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Volume 158,
Issue 12,
2012
Volume 158, Issue 12, 2012
- Cell and Molecular Biology of Microbes
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RpoH2 sigma factor controls the photooxidative stress response in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7
Bacteria belonging to the Alphaproteobacteria normally harbour multiple copies of the heat shock sigma factor (known as σ32, σH or RpoH). Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours five copies of rpoH genes, one of which is an rpoH2 homologue. The genes around the rpoH2 locus in A. brasilense show synteny with that found in rhizobia. The rpoH2 of A. brasilense was able to complement the temperature-sensitive phenotype of the Escherichia coli rpoH mutant. Inactivation of rpoH2 in A. brasilense results in increased sensitivity to methylene blue and to triphenyl tetrazolium chloride (TTC). Exposure of A. brasilense to TTC and the singlet oxygen-generating agent methylene blue induced several-fold higher expression of rpoH2. Comparison of the proteome of A. brasilense with its rpoH2 deletion mutant and with an A. brasilense strain overexpressing rpoH2 revealed chaperone GroEL, elongation factors (Ef-Tu and EF-G), peptidyl prolyl isomerase, and peptide methionine sulfoxide reductase as the major proteins whose expression was controlled by RpoH2. Here, we show that the RpoH2 sigma factor-controlled photooxidative stress response in A. brasilense is similar to that in the photosynthetic bacterium Rhodobacter sphaeroides, but that RpoH2 is not involved in the detoxification of methylglyoxal in A. brasilense.
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Functional amyloid formation by Streptococcus mutans
Dental caries is a common infectious disease associated with acidogenic and aciduric bacteria, including Streptococcus mutans. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars and tolerate acid end products. It has recently been recognized that micro-organisms can produce functional amyloids that are integral to biofilm development. We now show that the S. mutans cell-surface-localized adhesin P1 (antigen I/II, PAc) is an amyloid-forming protein. This conclusion is based on the defining properties of amyloids, including binding by the amyloidophilic dyes Congo red (CR) and Thioflavin T (ThT), visualization of amyloid fibres by transmission electron microscopy and the green birefringent properties of CR-stained protein aggregates when viewed under cross-polarized light. We provide evidence that amyloid is present in human dental plaque and is produced by both laboratory strains and clinical isolates of S. mutans. We provide further evidence that amyloid formation is not limited to P1, since bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates the covalent linkage of its substrates to the cell-wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with CR and does not form biofilms. Biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization, including CR, Thioflavin S and epigallocatechin-3-gallate, which also inhibited ThT uptake by S. mutans extracellular proteins. Taken together, these results indicate that S. mutans is an amyloid-forming organism and suggest that amyloidogenesis contributes to biofilm formation by this oral microbe.
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Characterization of the ROK-family transcriptional regulator RokA of Streptococcus pneumoniae D39
More LessThe Gram-positive human pathogen Streptococcus pneumoniae possesses an unusually high number of gene clusters specific for carbohydrate utilization. This provides it with the ability to use a wide array of sugars, which may aid during infection and survival in different environmental conditions present in the host. In this study, the regulatory mechanism of transcription of a gene cluster, SPD0424-8, putatively encoding a cellobiose/lactose-specific phosphotransferase system is investigated. We demonstrate that this gene cluster is transcribed as one transcriptional unit directed by the promoter of the SPD0424 gene. Upstream of SPD0424, a gene was identified encoding a ROK-family transcriptional regulator (RokA: SPD0423). DNA microarray and transcriptional reporter analyses with a rokA mutant revealed that RokA acts as a transcriptional repressor of the SPD0424-8 operon. Furthermore, we identified a 25 bp AT-rich DNA operator site (5′-TATATTTAATTTATAAAAAATAAAA-3′) in the promoter region of SPD0424, which was validated by promoter truncation studies, DNase I footprinting and electrophoretic mobility-shift assays. We tested a large range of different sugars for their effect on the expression of the SPD0424-8 operon, but only moderate variation in expression was observed in the conditions applied. Therefore, a co-factor for RokA-mediated transcriptional control could not be identified.
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Identification of the genes involved in the secretion and self-immunity of lacticin Q, an unmodified leaderless bacteriocin from Lactococcus lactis QU 5
More LessLacticin Q (LnqQ) produced by Lactococcus lactis QU 5 is an unmodified linear bacteriocin, which is synthesized without an N-terminal leader peptide. In vitro synthesis and in vivo expression of LnqQ have revealed the intracellular toxicity of this leaderless peptide, as well as the necessity of a dedicated secretion and self-immunity system of producer cells. Further DNA sequencing and analysis have discovered 11 putative orf genes at the LnqQ locus. None of the orf genes showed similarities to any of the bacteriocin biosynthetic genes characterized to date; however, six orf genes (orf 2q–7q), not including the structural gene (lnqQ), were highly conserved at the lacticin Z locus (orf2z–7z), which is a LnqQ homologue produced by L. lactis QU 14. ORF2q (ORF2z), the gene of which is located upstream of the structural gene, is a putative transcriptional regulator, whereas ORF6q and ORF7q (ORF6z and ORF7z) form a putative ATP-binding cassette transporter. The ORF3q–5q (ORF3z–5z) are all predicted to be membrane proteins with no clear functions. Co-expression of LnqQ and ORF3q–7q in a heterologous host allowed the extracellular production of LnqQ; additionally, the expression of ORF3q–7q rendered the host cells immune to LnqQ. This self-immunity was facilitated possibly by two means; firstly, by secreting the active LnqQ peptides, thus reducing the intracellular toxicity, and secondly, by protecting the host cells from extracellularly released LnqQ. This is the first report, to our knowledge, that describes intracellular toxicity of a leaderless bacteriocin and provides a rare example of biosynthetic genes that are required for bacteriocin secretion and immunity.
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- Environmental and Evolutionary Microbiology
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Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712
More LessThe widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, d-lactate dehydrogenase and α-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus.
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Anaerobic utilization of toluene by marine alpha- and gammaproteobacteria reducing nitrate
More LessAromatic hydrocarbons are among the main constituents of crude oil and represent a major fraction of biogenic hydrocarbons. Anthropogenic influences as well as biological production lead to exposure and accumulation of these toxic chemicals in the water column and sediment of marine environments. The ability to degrade these compounds in situ has been demonstrated for oxygen- and sulphate-respiring marine micro-organisms. However, if and to what extent nitrate-reducing bacteria contribute to the degradation of hydrocarbons in the marine environment and if these organisms are similar to their well-studied freshwater counterparts has not been investigated thoroughly. Here we determine the potential of marine prokaryotes from different sediments of the Atlantic Ocean and Mediterranean Sea to couple nitrate reduction to the oxidation of aromatic hydrocarbons. Nitrate-dependent oxidation of toluene as an electron donor in anoxic enrichment cultures was elucidated by analyses of nitrate, nitrite and dinitrogen gas, accompanied by cell proliferation. The metabolically active members of the enriched communities were identified by RT-PCR of their 16S rRNA genes and subsequently quantified by fluorescence in situ hybridization. In all cases, toluene-grown communities were dominated by members of the Gammaproteobacteria, followed in some enrichments by metabolically active alphaproteobacteria as well as members of the Bacteroidetes. From these enrichments, two novel denitrifying toluene-degrading strains belonging to the Gammaproteobacteria were isolated. Two additional toluene-degrading denitrifying strains were isolated from sediments from the Black Sea and the North Sea. These isolates belonged to the Alphaproteobacteria and Gammaproteobacteria. Serial dilutions series with marine sediments indicated that up to 2.2×104 cells cm–3 were able to degrade hydrocarbons with nitrate as the electron acceptor. These results demonstrated the hitherto unrecognized capacity of alpha- and gammaproteobacteria in marine sediments to oxidize toluene using nitrate.
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Population subdivision and the detection of recombination in non-typable Haemophilus influenzae
More LessThe disparity in diversity between unencapsulated (non-typable; NT) and encapsulated, serotypable Haemophilus influenzae (Hi) has been recognized for some time. It has previously been suggested that the wider diversity evidenced within NTHi compared with typable lineages may be due to different rates of recombination within the encapsulated and NT populations. To examine whether there is evidence for different levels of recombination within typable and NT lineages of Hi, we performed a statistical genetic analysis of 819 distinct genotypes of Hi to explore the congruence of serotype with population genetic clustering, and to identify patterns of recombination within the Hi population. We find that a significantly larger proportion of NT isolates show evidence of recombination, compared with typable isolates, and also that when admixture is present, the total amount of recombination per strain is greater within NT isolates, compared with the typable population. Furthermore, we demonstrate significant heterogeneity in the number of admixed individuals between NT lineages themselves, while such variation was not observed in typable lineages. This variability suggests that factors other than the presence of capsule are important determinants of recombination rate in the Hi population.
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Eukaryotic-type aromatic amino acid decarboxylase from the root colonizer Pseudomonas putida is highly specific for 3,4-dihydroxyphenyl-l-alanine, an allelochemical in the rhizosphere
Aromatic amino acid decarboxylases (AADCs) are found in various organisms and play distinct physiological roles. AADCs from higher eukaryotes have been well studied because they are involved in the synthesis of biologically important molecules such as neurotransmitters and alkaloids. In contrast, bacterial AADCs have received less attention because of their simplicity in physiology and in target substrate (tyrosine). In the present study, we found that Pseudomonas putida KT2440 possesses an AADC homologue (PP_2552) that is more closely related to eukaryotic enzymes than to bacterial enzymes, and determined the genetic and enzymic characteristics of the homologue. The purified enzyme converted 3,4-dihydroxyphenyl-l-alanine (DOPA) to dopamine with K m and k cat values of 0.092 mM and 1.8 s−1, respectively. The enzyme was essentially inactive towards other aromatic amino acids such as 5-hydroxy-l-tryptophan, l-phenylalanine, l-tryptophan and l-tyrosine. The observed strict substrate specificity is distinct from that of any AADC characterized so far. The proposed name of this enzyme is DOPA decarboxylase (DDC). Expression of the gene was induced by DOPA, as revealed by quantitative RT-PCR analysis. DDC is encoded in a cluster together with a LysR-type transcriptional regulator and a major facilitator superfamily transporter. This genetic organization is conserved among all sequenced P. putida strains that inhabit the rhizosphere environment, where DOPA acts as a strong allelochemical. These findings suggest the possible involvement of this enzyme in detoxification of the allelochemical in the rhizosphere, and the potential occurrence of a horizontal gene transfer event between the pseudomonad and its host organism.
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- Microbial Pathogenicity
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Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p
The bacterium Staphylococcus (St.) aureus and the opportunistic fungus Candida albicans are currently among the leading nosocomial pathogens, often co-infecting critically ill patients, with high morbidity and mortality. Previous investigations have demonstrated preferential adherence of St. aureus to C. albicans hyphae during mixed biofilm growth. In this study, we aimed to characterize the mechanism behind this observed interaction. C. albicans adhesin-deficient mutant strains were screened by microscopy to identify the specific receptor on C. albicans hyphae recognized by St. aureus. Furthermore, an immunoassay was developed to validate and quantify staphylococcal binding to fungal biofilms. The findings from these experiments implicated the C. albicans adhesin agglutinin-like sequence 3 (Als3p) in playing a major role in the adherence process. This association was quantitatively established using atomic force microscopy, in which the adhesion force between single cells of the two species was significantly reduced for a C. albicans mutant strain lacking als3. Confocal microscopy further confirmed these observations, as St. aureus overlaid with a purified recombinant Als3 N-terminal domain fragment (rAls3p) exhibited robust binding. Importantly, a strain of Saccharomyces cerevisiae heterologously expressing Als3p was utilized to further confirm this adhesin as a receptor for St. aureus. Although the parental strain does not bind bacteria, expression of Als3p on the cell surface conferred upon the yeast the ability to strongly bind St. aureus. To elucidate the implications of these in vitro findings in a clinically relevant setting, an ex vivo murine model of co-infection was designed using murine tongue explants. Fluorescent microscopic images revealed extensive hyphal penetration of the epithelium typical of C. albicans mucosal infection. Interestingly, St. aureus bacterial cells were only seen within the epithelial tissue when associated with the invasive hyphae. This differed from tongues infected with St. aureus alone or in conjunction with the als3 mutant strain of C. albicans, where bacterial presence was limited to the outer layers of the oral tissue. Collectively, the findings generated from this study identified a key role for C. albicans Als3p in mediating this clinically relevant fungal–bacterial interaction.
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MaHog1, a Hog1-type mitogen-activated protein kinase gene, contributes to stress tolerance and virulence of the entomopathogenic fungus Metarhizium acridum
Kai Jin, Yue Ming and Yu Xian XiaFungal biocontrol agents have great potential in integrated pest management. However, poor efficacy and sensitivity to various adverse factors have hampered their wide application. In eukaryotic cells, Hog1 kinase plays a critical role in stress responses. In this study, MaHog1 (GenBank accession no. EFY85878), encoding a member of the Hog1/Sty1/p38 mitogen-activated protein kinase family in Metarhizium (Me.) acridum, was identified. Targeted gene disruption was used to analyse the role of MaHog1 in virulence and tolerance of adverse factors. Mutants with MaHog1 depletion showed increased sensitivity to high osmotic stress, high temperature and oxidative stress, and exhibited remarkable resistance to cell wall-disturbing agents. These results suggest that Hog1 kinase has a conserved function in regulating multistress responses among fungi, and that MaHog1 might influence cell wall biogenesis in Me. acridum. Bioassays conducted with topical inoculation and intrahaemocoel injection revealed that MaHog1 is required for both penetration and postpenetration development of Me. acridum. MaHog1 disruption resulted in a significant reduction in virulence, likely due to the combination of a decrease in conidial germination, a reduction in appressorium formation and a decline in growth rate in insect haemolymph, which might be caused by impairing fungal tolerance of various stresses during infection.
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Antibiotic resistance plasmids spread among natural isolates of Escherichia coli in spite of CRISPR elements
Clustered, regularly interspaced, short palindromic repeats (CRISPRs) are implicated in defence against foreign DNA in various archaeal and bacterial species. They have also been associated with a slower spread of antibiotic resistance. However, experimental and evolutionary studies raise doubts about the role of CRISPRs as a sort of immune system in Escherichia coli. We studied a collection of 263 natural E. coli isolates from human and animal hosts, representative of the phylogenetic and lifestyle diversity of the species and exhibiting various levels of plasmid-encoded antibiotic resistance. We characterized the strains in terms of CRISPRs, performed replicon typing of the plasmids and tested for class 1 integrons to explore the possible association between CRISPRs and the absence of plasmids and mobile antibiotic resistance determinants. We found no meaningful association between the presence/absence of the cas genes, reflecting the activity of the CRISPRs, and the presence of plasmids, integrons or antibiotic resistance. No CRISPR in the collection contained a spacer that matched an antibiotic resistance gene or element involved in antibiotic resistance gene mobilization, and 79.8 % (210/263) of the strains lacked spacers matching sequences in the 2282 plasmid genomes available. Hence, E. coli CRISPRs do not seem to be efficient barriers to the spread of plasmids and antibiotic resistance, consistent with what has been reported for phages, and contrary to reports concerning other species.
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Manuka honey is bactericidal against Pseudomonas aeruginosa and results in differential expression of oprF and algD
More LessThe presence of Pseudomonas aeruginosa in cutaneous wounds is of clinical significance and can lead to persistent infections. Manuka honey has gained ground in clinical settings due to its effective therapeutic action and broad spectrum of antibacterial activity. In this study, the effect of manuka honey on P. aeruginosa was investigated using MIC, MBC, growth kinetics, confocal microscopy, atomic force microscopy and real-time PCR. A bactericidal mode of action for manuka honey against P. aeruginosa was deduced (12 %, w/v, MIC; 16 %, w/v, MBC) and confirmed by confocal and atomic force microscopy, which showed extensive cell lysis after 60 min exposure to inhibitory concentrations of manuka honey. The inability of honey-treated cells to form microcolonies was demonstrated and investigated using Q-PCR for three key microcolony-forming genes: algD, lasR and oprF. The expression of algD increased 16-fold whereas oprF expression decreased 10-fold following honey treatment; lasR expression remained unaltered. These findings confirm that manuka honey is effective at inducing cell lysis and identify two targets, at the genetic level, that might be involved in this process.
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- Physiology and Biochemistry
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The catabolite repression control protein Crc plays a role in the development of antimicrobial-tolerant subpopulations in Pseudomonas aeruginosa biofilms
Bacteria form complex surface-attached biofilm communities in nature. Biofilm cells differentiate into subpopulations which display tolerance towards antimicrobial agents. However, the signal transduction pathways regulating subpopulation differentiation in biofilms are largely unelucidated. In the present study, we show that the catabolite repression control protein Crc regulates the metabolic state of Pseudomonas aeruginosa cells in biofilms, and plays an important role in the development of antimicrobial-tolerant subpopulations in P. aeruginosa biofilms.
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The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum
More LessWe recently showed that the two-component system (TCS) HrrSA plays a central role in the control of haem homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study, ChrSA was shown to be crucial for haem resistance of C. glutamicum by activation of the putative haem-detoxifying ABC-transporter HrtBA in the presence of haem. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards haem. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA revealed that phosphorylated ChrA acts as an activator of hrtBA in the presence of haem. The haem oxygenase gene, hmuO, showed a decreased mRNA level in a chrSA deletion mutant but no significant binding of ChrA to the hmuO promoter was observed in vitro. In contrast, activation from P hmuO fused to eyfp was almost abolished in an hrrSA mutant, indicating that HrrSA is the dominant system for haem-dependent activation of hmuO in C. glutamicum. Remarkably, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralogous response regulator, whereas chrSA itself seemed to be repressed by HrrA. These data suggest a close interplay of HrrSA and ChrSA at the level of transcription and emphasize ChrSA as a second TCS involved in haem-dependent gene regulation in C. glutamicum, besides HrrSA.
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Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803
More LessThe biosynthesis of glycogen or starch is one of the main strategies developed by living organisms for the intracellular storage of carbon and energy. In phototrophic organisms, such polyglucans accumulate due to carbon fixation during photosynthesis and are used to provide maintenance energy for cell integrity, function and viability in dark periods. Moreover, it is assumed that glycogen enables cyanobacteria to cope with transient starvation conditions, as observed in most micro-organisms. Here, glycogen accumulates when an appropriate carbon source is available in sufficient amounts but growth is inhibited by lack of other nutrients. In this study, the role of glycogen in energy and carbon metabolism of phototrophic cyanobacteria was first analysed via a comparative physiological and metabolic characterization of knockout mutants defective in glycogen synthesis. We first proved the role of glycogen as a respiratory substrate in periods of darkness, the role of glycogen as a reserve to survive starvation periods such as nitrogen depletion and the role of glycogen synthesis as an ameliorator of carbon excess conditions in the model organism Synechocystis sp. PCC 6803. We provide striking new insights into the complex carbon and nitrogen metabolism of non-diazotrophic cyanobacteria: a phenotype of sensitivity to photomixotrophic conditions and of reduced glucose uptake, a non-bleaching phenotype based on an impaired acclimation response to nitrogen depletion and furthermore a phenotype of energy spilling. This study shows that the analysis of deficiencies in glycogen metabolism is a valuable tool for the identification of metabolic regulatory principles and signals.
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Characterization of volatile metabolites taken up by or released from Streptococcus pneumoniae and Haemophilus influenzae by using GC-MS
Volatile organic compounds (VOCs) released from or taken up by Streptococcus pneumoniae and Haemophilus influenzae cultures were analysed by means of GC–MS after adsorption of headspace samples on multi-bed sorption tubes. Sampling was performed at different time points during cultivation of bacteria to follow the dynamics of VOC metabolism. VOCs were identified not only by spectral library match but also based on retention times of native standards. As many as 34 volatile metabolites were released from S. pneumoniae and 28 from H. influenzae, comprising alcohols, aldehydes, esters, hydrocarbons, ketones and sulfur-containing compounds. For both species, acetic acid, acetaldehyde, methyl methacrylate, 2,3-butanedione and methanethiol were found in strongly elevated concentrations and 1-butanol and butanal in moderately elevated concentrations. In addition, characteristic volatile biomarkers were detected for both bacterial species and exclusively for S. pneumoniae, also catabolism of aldehydes (3-methylbutanal and hexanal) was found. The results obtained provide important input into the knowledge about volatile bacterial biomarkers, which may become particularly important for detection of pathogens in upper airways by breath-gas analysis in the future.
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Molecular characterization of ltp3 and ltp4, essential for C24-branched chain sterol-side-chain degradation in Rhodococcus rhodochrous DSM 43269
More LessA previously identified sterol catabolic gene cluster is widely dispersed among actinobacteria, enabling them to degrade and grow on naturally occurring sterols. We investigated the physiological roles of various genes by targeted inactivation in mutant RG32 of Rhodococcus rhodochrous, which selectively degrades sterol side-chains. The ltp3 and ltp4 deletion mutants were each completely blocked in side-chain degradation of β-sitosterol and campesterol, but not of cholesterol. These results indicated a role for ltp3 and ltp4 in the removal of C24 branches specifically. Bioinformatic analysis of the encoded Ltp3 and Ltp4 proteins revealed relatively high similarity to thiolase enzymes, typically involved in β-oxidation, but the catalytic residues characteristic for thiolase enzymes are not conserved in their amino acid sequences. Removal of the C24-branched side-chain carbons of β-sitosterol was previously shown to proceed via aldolytic cleavage rather than by β-oxidation. Our results therefore suggest that ltp3 and ltp4 probably encode aldol-lyases rather than thiolases. This is the first report, to our knowledge, on the molecular characterization of genes with specific and essential roles in carbon–carbon bond cleavage of C24-branched chain sterols in Rhodococcus strains, most likely acting as aldol-lyases. The results are a clear contribution to our understanding of sterol degradation in actinobacteria.
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A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12
We have developed a direct and efficient strategy, based on a three-step method, to select bacterial cell-envelope mutants resistant to bacteriophage infection. Escherichia coli K-12 strain W3110 underwent classical transposon mutagenesis followed by replica plating and selection for mutants resistant to infection by coliphage mEp213. To verify that phage resistance was due to mutations in the cell envelope, we transformed host cells with the viral genome using electroporation and selected those in which virions were subsequently detected in the supernatant. Among the nine mutants resistant to coliphage infection that we selected, six were in the fhuA gene, two were mutated in the waaC gene, and one was mutated in the gmhD gene. The latter two gene products are involved in the synthesis of lipopolysaccharide (LPS). The efficiency of plating and adsorption of phage mEp213 was affected in these mutants. We verified that LPS is required for the efficient infection of phage λ as well. We propose that this mutation-and-selection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.
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Direct surfactin–gramicidin S antagonism supports detoxification in mixed producer cultures of Bacillus subtilis and Aneurinibacillus migulanus
More LessAntibiotic production as a defence mechanism is a characteristic of a wide variety of organisms. In natural evolutionary adaptation, cellular events such as sporulation, biofilm formation and resistance to antibiotics enable some micro-organisms to survive environmental and antibiotic stress conditions. The two antimicrobial cyclic peptides in this study, gramicidin S (GS) from Aneurinibacillus migulanus and the lipopeptide surfactin (Srf) from Bacillus subtilis, have been shown to affect both membrane and intercellular components of target organisms. Many functions, other than that of antimicrobial activity, have been assigned to Srf. We present evidence that an additional function may exist for Srf, namely that of a detoxifying agent that protects its producer from the lytic activity of GS. We observed that Srf producers were more resistant to GS and could be co-cultured with the GS producer. Furthermore, exogenous Srf antagonized the activity of GS against both Srf-producing and non-producing bacterial strains. A molecular interaction between the anionic Srf and the cationic GS was observed with circular dichroism and electrospray MS. Our results indicate that the formation of an inactive complex between GS and Srf supports resistance towards GS, with the anionic Srf forming a chemical barrier to protect its producer. This direct detoxification combined with the induction of protective stress responses in B. subtilis by Srf confers resistance toward GS from A. migulanus and allows survival in mixed cultures.
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)
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