulose-binding domains found in microbial cellulases and xylanases. To investigate the role of the chitin-binding domain, clones producing versions of ChiA with or without this domain were examined. One version with the domain (ChiA1) bound to and hydrolysed chitin, whereas a truncated ChiA without the putative chitin-binding domain (ChiA2) did not bind to chitin but it could hydrolyse chitin, although not as well. ChiA1 diffused more slowly in agarose containing colloidal chitin than ChiA2, but diffusion of the Two proteins in agarose without colloidal chitin was similar. These results indicate that the chitin-binding domain helps determine the movement of chitinase along N-acetylglucosamine strands and within environments containing chitin.
Aspartate carbamoyltransferase from a psychrophilic deep-sea bacterium, Vibrio strain 2693: properties of the enzyme, genetic organization and synthesis in Escherichia coli
Authors: Xu Ying , Zhang Yuanfu , Liang Ziyuan , Van de Casteele Mark , Legrain Christianne , Glansdorff Nicolas
Published: June 2009

The aspartate carbamoyltransferase (ATCase) genes of psychrophilic Vibrio strain 2693 were cloned by complementation in Escherichia coli and the enzyme was partly characterized. The genes constitute a pyrBl operon homologous to the cognate structure in E. coli where pyrB and pyrl respectively encode the catalytic and the regulatory chains of ATCase. The strong sequence similarities noted between Vibrio and E. coli ATCases include extensive conservation of residues involved in interactions between subunits, suggesting that the two enzymes have very similar tertiary and quaternary structures. Vibrio ATCase is, however, not activated by ATP and not synergistically inhibited by CTP and UTP. It is also much more thermolabile than E. coli ATCase. With respect to Pyrococcus abyssi and E. coli ATCases, Vibrio ATCase presents marked differences in composition which could be related to its psychrophilic character. The results of these structural and functional comparisons indicate that Vibrio 2693 ATCase is a suitable model for biochemical studies on structure-function relationships in a ‘cold’ allosteric enzyme. The operon is expressed from a promoter which is immediately followed by a pyrimidine-rich leader ORF terminating within a putative transcription attenuator. These genetic and enzymic data strengthen the evolutionary relationship already noted between Vibrionaceae and Enterobacteriaceae.
Comparison of PhoP binding to the tuaA promoter with PhoP binding to other Pho-regulon promoters establishes a Bacillus subtilis Pho core binding site
Authors: Liu Wei , Hulett F. Marion
Published: June 2009

The phosphate-deficiency response in Bacillus subtilis is regulated by PhoP PhoR, a pair of two-component regulatory proteins. PhoR is a histidine kina and PhoP is a response regulator. Genetic evidence indicates that the Pho-regulon genes, which are induced or repressed under phosphate starvation conditions, are regulated by PhoP and PhoR at the transcriptional level. It has previously been shown that PhoP binds to four Pho-regulon promoters in both unphosphorylated and phosphorylated forms. This study demonstrates that another Pho-regulon gene promoter, the tuaA promoter preceding the operon which is responsible for cell wall teichuronic acid synthesis, is also transcriptionally regulated and is bound by PhoP. The binding affinity for phosphorylated PhoP was about 10-fold higher than that for unphosphorylated PhoP. Both unphosphorylated and phosphorylated PhoP bound upstream of the -20 region in the tuaA promoter. By aligning the Phop binding sites within the Pho-regulon promoters, a consensus core PhoP-binding region composed of four TT(A/T)ACA direct repeats, each separated by 5.2 non-conserved nucleotides was identified. PhoP, phosphorylated or unphosphorylated, binds to such a sequence in all Pho-regulon promoters studied. Phosphorylated PhoP binds to the core binding region with high affinity and to additional regions surrounding this region with similar or lower affinity.
Oxygen protection of nitrogen fixation in free-living Azorhizobium caulinodans: the role of cytochrome aa3
Authors: Boogerd Fred C. , Pronk Annemieke F. , Mashingaidze Cyril , Affourtit Charles , Stouthamer Adriaan H. , van Verseveld Henk W. , Westerhoff Hans V.
Published: June 2009

The growth properties of Azorhizobium caulinodans wild-type and a cytochrome aa3 mutant strain, both growing with N2 as N source at fixed dissolved partial oxygen pressures in the range 0.5--4.0 kPa, were studied by making use of continuous cultures (chemostats and pH-auxostats) and transient cultures. In succinate-limited chemostats, the wild-type exhibited a higher growth yield than the aa3 mutant at every dissolved oxygen tension tested, indicating activity of cytochrome aa3 in this entire oxygen regime. The growth yield of both the wild-type and the aa3 mutant declined when the dissolved oxygen tension was raised. In contrast, for growth on ammonia at the same dilution rate, the wild-type showed an increase in growth yield with increasing dissolved oxygen tension, whereas the growth yield of the aa3 mutant remained constant. The transient changes in growth properties observed in chemostat cultures after pulsing with succinate pointed to a negative effect of oxygen on the maximum specific growth rate. This was studied further in steady-state pH-auxostat cultures. The specific growth rate of both strains decreased with increasing dissolved oxygen tension. The less steep decline in growth rate of the wild-type compared to the aa3 mutant confirmed that cytochrome aa3 is active in the wild-type. Again, the growth yield of both strains decreased with the dissolved oxygen tension, but in contrast to the results obtained with chemostats, no difference in growth yield was observed between wild-type and mutant at any oxygen tension. In either type of continuous culture a decrease in the overall P/O ratio with increasing dissolved oxygen tension is improbable for the wild-type, and even more so for the aa3 mutant. Therefore, the adverse effects of oxygen on the growth of A. caulinodans are not readily explained by respiratory protection; alternatively, it is proposed that the catalytic oxidation of nitrogen-fixation-specific redox enzymes by oxygen (auto-protection) enables the bacterium to deal with intracellular oxygen at the expense of reducing equivalents and free energy. To compensate for the loss of free energy, respiration increases and an active cytochrome aa3 contributes to this by keeping the P/O ratio high.
The role of the Corynebacterium glutamicum rel gene in (p)ppGpp metabolism
Authors: Wehmeier Lutz , Schafer Andreas , Burkovski Andreas , Krmer Reinhard , Mechold Undine , Malke Horst , Phler Alfred , Kalinowski Jrn
Published: June 2009

To investigate the metabolism of (p)ppGpp in amino-acid-producing coryneform bacteria, a PCR-based strategy using degenerate consensus oligonucleotides was applied to isolate the rel gene of Corynebacterium glutamicum ATCC 13032. The gene consists of 2283 nucleotides and encodes a protein of 760 amino acids with a molecular mass of 84.4 kDa. The amino acid sequence revealed extensive similarities to the related proteins RelA and SpoT of Escherichia coli, which are known to be involved in (p)ppGpp biosynthesis and degradation. The C. glutamicum rel gene is located downstream of the apt gene encoding an adenine phosphoribosyltransferase, and an ORF with similarities to dciAE, which represents part of a dipeptide transport system in E. coli. A C. glutamicum mutant strain carrying a defined deletion in the rel gene was constructed. This mutant failed to accumulate (p)ppGpp in response to amino acid starvation. When overexpressed in E. coli, the C. glutamicum rel gene was able to reverse growth defects caused by an overexpressed relA gene. It is proposed that the C. glutamicum rel gene encodes a bifunctional enzyme with (p)ppGpp synthetase and (p)ppGpp-degrading activities.
A novel filamentous phage, fs-2, of Vibrio cholerae 0139
Authors: Ikema Masahide , Honma Yasuko
Published: June 2009

A novel filamentous bacteriophage, fs-2, was isolated from Vibrio cholerae O139 strain MDO14. The fs-2 phage was a long filamentous particle 1200 nm long and 7 nm wide. The purified phage formed a turbid plaque when spotted on a lawn of the host organisms. The plaque-formation activity was stable following heating to 70 � but was inhibited by treatment with chloroform. fs-2 had a single-stranded DNA genome and was converted to a double-stranded replicative form in the host cell. Almost all V. cholerae O139 and O1 El Tor biotype strains tested were sensitive to the phage, but most O1 classical strains and non-O1 non-O139 strains were resistant. The fs-2 genome comprised 8651 nucleotides containing nine open reading frames, five of which had predicted protein products partially homologous to the reported protein products of other filamentous phages. Although the extent of the homology was not particularly high, the genetic organization of other filamentous phages appears to be preserved in fs-2. The phage was not integrated into the chromosome of its host, but a 715 nucleotide fragment located in the large intergenic region of fs-2 was highly homologous to a part of region RS2 (repetitive sequence 2) of the V. cholerae CTX sequence which is speculated to be required for integration of the phage into the V. cholerae chromosome at a specific site.
Transcription of the trp operon in Lactococcus lactis is controlled by antitermination in the leader region
Authors: Frenkiel HéléeGne , Bardowski Jacek , Ehrlich S. Dusko , Chopin Alain
Published: June 2009

The regulatory functions of the leader region preceding the Lactococcus lactis trp operon have been studied by mutagenesis analysis. This leader presents striking similarity to ‘T-box’ leaders found upstream of many Gram-positive aminoacyl-tRNA synthetase genes and some amino acid biosynthesis operons, which are controlled by antitermination through interaction of the leader transcript with cognate uncharged tRNA. A region of the L. lactis leader transcript also contains a series of (G/U)AG repeats which, in Bacillus, are involved in the binding of the trp RNA-binding protein (TRAP) which controls trp transcription. A screen was developed for the isolation of regulatory mutants affected in the leader region. All spontaneous mutants contained deletions; point mutations were only obtained after UV-induced mutagenesis. All mutations affected the putative transcription terminator upstream of the trp operon, demonstrating that trp is indeed controlled by transcription antitermination.
Altered expression and modification of proteases from an avirulent mutant of Porphyromonas gingivalis W50 (W50/BE1)
Authors: Collinson Lucy M. , Rangarajan Minnie , Curtis Michael A.
Published: June 2009

Proteases ofPorphyromonas gingivalisare considered to be important factors in the virulence of this organism. A non-pigmenting mutant ofP. gingivalisW50 (W50/BE1) has been shown to be less virulent in animal models and to produce significantly less Arg-specific protease activity than the parent strain. Three proteases are present in the culture supernatant ofP. gingivalisW50: RI, RIA and RIB. All three proteases are derived fromprpR1,which encodes a polypeptide of 1706 amino acids that is organized into distinct domains (pro, α, β and γ). The aim of the present investigation was to purify and characterize the Arg-specific proteases produced by the avirulent W50/BE1 strain. Significant differences were observed between the proteases ofP. gingivalisW50 and W50/BE1. The levels of RI present in the culture supernatant of W50/BE1 were lower than those present in W50, and RIA and RIB were absent. RI from W50/BE1 was composed of three polypeptide chains, unlike the enzyme from W50, which is a heterodimer. The remainder of the Arg-specific protease activity in W50/BE1 was derived from a second gene,prR2,and was present in two fractions, RIIAs/BE (soluble) and RIIAv/BE (vesicle-bound). This activity contained two peptide chains: a ∼ 54 kDa chain corresponding to the protease domain and a ∼ 26 kDa chain, derived from the propeptide domain of the PrRII precursor. No enzyme with large glycan additions, equivalent to RIB in the vesicle fraction of the wild-type W50, was present. These data indicate that the reduced level of extracellular protease activity in W50/BE1 reflects reduced synthesis and/or export ofprpR1enzymes, which is only partially compensated by synthesis ofprR2-derived enzymes, and that all of these proteases undergo altered post-translational modification compared to the parent strain.
Evidence for a developmentally regulated prespore-specific glutamine synthetase in the cellular slime mould Dictyostelium discoideum
Authors: Dunbar Andrew J. , Wheldrake John F.
Published: June 2009

The enzyme glutamine synthetase (GS) is described for the first time in Dictyostelium discoideum. The appearance of this enzyme is developmentally regulated. The level of activity is low in vegetative cells and increases more than threefold during differentiation. Furthermore this enzyme is shown to be differentially localized in prespore cells, the specific activity being approximately fourfold higher than in prestalk cells. The enzyme has a pH optimum of 7.8 and 8.2 in the γ-glutamyltransferase and γ-glutamylsynthetase assays, respectively, and a temperature optimum of 45°C. Kinetic studies of GS revealed apparent Km values of 5.9 mM, 0.009 mM and 8.6 mM for glutamine, ADP and NH2OH, respectively, in the γ-glutamyltransferase assay, and of 2.2 mM, 0.12 mM and 0.64 mM for glutamate, ATP and NH2OH, respectively, in the γ-glutamylsynthetase assay.
Derepression of the glyoxylate cycle in mutants of Neurospora crassa accelerated for growth on acetate
Authors: Chaure P. T. , Connerton I. F.
Published: June 2009

Two spontaneous allelic mutations have been isolated with the unusual semi-dominant phenotype of faster-than-wild-type growth on acetate as sole carbon source. The mutants were designated Aag-1 (accelerated acetate growth) and mapped on linkage group II. Upon re-isolation of both the Aag-1 alleles from repeated back-crosses to wild-type, between 1 and 6% of the progeny were found to be acu (acetate non-utilizing) mutants. Ten of these were selected for heterokaryon complementation analysis with known acu mutants; nine proved to be new alleles of acu-5 (deficient in acetyl-CoA synthetase), and one was a new acetate non-utilizing class, designated acu-14. Although the Aag-1 mutants clearly have no acetate-growth-related enzyme deficiencies, they did exhibit significant constitutive enzyme levels for acetyl-CoA synthetase and the glyoxylate cycle enzymes (isocitrate lyase and malate synthase) on the non-inducing carbon source, sucrose. The derepression was restricted to these enzymes, as representative enzymes from other carbon-assimilatory pathways remained repressed and subject to carbon catabolite repression. Northern blot analysis of the mRNA levels of acetyl-CoA synthetase and the glyoxylate cycle enzymes from the mutants demonstrated the derepression to occur at the level of transcription. These data suggest that the physiological explanation for the accelerated acetate growth phenotype lies in the standing levels of the acetate-assimilatory enzymes, which enable the mutants to forgo some of the normal time required for adaption to growth on acetate.
The gene for Campylobacter trigger factor: evidence for multiple transcription start sites and protein products
Authors: Griffiths Phillippa L. , Park Robert W. A. , Connerton Ian F.
Published: June 2009

A gene encoding a protein of apparent molecular mass 56 kDa that shares 31 % identity with the amino acid sequence of trigger factor from Escherichia coli (a protein thought to be involved in cell division), was cloned from Campylobacter jejuni NCTC 11168. The clone was selected from a lambda ZAP II genomic DNA library following an immuno-screen using antiserum raised against glycine-extractable proteins from C. jejuni. The gene has two potential initiation codons, giving rise to two possible nested protein products. Complex differential growth-phase-dependent transcripts give rise to these products.
A catalase from Streptomyces coelicolor A3(2)
Authors: Walker G. E. , Dunbar Bryan , Hunter Iain S. , Nimmo Hugh G. , Coggins John R.
Published: June 2009

Catalase was purified from the Gram-positive bacterium Streptomyces coelicolor A3(2) in a three-step purification procedure comprising (NH4)2SO4, fractionation, Phenyl-Sepharose chromatography and Mono Q chromatography. The purification of catalase, as judged by the final specific activity of 110000 U mg-1 was 250-fold with a 35% yield. The native protein was a homotetramer with a subunit M
r 55000. N-terminal and internal peptide sequence analyses showed that there was a high degree of sequence similarity between the S. coelicolor catalase and other microbial and mammalian catalases. Southern blot analysis indicated that there was a single catalase gene in S. coelicolor. The specific activity of catalase throughout the growth of batch cultures was investigated and elevated catalase activity was found in stationary-phase cells.
Discovery of a ptsHl operon, which includes a third gene (ptsT), in the thermophile Bacillus stearothermophilus
Authors: Lai Xiaokuang , Ingram Lonnie O.
Published: June 2009

The discovery of a ptsHl operon in Bacillus stearothermophilus XL-65-6 coupled with our previous report of a cel operon (Lai Ingram, J Bacteriol 175, 6441-6450, 1993) demonstrates that this thermophilic organism contains all of the genes required for cellobiose uptake by the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Genes encoding the two general PTS proteins, HPr (ptsH) and enzyme l (ptsl), were cloned and sequenced. These form an operon which includes a third small gene (ptsT) of unknown function (encoded product M
r 18428). Both ptsH and ptsl were expressed at high levels from a single plasmid in Escherichia coli and complemented corresponding host mutations. Although the translated sequences for these genes were similar to homologues from Gram-positive mesophiles (64-77% identity), the B. stearothermophilus gene products were unusual in having a higher predicted pi and fewer negatively charged amino acid residues. Enzyme I also contained more alanine and leucine than mesophilic counterparts. Interestingly, ptsT inhibited the growth of E. coli ptsl mutants at 37°C. No such inhibition was observed during incubation at a lower temperature (30°C) or in E. coli DH5α, which is wild-type for ptsl. The predicted translation product from ptsT contained a high proportion of basic amino acids (27%) and had a high predicted pl (pH 117), properties similar to bacterial histone-like proteins, but did not exhibit homology to any sequences in the current database. Regions upstream and downstream from the ptsHl operon contain genes with homology to Bacillus subtilis ptsG and wapA (wall-associated protein), respectively.
Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase
Authors: Schauder Stephan , Schneider Karl-Heinz , Giffhorn Friedrich
Published: June 2009

A sorbitol dehydrogenase (SDH; L-iditol: NAD+ 2-oxidoreductase; EC1.1.1.14) was isolated from the phototrophic bacterium Rhodobacter sphaeroides strain M22, a transposon mutant of R. sphaeroides Si4 with the transposon inserted in the mannitol dehydrogenase (MDH) gene. SDH was purified 470-fold to apparent homogeneity by ammonium sulfate precipitation, chromatography on Phenyl-Sepharose, Q-Sepharose and Matrex Gel Red-A, and by gel filtration on Superdex 200. The relative molecular mass (M
r) of the native SDH was 61 000 as calculated from its Stokes’ radius (rs = 3.5 nm) and sedimentation coefficient (S
20,w = 4.235). SDS-PAGE resulted in one single band representing a polypeptide with a M
r of 29000, indicating that the native protein is a dimer. The isoelectric point of SDH was determined to be pH 4-8. The enzyme was specific for NAD+ and catalysed the oxidation of D-glucitol (sorbitol) to D-fructose, galactitol to D-tagatose and of L-iditol. The apparent K
m values were NAD+, 0-06 mM; D-glucitol, 6-2 mM; galactitol, 1-5 mM; NADH, 0-13 mM; D-fructose, 160 mM; and D-tagatose, 13 mM. The pH-optimum of substrate oxidation was 11-0 and that of substrate reduction 6-0-7-2. It was demonstrated that SDH is expressed in the wild-type strain R. sphaeroides Si4 together with MDH during growth on D-glucitol. Forty-four amino acids of the SDH N terminus were sequenced. This sequence exhibited 45-55% identity to the N-terminal sequence of 10 enzymes belonging to the short-chain alcohol dehydrogenase family.
Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli
Authors: George Anthony M. , Hall Ruth M. , Stokes H. W.
Published: June 2009

Spontaneous multidrug-resistant (Mdr) mutants of Klebsiella pneumoniae strain ECL8 arose at a frequency of 2-2 � 10-8 and showed increased resistance to a range of unrelated antibiotics, including chloramphenicol, tetracycline, nalidixic acid, ampicillin, norfloxacin, trimethoprim and puromycin. A chromosomal fragment from one such mutant was cloned, and found to confer an Mdr phenotype on Escherichia coli K12 cells that was essentially identical to that of the K. pneumoniae mutant. Almost complete loss of the OmpF porin in the E. coli transformant, and of the corresponding porin in the K. pneumoniae mutant, was observed. The presence of the Mdr mutation in K. pneumoniae or the cloned K. pneumoniae ramA (resistance antibiotic multiple) locus in E. coli also resulted in active efflux of tetracycline, and increased active efflux of chloramphenicol. After transformation of a ramA plasmid into E. coli, expression of chloramphenicol resistance occurred later than expression of resistance to tetracycline, puromycin, trimethoprim and nalidixic acid. The ramA gene was localized and sequenced. It encodes a putative positive transcriptional activator that is weakly related to the E. coli MarA and SoxS proteins. A ramA gene was also found to be present in an Enterobacter cloacae fragment that has previously been shown to confer an Mdr phenotype, and it appears that ramA, rather than the romA gene identified in that study, is responsible for multidrug resistance. The ramA gene from the wild-type K. pneumoniae was identical to that of the mutant strain and also conferred an Mdr phenotype on E. coli, indicating that the mutation responsible for Mdr in K. pneumoniae had not been cloned.
Detection of novel marine methanotrophs using phylogenetic and functional gene probes after methane enrichment
Authors: Holmes Andrew J. , Owens Nick J. P. , Murrell J. Colin
Published: June 2009

A major limitation of rRNA-targeted group-specific probes is that they may cross-react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences. We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology. Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients. The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries. Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected. A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas. Organisms containing this sequence were observed in enrichments by in situ hybridization. The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes.
A Rhizobium strain that nodulates and fixes nitrogen in association with alfalfa and soybean plants
Authors: Gao W. M. , Yang S. S.
Published: June 2009

A Rhizobium meliloti strain that forms effective nodules on soybean (Glycine max) and alfalfa (Medicago sativa) is described. The strain, 042B, was isolated from root nodules of alfalfa in Xinjiang Autonomous Region of China. Experiments showed that strain 042B was able to nodulate soybean as effectively as Bradyrhizobium japonicum USDA110, a widely used inoculant strain. Under hydroponic conditions, both strains performed similarly in many respects such as time required for the appearance of nodules, total nitrogenase activity, plant top dry weight and total plant nitrogen. In soil pot experiments, total nitrogen, seed weight and seed nitrogen of soybean plants inoculated with strains 042B or USDA110 were much higher than those of the uninoculated control, but there were no differences between plants with strain 042B relative to strain USDA110. However, when strain 042B was the inoculant, the number of nodules was greater, but the nodules were smaller than those of strain USDA110. Results from ELISA showed that nodule occupancy of strain 042B in soybean ranged from 82-90% and that of strain USDA110 ranged from 78-86%. Strain 042B can grow in the presence of 5% (w/v) NaCl, at 42 °C, and at pH 10·7. Strain 042B was shown to have two large plasmids (molecular sizes 200 kb and > 1000 kb, respectively). The DNA G + C mol% of strain 042B was 63·6. The DNA level of homology between strain 042B and type-strain R. meliloti USDA1002 was 76%, while DNA homologies of strain 042B were 10% and 12% with R. fredii USDA205 and B. japonicum USDA6, respectively.
Tritrichomonas foetus and Trichomonas vaginalis: the pattern of inactivation of hydrogenase activity by oxygen and activities of catalase and ascorbate peroxidase
Authors: Page-Sharp Madhu , Behm Carolyn A. , Smith Geoffrey D.
Published: June 2009

The concentration-dependence of the inhibition of whole-cell hydrogen formation by oxygen has been measured in the trichomonads Trichomonas vaginalis and Tritrichomonas foetus, and compared with the oxygen inhibition of the in situ hydrogenase activity as measured by a tritium exchange assay. The inhibition profiles closely paralleled each other, suggesting that hydrogenase is the primary site of inhibition of anaerobic fermentative metabolism. In addition the inhibition profile for isolated hydrogenosomes was measured and shown to be similar to that for whole organisms. Ascorbate peroxidase was shown to be present in both organisms whereas catalase was confirmed to be present only in Tritr. foetus. The kinetic parameters of both enzymes were measured and their respective roles in oxygen protection discussed.
Domain structure and function within the QUTA protein of Aspergillus nidulans: implications for the control of transcription
Authors: Levesley Ian , Newton Giles H. , Lamb Heather K. , van Schothorst Evert , Dalgleish Raymond W. M. , Samson Anthony C. R. , Roberts Clive F. , Hawkins Alastair R.
Published: June 2009

QUTA is a positively acting regulatory protein that regulates the expression of the eight genes comprising the quinic acid utilization gene (qut) gene cluster in Aspergillus nidulans. It has been proposed that the QUTA protein is composed of two domains that are related to the N-terminal two domains-dehydroquinate (DHQ) synthase and 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase - of the pentadomain AROM protein. The AROM protein is an enzyme catalysing five consecutive steps in the shikimate pathway, two of which are common to the qut pathway. A genetic and molecular analysis of non-inducible qutA mutants showed that all 23 mutations analysed map within the N-terminal half of the encoded QUTA protein. One dominant mutation (qutA382) introduces a stop codon at the boundary between the two domains that were identified on the basis of amino acid sequence alignments between the QUTA protein and the N-terminal two domains of the pentafunctional AROM protein. The truncated protein encoded by mutant qutA382 has DNA-binding ability but no transcription activation function. A second dominant mutation (in strain qutA214) is missense, changing 457E K in a region of localized high negative charge and potentially identifies a transcription activation domain in the N-terminus of the EPSP-synthase-like domain of the QUTA protein. A series of qualitative and quantitative Northern blot experiments with mRNA derived from wild-type and mutant qutA strains supported the view that the QUTA protein regulates the expression of the qut gene cluster, including the qutA gene which encodes it. A series of Western blot and zinc-binding experiments demonstrated that a putative zinc binuclear cluster motif located within the N-terminus of the QUTA protein is able to bind zinc in vitro.
Novel O-polysaccharide expression, as a lipid A-core-free form, in a lipopolysaccharide-core-defective mutant of Pseudomonas aeruginosa
Authors: Yokota Shin-ichi
Published: June 2009

Summary: Pseudomonas aeruginosa PML14e is a mutant strain, isolated from strain PML14 (Homma serotype I), that is resistant to all types of R-pyocins. PML14e completely lacked glucose and rhamnose as components of the lipopolysaccharide (LPS) outer core region. Whereas the O-polysaccharide attachment site on the LPS core was considered to be absent, PML14e was agglutinable with anti-serotype-I antibodies. The O-polysaccharide of PML14e was recovered in the supernatant after ultracentrifugation of the aqueous layer from a hot phenol/water extraction. Chromatographic behaviour and chemical analysis indicated that the PML14e O-polysaccharide was not linked to the lipid A. 1H-NMR spectroscopy indicated that the structure of the PML14e O-polysaccharide was the same as that of the O-polysaccharide from PML14. The above evidence indicated that the O-polysaccharide is expressed on the cell surface of the mutant strain PML14e as the lipid A-free form. To examine the nature of the cell surface, the accessibility of monoclonal antibodies (mAbs) against cell surface antigens was tested by enzyme-linked immunosorbent assay. An anti-lipid A mAb and an anti-outer-membrane protein mAb, the epitopes for which are considered to be exposed on rough strains, bound to a greater extent to the PML14e cells than to two other LPS-core-defective rough mutants, PML14b and PML14d. Whereas these mutants appeared to have lesser defects in the LPS core, they expressed less O-polysaccharide than PML14e. The results indicated that the epitopes exposed on rough strains, such as lipid A and outer-membrane proteins, were mainly hindered by covalently linked core oligosaccharide rather than by the O-polysaccharide chain.
The mercury resistance operon of the IncJ plasmid pMERPH exhibits structural and regulatory divergence from other Gramnegative mer operons
Authors: Osborn A. M. , Bruce K. D. , Ritchie D. A. , Strike P.
Published: June 2009

Summary: The bacterial mercury resistance determinant carried on the IncJ plasmid pMERPH has been characterized further by DNA sequence analysis. From the sequence of a 4097 bp Bg/II fragment which confers mercury resistance, it is predicted that the determinant consists of the genes merT, merP, merC and merA. The level of DNA sequence similarity between these genes and those of the mer determinant of Tn21 was between 56.4 and 62.4%. A neighbourjoining phylogenetic tree of merA gene sequences was constructed which suggested that pMERPH bears the most divergent Gram-negative mer determinant characterized to date. Although the determinant from pMERPH has been shown to be inducible, no regulatory genes have been found within the Bg/II fragment and it is suggested that a regulatory gene may be located elsewhere on the plasmid. The cloned determinant has been shown to express mercury resistance constitutively. Analysis of the pMERPH mer operator/promoter (O/P) region in vivo has shown constitutive expression from the mer PTCPA promoter, which could be partially repressed by the presence of a trans-acting MerR protein from a Tn21-like mer determinant. This incomplete repression of mer PTCPA promoter activity may be due to the presence of an extra base between the −35 and −10 sequences of the promoter and/or to variation in the MerR binding sites in the O/P region. Expression from the partially repressed mer PTCPA promoter could be restored by the addition of inducing levels of Hg2+ ions. Using the polymerase chain reaction with primers designed to amplify regions in the merP and merA genes, 1.37 kb pMERPH-like sequences have been amplified from the IncJ plasmid R391, the environmental isolate SE2 and from DNA isolated directly from non-cultivated bacteria in River Mersey sediment. This suggests that pMERPH-like sequences, although rare, are nevertheless persistent in natural environments.
Polyamines as Modulators of Microcycle Conidiation in Aspergillus Flavus
Authors: Khurana N. , Saxena R. K. , Gupta Rani , Rajam M. V.
Published: June 2009

Since polyamines (PAs) play a potential role in the regulation of growth and developmental processes in a wide variety of organisms, we have examined the influence of the PAs putrescine (Put) and spermidine (Spd) and the PA biosynthetic inhibitors α-difluoromethylornithine (DFMO), α-difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and cyclohexylamine (CHA), singly and in combinations on microcycle conidiation (MC) in Aspergillus flavus. The exogenous application of the diamine Put (concentrations ranging from 0·1 to 5 mM) caused a sharp decline of MC in a dose-dependent fashion, but induced vegetative growth. However, the triamine Spd (0·1-5 mM) had a minimal effect on MC and induced a shift from MC to normal conidiation. PA inhibitors, especially DFMO, MGBG and CHA, produced greater inhibition of MC and complete inhibition of MC was observed at 5 mM of these inhibitors. DFMA even at 5 mM had only a weak inhibitory effect on MC. DFMO also inhibited conidial germination and germ tube growth. MGBG and CHA, while having an inhibitory effect on MC, induced vegetative growth. The inhibitory effect of PA inhibitors was partially reversed by exogenous Put or Spd, with Spd being more effective than Put. The analysis of free PA levels during various phases of MC revealed that undifferentiated spores contained a high Put/Spd ratio and there was a dramatic decrease in Put/Spd ratio before and during microcycle conidiophore maturity. The change in spermine titres could not be detected. These observations imply that Put is essential for vegetative growth, while Spd is involved in MC, and that a low Put/Spd ratio seems to be important for spore differentiation to MC.
The Application of Serological Techniques to the Taxonomy of Arthrobacter and Related Organisms
Authors: Gray T. R. G. , Mansoor E. Y.
Published: June 2009

Antisera were raised against rods of 17 named Arthrobacter and Aureobacterium strains. Antigenic relationships between these strains, other soil bacteria and new Arthrobacter isolates from several soils were studied, using agglutination, immunodiffusion, immunofluorescence and ELISA techniques. Many of the named Arthrobacter species had common antigens, and there were also common antigens amongst named Arthrobacter strains and many fresh Arthrobacter isolates. Agglutination, ELISA and immunofluorescence tests revealed greater antigenic differences between the named strains than did immunodiffusion tests. Serological similarities between the 17 named strains and the fresh Arthrobacter isolates were calculated using S

J
coefficients. The occurrence of named strains in serogroups based on immunodiffusion data supported the taxonomic scheme for arthrobacters in Bergey’s Manual of Systematic Bacteriology. The distribution of soil isolates in serogroups resembled that in groups based on numerical analysis of diverse characters. This makes it possible to use serological tests to locate particular species in soil samples. Arthrobacter atrocyaneus was serologically distinct from other members of the Ar. globiformis/Ar. citreus group and did not cluster with them phenetically. Serological data suggest that Ar. aurescens, Ar. ureafaciens and Ar. histidinolovorans constitute a single species. Although A. simplex and A. tumescens have been placed in the genus Pimelobacter they are serologically distinct from one another and more closely resemble Ar. globiformis. Aureobacterium strains were serologically distinct from arthrobacters. Micrococcus roseus showed many cross-reactions with several antisera, supporting the placement of micrococci in the same family as arthrobacters.
The rRNA operons of Mycobacterium smegmatis and Mycobacterium tuberculosis : comparison of promoter elements and of neighbouring upstream genes
Authors: Gonzalez-y-Merchand Jorge A. , Colston M. Joseph , Cox Robert A.
Published: June 2009

Mycobacterium smegmatis has two rRNA (rrn) operons designated rrnA
f and rrnB
f. Appropriate restriction fragments of genomic DNA containing sequences immediately upstream from the 16S rRNA genes were cloned. We now report the nucleotide sequence of 552 bp upstream from the 5′-end of the Box AL antitermination element of the leader region of the rrnA
f operon. The 5′-end of this segment of DNA was found to comprise 113 codons of an ORF encoding a protein which is significantly similar to UDP-N-acetylglucosamine 1-carboxyvinyl-transferase (EC 2.5.1.7), which is important to cell wall synthesis. A homologous ORF is located immediately upstream from the single rrn (rrnA
s) operons of Mycobacterium tuberculosis and Mycobacterium leprae. Primer-extension analysis of the RNA fraction of M. smegmatis revealed four products which were related to transcription start points; the rrnB
f operon appears to have a single promoter whereas the rrnA
f operon has three (P1, P2 and P3). Analysis of M. tuberculosis RNA revealed two products corresponding to transcripts directed by promoters homologous with P1 and P3 of the rrnA
f of M. smegmatis. Thus, the promoter and upstream regions of the rrnA
f operon of M. smegmatis and the rrnA
s operon of M. tuberculosis are homologous. The presence of P2 in M. smegmatis and its absence from M. tuberculosis is attributable to insertions/deletions of 97 bp.
Dynamic expression of cell-surface antigens probed with Candida albicans-specific monoclonal antibodies
Authors: Deslauriers N. , Michaud J. , Carré B. , Léveillée C.
Published: June 2009

IgG hybridomas were produced which preferentially reacted with cell-surface antigens of either yeast cells or hyphae ofCandida albicans. Four mAbs were used in an immunostaining procedure to follow the expression dynamics of these antigens in media supplemented with glucose or galactose. Yeast cell growth was analysed during the lag phase, the early- and late-exponential phases and the stationary phase, and mycelium formation was analysed between 0.5 and 24 h induction at 370C. It appears that yeast cell-surface antigens 5C11 and 2E11 are expressed throughout all phases of yeast cell growth as well as on young hyphae after up to 1 h induction. Longer hyphae only faintly react with these two mAbs as they switch to hyphal cell-surface antigens 2G8 and 4E1 after 3 h induction. The reactivity to mAbs 2G8 and 4E1 was induced after a 3 h temperature shift and was confined to the terminal third of growing mycelia. Growth and hyphae induction in galactose prolonged the reactivity of young hyphae with the two anti-yeast-cell mAbs, whereas the expression of surface antigens 2G8 and 2E11 appeared delayed and desynchronized on hyphae. Whereas a similar reactivity was found with ten ATCC strains ofC. albicans, four clinical isolates had a unique pattern of reactivity. Immunoblot analyses of DTT extracts of cell-surface constituents indicated that the antigens were proteinaceous in nature and showed that yeast-cell antigens 5C11 and 2E11 are detected in four bands between 68 and 104 kDa, whereas mycelial antigens 4E1 and 2G8 are detected in 117 kDa and 104 kDa bands found in mycelial but not in yeast-cell extracts. Present data support the concept of a dynamic balance in the expression of phase-specific antigens inC. albicans.
The role of the outer membrane in formaldehyde tolerance in Escherichia coli VU3695 and Halomonas sp. MAC
Authors: Azachi Malkit , Henis Yigal , Shapira Roni , Oren Aharon
Published: June 2009

To investigate the mechanism of formaldehyde tolerance in Gram-negative bacteria, two formaldehyde-tolerant strains, Escherichia coli VU3695 and Halomonas sp. MAC (DSM 7328), and formaldehyde-sensitive revertants obtained by ethidium bromide or novobiocin treatment were studied. The presence of high levels of formaldehyde dehydrogenase activity alone proved insufficient to confer tolerance to high formaldehyde concentrations, as shown by the high activity displayed by formaldehyde-sensitive revertants of Halomonas MAC. Moreover, formaldehyde-tolerant strains also proved to be tolerant to high concentrations of acetaldehyde and glutaraldehyde, which are not oxidized by formaldehyde dehydrogenase. Treatment with sublethal concentrations of EDTA rendered the resistant strains highly sensitive to formaldehyde without affecting the activity of formaldehyde dehydrogenase. Comparison of the outer membrane proteins of formaldehyde-resistant strains with those of their sensitive revertants showed the presence of at least one additional high molecular mass protein in the tolerant strains. It is concluded that formaldehyde tolerance in the bacteria studied depends on the composition and structure of the outer membrane.