bluuebunny/crossref_metadata_2025 huggingface dataset:
Green fluorescent protein as a novel species-specific marker in enteric dual-species biofilms
Authors: Skillman Lucy C. , Sutherland Ian W. , Jones Martin V. , Goulsbra Amanda
Published: June 2009

Green fluorescent protein (GFP) was used as a tool to examine the interactions between pairs of bacterial species and their effects on subsequent biofilm development over 24 h. A plasmid encoding GFP from Aequorea victoria was transformed into strains of Enterobacter agglomerans and Escherichia coli ATCC 11229. The development of dual-species biofilms, containing one fluorescent and one non-fluorescent partner, was examined using viable counts. UV illumination of plates enabled both species to be identified in a mixture. The spatial distribution of each species was examined by UV microscopy, simultaneously staining the non-fluorescent strain with propidium iodide. GFP fluorescence was measured to quantify the adhesion of the strains to other cells or cell constituents or the invasion into pre-existing biofilms. Cooperation between Ent. agglomerans/GFP and Klebsiella pneumoniae G1 resulted in a 54 and a 23% increase in biofilm formation, respectively, compared with single-species biofilms. E. coli/GFP and Serratia marcescens 87b stably co-existed in biofilms but did not affect the growth of each other. The other bacterial partnerships examined were competitive, with the end result that one species dominated the biofilm. The methods described provide a convenient technique for the examination of mixed-species biofilm communities where the unique interactions between species determine the true properties of the resultant biofilms.
The gerC locus of Bacillus subtilis, required for menaquinone biosynthesis, is concerned only indirectly with spore germination
Authors: Leatherbarrow A. J. Howard , Yazdi Mohammed A. , Curson Janet P. , Moir Anne
Published: June 2009

The gerC region of Bacillus subtilis comprises a tricistronic operon, encoding enzymes that catalyse the late stages of menaquinone biosynthesis. The gerC58 mutation is responsible for a severe growth defect; unsuppressed mutant cells grow as very short rods, which sometimes septate aberrantly. Cultures grow only to a low cell density, rapidly lose viability, and never sporulate. Unlinked suppressor mutations can restore near-normal growth. Several independent suppressed isolates were examined; all grew to normal cell length, but they showed, to varying extents, a residual defect in the placement of the cell division septum. The germination properties of the suppressed derivatives varied from normal to significantly slow in germination in all germinants; therefore, the combination of the gerC mutation and different suppressor alleles resulted in spores with very different germination properties. This suggests that any relationship between the gerC gene products and spore germination is indirect. The gerCC58 mutation maps in a gene encoding the catalytic subunit of the heptaprenyl-diphosphate synthase, which is responsible for formation of the isoprenoid side chain of menaquinone-7, and it is proposed that the gerCA, gerCB and gerCC genes be renamed hepA, menG and hepB, respectively.
Glutamate and cyclic AMP regulate the expression of galactokinase in Mycobacterium smegmatis
Authors: Raychaudhuri Santanu , Basu Madhumita , Mandal Nitai C.
Published: June 2009

It was found thatMycobacterium smegmatisis unable to utilize galactose as the sole carbon source because the sugar alone cannot induce galactokinase. However, galactokinase was induced by glutamate alone, and was further stimulated by galactose. Rifampicin completely inhibited the glutamate-mediated expression ofgalKin both the absence and presence of galactose. Extracellular cAMP stimulated the expression of the enzyme only in the presence of glutamate plus galactose. ThegalKgene fromM. smegmatis, including its upstream promoter region, was cloned in a plasmid inEscherichia coli. The expression of kinase from these clones inE. coliwas dependent on cAMP and its receptor protein (CRP). The expression of UDP-galactose 4-epimerase was constitutive. This and other evidence suggests that thegalKgene is not linked togalTandgalEin the mycobacterial genome. In a glutamate-independent galactose-utilizing mutant (gin-1mutant) ofM. smegmatis, galKwas expressed in the absence of both galactose and glutamate, while in the presence of galactose this expression was increased twofold in the absence of glutamate and fourfold in its presence. Extracellularly added cAMP reduced the expression of the enzyme in the presence of galactose plus glutamate nearly to the basal level. It is proposed that inM. smegmatisthegalKgene is expressed from two different promoters; the expression from one promoter is dependent on glutamate but not on galactose and cAMP, while that from the other requires all three components. The role of galactose is possibly to derepress the latter promoter.
Attacin - an insect immune protein - binds LPS and triggers the specific inhibition of bacterial outer-membrane protein synthesis
Authors: Carlsson Anette , Nyström Thomas , de Cock Hans , Bennich Hans
Published: June 2009

Attacin is a 20 kDa antibacterial protein, originally isolated from the immune haemolymph of Hyalophora cecropia. It has been demonstrated previously that attacin causes increased permeability of the outer membrane of Escherichia coli and inhibition of outer-membrane protein synthesis at the transcriptional level. This is accompanied by inhibition of growth. Here, LPS is shown to serve as the receptor for attacin and evidence is presented that attacin does not need to enter the cell to exert its activity. The increase in outer-membrane permeability precedes any increase in inner-membrane permeability by at least one generation time (∼ 45 min), and the inhibiting effect of attacin on synthesis of outer-membrane proteins is detectable after only 10 min. It is also shown that attacin causes induction of several stress proteins and increased synthesis of LPS within, respectively, 25 and 60 min of treatment. Based on the results presented, it is proposed that attacin has the unique ability to specifically interfere with synthesis of outer-membrane proteins without entering the inner membrane or cytoplasm.
Conservation of the amino-terminal epitope of elongation factor Tu in eubacteria and archaea
Authors: Baensch Melanie , Frank Ronald , Khl Jrg
Published: June 2009

An epitope of elongation factor Tu (EF-Tu), which is found in organisms in both the bacterial and archaeal domains, was recently defined by mAb 900. To localize the conserved epitope within the EF-Tu molecule and to determine its sequence, SPOTScan analysis of synthetic peptides, Western blot analysis of purified EF-Tu domains and site-directed mutagenesis studies were used. Analysis of mAb 900 binding to overlapping 15-mer peptides encompassing the complete sequence of EF-Tu ofEscherichia coliwas inconclusive, suggesting three distinct regions may be epitopes. Western blot analysis of EF-Tu domains 1-3 ofThermus thermophilussuggested that the epitope was located at the N terminus. This was confirmed by site-directed mutagenesis of EF-Tu domain 1 ofMycoplasma hominis.By C-terminal truncation of the N-terminal 15-mer peptide the epitope was mapped to EF-Tu residues 1-6. Replacement of each of the residues in the epitope peptide demonstrated that only positions 5 and 6 were indispensable for antibody binding. These data provide evidence that the highly conserved epitope recognized by mAb 900 in the bacterial and archaeal domains is located at the very end of the N terminus of the EF-Tu molecule.
Phototrophic oxidation of ferrous iron by a Rhodomicrobium vannielii strain
Authors: Heising Silke , Schink Bernhard
Published: June 2009

Oxidation of ferrous iron was studied with the anaerobic phototrophic bacterial strain BS-1. Based on morphology, substrate utilization patterns, arrangement of intracytoplasmic membranes and the in vivo absorption spectrum, this strain was assigned to the known species Rhodomicrobium vannielii. Also, the type strain of this species oxidized ferrous iron in the light. Phototrophic growth of strain BS-1 with ferrous iron as electron donor was stimulated by the presence of acetate or succinate as cosubstrates. The ferric iron hydroxides produced precipitated on the cell surfaces as solid crusts which impeded further iron oxidation after two to three generations. The complexing agent nitrilotriacetate stimulated iron oxidation but the yield of cell mass did not increase stoichiometrically under these conditions. Other complexing agents inhibited cell growth. Ferric iron was not reduced in the dark, and manganese salts were neither oxidized nor reduced. It is concluded that ferrous iron oxidation by strain BS-1 is only a side activity of this bacterium that cannot support growth exclusively with this electron source over prolonged periods of time.
Cloning of the trkAH gene cluster and characterization of the Trk K+-uptake system of Vibrio alginolyticus
Authors: Nakamura Tatsunosuke , Yamamuro Naoya , Stumpe Stefan , Unemoto Tsutomu , Bakker Evert P.
Published: June 2009

K+-uptake genes of Vibrio alginolyticus were identified by cloning chromosomal DNA fragments of this organism into plasmids, followed by electroporation and selection for growth at low K+ concentrations of cells of an Escherichia coli strain defective in K+ uptake. A 4.1 kb DNA fragment contained a cluster of three ORFs on the same DNA strand: the previously identified trkA gene, a gene similar to E. coli trkH (V. alginolyticus trkH) and a new gene, orf1, whose function is not clear. Products of V. alginolyticus trkA and orf1 were detected in E. coli minicells. trkA and trkH from V. alginolyticus restored growth at low K+ concentrations of an E. coli ΔtrkA and an E. coli ΔtrkG ΔtrkH strain, respectively, suggesting that these V. alginolyticus genes can functionally replace their E. coli counterparts. In addition, a plasmid containing V. alginolyticus trkAH permitted growth of an E. coli ΔsapABCDF
(ΔtrkE) strain at low K+ concentrations. This effect was mainly due to V. alginolyticus trkH and was enhanced by trkA from this organism. Measurements of net K+-uptake rates indicated that the presence of these genes in E. coli renders the Trk systems independent of products from the E. coli sapABCDF (trkE) operon.
Overexpression of Candida albicans secretory aspartyl proteinase 2 and its expression in Saccharomyces cerevisiae do not augment virulence in mice
Authors: Dubois Nathalie , Colina Ana Rosa , Aumont Francine , Belhumeur Pierre , de Repentigny Louis
Published: June 2009

To elucidate the implications of secreted aspartyl proteinase (Sap)2p in the pathogenesis of Candida infections, the SAP2 gene was expressed in Saccharomyces cerevisiae and overexpressed in Candida albicans. The coding region of SAP2, including its signal sequence and propeptide, was amplified by PCR and cloned downstream of the S. cerevisiae or C. albicans ADH1 promoter. Plasmid expression of SAP2 in S. cerevisiae showed that the signal peptide was functional. Integrative transformation of S. cerevisiae and C. albicans was accomplished by homologous recombination within the URA3 locus for S. cerevisiae and the SAP2 locus for C. albicans. Negative control transformants carried plasmids either without the SAP2 insert or with mutated sap2. S. cerevisiae and C. albicans transformants showed similar growth rates to their parental strains or negative controls, when grown in medium containing amino acids. However, in medium with BSA as sole nitrogen source, constitutive expression of SAP2 enabled S. cerevisiae to grow and increased the growth rate of C. albicans. In both media, only S. cerevisiae transformants harbouring SAP2 secreted the enzyme, as confirmed by proteinase activity assays and immunoblotting. When C. albicans was grown in amino acids medium, the enzyme was detected exclusively in transformants constitutively expressing SAP2. However, in BSA medium these strains secreted enzyme earlier and secreted higher amounts of enzyme and total proteinase activity. In pathogenicity studies in intact mice, expression of Sap2p as a sole putative virulence factor did not cause S. cerevisiae to become virulent and constitutive overexpression of SAP2 did not augment virulence of C. albicans in experimental oral or systemic infection.
Intron polymorphism in small subunit rDNA of Nectria galligena
Authors: Crockard M. A. , Fulton C. E. , Bjourson A. J. , Brown Averil E.
Published: June 2009

PCR amplification of the small subunit (SSU) rDNA gene of 40 isolates of Nectria galligena revealed four length polymorphisms. PCR-RFLP analysis of the SSU rDNA gene divided the isolates into four categories similar, but not identical, to categories identified by Southern-RFLP analysis. Nucleotide sequence analysis revealed that isolates in three of the four SSU rDNA (18S) categories possess an intron of 363 bp, 1185 bp or 1423 bp at the NS 7 priming site. Isolates in the fourth category do not possess an intron. The nucleotide sequences of these introns did not contain the core elements characteristic of typical group I introns, nor did they exhibit a group I intron secondary structure. Homology between the introns indicates a common lineage, all three possibly having come from a larger intron and having been formed by subsequent deletions. PCR primers upstream of the SSU rDNA intron region and from within the internal transcribed spacer 1 region amplify a product specific to N. galligena, which will confirm the identity of the pathogen and reveal its 18S category in a single reaction.
The role of environmental factors in the regulation of virulence-determinant expression in Staphylococcus aureus 8325-4
Authors: Chan Pan F. , Foster Simon J.
Published: June 2009

Staphylococcus aureusis a major human pathogen, which produces a variety of virulence determinants. To study environmental regulation of virulence-determinant production, several transcriptional reporter gene fusions were constructed. Chromosomal fusions were made with the staphylococcal accessory regulator(sarA),α-haemolysin(hla),surface protein A(spa)and toxic-shock syndrome toxin-1(tst)genes. The effect of many different environmental conditions on the expression of the fusions was examined. Expression ofhla, tstandspawas strongly repressed in the presence of sodium chloride (1 M) or sucrose (20 mM), butsarAwas relatively unaffected. The global regulator of expression of virulence-determinant genes,agr(accessory gene regulator) was not involved in the salt or sucrose repression. Novobiocin, a DNA gyrase inhibitor, did not significantly increase the expression oftstin wild-type oragrbackgrounds and failed to relieve the salt suppression. Expression oftstwas strongly stimulated in several low-metal environments, independently ofagr,whilstspalevels were significantly reduced by EGTA. The complex, interactive role of environmental factors in the control of expression of the virulence determinants is discussed.
Mechanism of isoniazid uptake in Mycobacterium tuberculosis
Authors: Bardou Fabienne , Raynaud Catherine , Ramos Corinne , Lanéelle Marie Antoinette , Lanŕelle Gilbert
Published: June 2009

Initial transport kinetics of isoniazid (INH) and its uptake at the plateau were studied in Mycobacterium tuberculosis H37Rv under various experimental conditions. The initial uptake velocity increased linearly with INH concentration from 2 x 10-6 M to 10-2 M. It was modified neither by addition of a protonophore that abolished proline transport, nor following ATP depletion by arsenate, which inhibited glycerol uptake, two transport processes taken as controls for secondary active transport and facilitated diffusion, respectively. Microaerobiosis or low temperature (4 °) were without effect on initial uptake. It is thus likely that INH transport in M. tuberculosis proceeds by a passive diffusion mechanism, and that catalase-peroxidase (KatG) is not involved in the actual transport. However, conditions inhibiting KatG activity (high INH concentration, microaerobiosis, low temperature) decrease cell radioactivity at the uptake plateau. It is proposed that INH transport occurs by passive diffusion. KatG is involved only in the intracellular accumulation of oxidized derivatives of INH, especially of isonicotinic acid, which is trapped inside cells in its ionized form. This model explains observed and previously known characteristics of the accumulation of radioactivity in the presence of [14C]INH for various species and strains of mycobacteria.
Bacillus subtilis ORF yybQ encodes a manganese-dependent inorganic pyrophosphatase with distinctive properties: the first of a new class of soluble pyrophosphatase?
Authors: Young Tom W. , Kuhn Nicholas J. , Wadeson Albert , Ward Simon , Burges Dan , Cooke G Dunstan
Published: June 2009

The N-terminal 15 amino acids of the major protein associated with inorganic pyrophosphatase activity in Bacillus subtilis WB600 are identical to those of B. subtilis ORF yybQ. This ORF was amplified from B. subtilis WB600 DNA by PCR and cloned into an overexpression vector in Escherichia coli. Induction of overexpression produced a soluble protein of 34000 Da by SDS-PAGE and by matrix-assisted laser desorption and ionization mass spectrometry. The overexpressed protein had a high specific activity for the hydrolysis of magnesium pyrophosphate, and was specifically and reversibly activated by Mn2+ ions. These properties are identical to those of inorganic pyrophosphatase purified from B. subtilis WB600. No significant similarity was found between the derived sequence of the B. subtilis yybQ-encoded protein and published sequences of identified inorganic pyrophosphatases of Eukarya, Bacteria or Archaea domains. However, there is significant similarity to three putative proteins of unknown function from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from Streptococcus gordonii. The genomes of B. subtilis, M. jannaschii and A. fulgidus do not contain sequences similar to those of hitherto known soluble inorganic pyrophosphatases. The present findings, together with a survey of the properties of inorganic pyrophosphatases from 38 different sources, suggest that the B. subtilis yybQ-encoded protein is the first fully characterized member of a new class of inorganic pyrophosphatase.
Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae
Authors: ter Schure Eelko G. , Silljé Herman H. W. , Raeven Leon J. R. M. , Boonstra Johannes , Verkleij Arie J. , Verrips C. Theo
Published: June 2009

Variations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes. S. cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source. With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold. The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased. Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription. At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations. It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.
Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water availability
Authors: Hallsworth John E. , Magan Naresh
Published: June 2009

The insect pathogens Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus can be effective biocontrol agents when relative humidity (RH) is close to 100%. At reduced water availability, germination of propagules, and therefore host infection, cannot occur. Cultures of B. bassiana, M. anisopliae and P. farinosus were grown under different conditions to obtain conidia with a modified polyol and trehalose content. Conidia with higher intracellular concentrations of glycerol and erythritol germinated both more quickly and at lower water activity (aw) than those from other treatments. In contrast, conidia containing up to 235.7 mg trehalose g-1 germinated significantly (P < 0.05) more slowly than those with an equivalent polyol content but less trehalose, regardless of water availability. Conidia from control treatments did not germinate below 0.951-0.935 aw (. 95.1-93.5% RH). In contrast, conidia containing up to 164.6 mg glycerol plus erythritol g-1 germinated down to 0.887 aw (. 88.7% RH). These conidia germinated below the water availability at which mycelial growth ceases (0.930-0.920 aw). Germ tube extension rates reflected the percentage germination of conidia, so the most rapid germ tube growth occurred after treatments which produced conidia containing the most glycerol and erythritol. This study shows for the first time that manipulating polyol content can extend the range of water availability over which fungal propagules can germinate. Physiological manipulation of conidia may improve biological control of insect pests in the field.
Mutations in the glycerol kinase gene restore the ability of a ptsGHI mutant of Bacillus subtilis to grow on glycerol
Authors: Wehtje Christina , Beijer Lena , Nilsson Rune-Pär , Rutberg Blanka
Published: June 2009

Although glycerol is not taken up via the phosphotransferase system (PTS) in Bacillus subtilis, some mutations that affect the general components of the PTS impair the ability of cells to grow on glycerol. Five revertants of a pts deletion mutant that grow on glycerol were analysed. They were shown to carry mutations in the glycerol kinase gene. These are missense mutations located in parts of the glpK gene that could encode regions important for the activity of glycerol kinase. The results strongly suggest that the main effect of the PTS on glycerol utilization in B. subtilis is mediated via glycerol kinase.
Typing of Staphylococcus aureus strains by PCR-amplification of variable-length 16S-23S rDNA spacer regions: characterization of spacer sequences
Authors: Grtler Volker , Barrie Helen D.
Published: June 2009

To develop a rapid and accurate method of typing large numbers of clinical isolates of Staphylococcus aureus, the spacer region C of the rRNA operon [1391-507 (16S-23S)] was enzymically amplified from 322 strains. When the products were separated by denaturing PAGE, 15 variable-length rrn alleles were demonstrated, ranging in size from 906 to 1223 bp. The variable-length HpaII-digested region C [(region E; 1446-196 (16S-23S)] amplification products were cloned into M13mp18RF to sequence separate variable-length alleles. A total of 17 region E inserts were sequenced, aligned and divided into nine alleles by length (938-1174) and sequence properties. The 16S-23S spacer rDNA varied in length (303-551 bp) and in properties; three alleles contained a tRNAlle gene alone, two alleles contained a tRNAlle and a tRNAAla gene, and four alleles lacked tRNA genes. The sequences of two alleles showed less than 1% variation when isolated from two or three S. aureus strains. The 48 penicillin-and methicillin-sensitive strains were divided into 26 ribotypes; in contrast, the 274 methicillin-resistant S. aureus (MRSA) strains were divided into nine ribotypes (A-I) with 97% typing as either ribotype A or B (rrnL was missing in B). The sequence conservation of the rrn operons argues for the use of the 16S-23S spacer region as a stable and direct indicator of the evolutionary divergence of S. aureus strains.
Transcriptional and translational regulation of the expression of the major outer surface proteins in Lyme disease Borrelia strains
Authors: Jonsson Maria , Sven Bergström
Published: June 2009

The major outer surface proteins of Lyme disease spirochaetes are differentially expressed in different isolates.Borrelia afzeliistrain F1 expresses none, or very low amounts, of the OspA and OspB proteins. To elucidate the mechanisms that control the expression of these abundant surface proteins theospABoperon ofB. afzeliiF1 was cloned, sequenced and compared to the previously sequencedospABoperon ofB. afzeliiACAI andBorrelia burgdorferiB31. The twoB. afzeliistrains showed almost 100% identity at the DNA level, although Coomassie-stained gels and Western blot analyses showed significant variation in the Osp protein content. Transcriptional analysis revealed that the amount ofospABmRNA produced inB. afzeliiF1 varies more than the amount of protein, suggesting that the expression of OspA and OspB proteins is regulated at both the transcriptional and the translational level. Furthermore, the inverse relationship between the transcription ofospCand theospABoperon could indicate coregulation of these separately encoded operons.
Biosynthesis of fluorinated secondary metabolites by Streptomyces cattleya
Authors: Reid Karen A. , Bowden Roy D. , Dasaradhi Lakkaraju , Amin Muhammad R. , Harper David B.
Published: June 2009

The biosynthesis of organofluorine compounds byStreptomyces cattleyaNRRL 8057 was examined using19F NMR spectroscopy. The organism produced 12 mM fluoroacetate and 05 mM 4-fluorothreonine as secondary metabolites when cultured for 28 d on a chemically defined medium containing 2 mM fluoride. Cell suspensions from batch cultures harvested at the growth maximum of 4 d were not capable of fluoride uptake or fluorometabolite biosynthesis, but by 6 d had developed an efficient fluoride-uptake system and biosynthesized the two fluorometabolites in almost equal proportions. As the harvest age increased, the proportion of fluoroacetate to 4-fluorothreonine formed by cell suspensions rose progressively so that 16-d-old cells showed a ratio of 7626 for the two compounds. Fluoride uptake and fluorometabolite production by cell suspensions were highly dependent on pH, with both processes showing a maximum rate at pH 60 but declining rapidly at higher pH values. This decrease was particularly marked in the case of fluoroacetate biosynthesis which was barely detectable at pH 75. Fluoroacetate and 4-fluorothreonine showed only low levels of interconversion by cell suspensions, suggesting that the carbon skeleton of neither was derived by metabolism of the other. The limited interconversion observed is explicable in terms of a small degree of biological defluorination occurring with each compound, followed by reincorporation of the resulting fluoride ion into the organic form by the active fluorinating system, a phenomenon also noted on incubation of cell suspensions with a number of other fluorinated biochemical intermediates. Cell suspensions were supplemented with a variety of amino acids and tricarboxylic acid cycle intermediates to determine the identity of the carbon substrate of the fluorinating system. No compound tested significantly increased the total amount of fluorometabolites formed or altered their relative proportions. However, in studies with14-labelled precursors, the highest level of incorporation into fluoroacetate by cell suspensions was recorded with [U-14C]glycolate, suggesting that this compound or an activated derivative may be the substrate for the fluorinating system in the biosynthesis of fluoroacetate.
Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris
Authors: Hu Nien-tai , Hung Ming-Ni , Liao Chao-Tsai , Lin Ming-Huei
Published: June 2009

The last ORF of an xps gene cluster, designated xpsD, is required for the secretion of extracellular enzymes across the outer membrane in Xanthomonas campestris pv. campestris. It could encode a protein of 759 amino acid residues. A consensus N-terminal lipoprotein signal peptide was revealed from its deduced amino acid sequence. A [3H]palmitate labelling experiment indicated that XpsD was fatty-acylated. Differential extraction with Triton X-100 disclosed that XpsD was fractionated with the outer membrane. Sucrose gradient sedimentation analysis of total membranes also indicated that XpsD was mainly located in the outer membrane. At least part of XpsD is exposed to the cell surface as suggested by trypsin experiment results. Intact cells pretreated with antibody against XpsD could indirectly be labelled with fluorescent agent. When the N-terminal lipoprotein signal peptide was replaced with a nonlipoprotein signal peptide cleavable by signal peptidase I, non-fatty-acylated XpsD was synthesized. Its subcellular location was indistinguishable from that of the fatty-acylated XpsD. Complementation of an xpsD::Tn5 mutant of X. campestris pv. campestris indicated that this non-fatty-acylated XpsD remains functional in extracellular protein secretion. A stable, C-terminal truncated protein, XpsDd414-759, was synthesized from a mutated xpsD gene. Although it stayed associated with the outer membrane and exposed to the cell surface, it no longer could complement the xpsD::Tn5 mutant of X. campestris pv. campestris.
Isolation of regulatory mutants in photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 and partial complementation of a PrrB mutant by the HupT histidine-kinase
Authors: Gomelsky Mark , Kaplan Samuel
Published: June 2009

The photosynthetic bacterium Rhodobacter sphaeroides responds to the transition from aerobiosis to anaerobic photosynthesis by increasing the expression of the photosynthesis genes. Mutants have been isolated based on their inability, following such a transition, to increase transcription of the puc operon encoding the apoproteins of the light-harvesting complex II. Mutant D5, a representative of one mutant class, described here, although remaining photosynthetically competent, produced only low levels of the photosynthetic spectral complexes. Complementation analysis revealed that either the gene for the photosynthesis response regulator prrA or the gene encoding its cognate sensor kinase, prrB, was capable of rescuing this mutant. However, partial complementation of this mutant was achieved by placing in trans additional copies of other defined genes from the cosmid library of R. sphaeroides. We describe this effect in detail, attributable to the hupT gene, which has been proposed to encode a histidine-kinase for the hydrogen uptake system in Rhodobacter capsulatus. The effect of HupT on the expression of the photosynthesis genes was mediated through PrrA and independent of a functioning hydrogen uptake system. Thus, we raise the possibility that HupT can participate in phosphorylation of the heterologous response regulator PrrA by so-called cross-talk and therefore partially compensate for the defect in the mutant described. The observation of cross-talk, together with the complementation analysis, allowed us to assign the original mutation to the prrB gene; this was confirmed by DNA sequencing. Analysis of cross-talk in the wild-type, prrB and prrA genetic backgrounds suggested that besides kinase activity, PrrB may possess phosphatase activity toward PrrA. We also report the cloning, organization and structure of some of the hup genes from R. sphaeroides and construction of a Hup- strain.
Unusual effect of myo-inositol on phospholipid biosynthesis in Cryptococcus neoformans
Authors: Vincent Vicki L. , Klig Lisa S.
Published: June 2009

Cryptococcus neoformansis an opportunistic fungal pathogen which preferentially localizes to the inositol-rich environment of the central nervous system. One of its distinguishing traits is its capacity to catabolize inositol. Inositol is a precursor for the synthesis of phosphatidylinositol (PI). This study demonstrated thatC. neoformanssynthesizes inositol. Three inositolcontaining sphingolipids were identified inC. neoformans:ceramide-(P-inositol)2mannose, ceramide-P-inositol-mannose, and ceramide-P-inositol. These inositol-containing sphingolipids are typical of fungi but not higher eukaryotes. The effect of inositol on the membrane lipid composition ofC. neoformanswas also examined. In contrast to the nonpathogenic yeastSaccharomyces cerevisiae, neither the PI composition nor the synthesis of methylated phospholipids was altered by exogenous inositol. Hence,C. neoformansappears to have a metabolic mechanism for maintaining a steady lipid composition regardless of the inositol in its environment.
A 46 kDa integral membrane protein from Mycobacterium leprae resembles a number of bacterial and mammalian membrane transport proteins
Authors: Oskam Linda , Hartskeerl Rudy A. , Hermans Caroline J. , de Wit Madeleine Y. L. , Jarings Guus H. , Nicholls Robert D. , Klatser Paul R.
Published: June 2009

In this paper we describe the nucleotide sequence of a 3·4 kbp region of theMycobacterium lepraegenome. This region contains an open reading frame of 1290 bp with a coding capacity for a protein of 46179 Da, designated the 38L protein. Using antibodies against part of the 38L protein, we were able to demonstrate that the 38L protein is present in the membrane proteinfractionofM. leprae.The 38L protein showed significant matches with a number of integral membrane proteins involved in the transport of small molecules through the cellular membrane. Among these are a human and a murine protein involved in melanin biosynthesis. The 38L protein might play a role in the hypopigmentation observed in leprosy patients.
Accumulation of the angucycline antibiotic rabelomycin after disruption of an oxygenase gene in the jadomycin B biosynthetic gene cluster of Streptomyces venezuelae
Authors: Yang Keqian , Han Lei , Ayer Stephen W. , Vining Leo C.
Published: June 2009

DNA from a region downstream of and overlapping the polyketide synthase (PKS) gene cluster for jadomycin B biosynthesis in Streptomyces venezuelae was cloned and sequenced. Analysis of the nucleotide sequence located one complete ORF (ORF6), an incomplete one representing the 3' region of ORF4 in the PKS cluster, and a second incomplete one (ORF7). The deduced amino acid sequences for ORFs 6 and 7 resemble those of oxygenases. Since a plausible biosynthetic pathway for jadomycin B includes an angular polyketide intermediate that undergoes oxidative ring fission before condensation with an amino acid, we subcloned one of the presumptive oxygenase genes (ORF6) in a segregationally unstable shuttle vector (pHJL400) and disrupted it by inserting the gene for apramycin resistance. Transformation of S. venezuelae with the disruption vector and selection for apramycin resistance gave mutants blocked in jadomycin biosynthesis. Southern hybridization confirmed that gene replacement had occurred. Cultures of the mutants accumulated a metabolite identified by comparison with an authentic sample as rabelomycin, a non-nitrogenous polyketide-derived antibiotic originally isolated from Streptomyces olivaceus.
Physiological responses of Pseudomonas putida KT2442 to phosphate starvation
Authors: Eberl Leo , Givskov Michael , Sternberg Claus , Møller Søren , Christiansen Gunna , Molin Søren
Published: June 2009

The physiological responses of Pseudomonas putida KT2442 to phosphate starvation were examined with respect to cell morphology, qualitative demonstration of the accumulation of the intracellular storage component poly-3-hydroxyalkanoate (PHA), cellular ATP and ribosome content, and the rate of total protein synthesis. Upon prolonged incubation under phosphate-limiting conditions, the number of viable cells decreased by two to three orders of magnitude during the first 3 weeks. However, after this decline, viability of the cultures remained remarkably constant for many weeks. The cells remained rod-shaped under phosphate starvation conditions with a tendency to swell in parallel with the accumulation of PHA. Protein synthesis and ribosome concentration were gradually reduced, and ATP levels dropped to very low values after the onset of starvation; later, however, there was a return to near-normal ATP concentrations. Evidence was obtained that the strong selective pressure imposed by phosphate deprivation forces the selection of mutants with a competitive advantage. These mutants are able to grow, possibly utilizing nutrients derived from dead cells, and eventually take over the cultures. One frequently encountered mutant formed smaller colonies on rich solidified medium and displayed an altered cell morphology. This mutant was isolated and further characterized. By employing a bioluminescence-based marker system, we demonstrated that this mutant is able to replace wild-type cells in mixed culture experiments. Thus, long-term phosphate-deprived cultures represent dynamic regimes that can undergo population shifts.
Purification of meningococcal lipooligosaccharide by FPLC techniques
Authors: Evans J. S. , Maiden M. C. J.
Published: June 2009

A rapid and efficient method for the preparation of highly pure meningococcal lipo-oligosaccharide (LOS) was developed. This used a Superose 6 column on a FPLC system to purify LOS from phenol-water extracts of cell lysates ofNeisseria meningitidis.The purest LOS preparations, with no detectable protein contamination and less than 0.5% (w/w) residual RNA, were obtained when cell lysates had been treated with RNase ONE before phenol extraction and chromatographic separation. Preparations that had received no ribonuclease treatment had 2-3% residual RNA contamination and predigestion of samples with RNase A, which only partially degraded the RNA present in the crude extracts, resulted in LOS samples contaminated with 15-20% residual RNA. The LOS purified from RNase ONE-treated extracts was highly endotoxic, and showed no reduction in antibody binding or specific endotoxin activity compared to unpurified material. Approximately 80% of the LOS applied to the chromatography column was recovered as purified material.
Novel phosphotransferase genes revealed by bacterial genome sequencing: a gene cluster encoding a putative N-acetylgalactosamine metabolic pathway in Escherichia coli
Authors: Reizer Jonathan , Ramseier Tom M. , Reizer Aiala , Charbit Alain , Saier Milton H.
Published: June 2009

Summary: We have analysed a gene cluster in the 67.4-76.0 min region of theEscherichia colichromosome, revealed by recent systematic genome sequencing. The genes within this cluster include: (1) five genes encoding homologues of theE. colimannose permease of the phosphotransferase system (IIB, IIB', IIC, IIC' and IID); (2) genes encoding a putativeN-acetylgalactosamine 6-phosphate metabolic pathway including (a) a deacetylase, (b) an isomerizing deaminase, (c) a putative carbohydrate kinase, and (d) an aldolase; and (3) a transcriptional regulatory protein homologous to members of the DeoR family. Evidence is presented suggesting that the aldolase-encoding gene within this cluster is the previously designatedkbagene that encodes tagatose-1,6-bisphosphate aldolase. These proteins and a novel IIAMan-like protein encoded in the 2.4-4.1 min region are characterized with respect to their sequence similarities and phylogenetic relationships with other homologous proteins. A pathway for the metabolism ofN-acetylgalactosamine biochemically similar to that for the metabolism ofN-acetylglucosamine is proposed.
The second aconitase (AcnB) of Escherichia coli
Authors: Bradbury Alan J. , Gruer Megan J. , Rudd Kenneth E. , Guest John R.
Published: June 2009

Summary: The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kan
R mutant lacking AcnA, and the corresponding polypeptide identified by activity staining and weak cross-reactivity with AcnA antiserum. The acnB gene was located at 2.85 min (131.6 kb) in a region of the chromosome previously assigned to two unidentified ORFs. Aconitase specific activities were amplified up to fivefold by infection with λacnB phages from the Kohara λ-E. coli gene library, and up to 120-fold (50% of soluble protein) by inducing transformants containing a plasmid (pGS783) in which the acnB coding region is expressed from a regulated T7 promoter. The AcnB protein was purified to 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of M
r 100000 (SDS-PAGE) and 105000 (gel filtration analysis) compared with M
r 93500 predicted from the nucleotide sequence. The sequence identity between AcnA and AcnB is only 17% and the domain organization of AcnA and related proteins (1-2-3-linker-4) is rearranged in AcnB (4-1-2-3).
Inhibition of purified isocitrate lyase identified itaconate and oxalate as potential antimetabolites for the riboflavin overproducer Ashbya gossypii
Authors: Schmidt Georg , Stahmann K.-Peter , Sahm Hermann
Published: June 2009

Summary: A specific isocitrate lyase (ICL) activity of 0.17 U (mg protein)−1 was detected in cultures of the riboflavin-producing fungus Ashbya gossypii during growth on soybean oil. Enzyme activity was not detectable during growth on glucose [<0.005 U (mg protein)−1], indicating a regulation. The enzyme was purified 108-fold by means of ammonium sulphate fractionation, gel filtration and cation-exchange chromatography. SDS-PAGE of the purified protein showed a homogeneous band with an M
r of 66000. The M
r of 254000 determined by gel-filtration chromatography indicated a tetrameric structure of the native protein. The enzyme was found to have a pH optimum for the isocitrate cleavage of 7.0, and the K
m for threo-DL-isocitrate was determined as 550 μ. Enzyme activity was Mg2+− dependent. In regulation studies ICL was weakly inhibited by central metabolites. A concentration of 10 mM phosphoenolpyruvate or 6-phosphogluconate revealed a residual activity of more than 40%. On the other hand, oxalate (K
i: 4 μM) and itaconate (K
i: 170 μM) showed a strong inhibition and may therefore be interesting as antimetabolites.
Cytosine Accumulation as a Measure of the Proton Electrochemical Gradient Acting on the Overexpressed Cytosine Permease of Saccharomyces Cerevisiae
Authors: Eddy A. A. , Hopkins P.
Published: June 2009

The magnitude of the proton gradient (Δμ̄H+) driving solute accumulation inSaccharomyces Cerevisiaehas long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (Δψ). In the present work, the size of the cytosine gradient (Δμ̄cyt) that the yeast generated was used as a measure of the driving gradient (Δμ̄H+). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H+Zcytosine system, Δμ̄cyt, assayed in washed cell suspensions fermenting glucose and containing 0·5 or 50 mM KCI, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, –Δμ̄H+was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered Δμ̄cytto a value roughly equal to the magnitude of the pH gradient (ΔpH). A mathematical model was used to correct the first estimates of Δμ̄H+for the effect of cytosine leakage outside the symport. In such a system, Δμ̄cytcannot exceed the equivalent ratioVmax/KmL, whereVmaxandKmare kinetic parameters of the symport andLis the rate coefficient for leakage. The feasibility of assaying Δμ̄H+depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, –Δμ̄H+during fermentation was estimated to be up to 25 mV larger than Δμ̄cytand at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of ΔpH, Δψwas in turn deduced and compared with the maximum methylamine gradient (Δμ̄M) the yeast formed. The results supported the claim in the literature that, at acid pH, Δμ̄Mis a measure of Δψ.
The Behavioural Response of Anaerobic Rhodobacter Sphaeroides to Temporal Stimuli
Authors: Packer Helen L. , Gauden David E. , Armitage Judith P.
Published: June 2009

The behavioural response of Rhodobacter Sphaeroides to temporal changes in the concentration of chemoeffectors, and to stimuli affecting electron transport, was analysed using tethered cells. Populations of photosynthetically grown tethered cells of R. sphaeroides showed a transient response, a stop followed by adaptation, to a stepwise reduction in the concentration of chemoattractants (such as organic acids or sugars) and terminal electron acceptors. A step-down response was also measured in free swimming cells to a reduction in light intensity. As this response appears to apply to all effectors this suggests that there is a sensory pathway in anaerobically grown R. sphaeroides which responds primarily to a reduction in a stimulus. R. sphaeroides therefore responds when moving down a concentration gradient. This is the inverse of the classical Escherichia coli–Salmonella typhimurium model of bacterial sensory behaviour in which bacteria respond primarily when there is an increase in an attractant concentration, i.e. when moving up a gradient. R. sphaeroides does show a chemokinetic response to an increase in concentration of a limited number of compounds but this response is sustained and accompanied by an increase in the rate of flagellar rotation and therefore not simply equivalent to the transient increase in smooth swimming measured in E. coli on addition of an attractant.
A protein having similarity with methylmalonyl-CoA mutase is required for the assimilation of methanol and ethanol by Methylobacterium extorquens AM1
Authors: Smith Loraine M. , Meijer Wim G. , Dijkhuizen Lubbert , Goodwin Pat M.
Published: June 2009

A 4·0 kb region of Methylobacterium extorquens AM1 DNA which complements three mutants unable to convert acetyl-CoA to glyoxylate (and therefore defective in the assimilation of methanol and ethanol) has been isolated and sequenced. It contains two ORFs and the 3′-end of a third one. The mutations in all three mutants mapped within the first ORF, which was designated meaA ; it encodes a protein having similarity with methylmalonyl- CoA mutase. However, methylmalonyl-CoA mutase was measured in extracts of one of the mutants and the specific activity was found to be similar to that in extracts of wild-type cells. Furthermore, although the predicted meaA gene product has the proposed cobalamin-binding site, it does not contain a highly conserved sequence (RIARNT) which is present in all known methylmalonyl- CoA mutases; meaA may therefore encode a novel vitamin-B12-dependent enzyme. The predicted polypeptide encoded by the second ORF did not have similarity with any known proteins. The partial ORF encoded a protein with similarity with the 3-oxoacyl-[acyl-carrier-protein] reductases; it was not essential for growth on methanol or ethanol.