- Volume 149, Issue 12, 2003
Volume 149, Issue 12, 2003
- Mini-Review
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Facing extremes: archaeal surface-layer (glyco)proteins
More LessArchaea are best known in their capacities as extremophiles, i.e. micro-organisms able to thrive in some of the most drastic environments on Earth. The protein-based surface layer that envelopes many archaeal strains must thus correctly assemble and maintain its structural integrity in the face of the physical challenges associated with, for instance, life in high salinity, at elevated temperatures or in acidic surroundings. Study of archaeal surface-layer (glyco)proteins has thus offered insight into the strategies employed by these proteins to survive direct contact with extreme environments, yet has also served to elucidate other aspects of archaeal protein biosynthesis, including glycosylation, lipid modification and protein export. In this mini-review, recent advances in the study of archaeal surface-layer (glyco)proteins are discussed.
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- Microbiology Comment
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- Cell And Developmental Biology
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Involvement of DivIVA in the morphology of the rod-shaped actinomycete Brevibacterium lactofermentum
More LessIn Brevibacterium lactofermentum, as in many Gram-positive bacteria, a divIVA gene is located downstream from the dcw cluster of cell-division- and cell-wall-related genes. This gene (divIVABL ) is mostly expressed during exponential growth, and the protein encoded, DivIVABL, bears some sequence similarity to antigen 84 (Ag84) from mycobacteria and was detected with monoclonal antibodies against Ag84. Disruption experiments using an internal fragment of the divIVABL gene or a disrupted divIVABL cloned in a suicide conjugative plasmid were unsuccessful, suggesting that the divIVABL gene is needed for cell viability in Brev. lactofermentum. Transformation of Brev. lactofermentum with a multicopy plasmid containing divIVABL drastically altered the morphology of the corynebacterial cells, which became larger and bulkier, and a GFP fusion to DivIVABL mainly localized to the ends of corynebacterial cells. This localization pattern, together with the overproduction phenotype, suggests that DivIVA may be important in regulating the apical growth of daughter cells.
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- Biochemistry And Molecular Biology
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Use of a two-hybrid assay to study the assembly of a complex multicomponent protein machinery: bacterial septosome differentiation
More LessThe ability of each of the nine Escherichia coli division proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, FtsI, FtsN) to interact with itself and with each of the remaining eight proteins was studied in 43 possible combinations of protein pairs by the two-hybrid system previously developed by the authors' group. Once the presumed interactions between the division proteins were determined, a model showing their temporal sequence of assembly was developed. This model agrees with that developed by other authors, based on the co-localization sequence in the septum of the division proteins fused with GFP. In addition, this paper shows that the authors' assay, which has already proved to be very versatile in the study of prokaryotic and eukaryotic protein interaction, is also a powerful instrument for an in vivo study of the interaction and assembly of proteins, as in the case of septum division formation.
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Structural analysis by X-ray crystallography and calorimetry of a haemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum
More LessBotulism food poisoning is caused primarily by ingestion of the Clostridium botulinum neurotoxin (BoNT). The 1300 amino acid BoNT forms a progenitor toxin (PTX) that, when associated with a number of other proteins, increases its oral toxicity by protecting it from the low pH of the stomach and from intestinal proteases. One of these associated proteins, HA1, has also been suggested to be involved with internalization of the toxin into the bloodstream by binding to oligosaccharides lining the intestine. Here is reported the crystal structure of HA1 from type C Clostridium botulinum at a resolution of 1·7 Å. The protein consists of two β-trefoil domains and bears structural similarities to the lectin B-chain from the deadly plant toxin ricin. Based on structural comparison to the ricin B-chain lactose-binding sites, residues of type A HA1 were selected and mutated. The D263A and N285A mutants lost the ability to bind carbohydrates containing galactose moieties, implicating these residues in carbohydrate binding.
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High-affinity myo-inositol transport in Candida albicans: substrate specificity and pharmacology
More LessInositol is considered a growth factor in yeast cells and it plays an important role in Candida as an essential precursor for phospholipomannan, a glycophosphatidylinositol (GPI)-anchored glycolipid on the cell surface of Candida which is involved in the pathogenicity of this opportunistic fungus and which binds to and stimulates human macrophages. In addition, inositol plays an essential role in the phosphatidylinositol signal transduction pathway, which controls many cell cycle events. Here, high-affinity myo-inositol uptake in Candida albicans has been characterized, with an apparent K m value of 240±15 μM, which appears to be active and energy-dependent as revealed by inhibition with azide and protonophores (FCCP, dinitrophenol). Candida myo-inositol transport was sodium-independent but proton-coupled with an apparent K m value of 11·0±1·1 nM for H+, equal pH 7·96±0·05, suggesting that the C. albicans myo-inositol–H+ transporter is fully activated at physiological pH. C. albicans inositol transport was not affected by cytochalasin B, phloretin or phlorizin, an inhibitor of mammalian sodium-dependent inositol transport. Furthermore, myo-inositol transport showed high substrate specificity for inositol and was not significantly affected by hexose or pentose sugars as competitors, despite their structural similarity. Transport kinetics in the presence of eight different inositol isomers as competitors revealed that proton bonds between the C-2, C-3 and C-4 hydroxyl groups of myo-inositol and the transporter protein play a critical role for substrate recognition and binding. It is concluded that C. albicans myo-inositol–H+ transport differs kinetically and pharmacologically from the human sodium-dependent myo-inositol transport system and constitutes an attractive target for delivery of cytotoxic inositol analogues in this pathogenic fungus.
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Differential regulation of the Proteus mirabilis urease gene cluster by UreR and H-NS
More LessProteus mirabilis, a cause of catheter-associated urinary tract infection, relies on several virulence factors to colonize the urinary tract. Among these, urease contributes to the development of urinary stones resulting from the increase in local pH due to urease-mediated hydrolysis of urea to NH3 and CO2. UreR, an AraC-like transcriptional activator, activates transcription of the genes encoding the urease subunits and accessory proteins (ureDABCEFG) in the presence of urea. UreR also initiates transcription of its own gene in a urea-inducible manner by binding to the intergenic region between ureR and ureD. The intergenic region contains poly(A) tracts that appear to be the target of H-NS. It has been shown that Escherichia coli and P. mirabilis H-NS acts to repress transcription of ureR in an E. coli model system. It was hypothesized that H-NS represses urease gene expression in the absence of UreR and urea by binding to the intergenic region. To demonstrate this the P. mirabilis hns gene was cloned and the 15·6 kDa H-NS was overexpressed and purified as a myc-His tail fusion. Using a gel shift assay, purified H-NS-myc-His bound preferentially to a 609 bp DNA fragment containing the entire ureR-ureD intergenic region. H-NS and UreR were able to displace each other from the ureR-ureD intergenic region. Circular permutation analysis revealed that the intergenic region is bent. Moreover, H-NS recognizes this curvature, binds the DNA fragment and induces further bending of the DNA as shown by a circular ligation assay. The effects of H-NS, urea and temperature (25 vs 37 °C) on urease expression were shown in E. coli containing an hns knockout and P. mirabilis where expression was increased at 37 °C. Increased transcription from pureR was seen in the E. coli hns knockout when temperature was increased from 25 to 37 °C. These findings suggest H-NS and UreR differentially regulate urease in a negative and positive manner, respectively.
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The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration
More LessThe napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66·5 bp downstream of the centre of a putative FNR-like binding site.
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Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans
More LessIn Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, β-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of β-galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic β-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N2O. Additions of nitrate or nitroprusside lowered β-galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.
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Two MerR homologues that affect copper induction of the Bacillus subtilis copZA operon
More LessCopper ions induce expression of the Bacillus subtilis copZA operon encoding a metallochaperone, CopZ, and a CPx-type ATPase efflux protein, CopA. The copZA promoter region contains an inverted repeat sequence similar to that recognized by the mercury-sensing MerR protein. To investigate the possible involvement of MerR homologues in copZA regulation, null mutations were engineered affecting each of four putative MerR-type regulators: yyaN, yraB, yfmP and yhdQ. Two of these genes affected copper regulation. Mutation of yhdQ (hereafter renamed cueR) dramatically reduced copper induction of copZA, and purified CueR bound with high affinity to the copZA promoter region. These results suggest that CueR is a direct regulator of copZA transcription that mediates copper induction. Surprisingly, a yfmP mutation also reduced copper induction of copZA. Sequence analysis suggested that yfmP was cotranscribed with yfmO, encoding a putative multidrug efflux protein. The yfmPO operon is autoregulated: a yfmP mutation derepressed the yfmP promoter and purified YfmP bound the yfmP promoter region, but not the copZA promoter region. Since the yfmP mutant strain was predicted to express elevated levels of the YfmO efflux pump, it was hypothesized that copper efflux might be responsible for the reduced copZA induction. Consistent with this model, in a yfmP yfmO double mutant copper induction of copZA was normal. The results demonstrate the direct regulation of the B. subtilis copper efflux system by CueR, and indirect regulation by a putative multidrug efflux system.
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Identification of functionally important regions of a haemoglobin receptor from Neisseria meningitidis
More LessThe HmbR outer-membrane receptor enables Neisseria meningitidis to use haemoglobin (Hb) as a source of iron. This protein functions by binding Hb, removing haem from it, and releasing the haem into the periplasm. Functionally important HmbR receptor domains were discerned using a series of HmbR deletions and site-directed mutations. Mutations exhibiting similar defective phenotypes in N. meningitidis fell into two groups. The first group of mutations affected Hb binding and were located in putative extracellular loops (L) L2 (amino acid residues (aa) 192–230) and L3 (aa 254–284). The second group of mutations resulted in a failure to utilize Hb but proficiency in Hb binding was retained. These mutations localized to the putative extracellular loops L6 (aa 420–462) and L7 (aa 486–516). A highly conserved protein motif found in all haem/Hb receptors, within putative extracellular loop L7 of HmbR, is essential for Hb utilization but not required for Hb binding. This finding suggests a mechanistic involvement of this motif in haem removal from Hb. In addition, an amino-terminal deletion in the putative cork-like domain of HmbR affected Hb usage but not Hb binding. This result supports a role of the cork domain in utilization steps that are subsequent to Hb binding.
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PepN is the major aminopeptidase in Escherichia coli: insights on substrate specificity and role during sodium-salicylate-induced stress
More LessPepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5αΔpepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.
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The LysR-type transcriptional regulator CysB controls the repression of hslJ transcription in Escherichia coli
More LessThe LysR-type transcriptional regulator (LTTR) CysB is a transcription factor in Escherichia coli cells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription. The hslJ gene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium. cysB mutants showed upregulation of the hslJ : : lacZ gene fusion and exhibited increased novobiocin resistance. In this study the hslJ transcription start point and the corresponding putative σ 70 promoter were determined. The hslJ promoter region was defined by employing different hslJ–lacZ operon fusions, and transcription of the hslJ gene was shown to be subject to both repression imposed by the CysB regulator and direct or indirect autogenous negative control. These two regulations compete to some extent but they are not mutually exclusive. CysB acts as a direct repressor of hslJ transcription and binds the hslJ promoter region that carries the putative CysB repressor site. This CysB binding, apparently responsible for repression, is enhanced in the presence of the ligand N-acetylserine (NAS), hitherto considered to be a positive cofactor in CysB-mediated gene regulations. Interallelic complementation of characterized CysB mutants I33N and S277Ter partially restored the repression of hslJ transcription and the consequent novobiocin sensitivity, but did not complement the cysteine auxotrophy.
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Pathways for phosphatidylcholine biosynthesis in bacteria
More LessPhosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti and Legionella pneumophila have both PMT and PCS activities. In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities. Genes from M. loti and L. pneumophila encoding a Pmt or a Pcs activity and the genes from P. aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such as Brucella melitensis, P. aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.
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Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system
More LessAcinetobacter baumannii causes severe infections in compromised patients, survives on abiotic surfaces in hospital environments and colonizes different medical devices. In this study the analysis of the processes involved in surface attachment and biofilm formation by the prototype strain 19606 was initiated. This strain attaches to and forms biofilm structures on plastic and glass surfaces, particularly at the liquid–air interface of cultures incubated stagnantly. The cell aggregates, which contain cell stacks separated by water channels, formed under different culture conditions and were significantly enhanced under iron limitation. Electron and fluorescence microscopy showed that pili and exopolysaccharides are part of the cell aggregates formed by this strain. Electron microscopy of two insertion derivatives deficient in attachment and biofilm formation revealed the disappearance of pili-like structures and DNA sequencing analysis showed that the transposon insertions interrupted genes with the highest similarity to hypothetical genes found in Pseudomonas aeruginosa, Pseudomonas putida and Vibrio parahaemolyticus. Although the products of these genes, which have been named csuC and csuE, have no known functions, they are located within a polycistronic operon that includes four other genes, two of which encode proteins related to chaperones and ushers involved in pili assembly in other bacteria. Introduction of a copy of the csuE parental gene restored the adherence phenotype and the presence of pili on the cell surface of the csuE mutant, but not that of the csuC derivative. These results demonstrate that the expression of a chaperone-usher secretion system, some of whose components appear to be acquired from unrelated sources, is required for pili formation and the concomitant attachment to plastic surfaces and the ensuing formation of biofilms by A. baumannii cells.
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- Biodiversity And Evolution
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Gradual evolution in bacteria: evidence from Bacillus systematics
More LessThe bacterial genome projects have suggested a central role for horizontal transfer in bacterial adaptation, but it is difficult to rule out an adaptive role for ordinary genetic change in existing genes. The bacterial systematics literature can readily address the importance of gene acquisition in adaptive evolution, since phenotypic characterization typically assesses presence versus absence of metabolic capabilities, and metabolic gains and losses are most likely due to horizontal transfer and/or gene loss. Bacterial systematists have not geared their studies toward quantitative differences in metabolic capabilities, which are more likely to involve adjustments of existing genes. Here, quantitative variation in metabolism within and between three closely related Bacillus taxa has been assayed. While these taxa show no qualitative (i.e. presence versus absence) differences in resource utilization, they are quantitatively different in utilization of 8 % of 95 resources tested. Moreover, 93 % of the resources tested showed significant quantitative variation among strains within a single taxon. These results suggest that ordinary genetic changes in existing genes may play an important role in adaptation. If these results are typical, future genomically based assays of quantitative variation in phenotype (e.g. microarray analysis of mRNA concentrations) may identify hundreds of genes whose expression has been modified. A protocol is presented for identifying those modifications of gene expression and those gene acquisitions that are most likely to have played a role in adaptive evolution.
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The distribution and genetic structure of Escherichia coli in Australian vertebrates: host and geographic effects
More LessEscherichia coli was isolated from more than 2300 non-domesticated vertebrate hosts living in Australia. E. coli was most prevalent in mammals, less prevalent in birds and uncommon in fish, frogs and reptiles. Mammals were unlikely to harbour E. coli if they lived in regions with a desert climate and less likely to have E. coli if they lived in the tropics than if they lived in semi-arid or temperate regions. In mammals, the likelihood of isolating E. coli from an individual depended on the diet of the host and E. coli was less prevalent in carnivores than in herbivores or omnivores. In both birds and mammals, the probability of isolating E. coli increased with the body mass of the host. Hosts living in close proximity to human habitation were more likely to harbour E. coli than hosts living away from people. The relative abundance of E. coli groups A, B1, B2 and D strains in mammals depended on climate, host diet and body mass. Group A strains were uncommon, but were isolated from both ectothermic and endothermic vertebrates. Group B1 strains could also be isolated from any vertebrate group, but were predominant in ectothermic vertebrates, birds and carnivorous mammals. Group B2 strains were unlikely to be isolated from ectotherms and were most abundant in omnivorous and herbivorous mammals. Group D strains were rare in ectotherms and uncommon in endotherms, but were equally abundant in birds and mammals. The results of this study suggest that, at the species level, the ecological niche of E. coli is mammals with hindgut modifications to enable microbial fermentation, or in the absence of a modified hindgut, E. coli can only establish a population in ‘large-bodied’ hosts. The non-random distribution of E. coli genotypes among the different host groups indicates that strains of the four E. coli groups may differ in their ecological niches and life-history characteristics.
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- Environmental Microbiology
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A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus
More LessPlasmid analysis of isolates from a small Paracoccus population revealed that all 15 representatives carried at least one endogenous plasmid of 23 or 15 kb in size, in addition to further plasmids of different sizes. It was shown by restriction analysis and hybridization that the 23 and 15 kb plasmids from the different isolates were identical or very similar to each other. By partial sequencing of pOL18/23, one of the 23 kb plasmids, a complete rrn operon with the structural genes for 16S, 23S and 5S rRNA, two genes for tRNAIle and tRNAAla within the spacer between the 16S and 23S rRNA genes, and a final tRNAfMet at the end of the operon were discovered. Expression of a green fluorescent protein gene (gfp) after insertion of a DNA fragment from the region upstream of the rRNA genes into a promoter-probe vector demonstrated that the rrn promoter region is functional. The rrn operon encoded by plasmid pOL18/23 is the first complete rrn operon sequenced from a strain of the genus Paracoccus, and only the second example of an rrn operon on a small plasmid.
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A dual lethal system to enhance containment of recombinant micro-organisms
More LessActive containment systems based on the controlled expression of a lethal gene are designed to increase containment of recombinant micro-organisms used for environmental applications. A major drawback in containment is the existence of mutations that generate surviving cells that cease to respond to the toxic effect of the lethal function. In this work the authors have developed for the first time a strategy to reduce the problem of mutations and increase the efficiency of containment based on the combination of two lethal functions acting on different cellular targets of major concern in containment, DNA and RNA, and whose expression is under control of different regulatory signals. To engineer the dual gene containment circuit, two toxin–antitoxin pairs, i.e. the colicin E3–immunity E3 and the EcoRI restriction–modification systems, were combined. The genes encoding the immunity E3 and the EcoRI methyltransferase proteins (antitoxins) were stably inserted into the chromosome of the host cell, whereas the broad-host-range lethal genes encoding the colicin E3 RNase and the EcoRI restriction endonuclease (toxins) were flanking the contained trait in a plasmid. This dual lethal cassette decreased gene transfer frequencies, through killing of the recipient cells, by eight orders of magnitude, which provides experimental evidence that the anticipated containment level due to the combination of single containment systems is generally achieved. Survivors that escaped killing were analysed and the mutational events involved were characterized.
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- Genes And Genomes
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A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus
More LessOchratoxin A is an important nephrotoxic and nephrocarcinogenic mycotoxin, produced by Aspergillus ochraceus as a polyketide-derived secondary metabolite. A portion of a putative polyketide synthase gene (pks) involved in the biosynthesis of this mycotoxin was cloned by using a suppression subtractive hybridization PCR-based approach. The predicted amino acid sequence of the 1·4 kb clone shared 28–35 % identity to acyl transferase regions from fungal polyketide synthases found in the databases. Based on reverse transcription PCR studies, the pks gene is expressed only under ochratoxin A permissive conditions and only during the early stages of the mycotoxin synthesis. A mutant in which the pks gene has been interrupted cannot synthesize ochratoxin A. This report is the first of the cloning and characterization of a gene involved in ochratoxin A biosynthesis.
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Phage display reveals 52 novel extracellular and transmembrane proteins from Lactobacillus reuteri DSM 20016T
More LessExtracellular and transmembrane proteins are important for the binding of bacteria to intestinal surfaces and for their interaction with the host. The aim of this study was to identify genes encoding extracellular and transmembrane proteins from the probiotic bacterium Lactobacillus reuteri by construction and screening of a phage display library. This library was constructed by insertion of randomly fragmented DNA from L. reuteri into the phagemid vector pG3DSS, which was previously developed for screening for extracellular proteins. After affinity selection of the library, the L. reuteri inserts were sequenced and analysed with bioinformatic tools. The screening resulted in the identification of 52 novel genes encoding extracellular and transmembrane proteins. These proteins were classified as: transport proteins; enzymes; sensor–regulator proteins; proteins involved in host/microbial interactions; conserved hypothetical proteins; and unconserved hypothetical proteins. Further characterization of the extracellular and transmembrane proteins identified should contribute to the understanding of the probiotic properties of L. reuteri.
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Extracting phylogenetic information from whole-genome sequencing projects: the lactic acid bacteria as a test case
More LessThe availability of an ever increasing number of complete genome sequences of diverse prokaryotic taxa has led to the introduction of novel approaches to infer phylogenetic relationships among bacteria. In the present study the sequences of the 16S rRNA gene and nine housekeeping genes were compared with the fraction of shared putative orthologous protein-encoding genes, conservation of gene order, dinucleotide relative abundance and codon usage among 11 genomes of species belonging to the lactic acid bacteria. In general there is a good correlation between the results obtained with various approaches, although it is clear that there is a stronger phylogenetic signal in some datasets than in others, and that different parameters have different taxonomic resolutions. It appears that trees based on different kinds of information derived from whole-genome sequencing projects do not provide much additional information about the phylogenetic relationships among bacterial taxa compared to more traditional alignment-based methods. Nevertheless, it is expected that the study of these novel forms of information will have its value in taxonomy, to determine which genes are shared, when genes or sets of genes were lost in evolutionary history, to detect the presence of horizontally transferred genes and/or confirm or enhance the phylogenetic signal derived from traditional methods. Although these conclusions are based on a relatively small dataset, they are largely in agreement with other studies and it is anticipated that similar trends will be observed when comparing other genomes.
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Mitochondrial-type iron–sulfur cluster biosynthesis genes (IscS and IscU) in the apicomplexan Cryptosporidium parvum
More LessSeveral reports have indicated that the iron–sulfur cluster [Fe–S] assembly machinery in most eukaryotes is confined to the mitochondria and chloroplasts. The best-characterized and most highly conserved [Fe–S] assembly proteins are a pyridoxal-5′-phosphate-dependent cysteine desulfurase (IscS), and IscU, a protein functioning as a scaffold for the assembly of [Fe–S] prior to their incorporation into apoproteins. In this work, genes encoding IscS and IscU homologues have been isolated and characterized from the apicomplexan parasite Cryptosporidium parvum, an opportunistic pathogen in AIDS patients, for which no effective treatment is available. Primary sequence analysis (CpIscS and CpIscU) and phylogenetic studies (CpIscS) indicate that both genes are most closely related to mitochondrial homologues from other organisms. Moreover, the N-terminal signal sequences of CpIscS and CpIscU predicted in silico specifically target green fluorescent protein to the mitochondrial network of the yeast Saccharomyces cerevisiae. Overall, these findings suggest that the previously identified mitochondrial relict of C. parvum may have been retained by the parasite as an intracellular site for [Fe–S] assembly.
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- Pathogens And Pathogenicity
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Variation of the natural transformation frequency of Campylobacter jejuni in liquid shake culture
Natural transformation, a mechanism that generates genetic diversity in Campylobacter jejuni, was studied in a novel liquid shake culturing system that allowed an approximately 10 000-fold increase in cell density. C. jejuni transformation frequency was analysed in this system under 10 %, 5·0 % and 0·7 % CO2 atmospheres. At 5·0 % and 10 % CO2 concentrations, when purified isogenic chromosomal DNA was used to assess competence, transformation frequency ranged from 10−3 to 10−4 at low cell concentrations and declined as cell density increased. Transformation frequency under a 0·7 % CO2 atmosphere was more stable, maintaining 10−3 levels at high cell densities, and was 10- to 100-fold higher than that under a 10 % CO2 atmosphere. Three of four C. jejuni strains tested under a 5·0 % CO2 atmosphere were naturally competent for isogenic DNA; competent strains demonstrated a lack of barriers to intraspecies genetic exchange by taking up and incorporating chromosomal DNA from multiple C. jejuni donors. C. jejuni showed a preference for its own DNA at the species level, and co-cultivation demonstrated that DNA transfer via natural transformation occurred between isogenic populations during short periods of exposure in liquid medium when cell density and presumably DNA concentrations were low. Transformation frequency during co-cultivation of isogenic populations was also influenced by CO2 concentration. Under a 0·7 % CO2 atmosphere, co-cultivation transformation frequency increased approximately 500-fold in a linear fashion with regard to cell density, and was 1000- to 10 000-fold higher during late-exponential-phase growth when compared to cultures grown under a 10 % CO2 atmosphere.
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The surface (S-) layer is a virulence factor of Bacteroides forsythus
M. Sabet, S.-W. Lee, R. K. Nauman, T. Sims and H.-S. UmBacteroides forsythus has emerged as a crucial periodontal pathogen with possible implications for systemic disease. The aim of this study was to isolate the S-layer from B. forsythus and examine its virulence potential as a part of efforts to characterize virulence factors of B. forsythus. The role of the S-layer in the haemagglutinating and adherent/invasive activities was evaluated. It was observed that the S-layer alone was able to mediate haemagglutination. In adherent and invasive studies, transmission electron microscopy clearly revealed that B. forsythus cells were able to attach to and invade KB cells, showing the formation of a microvillus-like extension around adherent and intracellular bacteria. The quantitative analysis showed that five different B. forsythus strains exhibited attachment (1·9–2·3 %) and invasion (0·4–0·7 %) capabilities. It was also observed through antibody inhibition assays that adherent/invasive activities of B. forsythus are mediated by the S-layer. Furthermore, an in vivo immunization study adopting a murine abscess model was used to prove that the S-layer is involved in the infectious process of abscess formation. While mice immunized with purified S-layer and B. forsythus whole cells did not develop any abscesses when challenged with viable B. forsythus cells, unimmunized mice developed abscesses. Collectively, the data obtained from these studies indicate that the S-layer of B. forsythus is a virulence factor.
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Genetic control of chlamydospore formation in Candida albicans
More LessThe chlamydospore is a distinctive morphological feature of the fungal pathogen Candida albicans that can be induced to form in oxygen-limited environments and has been reported in clinical specimens. Chlamydospores are not produced by the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, so there is limited understanding of the pathways that govern their development. Here, the results of a forward genetic approach that begins to define the genetic control of chlamydospore formation are described. Six genes – ISW2, MDS3, RIM13, RIM101, SCH9 and SUV3 – are required for efficient chlamydospore formation, based on the phenotypes of homozygous insertion mutants and reconstituted strains. Mutations in ISW2, SCH9 and SUV3 completely abolish chlamydospore formation. Mutations in RIM13, RIM101 and MDS3 delay normal chlamydospore formation. The involvement of alkaline pH-response regulators Rim13p and Mds3p in chlamydospore formation is unexpected in view of the fact that chlamydospores in the inducing conditions used here are repressed in alkaline media.
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CesAB is an enteropathogenic Escherichia coli chaperone for the type-III translocator proteins EspA and EspB
Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that colonize mucosal surfaces of the intestine via formation of attaching and effacing (A/E) lesions. The genes responsible for induction of the A/E lesions are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes the adhesin intimin and the type III secretion system needle complex, translocator and effector proteins. One of the major EPEC translocator proteins, EspA, forms a filamentous conduit along which secreted proteins travel before they arrive at the translocation pore in the plasma membrane of the host cell, which is composed of EspB and EspD. Prior to secretion, many type III proteins, including translocators, are maintained in the bacterial cytoplasm by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins Tir, Map and EspF, and the translocator proteins EspD and EspB. In this study, CesAB (Orf3 of the LEE) was identified as a chaperone for EspA and EspB. Specific CesAB–EspA and CesAB–EspB protein interactions are demonstrated. CesAB was essential for stability of EspA within the bacterial cell prior to secretion. Furthermore, a cesAB mutant failed to secrete EspA, as well as EspB, to assemble EspA filaments, to induce A/E lesion following infection of HEp-2 cells and to adhere to, or cause haemolysis of, erythrocytes.
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The CepIR quorum-sensing system contributes to the virulence of Burkholderia cenocepacia respiratory infections
More LessThe cepIR genes encode an N-acyl homoserine lactone (AHL)-dependent quorum-sensing system consisting of an AHL synthase that directs the synthesis of N-octanoyl-l-homoserine lactone (ohl) and n-hexanoyl-l-homoserine lactone and a transcriptional regulator. The virulence of cepIR mutants was examined in two animal models. Rats were infected with agar beads containing Burkholderia cenocepacia K56-2, K56-I2 (cepI : : Tpr) or K56-R2 (cepR : : Tn5-OT182). At 10 days post-infection, the extent of lung histopathological changes was significantly lower in lungs infected with K56-I2 or K56-R2 compared to the parent strain. Intranasal infections were performed in Cftr (−/−) mice and their wild-type siblings. K56-2 was more virulent in both groups of mice. K56-I2 was the least virulent strain and was not invasive in the Cftr (−/−) mice. OHL was readily detected in lung homogenates from Cftr (−/−) mice infected with K56-2 but was only detected at levels slightly above background in a few mice infected with K56-I2. Lung homogenates from mice infected with K56-2 had significantly higher levels of the inflammatory mediators murine macrophage inflammatory protein-2, KC/N51, interleukin-1β and interleukin-6 than those from K56-I2-infected animals. These studies indicate that a functional CepIR quorum-sensing system contributes to the severity of B. cenocepacia infections. A zinc metalloprotease gene (zmpA) was shown to be regulated by CepR and may be one of the factors that accounts for the difference in virulence between the cepI mutant and the parent strain.
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- Physiology
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Co-ordination of iron acquisition, iron porphyrin chelation and iron–protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens
More LessThe cytoplasmic membrane protein CcmC is, together with other Ccm proteins, a component for the maturation of c-type cytochromes in Gram-negative bacteria. A Pseudomonas fluorescens ATCC 17400 ccmC mutant is cytochrome c-deficient and shows considerably reduced production of the two siderophores pyoverdine and quinolobactin, paralleled by a general inability to utilize various iron sources, with the exception of haem. The ccmC mutant accumulates in a 5-aminolevulinic acid-dependent synthesis a reddish, fluorescent pigment identified as protoporphyrin IX. As a consequence a visA phenotype similar to that of a ferrochelatase-deficient hemH mutant characterized by drastically reduced growth upon light exposure was observed for the ccmC mutant. The defect of iron–protoporphyrin formation was further demonstrated by the failure of ccmC cell-free proteinase K-treated extracts to stimulate the growth of a haem auxotrophic hemH indicator strain, compared to similarly prepared wild-type extracts. In addition, the ccmC mutant did not sustain hemH growth in cross-feeding experiments while the wild-type did. Significantly reduced resistance to oxidative stress mediated by haem-containing catalases was observed for the ccmC mutant. A double hemH ccmC mutant could not be obtained in the presence of external haem without the hemH gene in trans, indicating that the combination of the two mutations is lethal. It was concluded that CcmC, apart from its known function in cytochrome c biogenesis, plays a role in haem biosynthesis. A function in the regulatory co-ordination of iron acquisition via siderophores, iron insertion into porphyrin via ferrochelatase and iron–protoporphyrin export for cytochrome c formation is predicted.
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Compaction of the Escherichia coli nucleoid caused by Cyt1Aa
More LessCompaction of the Escherichia coli nucleoid in the cell's centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein from Bacillus thuringiensis subsp. israelensis. Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused by chloramphenicol. The possibility that small, compact nucleoids in Cyt1Aa-expressing cells resulted in DNA replication run-out and segregation following cell division was ruled out by measuring relative nucleoid length. Treatments with membrane-perforating substances other than Cyt1Aa did not cause such compaction of the nucleoids, but rather the nucleoids overexpanded to occupy nearly all of the cell volume. These findings support the suggestion that, in addition to its perforating ability, Cyt1Aa causes specific disruption of nucleoid associations with the cytoplasmic membrane. In situ immunofluorescence labelling with Alexa did not demonstrate a great amount of Cyt1Aa associated with the membrane. Clear separation between Alexa-labelled Cyt1Aa and 4′,6-diamidino-2-phenylindole (DAPI)-stained DNA indicates that the nucleoid does not bind Cyt1Aa. Around 2 h after induction, nucleoids in Cyt1Aa-expressing cells started to decompact and expanded to fill the whole cell volume, most likely due to partial cell lysis without massive peptidoglycan destruction.
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