- Volume 156, Issue 3, 2010
Volume 156, Issue 3, 2010
- Mini-Review
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The microbiology of Lascaux Cave
More LessLascaux Cave (Montignac, France) contains paintings from the Upper Paleolithic period. Shortly after its discovery in 1940, the cave was seriously disturbed by major destructive interventions. In 1963, the cave was closed due to algal growth on the walls. In 2001, the ceiling, walls and sediments were colonized by the fungus Fusarium solani. Later, black stains, probably of fungal origin, appeared on the walls. Biocide treatments, including quaternary ammonium derivatives, were extensively applied for a few years, and have been in use again since January 2008. The microbial communities in Lascaux Cave were shown to be composed of human-pathogenic bacteria and entomopathogenic fungi, the former as a result of the biocide selection. The data show that fungi play an important role in the cave, and arthropods contribute to the dispersion of conidia. A careful study on the fungal ecology is needed in order to complete the cave food web and to control the black stains threatening the Paleolithic paintings.
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- Sgm Special Lecture
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Metals, minerals and microbes: geomicrobiology and bioremediation
More LessMicrobes play key geoactive roles in the biosphere, particularly in the areas of element biotransformations and biogeochemical cycling, metal and mineral transformations, decomposition, bioweathering, and soil and sediment formation. All kinds of microbes, including prokaryotes and eukaryotes and their symbiotic associations with each other and ‘higher organisms’, can contribute actively to geological phenomena, and central to many such geomicrobial processes are transformations of metals and minerals. Microbes have a variety of properties that can effect changes in metal speciation, toxicity and mobility, as well as mineral formation or mineral dissolution or deterioration. Such mechanisms are important components of natural biogeochemical cycles for metals as well as associated elements in biomass, soil, rocks and minerals, e.g. sulfur and phosphorus, and metalloids, actinides and metal radionuclides. Apart from being important in natural biosphere processes, metal and mineral transformations can have beneficial or detrimental consequences in a human context. Bioremediation is the application of biological systems to the clean-up of organic and inorganic pollution, with bacteria and fungi being the most important organisms for reclamation, immobilization or detoxification of metallic and radionuclide pollutants. Some biominerals or metallic elements deposited by microbes have catalytic and other properties in nanoparticle, crystalline or colloidal forms, and these are relevant to the development of novel biomaterials for technological and antimicrobial purposes. On the negative side, metal and mineral transformations by microbes may result in spoilage and destruction of natural and synthetic materials, rock and mineral-based building materials (e.g. concrete), acid mine drainage and associated metal pollution, biocorrosion of metals, alloys and related substances, and adverse effects on radionuclide speciation, mobility and containment, all with immense social and economic consequences. The ubiquity and importance of microbes in biosphere processes make geomicrobiology one of the most important concepts within microbiology, and one requiring an interdisciplinary approach to define environmental and applied significance and underpin exploitation in biotechnology.
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- Comment
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Genome update: the 1000th genome – a cautionary tale
More LessThere are now more than 1000 sequenced prokaryotic genomes deposited in public databases and available for analysis. Currently, although the sequence databases GenBank, DNA Database of Japan and EMBL are synchronized continually, there are slight differences in content at the genomes level for a variety of logistical reasons, including differences in format and loading errors, such as those caused by file transfer protocol interruptions. This means that the 1000th genome will be different in the various databases. Some of the data on the highly accessed web pages are inaccurate, leading to false conclusions for example about the largest bacterial genome sequenced. Biological diversity is far greater than many have thought. For example, analysis of multiple Escherichia coli genomes has led to an estimate of around 45 000 gene families — more genes than are recognized in the human genome. Moreover, of the 1000 genomes available, not a single protein is conserved across all genomes. Excluding the members of the Archaea, only a total of four genes are conserved in all bacteria: two protein genes and two RNA genes.
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- Cell And Molecular Biology Of Microbes
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Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp. longum CRC 002
More LessThe effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp. longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080±120 mg EPS l−1 (P<0.01) after 24 h of incubation. For fructose, galactose and glucose, EPS production was similar, at 512±63, 564±165 and 616±93 mg EPS l−1, respectively. The proposed repeating unit composition of the EPS is 2 galactose to 3 glucose. The effect of sugar and fermentation time on expression of genes involved in sugar nucleotide production (galK, galE1, galE2, galT1, galT2, galU, rmlA, rmlB1 and rmlCD) and the priming glycosyltransferase (wblE) was quantified using real-time reverse transcription PCR. A significantly higher transcription level of wblE (9.29-fold) and the genes involved in the Leloir pathway (galK, 4.10-fold; galT1, 2.78-fold; and galE2, 4.95-fold) during exponential growth was associated with enhanced EPS production on lactose compared to glucose. However, galU expression, linking glucose metabolism with the Leloir pathway, was not correlated with EPS production on different sugars. Genes coding for dTDP-rhamnose biosynthesis were also differentially expressed depending on sugar source and growth phase, although rhamnose was not present in the composition of the EPS. This precursor may be used in cell wall polysaccharide biosynthesis. These results contribute to understanding the changes in gene expression when different sugar substrates are catabolized by B. longum subsp. longum CRC 002.
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Autophagy is involved in starvation response and cell death in Blastocystis
More LessPrevious studies have demonstrated that colony forms of Blastocystis undergo cell death with numerous membrane-bound vesicles containing organelles located within the central vacuole, resembling morphological features of autophagy. In this study, we investigated whether Blastocystis underwent autophagy upon amino acid starvation and rapamycin treatment. Concurrently, we provide new insight into a possible function of the central vacuole. The use of the autophagy marker monodansylcadaverine, and the autophagy inhibitors3-methyladenine and wortmannin, showed the existence of autophagy in amino-acid-starved and rapamycin-treated Blastocystis. Confocal microscopy and transmission electron microscopy studies also showed morphological changes that were suggestive of autophagy. The unusually large size of the autophagic compartments within the parasite central vacuole was found to be unique in Blastocystis. In addition, autophagy was found to be triggered when cells were exposed to the cytotoxic antibody mAb 1D5, and autophagy was intensified in the presence of the caspase inhibitor zVAD.fmk. Taken together, our results suggest that the core machinery for autophagy is conserved in Blastocystis, and that it plays an important role in the starvation response and cell death of the parasite.
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The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin
More LessThe LysR-type transcriptional regulator (LTTR) OxyR orchestrates the defence of the opportunistic pathogen Pseudomonas aeruginosa against reactive oxygen species. In previous work we also demonstrated that OxyR is needed for the utilization of the ferrisiderophore pyoverdine, stressing the importance of this regulator. Here, we show that an oxyR mutant is unable to swarm on agar plates, probably as a consequence of absence of production of rhamnolipid surfactant molecules. Another obvious phenotypic change was the increased production of the phenazine redox-active molecule pyocyanin in the oxyR mutant. As already described, the oxyR mutant could not grow in LB medium, unless high numbers of cells (>108 ml−1) were inoculated. However, its growth in Pseudomonas P agar (King's A), a medium inducing pyocyanin production, was like that of the wild-type, suggesting a protective action of this redox-active phenazine compound. This was confirmed by the restoration of the capacity to grow in LB medium upon addition of pure pyocyanin. Although both rhamnolipid and pyocyanin production are controlled by quorum sensing, no obvious changes were observed in the production of N-acylhomoserine lactones or the Pseudomonas quinolone signal (PQS). Complementation of rhamnolipid production and motility, and restoration of normal pyocyanin levels, was only possible when the oxyR gene was in single copy, while pyocyanin levels were increased when oxyR was present in a multicopy vector. Conversely, plating efficiency was increased only when the oxyR gene was present in multicopy, but not when in single copy in the chromosome, due to lower expression of oxyR compared with the wild-type, suggesting that some phenotypes are differently affected in function to the levels of OxyR molecules in the cell. Analysis of transcripts of oxidative stress-response enzymes showed a strong decrease of katB, ahpC and ahpB expression in the oxyR mutant grown in LB, but this was not the case when the mutant was grown on P agar, suggesting that the OxyR dependency for the transcription of these genes is not total.
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Genotyping of Mycobacterium avium complex organisms using multispacer sequence typing
More LessMycobacterium avium complex (MAC) currently comprises eight species of environmental and animal-associated, slowly-growing mycobacteria: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense, Mycobacterium bouchedurhonense, Mycobacterium marseillense and Mycobacterium timonense. In humans, MAC organisms are responsible for opportunistic infections whose unique epidemiology remains poorly understood, in part due to the lack of a genotyping method applicable to all eight MAC species. In this study we developed multispacer sequence typing (MST), a sequencing-based method, for the genotyping of MAC organisms. An alignment of the genome sequence of M. avium subsp. hominissuis strain104 and M. avium subsp. paratuberculosis strain K-10 revealed621 intergenic spacers <1000 bp. From these, 16 spacers were selected that ranged from 300 to 800 bp and contained a number of variable bases, <50 within each of the 16 spacers. Four spacers were successfully PCR-amplified and sequenced in 11 reference strains. Combining the sequence of these four spacers in 106 MAC organisms, including 83 M. avium, 11 M. intracellulare, six M. chimaera, two M. colombiense and one each of M. arosiense, M. bouchedurhonense, M. marseillense and M. timonense, yielded a total of 45 spacer types, with an index of discrimination of 0.94. Each spacer type was specific for a species and certain spacer types were specific for subspecies of M. avium. MST is a new method for genotyping of organisms belonging to any one of the eight MAC species tested in this study.
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σ 54-mediated control of the mannose phosphotransferase sytem in Lactobacillus plantarum impacts on carbohydrate metabolism
More LessSigma factors direct specific binding of the bacterial RNA polymerase to the promoter. Here we present the elucidation of the σ 54 regulon in Lactobacillus plantarum. A sequence-based regulon prediction of σ 54-dependent promoters revealed an operon encoding a mannose phosphotransferase system (PTS) as the best candidate for σ 54-mediated control. A σ 54 (rpoN) mutant derivative did not grow on mannose, confirming this prediction. Additional mutational analyses established the presence of one functional mannose PTS in L. plantarum, the expression of which is controlled by σ 54 in concert with the σ 54-activator ManR. Genome-wide transcription comparison of the wild-type and the rpoN-deletion strain revealed nine upregulated genes in the wild-type, including the genes of the mannose PTS, and 21 upregulated genes in the rpoN mutant. The σ 54-controlled mannose PTS was shown also to transport glucose in L. plantarum wild-type cells, and its presence causes a lag phase when cultures are transferred from glucose- to galactose-containing media. The mannose PTS appeared to drain phosphoenolpyruvate (PEP) pools in resting cells, since no PEP could be detected in resting wild-type cells, while mannose PTS mutant derivatives contained 1–3 μM PEP (mg protein)−1. Our data provide new insight into the role of σ 54 in L. plantarum and possibly other Gram-positive bacteria in the control of expression of an important glucose transporter that contributes to glucose-mediated catabolite control via modulation of the PEP pool.
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Characterization of two different types of UDP-glucose/-galactose4-epimerase involved in galactosylation in fission yeast
More LessSchizosaccharomyces species are currently the only known organisms with two types of genes encoding UDP-glucose/-galactose 4-epimerase, uge1 + and gal10 +. A strain deleted for uge1+ exhibited a severe galactosylation defect and a decrease in activity and in UDP-galactose content when grown in glucose-rich medium (2 % glucose), indicating that Uge1p is a major UDP-glucose/-galactose 4-epimerase under these growth conditions. In contrast, gal10 + was efficiently expressed and involved in galactosylation of cell-surface proteins in low-glucose medium (0.1 % glucose and 2 % glycerol), but not in galactose-containing medium. In a uge1Δgal10Δ strain, the galactosylation defect was suppressed and UDP-galactose content restored to wild-type levels in galactose-containing medium. Disruption of gal7 +, encoding galactose-1-phosphate uridylyltransferase, in the uge1Δgal10Δ strain reversed suppression of the galactosylation defect and reduced levels of UDP-galactose, indicating that galactose is transported from the medium to the cytosol and is converted into UDP-galactose via galactose 1-phosphate by Gal7p in Sch. pombe.
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Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7
More LessAlternative sigma factor 54 (RpoN) is an important regulator of stress resistance and virulence genes in many bacterial species. In this study, we report on the gene expression alterations that follow rpoN inactivation in Escherichia coli O157 : H7 strain Sakai (SakairpoN : : kan), and the influence of RpoN on the acid resistance phenotype. Microarray gene expression profiling revealed the differential expression of 103 genes in SakairpoN : : kan relative to Sakai. This included the growth-phase-dependent upregulation of genes required for glutamate-dependent acid resistance (GDAR) (gadA, gadB, gadC and gadE), and the downregulation of locus of enterocyte effacement (LEE) genes, which encode a type III secretion system. Upregulation of gad genes in SakairpoN : : kan during exponential growth correlated with increased GDAR and survival in a model stomach system. Complementation of SakairpoN : : kan with a cloned version of rpoN restored acid susceptibility. Genes involved in GDAR regulation, including rpoS (sigma factor 38) and gadE (acid-responsive regulator), were shown to be required for the survival of SakairpoN : : kan by the GDAR mechanism. This study describes the contribution of rpoN to acid resistance and GDAR gene regulation, and reveals RpoN to be an important regulator of stress resistance and virulence genes in E. coli O157 : H7.
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FdTonB is involved in the photoregulation of cellular morphology during complementary chromatic adaptation in Fremyella diplosiphon
More LessWe have characterized a Fremyella diplosiphon TonB protein (FdTonB) and investigated its function during complementary chromatic adaptation. Sequence similarity analysis of FdTonB (571 aa) led to identification of several conserved domains characteristic of TonB proteins, including an N-terminal transmembrane domain, a central proline-rich spacer and a C-terminal TonB-related domain (TBRD). We identified a novel glycine-rich domain containing (Gly-X) n repeats. To assess FdTonB function, we constructed a ΔtonB mutant through homologous recombination based upon truncation of the central proline-rich spacer, glycine-rich domain and TBRD. Our ΔtonB mutant exhibited an aberrant cellular morphology under green light, with expanded cell width compared to the parental wild-type (WT) strain. The cellular morphology of the ΔtonB mutant recovered upon WT tonB expression. Interestingly, tonB expression was found to be independent of RcaE. As ΔtonB and WT strains respond in the same way when grown under iron-replete versus iron-limited conditions, our results suggest that FdTonB is not involved in the classic TonB function of mediating cellular adaptation to iron limitation, but exhibits a novel function related to the photoregulation of cellular morphology in F. diplosiphon.
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The BAM complex subunit BamE (SmpA) is required for membrane integrity, stalk growth and normal levels of outer membrane β-barrel proteins in Caulobacter crescentus
More LessThe outer membrane of Gram-negative bacteria is an essential compartment containing a specific complement of lipids and proteins that constitute a protective, selective permeability barrier. Outer membrane β-barrel proteins are assembled into the membrane by the essential hetero-oligomeric BAM complex, which contains the lipoprotein BamE. We have identified a homologue of BamE, encoded by CC1365, which is located in the outer membrane of the stalked alpha-proteobacterium Caulobacter crescentus. BamE associates with proteins whose homologues in other bacteria are known to participate in outer membrane protein assembly: BamA (CC1915), BamB (CC1653) and BamD (CC1984). Caulobacter cells lacking BamE grow slowly in rich medium and are hypersensitive to anionic detergents, some antibiotics and heat exposure, which suggest that the membrane integrity of the mutant is compromised. Membranes of the ΔbamE mutant have normal amounts of the outer membrane protein RsaF, a TolC homologue, but are deficient in CpaC*, an aggregated form of the outer membrane secretin for type IV pili. ΔbamE membranes also contain greatly reduced amounts of three TonB-dependent receptors that are abundant in wild-type cells. Cells lacking BamE have short stalks and are delayed in stalk outgrowth during the cell cycle. Based on these findings, we propose that Caulobacter BamE participates in the assembly of outer membrane β-barrel proteins, including one or more substrates required for the initiation of stalk biogenesis.
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Carotenoids of Gemmatimonas aurantiaca (Gemmatimonadetes): identification of a novel carotenoid, deoxyoscillol 2-rhamnoside, and proposed biosynthetic pathway of oscillol 2,2′-dirhamnoside
More LessGemmatimonas aurantiaca strain T-27T is an orange-coloured, Gram-negative, facultatively aerobic, polyphosphate-accumulating bacterium belonging to a recently proposed phylum, Gemmatimonadetes. We purified its pigments and identified them as carotenoids and their glycoside derivatives using spectral data. The major carotenoid was (2S,2′S)-oscillol 2,2′-di-(α-l-rhamnoside), and the minor carotenoids were (2S)-deoxyoscillol 2-(α-l-rhamnoside) and didemethylspirilloxanthin. Deoxyoscillol2-rhamnoside is a novel carotenoid. Oscillol 2,2′-diglycosides have hitherto only been reported in a limited number of cyanobacteria, and this is believed to be the first finding of such carotenoids in another bacterial phylum. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence, we propose a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. We propose the involvement of geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI) for lycopene synthesis; and of carotenoid1,2-hydratase (CruF) and carotenoid 2-O-rhamnosyltransferase (CruG) for oscillol 2,2′-dirhamnoside synthesis. Further, isopentenyl pyrophosphate could be synthesized by a non-mevalonate pathway (DXP pathway).
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Regulation of dsr genes encoding proteins responsible for the oxidation of stored sulfur in Allochromatium vinosum
More LessSulfur globules are formed as obligatory intermediates during the oxidation of reduced sulfur compounds in many environmentally important photo- and chemolithoautotrophic bacteria. It is well established that the so-called Dsr proteins are essential for the oxidation of zero-valent sulfur accumulated in the globules; however, hardly anything is known about the regulation of dsr gene expression. Here, we present a closer look at the regulation of the dsr genes in the phototrophic sulfur bacterium Allochromatium vinosum. The dsr genes are expressed in a reduced sulfur compound-dependent manner and neither sulfite, the product of the reverse-acting dissimilatory sulfite reductase DsrAB, nor the alternative electron donor malate inhibit the gene expression. Moreover, we show the oxidation of sulfur to sulfite to be the rate-limiting step in the oxidation of sulfur to sulfate as sulfate production starts concomitantly with the upregulation of the expression of the dsr genes. Real-time RT-PCR experiments suggest that the genes dsrC and dsrS are additionally expressed from secondary internal promoters, pointing to a special function of the encoded proteins. Earlier structural analyses indicated the presence of a helix–turn–helix (HTH)-like motif in DsrC. We therefore assessed the DNA-binding capability of the protein and provide evidence for a possible regulatory function of DsrC.
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Treponema denticola biofilm-induced expression of a bacteriophage, toxin–antitoxin systems and transposases
Treponema denticola is an oral spirochaete that has been strongly associated with chronic periodontitis. The bacterium exists as part of a dense biofilm (subgingival dental plaque) accreted to the tooth. To determine T. denticola gene products important for persistence as a biofilm we developed a continuous-culture biofilm model and conducted a genome-wide transcriptomic analysis of biofilm and planktonic cells. A total of 126 genes were differentially expressed with a fold change of 1.5 or greater. This analysis identified the upregulation of putative prophage genes in the T. denticola 35405 genome. Intact bacteriophage particles were isolated from T. denticola and circular phage DNA was detected by PCR analysis. This represents the first, to our knowledge, functional bacteriophage isolated from T. denticola, which we have designated φtd1. In biofilm cells there was also an upregulation of genes encoding several virulence factors, toxin–antitoxin systems and a family of putative transposases. Together, these data indicate that there is a higher potential for genetic mobility in T. denticola when growing as a biofilm and that these systems are important for the biofilm persistence and therefore virulence of this bacterium.
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Gα5 subunit-mediated signalling requires a D-motif and the MAPK ERK1 in Dictyostelium
More LessThe Dictyostelium Gα5 subunit has been shown to reduce cell viability, inhibit folate chemotaxis and accelerate tip morphogenesis and gene expression during multicellular development. Alteration of the D-motif (mitogen-activated protein kinase docking site) at the amino terminus of the Gα5 subunit or the loss of extracellular signal-regulated kinase (ERK)1 diminished the lethality associated with the overexpression or constitutive activation of the Gα5 subunit. The amino-terminal D-motif of the Gα5 subunit was also found to be necessary for the reduced cell size, small aggregate formation and precocious developmental gene expression associated with Gα5 subunit overexpression. This D-motif also contributed to the aggregation delay in cells expressing a constitutively active Gα5 subunit, but the D-motif was not necessary for the inhibition of folate chemotaxis. These results suggest that the amino-terminal D-motif is required for some but not all phenotypes associated with elevated Gα5 subunit functions during growth and development and that ERK1 can function in Gα5 subunit-mediated signal transduction.
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- Environmental And Evolutionary Microbiology
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An observational study of the microbiome of the maternal pouch and saliva of the tammar wallaby, Macropus eugenii, and of the gastrointestinal tract of the pouch young
More LessMarsupial mammals, born in an extremely atricial state with no functional immune system, offer a unique opportunity to investigate both the developing microbiome and its relationship to that of the mother and the potential influence of this microbiome upon the development of the immune system. In this study we used a well-established marsupial model animal, Macropus eugenii, the tammar wallaby, to document the microbiome of three related sites: the maternal pouch and saliva, and the gastrointestinal tract (GIT) of the young animal. We used molecular-based methods, targeting the 16S rDNA gene to determine the bacterial diversity at these study sites. In the maternal pouch, 41 unique phylotypes, predominantly belonging to the phylum Actinobacteria, were detected, while in the saliva, 48 unique phylotypes were found that predominantly belonged to the phylum Proteobacteria. The GIT of the pouch young had a complex microbiome of 53 unique phylotypes, even though the pouch young were still permanently attached to the teat and had only been exposed to the external environment for a few minutes immediately after birth while making their way from the birth canal to the maternal pouch. Of these 53 phylotypes, only nine were detected at maternal sites. Overall, the majority of bacteria isolated were novel species (<97 % identity to known 16S rDNA sequences), and each study site (i.e. maternal pouch and saliva, and the GIT of the pouch young) possessed its own unique microbiome.
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Listeria monocytogenes does not survive ingestion by Acanthamoeba polyphaga
More LessListeria monocytogenes is a ubiquitous bacterium capable of infecting humans, particularly pregnant women and immunocompromised individuals. Although the intracellular invasion and pathogenesis of listeriosis in mammalian tissues has been well studied, little is known about the ecology of L. monocytogenes, and in particular the environmental reservoir for this bacterium has not been identified. This study used short-term co-culture at 15, 22 and 37 °C to examine the interaction of L. monocytogenes strains with Acanthamoeba polyphaga ACO12. Survival of L. monocytogenes cells phagocytosed by monolayers of trophozoites was assessed by culture techniques and microscopy. A. polyphaga trophozoites eliminated bacterial cells within a few hours post-phagocytosis, irrespective of the incubation temperature used. Wild-type L. monocytogenes and a phenotypic listeriolysin O mutant were unable to either multiply or survive within trophozoites. By contrast, Salmonella enterica serovar Typhimurium C5 cells used as controls were able to survive and multiply within A. polyphaga trophozoites. The data presented indicate that A. polyphaga ACO12 is unlikely to harbour L. monocytogenes, or act as an environmental reservoir for this bacterium.
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Aerobic carboxydotrophy under extremely haloalkaline conditions in Alkalispirillum/Alkalilimnicola strains isolated from soda lakes
More LessAerobic enrichments from soda lake sediments with CO as the only substrate resulted in the isolation of five bacterial strains capable of autotrophic growth with CO at extremely high pH and salinity. The strains belonged to the Alkalispirillum/Alkalilimnicola cluster in the Gammaproteobacteria, where the ability to oxidize CO, but not growth with CO, has been demonstrated previously. The growth with CO was possible only at an oxygen concentration below 5 % and CO concentration below 20 % in the gas phase. The isolates were also capable of growth with formate but not with H2. The carboxydotrophic growth occurred within a narrow pH range from 8 to 10.5 (optimum at 9.5) and a broad salt concentration from0.3 to 3.5 M total Na+ (optimum at 1.0 M). Cells grown on CO had high respiration activity with CO and formate, while the cells grown on formate actively oxidized formate alone. In CO-grown cells, CO-dehydrogenase (CODH) activity was detectable both in soluble and membrane fractions, while the NAD-independent formate dehydrogenase (FDH) resided solely in membranes. The results of total protein profiling and the failure to detect CODH with conventional primers for the coxL gene indicated that the CO-oxidizing enzyme in haloalkaliphilic isolates might differ from the classical aerobic CODH complex. A single cbbL gene encoding the RuBisCO large subunit was detected in all strains, suggesting the presence of the Calvin cycle of inorganic carbon fixation. Overall, these results demonstrated the possibility of aerobic carboxydotrophy under extremely haloalkaline conditions.
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- Genes And Genomes
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SanG, a transcriptional activator, controls nikkomycin biosynthesis through binding to the sanN–sanO intergenic region in Streptomyces ansochromogenes
More LessStreptomyces ansochromogenes SanG is a pathway-specific regulator that mainly controls the transcription of two transcriptional units involved in nikkomycin biosynthesis. SanG consists of three major functional domains: an N-terminal Streptomyces antibiotic regulatory protein (SARP) domain, a central ATPase domain, and a C-terminal half homologous to guanylate cyclases belonging to the LuxR family. SanG was expressed in Escherichia coli as a C-terminally His6-tagged protein. The purified SanG-His6 was shown to be a dimer in solution by dynamic light scattering. An electrophoretic mobility-shift assay showed that the purified SanG protein could bind to the DNA fragment containing the bidirectional sanN–sanO promoter region. The SanG-binding sites within the bidirectional sanN–sanO promoter region were determined by footprinting analysis and identified a consensus-directed repeat sequence 5′-CGGCAAG-3′. SanG showed significant ATPase/GTPase activity in vitro, and addition of ATP/GTP enhanced the affinity of SanG for target DNA, but ATP/GTP hydrolysis was not essential for SanG binding to the target DNA. However, real-time reverse transcription PCR showed that mutation of the ATPase/GTPase domain of SanG significantly decreased the transcriptional level of sanN–I and sanO–V. These results indicated that the ATPase/GTPase activity of SanG modulated the transcriptional activation of SanG target genes during nikkomycin biosynthesis.
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Phenotypic and genomic characterization of pneumococcus-like streptococci isolated from HIV-seropositive patients
Accurate differentiation between pneumococci and other viridans streptococci is essential given their differences in clinical significance. However, classical phenotypic tests are often inconclusive, and many examples of atypical reactions have been reported. In this study, we applied various phenotypic and genotypic methods to discriminate between a collection of 12 streptococci isolated from the upper respiratory tract of HIV-seropositive individuals in 1998 and 1999. Conventional phenotypic characterization initially classified these streptococci as Streptococcus pneumoniae, as they were all sensitive to optochin and were all bile soluble. However, they did not agglutinate with anti-pneumococcal capsular antibodies and were also far more resistant to antimicrobial agents than typeable pneumococci isolated in the same period. Genotypic characterization of these isolates and control isolates by both multilocus sequence analysis (MLSA) and comparative genomic hybridization (CGH) showed that only a single isolate was genetically considered to be a true S. pneumoniae isolate, and that the remaining 11 non-typable isolates were indeed distinct from true pneumococci. Of these, 10 most closely resembled a subgroup of Streptococcus mitis isolates genetically, while one strain was identified as a Streptococcus pseudopneumoniae isolate. CGH also showed that a considerable part of the proposed pneumococcal core genome, including many of the known pneumococcal virulence factors, was conserved in the non-typable isolates. Sequencing of part of the 16S rRNA gene and investigation for the presence of ply by PCR corroborated these results. In conclusion, our findings confirm the close relationship between streptococci of the Mitis group, and show that both MLSA and CGH enable pneumococci to be distinguished from other Mitis group streptococci.
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Connecting parts with processes: SubtiWiki and SubtiPathways integrate gene and pathway annotation for Bacillus subtilis
Bacillus subtilis is the model organism for a large group of Gram-positive bacteria, the Firmicutes. Several online databases have been established over time to manage its genetic and metabolic information, but they differ greatly in their rate of update and their focus on B. subtilis. Therefore, a European systems biology consortium called for an integrated solution that empowers its users to enrich online content. To meet this goal we created SubtiWiki and SubtiPathways, two complementary online tools for gene and pathway information on B. subtilis 168. SubtiWiki (http://subtiwiki.uni-goettingen.de/) is a scientific wiki for all genes of B. subtilis and their protein or RNA products. Each gene page contains a summary of the most important information; sections on the gene, its product and expression; sections concerning biological materials and laboratories; and a list of references. SubtiWiki has been seeded with key content and can be extended by any researcher after a simple registration, thus keeping it always up to date. As a complement, SubtiPathways (http://subtipathways.uni-goettingen.de/) is an online tool for navigation of the metabolism of B. subtilis and its regulation. Each SubtiPathways diagram presents a metabolic pathway with its participating enzymes, together with the regulatory mechanisms that act on their expression and activity, in an intuitive interface that is based on Google Maps. Together, SubtiWiki and SubtiPathways provide an integrated view of the processes that make up B. subtilis and its components, making it the most comprehensive web resource for B. subtilis researchers.
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- Microbial Pathogenicity
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Epidemiology, variable genetic organization and regulation of the EDIN-B toxin in Staphylococcus aureus from bacteraemic patients
More LessEDIN-B (epidermal cell differentiation inhibitor-B; also termed C3Stau) is an exotoxin of Staphylococcus aureus which ADP-ribosylates and inactivates Rho GTP binding proteins. The EDIN-B gene (edin-B) and the gene for exfoliative toxin D (etd) make up the central part of a recently described pathogenicity island. Here we evaluated the prevalence and genetic organization of the edin-B/etd pathogenicity island in invasive S. aureus isolates, and characterized edin-B transcription and EDIN-B production using artificial constructs transduced in S. aureus strains RN6390 and Newman. We found that eight out of121 (7 %) S. aureus blood culture isolates harbour edin-B, which is organized in three novel variants of the original edin-B/etd pathogenicity island. In the serum of patients infected with edin-B-positive S. aureus, significant titres of anti-EDIN-B antibodies could be detected. Regulation of edin-B transcription depended on the sarA but not on the agr regulatory system. Furthermore, retrieval of EDIN-B protein secreted by S. aureus RN6390 required the presence of α2-macroglobulin to inhibit the activity of extracellular proteases. These data suggest that the EDIN-B toxin is produced during human infection, is part of a highly variable pathogenicity island and can be controlled by the sarA gene regulon and secreted bacterial proteases.
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Role of an RNA polymerase interacting protein, MsRbpA, from Mycobacterium smegmatis in phenotypic tolerance to rifampicin
More LessRifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the β subunit of RNA polymerase. However, it has been observed that protein–protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the β subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective IC50.
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Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection
Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20–25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro-expressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.
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The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans
More LessThe Burkholderia cepacia complex (Bcc) emerged as problematic opportunistic pathogens to cystic fibrosis (CF) patients. Although several virulence factors have been identified in Bcc, the knowledge of their relative contribution to Bcc pathogenicity remains scarce. In this work, we describe the identification and characterization of a B. cepacia IST408 mutant containing a disruption in the hfq gene. In other bacteria, Hfq is a global regulator of metabolism, acting as an RNA chaperone involved in the riboregulation of target mRNAs by small regulatory non-coding RNAs (sRNAs). The B. cepacia Hfq protein was overproduced as a histidine-tagged derivative, and we show evidence that the protein forms hexamers and binds sRNAs. When provided in trans, the B. cepacia IST408 hfq gene complemented the Escherichia coli hfq mutant strain GS081. Our results also show that the B. cepacia hfq mutant is more susceptible to stress conditions mimicking those faced by Bcc bacteria when infecting the CF host. In addition, the B. cepacia hfq mutant and two hfq mutants derived from B. dolosa and B. ambifaria clinical isolates also exhibited a reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as an infection model. These data, together with the conservation of Hfq orthologues among Bcc, strongly suggest that Hfq plays a major role in the survival of Bcc under stress conditions, contributing to the success of Bcc as CF pathogens.
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Candida albicans biofilm formation in a new in vivo rat model
Device-associated microbial growth, including Candida biofilms, represents more than half of all human microbial infections and, despite a relatively small risk of implant-associated diseases, this type of infection usually leads to high morbidity, increased health-care costs and prolonged antimicrobial therapy. Animal models are needed to elucidate the complex host–pathogen interactions that occur during the development of attached and structured biofilm populations. We describe here a new in vivo model to study Candida biofilm, based on the avascular implantation of small catheters in rats. Polyurethane biomaterials challenged with Candida cells were placed underneath the skin of immunosuppressed animals following only minor surgery. The model allowed the study of up to ten biofilms at once, and the recovery of mature biofilms from 2 days after implantation. The adhering inoculum was adjusted to the standard threshold of positive diagnosis of fungal infection in materials recovered from patients. Wild-type biofilms were mainly formed of hyphal cells, and they were unevenly distributed across the catheter length as observed in infected materials in clinical cases. The hyphal multilayered structure of the biofilms of wild-type strains was observed by confocal microscopy and compared to the monolayer of yeast or hyphal cells of two well-known biofilm-deficient strains, efg1Δ/efg1Δ cph1Δ/cph1Δ and bcr1Δ/bcr1Δ, respectively. The subcutaneous Candida biofilm model relies on the use of implanted catheters with accessible, fast and minor surgery to the animals. This model can be used to characterize the ability of antimicrobial agents to eliminate biofilms, and to evaluate the prophylactic effect of antifungal drugs and biomaterial coatings.
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Direct interaction of iron-regulated surface determinant IsdB of Staphylococcus aureus with the GPIIb/IIIa receptor on platelets
The interaction of bacteria with platelets is implicated in the pathogenesis of endovascular infections, including infective endocarditis, of which Staphylococcus aureus is the leading cause. Several S. aureus surface proteins mediate aggregation of platelets by fibrinogen- or fibronectin-dependent processes, which also requires specific antibodies. In this study S. aureus was grown in iron-limited medium to mimic in vivo conditions in which iron is unavailable to pathogens. Under such conditions, a S. aureus mutant lacking the known platelet-activating surface proteins adhered directly to platelets in the absence of plasma proteins and triggered aggregation. Platelet adhesion and aggregation was prevented by inhibiting expression of iron-regulated surface determinant (Isd) proteins. Mutants defective in IsdB, but not IsdA or IsdH, were unable to adhere to or aggregate platelets. Antibodies to the platelet integrin GPIIb/IIIa inhibited platelet adhesion by IsdB-expressing strains, as did antagonists of GPIIb/IIIa. Surface plasmon resonance demonstrated that recombinant IsdB interacts directly with GPIIb/IIIa.
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Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2Δ) mutants is influenced by the carbon source and rapamycin
More LessThe isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Δ mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Δ mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Δ mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Δ starvation-cidal defects in either species, the cidal phenotype was not due to α-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Δ starvation viability, it increased Ca. albicans ilv1Δ and ilv2Δ viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Δ mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Δ and ilv2Δ viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.
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- Physiology And Biochemistry
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Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis
More LessThe incorporation of dUMP during replication or the deamination of cytosine in DNA results in the occurrence of uracils in genomes. To maintain genomic integrity, uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base-excision repair pathway. Here, we cloned, purified and biochemically characterized a family 5 UDG, UdgB, from Mycobacterium smegmatis to allow us to use it as a model organism to investigate the physiological significance of the novel enzyme. Studies with knockout strains showed that compared with the wild-type parent, the mutation rate of the udgB − strain was approximately twofold higher, whereas the mutation rate of a strain deficient in the family 1 UDG (ung −) was found to be ∼8.4-fold higher. Interestingly, the mutation rate of the double-knockout (ung −/udgB −) strain was remarkably high, at ∼19.6-fold. While CG to TA mutations predominated in the ung − and ung −/udgB − strains, AT to GC mutations were enhanced in the udgB − strain. The ung −/udgB − strain was notably more sensitive to acidified nitrite and hydrogen peroxide stresses compared with the single knockouts (ung − or udgB −). These observations reveal a synergistic effect of UdgB and Ung in DNA repair, and could have implications for the generation of attenuated strains of Mycobacterium tuberculosis.
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Phosphoribosylpyrophosphate synthetase (PrsA) variants alter cellular pools of ribose 5-phosphate and influence thiamine synthesis in Salmonella enterica
More LessPhosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of the purF gene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA independent of PurF are present in Salmonella enterica. Here, we describe mutant alleles of the essential prsA gene, which encodes PRPP synthetase (E.C.2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation and allowed non-enzymic formation of PRA due to a build-up of ribose 5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network.
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Superoxide radical induces sclerotial differentiation in filamentous phytopathogenic fungi: a superoxide dismutase mimetics study
More LessThis study shows that the superoxide radical (O2 •−), a direct indicator of oxidative stress, is involved in the differentiation of the phytopathogenic filamentous fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii and Sclerotinia minor, shown by using superoxide dismutase (SOD) mimetics to decrease their sclerotial differentiation. The production rate of O2 •− and SOD levels in these fungi, as expected, were significantly lowered by the SOD mimetics, with concomitant decrease of the indirect indicator of oxidative stress, lipid peroxidation.
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Racemase activity effected by two dehydrogenases in sulfolactate degradation by Chromohalobacter salexigens: purification of (S)-sulfolactate dehydrogenase
More LessChromohalobacter salexigens DSM 3043, whose genome has been sequenced, is known to degrade (R,S)-sulfolactate as a sole carbon and energy source for growth. Utilization of the compound(s) was shown to be quantitative, and an eight-gene cluster (Csal_1764–Csal_1771) was hypothesized to encode the enzymes in the degradative pathway. It comprised a transcriptional regulator (SuyR), a Tripartite Tricarboxylate Transporter-family uptake system for sulfolactate (SlcHFG), two sulfolactate dehydrogenases of opposite sulfonate stereochemistry, namely novel SlcC and ComC [(R)-sulfolactate dehydrogenase] [EC1.1.1.272] and desulfonative sulfolactate sulfo-lyase (SuyAB) [EC4.4.1.24]. Inducible reduction of 3-sulfopyruvate, inducible SuyAB activity and induction of an unknown protein were detected. Separation of the soluble proteins from induced cells on an anion-exchange column yielded four relevant fractions. Two different fractions reduced sulfopyruvate with NAD(P)H, a third yielded SuyAB activity, and the fourth contained the unknown protein. The latter was identified by peptide-mass fingerprinting as SlcH, the candidate periplasmic binding protein of the transport system. Separated SuyB was also identified by peptide-mass fingerprinting. ComC was partially purified and identified by peptide-mass fingerprinting. The (R)-sulfolactate that ComC produced from sulfopyruvate was a substrate for SuyAB, which showed that SuyAB is (R)-sulfolactate sulfo-lyase. SlcC was purified to homogeneity. This enzyme also formed sulfolactate from sulfopyruvate, but the latter enantiomer was not a substrate for SuyAB. SlcC was obviously (S)-sulfolactate dehydrogenase.
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Volumes and issues
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Volume 170 (2024)
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