- Volume 146, Issue 8, 2000
Volume 146, Issue 8, 2000
- Review Article
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Families of transmembrane transporters selective for amino acids and their derivatives
More LessThe information presented in this review was initially prepared for presentation at the FASEB meeting on amino acid transport held in Copper Mountain, Colorado, June 26–July 1, 1999 and was updated in January 2000 following the meeting of the Transport Nomenclature Panel of the International Union of Biochemistry and Molecular Biology (IUBMB) in Geneva, November 28–30, 1999. The system of classification described in this review reflects the recommendations of that panel.
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- Microbiology Comment
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- Biochemistry
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Enzymically mediated bioprecipitation of uranium by a Citrobacter sp.: a concerted role for exocellular lipopolysaccharide and associated phosphatase in biomineral formation
More LessA Citrobacter sp. accumulated uranyl ion () via precipitation with phosphate ligand liberated by phosphatase activity. The onset and rate of uranyl phosphate deposition were promoted by , forming NH4UO2PO4, which has a lower solubility product than NaUO2PO4. This acceleration decoupled the rate-limiting chemical crystallization process from the biochemical phosphate ligand generation. This provided a novel approach to monitor the cell-surface-associated changes using atomic-force microscopy in conjunction with transmission electron microscopy and electron-probe X-ray microanalysis, to visualize deposition of uranyl phosphate at the cell surface. Analysis of extracted surface materials by 31P NMR spectroscopy showed phosphorus resonances at chemical shifts of 0·3 and 2·0 p.p.m., consistent with monophosphate groups of the lipid A backbone of the lipopolysaccharide (LPS). Addition of to the extract gave a yellow precipitate which contained uranyl phosphate, while addition of Cd2+ gave a chemical shift of both resonances to a single new resonance at 3 p.p.m. Acid-phosphatase-mediated crystal growth exocellularly was suggested by the presence of acid phosphatase, localized by immunogold labelling, on the outer membrane and on material exuded from the cells. Metal deposition is proposed to occur via an initial nucleation with phosphate groups localized within the LPS, shown by other workers to be produced exocellularly in association with phosphatase. The crystals are further consolidated with additional, enzymically generated phosphate in close juxtaposition, giving high loads of LPS-bound uranyl phosphate without loss of activity and distinguishing this from simple biosorption, or periplasmic or cellular metal accumulation mechanisms. Accumulation of ‘tethered’ metal phosphate within the LPS is suggested to prevent fouling of the cell surface by the accumulated precipitate and localization of phosphatase exocellularly is consistent with its possible functions in homeostatis and metal resistance.
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- Development And Structure
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The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum
The terminal event of spore differentiation in the cellular slime mould Dictyostelium discoideum is the assembly of the spore coat, which surrounds the dormant amoeba and allows the organism to survive during extended periods of environmental stress. The spore coat is a polarized extracellular matrix composed of glycoproteins and cellulose. The process of spore coat formation begins by the regulated secretion of spore coat proteins from the prespore vesicles (PSVs). Four of the major spore coat proteins (SP96, PsB/SP85, SP70 and SP60) exist as a preassembled multiprotein complex within the PSVs. This complete complex has an endogenous cellulose-binding activity. Mutant strains lacking either the SP96 or SP70 proteins produce partial complexes that do not have cellulose-binding activity, while mutants lacking SP60 produce a partial complex that retains this activity. Using a combination of immunofluorescence microscopy and biochemical methods we now show that the lack of cellulose-binding activity in the SP96 and SP70 mutants results in abnormally assembled spore coats and spores with greatly reduced viability. In contrast, the SP60 mutant, in which the PsB complex retains its cellulose-binding activity, produces spores with apparently unaltered structure and viability. Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure. These results support the idea that the cellulose-binding activity associated with the complete PsB complex plays an active role in the assembly of the spore coat.
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Fruiting body development in Coprinus cinereus: regulated expression of two galectins secreted by a non-classical pathway
More LessThe GenBank accession number for the sequence reported in this paper is AF130360
Fruiting body formation in the basidiomycete Coprinus cinereus is a developmental process that occurs as a response of the mycelium to external stimuli. First, localized, highly branched hyphal structures (knots) are formed as a reaction to nutritional depletion. Hyphal-knot formation is repressed by light; however, light signals are essential for the development of the hyphal knot into an embryonic fruiting body (primordium) as well as karyogamy, meiosis and fruiting body maturation. The role of the different environmental signals in the initial phases of fruiting body development was analysed. It was observed that two fungal galectins, Cgl1 and Cgl2, are differentially regulated during fruiting body formation. cgl2 expression initiated in early stages of fruiting body development (hyphal knot formation) and was maintained until maturation of the fruiting body, whereas cgl1 was specifically expressed in primordia and mature fruiting bodies. Immunofluorescence and immuno-electron microscopy studies detected galectins within specific fruiting body tissues. They localized in the extracellular matrix and the cell wall but also in membrane-bound bodies in the cytoplasm. Heterologous expression of Cgl2 in Saccharomyces cerevisiae indicated that secretion of this protein occurred independently of the classical secretory pathway.
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- Environmental Microbiology
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Gas vesicle genes in Planktothrix spp. from Nordic lakes: strains with weak gas vesicles possess a longer variant of gvpC
More LessThe GenBank accession numbers for the new sequences in this paper are AJ253125–253133.
In cyanobacteria of the genus Planktothrix, there are three length variants of gvpC, the gene that encodes the outer protein of the gas vesicle. Sequence analyses indicated that the three allelic variants of gvpC differ principally in the presence or absence of a 99 nt and a 213 nt section. Strains with the new variant, gvpC 28, which encodes a 28 kDa form of GvpC, produce gas vesicles that collapse at the relatively low critical pressure (p c) of 0·61–0·75 MPa. The authors have identified 12 classes of gvp genotypes that differ in the number and arrangement of alternating gvpA–gvpC genes and in the presence of ΩC, a fragment of gvpC. The gvpC 28 gene was found to be the most common variant of gvpC amongst 71 strains of Planktothrix isolated from Nordic lakes: 34 strains contained only gvpC 28; 22 strains, which possessed only the shorter gvpC 20 gene, produced gas vesicles with a higher p c of 0·76–0·91 MPa; and 15 strains, which possessed both gvpC 20 and gvpC 28, also produced the stronger gas vesicles. Genotypes with only the gvpC 28 genes were more common amongst green Planktothrix strains (33 out of 38) than red strains (one out of 33). It is suggested that there is competition between the strains producing the two types of gas vesicles, with the stronger forms favoured in lakes deeper than 60 m, in which the combination of cell turgor pressure and hydrostatic pressure can collapse the weaker gas vesicles. The fact that none of the Nordic lakes are deeper than 67 m would explain the absence of the gvpC 16-containing strains that produce even narrower gas vesicles of p c 1·0–1·2 MPa, which are common in the much deeper Lake Zürich.
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Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR
More LessColony PCR and semi-nested PCR techniques were employed for screening polyhydroxyalkanoate (PHA) producers isolated from the environment. Three degenerate primers were designed based on multiple sequence alignment results and were used as PCR primers to detect PHA synthase genes. Optimized colony PCR conditions were achieved by adding 3% DMSO combined with 1 M betaine to the reaction mixture. The sensitivity limit of the colony PCR was 1× 105 viable cells for Ralstonia eutropha. Nineteen PHA-positive bacteria were used to evaluate this PCR protocol; fifteen of the nineteen could be detected by colony PCR, and the other four could be detected by applying semi-nested PCR detection following colony PCR. In a preliminary screening project, 38 PHA-positive strains were isolated from environmental samples by applying the PCR protocol, and their phenotype was further confirmed by Nile blue A staining assay. By combining the colony PCR and semi-nested PCR techniques, a rapid, reliable and highly accurate detection method has been developed for detecting PHA producers. This protocol is suitable for screening large numbers of environmental isolates. The PHA accumulation ability of well-separated colonies isolated from environmental samples can be directly validated by PCR with no further culturing or chromosomal DNA extraction procedures. In addition to its application to the screening of wild-type isolates, the individual PCR-amplified product is also suitable as a specific probe for PHA operon cloning. The results suggest that the application of this PCR protocol for rapid detection of PHA producers from the environment is plausible.
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- Genetics And Molecular Biology
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A novel Cellvibrio mixtus family 10 xylanase that is both intracellular and expressed under non-inducing conditions
The GenBank accession numbers for the sequences described in this paper are AF049493 and AF168359 for xynC and xynG, respectively.
Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in λZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M r of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M r of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.
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Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement
More LessProgress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed: a marked deletion of the plcABC cluster, which encodes three phospholipases C; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin); and a double unmarked mutant tlyAΔ plcABCΔ. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by PacI restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PacI site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.
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Phylogenetic associations of ISAa1 and IS150-like insertion sequences in Actinobacillus actinomycetemcomitans
More LessThe distribution and number of two insertion sequences (ISs), ISAa1 and an IS150-like element, in the genomes of a collection of Actinobacillus actinomycetemcomitans strains previously subjected to population genetic analysis were determined to obtain information about their stability and biological significance. The hybridization patterns revealed that these IS elements are widespread in the genome of A. actinomycetemcomitans strains and that their occurrence agrees with the overall population structure of the species. While the patterns of ISAa1 showed significant evolutionary stability, the IS150-like element showed evidence of intra-genomic variability even within members of the previously identified high-toxicity JP2 clone. Searching of the available genome sequence of strain HK1651 of the JP2 clone (www.genome.ou.edu/act.html) revealed close proximity of the IS elements to housekeeping genes, but no evidence of structural disruption of genes or integrations that may be presumed to influence pathogenic potential.
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The ABC transporter AtrB from Aspergillus nidulans mediates resistance to all major classes of fungicides and some natural toxic compounds
More LessThis paper reports the functional characterization of AtrBp, an ABC transporter from Aspergillus nidulans. AtrBp is a multidrug transporter and has affinity to substrates belonging to all major classes of agricultural fungicides and some natural toxic compounds. The substrate profile of AtrBp was determined by assessing the sensitivity of deletion and overexpression mutants of atrB to several toxicants. All mutants showed normal growth as compared to control isolates. ΔatrB mutants displayed increased sensitivity to anilinopyrimidine, benzimidazole, phenylpyrrole, phenylpyridylamine, strobirulin and some azole fungicides. Increased sensitivity to the natural toxic compounds camptothecin (alkaloid), the phytoalexin resveratrol (stilbene) and the mutagen 4-nitroquinoline oxide was also found. Overexpression mutants were less sensitive to a wide range of chemicals. In addition to the compounds mentioned above, decreased sensitivity to a broader range of azoles, dicarboximides, quintozene, acriflavine and rhodamine 6G was observed. Decreased sensitivity in overexpression mutants negatively correlated with levels of atrB expression. Interestingly, the overexpression mutants displayed increased sensitivity to dithiocarbamate fungicides, chlorothalonil and the iron-activated antibiotic phleomycin. Accumulation of the azole fungicide [14C]fenarimol by the overexpression mutants was lower as compared to the parental isolate, demonstrating that AtrBp acts by preventing intracellular accumulation of the toxicant. Various metabolic inhibitors increased accumulation levels of [14C]fenarimol in the overexpression mutants to wild-type levels, indicating that reduced accumulation of the fungicide in these mutants is due to increased energy-dependent efflux as a result of higher pump capacity of AtrBp.
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Primary sequence and enzymic properties of two modular endoglucanases, Cel5A and Cel45A, from the anaerobic fungus Piromyces equi
More LessThe EMBL accession numbers for the sequences reported in this paper are AJ277482 and AJ277483.
Two endoglucanase cDNAs, designated cel5A and cel45A, were isolated from a cDNA library of the anaerobic fungus Piromyces equi. Sequence analysis revealed that cel5A has an open reading frame of 5142 bp and encodes a 1714 amino acid modular enzyme, Cel5A, with a molecular mass of 194847 Da. Cel5A consists of four catalytic domains homologous to family-5 glycosyl hydrolases, two C-terminal dockerins and one N-terminal dockerin. This is the first report of a complete gene containing tandem repeats of family-5 catalytic domains. The cDNA cel45A has an open reading frame of 1233 bp and encodes a 410 amino acid modular enzyme, Cel45A, with a molecular mass of 44380 Da. The catalytic domain, located at the C terminus, is homologous to the family-45 glycosyl hydrolases. Cel45A is the first family-45 enzyme to be described in an anaerobe. The presence of dockerins at the N and C termini of Cel5A and at the N terminus of Cel45A implies that both enzymes are part of the high-molecular-mass cellulose-degrading complex produced by Piromyces equi. The catalytic domain nearest the C terminus of Cel5A and the catalytic domain of Cel45A were hyperexpressed as thioredoxin fusion proteins, Trx-Cel5A′ and Trx-Cel45A′, and subjected to biochemical analysis. Trx-Cel5A′ has a broad substrate range, showing activity against carboxymethylcellulose, acid-swollen cellulose, barley β-glucan, lichenin, carob galactomannan,p-nitrophenyl β-D-cellobiopyranoside and xylan. Trx-Cel45A′ is active against carboxymethylcellulose, acid-swollen cellulose and the mixed linkage glucans, barley β-glucan and lichenin.
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- Genomics
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The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations
More LessIn this paper an analysis of 175 currently sequenced transport proteins that comprise the amino acid/polyamine/organocation (APC) superfamily is reported. Members of this superfamily fall into 10 well-defined families that are either prokaryote specific, eukaryote specific or ubiquitous. Most of these proteins exhibit 12 probable transmembrane spanners (TMSs), but members of two of these families deviate from this pattern, exhibiting 10 and 14 TMSs. All members of these families are tabulated, their functional properties are reviewed and phylogenetic/sequence analyses define the evolutionary relationships of the proteins to each other. Evidence is presented that the APC superfamily may include two other currently recognized families that exhibit greater degrees of sequence divergence from APC superfamily members than do the proteins of the 10 established families from each other. At least some of the protein members of these two distantly related families exhibit 11 established TMSs. Altogether, the APC superfamily probably includes 12 currently recognized families with members that exhibit exclusive specificity for amino acids and their derivatives but which can possess 10, 11, 12 or 14 TMSs per polypeptide chain.
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Phylogeny of related functions: the case of polyamine biosynthetic enzymes
More LessGenome annotation requires explicit identification of gene function. This task frequently uses protein sequence alignments with examples having a known function. Genetic drift, co-evolution of subunits in protein complexes and a variety of other constraints interfere with the relevance of alignments. Using a specific class of proteins, it is shown that a simple data analysis approach can help solve some of the problems posed. The origin of ureohydrolases has been explored by comparing sequence similarity trees, maximizing amino acid alignment conservation. The trees separate agmatinases from arginases but suggest the presence of unknown biases responsible for unexpected positions of some enzymes. Using factorial correspondence analysis, a distance tree between sequences was established, comparing regions with gaps in the alignments. The gap tree gives a consistent picture of functional kinship, perhaps reflecting some aspects of phylogeny, with a clear domain of enzymes encoding two types of ureohydrolases (agmatinases and arginases) and activities related to, but different from ureohydrolases. Several annotated genes appeared to correspond to a wrong assignment if the trees were significant. They were cloned and their products expressed and identified biochemically. This substantiated the validity of the gap tree. Its organization suggests a very ancient origin of ureohydrolases. Some enzymes of eukaryotic origin are spread throughout the arginase part of the trees: they might have been derived from the genes found in the early symbiotic bacteria that became the organelles. They were transferred to the nucleus when symbiotic genes had to escape Muller’s ratchet. This work also shows that arginases and agmatinases share the same two manganese-ion-binding sites and exhibit only subtle differences that can be accounted for knowing the three-dimensional structure of arginases. In the absence of explicit biochemical data, extreme caution is needed when annotating genes having similarities to ureohydrolases.
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- Pathogenicity And Medical Microbiology
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Survival in experimental Candida albicans infections depends on inoculum growth conditions as well as animal host
More LessEvidence is presented that the growth medium used to prepare a Candida albicans challenge inoculum is a significant factor determining the ability of a fungus strain to gain an initial invasive hold immediately after injection into an animal host, and thus determining gross strain lethality. Three C. albicans strains, one known to be attenuated in virulence, were grown in two broth media and injected intravenously at different doses into female NMRI mice and male albino guinea pigs. For each fungus strain and challenge dose, survival was longer from inocula grown in a diluted, buffered peptone-based broth than from inocula grown in Sabouraud glucose broth. When animals were challenged intravenously with yeast doses adjusted to give the same mean survival time regardless of strain or growth medium, the progression of fungus tissue burdens (c.f.u. g−1) in kidneys, lungs, liver, spleen and brain samples was broadly similar for all three C. albicans strains but differed between the two animal hosts. The morphological form of C. albicans recovered from infected tissues differed at the level of both the fungus strain and the host tissue. Use of survival-standardized inocula provides a means of distinguishing differences in progression of experimental disseminated Candida infections that are related to the infecting strain from those related to the animal host.
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Pseudomonas aeruginosa cystic fibrosis clinical isolates produce exotoxin A with altered ADP-ribosyltransferase activity and cytotoxicity
More LessThe GenBank accession numbers for the toxA sequences are: strain 4384, AF227419; strain 5154, AF227420; strain 5166, AF227421; strain 5552, AF227422; strain 5585, AF227423; strain 5588, AF227424.
The role of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in the lung infections of cystic fibrosis (CF) patients is not well understood. Transcript-accumulation studies of bacterial populations in sputum reveal high levels of transcription of toxA, which encodes ETA, in some patients with CF. However, in general, tissue damage in the lungs of patients with CF does not seem to be consistent with a high level of expression of active ETA. To address this discrepancy the authors analysed the production and activity of ETA produced by a number of P. aeruginosa CF isolates. One CF isolate, strain 4384, transcribed toxA at levels similar to the hypertoxigenic strain PA103 but produced an ETA with reduced ADP-ribosyltransferase (ADPRT) activity. Complementation in trans of strain 4384 with the wild-type toxA and a mixed toxin experiment suggested the absence of inhibitory accessory factors within this strain. The toxA gene from strain 4384 was cloned and sequenced, revealing only three mutations in the gene, all within the enzymic domain. The first mutation changed Ser-410 to Asn. The second mutation was located within an α-helix, altering Ala-476 to Glu. The third mutation, Ser-515 to Gly, was found at the protein surface. To date, Ser-410, Ala-476 and Ser-515 have not been reported to play a role in the ADPRT activity of ETA. However, it may be the combination of these mutations that reduces the enzymic activity of ETA produced by strain 4384. Expression of 4384 toxA and wild-type toxA in an isogenic strain revealed that 4384 ETA had 10-fold less ADPRT activity than wild-type ETA. ETA purified from strain 4384 also demonstrated 10-fold less ADPRT activity as compared to wild-type ETA. Cytotoxicity assays of purified ETA from strain 4384 indicated that the cytotoxicity of 4384 ETA is not reduced; it may be slightly more toxic than wild-type ETA. Analysis of five other CF isolates revealed a similar reduction in ADPRT activity to that seen in strain 4384. Sequence analysis of the enzymic domain of toxA from the five CF strains identified a number of mutations that could account for the reduction in ADPRT activity. These results suggest that some CF isolates produce an ETA with reduced enzymic activity and this may partially explain the pathogenesis of chronic lung infections of CF due to P. aeruginosa.
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The membrane phospholipids of Neisseria meningitidis and Neisseria gonorrhoeae as characterized by fast atom bombardment mass spectrometry
The phospholipids of Neisseria meningitidis and Neisseria gonorrhoeae were characterized by fast atom bombardment (FAB)-MS and GLC-MS. The major phospholipids were phosphatidylethanolamine (PE), followed by phosphatidylglycerol (PG), with minor amounts of phosphatidic acid (PA) and trace levels of cardiolipin (DPG). All of the phospholipid preparations were variable in their fatty acyl substituents, which included C16:1, C16:0, C18:1, C14:0, C14:1 and C12:0. By MS/MS analysis, all pathogenic Neisseria spp. phospholipids contained a saturated fatty acyl substituent and either a saturated or unsaturated fatty acyl substituent in the sn-1 and sn-2 positions, respectively. Compared with enteric bacterial species, the phospholipids of N. meningitidis and N. gonorrhoeae have increased levels of phospholipids with short-chain fatty acyl residues (i.e. increases in C12:0, C14:1 and C14:0) and variable amounts of C18:1. The percentage of total PE and PG molecules with the shorter-chain fatty acids ranges from 35 to 47% and 42 to 66%, respectively, for N. meningitidis while these respective values are <10% and <5% for Escherichia coli. The variability and variety of meningococcal and gonococcal phospholipids suggest novel genetic mechanisms of neisserial phospholipid assembly and regulation, which may be important for the biology and pathogenesis of N. meningitidis and N. gonorrhoeae.
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Interactions between Streptococcus suis serotype 2 and different epithelial cell lines
More LessStreptococcus suis is an important swine pathogen responsible for cases of sudden death, septicaemia, meningitis, endocarditis and pneumonia. It is also recognized as a zoonotic agent in people occupationally exposed to pigs or pig products. Knowledge on virulence factors of S. suis serotype 2 is limited and the pathogenesis of the infection is poorly understood. It has been suggested that the disease due to S. suis serotype 2 begins with colonization of the nasopharyngeal epithelium, followed by either spread within the respiratory tract or invasion of the bloodstream. The mechanisms involved in the access of bacteria from the bloodstream to the central nervous system are unknown. It is possible that epithelial cells of the choroid plexus also play an important role in the pathogenesis of the meningitis. Different interactions (adhesion, invasion and toxic effects) of S. suis serotype 2 with epithelial cell lines [LLC-PK1, PK(15), A549, HeLa and MDCK] were studied and compared to those of a human pathogen which also causes meningitis, group B Streptococcus (GBS). The results showed that S. suis serotype 2, in contrast to GBS, is able to adhere to but not to invade epithelial cells. The adhesin(s) involved seem(s) to be partially masked by the capsule and are a part of the cell wall. The haemolysin produced by S. suis serotype 2 is responsible for a toxic effect observed on epithelial cells. The results described give additional evidence that pathogenesis of the infection differs between S. suis and GBS. In particular, it is possible that suilysin-positive S. suis strains use adherence and cell injury, as opposed to direct cellular invasion, as part of a complicated multistep process which leads to bacteraemia and meningitis in pigs.
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- Physiology And Growth
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Heterogeneous distribution of lysine 6-aminotransferase during cephamycin C biosynthesis in Streptomyces clavuligerus demonstrated using green fluorescent protein as a reporter
More LessThe cellular distribution of the cephamycin biosynthetic enzyme lysine 6-aminotransferase (LAT) has been studied in Streptomyces clavuligerus hyphae by confocal microscopy using the S65T mutant of green fluorescent protein (GFP) as a reporter. LAT mediates the first committed step in the biosynthesis of the secondary metabolite cephamycin C by S. clavuligerus. The enzymic activity of LAT varies with time during the growth of S. clavuligerus in liquid medium. To investigate if this temporal variation occurs uniformly amongst all hyphae, S. clavuligerus was transformed with a plasmid containing the LAT-encoding gene translationally fused to the GFP-encoding gene. The LAT–GFP fusion product displayed fluorescence spectral characteristics of GFP, and showed similar temporal characteristics of LAT activity compared to the wild-type strain of S. clavuligerus. The transformed strain exhibited a heterogeneous distribution of fluorescence in mycelia grown in liquid cultures. This distribution varied significantly as the batch progressed: only a fraction of the mycelia fluoresced in the early growth phase, whereas nearly all hyphae fluoresced by the late growth phase. Thereafter, a non-uniform distribution of fluorescence was again observed in the declining growth phase. A large fraction of the non-fluorescent cells in the declining growth phase were found to be non-viable. Observations of S. clavuligerus colonies grown on solid agar also showed variation of LAT–GFP expression at different stages of growth. These observations in the solid phase can be explained in terms of nutrient deprivation and signalling molecules. The results suggest that physiological differentiation of S. clavuligerus mycelia leading to cephamycin C biosynthesis is both temporally and spatially distributed. The findings also revealed that the observed heterogeneity was independent of the position of individual cell compartments within the hypha. The potential of GFP as a reporter for the quantitative study of cephamycin biosynthesis at the cellular level has also been demonstrated.
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Environmental regulation of glycosidase and peptidase production by Streptococcus gordonii FSS2
More LessThe synthesis of cell-associated and secreted proteins by Streptococcus gordonii FSS2, an infective endocarditis (IE) isolate, was influenced by both environmental pH and carbon source. Controlling the pH at 7·5 in stirred batch cultures showed that cell-associated and secreted protein concentrations were increased during late exponential and stationary phase by 68% and 125%, respectively, compared with similar cultures without pH control. The expression of five glycosidase and eight peptidase activities were examined using fluorogen-labelled synthetic substrates. Enzyme activities were significantly down-regulated during exponential growth, increasing during stationary phase (P<0·01) whether the culture pH was controlled at pH 7·5 or allowed to fall naturally to pH 4·4. Culture-supernatant activities were significantly increased (P<0·05) when the pH was maintained at 6·0 or 7·5, indicating modulation of enzyme activity by pH. Growth under nitrogen-limitation/glucose-excess conditions resulted in a significant repression of cell-associated glycosidase activities (P<0·01), whilst in the supernatant, activities were generally reduced. The expression of peptidase activities in the culture supernatant did not significantly change. The results suggest a possible role for catabolite repression by glucose in regulating enzyme expression. When S. gordonii FSS2 was cultured with 50% (v/v) added heat-inactivated foetal bovine serum, several cell-associated enzyme activities increased initially but were then reduced as the culture time was extended to 116 h. Culture-supernatant enzyme activities (N-acetyl-β-D-glucosaminidase, N-acetyl-β-D-galactosaminidase, thrombin, Hageman factor, collagenase and chymotrypsin), however, were significantly increased (P<0·01) over the same time period. The findings indicated that most of the important glycosidases synthesized by S. gordonii FSS2 were down-regulated by acid growth conditions and may also be subject to catabolite repression by glucose but conversely may be up-regulated by growth in serum. These results may have implications for streptococcal growth in an IE vegetation and in the mouth between meals or during sleep.
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Generation of Lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities
Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N- and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in the processing of KGP. A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antibiotics, in both complex and chemically defined media. The KGP activity of E8, grown in the absence of tetracycline, in whole cultures and in culture supernatants (up to 6 d) was identical to levels in W50. However, in the presence of tetracycline in the growth medium, the level of KGP was reduced to 50% of levels present in whole cultures of W50. Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this effect is most likely to be on the synthesis of Kgp polypeptide. K1A was also stable in the absence of antibiotics but was unable to pigment, and remained straw-coloured throughout growth. RGP activity in whole cultures of K1A was identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50% of levels present in W50. Thus, although KGP is not required for generation of RGP activity from RgpA and RgpB polypeptides, its absence affects the release/transport of RGP into culture supernatant.
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Expression of the phospho-β-glycosidase ArbZ from Lactobacillus delbrueckii subsp. lactis in Lactobacillus helveticus: substrate induction and catabolite repression
More LessArbZ from Lactobacillus delbrueckii subsp. lactis was previously shown to enable utilization of the β-glucoside arbutin by Escherichia coli. The arbZ gene was cloned and expressed in the industrially used β-glucoside-negative strain Lactobacillus helveticus 3036(62). The transformants were able to ferment not only arbutin, but also cellobiose, salicin and methyl-β-glucoside (MβGlc). Cleavage of β-glucosides by the transformants depended on the integrity of the cytoplasmic membrane, whereas in cell-free extracts only C6-phosphorylated substrates were hydrolysed. This suggested that ArbZ is a phospho-β-glycosidase. ArbZ activity in transformants of Lb. helveticus was subject to substrate induction mediated by the β-glucosides arbutin, salicin and MβGlc, whereas cellobiose or the β-galactoside lactose had no inducing effect. Northern blot analysis proved that induction by MβGlc was due to enhanced transcription of arbZ. Catabolite repression of arbZ induction was observed with glucose, mannose, fructose and galactose. The induction kinetics observed in the presence of these sugars indicated that at least two different mechanisms are operative in catabolite repression of arbZ in Lb. helveticus.
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Threshold level of protein kinase A activity and polarized growth in Mucor rouxii
More LessA model system to study the involvement of cAMP-mediated regulation of a cellular process such as hyphal morphogenesis was investigated. Impairment of polarized growth was observed when Mucor rouxii sporangiospores were grown in the presence of N 6-cAMP analogues and of SQ 65,442, a cAMP phosphodiesterase inhibitor. Together with an effect on isodiametric growth, there was increased pigmentation, increased cell fragility and loss of cell adhesiveness. The total effect on morphology was attained even after adding the compounds shortly before germ-tube emergence; when added after this time growth continued in a non-polarized form and rounding of the germ tip was observed. The morphological effect was observed under all the nutritional and environmental conditions studied (aerobic conditions and defined medium with maltose or glucose, Casamino acids medium with glucose, or rich medium; anaerobic conditions with rich medium; and following a shift from anaerobiosis to aerobiosis). The time of germ-tube emergence, and the size of the cell at this time, was dependent on the growth medium. Protein kinase A (PKA) specific activity was followed during the germination process under three growth conditions. It was found that the total activity of PKA correlated with differentiation and not with growth, and that the total specific activity at the time of germination was the same, independent of the culture medium. The time of germ-tube emergence correlated with the time of attainment of a threshold level of PKA total specific activity. The concentration of dibutyryl-cAMP needed to promote isodiametric growth correlated with the total units of activity of PKA to be activated per cell. It was concluded that PKA is involved in the morphogenetic process of the fungus grown under all the nutritional and ambient conditions tested.
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- Plant-Microbe Interactions
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Characterization of a tonB mutation in Erwinia chrysanthemi 3937: TonB Ech is a member of the enterobacterial TonB family
More LessThe GenBank accession number for the sequence reported in this paper is Y15888.
The pectinolytic enterobacterium Erwinia chrysanthemi 3937 causes a systemic disease in its natural host, the African violet (Saintpaulia ionantha). It produces two structurally unrelated siderophores, chrysobactin and achromobactin. Chrysobactin makes a large contribution to invasive growth of the bacterium in its host. Insertion mutants of a chrysobactin-defective strain were constructed and screened on the universal CAS-agar medium used for siderophore detection. A set of mutants affected in the production of achromobactin were identified. This paper describes a mutant affected in the transport of all the ferrisiderophores used by the bacterium as iron sources. Molecular analysis revealed that the insertion mutation disrupts the tonB gene. The predicted Er. chrysanthemi TonB protein has a molecular mass of 27600 Da and shares 20–58% identity with the TonB proteins from 20 other bacterial species. The pathogenicity of the tonB mutant was assessed by inoculation of African violets. The impairment in the spread of symptoms was similar in the tonB mutant to that in chrysobactin-defective mutants. However, the pectinolytic activity, the major pathogenicity determinant in Er. chrysanthemi, appeared to be stimulated twofold in the tonB mutant.
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Analysis of aberrant virulence of Gibberella zeae following transformation-mediated complementation of a trichothecene-deficient (Tri5) mutant
More LessGibberella zeae causes wheat ear blight and produces trichothecene toxins in infected grain. In previous studies, trichothecene production in this fungus was disabled by specific disruption of the trichodiene synthase gene (Tri5) and was restored by two methods: gene reversion and transformation-mediated mutant complementation. In previous field tests of wheat ear blight, trichothecene-nonproducing mutants were less virulent than the wild-type progenitor strain from which they were derived. Trichothecene-producing revertants also were restored to wild-type levels of virulence. In contrast, in the field test of wheat ear blight reported here, trichothecene-producing strains obtained by Tri5 mutant complementation were not restored to wild-type levels of virulence. The complemented mutants showed a slightly reduced radial growth compared to the wild-type strain, but otherwise appeared normal in morphology, pigmentation and sexual fertility. Genetic analysis indicated that the aberrant virulence of a complemented mutant was likely due to non-target effects that occurred during the process of transforming the trichothecene-nonproducing mutant with Tri5. These results confirm previous findings that trichothecenes contribute to the virulence of G. zeae, but also demonstrate that manipulating this fungus in the laboratory may cause it to undergo subtle changes that reduce its virulence.
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Overproduction of an inducible extracellular serine protease improves biological control of Pythium ultimum by Stenotrophomonas maltophilia strain W81
More LessStenotrophomonas maltophilia W81 can protect sugar beet against Pythium-mediated damping-off disease through the production of an extracellular protease. Here, the proteolytic enzyme of W81 was purified by anion-exchange chromatography and characterized as a serine protease. The purified enzyme was fungicidal against Pythium ultimum in vitro. Its synthesis was inducible by casein in W81, and mutagenesis of this strain using the luciferase (luxAB) reporter transposon Tn5-764cd resulted in the isolation of two mutant derivatives (W81M3 and W81M4) capable of producing significantly increased levels of extracellular protease in the presence of casein. Strain W81M4 also exhibited increased chitinolytic activity. The luxAB fusions in strains W81M3 and W81M4 were highly expressed in the absence of casein but not in its presence, suggesting that the corresponding loci were involved in down-regulating extracellular protease production. Extracellular protease production in the W81 wild-type strain and protease overproduction in mutants W81M3 and W81M4 were also induced in the presence of the autoclaved fungal mycelium. In soil microcosms naturally infested by Pythium spp., inoculation of sugar beet seeds with W81M3 or W81M4 resulted in improved biocontrol of Pythium-mediated damping-off disease compared with W81, and the level of protection achieved was equivalent to that conferred by chemical fungicides. The wild-type W81 and its mutant derivatives did not differ in rhizosphere colonization. Therefore, the improved biocontrol ability of W81M3 and W81M4 resulted from their capacity to overproduce extracellular serine protease.
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Induction of the sexual stage of Pestalotiopsis microspora, a taxol-producing fungus
The GenBank accession numbers for the sequences determined in this work are: Pestalotiopsis microspora NE-32 18S rDNA, AF104356; Pestalosphaeria hansenii ATCC 48245 18S rDNA, AF242846.
Pestalotiopsis microspora, isolate NE-32, is an endophyte of the Himalayan yew (Taxus wallichiana) that produces taxol, an important chemotherapeutic drug used in the treatment of breast and ovarian cancers. Conditions were determined to induce the perfect stage (teleomorph) of this organism in the laboratory as a critical first step to study inheritance of taxol biosynthetic genes. The perfect stage of Pestalotiopsis microspora NE-32 forms in a period of 3–6 weeks on water agarose with dried yew needles at 16–20 °C with 12 h of light per day. Morphological analysis of the teleomorph and sequencing of the 18S rDNA indicates that Pestalosphaeria hansenii is the perfect stage of Pestalotiopsis microspora. Only certain plants (e.g. yews, some pines, pecan, oat and some barley cultivars) allow the production of perithecia. Exhaustive methylene chloride extraction of yew (Taxus cuspidata) needles removes their capacity to induce production of perithecia. The methylene chloride extract is able to induce formation of perithecia by strain NE-32 in a bioassay system utilizing the sterilized sheaths of the Cholla cactus (Opuntia bigelovii) spine, indicating that a chemical compound(s) in yew stimulates the formation of the perfect stage. This hydrophobic plant compound(s) has been designated the perithecial-stimulating factor (PSF). The data suggest that plant products may play a role in regulating the biology of endophytic microbes.
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- Systematics And Evolution
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Genomic subpopulations within the species Pediococcus acidilactici detected by multilocus typing analysis: relationships between pediocin AcH/PA-1 producing and non-producing strains
More LessThe GenBank accession numbers for the sequences determined in this work are given in Table 4 T4 .
A high degree of genetic polymorphism among P. acidilactici strains was highlighted by a multilocus typing approach analysing several housekeeping genes and by sampling the whole genome using random amplified polymorphic DNA (RAPD) fingerprint analysis performed by using a single primer pedA gene targeted in low-stringency amplification conditions. Restriction fragment length polymorphism of the rpoC, ldhD/L and mle genes, and a modified RAPD analysis, permitted the grouping of Pediococcus acidilactici strains in seven genotypes (I–VII). Genotypic results obtained by analysing housekeeping genes involved in the transcription/translation machinery and in primary metabolism were supported by phylogenetic analysis based on the partial 16S rDNA sequencing of a reference strain of each of the seven clusters obtained. Three of the seven genotypes detected showed relationships with pediocin AcH/PA-1 production and carbohydrate fermentation patterns: all pediocin-producing and sucrose-positive strains were grouped in genotype VII, melibiose-, sucrose- and raffinose-positive strains in genotype VI, and arabinose-positive strains in genotype V.
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Analysis of the β-glucoside utilization (bgl) genes of Shigella sonnei: evolutionary implications for their maintenance in a cryptic state
More LessThe GenBank accession number for the bglR–bglG sequence reported in this paper is AF183894.
The pattern of expression of the genes involved in the utilization of aryl β-glucosides such as arbutin and salicin is different in the genus Shigella compared to Escherichia coli. The results presented here indicate that the homologue of the cryptic bgl operon of E. coli is conserved in Shigella sonnei and is the primary system involved in β-glucoside utilization in the organism. The organization of the bgl genes in S. sonnei is similar to that of E. coli; however there are three major differences in terms of their pattern of expression. (i) The bglB gene, encoding phospho-β-glucosidase B, is insertionally inactivated in S. sonnei. As a result, mutational activation of the silent bgl promoter confers an Arbutin-positive (Arb+) phenotype to the cells in a single step; however, acquiring a Salicin-positive (Sal+) phenotype requires the reversion or suppression of the bglB mutation in addition. (ii) Unlike in E. coli, a majority of the activating mutations (conferring the Arb+ phenotype) map within the unlinked hns locus, whereas activation of the E. coli bgl operon under the same conditions is predominantly due to insertions within the bglR locus. (iii) Although the bgl promoter is silent in the wild-type strain of S. sonnei (as in the case of E. coli), transcriptional and functional analyses indicated a higher basal level of transcription of the downstream genes. This was correlated with a 1 bp deletion within the putative Rho-independent terminator present in the leader sequence preceding the homologue of the bglG gene. The possible evolutionary implications of these differences for the maintenance of the genes in the cryptic state are discussed.
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