- Volume 154, Issue 5, 2008
Volume 154, Issue 5, 2008
- Cell And Developmental Biology
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Rapid tip-directed movement of Golgi equivalents in growing Aspergillus nidulans hyphae suggests a mechanism for delivery of growth-related materials
More LessGolgi equivalents (GEs) process materials in the fungal secretory pathway. Despite the importance of localized secretion in fungal tip growth, GE behaviour in living hyphae has not been documented. The distribution was monitored of an Aspergillus nidulans putative GE-associated protein, CopA, tagged with GFP (CopA–GFP). This co-localized with a Golgi body/GE marker established in other systems, α-2,6-sialyltransferase, tagged with red fluorescent protein (ST–RFP). CopA–GFP and ST–RFP distributions responded similarly to brefeldin A, which impairs Golgi/GE trafficking. We used a CopA–GFP, hypA1 strain to study GE distribution and behaviour in growing A. nidulans hyphae. This strain has a wild-type phenotype at 28 °C, can be manipulated by changing growth temperature or by use of cytoskeleton inhibitors, and its GE behaviour is consistent with that in a wild-type-morphology strain. A. nidulans GEs were more abundant at hyphal tips than subapically, and showed saltatory motility in all directions. Anterograde GE movements predominated. These were positively correlated with, but at least 10-fold faster than, hyphal growth rate, under all growth and experimental conditions investigated. The actin inhibitor latrunculin B reduced both anterograde GE movement and hyphal growth rate, whereas the microtubule (MT) depolymerizer benomyl increased anterograde GE movement and decreased hyphal growth rate. The MT stabilizer taxol increased A. nidulans GE movement but not hyphal growth rate. A. nidulans GE motility appears to have a complex dependence on both actin and MTs. We present a model for apical delivery of growth materials in which A. nidulans GEs play a role in long-distance transport.
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- Biochemistry And Molecular Biology
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A global modulatory role for the Yersinia enterocolitica H-NS protein
More LessThe H-NS protein plays a significant role in the modulation of gene expression in Gram-negative bacteria. Whereas isolation and characterization of hns mutants in Escherichia coli, Salmonella and Shigella represented critical steps to gain insight into the modulatory role of H-NS, it has hitherto not been possible to isolate hns mutants in Yersinia. The hns mutation is considered to be deleterious in this genus. To study the modulatory role of H-NS in Yersinia we circumvented hns lethality by expressing in Y. enterocolitica a truncated H-NS protein known to exhibit anti-H-NS activity in E. coli (H-NSTEPEC). Y. enterocolitica cells expressing H-NSTEPEC showed an altered growth rate and several differences in the protein expression pattern, including the ProV protein, which is modulated by H-NS in other enteric bacteria. To further confirm that H-NSTEPEC expression in Yersinia can be used to demonstrate H-NS-dependent regulation in this genus, we used this approach to show that H-NS modulates expression of the YmoA protein.
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Acidic stress induces autolysis by a CSP-independent ComE pathway in Streptococcus pneumoniae
More LessIn Streptococcus pneumoniae, autolysis is considered a programmed cell-death process executed principally by the major autolysin (LytA), and the underlying mechanism causing its activation is not completely understood. It is known that autolysis is triggered by competence development at alkaline pH and regulated by a two-component system, ComDE, which senses a competence-stimulating peptide (CSP) and behaves as a quorum-sensing mechanism. In this work, we found that acidic stress triggered a LytA-mediated autolysis and, curiously, this phenomenon was regulated by a CSP-independent ComE pathway. A further analysis of a hyperactive ComD mutant revealed that ComE needs to be phosphorylated to activate acidic stress-induced lysis (ASIL). The comE transcripts were induced by acidic culture conditions, suggesting that ComE could be sensing acidic stress. We also investigated CiaRH, a two-component system whose null mutants show a comE derepression and a CSP-dependent autolysis induction at alkaline pH. By analysis of cia comE double mutants, we demonstrated that CiaRH protected cells from ASIL by a ComE-independent pathway. Here, we propose that ComE is the principal route of the signalling pathway that determines a global stress response, and clearly regulates the induction of the LytA-mediated programmed cell death in S. pneumoniae. Acidic stress may represent for S. pneumoniae an alternative condition, in addition to competence and antibiotics, to assure the release of virulence factors, DNA and cell-wall compounds by autolysis, favouring genetic exchange and contributing to its pathogenesis.
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The importance of chitobiase and N-acetylglucosamine (GlcNAc) uptake in N,N′-diacetylchitobiose [(GlcNAc)2] utilization by Serratia marcescens 2170
More LessN,N′-diacetylchitobiose [(GlcNAc)2] is the main degradation product from chitin by the action of chitinases of Serratia marcescens 2170. Uptake of (GlcNAc)2 via a (GlcNAc)2-specific enzyme II permease by this bacterium has been demonstrated previously. Here, we report the contribution of chitobiase and N-acetylglucosamine (GlcNAc) uptake to the utilization of (GlcNAc)2. When S. marcescens 2170 was cultivated in a medium containing chitin, chitobiase activity was detected both inside and outside the cells; intracellular chitobiase was more abundant and suggested to be mainly located in the periplasm. Production of chitobiase was induced by GlcNAc and more effectively by (GlcNAc)2. For induction of chitobiase, uptake of (GlcNAc)2 was essential but ChiR, an essential regulator of chitinase induction, was not required. S. marcescens 2170 grew well on both GlcNAc and (GlcNAc)2 but mutants defective in either chitobiase or NagE, the GlcNAc-specific enzyme II permease, showed reduced growth on (GlcNAc)2. These results suggest that, in addition to uptake as (GlcNAc)2, a proportion of the (GlcNAc)2 is converted to GlcNAc by chitobiase, mainly in the periplasm, and incorporated into the cytoplasm by NagE. The mutant defective in chitobiase grew more slowly on (GlcNAc)2 than on GlcNAc, indicating that (GlcNAc)2 is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. Therefore, uptake as both (GlcNAc)2 and GlcNAc is important for efficient utilization of (GlcNAc)2 in S. marcescens.
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The role of a purine-specific nucleoside hydrolase in spore germination of Bacillus thuringiensis
More LessA homologous gene (iunH) of a putative nucleoside hydrolase (NH), which had been identified from the exosporia of Bacillus cereus and Bacillus anthracis spores, was cloned from Bacillus thuringiensis subsp. kurstaki. Disruption of iunH did not affect the vegetative growth and sporulation of Bacillus thuringiensis, but promoted both inosine- and adenosine-induced spore germination. The inosine- or adenosine-induced germination rate decreased when the wild-type iunH gene was overexpressed in Bacillus thuringiensis. The iunH gene product was characterized as a purine-specific NH. The kinetic parameters of IunH with inosine as substrate were K m=399±115 μM, k cat=48.9±8.5 s−1 and k cat/K m=1.23×105 M−1 s−1. The optimal pH and temperature for IunH were found to be pH 6 and 80 °C. Meanwhile, the specific activity of inosine hydrolase in intact spores of the wild-type strain with inosine as substrate was 2.89±0.23×10−2 μmol min−1 (mg dry wt)−1. These results indicate that IunH is important in moderating inosine- or adenosine-induced germination of Bacillus thuringiensis spores.
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The S.ma.I2 class C group II intron inserts at integron attC sites
More LessWe previously found the class C S.ma.I2 group II (GII) intron in Serratia marcescens SCH909 inserted into the variable region of a class 1 integron within the attC site of the ant(2′′)-Ia gene cassette. Here, we demonstrate that this ant(2′′)-Ia : : S.ma.I2 gene cassette is a recombinationally active element despite the presence of the S.ma.I2 intron. In addition, S.ma.I2 is an active GII intron capable of performing self-splicing and invading specific target sites. Intron homing to a DNA target site is RecA-independent and recognizes the intron binding site (IBS)1 and IBS3 regions, formed by the 5′ TTGTT 3′ consensus sequence located within the inverse core site of attC integrons. Our results also indicate that the process for S.ma.I2 intron mobilization involves a secondary structure provided by the folding of the complete attC site. Moreover, phylogenetic analysis of the class C GII introns showed a clear divergent clade formed by introns that insert within specific sites usually associated with lateral gene transfer.
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Biosynthetic and regulatory elements involved in the production of the siderophore vanchrobactin in Vibrio anguillarum
More LessSome Vibrio anguillarum strains produce a catechol-type siderophore named vanchrobactin, whose biosynthetic pathway has not been completely elucidated. In addition to the previously described genes vabA, vabC, vabB, vabE, vabF, vabS and vabH, in the present study we have identified the genes encoding a DAHP (3-deoxy-d-arabino-heptulosonate-7-phosphate) synthetase (vabG), a phosphopantheteinyl transferase (vabD), a LysR-family transcriptional regulator (vabR) and a putative siderophore receptor (fvtA). A deletion affecting vabG or vabD greatly reduced growth under iron-limiting conditions, whereas deletion of vabR did not have significant effects. Vanchrobactin production was abolished in the vabD mutant, whereas the vabG mutant retained a residual vanchrobactin production ability. Reverse transcriptase-mediated PCR indicated that this 11-gene cluster is organized into six iron-regulated transcriptional units. Transcriptional lacZ fusions demonstrated that the ferric uptake regulator (Fur) protein is the main iron-responsive regulator of these genes. Interestingly, the vabG gene was strongly iron-repressed, but Fur was not essential for this repression. In addition, the maximal expression from the vabG promoter was achieved only in the presence of an intact copy of vabR. Analysis of the β-galactosidase activities of a fvtA : : lacZ fusion in a vabB mutant and in the presence of added vanchrobactin suggested that a ferric-vanchrobactin-dependent activator plays a positive regulatory role in transcription of the fvtA–vabD operon. This possibility is reinforced by the presence of a predicted AraC box upstream of fvtA. We propose that vanchrobactin biosynthesis is subjected to a complex regulatory circuitry aimed at adjusting vanchrobactin production for the maintenance of iron homeostasis in V. anguillarum.
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Sulphur and nitrogen regulation of the protease-encoding ACP1 gene in the fungus Botrytis cinerea: correlation with a phospholipase D activity
More LessSulphur and nitrogen catabolic repressions are regulations that have long been recognized in fungi, but whose molecular bases remain largely elusive. This paper shows that catabolic repression of a protease-encoding gene correlates with the modulation of a phosphatidylethanolamine (PE)-specific phospholipase D (PLD) activity in the pathogenic fungus Botrytis cinerea. Our results first demonstrate that the ACP1 gene is subject to sulphur catabolic repression, with sulphate and cysteine inhibiting its expression. Sulphate and cysteine also cause a decrease of the total cellular PLD activity and, reciprocally, the two PLD inhibitors AEBSF [4-(2-aminoethyl)benzenesulphonyl fluoride] and curcumin negatively affect ACP1 expression in vivo. Cysteine moreover inhibits the PE-specific PLD activity in cell extracts. ACP1 is regulated by nitrogen, but here we show that this regulation does not rely on the proximal AREA binding site in its promoter, and that glutamine does not play a particular role in the process. A decrease in the total cellular PLD activity is also observed when the cells are fed ammonia, but this effect is smaller than that produced by sulphur. RNA-interference experiments finally suggest that the enzyme responsible for the PE-specific PLD activity is encoded by a gene that does not belong to the known HKD gene family of PLDs.
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- Biodiversity And Evolution
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Long-distance dispersal and recombination in environmental populations of Cryptococcus neoformans var. grubii from India
The basidiomycete yeast Cryptococcus neoformans is a cause of significant morbidity and mortality in immunocompromised hosts throughout the world. The sporadic nature of the infection and the limited empirical evidence for direct human-to-human transmission have led to the belief that infections in humans are predominantly caused by the inhalation of basidiospores from environmental sources. Therefore, analysing the structure of environmental populations of C. neoformans can significantly increase our understanding of its ecology, evolution and epidemiology. Decaying wood is a rich source of organic and inorganic compounds and is known to be a suitable ecological niche for many micro-organisms, including C. neoformans. However, relatively little is known about the population structure of C. neoformans sampled from decaying wood. In this study, we analysed samples of C. neoformans var. grubii colonizing decaying wood in tree hollows of nine tree species in five geographical locations (Delhi, Bulandshahar, Hathras, Amritsar and Amrouli) in north-western India. Multilocus sequence typing was conducted using five gene fragments for each of 78 isolates. All isolates belonged to mating type α. Population-genetic analyses identified no evidence for significant differentiation among populations belonging to either different geographical areas or different host tree species. Interestingly, despite the lack of mating type a strains in our survey, we found unambiguous evidence for recombination in our population analyses. Our results are consistent with the hypothesis of long-distance dispersal and recombination in environmental populations of this species in India.
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Variation in the Neisseria lactamica porin, and its relationship to meningococcal PorB
More LessOne potential vaccine strategy in the fight against meningococcal disease involves the exploitation of outer-membrane components of Neisseria lactamica, a commensal bacterium closely related to the meningococcus, Neisseria meningitidis. Although N. lactamica shares many surface structures with the meningococcus, little is known about the antigenic diversity of this commensal bacterium or the antigenic relationships between N. lactamica and N. meningitidis. Here, the N. lactamica porin protein (Por) was examined and compared to the related PorB antigens of N. meningitidis, to investigate potential involvement in anti-meningococcal immunity. Relationships among porin sequences were determined using distance-based methods and FST , and maximum-likelihood analyses were used to compare the selection pressures acting on the encoded proteins. These analyses demonstrated that the N. lactamica porin was less diverse than meningococcal PorB and although it was subject to positive selection, this was not as strong as the positive selection pressures acting on the meningococcal porin. In addition, the N. lactamica porin gene sequences and the protein sequences of the loop regions predicted to be exposed to the human immune system were dissimilar to the corresponding sequences in the meningococcus. This suggests that N. lactamica Por, contrary to previous suggestions, may have limited involvement in the development of natural immunity to meningococcal disease and might not be effective as a meningococcal vaccine component.
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The microbiome of the cloacal openings of the urogenital and anal tracts of the tammar wallaby, Macropus eugenii
More LessThe bacterial diversity of the openings of the urogenital and anal tracts of the adult female tammar wallaby, Macropus eugenii, was determined in order to ascertain whether the physical proximity of the openings of these tracts within the cloaca affected the two populations of bacteria. Terminal restriction fragment length polymorphism (T-RFLP) analyses of 42 wallabies identified 81 different terminal fragments, indicative of diverse and complex microbiomes at these anatomical locations. Subsequent amplified rDNA restriction analysis (ARDRA) identified 72 phylotypes from the urogenital tract and 50 from the anal tract. Twenty-two of these phylotypes were common to both tracts. Phylogenetic analysis of sequenced 16S rDNA showed that 83 % of the phylotypes were unidentified species based on the premise that any sequence possessing <97 % homology to a known bacterial species or phylotype was novel. Thus, despite the close proximity of the openings of the urogenital and anal tracts within the cloaca, the two sites retained a diverse range of distinct bacteria, with only a small percentage of overlapping species.
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- Environmental Microbiology
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Biofilm formation by saprophytic and pathogenic leptospires
Leptospires exist as saprophytic organisms that are aquatic or as pathogens that are able to survive in water. Leptospirosis is transmitted to humans through environmental surface waters contaminated by the urine of mammals, usually rodents, which are chronically infected by pathogenic strains. The ecology of Leptospira spp. prompted us to evaluate if these spirochaetes were able to form biofilms. This study investigated the characteristics of biofilm development by both saprophytic and pathogenic Leptospira species using microscopic examinations and a polystyrene plate model. Biofilms were formed preferentially on glass and polystyrene surfaces. Electron microscopic images showed cells embedded in an extracellular matrix. The formation of such a biofilm is consistent with the life of saprophytic strains in water and may help pathogenic strains to survive in environmental habitats and to colonize the host.
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Salmonella enterica serovar Typhimurium typing by prophage-specific PCR
More LessRecent data from microarray analysis have shown that integrated prophages are the most frequent sources of genomic variation between different strains of Salmonella enterica serovar Typhimurium (S. Typhimurium). This led us to hypothesize that PCR detection of the integrated prophages might be an efficient typing tool that could be used as an alternative to PFGE. In this study, we optimized four triplex PCRs specific for 12 target sequences of mostly prophage origin, and tested them in 102 field strains. The same set of strains was also characterized by PFGE. Among the strains, 22 different multiplex PCR, and 25 different PFGE profiles, were identified. Despite the fact that the PFGE was slightly more discriminatory, multiplex PCR typing, owing to its simplicity and potential of simple data sharing between laboratories, represents an interesting user-friendly alternative to PFGE typing of S. Typhimurium.
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Genes for two multicopper proteins required for Fe(III) oxide reduction in Geobacter sulfurreducens have different expression patterns both in the subsurface and on energy-harvesting electrodes
Previous studies have shown that Geobacter sulfurreducens requires the outer-membrane, multicopper protein OmpB for Fe(III) oxide reduction. A homologue of OmpB, designated OmpC, which is 36 % similar to OmpB, has been discovered in the G. sulfurreducens genome. Deletion of ompC inhibited reduction of insoluble, but not soluble Fe(III). Analysis of multiple Geobacter and Pelobacter genomes, as well as in situ Geobacter, indicated that genes encoding multicopper proteins are conserved in Geobacter species but are not found in Pelobacter species. Levels of ompB transcripts were similar in G. sulfurreducens at different growth rates in chemostats and during growth on a microbial fuel cell anode. In contrast, ompC transcript levels increased at higher growth rates in chemostats and with increasing current production in fuel cells. Constant levels of Geobacter ompB transcripts were detected in groundwater during a field experiment in which acetate was added to the subsurface to promote in situ uranium bioremediation. In contrast, ompC transcript levels increased during the rapid phase of growth of Geobacter species following addition of acetate to the groundwater and then rapidly declined. These results demonstrate that more than one multicopper protein is required for optimal Fe(III) oxide reduction in G. sulfurreducens and suggest that, in environmental studies, quantifying OmpB/OmpC-related genes could help alleviate the problem that Pelobacter genes may be inadvertently quantified via quantitative analysis of 16S rRNA genes. Furthermore, comparison of differential expression of ompB and ompC may provide insight into the in situ metabolic state of Geobacter species in environments of interest.
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Proteomic analysis of Haloferax volcanii reveals salinity-mediated regulation of the stress response protein PspA
More LessA proteomic survey of the halophilic archaeon Haloferax volcanii was performed by comparative two-dimensional gel electrophoresis in order to determine the molecular effects of salt stress on the organism. Cells were grown under optimal (2.1 M) and high (3.5 M) NaCl conditions. From this analysis, over 44 protein spots responsive to these conditions were detected. These spots were excised, digested in-gel with trypsin, subjected to QSTAR tandem mass spectrometry (LC/MS/MS) analysis, and identified by comparing the MS/MS-derived peptide sequence to that deduced from the H. volcanii genome. Approximately 40 % of the proteins detected (18 in total) displayed differential abundance based on the detection of at least two peptide fragments per protein and overall MOWSE scores of ≥75 per protein. All of these identified proteins were either uniquely present or 2.3- to 26-fold higher in abundance under one condition compared to the other. The majority of proteins identified in this study were preferentially displayed under optimal salinity and primarily involved in translation, transport and metabolism. However, one protein of interest whose transcript levels were confirmed in these studies to be upregulated under high salt conditions was identified as a homologue of the phage shock protein PspA. The pspA gene belongs to the psp stress-responsive regulon commonly found among Gram-negative bacteria where its transcription is stimulated by a wide variety of stressors, including heat shock, osmotic shock and prolonged stationary-phase incubation. Homologues of PspA are also found among the genomes of cyanobacteria, higher plants and other Archaea, suggesting that this protein may retain some aspects of functional conservation across the three domains of life. Given its integral role in sensing a variety of membrane stressors in bacteria, these results suggest that PspA may play an important role in hypersaline adaptation in H. volcanii.
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Sulfidogenesis under extremely haloalkaline conditions by Desulfonatronospira thiodismutans gen. nov., sp. nov., and Desulfonatronospira delicata sp. nov. – a novel lineage of Deltaproteobacteria from hypersaline soda lakes
High rates of sulfidogenesis were observed in sediments from hypersaline soda lakes. Anaerobic enrichment cultures at 2 M Na+ and pH 10 inoculated with sediment samples from these lakes produced sulfide most actively with sulfite and thiosulfate as electron acceptors, and resulted in the isolation of three pure cultures of extremely natronophilic sulfidogenic bacteria. Strain ASO3-1 was isolated using sulfite as a sole substrate, strain AHT 8 with thiosulfate and formate, and strain AHT 6 with thiosulfate and acetate. All strains grew in a mineral soda-based medium by dismutation of either sulfite or thiosulfate, as well as with sulfite, thiosulfate and sulfate as acceptors, and H2 and simple organic compounds as electron donors. The acetyl-CoA pathway was identified as the pathway for inorganic carbon assimilation by strain ASO3-1. All strains were obligately alkaliphilic, with an optimum at pH 9.5–10, and grew in soda brines containing 1–4 M total Na+ (optimum at 1.0–2.0 M). The cells accumulated high amounts of the organic osmolyte glycine betaine. They formed a new lineage within the family Desulfohalobiaceae (Deltaproteobacteria), for which the name Desulfonatronospira gen. nov. is proposed. Strains ASO3-1T and AHT 8 from Kulunda Steppe formed Desulfonatronospira thiodismutans sp. nov., and strain AHT 6T from Wadi al Natrun is suggested as Desulfonatronospira delicata sp. nov.
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- Genes And Genomes
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Bioinformatic insights into the biosynthesis of the Group B carbohydrate in Streptococcus agalactiae
More LessStreptococcus agalactiae is a major human and animal pathogen, most notable as a cause of life-threatening disease in neonates. S. agalactiae is also called the Group B Streptococcus in reference to the diagnostically significant Lancefield Group B typing antigen. Although the structure of this complex carbohydrate antigen has been solved, little is known of its biosynthesis beyond the identification of a relevant locus in sequenced S. agalactiae genomes. Analysis of the sugar linkages present in the Group B carbohydrate (GBC) structure has allowed us to deduce the minimum enzymology required to complete its biosynthesis. Most of the enzymes required to complete this biosynthesis can be identified within the putative biosynthetic locus. Surprisingly, however, three crucial N-acetylglucosamine transferases and enzymes required for activated precursor synthesis are not apparently located in this locus. A model for GBC biosynthesis wherein the complete polymer is assembled at the cytoplasmic face of the plasma membrane before translocation to the cell surface is proposed. These analyses also suggest that GBC is the major teichoic acid-like polymer in the cell wall of S. agalactiae, whereas lipoteichoic acid is the dominant poly(glycerophosphate) antigen. Genomic analysis has allowed us to predict the pathway leading to the biosynthesis of GBC of S. agalactiae.
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The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity
dsDNA bacteriophages use the dual system endolysin–holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His6-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His6-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C4–C18), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C16 and C18). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 °C and pH 7.5–8.0. Activity was increased in the presence of Ca2+ and Mn2+. To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.
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Instability of the Salmonella RcsCDB signalling system in the absence of the attenuator IgaA
More LessIgaA is a Salmonella enterica membrane protein that attenuates the response of the RcsCDB signalling system to envelope stress. This protein is essential unless the RcsCDB system is inactivated, suggesting that IgaA may constantly adjust the magnitude of the response. Such a functional link is also supported by the concurrence of the igaA and rcsD-rcsB-rcsC loci in genomes of enteric bacteria and the selection of spontaneous mutations in the RcsCDB system following IgaA deprivation. However, the exact nature of the spontaneous mutations rendering IgaA dispensable remains undefined. In this work, we examined how the transduction of an igaA null allele affects the status of the RcsCDB system. Loss of RcsCDB response was registered in ∼90 % of the IgaA-defective clones, which failed to produce the capsule material positively regulated by this system. About half of these non-mucoid clones suppressed the loss of IgaA with large deletions encompassing variable regions of the rcsD-rcsB-rcsC locus. Unexpectedly, mucoid transductants were also reproducibly obtained and indicated the capacity of S. enterica to retain a functional RcsCDB system in the absence of IgaA. Decreased levels of either RcsC or RcsD were shown in ‘mucoid’ clones lacking IgaA and displaying low responsiveness to stimuli. Taken together, these data demonstrate that the stability and responsiveness of the RcsCDB system relies on its attenuator IgaA. The type of suppressions found also support a model with IgaA controlling the level of signal flowing through RcsC and RcsD.
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A chromosome map of the Flavescence dorée phytoplasma
The Flavescence dorée phytoplasma (FD-P), a non-cultivable, plant-pathogenic bacterium of the class Mollicutes, is the causal agent of a quarantine disease affecting vineyards of southern Europe, mainly in southern France and northern Italy. To investigate FD-P diversity and phytoplasma genetic determinants governing the FD-P life cycle, a genome project has been initiated. A physical map of the chromosome of FD-P strain FD92, purified from infected broad beans, was constructed by performing restriction digests of the chromosome and resolving the fragments by PFGE. Single and double digestions of the chromosome with the enzymes SalI, BssHII, MluI and EagI were performed and used to map 13 restriction sites on the FD-P chromosome. The size of the chromosome was calculated to be 671 kbp. Southern blot analyses using cloned phytoplasma probes were carried out to assist in the arrangement of contiguous restriction fragments and to map eight genetic loci, including the two rRNA operons, the tuf, uvrB-degV and secY-map (FD9) genes, the FD2 marker and two orphan sequences (FDDH29 and FDSH05) isolated through subtractive suppression hybridization.
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- Pathogens And Pathogenicity
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A missense mutation causes aspartase deficiency in Yersinia pestis
More LessIt is established that cells of Yersinia pestis, the causative agent of bubonic plague, excrete l-aspartic acid at the expense of exogenous l-glutamic acid during expression of the low-calcium response. Results of enzymic analysis provided here suggest that a previously defined deficiency of aspartase (AspA) accounts for this phenomenon rather than an elevated oxaloacetate pool. The only known distinction between most sequenced isolates of aspA from Y. pestis and the active gene in Yersinia pseudotuberculosis (the immediate progenitor of Y. pestis) is a single base transversion (G·C→T·A) causing replacement of leucine (encoded by UUG) for valine (encoded by GUG) at amino acid position 363. The gene from Y. pestis KIM possesses a unique second transversion (G·C→T·A) at amino acid 146 causing substitution of aspartic acid (encoded by GAU) with tyrosine (encoded by UAU). We show in this study that Y. pestis expresses aspA as cross-reacting immunological material (CRIM). Functional and inactive aspA of Y. pseudotuberculosis PB1 and Y. pestis KIM, respectively, were then cloned and expressed in AspA-deficient Escherichia coli. After purification to near homogeneity, the products were subjected to biochemical analysis and found to exhibit similar secondary, tertiary and quaternary (tetrameric) structures as well as comparable Michaelis constants for l-aspartic acid. However, the k cat of the Y. pestis CRIM of strain KIM is only about 0.1 % of that determined for the active AspA of Y. pseudotuberculosis. Return of valine for leucine at position 363 of the Y. pestis enzyme restored normal turnover (k cat 86±2 s−1) provided that the amino acid substitution at position 146 was also reversed. These observations have important implications for understanding the nature of the stringent low-calcium response of Y. pestis and its role in promoting acute disease.
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The host adherens junction molecule nectin-1 is downregulated in Chlamydia trachomatis-infected genital epithelial cells
More LessNectin-1, a member of the immunoglobulin superfamily, is a Ca2+-independent cell adhesion protein implicated in the organization of E-cadherin-based adherens junctions (AJs) and claudin-based tight junctions (TJs) in epithelial cells. Nectin-1 also regulates cell–cell adhesion and cell polarization in a Cdc42- and Rac-dependent manner. Western blot analyses demonstrated that accumulation of host nectin-1 is decreased by 85 % at 48 hours post-infection (h.p.i.) in Chlamydia trachomatis serovar E-infected HeLa cells. Time-course experiments demonstrated that this decrease was sustained to 60 h.p.i. Nectin-1 downregulation in C. trachomatis-infected cells was prevented by both chloramphenicol exposure and prior inactivation of the chlamydiae with UV light, demonstrating that active C. trachomatis replication was required. Penicillin G-exposure studies demonstrated that nectin-1 accumulation was also altered during persistent infection. Finally, RT-PCR analyses indicated that chlamydial infection did not alter accumulation of any nectin-1 transcripts, demonstrating that nectin-1 accumulation is reduced at a post-transcriptional level. Intesrestingly, N-cadherin-dependent cell–cell junctions can be disrupted by C. trachomatis infection, as reported by Prozialeck et al. (2002) . Because interaction of nectin molecules on adjacent cells is essential for AJ formation, these data suggest that C. trachomatis may disrupt AJs, at least in part, by diminishing nectin-1 accumulation. Notably, release of chlamydiae-infected epithelial cells has been observed both in vitro from polarized monolayers and in vivo from tissues, suggesting that chlamydia-modulated downregulation of adhesion molecules and the subsequent disruption of host cell adherence may be involved in chlamydial dissemination or pathogenesis.
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Ureaplasma parvum lipoproteins, including MB antigen, activate NF-κB through TLR1, TLR2 and TLR6
More LessUreaplasma species (Ureaplasma parvum and Ureaplasma urealyticum) are commonly isolated pathogens from the female reproductive tract and are associated with perinatal diseases in humans. Inappropriate induction of inflammatory responses may be involved in the occurrence of such diseases; however, pathogenic agents that induce the inflammatory response have not been identified in ureaplasmas. In this study, we examined the involvement of Toll-like receptors (TLRs) in the activation of the immune response by U. parvum lipoproteins, as well as the U. parvum components responsible for nuclear factor κB (NF-κB) activation. The Triton X-114 (TX-114) detergent phase of U. parvum was found to induce NF-κB through TLR2. The active components of the TX-114 detergent phase were lipoproteins, such as multiple banded (MB) antigen, UU012 and UU016 of U. parvum. The activation of NF-κB by these lipoproteins was inhibited by dominant negative (DN) constructs of TLR1 and DN TLR6. Thus, the lipoproteins from U. parvum were found to activate NF-κB through TLR1, TLR2 and TLR6. Furthermore, these lipoproteins possessed an ability to induce tumour necrosis factor-alpha (TNF-α) in mouse peritoneal macrophages.
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Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae
More LessOne of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.
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Copper-dependent transcriptional regulation by Candida albicans Mac1p
More LessWe have previously shown that copper uptake and regulation in the opportunistic pathogen Candida albicans has some similarities to those in Saccharomyces cerevisiae, including the activation of the copper transporter gene CaCTR1 under low-copper conditions by the transcription factor CaMac1p. However, in this study, further analysis has shown that the actual mechanism of regulation by CaMac1p is different from that of its S. cerevisiae homologue. We demonstrate for the first time, to our knowledge, that the CaMAC1 gene is transcriptionally autoregulated in a copper-dependent manner, in contrast to ScMAC1, which is constitutively transcribed. We also demonstrate that the presence of one copper response element in the promoters of CaCTR1, CaMAC1 and the ferric/cupric reductase gene CaFRE7 is sufficient for normal levels of copper-responsive transcription. In contrast, two promoter elements are essential for normal levels of copper-dependent transcriptional activation by ScMac1p. CaMac1p is also involved in the regulation of the iron-responsive transcriptional repressor gene SFU1 and the alternative oxidase gene AOX2. This work describes a key feature of the copper uptake system in C. albicans that distinguishes it from similar processes in the model yeast S. cerevisiae. The importance of copper uptake in the environment of the human host and the implications for the disease process are discussed.
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- Physiology
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Collaboration of FlhF and FlhG to regulate polar-flagella number and localization in Vibrio alginolyticus
Precise regulation of the number and placement of flagella is critical for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. We have shown previously that the number of polar flagella is positively regulated by FlhF and negatively regulated by FlhG. We now show that ΔflhF cells are non-flagellated as are most ΔflhFG cells; however, some of the ΔflhFG cells have several flagella at lateral positions. We found that FlhF–GFP was localized at the flagellated pole, and its polar localization was seen more intensely in ΔflhFG cells. On the other hand, most of the FlhG–GFP was diffused throughout the cytoplasm, although some was localized at the pole. To investigate the FlhF–FlhG interaction, immunoprecipitation was performed by using an anti-FlhF antibody, and FlhG co-precipitated with FlhF. From these results we propose a model in which FlhF localization at the pole determines polar location and production of a flagellum, FlhG interacts with FlhF to prevent FlhF from localizing at the pole, and thus FlhG negatively regulates flagellar number in V. alginolyticus cells.
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Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L108
More LessGrowth of Aquincola tertiaricarbonis L108 on the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), as well as on their main metabolites tert-butyl alcohol (TBA), tert-amyl alcohol (TAA) and 2-hydroxyisobutyrate (2-HIBA) was systematically investigated to characterize the range and rates of oxygenate degradation by this strain. The effective maximum growth rates for MTBE, ETBE and TAME at pH 7 and 30 °C were 0.045 h−1, 0.06 h−1 and 0.055 h−1, respectively, whereas TAA, TBA and 2-HIBA permitted growth at rates up to 0.08 h−1, 0.1 h−1 and 0.17 h−1, respectively. The experimental growth yields with all these substrates were high. Yields of 0.55 g dry mass (dm) (g MTBE)−1, 0.53 g dm (g ETBE)−1, 0.81 g dm (g TAME)−1, 0.48 g dm (g TBA)−1, 0.76 g dm (g TAA)−1 and 0.54 g dm (g 2-HIBA)−1 were obtained. Maximum specific degradation rates were 0.92 mmol MTBE h−1 (g dm)−1, 1.11 mmol ETBE h−1 g−1, 0.66 mmol TAME h−1 g−1, 1.19 mmol TAA h−1 g−1, 2.82 mmol TBA h−1 g−1, and 3.27 mmol 2-HIBA h−1 g−1. The relatively high rates with TBA, TAA and 2-HIBA indicate that the transformations of these metabolites did not limit the metabolism of MTBE and the related ether compounds. Despite the fact that these metabolites still carry a tertiary carbon atom that is commonly suspected to confer recalcitrance to the ether oxygenates, the transformation rates were in the same range as those with succinate and fructose. With MTBE, strain L108 grew at pHs between 5.5 and 8.0 at near-maximal rate, whereas no growth was found below pH 5.0 and above pH 9.0. The optimum growth temperature was 30 °C, but at 5 °C still about 15 % of the maximum rate remained, whereas no growth occurred at 42 °C. This indicates that MTBE metabolites are valuable substrates and that A. tertiaricarbonis L108 is a good candidate for bioremediation purposes. The possible origin of its exceptional metabolic capability is discussed in terms of the evolution of enzymic activities involved in the conversion of compounds carrying tertiary butyl groups.
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- Plant-Microbe Interactions
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Influence of the chloride channel of Fusarium oxysporum on extracellular laccase activity and virulence on tomato plants
More LessCLC-type voltage-gated chloride channels are a family of proteins which mediate chloride transport across the plasma and intracellular membranes. A clc1 gene from the vascular wilt fungus Fusarium oxysporum was characterized and disrupted. The predicted Clc1 protein contained highly conserved transmembrane and CBS domains of this protein family and showed significant identities to the Saccharomyces cerevisiae GEF1 and the Cryptococcus neoformans CLC-A chloride channels. Inactivation of clc1 caused a deficiency in laccase activity which was more severe than that found in any of the structural laccase mutants previously described. The addition of copper sulphate to the growth medium resulted in total recovery of extracellular laccase activity in Δclc1 mutants, although it did not activate transcription of any laccase genes. The pleiotropic phenotype displayed by the Fusarium chloride channel-deficient mutants included a significant delay in the development of disease on tomato plants, with a higher sensitivity to oxidative stress compounds as well as a significant decrease in laccase activity, thus suggesting a possible connection between virulence and the two processes. Nevertheless, we cannot rule out that additional phenotypes present in the Δclc1 mutants could play an essential role in the full virulence of Fusarium.
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Internuclear gene silencing in Phytophthora infestans is established through chromatin remodelling
In the plant pathogen Phytophthora infestans, nuclear integration of inf1 transgenic DNA sequences results in internuclear gene silencing of inf1. Although silencing is regulated at the transcriptional level, it also affects transcription from other nuclei within heterokaryotic cells of the mycelium. Here we report experiments exploring the mechanism of internuclear gene silencing in P. infestans. The DNA methylation inhibitor 5-azacytidine induced reversion of the inf1-silenced state. Also, the histone deacetylase inhibitor trichostatin-A was able to reverse inf1 silencing. inf1-expression levels returned to the silenced state when the inhibitors were removed except in non-transgenic inf1-silenced strains that were generated via internuclear gene silencing, where inf1 expression was restored permanently. Therefore, inf1-transgenic sequences are required to maintain the silenced state. Prolonged culture of non-transgenic inf1-silenced strains resulted in gradual reactivation of inf1 gene expression. Nuclease digestion of inf1-silenced and non-silenced nuclei showed that inf1 sequences in silenced nuclei were less rapidly degraded than non-silenced inf1 sequences. Bisulfite sequencing of the endogenous inf1 locus did not result in detection of any cytosine methylation. Our findings suggest that the inf1-silenced state is based on chromatin remodelling.
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