- Volume 145, Issue 10, 1999
Volume 145, Issue 10, 1999
- Review Article
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- Microbiology Comment
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- Antigens And Immunity
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Antibodies to a synthetic 1–9-N-terminal amino acid fragment of mature pediocin PA-1: sensitivity and specificity for pediocin PA-1 and cross-reactivity against Class IIa bacteriocins
Polyclonal antibodies specific for pediocin PA-1 (PedA1) were generated by immunization of rabbits with a chemically synthesized 1–9-N-terminal amino acid fragment of this bacteriocin (PH1) conjugated to the carrier protein keyhole limpet haemocyanin (KLH). The PH1 fragment holds a highly conserved amino acid sequence with closely related Class IIa bacteriocins. The sensitivity and specificity of the PH1–KLH-generated rabbit polyclonal antibodies were evaluated by the development of various ELISAs, such as a non-competitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), a competitive direct ELISA (CD-ELISA) and a sandwich ELISA (S-ELISA), and by protein slot-blotting and Western blotting. NCI- and CI-ELISA were valuable for detecting the existence of PedA1-specific antibodies in the sera of immunized rabbits. The limit of detection of PedA1 in MRS medium was found to be 0·5 μg ml−1 in NCI-ELISA, while CI-ELISA on plates coated with purified PedA1 increased the affinity of the PH1–KLH-generated antibodies for PedA1; the limit of detection of PedA1 was less than 0·01 μg ml−1 and 50% binding inhibition was achieved with 0·1 μg PedA1 ml−1. Similarly, the limits of detection of PedA1 in MRS medium were found to be 5 μg ml−1 by protein slot-blotting and 0·01 μg ml−1 by Western blotting. Most importantly, PH1–KLH-generated polyclonal antibodies detected the presence of PedA1 in the supernatants of the producing strains of Pediococcus acidilactici 347, Z102, A172, X13 and P20, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing closely related or different bacteriocins. The approaches taken for the selection of the bacteriocin peptide fragment, the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of adequate specificity for other bacteriocins of interest in the food industry.
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- Biochemistry
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A low-M r lipase activation factor cooperating with lipase modulator protein LimL in Pseudomonas sp. strain 109
More LessPseudomonas sp. strain 109 produces a unique lipase (LipL) which efficiently catalyses intramolecular transesterification of ω-hydroxyesters to form macrocyclic lactones. In vivo production of enzymically active LipL requires lipase modulator protein (LimL), which functions as a molecular chaperone for the correct folding of LipL. However, previous work has shown that LipL forms a tight complex with LimL in vitro and the resulting LipL–LimL complex is only partially active, suggesting an additional mechanism that facilitates the dissociation of the complex to form enzymically active LipL. In the present work, a low-M r compound (lipase activation factor, LAF) was found in Pseudomonas sp. strain 109 that when added to the LipL–LimL complex resulted in the activation of LipL. Ca2+ ions also enhanced lipase activity, but the instantaneous activation by Ca2+ was different from the gradual and time-dependent activation by LAF, indicating the novel nature of this compound. LAF passed through an ultrafiltration membrane with an M r cut-off of 3000 and showed an apparent M r of 330±30 on Superdex Peptide gel-filtration chromatography. Treatment of the LipL–LimL complex with LAF liberated free active LipL, indicating that LAF was necessary to dissociate the LipL–LimL complex.
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- Bioenergetics And Transport
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Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system – two highly similar glucose permeases in Staphylococcus carnosus with different glucoside specificity: protein engineering in vivo?
More LessPrevious sequence analysis of the glucose-specific PTS gene locus from Staphylococcus carnosus revealed the unexpected finding of two adjacent, highly similar ORFs, glcA and glcB,each encoding a glucose-specific membrane permease EIICBAGlc. glcA and glcB show 73% identity at the nucleotide level and glcB is located 131 bp downstream from glcA. Each of the genes is flanked by putative regulatory elements such as a termination stem–loop, promoter and ribosome-binding site, suggesting independent regulation. The finding of putative cis-active operator sequences, CRE (catabolite-responsive elements) suggests additional regulation by carbon catabolite repression. As described previously by the authors, both genes can be expressed in Escherichia coli under control of their own promoters. Two putative promoters are located upstream of glcA, and both were found to initiate transcription in E. coli. Although the two permeases EIICBAGlc1 and EIICBAGlc2 show 69% identity at the protein level, and despite the common primary substrate glucose, they have different specificities towards glucosides as substrate. EIICBAGlc1 phosphorylates glucose in a PEP-dependent reaction with a K m of 12 μM; the reaction can be inhibited by 2-deoxyglucose and methyl β-D-glucoside. EIICBAGlc2 phosphorylates glucose with a K m of 19 μM and this reaction is inhibited by methyl α-D-glucoside, methyl β-D-glucoside, p-nitrophenyl α-D-glucoside, o-nitrophenyl β-D-glucoside and salicin, but unlike other glucose permeases, including EIICBAGlc1, not by 2-deoxyglucose. Natural mono- or disaccharides, such as mannose or N-acetylglucosamine, that are transported by other glucose transporters are not phosphorylated by either EIICBAGlc1 nor EIICBAGlc2, indicating a high specificity for glucose. Together, these findings support the suggestion of evolutionary development of different members of a protein family, by gene duplication and subsequent differentiation. C-terminal fusion of a histidine hexapeptide to both gene products did not affect the activity of the enzymes and allowed their purification by Ni2+-NTA affinity chromatography after expression in a ptsG (EIICBGlc) deletion mutant of E. coli. Upstream of glcA, the 3’ end of a further ORF encoding 138 amino acid residues of a putative antiterminator of the BglG family was found, as well as a putative target DNA sequence (RAT), which indicates a further regulation by glucose specific antitermination.
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Substrate specificity of the periplasmic dipeptide-binding protein from Escherichia coli: experimental basis for the design of peptide prodrugs
More LessPure dipeptide-binding protein (DppA) from Escherichia coli was studied in a filter binding assay to determine its binding specificity. A substrate:DppA stoichiometry of 1:1 was found with both [14C]AlaAla and Ala[14C]Phe. Surprisingly, substrate binding did not vary over the pH range pH 3–9·5. Different dipeptides yielded liganded protein with various pI values, implying that DppA can undergo subtly different conformational changes to accommodate different substrates. Using [125I]Tyr-peptides as substrates in competition assays, the relative binding affinities for a range of dipeptides were found to parallel their overall transport rates into E. coli through the dipeptide permease (Dpp), showing that DppA alone controls the specificity of Dpp. With a series of substituted glycyl peptides, binding affinity was progressively enhanced by alkylation (with methyl to butyl) of the N-terminal α-amino group. Thus, results from this approach provide an essential experimental basis, which complements the information from the crystal structure of DppA, for the design of peptidomimetic antibacterials targeted for transport through Dpp.
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- Development And Structure
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A re-examination of twitching motility in Pseudomonas aeruginosa
More LessTwitching motility is a form of solid surface translocation which occurs in a wide range of bacteria and which is dependent on the presence of functional type IV fimbriae or pili. A detailed examination of twitching motility in Pseudomonas aeruginosa under optimal conditions in vitro was carried out. Under these conditions (at the smooth surface formed between semi-solid growth media and plastic or glass surfaces) twitching motility is extremely rapid, leading to an overall radial rate of colony expansion of 0·6 mm h−1 or greater. The zones of colony expansion due to twitching motility are very thin and are best visualized by staining. These zones exhibit concentric rings in which there is a high density of microcolonies, which may reflect periods of expansion and consolidation/cell division. Video microscopic analysis showed that twitching motility involves the initial formation of large projections or rafts of aggregated cells which move away from the colony edge. Behind the rafts, individual cells move rapidly up and down trails which thin and branch out, ultimately forming a fine lattice-like network of cells. The bacteria in the lattice network then appear to settle and divide to fill out the colonized space. Our observations redefine twitching motility as a rapid, highly organized mechanism of bacterial translocation by which P. aeruginosa can disperse itself over large areas to colonize new territories. It is also now clear, both morphologically and genetically, that twitching motility and social gliding motility, such as occurs in Myxococcus xanthus, are essentially the same process.
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- Environmental Microbiology
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The distribution of enteric bacteria from Australian mammals: host and geographical effects
More LessBacteria of the family Enterobacteriaceae were isolated from 642 mammalian hosts, representing 16 families and 79 species, collected from throughout Australia. Escherichia coli was the most common of the 24 enteric species recovered and represented almost half of the isolates. Association analysis revealed that most other species of bacteria were less likely to be recovered from hosts in which E. coli was present. The composition of the enteric community of a host was found to be determined by both the taxonomic family to which the host belonged and the geographical area from which the host was collected. Hosts collected from the northern areas of Queensland and the Northern Territory had more diverse enteric communities than hosts collected from New South Wales or Western Australia. Hosts of the families Petauridae and Vespertilionidae had more diverse enteric communities than did members of the Macropodidae or Phalangeridae. The probability of occurrence of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca and K. pneumoniae in a host was found to vary with respect to host family and/or host locality. The non-random distribution of these species demonstrates the presence of extensive population structure and may suggest the existence of adaptations specific to both the primary and secondary habitats of these enteric bacteria.
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The genetic structure of enteric bacteria from Australian mammals
More LessA total of 246 isolates representing five species of the family Enterobacteriaceae, taken from a variety of Australian mammal species, were characterized using multi-locus enzyme electrophoresis. Genome diversity estimates varied significantly among species, with the Klebsiella pneumoniae sample exhibiting the lowest diversity and the Citrobacter freundii sample the highest. Multi-locus linkage disequilibrium estimates revealed that alleles were non-randomly associated in all five species samples, but the magnitude of the estimates differed significantly among species. Escherichia coli had the lowest linkage disequilibrium estimate and Klebisella oxytoca the largest. Molecular analyis of variance was used to determine the extent to which population structure explained the observed genetic variation in a species. Two population levels were defined: the taxonomic family of the host from which the isolate was collected and the geographical locality where the host was collected. The amount of explained variation varied from 0% for K. oxytoca to 22% for K. pneumoniae. Host locality explained a significant amount of the genetic variation in the C. freundii (12%), E. coli (5%), Hafnia alvei (17%) and K. pneumoniae (22%) samples. Host family explained a significant fraction of the variation in E. coli (6%) H. alvei (7%) and K. pneumoniae (20%). Estimates of effective population size for all five species, based on the probability that two randomly chosen isolates will be identical, failed to reveal any relationship between the effective population size and the genetic diversity of a species.
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The relationship between critical pressure and width of gas vesicles in isolates of Planktothrix rubescens from Lake Zürich
More LessThe mean critical collapse pressure (p c) of gas vesicles in 81 strains of the cyanobacterium Planktothrix rubescens from Lake Zürich, Switzerland, was bimodally distributed between a minimum of 0·86 MPa and a maximum of 1·17 MPa. Measurements were made of the cylinder diameter (d) of gas vesicles isolated from seven of the strains. The mean diameter, which varied from 48 to 61 nm, was inversely related to p c, in keeping with the theory of strength of thin-walled rigid cylinders. These measurements extended the range of p c–width relationship of gas vesicles, which can be described by the expression p c=461(d/nm)−1·53 MPa. p c was correlated with gas vesicle genotype (see the accompanying paper by S. J. Beard, B. A. Handley, P. K. Hayes & A. E. Walsby, Microbiology 145, 2757–2768): of the 81 strains investigated, all those with the gas vesicle genotype GV2 produced gas vesicles with a mean p c of less than 1·0 MPa, whereas those of GV3 had a mean p c of greater than 1·0 MPa. It is suggested that gas vesicles of the GV3 strains, which are narrower and stronger than any previously recorded in freshwater cyanobacteria, have evolved to withstand the high hydrostatic pressures during deep winter mixing in Lake Zürich.
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The effect of motility and cell-surface polymers on bacterial attachment
More LessRecently it was shown that motility of Vibrio alginolyticus facilitated cell attachment to glass surfaces. In the present study the same relationship between motility and cell attachment was confirmed for Alcaligenes and Alteromonas spp. These findings clearly answer a long-standing question: does motility facilitate attachment? However, they are contradictory to a general view on cell attachment that the energy barrier due to electrostatic repulsion between negatively charged bacterial cells and a glass surface is much greater than both the thermal kinetic energy of the bacterial cell and the bacterial swimming energy. It is shown that the energy barrier becomes far less than that usually estimated when bacterial cells are rich in polymers at their surfaces. This finding reasonably explains the dependence of bacterial attachment rate on cell motility and demands reconsideration of the mechanism of bacterial attachment.
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Genetic organization and characteristics of the 3-(3-hydroxyphenyl)propionic acid degradation pathway of Comamonas testosteroni TA441
More LessComamonas testosteroni TA441 degrades 3-(3-hydroxyphenyl)propionate (3HPP) via the meta pathway. A gene cluster required for degradation of 3HPP was cloned from strain TA441 and sequenced. The genes encoding six catabolic enzymes, a flavin-type hydroxylase (mhpA), extradiol dioxygenase (mhpB), 2-keto-4-pentenoate hydratase (mhpD), acetaldehyde dehydrogenase (acylating) (mhpF), 4-hydroxy-2-ketovalerate aldolase (mhpE) and the meta cleavage compound hydrolase (mhpC), were found in this cluster, encoded in this order. mhpD and mhpF were separated by two genes, orf4 and orf5, which were not necessary for growth on 3HPP. The gene mhpR, encoding a putative transcriptional activator of the IclR family, was located adjacent to mhpA in the opposite orientation. Disruption of the mhpB or mhpR genes affected growth on 3HPP or trans-3-hydroxycinnamate. The mhpB and mhpC gene products showed high specificity for 3-(2,3-dihydroxyphenyl)propionate (DHPP) and the meta cleavage compound produced from DHPP, respectively.
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Occurrence and expression of glutathione-S-transferase-encoding bphK genes in Burkholderia sp. strain LB400 and other biphenyl-utilizing bacteria
More LessThe gene bphK of Burkholderia sp. strain LB400 has previously been shown to be located within the bph locus, which specifies the degradation of biphenyl (BP) and chlorobiphenyls, and to encode a glutathione S-transferase (GST) which accepts 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The specific physiological role of this gene is not known. It is now shown that the gene is expressed in the parental organism and that GST activity is induced more than 20-fold by growth of the strain on BP relative to succinate when these compounds serve as sole carbon source. Approximately the same induction factor was observed for 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, which is encoded by the 5′-adjacent bphC gene. This suggests that the expression of bphK is coregulated with the expression of genes responsible for the catabolism of BP. A bphK probe detected only a single copy of the gene in strain LB400. A spontaneous BP− mutant of the organism neither gave a signal with the bphK probe nor showed CDNB-accepting GST activity, suggesting that this activity is solely encoded by bphK. Complementation of the mutant with a bph gene cluster devoid of bphK restored the ability to grow on BP, indicating that bphK is not essential for utilization of this carbon source. BphK activity proved to be almost unaffected by up to 100-fold differences in proton concentration or ionic strength. The enzyme showed a narrow range with respect to a variety of widely used electrophilic GST substrates, accepting only CDNB. A number of established laboratory strains as well as novel isolates able to grow on BP as sole carbon and energy source were examined for BphK activity and the presence of a bphK analogue. CDNB assays, probe hybridizations and PCR showed that several, but not all, BP degraders possess this type of GST activity and/or a closely related gene. In all bacteria showing BphK activity, this was induced by growth on BP as sole carbon source, although activity levels differed by up to 10-fold after growth on BP and by up to 60-fold after growth on succinate. This resulted in a variation of induction factors between 2 and 30. In the majority of bphK + bacteria examined, the gene appeared to be part of LB400-like bph gene clusters. DNA sequencing revealed almost complete identity of bphK genes from five different bph gene clusters. These results suggest that bphK genes, although not essential, fulfil a strain-specific function related to the utilization of BPs by their host organisms. The usefulness of BphK as a reporter enzyme for monitoring the expression of catabolic pathways is discussed.
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- Genetics And Molecular Biology
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Expression of leading region genes on IncI1 plasmid ColIb-P9: genetic evidence for single-stranded DNA transcription
More LessThe leading region of a plasmid is the first sector to enter the recipient cell in bacterial conjugation. This sector of IncI1 plasmid ColIb-P9 includes genes that are transcribed in a transient pulse early in the conjugatively infected cell to promote establishment of the immigrant plasmid. Evidence is presented that the burst of gene expression is regulated by a process which is independent of a repressor but dependent on the orientation of the genes on the unique plasmid strand transferred in conjugation. The nucleotide sequence of 11·7 kb of the leading region was determined and found to contain 10 ORFs; all are orientated such that the template strand for transcription corresponds to the transferred strand. The leading region contains three dispersed repeats of a sequence homologous to a novel promoter in ssDNA described by H. Masai & K. Arai (1997 R20 , Cell 89, 897–907). It is proposed that the repeats are promoters that form in the transferring strand of ColIb to support transient transcription of genes transferred early in conjugation.
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The diversity of gas vesicle genes in Planktothrix rubescens from Lake Zürich
More LessPart of the gas vesicle gene cluster was amplified by PCR from three strains of Planktothrix rubescens isolated from Lake Zürich, Switzerland. Each contains multiple alternating copies of gvpA and gvpC. All of the gvpA sequences in the different strains are identical. There are two types of gvpC: gvpC 20, of length 516 bp, encodes a 20 kDa protein of 172 amino acid residues (whose N-terminal amino acid sequence is homologous with the sequence of GvpC in Planktothrix [Oscillatoria] agardhii); gvpC 16, of length 417 bp, encodes a 16 kDa protein of 139 amino acid residues that differs in lacking an internal 33-residue section. An untranslated 72 bp fragment from the 3′ end of gvpC, designated ΩC, is also present in some strains. The two types of gvpC and presence of ΩC could be distinguished by the different lengths of PCR amplification products obtained using pairs of oligonucleotide primers homologous to internal sequences in gvpC and gvpA. Three genotype classes were found: GV1, containing only gvpC 20; GV2, containing gvpC 20 and ΩC; and GV3, containing gvpC 16, gvpC 20 and ΩC. Subclasses of GV2 and GV3 contained either one or two copies of ΩC. The accompanying paper by D. I. Bright & A. E. Walsby (Microbiology 145, 2769–2775) shows that strains of the GV3 genotype produce gas vesicles with a higher critical pressure than those of GV1 and GV2. A PCR survey of 185 clonal cultures of P. rubescens isolated from Lake Zürich revealed that 3 isolates were of genotype GV1, 73 were of GV2 and 109 were of GV3. The PCR technique was used to distinguish the gas vesicle genotype, and thence the associated critical-pressure phenotype, of single filaments selected from lakewater samples. Sequence analysis of the 16S rDNA and of regions within the operons encoding phycoerythrin, phycocyanin and Rubisco confirmed that these strains of Planktothrix form a tight phylogenetic group.
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Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli
More LessSynthesis of the [NiFe] hydrogenases 1 and 2 of Escherichia coli is induced in response to anaerobiosis and is repressed when nitrate is present in the growth medium. The hydrogenase 1 and hydrogenase 2 enzymes are encoded by the polycistronic hyaABCDEF and hybOABCDEFG operons, respectively. Primer extension analysis was used to determine the initiation site of transcription of both operons. This permitted the construction of single-copy lacZ operon fusions, which were used to examine the transcriptional regulation of the two operons. Expression of both was induced by anaerobiosis and repressed by nitrate, which is in complete accord with earlier biochemical studies. Anaerobic induction of the hyb operon was only partially dependent on the FNR protein and, surprisingly, was enhanced by an arcA mutation. This latter result indicated that ArcA suppresses anaerobic hyb expression and that a further factor, which remains to be identified, is involved in controlling anaerobic induction of operon expression. Nitrate repression of hyb expression was mediated by the NarL/NarX and NarP/NarQ two-component regulatory systems. Remarkably, a narP mutant lacked anaerobic induction of hyb expression, even in the absence of added nitrate. Anaerobic induction of hya expression was dependent on the ArcA and AppY regulators, which confirms earlier observations by other authors. Nitrate repression of the hya operon was mediated by both NarL and NarP. Taken together, these data indicate that although the hya and hyb operons share common regulators, there are important differences in the control of expression of the individual operons.
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The dnaA gene region of Mycobacterium avium and the autonomous replication activities of its 5′ and 3′ flanking regions
More LessA 3·9 kb DNA fragment containing the dnaA gene region of Mycobacterium avium was cloned and its nucleotide sequence was determined. Nucleotide sequence analyses indicated that this region encodes three genes in the order rpmH (ribosomal protein L34), dnaA (the putative initiator protein) and dnaN (the β subunit of DNA polymerase III). The intergenic regions between the rpmH–dnaA and dnaA–dnaN genes were found to contain several putative DnaA boxes, 9 nt long DnaA protein recognition sequences. A DNA fragment containing the 3′ but not the 5′ flanking region of the M. avium dnaA gene when cloned in Escherichia coli plasmids, which are otherwise non-replicative in mycobacteria, exhibited autonomous replication activity in M. avium but not in Mycobacterium bovis BCG and Mycobacterium smegmatis. The 5′ flanking region of dnaA, on the other hand, exhibited autonomous replication activity in M. bovis BCG but not in M. avium and M. smegmatis. The implications of these results for the understanding of the M. avium oriC replication initiation process are discussed.
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Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells
More LessThe identification of Mycobacterium tuberculosis genes expressed within host cells would contribute greatly to the development of new strategies to combat tuberculosis. By combining the natural fluorescence of the Aequoria victoria green fluorescent protein (GFP) with the counterselectable property of the Bacillus subtilis SacB protein, M. tuberculosis promoters displaying enhanced in vivo activity have been isolated. Macrophages were infected with recombinant Mycobacterium bovis bacille Calmette–Guérin containing a library of M. tuberculosis promoters controlling gfp and sacB expression, and fluorescent bacteria recovered by fluorescence-activated cell sorting. The expression of sacB was used to eliminate clones with strong promoter activity outside the macrophage, resulting in the isolation of seven clones containing M. tuberculosis promoters with greater activity intracellularly. The gene products identified displayed similarity to proteins from other organisms whose functions include nutrient utilization, protection from oxidative stress and defence against xenobiotics. These proposed functions are consistent with conditions encountered within the host cell and thus suggest that the augmented activity of the isolated promoters/genes may represent strategies employed by M. tuberculosis to enhance intracellular survival and promote infection.
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Regulation of polyphosphate kinase gene expression in Acinetobacter baumannii 252
More LessA strain of Acinetobacter baumannii cultured in butyric acid media was found to take up phosphate following a period of phosphate release. PCR was used to clone the polyphosphate kinase (ppk) gene from the strain. The promoter for the ppk gene was functional in the heterologous Escherichia coli host. Using RT-PCR, transcription of the ppk gene was found to be regulated by phosphate concentration.
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Two fatty acid Δ9-desaturase genes, ole1 and ole2, from Mortierella alpina complement the yeast ole1 mutation
Genes encoding two distinct fatty acid Δ9-desaturases were isolated from strains of the oleaginous fungus Mortierella alpina. Two genomic sequences, Δ9-1 and Δ9-2, each containing a single intron, were cloned from strain CBS 528.72 while one cDNA clone, LM9, was isolated from strain CBS 210.32. The Δ9-1 gene encoded a protein of 445 aa which shared 99% identity with the LM9 gene product. These proteins also showed 40–60% identity to the Δ9-desaturases (Ole1p) of other fungi and contained the three conserved histidine boxes, C-terminal cytochrome b 5 fusion and transmembrane domains characteristic of endoplasmic reticulum membrane-bound Δ9-desaturases. LM9 and Δ9-1 are therefore considered to represent the same gene (ole1). The ole1 gene was transcriptionally active in all M. alpina strains tested and its function was confirmed by complementation of the Saccharomyces cerevisiae ole1 mutation. Fatty acid analysis of yeast transformants expressing the CBS 210.32 ole1 gene showed an elevated level of oleic acid (18:1) compared to palmitoleic acid (16:1), the major fatty acid component of wild-type S. cerevisiae. This indicated that the M. alpina Δ9-desaturase had a substrate preference for stearic acid (18:0) rather than palmitic acid (16:0). Genomic clone Δ9-2 (ole2) also encoded a protein of 445 aa which had 86% identity to the Δ9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation. The transcript from this gene could only be detected in one of the six M. alpina strains tested, suggesting that its expression may be strain-specific or induced under certain physiological conditions.
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Identification of structural and functional domains of the tetracycline efflux protein TetA(P) from Clostridium perfringens
More LessThe Clostridium perfringens tetracycline-resistance protein, TetA(P), is an integral inner-membrane protein that mediates the active efflux of tetracycline from the cell. TetA(P) acts as an antiporter, presumably transporting a divalent cation–tetracycline complex in exchange for a proton, and is predicted to have 12 transmembrane domains (TMDs). Two glutamate residues that are located in predicted TMD 2 were previously shown to be required for the active efflux of tetracycline by TetA(P). To identify additional residues that are required for the structure or function of TetA(P), a random mutagenesis approach was used. Of the 61 tetracycline-susceptible mutants that were obtained in Escherichia coli, 31 different derivatives were shown to contain a single amino acid change that resulted in reduced tetracycline resistance. The stability of the mutant TetA(P) proteins was examined by immunoblotting and 19 of these strains were found to produce a detectable TetA(P) protein. The MIC of these derivatives ranged from 2 to 15 μg tetracycline ml−1, compared to 30 μg tetracycline ml−1 for the wild-type. The majority of these mutants clustered into three potential loop regions of the TetA(P) protein, namely the cytoplasmic loops 2–3 and 4–5, and loop 7–8, which is predicted to be located in the periplasm in E. coli. It is concluded that these regions are of functional significance in the TetA(P)-mediated efflux of tetracycline from the bacterial cell.
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- Genomics
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Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive sequence RPS at specific genomic sites
More LessThe 11 kb complex DNA fingerprinting probe Ca3 is effective both in cluster analyses of Candida albicans isolates and in identifying microevolutionary changes in the size of hypervariable genomic fragments. A 2·6 kb EcoRI fragment of Ca3, the C fragment, retains the capacity to identify these microevolutionary changes, and when the C fragment is cleaved with SacI, the capacity is retained exclusively by a 1 kb subfragment, C1, which contains a partial RPS repeat element. The microevolutionary changes identified by Ca3, therefore, may involve reorganization of RPS elements dispersed throughout the genome. To test this possibility, hypervariable fragments from several strains of C. albicans were sequenced and compared. The results demonstrate that the microevolutionary changes identified by Ca3 are due to the insertion and deletion of full-length tandem RPS elements at specific genomic sites dispersed throughout the C. albicans genome. The RPS elements at these dispersed sites are bordered by the same upstream and downstream sequences. The frequency of recombination was estimated to be one recombination per 1000 cell divisions by following RPS reorganization in vitro. The results are inconsistent with unequal recombination between homologous or heterologous chromosomes, but consistent with intrachromosomal recombination. Two alternative models of intrachromosomal recombination are proposed: unequal sister-chromatid exchange and slipped misalignment at the replication fork.
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Repeated extragenic sequences in prokaryotic genomes: a proposal for the origin and dynamics of the RUP element in Streptococcus pneumoniae
More LessA survey of all Streptococcus pneumoniae GenBank/EMBL DNA sequence entries and of the public domain sequence (representing more than 90% of the genome) of an S. pneumoniae type 4 strain allowed identification of 108 copies of a 107-bp-long highly repeated intergenic element called RUP (for repeat unit of pneumococcus). Several features of the element, revealed in this study, led to the proposal that RUP is an insertion sequence (IS)-derivative that could still be mobile. Among these features are: (1) a highly significant homology between the terminal inverted repeats (IRs) of RUPs and of IS630-Spn1, a new putative IS of S. pneumoniae; and (2) insertion at a TA dinucleotide, a characteristic target of several members of the IS630 family. Trans-mobilization of RUP is therefore proposed to be mediated by the transposase of IS630-Spn1. To account for the observation that RUPs are distributed among four subtypes which exhibit different degrees of sequence homogeneity, a scenario is invoked based on successive stages of RUP mobility and non-mobility, depending on whether an active transposase is present or absent. In the latter situation, an active transposase could be reintroduced into the species through natural transformation. Examination of sequences flanking RUP revealed a preferential association with ISs. It also provided evidence that RUPs promote sequence rearrangements, thereby contributing to genome flexibility. The possibility that RUP preferentially targets transforming DNA of foreign origin and subsequently favours disruption/rearrangement of exogenous sequences is discussed.
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- Pathogenicity And Medical Microbiology
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In vivo detection of Escherichia coli type 1 fimbrial expression and phase variation during experimental urinary tract infection
More LessAdhesion mediated by fimbriae is thought to play an important role in the pathogenesis of urinary tract infections (UTI) by Escherichia coli. The majority of clinical isolates of E. coli from UTI are able to express type 1 fimbriae. However, the importance of these fimbriae as a virulence factor has been controversial. To investigate the expression of type 1 fimbriae in vivo during UTI, mice were transurethrally infected with uropathogenic E. coli C175-94 and type 1 fimbrial expression was determined directly by two independent methods at 2 h, 1 d and 3 d after infection. By use of an assay combining in situ rRNA hybridization and immunofluorescence, all bacterial cells detected in urine, bladders and kidneys from mice sacrificed 1 and 3 d after onset of infection were found to express type 1 fimbriae. In contrast, the majority of cells in the suspension used for infection of mice and specimens from mice sacrificed 2 h after inoculation were found to be non-fimbriated. Similar results were obtained with a PCR assay revealing the orientation of the invertible promoter driving the transcription of type 1 fimbrial genes. Whilst the promoter in both ON and OFF positions could be amplified from the suspension used for infection and specimens from mice sacrificed 2 h after inoculation, at 1 and 3 d after onset of infection only the promoter in the ON orientation could be amplified. These results show that introduction of E. coli C175-94 into the mouse urinary tract leads to markedly enhanced expression of type 1 fimbriae.
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Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans
The cytochrome P450 14α-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G465S). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.
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Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans
More LessLanosterol 14α-demethylase (14DM) is the target of the azole antifungals, and alteration of the 14DM sequence leading to a decreased affinity of the enzyme for azoles is one of several potential mechanisms for resistance to these drugs in Candida albicans. In order to identify such alterations the authors investigated a collection of 19 C. albicans clinical isolates demonstrating either frank resistance (MICs ≤32 μg ml−1) or dose-dependent resistance (MICs 8–16 μg ml−1) to fluconazole. In cell-free extracts from four isolates, including the Darlington strain ATCC 64124, sensitivity of sterol biosynthesis to inhibition by fluconazole was greatly reduced, suggesting that alterations in the activity or affinity of the 14DM could contribute to resistance. Cloning and sequencing of the 14DM gene from these isolates revealed 12 different alterations (two to four per isolate) leading to changes in the deduced amino acid sequence. Five of these mutations have not previously been reported. To demonstrate that these alterations could affect fungal susceptibility to azoles, the 14DM genes from one sensitive and three resistant C. albicans strains were tagged at the carboxyl terminus with a c-myc epitope and expressed in Saccharomyces cerevisiae under control of the endogenous promoter. Transformants receiving 14DM genes from resistant strains had fluconazole MICs up to 32-fold higher than those of transformants receiving 14DM from a sensitive strain, although Western blot analysis indicated that the level of expressed 14DM was similar in all transformants. Amino acid substitutions in the 14DM gene from the Darlington strain also conferred a strong cross-resistance to ketoconazole. In conclusion, multiple genetic alterations in C. albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates.
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The relationship between Helicobacter pylori motility, morphology and phase of growth: implications for gastric colonization and pathology
More LessTo explore the relationship between Helicobacter pylori motility, morphology and phase of growth, bacteria were isolated from antral biopsies of patients with duodenal ulcer or non-ulcer dyspepsia, and grown in liquid medium in batch and continuous culture systems. Motilities and morphologies of H. pylori in different phases of growth were examined with a Hobson BackTracker and by transmission electron microscopy. Morphologies of bacteria grown in vitro were also compared with those of bacteria in antral biopsies from patients with non-autoimmune gastritis. H. pylori had poor motility in lag phase, became highly motile in mid-exponential phase and lost motility in the decline phase of growth. Motilities of bacteria in the same phase of growth from patients with duodenal ulcer or non-ulcer dyspepsia were not significantly different. In the mid/late-exponential phase of growth bacteria had helical morphologies and multiple polar flagella, typical of H. pylori in the gastric mucus layer. In the decline phase of growth bacteria shed flagella, and had precoccoidal or coccoidal morphologies. These findings support the view that helical and coccoidal H. pylori are in different phases of growth with different roles in gastric colonization, indicate that bacterial motility per se is unlikely to be a determinant of H. pylori pathology, and suggest that H. pylori in the antral mucus layer is in a state of continuous (exponential phase) growth.
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Trichomonas vaginalis interactions with fibronectin and laminin
More LessThe sexually transmitted protozoan Trichomonas vaginalis cytoadheres to vaginal epithelial cells and causes contact-dependent cytotoxicity which, when combined with the normal exfoliation process, leads to erosion of the epithelium, which may allow trichomonads into extracellular matrix and basement membrane sites. Therefore, the association of T. vaginalis with immobilized fibronectin (FN) and laminin (LM) on cover-slips was examined. Binding of live parasites to coated cover-slips was time- and parasite-density-dependent. Coincubation with an inhibitor of trichomonad cysteine proteinases resulted in an increased attachment of parasites to FN but had no effect on binding to LM, denoting that protease activity influenced optimal FN associations. Further, 20 h mid-exponential phase trichomonads placed in fresh culture medium for 3 h gave higher levels of binding to FN, suggesting that changes during growth in vitro to T. vaginalis organisms affect maximal levels of binding to FN. Extended incubation with substrates diminished the capacity of parasites to bind FN and LM. Treatment of live organisms with periodate reduced binding to LM but not FN, suggesting a role for carbohydrates. In addition, trypsinization of live parasites decreased numbers bound to both substrates. Placement of trypsinized parasites in medium for 2 h fully regenerated binding to FN but not LM. Incubation of trypsinized parasites with cycloheximide abrogated regeneration of attachment to FN, affirming a role for synthesized surface proteins in FN binding. Importantly, the T. vaginalis adhesin proteins that mediate cytoadherence, and iron, a factor that regulates adhesin synthesis, were not involved in FN and LM recognition. These results suggest a role for surface proteins and carbohydrates in trichomonal associations with FN and LM, respectively.
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The site-specific integration of genetic elements may modulate thermostable protease production, a virulence factor in Dichelobacter nodosus, the causative agent of ovine footrot
More LessThe Gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. The authors have previously characterized two genetic elements, the intA (vap) and intB elements, which integrate into the genome of D. nodosus. In the virulent strain A198 there are two copies of the intA element. One copy is integrated into the 3’ end of the tRNA-ser GCU gene, close to the aspartokinase (askA) gene, and the second copy is integrated into the 3’ end of the tRNA-ser GGA gene, next to the polynucleotide phosphorylase (pnpA) gene. In this study, a new genetic element was identified in the benign strain C305, the intC element, integrated into the 3’ end of the tRNA-ser GCU gene, next to askA. The intC element was found in most D. nodosus strains, both benign and virulent, which were examined, and was integrated into tRNA-ser GCU in most strains. Between the askA and tRNA-ser GCU genes, a gene (designated glpA), was identified whose predicted protein product has very high amino acid identity with RsmA from the plant pathogen Erwinia carotovora. RsmA acts as a global repressor of pathogenicity in E. carotovora, by repressing the production of extracellular enzymes. In virulent strains of D. nodosus the intA element was found to be integrated next to pnpA, and either the intA or intC element was integrated next to glpA. By contrast, all but one of the benign strains had intB at one or both of these two positions, and the one exception had neither intA, intB nor intC at one position. The loss of the intC element from the virulent strain 1311 resulted in loss of thermostable protease activity, a virulence factor in D. nodosus. A model for virulence is proposed whereby integration of the intA and intC genetic elements modulates virulence by altering the expression of glpA, pnpA, tRNA-ser GCU and tRNA-ser GGA.
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Emergence of multidrug-resistant mutants is increased under antibiotic selective pressure in Pseudomonas aeruginosa
More LessPseudomonas aeruginosa is one of the most important opportunistic pathogens involved in nosocomial infections, cystic fibrosis patients included. Hospital isolates frequently present multidrug-resistance (MDR) phenotypes as the consequence of constant antibiotic selective pressure. The kinetics of emergence of P. aeruginosa MDR mutants under antibiotic selective pressure indicated that long-term incubation in the presence of the bacteriostatic antibiotic tetracycline increases the mutation rate per cell per day of P. aeruginosa PAO1 by several orders of magnitude. The tetracycline-resistant mutants obtained were stable, showed decreased susceptibility to antibiotics belonging to different structural families, and contained an outer-membrane protein not present in the wild-type P. aeruginosa strain PAO1. These data are consistent with the hypothesis that incubation in the presence of tetracycline favours the emergence of MDR mutants in P. aeruginosa. The results are relevant for understanding the rapid emergence of antibiotic-resistant mutants among bacterial populations during infections. Their relationship to other models of increased mutagenesis under stress is discussed with respect to the adaptive mutation phenomenon.
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- Physiology And Growth
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The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity
More LessAmbient pH regulates the expression of virulence genes of Metarhizium anisopliae, but it was unknown if M. anisopliae can regulate ambient pH. Mutants of M. anisopliae altered in production of oxalic acid were evaluated for the interrelationship of ambient pH, buffering capacity added to media, growth, and generation of extracellular proteases and ammonia. Wild-type and acid-overproducing mutants [Acid(+)] grew almost as well at pH 8 as at pH 6, but acid-non-producing [Acid(−)] mutants showed limited growth at pH 8, indicating that acid production is linked to the ability to grow at higher pH. Production of ammonia by M. anisopliae was strongly stimulated by low levels of amino acids in the medium when cells were derepressed for nitrogen and carbon. Likewise, although Aspergillus fumigatus and Neurospora crassa produced some ammonia in minimal media, addition of low levels of amino acids enhanced production. Ammonia production by A. fumigatus, N. crassa and M. anisopliae increased the pH of the medium and allowed production of subtilisin proteases, whose activities are observed only at basic pH. In contrast, protease production by the Acid(+) mutants of M. anisopliae was greatly reduced because of the acidification of the medium. This suggests that alkalinization by ammonia production is adaptive by facilitating the utilization of proteinaceous nutrients. Collectively, the data imply that ammonia may have functions related to regulation of the microenvironment and that it represents a previously unconsidered virulence factor in diverse fungi with the potential to harm tissues and disturb the host’s immune system.
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Candida albicans and Yarrowia lipolytica as alternative models for analysing budding patterns and germ tube formation in dimorphic fungi
More LessThe site for bud selection and germ tube emission in two yeasts, Candida albicans and Yarrowia lipolytica, was analysed. Both dimorphic organisms display different patterns of budding, which also differ from those described for Saccharomyces cerevisiae. C. albicans, which is diploid and (until now) lacks a known sexual cycle, buds in an axial budding pattern. During the yeast–hypha transition induced by pH, serum, N-acetylglucosamine (GlcNAc) or temperature, germ tube emergence occurs at approximately 50% in a polar manner, while the other 50% of cells show non-polar germ tube emission. Y. lipolytica, in which most of the natural isolates are haploid and which has a well characterized sexual cycle, buds with a polar budding pattern independently of the degree of ploidy. Germ tube emission during the yeast–hypha transition in both haploid and diploid cells generally occurs at the pole distal from the division site (bipolar). The addition of hydroxyurea (HU), an inhibitor of DNA synthesis, also produces different effects. In its presence, and therefore in the absence of DNA synthesis, the yeast–hypha transition is completely abolished in Y. lipolytica. By contrast, in C. albicans germ tube emission in the presence of HU is similar to that observed in control cultures for at least 90 min under induction conditions. These results demonstrate that, rather than a single developmental model, several models of development should be invoked to account for the processes involved in the morphological switch in yeasts (the yeast–hypha transition).
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Involvement of glutathione in the regulation of respiratory oscillation during a continuous culture of Saccharomyces cerevisiae
More LessRespiratory oscillation occurred during aerobic continuous culture of Saccharomyces cerevisiae. During oscillation, phase-related changes in NAD(P)H and GSH levels occur. Perturbation of oscillation and inhibition of respiration occurred when GSH or GSSG was injected; however, there was a phase delay in perturbation in the case of an injection during high respiration. The perturbation phase delay was not apparent when a combination of dl-buthionine-(S,R)-sulphoximine, GSH and 5-nitro-2-furaldehyde was injected. Perturbation by GSH injection caused the intracellular GSH concentration to increase, the GSSG concentration to decrease and the cessation of ethanol uptake. NAD(P)H during perturbation was inversely related to dissolved oxygen. Perturbation by calcium pantothenate and pyridoxal-HCl caused a period of enhanced respiration before oscillation returned. These results suggest that the NAD+/NADH redox is not directly involved in oscillation control and regulation involves glutathione metabolism. Possible regulation points include alcohol dehydrogenase inhibition and/or respiratory-chain inhibition.
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Morphogenesis is coordinated with nuclear division in germinating Aspergillus nidulans conidiospores
More LessGerminating Aspergillus nidulans conidiospores switch to polarized apical growth following an initial period of isotropic expansion. At the same time, they re-enter the nuclear division cycle. The relationship between spore polarization and nuclear division was investigated by testing the effect of cell cycle inhibitors and temperature-sensitive cell cycle mutations on spore morphogenesis. On rich media, it was found that spore polarization is delayed if completion of the first mitosis is blocked. The observed delay may be dependent upon the activity of the mitosis-promoting NIMA kinase. An additional mechanism appears to prevent polarization as the spore progresses through its first S phase. In contrast, on poor media, spore polarization does not require completion of the first mitosis. These observations suggest that spore morphogenesis is influenced by cell cycle signals in a growth-dependent manner.
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Nucleosides as a carbon source in Bacillus subtilis: characterization of the drm–pupG operon
More LessIn Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli.
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Neisseria gonorrhoeae bacterioferritin: structural heterogeneity, involvement in iron storage and protection against oxidative stress
More LessThe iron-storage protein bacterioferritin (Bfr) from Neisseria gonorrhoeae strain F62 was identified in cell-free extracts and subsequently purified by column chromatography. Gonococcal Bfr had an estimated molecular mass of 400 kDa by gel filtration; however, analysis by SDS-PAGE revealed that it was composed of 18 kDa (BfrA) and 22 kDa (BfrB) subunits. DNA encoding BfrB was amplified by PCR using degenerate primers derived from the N-terminal amino acid sequence of BfrB and from a C-terminal amino acid sequence of Escherichia coli Bfr. The DNA sequence of bfrA was subsequently obtained by genome walking using single-specific-primer PCR. The two Bfr genes were located in tandem with an intervening gap of 27 bp. A potential Fur-binding sequence (12 of 19 bp identical to the consensus neisserial fur sequence) was located within the 5’ flanking region of bfrA in front of a putative −35 hexamer. The homology between the DNA sequences of bfrA and bfrB was 55·7%; the deduced amino acid sequences of BfrA (154 residues) and BfrB (157 residues) showed 39·7% identity, and showed 41·3% and 56·1% identity, respectively, to E. coli Bfr. Expression of recombinant BfrA and BfrB in E. coli strain DH5α was detected on Western blots probed with polyclonal anti-E. coli Bfr antiserum. Most Bfrs are homopolymers with identical subunits; however, the evidence presented here suggests that gonococcal Bfr was composed of two similar but not identical subunits, both of which appear to be required for the formation of a functional Bfr. A Bfr-deficient mutant was constructed by inserting the Ω fragment into the BfrB gene. The growth of the BfrB-deficient mutant in complex medium was reduced under iron-limited conditions. The BfrB-deficient mutant was also more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These results demonstrate that gonococcal Bfr plays an important role in iron storage and protection from iron-mediated oxidative stress.
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Physiological characterization of Streptococcus bovis mutants that can resist 2-deoxyglucose-induced lysis
More LessStreptococcus bovis JB1 does not normally lyse, but stationary phase lysis can be induced by including 2-deoxyglucose (2DG) in the growth medium. Isolates deficient in glucose/2DG phosphotransferase activity (PTS−) also lysed when 2DG was present (Lys+) and this result indicated that 2DG phosphorylation via the PTS was not an obligate requirement for 2DG-induced lysis. Cells and cell walls from 2DG-grown cultures lysed faster when proteinase K was added, but glucose-grown cultures and cell walls were not affected. A lipoteichoic acid (LTA) extract (aqueous phase from hot phenol treatment) from glucose-grown cells inhibited the lysis of 2DG-grown cultures, but a similar extract prepared from 2DG-grown cells was without effect. Thin-layer chromatography and differential staining indicated that wild-type and Lys+ PTS− cells incorporated 2DG into LTA, but lysis-resistant cultures (Lys− PTS+ and Lys− PTS−) did not. LTA from lysis-resistant (Lys− PTS+ and Lys− PTS−) cells grown with glucose and 2DG also prevented 2DG-dependent lysis of the wild-type. LTA could not inhibit degradation of cell walls isolated from 2DG-grown cultures, but LTA inhibited the lysis of Micrococcus lysodeikticus (Micrococcus luteus) cells that were exposed to supernatants from 2DG-grown S. bovis cultures. Group D streptococci (including S. bovis) normally have an α-1,2 linked glucose disaccharide (kojibiose) in their LTA, but kojibiose cannot be synthesized from 2DG. This observation suggested that the kojibiose moiety of LTA was involved in autolysin inactivation. Wild-type S. bovis had ATP- as well as PEP-dependent mechanisms of 2DG phosphorylation and one lysis-resistant phenotype (Lys− PTS−) had reduced levels of both activities. However, the Lys− PTS+ phenotype was still able to phosphorylate 2DG via ATP and PEP and this result indicated that some other step of 2DG incorporation into LTA was being inhibited. Based on these results, growth in the presence of 2DG appears to prevent synthesis of normal LTA, which is involved in the regulation of autolytic enzymes.
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Extracellular metal-binding activity of the sulphate-reducing bacterium Desulfococcus multivorans
More LessPolarography was used to measure the copper-binding ability of culture filtrates from a range of sulphate-reducing bacteria (SRB), including pure cultures and environmental isolates. Of those tested, Desulfococcus multivorans was shown to have the greatest copper-binding capacity and this organism was used for further experiments. Extracellular copper- and zinc-binding activities of Dc. multivorans culture filtrates from batch cultures increased over time and reached a maximum after 10 d growth. The culture filtrate was shown to bind copper reversibly and zinc irreversibly. Twelve-day-old Dc. multivorans culture filtrates were shown to have a copper-binding capacity of 3·64±0·33 μmol ml−1 with a stability constant, log10 K, of 5·68±0·64 (n=4). The metal-binding compound was partially purified from culture growth media by dichloromethane extraction followed by HPLC using an acetonitrile gradient.
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Lovastatin inhibits the production of gibberellins but not sterol or carotenoid biosynthesis in Gibberella fujikuroi
Sterols, carotenoids and gibberellins are synthesized after the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate in different subcellular compartments of the fungus Gibberella fujikuroi. Lovastatin inhibits growth in many organisms, presumably because of the inhibition of the synthesis of essential terpenoids. However, in G. fujikuroi growth of the mycelia and sterol and carotenoid content were not affected by the presence of lovastatin. Nevertheless, lovastatin did inhibit the accumulation of gibberellins in the culture medium; this inhibition, however, was counteracted by the addition of mevalonate to the medium. The conversion of HMG-CoA to mevalonate in cell-free extracts was inhibited by 10 nM lovastatin. Since G. fujikuroi apparently possesses a single gene for HMG-CoA reductase, as shown by Southern hybridization and PCR amplification, it was concluded that the biosynthesis of sterols, carotenoids and gibberellins shares a single HMG-CoA reductase, but the respective subcellular compartments are differentially accessible to lovastatin.
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- Systematics And Evolution
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Galactomannans from the cell walls of species of Paecilomyces sect. Paecilomyces and their teleomorphs as immunotaxonomic markers
An alkali-extractable and water-soluble fraction (F1S) was obtained from cell walls of Paecilomyces variotii and species of the related genera Talaromyces, Byssochlamys and Thermoascus. The structure of the main polysaccharide of these fractions was studied and found to consist of a core of (1→6)-α-mannopyranose partially substituted at O-2 by chains of galactofuranose and shorter chains of mannopyranose. The differences in the regularity of the branching points and the length of the galactofuranose side chains are useful to distinguish between species. These differences were detected by immunological methods, since highly specific polyclonal antibodies were raised against these polysaccharides. Mycelium of P. variotii CBS 990.73A was stained by indirect immunofluorescence. The polysaccharides studied in this work differ from the one described for species from section Isarioidea, and this is another indication of the heterogeneity of the genus Paecilomyces.
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