- Volume 153, Issue 8, 2007
Volume 153, Issue 8, 2007
- Cell And Developmental Biology
-
-
-
Essential roles of class E Vps proteins for sorting into multivesicular bodies in Schizosaccharomyces pombe
The multivesicular body (MVB) sorting pathway is required for a number of biological processes, including downregulation of cell-surface proteins and protein sorting into the vacuolar lumen. The function of this pathway requires endosomal sorting complexes required for transport (ESCRT) composed of class E vacuolar protein sorting (Vps) proteins in Saccharomyces cerevisiae, many of which are conserved in Schizosaccharomyces pombe. Of these, sst4/vps27 (homologous to VPS27) and sst6 (similar to VPS23) have been identified as suppressors of sterility in ste12Δ (sst), although their functions have not been uncovered to date. In this report, these two sst genes are shown to be required for vacuolar sorting of carboxypeptidase Y (CPY) and an MVB marker, the ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein, despite the lack of the ubiquitin E2 variant domain in Sst6p. Disruption mutants of a variety of other class E vps homologues also had defects in sorting of CPY and Ub–GFP–CPS. Sch. pombe has a mammalian AMSH homologue, sst2. Phenotypic analyses suggested that Sst2p is a class E Vps protein. Taken together, these results suggest that sorting into multivesicular bodies is dependent on class E Vps proteins, including Sst2p, in Sch. pombe.
-
-
- Biochemistry And Molecular Biology
-
-
-
Mutations in yhiT enable utilization of exogenous pyrimidine intermediates in Salmonella enterica serovar Typhimurium
Mutants capable of utilizing the pyrimidine biosynthetic intermediates carbamoylaspartate and dihydroorotate for growth were derived from pyrimidine auxotrophs of Salmonella enterica serovar Typhimurium LT2. The gain-of-function phenotypes both resulted from mutations in a single gene, yhiT, the third gene of a putative four-gene operon, yhiVUTS, for which there is no homologous region in Escherichia coli. Notably, when a mutant yhiT allele was transferred to a pyrimidine-requiring E. coli strain, the transformant was then capable of using carbamoylaspartate or dihydrorotate as a pyrimidine source. The operon arrangement of the yhiVUTS genes was supported by genetic analyses and studies employing RT-PCR, coupled to the determination of the transcriptional start site using 5′-random amplification of cDNA ends (RACE). Computer-generated predictions indicated that YhiT is an integral membrane protein with 12 putative transmembrane domains typical of bacterial transport proteins. Competition experiments showed that mutant YhiT interacts with the C4-dicarboxylates succinate and malate, as well as the amino acids aspartate and asparagine. The native function of wild-type YhiT remains undetermined, but the collective results are consistent with a role as a general transporter of C4-dicarboxylates and other compounds with a similar basic structure.
-
-
-
-
Heterologous expression of linoleic acid isomerase from Propionibacterium acnes and anti-proliferative activity of recombinant trans-10, cis-12 conjugated linoleic acid
More LessThe linoleic acid isomerase enzyme from Propionibacterium acnes responsible for bioconversion of linoleic acid to trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA) was cloned and overexpressed in Lactococcus lactis and Escherichia coli, resulting in between 30 and 50 % conversion rates of the substrate linoleic acid to t10, c12 CLA. The anti-proliferative activities of the fatty acids produced following isomerization of linoleic acid by L. lactis and E. coli were assessed using the human SW480 colon cancer cell line. Fatty acids generated from both L. lactis and E. coli contained a mixture of linoleic acid and t10, c12 CLA at a ratio of ∼1.35 : 1. Following 5 days of incubation of SW480 cells with 5–20 μg ml−1 (17.8–71.3 μM) of the t10, c12 CLA, there was a significant (P<0.001) reduction in growth of the SW480 cancer cells compared with the linoleic acid control. Cell viability after treatment with the highest concentration (20 μg ml−1) of the t10, c12 CLA was reduced to 7.9 % (L. lactis CLA) and 19.6 % (E. coli CLA), compared with 95.4 % (control linoleic acid) and 31.7 % (pure t10, c12 CLA). In conclusion, this is believed to represent the first report in which recombinant strains are capable of producing CLA with an anti-proliferative potential.
-
-
-
Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions
A transcriptional profiling of the metabolism of Corynebacterium glutamicum under oxygen deprivation conditions is reported. It was observed that the glucose consumption rate per cell when C. glutamicum cells were incubated under oxygen deprivation conditions was higher than that achieved by cells incubated under aerobic growth conditions. Furthermore, DNA microarray and quantitative RT-PCR analyses revealed that the genes of several key enzymes of the glycolytic and organic acid production pathways, including gapA, pgk, tpi, ppc, ldhA and mdh, were significantly upregulated under oxygen deprivation conditions. The corresponding enzymic activities consistently correlated with the regulation patterns of the genetic expression observed at the transcriptional level. Studies of lacZ fusions with the gapA, ldhA and mdh genes indicated not only that these genes are strongly induced at the onset of the stationary phase under aerobic growth conditions, but also that high expression levels are maintained under oxygen deprivation conditions. These results indicate that the genetic expression of several key metabolic enzymes in C. glutamicum cells incubated under oxygen deprivation conditions is chiefly regulated at the transcriptional level. The physiological consequence of the observed increased transcription under oxygen deprivation conditions is an increased rate of carbon source consumption, which is accompanied by a concomitant increase in organic acid production.
-
-
-
Comparative proteomics of cell division mutants and wild-type of Synechococcus sp. strain PCC 7942
More LessBacterial cell division is a highly co-ordinated and fine-tuned process. In the unicellular cyanobacterium Synechococcus sp. strain PCC 7942, inactivating mutations in the ftn2 and ftn6 genes block cell division and result in a phenotype with extensively elongated cells. In order to establish the pleiotropic responses induced and cellular processes affected by blocked cell division, the proteomes of wild-type and the cell division mutants Ftn2 and Ftn6 of Synechococcus sp. strain PCC 7942 were characterized and compared. By separating soluble extracted proteins on 2D gels, more than 800 protein spots were visualized on each SYPRO Ruby-stained gel. Quantitative differences in protein composition were detected by using the PDQuest software, and comparative analysis revealed that 76 protein spots changed significantly in the cell division mutants. These protein spots were selected for identification using peptide mass fingerprints generated by MALDI-TOF MS. Fifty-three protein spots were successfully identified, representing 44 different proteins. The upregulated proteins include proteins involved in cell division/cell morphogenesis, protein synthesis and processing, oxidative stress response, amino acid metabolism, nucleotide biosynthesis, and glycolysis, as well as unknown proteins. Among the downregulated proteins are those involved in chromosome segregation, protein processing, photosynthesis, redox regulation, carbon dioxide fixation, nucleotide biosynthesis, the biosynthetic pathway to fatty acids, and energy production. Besides eliciting common responses, inactivation of Ftn2 and Ftn6 in the mutants may result in different responses in protein levels between the mutants. Among 18 identified differentially affected protein spots, 75 % (9/12) of the protein spots affected in the Ftn2 mutant were upshifted, whereas in the Ftn6 mutant 70 % (7/10) of the affected protein spots were downshifted. Identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in cyanobacterial cell division.
-
-
-
An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling
More LessUndecaprenyl phosphate (Und-P) is a universal lipid carrier of glycan biosynthetic intermediates for carbohydrate polymers that are exported to the bacterial cell envelope. Und-P arises from the dephosphorylation of undecaprenyl pyrophosphate (Und-PP) molecules produced by de novo synthesis and also from the recycling of released Und-PP after the transfer of the glycan component to other acceptor molecules. The latter reactions take place at the periplasmic side of the plasma membrane, while cytoplasmic enzymes catalyse the de novo synthesis. Four Und-PP pyrophosphatases were recently identified in Escherichia coli. One of these, UppP (formerly BacA), accounts for 75 % of the total cellular Und-PP pyrophosphatase activity and has been suggested to participate in the Und-P de novo synthesis pathway. Unlike UppP, the other three pyrophosphatases (YbjG, YeiU and PgpB) have a typical acid phosphatase motif also found in eukaryotic dolichyl-pyrophosphate-recycling pyrophosphatases. This study shows that double and triple deletion mutants in the genes uppP and ybjG, and uppP, ybjG and yeiU, respectively, are supersensitive to the Und-P de novo biosynthesis inhibitor fosmidomycin. In contrast, single or combined deletions including pgpB have no effect on fosmidomycin supersensitivity. Experimental evidence is also presented that the acid phosphatase motifs of YbjG and YeiU face the periplasmic space. Furthermore, the quadruple deletion mutant ΔuppP-ΔybjG-ΔyeiU-ΔwaaL has a growth defect and abnormal cell morphology, suggesting that accumulation of unprocessed Und-PP-linked O antigen polysaccharides is toxic for these cells. Together, the results support the notion that YbjG, and to a lesser extent YeiU, exert their enzymic activity on the periplasmic side of the plasma membrane and are implicated in the recycling of periplasmic Und-PP molecules.
-
-
-
LiaRS-dependent gene expression is embedded in transition state regulation in Bacillus subtilis
Maintaining envelope integrity is crucial for the survival of any bacterial cell, especially those living in a complex and ever-changing habitat such as the soil ecosystem. The LiaRS two-component system is part of the regulatory network orchestrating the cell-envelope stress response in Bacillus subtilis. It responds to perturbations of the cell envelope, especially the presence of antibiotics that interfere with the lipid II cycle, such as bacitracin or vancomycin. LiaRS-dependent regulation is strictly repressed by the membrane protein LiaF in the absence of inducing conditions. Here, it is shown that the LiaR-dependent liaI promoter is induced at the onset of stationary phase without addition of exogenous stresses. Its activity is embedded in the complex regulatory cascade governing adaptation at the onset of stationary phase. The liaI promoter is directly repressed by the transition state regulator AbrB and responds indirectly to the activity of Spo0A, the master regulator of sporulation. The activity of the liaI promoter is therefore tightly regulated by at least five regulators to ensure an appropriate level of liaIH expression.
-
-
-
Region 2.1 of the Escherichia coli heat-shock sigma factor RpoH (σ 32) is necessary but not sufficient for degradation by the FtsH protease
More LessThe cellular level of the Escherichia coli heat-shock sigma factor RpoH (σ 32) is negatively controlled by chaperone-mediated proteolysis through the essential metalloprotease FtsH. Point mutations in the highly conserved region 2.1 stabilize RpoH in vivo. To assess the importance of this turnover element, hybrid proteins were constructed between E. coli RpoH and Bradyrhizobium japonicum RpoH1, a stable RpoH protein that differs from region 2.1 of E. coli RpoH at several positions. Nine amino acids forming a putative α-helix were exchanged between the two proteins. Both hybrids were active sigma factors and showed intermediate protein stability. Introduction of RpoH region 2.1 into the general stress sigma factor RpoS, which is a substrate of the ClpXP protease, did not render RpoS susceptible to FtsH. Hence, region 2.1 alone is not sufficient to confer FtsH sensitivity to other proteins. Region 2.1 is not a major chaperone-binding site since DnaK and DnaJ bound efficiently to all RpoH variants. The in vivo stability of the mutated RpoH proteins correlated with their stability in a purified in vitro degradation system, suggesting that region 2.1 might be directly involved in the interaction with the FtsH protease.
-
-
-
Analysis of the AAA+ chaperone clpB gene and stress-response expression in the halophilic methanogenic archaeon Methanohalophilus portucalensis
More LessClpB is a member of the protein-disaggregating chaperone machinery belonging to the AAA+ superfamily. This paper describes a new clpB gene from the halophilic methanoarchaeon Methanohalophilus portucalensis, which has not been reported previously in Archaea. The partial sequence of clpB was identified from the investigation of the salt-stress response of Meh. portucalensis by differential-display RT-PCR (DDRT-PCR). Furthermore, the complete clpB sequence (2610 nt) and its upstream genes encoding the type I chaperonin GroEL/ES were obtained through inverse PCR, Southern hybridization and sequencing. The G+C ratio of clpB is 49.6 mol%. The predicted ClpB polypeptide contains 869 aa and possesses a long central domain and a predicted distinctly discontinuous coiled-coil motif separating two nucleotide-binding domains (NBD1 and NBD2). NBD1 has a single Walker A and two Walker B motifs and NBD2 has only one of each Walker motif, a characteristic of HSP100 proteins. Two repeated Clp amino-terminal domain motifs (ClpN) were identified in ClpB. The putative amino acid sequence shared 75.6 % identity with the predicted clpB homologue annotated as ATPase AAA-2 of Methanococcoides burtonii DSM 6242. Preliminary phylogenetic analysis clustered Meh. portucalensis ClpB (MpClpB) with the low G+C Gram-positive bacteria. Stress response analysis of clpB by Northern blotting showed up to 1.5-fold increased transcription levels in response to both salt up-shock (from 2.1 to 3.1 M NaCl) and down-shock (from 2.1 to 0.9 M NaCl). Both clpB and groEL/ES transcript levels increased when the temperature was shifted from 37 °C to 55 °C. Under heat stress clpB transcription was repressed by the addition of the osmolyte betaine (1 mM). In conclusion, a novel AAA+ chaperone clpB gene from a halophilic methanogen that responded to the fluctuations in temperature, salt concentration and betaine has been identified and analysed for the first time.
-
-
-
The Escherichia coli AraC-family regulators GadX and GadW activate gadE, the central activator of glutamate-dependent acid resistance
More LessEscherichia coli can survive pH 2 acid stress by using several acid resistance systems. The most efficient of these employs glutamate decarboxylase (GadA/GadB) to consume protons, and an antiporter (GadC) to exchange the intracellular decarboxylation product for external glutamic acid. Expression of the essential transcriptional activator of this system, GadE, is controlled by several regulators in a hierarchical fashion. In this study, two additional activators have been identified. The AraC-family regulators GadX and GadW, previously found to activate gadA/BC in vitro, are now shown in vivo to directly activate gadE expression, which, in turn, activates the gadA/BC genes. In vivo results using E. coli and Salmonella enterica show that these regulators actually have little direct effect on gadA and gadBC promoters. The numerous gadE induction pathways converge on a 798 bp control region situated upstream of the gadE promoter region. Deletions of this control region exposed the region between −798 and −360 nt (relative to the translational start) to be required for maximum gadE–lacZ expression in Luria–Bertani (LB) medium and to be the primary focus of GadX and GadW control. The GadE protein itself, which binds to three GAD box sequences present between −233 and −42 nt, helped activate GadE expression in LB, but only when the −798 to −360 region was absent. These regulatory regions and proteins appear to integrate a variety of physiological signals that forecast a need for GadE-dependent gene expression and acid resistance.
-
-
-
Structural characterization of a partially arabinosylated lipoarabinomannan variant isolated from a Corynebacterium glutamicum ubiA mutant
Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan lipoarabinomannan (LAM). This study shows by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, Cg-LAM, characterized by single t-Araf residues linked to the α(1→6)-linked mannan backbone. MALDI-TOF MS showed an average molecular mass of 13 800–15 400 Da for Cg-LAM. The biosynthetic origin of Araf residues found in the extracytoplasmic arabinan domain of AG and LAM is well known to be provided by decaprenyl-monophosphoryl- d -arabinose (DPA). However, the characterization of LAM in a C. glutamicum : : ubiA mutant devoid of prenyltransferase activity and devoid of DPA-dependent arabinan deposition into AG revealed partial formation of LAM, albeit with a slightly altered molecular mass. These data suggest that in addition to DPA utilization as an Araf donor, alternative pathways exist in Corynebacterianeae for Araf delivery, possibly via an unknown sugar nucleotide.
-
-
-
New bacteriophages that infect the phytopathogen Ralstonia solanacearum
More LessFour kinds of bacteriophage (φRSL, φRSA, φRSM and φRSS) were isolated from Ralstonia solanacearum, a soil-borne Gram-negative bacterium that is the causative agent of bacterial wilt in many important crops. The Myovirus-type phages φRSL1 and φRSA1 contained dsDNA genomes of 240 kbp and 39 kbp, respectively. These phages have relatively wide host ranges and gave large clear plaques with various host strains; especially φRSA1 was able to infect all 15 R. solanacearum strains of different races or different biovars tested in this study. Three host strains contained φRSA1-related sequences in their genomic DNAs, suggesting a lysogenic cycle of φRSA1. Two phages, φRSM1 and φRSS1, were characterized as Ff-type phages (Inovirus) based on their particle morphology, genomic ssDNA and infection cycle. However, despite their similar fibrous morphology, their genome size (9.0 kb for φRSM1 and 6.6 kb for φRSS1) and genome sequence were different. Strains of R. solanacearum that were sensitive to φRSM1 were resistant to φRSS1 and vice versa. Several R. solanacearum strains contained φRSM1-related sequences and at least one strain produced φRSM1 particles, indicating the lysogenic state of this phage. These phages may be useful as a tool not only for molecular biological studies of R. solanacearum pathogenicity but also for specific and efficient detection (φRSM1 and φRSS1) and control of harmful pathogens (φRSL and φRSA) in cropping ecosystems as well as growing crops.
-
-
-
Genetic characterization of the hdrRM operon: a novel high-cell-density-responsive regulator in Streptococcus mutans
More LessMany species of bacteria can adhere to surfaces and grow as sessile communities. The continued accumulation of bacteria can eventually lead to the extremely high-cell-density environment characteristic of many biofilms or cell colonies. This is the normal habitat of the cariogenic species Streptococcus mutans, which normally resides in the high-cell-density, multispecies community commonly referred to as dental plaque. Previous work has demonstrated that the transcription of two separate bacteriocins can be activated by the high-cell-density conditions created through the centrifugation and incubation of cell pellets. In this study, we identified an uncharacterized two-gene operon that was induced >10-fold by conditions of high cell density. The genes of the operon encode a putative transcription regulator and a membrane protein, which were renamed as hdrR and hdrM, respectively. A transcription fusion to the hdrRM operon confirmed its induction by high cell density. Mutation of hdrM abolished bacteriocin production, greatly increased natural competence, reduced the growth rate, and severely affected biofilm formation. Interestingly, no obvious phenotypes were observed from a non-polar mutation of hdrR or mutations affecting the entire operon. These data suggest that the hdrRM operon may constitute a novel regulatory system responsible for mediating a cellular response to a high-cell-density environment.
-
-
-
Dioctatin A is a strong inhibitor of aflatoxin production by Aspergillus parasiticus
Dioctatin A (DotA), a metabolite of Streptomyces, is known to be an inhibitor of human dipeptidyl aminopeptidase II. Here, it was found that DotA strongly inhibited aflatoxin production by Aspergillus parasiticus, with an IC50 value of 4.0 μM. The mycelial growth of the fungus was not affected by the addition of DotA at a concentration of 50 μM, but inhibition of conidiation was observed at the same concentration. DotA inhibited production of norsolorinic acid, an early biosynthetic intermediate of aflatoxin, and it strongly reduced the mRNA levels of genes encoding aflatoxin biosynthetic enzymes, and significantly decreased the mRNA level of aflR, which encodes a key regulatory protein for aflatoxin biosynthesis. In addition to these genes, the mRNA level of brlA, which encodes a conidiation-specific transcription factor, was also reduced by the addition of DotA. It was also found that DotA dramatically enhanced kojic acid production by the fungus. Furthermore, DotA inhibited production of sterigmatocystin, which is a toxic aflatoxin biosynthetic intermediate, and it also inhibited conidiation in Aspergillus nidulans. These results indicate that DotA has pleiotropic effects on regulatory mechanisms of fungal secondary metabolite production and differentiation, leading to inhibition of aflatoxin production.
-
-
-
Functional characterization of three genes encoding putative oxidoreductases required for cercosporin toxin biosynthesis in the fungus Cercospora nicotianae
More LessCercosporin is a non-host-selective, photoactivated polyketide toxin produced by many phytopathogenic Cercospora species, which plays a crucial role during pathogenesis on host plants. Upon illumination, cercosporin converts oxygen molecules to toxic superoxide and singlet oxygen that damage various cellular components and induce lipid peroxidation and electrolyte leakage. Three genes (CTB5, CTB6 and CTB7) encoding putative FAD/FMN- or NADPH-dependent oxidoreductases in the cercosporin toxin biosynthetic pathway of C. nicotianae were functionally analysed. Replacement of each gene via double recombination was utilized to create null mutant strains that were completely impaired in cercosporin production as a consequence of specific interruption at the CTB5, CTB6 or CTB7 locus. Expression of CTB1, CTB5, CTB6, CTB7 and CTB8 was drastically reduced or nearly abolished when CTB5, CTB6 or CTB7 was disrupted. Production of cercosporin was revived when a functional gene cassette was introduced into the respective mutants. All ctb5, ctb6 and ctb7 null mutants retained wild-type levels of resistance against toxicity of cercosporin or singlet-oxygen-generating compounds, indicating that none of the genes plays a role in self-protection.
-
-
-
The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits
More LessHexose kinases play a central role in the initiation of sugar metabolism of living organisms and have also been implicated in carbon catabolite repression in yeasts and plants. In this study, the genes encoding glucokinase (Glk1) and hexokinase (Hxk1) from the plant-pathogenic ascomycete Botrytis cinerea were isolated and functionally characterized. Glk1-deficient mutants were indistinguishable from the wild-type in all growth parameters tested. In contrast, Δhxk1 mutants lacking Hxk1 showed a pleiotropic growth defect. On artificial media, vegetative growth was retarded, and conidia formation strongly reduced. No or only marginal growth of Δhxk1 mutants was observed when fructose, galactose, sucrose or sorbitol were used as carbon sources, and fructose inhibited growth of the mutant in the presence of other carbon sources. B. cinerea mutants containing hxk1 alleles with point mutations leading to enzymically inactive enzymes showed phenotypes similar to the Δhxk1 disruption mutant, indicating that loss of hexose phosphorylation activity of Hxk1 is solely responsible for the pleiotropic growth defect. Virulence of the Δhxk1 mutants was dependent on the plant tissue: on leaves, lesion formation was only slightly retarded compared to the wild-type, whereas only small lesions were formed on apples, strawberries and tomatoes. The low virulence of Δhxk1 mutants on fruits was correlated with their high contents of sugars, in particular fructose. Heterologous expression of Hxk1 and Glk1 in yeast allowed their enzymic characterization, revealing kinetic properties similar to other fungal hexokinases and glucokinases. Both Δglk1 and Δhxk1 mutants showed normal glucose repression of secreted lipase 1 activity, indicating that, in contrast to yeast, B. cinerea hexose kinases are not involved in carbon catabolite repression.
-
- Biodiversity And Evolution
-
-
-
Intragenomic heterogeneity and intergenomic recombination among Vibrio parahaemolyticus 16S rRNA genes
More LessVibrio parahaemolyticus is a marine bacterium bearing 11 copies of ribosomal operons. In some strains, such as RIMD2210633, the genome includes identical copies of 16S rRNA genes (rrs). However, it is known that other strains of the species, such as strains ATCC 17802 and RIMD2210856, show conspicuous intragenomic rrs heterogeneity. The extent and diversity of the rrs heterogeneity in V. parahaemolyticus were studied in further detail by characterization of the rrs copies in environmental isolates belonging to 21 different genotype groups. Thirteen of these groups showed intragenomic heterogeneity, containing altogether 16 sequences differing within a 25 bp segment of their rrs. These sequences grouped into four clusters differing in at least four nucleotide sites. Some isolates contained rrs alleles from up to three different clusters. Each segment sequence conserved the stem–loop characteristic of the 16S rRNA structure of this 25 bp sequence. The double-stranded stem sequence was quite variable, but almost every variation had a compensatory change to maintain seven to eight paired bases. Conversely, the single-strand loop sequence was conserved. The results may be explained as a consequence of recombination among rrs evolving in different bacteria. The results suggest that intergenomic rrs recombination is very high in V. parahaemolyticus and that it occurs solely among Vibrio species. This high rrs homologous intergenomic recombination could be an effective mechanism to maintain intragenomic rrs cohesion, mediating the dispersal of the most abundant rrs version among the 11 intragenomic loci.
-
-
-
-
Phylogeny and shared conserved inserts in proteins provide evidence that Verrucomicrobia are the closest known free-living relatives of chlamydiae
More LessThe evolutionary relationships of Chlamydiales, Verrucomicrobia and Planctomycetes were studied based on phylogenetic trees for a concatenated dataset of 11 widely distributed proteins, as well as conserved inserts in several proteins. In phylogenetic trees, a close relationship of chlamydiae to Verrucomicrobium was supported by different phylogenetic methods. Although the Planctomycetes branched close to the chlamydiae-Verrucomicrobia clade, their specific affiliation to these groups was generally not supported. Results are also presented for two conserved inserts, a 6 aa insert in the lysyl-tRNA synthetase and a 3 aa insert in the RNA polymerase β subunit (RpoB), that are uniquely shared by Verrucomicrobium spinosum and all available Chlamydiales homologues, but which are not found in any of the available Planctomycetes or other bacterial homologues. Signature sequences in a number of other proteins [including a large insert (>150 aa) in DNA gyrase B] provide information regarding the branching position of these groups relative to other bacterial phyla. A close and specific relationship of V. spinosum to the Chlamydiales species, seen both in phylogenetic trees and by means of uniquely shared inserts in protein sequences, strongly indicates that these two groups of species shared a common ancestor exclusive of all other known bacteria. These results suggest that Verrucomicrobia may be the closest free-living relatives of the parasitic chlamydiae.
-
-
-
Genotypic and phenotypic characterization of Lactobacillus casei strains isolated from different ecological niches suggests frequent recombination and niche specificity
More LessLactobacillus casei strains are lactic acid bacteria (LAB) that colonize diverse ecological niches, and have broad commercial applications. To probe their evolution and phylogeny, 40 L. casei strains were characterized; the strains included isolates from plant materials (n=9), human gastrointestinal tracts (n=7), human blood (n=1), cheeses from different geographical locations (n=22), and one strain of unknown origin. API biochemical testing identified niche-specific carbohydrate fermentation profiles. A multilocus sequence typing (MLST) scheme was developed for L. casei. Partial sequencing of six housekeeping genes (ftsZ, metRS, mutL, nrdD, pgm and polA) revealed between 11 (nrdD) and 20 (mutL) allelic types, as well as 36 sequence types. Phylogenetic analysis of MLST data by Reticulate and split decomposition analysis indicated frequent intra-species recombination. Purifying selection was detected, and is likely to have contributed to the evolution of certain L. casei genes. Pulsed-field gel electrophoresis (PFGE) using SfiI was able to discriminate all the isolates, even those not differentiated by MLST. Phylogenetic trees reconstructed based on the MLST data using minimum evolution algorithm, and the SfiI-PFGE restriction patterns using the unweighted-pair group method with arithmetic mean (UPGMA), revealed consensus clusters of strains specific to cheese and silage. Topological discrepancies between the MLST and PFGE trees were also observed, suggesting that intragenic point mutations have accumulated at a slower rate than indels and genome rearrangements in L. casei. The L. casei population analysed in this study demonstrated both a high level of phenotypic and genotypic diversity, as well as specificity to different ecological niches.
-
-
-
Recombination and positive selection contribute to evolution of Listeria monocytogenes inlA
More LessThe surface molecule InlA interacts with E-cadherin to promote invasion of Listeria monocytogenes into selected host cells. DNA sequencing of inlA for 40 L. monocytogenes isolates revealed 107 synonymous and 45 nonsynonymous substitutions. A frameshift mutation in a homopolymeric tract encoding part of the InlA signal peptide was identified in three lineage II isolates, which also showed reduced ability to invade human intestinal epithelial cells. Phylogenies showed clear separation of inlA sequences into lineages I and II. Thirteen inlA recombination events, predominantly involving lineage II strains as recipients (12 events), were detected and a number of amino acid residues were shown to be under positive selection. Four of the 45 non-synonymous changes were found to be under positive selection with posterior probabilities >95 %. Mapping of polymorphic and positively selected amino acid sites on the partial crystal structure for InlA showed that the internalin surface of the leucine-rich repeat (LRR) region that faces the InlA receptor E-cadherin does not include any polymorphic sites; all polymorphic and positively selected amino acids mapped to the outer face of the LRR region or to other InlA regions. The data show that (i) inlA is highly polymorphic and evolution of inlA involved a considerable number of recombination events in lineage II isolates; (ii) positive selection at specific amino acid sites appears to contribute to evolution of inlA, including fixation of recombinant events; and (iii) single-nucleotide deletions in a lineage II-specific 3′ homopolymeric tract in inlA lead to complete loss of InlA or to production of truncated InlA, which conveys reduced invasiveness. In conclusion, inlA has a complex evolutionary history, which is consistent with L. monocytogenes’ natural history as an environmental pathogen with broad host-range, including its adaptation to environments and hosts where different inlA alleles may provide a selective advantage or where inlA may not be required.
-
-
-
Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region
More LessMycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 5′ end of the variable lipoprotein and haemagglutinin gene (vlhA), amplicons of ∼400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A–J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A–J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.
-
- Environmental Microbiology
-
-
-
Mycobacterium smegmatis mc2 155 fbiC and MSMEG_2392 are involved in triphenylmethane dye decolorization and coenzyme F420 biosynthesis
More LessMycobacteria can tolerate relatively high concentrations of triphenylmethane dyes such as malachite green and methyl violet. To identify mycobacterial genes involved in the decolorization of malachite green, a transposon mutant library of Mycobacterium smegmatis mc2 155 was screened for mutants unable to decolorize this dye. One of the genes identified was MSMEG_5126, an orthologue of Mycobacterium bovis fbiC encoding a 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) synthase, which is essential for the biosynthesis of the electron carrier coenzyme F420. The other gene identified was MSMEG_2392, encoding an alanine-rich protein with a DUF121 domain. The minimum inhibitory concentrations (MICs) for malachite green and methyl violet of the six fbiC mutants and two MSMEG_2392 mutants were one-third and one-fifth, respectively, of the MIC of the parent strain M. smegmatis mc2 155. Representative fbiC and MSMEG_2392 mutant strains were also sensitive to oxidative stress caused by the redox-cycling agents plumbagin and menadione, and the sensitivity was reversed in the complemented strains. HPLC analysis of representative fbiC and MSMEG_2392 strains revealed that, while the fbiC mutant lacked both coenzyme F420 and FO, the MSMEG_2392 mutant contained FO but not coenzyme F420. These results indicate that MSMEG_2392 is involved in the biosynthesis of coenzyme F420.
-
-
- Genes And Genomes
-
-
-
OmpR negatively regulates expression of invasin in Yersinia enterocolitica
More LessInvasin, the major adhesion and invasion factor of Yersinia enterocolitica, is encoded by the inv gene, which is regulated by growth phase and in response to a variety of environmental conditions such as temperature, pH and osmolarity. So far, three proteins, RovA, H-NS and YmoA, have been identified as factors regulating the expression of the inv gene in enteropathogenic Yersinia. Here, data from inv′ : : lacZYA chromosomal gene fusion studies are presented indicating that OmpR, the response regulator of the EnvZ/OmpR two-component system, acts to negatively regulate inv expression at the transcriptional level at 25 °C, and that high osmolarity enhances the inhibitory effect of this protein. In a strain lacking OmpR the expression of inv at 25 °C was increased sixfold, but at 37 °C, a temperature known to repress inv expression, this effect was not observed, suggesting that temperature regulation of inv is OmpR-independent. Furthermore, the expression of inv in the ompR background was no longer responsive to increased osmolarity. Complementation with the active ompR allele restored wild-type inv expression in the ompR mutant. In silico analysis of the Y. enterocolitica O : 9 inv promoter sequence revealed the presence of an OmpR consensus binding site located in the −15 to −33 region. OmpR was able to specifically bind to a fragment of the inv promoter containing this putative binding site in electrophoretic mobility shift assays. Thus, OmpR seems to be a repressor of inv in Y. enterocolitica.
-
-
-
-
Development of an intermolecular transposition assay system in Bacillus subtilis 168 using IS4Bsu1 from Bacillus subtilis (natto)
More LessMost of the spontaneous poly-γ-glutamate (γ-PGA)-deficient mutants of Bacillus subtilis (natto) appear to have resulted from the insertion of IS4Bsu1 exclusively into the comP gene. However, complete genomic analysis of B. subtilis 168, a close relative of B. subtilis (natto), revealed no IS4Bsu1 insertion. Preliminary experiments using a transformable ‘natto’ strain indicated that the frequency of transposition of IS4Bsu1 was exceptionally high under competence-developing conditions. On the other hand, such high-frequency transposition was not observed when cells were grown in a rich medium, such as LB medium, suggesting that there must be suitable environmental conditions that give rise to the transposition of IS4Bsu1. To assess the behaviour of IS4Bsu1 and explore any host factors playing roles in IS transposition, an intermolecular transposition assay system was constructed using a modified IS4Bsu1 element in B. subtilis 168. Here, the details of the intermolecular transposition assay system are given, and the increase in transposition frequency observed under high-temperature and competence-inducing conditions is described.
-
-
-
Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria
More LessMycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP–int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a tRNALys gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions.
-
-
-
A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium
More LessThe mechanisms that promote and regulate transcription in mycoplasmas are poorly understood. Here, a promoter-probe vector based on the pMTnTetM438 minitransposon and containing a promoterless lacZ reporter gene was constructed to analyse Mycoplasma genitalium transcription in vivo. Recovered transposon insertions were in monocopy, with 16 % expressing enough β-galactosidase (β-Gal) to yield colonies exhibiting a detectable blue colour. A sample of 52 blue colonies was propagated and selected for further analyses. The β-Gal activity of the corresponding cultures was measured to quantify, in a reproducible way, the transcription levels of the interrupted ORFs. Several insertions were found in sense with the interrupted ORF, but surprisingly there was also a number of insertions in non-coding regions, many of them in repetitive DNA regions known as MgPa islands. Moreover, 30 % of the analysed transposon insertions had the lacZ gene in the opposite orientation to the coding frame, suggesting the existence of antisense transcripts that may be involved in the control of gene expression in M. genitalium.
-
- Pathogens And Pathogenicity
-
-
-
Temporal analysis of Candida albicans gene expression during biofilm development
Microarrays were used to identify changes in gene expression associated with Candida albicans biofilm development. Two biofilm substrates (denture and catheter), and two C. albicans strains for each substrate, were tested to remove model- and strain-dependent variability from the overall dataset. Three biofilm developmental phases were examined: early (6 h), intermediate (12 h), and mature (48 h). Planktonic specimens were collected at the same time points. Data analysis focused primarily on gene expression changes over the time-course of biofilm development. Glycolytic and non-glycolytic carbohydrate assimilation, amino acid metabolism, and intracellular transport mechanisms were important during the early phase of biofilm formation. These early events increase intracellular pools of pyruvate, pentoses and amino acids, which prepare the biofilm for the large biomass increase that begins around 12 h of development. This developmental stage demands energy and utilizes specific transporters for amino acids, sugars, ions, oligopeptides and lactate/pyruvate. At mature phase (48 h), few genes were differentially expressed compared with the 12 h time point, suggesting a relative lack of initiation of new metabolic activity. Data analysis to assess biofilm model-specific gene expression showed more dynamic changes in the denture model than in the catheter model. Data analysis to identify gene expression changes that are associated with each strain/substrate combination identified the same types of genes that were identified in the analysis of the entire dataset. Collectively, these data suggest that genes belonging to different, but interconnected, functional categories regulate the morphology and phenotype of C. albicans biofilm.
-
-
-
-
Mapping of the proinflammatory domains of MspTL of Treponema lecithinolyticum
More LessThe major surface protein (MspTL) of Treponema lecithinolyticum, associated with periodontitis and endodontic infections, has been reported to induce proinflammatory mediators such as intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-1β, IL-6 and IL-8. The purpose of this study was to examine the role of MspTL in cell adhesion/migration and to identify its proinflammatory domains. Using the human monocytic cell line THP-1 and human dermal microvascular endothelial cells (HMEC-1), it was demonstrated that MspTL increased adhesion of monocytes to endothelial cells and transendothelial migration. To analyse the proinflammatory domains of the protein, four gene constructs covering different regions of MspTL were designed and expressed in Escherichia coli using the expression vector pQE-30. Histidine-tagged recombinant proteins were purified using Ni-NTA agarose and polymyxin B agarose to remove LPS contamination. Recombinant truncated polypeptides were assessed for the ability to induce ICAM-1 and proinflammatory factors in THP-1 cells by real-time RT-PCR and ELISA. Of the four polypeptides, the one spanning the N-terminal 86 amino acids significantly induced ICAM-1, IL-1β, IL-6, IL-8, tumour necrosis factor-α (TNF-α), cyclooxygenase (COX)-2, and prostaglandin E2 (PGE2). The results indicate that MspTL may induce cell adhesion and inflammation via its N-terminal region.
-
-
-
Mesophilic Aeromonas UDP-glucose pyrophosphorylase (GalU) mutants show two types of lipopolysaccharide structures and reduced virulence
A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and three l-glycero-d-manno-heptose residues. galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and O44 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1 % the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.
-
-
-
Identification of minor inner-membrane components of the Shigella type III secretion system ‘needle complex’
Type III secretion systems (T3SSs or secretons) are central virulence factors of many Gram-negative bacteria, used to inject protein effectors of virulence into eukaryotic host cells. Their overall morphology, consisting of a cytoplasmic region, an inner- and outer-membrane section and an extracellular needle, is conserved in various species. A portion of the secreton, containing the transmembrane regions and needle, has been isolated biochemically and termed the ‘needle complex’ (NC). However, there are still unsolved questions concerning the nature and relative arrangement of the proteins assembling the NC. Until these are resolved, the mode of function of the NC cannot be clarified. This paper describes an affinity purification method that enables highly efficient purification of Shigella NCs under near-physiological conditions. Using this method, three new minor components of the NC were identified by mass spectrometry: IpaD, a known component of the needle tip complex, and two predicted components of its central inner-membrane export apparatus, Spa40 and Spa24. A further minor component of the NC, MxiM, is only detected by immunoblotting. MxiM is a ‘pilotin’-type protein for the outer-membrane ‘secretin’ ring formed of MxiD. As expected, it localized to the outer rim of the upper ring of NCs, validating the other findings.
-
-
-
A putative DNA adenine methyltransferase is involved in Yersinia pseudotuberculosis pathogenicity
More LessSome adenine methyltransferases have been shown not only to protect specific DNA restriction sites from cleavage by a restriction endonuclease, but also to play a role in various bacterial processes and sometimes in bacterial virulence. This study focused on a type I restriction–modification system (designated yrmI) of Y. pseudotuberculosis. This system is composed of three adjacent genes which could potentially encode an N 6-adenine DNA methylase (YamA), an enzyme involved in site-specific recognition (YrsA) and a restriction endonuclease (YreA). Screening of 85 isolates of Y. pestis and Y. pseudotuberculosis indicated that the yrmI system has been lost by Y. pestis and that yamA (but not yrsA or yreA) is present in all Y. pseudotuberculosis strains tested, suggesting that it may be important at some stages of the epidemiological cycle of this species. To further investigate the role of yamA in Y. pseudotuberculosis survival, multiplication or virulence, a ΔyamA mutant of Y. pseudotuberculosis IP32953 was constructed by allelic exchange with a kanamycin cassette. The fact that ΔyamA mutants were obtained indicated that this gene is not essential for Y. pseudotuberculosis viability. The IP32953ΔyamA mutant strain grew as well as the wild-type in a rich medium at both 28 °C and 37 °C. It also grew normally in a chemically defined medium at 28 °C, but exhibited a growth defect at 37 °C. In contrast to the Dam adenine methyltransferase, a mutation in yamA did not impair the functions of DNA repair or resistance to detergents. However, the ΔyamA mutant exhibited a virulence defect in a mouse model of intragastric infection. The in silico analysis indicated that the chromosomal region carrying the Y. pseudotuberculosis yrmI locus has been replaced in Y. pestis by a horizontally acquired region which potentially encodes another methyltransferase. YamA might thus be dispensable for Y. pestis growth and virulence because this species has acquired another gene fulfilling the same functions.
-
-
-
The role of Staphylococcus aureus surface protein SasG in adherence and biofilm formation
More LessStaphylococcus aureus colonizes the moist squamous epithelium of the anterior nares. One of the adhesins likely to be responsible is the S. aureus surface protein G (SasG), which has sequence similarity with the proteins Pls (plasmin sensitive) of S. aureus and Aap (accumulation associated protein) of Staphylococcus epidermidis. Expression of SasG by a laboratory strain of S. aureus could not be detected by Western immunoblotting. To enable investigation of SasG, the gene was cloned into two expression vectors, the IPTG-inducible pMUTIN4 and the tetracycline-inducible pALC2073, and introduced into S. aureus. Expression of SasG masked the ability of exponentially grown S. aureus cells expressing protein A (Spa), clumping factor B (ClfB) and the fibronectin binding proteins A and B (FnBPA and FnBPB) to bind to IgG, cytokeratin 10 and fibronectin, respectively. SasG also masked binding to fibrinogen mediated by both ClfB and the FnBPs. Western immunoblotting showed no reduction in expression of the blocked adhesins following induction of SasG. SasG size variants with eight, six or five B repeats masked binding to the ligands, whereas variants with four, two or one repeats had no effect. SasG-expressing strains formed peritrichous fibrils (53.47±2.51 nm long) of varying density on the cell wall, which were labelled by immunogold negative staining with anti-SasG antibodies. SasG-expressing strains of S. aureus also formed biofilm independently of the polysaccharide intercellular adhesin (PIA). SasG variants with eight, six and five repeats formed biofilm, whereas variants with four, two or one repeats did not. It was concluded that the fibrillar nature of SasG explains its ability to mask binding of S. aureus microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to their ligands and to promote formation of biofilm. In addition, the strong adhesion of SasG to desquamated nasal epithelial cells likely compensates for its blocking of the binding of S. aureus ClfB to cytokeratin 10, which is important in adhesion to squames by cells lacking SasG. Several clinical isolates expressed SasG at levels similar to those of SH1000 sasG : : pMUTIN4, indicating that the properties described in the laboratory strain SH1000 may be relevant in vivo.
-
-
-
Molecular heterogeneity of EmaA, an oligomeric autotransporter adhesin of Aggregatibacter (Actinobacillus) actinomycetemcomitans
More LessAdhesion of Aggregatibacter actinomycetemcomitans to extracellular matrix proteins is mediated by antennae-like surface structures composed of EmaA oligomers. EmaA is an outer-membrane protein orthologous to the autotransporter YadA, a virulence determinant of Yersinia. emaA was present in the 27 strains examined, covering the six serotypes of A. actinomycetemcomitans. Ten individual genotypes and three different forms of the protein (full-length, intermediate and truncated) were predicted. The prototypic, full-length EmaA (202 kDa) was only associated with serotypes b and c, which displayed antennae-like surface structures. These strains bound to collagen embedded in a 3D matrix. The intermediate form of EmaA (173 kDa) was exclusively associated with serotypes d and a, which contained a 279 aa in-frame deletion, as well as a different N-terminal head domain sequence. These differences modified the appearance of the EmaA structures on the cell surface but maintained collagen-binding activity. Strains containing the truncated form of EmaA had single or multiple substitutions, deletions or insertions in the sequences, which resulted in the absence of EmaA molecules on the outer membrane and loss of collagen-binding activity. Population structure analyses of this organism, based on emaA, indicated that serotypes b and c belonged to one subpopulation, which was independent of the other serotypes. The main divergence was found in the functional head domain. The conserved emaA genotype within serotypes suggests a stable clonal linkage between this autotransporter protein and other virulence determinants.
-
-
-
M protein from Streptococcus pyogenes induces tissue factor expression and pro-coagulant activity in human monocytes
More LessInvasive infections caused by the important pathogen Streptococcus pyogenes are often associated with disturbed blood coagulation in the human host, and may in severe cases develop into the life-threatening condition disseminated intravascular coagulation. In this study, the addition of M1 protein to human blood or purified peripheral blood mononuclear cells led to a dose-dependent increase of pro-coagulant activity, which was mediated by an upregulation of tissue factor on monocytes. Analysis of the resulting clots by transmission electron microscopy revealed that the cells were covered with a fibrin network that seemed to originate from the cell surface. Taken together, the results imply an important role for M proteins in the induction of haemostatic disorders in invasive streptococcal infectious diseases.
-
-
-
In vitro expression of the first capsule gene of Streptococcus pneumoniae, cpsA, is associated with serotype-specific colonization prevalence and invasiveness
More LessThe polysaccharide capsule protects Streptococcus pneumoniae from phagocytosis during invasive infection, but inhibits adherence. Serotypes vary in their tendency to colonize the nasopharynx or cause invasive infection, and differences in capsule expression may play a role. Expression of the first gene of the capsule operon, cpsA, during in vitro growth of 43 clinical isolates representing 14 common pneumococcal serotypes was compared using quantitative RT-PCR. Serotypes associated with invasive infection (1, 4, 5, 7F, 8 and 14) expressed an average of twofold (P=0.0003) more cpsA than serotypes associated with nasopharyngeal colonization (6A, 6B, 9V, 15, 18C, 19F, 23F and 33). There was no difference in cpsA expression in response to growth under environmental oxygen or anaerobic conditions between the invasive and colonizing transparent strains tested: oxygen concentration did not affect cpsA expression in either the invasive or the colonizing transparent strains. Expression of cpsA at OD600 0.6 tended to be greater in strains with a longer lag phase during in vitro growth (P=0.07). Therefore, cpsA expression under ambient oxygen concentrations correlates with serotype-specific invasiveness and is inversely associated with the prevalence of serotype-specific carriage.
-
-
-
In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages
More LessThe Gram-negative proteobacterium Burkholderia pseudomallei can survive and multiply within a variety of eukaryotic cells, including macrophages. This property is believed to be important for its ability to cause the disease melioidosis in a wide range of animal species, including humans. To identify determinants that are important for the ability of B. pseudomallei to survive within macrophages, in vivo expression technology (IVET) was employed. Several putative macrophage-inducible genes were identified that are likely to contribute to the virulence of B. pseudomallei, including three genes (tssH-5, tssI-5 and tssM-5) located within the same type VI secretion system cluster (tss-5), mntH, encoding a natural resistance-associated macrophage protein (NRAMP)-like manganese ion transporter, and a haem acquisition gene, bhuT. The macrophage-inducibility of the tss-5 gene cluster was confirmed by reporter gene analysis. Construction of tssH-5 and bhuT null mutants indicated that expression of the tss-5 unit and the bhu operon were not required for intramacrophage survival. A further five tss units were identified within the B. pseudomallei genome that, together with tss-5, account for approximately 2.3 % of the total genome size. The presence of six type VI secretion systems in this organism is likely to be an important factor in making this bacterium such a versatile pathogen.
-
-
-
Membrane–cytosolic translocation of verotoxin A1 subunit in target cells
More LessIn sensitive cells, verotoxin 1 (VT1) utilizes a globotriaosylceramide receptor-dependent retrograde transport pathway from the cell surface to the Golgi/endoplasmic reticulum (ER). The VT1 A subunit (VTA) is an RNA glycanase. Although translocation of VTA from the ER to the cytosol is considered the route for protein synthesis inhibition, cell-based evidence is lacking. A dual-fluorescent-labelled VT1 holotoxin was constructed to simultaneously monitor VTA and VT1 B subunit (VTB) intracellular transport. By confocal microscopy, VTA/VTB subunits remained associated throughout the retrograde transport pathway without cytosolic staining. However, in [125I]VT1-treated cells, the selective cytosolic translocation (4 %) of the activated form of VTA, VTA1, was demonstrated for the first time by monitoring [125I]VTA1 release after plasma membrane permeabilization by streptolysin O (SLO). Lactacystin, a proteasome inhibitor, increased cytosolic VTA1 and enhanced VT1 cytotoxicity. VT1 ER arrival coincided with cytosolic VTA1 detection. Brefeldin A and 16 °C, conditions which inhibit VT1 retrograde transport to the Golgi/ER, prevented VTA1 cytosolic translocation; however, these treatments did not completely prevent VT1-induced protein synthesis inhibition. Thus, efficient cytosolic translocation of VTA1 requires transport to the Golgi/ER, but alternative minor escape pathways for protein synthesis inhibition may operate when transport to the Golgi/ER is prevented. Inhibition of protein synthesis was time and dose dependent, and not necessarily a valid index of subsequent cytopathology. Only protein synthesis inhibition following >3 h VT1 exposure correlated with eventual cell cytotoxicity. Extrapolation of translocated cytosolic VTA1 values indicates that about one molecule of translocated VTA1 per cell is sufficient to inhibit protein synthesis and kill a cell.
-
-
-
DAF- and collagen-binding properties of chimeric Dr fimbriae
More LessDisplay of heterologous proteins on the surface of micro-organisms has become an increasingly used strategy for a range of applications in microbiology, biotechnology and vaccinology. In this study, the potential of the major structural protein DraE of Escherichia coli Dr fimbriae as a display system for heterologous sequences was tested. One copy of a heterologous sequence mimicking a small Pk epitope of simian virus 5 was inserted into the draE gene, replacing the N-terminal region of the surface-exposed domain 2 as previously done with the glycoprotein D of herpes simplex virus type 1. The exposure of chimeric proteins on the bacterial surface was detected by immunofluorescence microscopy. Insertion of the heterogenic peptides had no detectable effect on the Ig-barrel structure of the DraE fimbrial subunits, as confirmed by FTIR spectroscopy. Additionally, the affinity of the chimeric fimbriae for DAF and type IV collagen was similar to that of the wild-type Dr fimbriae.
-
- Physiology
-
-
-
FlhF, a signal recognition particle-like GTPase, is involved in the regulation of flagellar arrangement, motility behaviour and protein secretion in Bacillus cereus
Flagellar arrangement is a highly conserved feature within bacterial species. However, only a few genes regulating cell flagellation have been described in polar flagellate bacteria. This report demonstrates that the arrangement of flagella in the peritrichous flagellate Bacillus cereus is controlled by flhF. Disruption of flhF in B. cereus led to a reduction in the number of flagella from 10–12 to 1–3 filaments per cell in the insertion mutant MP06. Moreover, compared to the parental strain, MP06 exhibited: (i) shorter smooth swimming phases, causing reduced swimming motility but not affecting chemotaxis; (ii) complete inhibition of swarming motility, as differentiated swarm cells were never detected; (iii) an increased amount of extracellular proteins; and (iv) differential export of virulence determinants, such as haemolysin BL (HBL), phosphatidylcholine-preferring phospholipase C (PC-PLC) and non-haemolytic enterotoxin (NHE). Introduction of a plasmid harbouring flhF (pDGflhF) into MP06 completely restored the wild-type phenotype in the trans-complemented strain MP07. B. cereus flhF was found to constitute a monocistronic transcriptional unit and its overexpression did not produce abnormal features in the wild-type background. Characterization of a B. cereus mutant (MP05) carrying a partial flhF deletion indicated that the last C-terminal domain of FlhF is involved in protein export while not required for flagellar arrangement and motility behaviour. Taken together, these data suggest that B. cereus FlhF is a promising candidate for connecting diverse cellular functions, such as flagellar arrangement, motility behaviour, pattern of protein secretion and virulence phenotype.
-
-
-
-
The acid-resistance pathways of Shigella flexneri 2457T
More LessThe stationary-phase acid-resistance pathways of Shigella flexneri 2457T have not previously been studied. The two acid-resistance systems, the glutamate-dependent acid-resistance (GDAR) and the oxidative pathways, reported elsewhere for Escherichia coli and S. flexneri 3136, were both detected in S. flexneri 2457T. However, S. flexneri 2457T cells grown overnight under fermentative conditions and acid-shocked in minimal media in the absence of glutamate, an acid test often described as a negative control for both pathways, were capable of surviving acid challenge. It is possible that this resistance is due to the oxidative pathway operating in a non-glucose-repressible manner, or to a novel pathway present in S. flexneri 2457T. The construction of gadB and gadC mutants ruled out any contribution by the GDAR pathway, whilst further characterizing the GDAR properties of S. flexneri 2457T. Interestingly, study of the role of rpoS in the oxidative pathway and the unusual acid-resistance phenotype revealed that the frameshift present in the 2457T rpoS gene results in expression of a truncated RpoS protein, which may be reduced in activity and is not essential for the acid-resistance phenotype of S. flexneri 2457T.
-
-
-
Plasma-membrane Cnh1 Na+/H+ antiporter regulates potassium homeostasis in Candida albicans
More LessThe physiological role of Candida albicans Cnh1, a member of the Na+/H+ antiporter family, was characterized. Though CaCnh1p had broad substrate specificity and mediated efflux of at least four alkali metal cations upon heterologous expression in Saccharomyces cerevisiae, its presence in C. albicans cells was important especially for potassium homeostasis. In C. albicans, CaCnh1p tagged with GFP was localized in the plasma membrane of cells growing as both yeasts and hyphae. Deletion of CNH1 alleles did not affect tolerance to NaCl, LiCl or CsCl, but resulted in increased sensitivity to high external concentrations of KCl and RbCl. The potassium and rubidium tolerance of a cnh1 homozygous mutant was fully restored by reintegration of CNH1 into the genome. The higher sensitivity of the cnh1/cnh1 mutant to external KCl was caused by a lower K+ efflux from these cells. Together, the functional characterization of the CaCnh1 antiporter in C. albicans revealed that this antiporter plays a significant role in C. albicans physiology. It ensures potassium and rubidium tolerance and participates in the regulation of intracellular potassium content of C. albicans cells.
-
- Plant-Microbe Interactions
-
-
-
Muscodor albus E-6, an endophyte of Guazuma ulmifolia making volatile antibiotics: isolation, characterization and experimental establishment in the host plant
More LessMuscodor albus is an endophytic fungus, represented by a number of isolates from tropical tree and vine species in several of the world's rainforests, that produces volatile organic compounds (VOCs) with antibiotic activity. A new isolate, E-6, of this organism, with unusual biochemical and biological properties, has been obtained from the branches of a mature Guazuma ulmifolia (Sterculiaceae) tree growing in a dry tropical forest in SW Ecuador. This unique organism produces many VOCs not previously observed in other M. albus isolates, including butanoic acid, 2-methyl-; butanoic acid, 3-methyl-; 2-butenal, 2-methyl-; butanoic acid, 3-methylbutyl ester; 3-buten-1-ol, 3-methyl; guaiol; 1-octene, 3-ethyl-; formamide, N-(1-methylpropyl); and certain azulene and naphthalene derivatives. Some compounds usually seen in other M. albus isolates also appeared in the VOCs of isolate E-6, including caryophyllene; phenylethyl alcohol; acetic acid, 2-phenylethyl ester; bulnesene; and various propanoic acid, 2-methyl- derivatives. The biological activity of the VOCs of E-6 appears different from the original isolate of this fungus, CZ-620, since a Gram-positive bacterium was killed, and Sclerotinia sclerotiorum and Rhizoctonia solani were not. Scanning electron micrographs of the mycelium of isolate E-6 showed substantial intertwining of the hyphal strands. These strands seemed to be held together by an extracellular matrix accounting for the strong mat-like nature of the mycelium, which easily lifts off the agar surface upon transfer, unlike any other isolate of this fungus. The ITS-5.8S rDNA partial sequence data showed 99 % similarity to the original M. albus strain CZ-620. For the first time, successful establishment of M. albus into its natural host, followed by recovery of the fungus, was accomplished in seedlings of G. ulmifolia. Overall, isolates of M. albus, including E-6, have chemical, biological and structural characteristics that make them potentially useful in medicine, agricultural and industrial applications.
-
-
- Theoretical Microbiology
-
-
-
Modelling the spatial dynamics of plasmid transfer and persistence
More LessBacterial plasmids are extra-chromosomal genetic elements that code for a wide variety of phenotypes in their bacterial hosts and are maintained in bacterial communities through both vertical and horizontal transfer. Current mathematical models of plasmid–bacteria dynamics, based almost exclusively on mass-action differential equations that describe these interactions in completely mixed environments, fail to adequately explain phenomena such as the long-term persistence of plasmids in natural and clinical bacterial communities. This failure is, at least in part, due to the absence of any spatial structure in these models, whereas most bacterial populations are spatially structured in microcolonies and biofilms. To help bridge the gap between theoretical predictions and observed patterns of plasmid spread and persistence, an individual-based lattice model (interacting particle system) that provides a predictive framework for understanding the dynamics of plasmid–bacteria interactions in spatially structured populations is presented here. To assess the accuracy and flexibility of the model, a series of experiments that monitored plasmid loss and horizontal transfer of the IncP-1β plasmid pB10 : : rfp in Escherichia coli K12 and other bacterial populations grown on agar surfaces were performed. The model-based visual patterns of plasmid loss and spread, as well as quantitative predictions of the effects of different initial parental strain densities and incubation time on densities of transconjugants formed on a 2D grid, were in agreement with this and previously published empirical data. These results include features of spatially structured populations that are not predicted by mass-action differential equation models.
-
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)