bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli
Authors: Louis Petra , Galinski Erwin A.
Published: June 2009

The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli. These genes were not only expressed, but also osmoregulated in E. coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4∙4 kb fragment revealed four major ORFs, which were designated ectA, ectB, ectC and orfA. The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into E. coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that ectA codes for l -2,4-diaminobutyric acid acetyltransferase, ectB for l -2,4-diaminobutyric acid transaminase and ectC for l -ectoine synthase. A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.
The Bacillus subtilis clpC operon encodes DNA repair and competence proteins
Authors: Krüger EIke , Msadek Tarek , Ohlmeier Steffen , Hecker Michael
Published: June 2009

ClpC of Bacillus subtilis, controlling competence gene expression and survival under stress conditions, is encoded by the fourth gene of a six-gene operon. The product of orf1 contains a potential helix-turn-helix motif, but shows no significant similarities with known protein sequences. The second and third genes encode proteins with similarities to zinc-finger proteins (orf2) and arginine kinases (orf3), respectively. The product of orf5 contains a zinc-finger motif and an ATP-binding domain, and is highly similar to the product of the Escherichia coli sms gene. A strain bearing a disruption of orf5 showed increased sensitivity to the alkylating agent methyl methanesulfonate. Furthermore, this mutant strain displayed decreased capacity for genetic recombination as measured by transformation experiments. The last open reading frame, orf6, encodes a protein with limited similarity in its C-terminal part to the B. subtilis comEA gene product and to the UvrC DNA repair excinuclease. Inactivation of orf5 resulted in strongly diminished transformation with all types of DNA. Mutations affecting either orf5 or orf6 resulted in strains with decreased resistance to UV-irradiation in the stationary phase, indicating that these proteins play a role in the development of a nonspecific stationary-phase resistance to UV-irradiation. Moreover, these results suggest an involvement of both proteins in transformation and presumably in DNA repair.
The soybean cultivar specificity gene noIX is present, expressed in a nodD-dependent manner, and of symbiotic significance in cultivar-nonspecific strains of Rhizobium (Sinorhizobium) fredii
Authors: Bellato C. , Krishnan H. B. , Cubo T. , Temprano F. , Pueppke S. G.
Published: June 2009

Rhizobium (now Sinorhizobium) fredii is a symbiotic nitrogen-fixing bacterium that can nodulate soybean in a cultivar-specific manner. This process is governed by a set of negatively acting nodulation genes termed noIXWBTUV. These genes prevent R. fredii strain USDA257 from infecting soybean cultivars such as McCall, but they do not block nodulation of cultivar Peking. R. fredii strain USDA191 contains DNA sequences that hybridize to noIXWBTUV, yet it forms normal nitrogen-fixing nodules on both McCall and Peking soybean. These sequences were isolated and their structure and function examined in comparison to noIXWBTUV of strain USDA257. Restriction maps of the two loci are identical, as is a 2∙4 kb DNA sequence that corresponds to noIX and its promoter region. Expression of noIX by strain USDA191 is flavonoid-dependent in culture and readily detectable in nodules. The gene is not inducible in a mutant of strain USDA191 that lacks the regulatory nodD1 gene, and its expression is greatly attenuated in a nodD2 mutant. noIX is also present and flavonoid-inducible in HH103, a second R. fredii strain that nodulates McCall soybean normally. Inactivation of noIX in strain HH103, USDA191 or USDA257 leads to retardation of initial nodulation rates on soybean cultivars such as Peking and to acquisition of the capacity to form nitrogen-fixing nodules on two species of Erythrina. noIX is thus of symbiotic significance in all three strains, even though it regulates soybean cultivar specificity only in strain USDA257.
Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family
Authors: Rodríguez-Peña José M. , Alvarez Isabel , Ibáñez Magdalena , Rotger Rafael
Published: June 2009

The nucleotide sequence relatedness between the chromosome of Salmonella typhi and the virulence plasmid of Salmonella enteritidis was investigated using short DNA probes of < 2 kb covering the whole virulence plasmid sequence. Only one homologous region was detected. This region was subsequently cloned and partially sequenced. Sequences closely related to the pefl gene and the ORFs orf7, orf8 and orf9, which are located downstream of the fimbrial pef operon of the Salmonella typhimurium virulence plasmid, were detected. Sequencing of the cloned S. typhi DNA fragment also revealed identity with genes of the fimbrial sef operon characterized in the chromosome of S. enteritidis. These nucleotide sequences mapped upstream of the S. typhi chromosomal region homologous to the S. enteritidis virulence plasmid. The general organization of the cloned S. typhi chromosomal fragment was similar to the fimbriae-encoding region of the S. typhimurium virulence plasmid. The deduced product of orf8 in the S. typhimurium virulence plasmid, as well as those of the corresponding ORFs in the homologous region of the S. typhi chromosome and in the S. enteritidis virulence plasmid (designated dlt and dlp, respectively), appeared to be related to the thioredoxin family of thiol:disulphide oxidoreductases. The dlp gene was able to complement the DTT-sensitive phenotype, the inability to metabolize glucose 1-phosphate and the low alkaline phosphatase activity of a dsbA mutant of Escherichia coli. The dlt gene partially complemented the lack of alkaline phosphatase activity, but not the other mutant phenotypes. The products of both genes could be detected using the T7 RNA polymerase promoter expression system. The estimated molecular masses of the products of the dlt and dlp genes by SDS-PAGE were 26 and 23 kDa, respectively, the first being in agreement with the deduced amino acid sequence and the latter, somewhat smaller. The processing of a possible leader peptide in the Dlp protein, but not in the Dlt protein, could be responsible for this difference. The Dlp protein appeared as a doublet band on SDS-PAGE, which is characteristic of the oxidized and reduced states of this kind of protein.

The EMBL accession numbers for the sequences of dlt and dlp reported in this paper are X94325 and X94326, respectively.
The outer membrane of lipid A-deficient Escherichia coli mutant LH530 has reduced levels of OmpF and leaks periplasmic enzymes
Authors: Nurminen Marjatta , Hirvas Laura , Vaara Martti
Published: June 2009

We have previously described a new Escherichia coli K-12 mutant, LH530, which has a defective outer membrane. LH530 is very sensitive to hydrophobic antibiotics, does not grow at 42 ° and synthesizes reduced amounts of lipid A. Phenotypically LH530 is very similar to the known lipid A biosynthesis mutants of E. coli and Salmonella typhimurium. Its genetic defect is not known, but the defect is suppressed by multiple copies of ORF195. Here we show that at 37 ° LH530 contains a reduced amount of the OmpF porin and that it leaks periplasmic °-lactamase at 37 °. and 42 °. We further show that ORF195, when present at low copy number, restores the antibiotic resistance and lipid A biosynthesis of LH530 at 28 °, but not at higher temperatures. In contrast, OmpF expression is restored at 37 °.
The flagellin N-methylase gene fliB and an adjacent serovar-specific IS200 element in Salmonella typhimurium
Authors: Burnens André P. , Stanley John , Sack Ragna , Hunziker Peter , Brodard Isabelle , Nicolet Jacques
Published: June 2009

The cloning and molecular genetic analysis of a locus mapping within the flagellar gene (fli) complex ofSalmonella typhimuriumis reported. A copy of the insertion element IS200was located in a noncoding stretch of DNA upstream of thefliAgene. Comparative nucleotide sequence analysis showed that this copy of IS200was 711 bp long and that its flanking regions contained no features common to other characterized insertion sites of this element. The element was located 37 bp downstream of an ORF whose product was shown by interspecific transfer and amino acid analysis to carry outN-methylation of selected lysine residues inSalmonella flagellin.The sequence and phenotype of this ORF identified it asfliB,encoding the only prokaryoticN-methylase acting on amino groups to have been characterized to date. It was found to be conserved among all clinically significant serovars ofSalmonella.The IS200insertion site is of particular interest since it was conserved in all but two rare evolutionary lines ofS. typhimurium,and was absent from 85Salmonellastrains belonging to 37 other serovars. It is thus a phylogenetically significant marker at the serovar level.
Use of a glycerol-limited, long-term chemostat for isolation of Escherichia coli mutants with improved physiological properties
Authors: Weikert Christian , Sauer Uwe , Bailey James E.
Published: June 2009

The evolution of Escherichia coli MG1655 mutants was followed over 126 d in a glycerol-limited chemostat at a dilution rate of 0.05 h-1. This corresponds to a total of 217 generations at a doubling time of 13.9 h. After this time, nearly 90% of the chemostat population consisted of evolved mutant strains as determined by their altered colony morphologies on plates. Two mutants were isolated that exhibited generally improved growth phenotypes in batch cultivations on glycerol, glucose or the gluconeogenic substrate acetate. Higher specific growth rates and increased biomass yields were found for both mutants. For one mutant, this behaviour was combined with significantly reduced secretion of overflow metabolites when either glycerol or glucose was the carbon source. Additionally, during all growth phases of a batch cultivation, this mutant exhibited increased resistance to a variety of adverse conditions including heat shock, osmotic stress and nutrient deprivation. It also displayed significantly shorter lag phases.
Paracoccidioides brasiliensis by ajoene is associated with blockade of phosphatidylcholine biosynthesis
Authors: San-Bias Gioconda , Urbina Julio A. , Marchán Edgar , Contreras Lellys M. , Sorais Françoise , San-Blas Felipe
Published: June 2009

In Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans, no significant differences were observed in the phospholipid species of both morphological phases. The species observed were phosphatidylcholine (PC, 30–40%), phosphatidylethanolamine (PE, 27-28%), phosphatidylserine (16–19%), phosphatidylinositol (13–17%) and sphingomyelin (3–5–0025;). The main fatty acids found in the yeast (Y) phase were palmitate (56%), linoleate (18%) and oleate (15%), while linoleate predominated (61 %) in the mycelial (M) phase, followed by palmitate (27%) and oleate (7%). In the Y phase the main free sterol was ergosta-5,22-dien-3β-ol (82%) plus some lanosterol (12%) and ergosterol (6%), while in the M phase, the latter predominated (88%), followed by low levels of ergosta-5,22-dien-3β-ol (12%). Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a platelet aggregation inhibitor derived from garlic, induced alterations in phospholipid and fatty acid proportions such that PC was reduced to about 18% in both phases and PE increased to 38% (Y phase) or 44% (M phase), suggesting inhibition of PC synthesis. Ajoene also reduced saturated fatty acids (16:0 and 18:0) from 67 to 35% in the Y phase, with a corresponding increase in the unsaturated components. This effect was not seen in the M phase.
Stimulation of sexual reproduction in Phytophthora cactorum by phospholipids is not due to sterol contamination
Authors: Jee Hyeong-Jin , Tang Chung-Shih , Ko Wen-Hsiung
Published: June 2009

Phytophthora cactorum did not form oospores on basal medium unless phosphatidylcholine (lecithin) or phosphatidylethanolamine (cephalin) was added. After removal of putative sterols by aminopropyl column chromatography, the activities of lecithin and cephalin were increased 47- and 2.8-fold, respectively, thus confirming the previous reports that sterols are not essential for sexual reproduction in this organism. Thin-layer chromatography (TLC) of the commercial lecithin revealed the presence of an unknown inhibitory substance which, when added to the purified lecithin, caused a 50% reduction of oospore formation. Commercial cephalin also showed a twofold increase in activity after removal of putative sterols and the existence of an unknown inhibitor when it was subjected to TLC. Addition of the inhibitor to the purified cephalin completely inhibited the growth of the test organism. One sample of lecithin tested was not stimulatory to oospore formation. However, after washing with deionized water or NaCl solution, it induced the production of 17300 and 24450 oospores (100 μg)-1, respectively. The ability of cephalin to induce oospore formation was increased 2⋅3-fold by washing with deionized water and 8⋅3-fold by washing with NaCl solution. Like sterols, the digitonin precipitable component (digitonide) of the non-phospholipid fraction of commercial lecithin or cephalin was stimulatory to oospore formation of P. cactorum but not Phytophthora parasitica. However, the non-digitonide component was not only more active than the digitonide component, but also stimulatory to P. parasitica. Gas chromatography and mass spectrometry (GC-MS) analysis of the digitonide component from lecithin failed to detect any putative sterol contaminant. The amount of the putative sterol contaminant in the digitonide component from cephalin was also below the detection limit of GC-MS. When 0.01-10 ng cholesterol was added to basal medium discs each containing 100 fig cephalin, the numbers of oospores produced by P. cactorum and P. parasitica were not significantly changed. It is concluded that, in the fungi tested, sterols did not play any significant role in the stimulation of sexual reproduction by highly purified phospholipids.
Construction and properties of aconitase mutants of Escherichia coli
Authors: Gruer Megan J. , Bradbury Alan J. , Guest John R.
Published: June 2009

Escherichia coli contains two genes (acnA and acnB) encoding aconitase activities. An acnB mutant was engineered by replacing the chromosomal acnB gene by an internally deleted derivative containing a tet
R cassette. An acnB double mutant was then made by transducing a previously constructed acnA::kan
R mutation into the acnB::tet
R strain. Western blotting confirmed that the AcnA and AcnB proteins were no longer produced by the corresponding mutants and PCR analysis showed that the chromosomal acnB gene had been replaced by the disrupted gene. Aerobic and anaerobic growth in glucose minimal medium were impaired but not abolished by the acnB mutation, indicating that the lesion is partially complemented by the acnA
+ gene, and growth was enhanced by glutamate. The acnAB double mutant would not grow on unsupplemented glucose minimal medium and although it responded to glutamate like a typical auxotroph under anaerobic conditions, under aerobic conditions no response to glutamate was observed before it was over-grown by ‘revertants’ lacking citrate synthase (acnAB gltA). The acnAB double mutant retained a low but significant aconitase activity (< 5% of wild-type), designated AcnC. Enzymological and regulatory studies with acn-lacZ fusions indicated that AcnB is the major aconitase, which is synthesized earlier in the growth cycle than AcnA, and subject to catabolite and anaerobic repression.
The Bacillus subtilis 168 chromosome from sspE to katA
Authors: Cummings N. J. , Connerton I. F.
Published: June 2009

We have cloned and sequenced a 24.5 kb region of the Bacillus subtilis 168 chromosome spanning the sspE and katA genes. The region contains a ribosomal RNA operon, rrnD, a tRNA gene set, trnD and 17 ORFs, 16 with putative ribosome-binding sites. Four of the ORFs (ORF2, ORF14, ORF16 and ORF17) match to known B. subtilis genes (sspE, thiA, senS and katA). Eight of the remaining ORF products show similarities with proteins present in the databases, including an ATP-binding transport protein, a glutamate-1-semialdehyde aminotransferase, a thiol-specific antioxidant protein, a mitomycin radical oxidase and a ferric uptake regulation protein.
An important role for glutathione and γ-glutamyltranspeptidase in the supply of growth requirements during nitrogen starvation of the yeast
Saccharomyces cerevisiae
Authors: Mehdi Karim , Penninckx Michel J.
Published: June 2009

When the yeast
Saccharomyces cerevisiae
Σ1278b was starved for nitrogen, the total glutathione (GSH) pool increased from 7 to 17 nmol (mg dry wt)-1 during the first 2 h and then declined. More than 90％ of the total GSH shifted towards the central vacuole during this time. This transient stimulation was not observed in the presence of buthionine-(S,R)-sulphoximine (BSO), a specific transition-state-analogue inhibitor of γ-glutamylcysteine synthase (γ-GCS), nor in a mutant strain deficient in this enzyme. γ-Glutamyltranspeptidase (γ-GT), a vacuolar enzyme responsible for the initial step of GSH degradation, was derepressed during nitrogen starvation. This mechanism can apparently enable the starved yeast cell to use the constituent amino acids from GSH which accumulate in the vacuole to satisfy its growth requirements for nitrogen.
A Neurospora crassa mutant altered in the regulation of L-amino acid oxidase
Authors: Calderón Jorge , Olvera Leticia , Martínez Luz María , Dávila Guillermo
Published: June 2009

The isolation and characterization of a Neurospora crassa mutant altered in l-amino oxidase regulation is reported. The previously isolated gln-1bR8 strain, which only synthesizes the glutamine synthetase α monomer and lacks the β monomer, was used as parental strain. A mutant derivative of strain was selected for its ability to grow on minimal medium in the presence of DL-methionine-SR-sulfoximine (MSO), an inhibitor of glutamine synthetase activity. This gln-1bR8;MSO
R mutant overcame the inhibitory effect of MSO by increasing the activity of L-amino acid oxidase, an enzyme capable of degrading this compound. In contrast with the wild-type strain, the L-amino acid oxidase of the MSOR mutant was resistant to glutamine repression; in fact, it was induced by this amino acid but repressed by ammonium. This mutant is different from other nitrogen regulatory N. crassa mutants reported and is only altered in the regulation of L-amino acid oxidase. The MSOR mutation is epistatic to nit-2 since the nit2;MSO
R double mutant regulated the L-amino acid oxidase in the same way as the MSO
R single mutant.
Isolation of developmentally regulated genes from the edible mushroom Agaricus bisporus
Authors: De Groot Piet W. J. , Schaap Peter J. , Van Griensven Leo J. L. D. , Visser Jaap
Published: June 2009

From a cDNA library, constructed from mushroom primordia, nine cDNAs were isolated which were either induced or specifically expressed during fruit body development and maturation of the basidiomycete Agaricus bisporus. These cDNAs varied in size from 372 to 1019 bp and hybridized to transcripts of 400-1600 nt. Four of the cDNAs were only expressed in the generative phase o: the life cycle while the other five cDNAs were strongly induced but had low steady-state mRNA levels in vegetatively grown mycelium of the hybrid strain Horst U1. An apparent full-length cDNA could be identified by sequence analysis and specified a putative protein homologous to the δ-subunit of the mitochondrial ATP synthase complex of Saccharomyces cerevisiae and Neurospora crassa. For one of the partial cDNAs, significant homology was found with a family of cell division control proteins, while another partial cDNA appeared to encode a cytochrome P450. All cDNAs, except the presumed cytochrome-P450-specifying cDNA (cypA), hybridized with single copy genes scattered over the Agaricus genome. For the cypA gene, the presence of several additional copies was shown by heterologous hybridizations. Based on changes in expression levels of the fruit-body-induced genes during development coinciding with alterations in morphological appearance of mushrooms, four stages of development were distinguished during growth and maturation of A. bisporus fruit bodies.
β-Tubulin genes and the basts for benzimidazole sensitivity of the opportunistic fungus Cryptococcus neoformans
Authors: Cruz Maria Cristina , Edlind Thomas
Published: June 2009

The basidiomycete Cryptococcus neoformans causes life-threatening infections in immunocompromised patients, and available chemotherapeutic agents are potentially toxic or have limited efficacy. in vitro, C. neoformans is very sensitive to selected benzimidazole compounds (e.g. albendazole), which act by disrupting microtubules through binding to the β-tubulin subunit. To understand the basis for this benzimidazole sensitivity, we have characterized C. neoformans β-tubulin genes and their expression. Analysis of PCR amplification products, genomic and cDNA clones and Southern blots identified two β-tubulin genes. TUB1 contains seven introns, including one that splits the start codon, and encodes a 447 amino acid protein with >80% identity to most other β-tubulins. A partial sequence of TUB2 revealed a higher density of introns and a considerably more divergent β-tubulin. The relative expression of TUB1 to TUB2 determined by reverse-transcription PCR was about 3:1, consistent with a more limited role for the TUB2 product. Comparisons of β-tubulin sequences from C. neoformans and from various benzimidazole-sensitive and -resistant organisms strongly suggest that the TUB1 product represents the primary benzimidazole target. This was supported by the identification of a His6 to Gin change in TUB1 from three independently isolated albendazole-resistant mutants.
The pyruvate dehydrogenase complex of the chemolithoautotrophic bacterium Thiobacillus ferrooxidans has an unusual E2-E3 subunit fusion
Authors: Powles Rosamund , Rawlings Douglas
Published: June 2009

Summary: The genes encoding pyruvate dehydrogenase (PDH) of Thiobacillus ferrooxidans were previously located by cloning and sequence analysis of the region upstream of the genes encoding the citrate synthase and -glutamylcysteine synthetase genes. The pdh genes of T. ferrooxidans were able to complement an Escherichia coli aroP-lpd mutant for growth on minimal medium lacking acetate, indicating that the T. ferrooxidans PDH complex was functional in E. coli. The predicted amino acid sequence of the T. ferrooxidans PDH complex contained three ORFs. The first ORF encoded a 36.7 kDa homologue of the PDH complex E1α subunit, the second ORF a 37.4 kDa E1α subunit and the third ORF an unusual 102 kDa fusion of the E2 and E3 subunits. In spite of T. ferrooxidans being a Gram-negative bacterium, its PDH complex had more features in common with Gram-positive bacteria and eukaryotes.
The starvation-stress response of Vibrio (Listoneila) anguillarum
Authors: Nelson David R. , Sadlowski Yola , Eguchi Mitsuru , Kjelleberg Staffan
Published: June 2009

Summary: The starvation-stress response of Vibrio (Listonella) anguillarum was investigated and characterized with regard to changes in cell morphology and the ability of V. anguillarum to survive starvation, heat shock, exposure to H2O2 and exposure to ethanol. The ability of V. anguillarum to survive exposal to the latter three stresses after initiation of starvation was also examined. Results of these experiments indicated that when starved for carbon, nitrogeand phosphorus, the c.f.u. of V. anguillarum declined by about one order of magnitude over the first 5-7 d of starvation; starvation for an additional 3-4 weeks resulted in a gradual decline in c.f.u. by another order of magnitude. Examination of starved cells by electron microscopy revealed that while most cells formed spherical ultramicrocells during starvation, some of the cells elongated to form short spirals. While cross-protection against other stresses such as oxidative stress (exposure to H2O2) and exposure to ethanol developed only a small degree of resistance to heat shock developed. Moreover, in all cases these resistances disappeared during prolonged starvation (usually > 5 d). Additionally, the rate of protein synthesis per c.f.u., measured by [35S]methionine incorporation, declined during the initial 6 h of starvation and increased to over 70% of the rate measured in exponentially growing cells by 5 d of starvation. It was concluded that the starvation-stress response of V. anguillarum differs significantly from those starvation responses reported for other bacteria, including responses displayed by other Vibrio species.
Energetics of methanogenic benzoate degradation by Syntrophus gentianae in syntrophic coculture
Authors: Schöcke Ludger , Schink Bernhard
Published: June 2009

Summary: Growing cocultures of Syntrophus gentianae with Methanospirillum hungatei degraded benzoate to CH4 and acetate. During growth, the change of free energy available for Syntrophus gentianae ranged between -50 and -55 kJ mol−1. At the end-point of benzoate degradation, a residual concentration of benzoate of 0.2 mM was found, correlating with a free energy change of -45 kJ mol−1 available to the fermenting bacterium. Benzoate thresholds were also observed in dense cell suspensions. They corresponded 1 a final energy situation in the range -31.8 to -45.8 kJ mol−1 for the fermentin bacterium. Addition of a H2-oxidizing sulfate reducer to the methanogenic coculture inhibited by bromoethanesulfonate (BES) resulted in benzoate degradation to below the limit of benzoate detection (10 μM). Accumulated acetate proved to be thermodynamically inhibitory; removal of acetate by Methanosaeta concilii in methanogenic or molybdate-inhibited sulfate-reducing cocultures led to degradation of residual benzoate with a final δG’ -45.8 kJ mol−1. In methanogenic cocultures, the residual Gibbs free energy (δG’) available for the fermenting bacterium at the end of benzoate degradation correlated with the concentration of acetate built up during the course of benzoate degradation; higher concentrations led to more positive values for δG’. Addition of different concentrations of propionate resulted in different values for δG when benzoate degradation had ceased; higher concentrations led to more positive values for δG’. Addition of acetate or propionate to benzoate-degrading cocultures also lowered the rate of benzoate degradation. The protonophore carbonylcyanide chlorophenylhydrazone (CCCP) facilitated further benzoate degradation in methanogenic BES-inhibited cocultures until a δG’ of -31 kJ mol−1 was reache We conclude that the minimum energy required for growth and energy conservation of the benzoate-fermenting bacterium S. gentianae is approximately -45 kJ (mol benzoate)−1, equivalent to two-thirds of an ATP unit. Both hydrogen and acetate inhibit benzoate degradation thermodynamically, and acetate also partly uncouples substrate degradation from energy conservation.
Degradation of chlorophenoxyacetic acids by the lignin-degrading fungus Dichomitus squalens
Authors: Bhasker Reddy G. Vijay , Joshi Dinesh K. , Gold Michael H.
Published: June 2009

Summary: We have examined the degradation of 14C ring- and side-chain-labelled 2,4,5-trichlorophenoxyacetic acid by Dichomitus squalens and Phanerochaete chrysosporium. The effects of Mn2+ on the degradation of these radiolabeled substrates by D. squalens and the effect of nitrogen limitation on their degradation by D. chrysosporium suggested that in both fungi, side-chain cleavage was catalysed by a mechanism independent of the lignin degradation system, whereas the degradation of the aromatic ring was dependent on the lignin degradative system. Using unlabelled substrates, a pathway for the degradation of chlorophenoxyacetic acids was elucidated in D. squalens. Time courses for the degradation of unlabelled chlorophenoxyacetic acids by D. squalens demonstrated that the corresponding chlorophenol was the initial product formed. The chlorophenol intermediate was xylosylated to form the chlorophenolxyloside. In turn, the chlorophenolxyloside could be hydrolysed by an intracellular -xylosidase to regenerate the chlorophenol. The chlorophenol product of the xylosidase reaction was oxidatively dechlorinated to form 2-chloro-p-benzoquinone which could undergo subsequent further dechlorination and ring-opening reactions, as has been reported previously for P. chrysosporium.
The effects of adherence to silicone surfaces on antibiotic susceptibility in Staphylococcus aureus
Authors: Williams Ian , Venables W. Alfred , Lloyd David , Paul Frank , Critchley Ian
Published: June 2009

Summary: Sensitivity of Staphylococcus aureus to the antibiotics tetracycline, benzylpenicillin and vancomycin was found to decrease by 2-10-fold when cells were grown adherent to silicone catheter surfaces. Sensitivity to rifampicin and fusidic acid was not significantly altered in adherent cells. Susceptibility further decreased with increased adherence time prior to antibiotic challenge. The resistance observed was not genotypic, or due to the presence of a specialized subpopulation of bacteria, as it disappeared when the bacteria were removed from the catheter, subcultured and retested. Also, adherent bacteria were found to grow more slowly than bacteria growing planktonically. It is concluded that the decrease in antibiotic susceptibility of adherent bacteria is a function of the physiological status of the individual cells rather than a function of biofilm formation or slime production. The decrease in growth rate of the adherent bacteria is a result of the adherence process rather than a result of nutrient depletion. The decrease in growth rate is implicated, but is not the sole factor, in the decreased antibiotic susceptibility of adherent bacteria.
Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210: limitations and regulations
Authors: Barbirato Fabien , Astruc Suzette , Soucaille Philippe , Camarasa Carole , Salmon Jean Michel , Bories André
Published: June 2009

Summary: Continuous cultures ofEnterobacter agglomeransCNCM 1210 were performed under regulated pH conditions (pH 7.0) with glycerol or glucose (20 gl−1) as carbon source. Cultures grown on glucose produced mainly acetate, ethanol and formate. In contrast, 1,3-propanediol (PPD) was the main product with glycerol. The carbon flow distribution at branching metabolic points was investigated. Higher PPD yields with increased dilution rate were correlated with an important increase in the relative ratio of glycerol dehydratase to glycerol dehydrogenase. Determination of intracellular triose-phosphate and fructose 1,6-biphosphate concentrations demonstrated that glyceraldehyde-3-phosphate dehydrogenase is the limiting step in glycerol dissimilation. At the pyruvate branching point, pyruvate dehydrogenase (PDH) activity was systematically detected. The pyruvate flow shifted to PDH is suspected to represent up to 22% of the acetyl-CoA formed. In addition, this enzyme pattern combined with the enhancedin vivolactate dehydrogenase activity at high growth rates, was correlated with a decrease in the pyruvate formate-lyase activity. A regulation of this latter enzyme by the accumulation of triose-phosphate is suspected.
Mitogenic factor secreted by Streptococcus pyogenes is a heat-stable nuclease requiring His122 for activity
Authors: Iwasaki Makoto , Igarashi Hisanaga , Yutsudo Takashi
Published: June 2009

Summary: The gene encoding a mitogenic factor, termed MF, was cloned from Streptococcus pyogenes and the recombinant MF was overexpressed in Escherichia coli. Both the natural and recombinant MF had heat-resistant nuclease activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultaneous presence of two divalent cations, Mg2+ and Ca2+, compared to either alone and was inhibited by EDTA or NaCI. The heat stability of MF was biphasic; the DNase activity was heat-stable from 0 to 50 °C and over 80 °C but very unstable at around 60 °C. DNA digested by MF possessed 5′-phosphorylated and 3′-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyribonuclease I. A mutant clone revealed that His122 was a residue essential to the nuclease activity.
A cytoplasmic lectin produced by the fungus Arthrobotrys oligospora functions as a storage protein during saprophytic and parasitic growth
Authors: Rosén Stefan , Sjollema Klaas , Veenhuis Marten , Tunlid Anders
Published: June 2009

Summary: It was recently shown that the nematode-infecting fungus Arthrobotrys oligospora contains a saline-soluble lectin (designated AOL) that is a member of a novel family of fungal lectins sharing similar primary sequences and binding specificities. During saprophytic growth in liquid cultures, levels of AOL and AOL mRNA were found to vary depending on the growth phase of the mycelium and the carbon/nitrogen (C/N) ratio of the medium. AOL was not detected in young mycelium. In older mycelium (stationary growth phase) grown in media with low C/N ratios (1 or 6), AOL comprised 5-20% of the total amount of saline-soluble proteins present in the mycelium. Neither the lectin nor its transcript was detected in mycelia grown in medium with higher C/N ratios (≥150). Under conditions of nitrogen starvation, AOL was preferentially degraded in relation to the total amount of saline-soluble proteins present in the mycelium. During the infection of nematodes, the level of AOL protein and AOL mRNA increased significantly once the nematodes had been penetrated and digested. Large amounts of AOL accumulated in the trophic hyphae growing inside the nematode as visualized by immunofluorescence microscopy. Later, AOL labelling was detected outside the digested nematodes, preferentially in strands of aggregated hyphae and in newly developed trap cells. Electron microscopy showed that AOL was localized to the cytoplasm and the nucleus of both vegetative mycelium and trap cells, and in the trophic hyphae growing inside the infected nematodes. These results indicate that AOL functions as a storage protein during both saprophytic and parasitic growth.
Efficient secretion of the model antigen M6-gp41E in Lactobacillus plantarum NCIMB 8826
Authors: Hols Pascal , Slos Philippe , Dutot Philippe , Reymund Jacqueline , Chabot Paul , Delplace Brigitte , Delcour Jean , Mercenier Annick
Published: June 2009

Summary: FourLactobacillusstrains (Lb. plantarumNCIMB 8826,Lb. paracaseiLbTGS1.4,Lb. caseiATCC 393 andLb. fermentumKLD) were tested for their ability to produce and secrete heterologous proteins. These strains were first screened with an α-amylase reporter under the control of a set of expression or expression/secretion signals from various lactic acid bacteria. With most of the constructions tested, the level of extracellular production was highest inLb. plantarumNCIMB 8826, and lowest inLb. paracaseiLbTGS1.4. These two strains were next assayed using a model antigen consisting of the N-terminal part of the M6 protein fromStreptococcus pyogenesfused to the linear epitope ELDKWAS from human immunodeficiency virus gp41 protein. Secretion of this heterologous protein was inefficient inLb. paracaseiLbTGS1.4, which accumulated a large intracellular pool of the unprocessed precursor, whereasLb. plantarumNCIMB 8826 was able to secrete the antigen to a level as high as 10 mg I−1.
A new nos gene downstream from nosDFY is essential for dissimilatory reduction of nitrous oxide by Rhizobium (Sinorhizobium) meliloti
Authors: Chan Yiu-Kwok , McCormic Wayne A. , Watson Robert J.
Published: June 2009

Summary: Rhizobium (Sinorhizobium) meliloti strains capable of dissimilatory nitrous oxide reduction (Nos+) carry a nosRZDFY gene cluster on a 10.1 kb EcoRI fragment of the nod megaplasmid near the fixGHIS genes. These nos genes are arranged in three complementation groups and the 10.1 kb EcoRI fragment is sufficient to confer Nos activity to R. meliloti strains lacking such activity. An overlapping HindIII fragment containing the nosRZDFY genes but missing a 0.6 kb Hin dIII-EcoRI downstream segment was found incapable of imparting Nos activity to strains unable to reduce nitrous oxide, suggesting the presence of other nos gene(s) in this region. Tn5 introduced near the HindIII site resulted in mutants with a Nos− phenotype. Complete sequence analysis of nosY showed that it was well-conserved with respect to that of Pseudomonas stutzeri. Two previously unreported genes downstream of nosY in R. meliloti were also revealed. Contiguous with nosY was a sequence showing 63% identity with the ORFL protein of P. stutzeri. It appeared to be in the same operon as nosDFY and was predicted to encode a membrane lipoprotein similar to the putative NosL of P. stutzeri. Unlike the latter protein, however, amino acid sequences typical of metal-binding sites and cysteine residues indicative of the active site of protein disulphide isomerase were absent in the predicted NosL of R. meliloti. The Tn5 mutations resulting in a Nos− phenotype were localized within a 966 nucleotide gene 31 nucleotides downstream of nosDFYL with the same orientation. The new gene, nosX, was determined to be in a separate complementation group. It encoded a periplasmic protein with homology in the C-terminal domain with RnfF of Rhodobacter capsulatus and with a hypothetical Escherichia coli protein, YOJK. It was concluded that there are seven genes constituting the nos cluster in R. meliloti. They are organized in four complementation groups and in the same orientation, spanning a distance of about 9 kb on the nod megaplasmid.
Genetic relationships among Pasteurella trehalosi isolates based on multilocus enzyme electrophoresis
Authors: Davies Robert L. , Arkinsaw Scott , Selander Robert K.
Published: June 2009

Summary: Genetic diversity among 60 British Pasteurella trehalosi isolates representing the four recognized capsular serotypes, T3, T4, T10 and T15, and recovered predominantly from sheep suffering from systemic pasteurellosis, was estimated by analysing electrophoretically demonstrable allelic variation at structural genes encoding 19 enzymes. Thirteen of the loci were polymorphic and 20 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The population structure of P. trehalosi is clonal and its genetic diversity is limited compared with most other pathogenic bacteria. ETs represent clones, and isolates of the same ET were generally associated with the same combination of serotype, LPS type and outer-membrane protein (OMP) type. The genetic diversity of isolates within each of the capsular serotypes varied. Serotype T10 was represented by 18 isolates in two related ETs and exhibited little diversity. By contrast, serotype T15 was represented by 18 isolates in nine ETs and was almost as diverse as the species as a whole. Serotype T4 was represented by 18 isolates in five ETs and was less diverse than serotype T15. Although serotype T3 was more diverse than serotype T15 it was represented by only three isolates. With the exception of the T10 isolates and those recovered from healthy sheep, 35 disease isolates belonged to 16 ETs, each of which was represented by only one to four isolates. The fact that a high proportion of disease is caused by a relatively large number of clones suggests that P. trehalosi is essentially an opportunistic pathogen. In addition to having the same capsular structure, isolates belonging to the two T10 clones were characterized by possession of similar, if not identical, O-antigens (LPS types 2 and 4). The occurrence of 18 serotype T10 isolates in only two ETs suggests that the T10 capsule and type 2/4 O-antigen confer enhanced virulence on members of these two clones. Multilocus enzyme electrophoresis (MLEE) had greater resolving power than did capsule/LPS/OMP analysis, being able to distinguish 20 rather than 14 sub-divisions within P. trehalosi. The technique demonstrated genetic identity or non-identity among strains of the same or different serotypes from different geographic localities within the UK and was a useful epidemiological tool.
Activation of the CheA kinase by asparagine in Bacillus subtilis chemotaxis
Authors: Garrity Liam F. , Ordal George W.
Published: June 2009

Summary: Past experiments have shown that CheA and CheY are required to generate smooth swimming signals in Bacillus subtilis chemotaxis. This study, as anticipated from in vivo experiments, demonstrates in vitro that an attractant-bound chemoreceptor leads to an increase in CheA activity, which in turn leads to an increase in the Che Y-P pool that ultimately causes a behavioural change in the bacteria. Asparagine has been found to increase the rate of Che Y-P formation in the presence of McpB-containing membranes, CheA, and an excess of CheY. This asparagine effect requires the presence of both CheA and McpB, the latter of which has been shown to be the sole receptor for this attractant. Utilizing membranes from a number of B. subtilis null mutant strains, insight has also been gained into the potential roles of a number of unique chemotaxis proteins in the regulation of CheA activity in the presence and absence of this attractant.
pH-regulated expression of the acid and alkaline extracellular proteases of Yarrowia lipolytica
Authors: Glover David J. , McEwen Robert K. , Thomas Colin R. , Young Thomas W.
Published: June 2009

Summary: The pH-regulated expression of the acid (AXP) and alkaline (AEP) extracellular proteases of the yeast Yarrowia lipolytica 148 was analysed. Expression in batch and continuous cultures was determined at the mRNA level by Northern blotting, and at the enzyme level by enzyme assays and Western blotting. Culture pH regulated AEP and AXP expression predominantly at the level of mRNA content. Highest levels of AEP mRNA were detected at pH 6.5 whereas highest levels of AXP mRNA were detected at pH 5.5. At pH values either side of these maxima AEP and AXP expression were progressively down-regulated. For both enzymes, the variation in mRNA levels with culture pH occurred progressively rather than by discrete steps. AXP expression did not occur above pH 7.0. Some degree of AEP expression occurred at all pH values tested in two unrelated strains of Y. lipolytica.
Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans
Authors: Torralba Sara , Raudaskoski Marjatta , Pedregosa Ana María , Laborda Fernando
Published: June 2009

Summary: The role of actin in apical growth and enzyme secretion in the filamentous fungusAspergillus nidulanswas studied by treating the hyphae with cytochalasin A (CA), which inhibits actin polymerization. Indirect immunofluorescence microscopy revealed actin at the tips of main hyphae and branches, and at the sites of developing septa. CA inhibited the growth of the fungus and changed the growth pattern of hyphal tips from cylindrical tubes to spherical beads. The regions with swellings showed no actin fluorescence, and neither was actin seen in association with septa. After 4 h exposure, hyphae were able to resume the normal tip growth pattern in the presence of CA for a short period of time and new cylindrical hyphae, with actin fluorescence at the apex, emerged from the swollen tips. Later, the tips of the hyphae swelled again, which led to a beaded apperance. We also studied the effect of CA on the secretion of α- and β-galactosidase. α-Galactosidase is secreted into the culture medium, whereas β-galactosidase remains in the mycelium, with part of its activity bound to the cell wall. WhenA. nidulansmycelium was incubated in the presence of CA, a reduction in the secretion of α-galactosidase into the culture medium and a decrease in the α- and β-galactosidase activities bound to the cell wall was detected. However, the CA dose used for the hyphae did not modify the secretion of the enzymes from protoplasts. Results described here provide evidence that a polymerized actin cytoskeleton is required for normal apical growth, hyphal tip shape and polarized enzyme secretion inA. nidulans.Cytochalasin-induced disruptions of the actin cytoskeleton could result in the alterations of apical growth and inhibition of enzyme secretion observed by blocking secretory vesicle transport to the apex.
Response of Bacillus subtilis to high osmolarity: uptake of carnitine, crotonobetaine and γ-butyrobetaine via the ABC transport system OpuC
Authors: Kappes Rainer M. , Bremer Erhard
Published: June 2009

Summary: It was found that low concentrations of the naturally occurring and structurally related betaines L-carnitine, crotonobetaine and γ-butyrobetaine conferred a high degree of osmotic tolerance to Bacillus subtilis. Kinetic analysis of L-[N-methyl
−14C]carnitine uptake in cells grown in minimal medium revealed the presence of a high-affinity transport system with a K
m value of 5 μM and a maximum rate of transport (V
max) of 41 nmol min−1 (mg protein)−1. A rise in medium osmolarity moderately increased the maximum velocity [V
max 71 nmol min−1 (mg protein)−1] of this transport system, but had little effect on its affinity. Growth and transport studies with a set of strains that carried defined mutations in the previously identified glycine betaine transport systems OpuA, OpuC and OpuD allowed the identification of the ATP-binding cassette (ABC) transport system OpuC as the only uptake route for L-carnitine in B. subtilis. Competition experiments with crotonobetaine and γ-butyrobetaine revealed that the OpuC system also exhibited a high affinity for these trimethylammonium compounds with K
i values of 6.4 μM. Tracer experiments with radiolabelled L-carnitine and 13C-NMR tracings of cell extracts demonstrated that these betaines are accumulated by B. subtilis in an unmodified form. In contrast, the β-substituted acylcarnitine esters acetylcarnitine and octanoylcarnitine both functioned as osmoprotectants for B. subtilis but were found to be accumulated as carnitine by the cells. None of these trimethylammonium compounds were used as sole carbon or nitrogen sources. The results thus characterize L-carnitine, crotonobetaine and γ-butyrobetaine as effective compatible solutes for B. subtilis and establish a crucial role of the ABC transport system OpuC for the supply of B. subtilis with a variety of osmoprotectants.