Showing papers in "Archives of Microbiology in 2012"
TL;DR: The results of this study show that the hybrid offspring Yuyu 23, Zhengdan958, Jingdan 28 and Jingyu 11 had 3, 33, 38 and 2 OTUs of bacteria, respectively, while the parents Ye 478, Chang 7-2, Zheng 58, Jing 24 and Jing 89 had 12, 36, 6, 12 and 1 OTUs, respectively.
Abstract: The seeds of plants are carriers of a variety of beneficial bacteria and pathogens. Using the non-culture methods of building 16S rDNA libraries, we investigated the endophytic bacterial communities of seeds of four hybrid maize offspring and their respective parents. The results of this study show that the hybrid offspring Yuyu 23, Zhengdan958, Jingdan 28 and Jingyu 11 had 3, 33, 38 and 2 OTUs of bacteria, respectively. The parents Ye 478, Chang 7-2, Zheng 58, Jing 24 and Jing 89 had 12, 36, 6, 12 and 2 OTUs, respectively. In the hybrid Yuyu 23, the dominant bacterium Pantoea (73.38 %) was detected in its female parent Ye 478, and the second dominant bacterium of Sphingomonas (26.62 %) was detected in both its female (Ye 478) and male (Chang 7-2) parent. In the hybrid Zhengdan 958, the first dominant bacterium Stenotrophomonas (41.67 %) was detected in both the female (Zheng 58) and male (Chang 7-2) parent. The second dominant bacterium Acinetobacter (9.26 %) was also the second dominant bacterium of its male parent. In the hybrid Jingdan 28, the second dominant bacterium Pseudomonas (12.78 %) was also the second dominant bacterium of its female parent, and its third dominant bacterium Sphingomonas (9.90 %) was the second dominant bacterium of its male parent and detected in its female parent. In the hybrid Jingyu 11, the first dominant bacterium Leclercia (73.85 %) was the third dominant bacterium of its male parent, and the second dominant bacterium Enterobacter (26.15 %) was detected in its male parent. As far as we know, this was the first research reported in China on the diversity of the endophytic bacterial communities of the seeds of various maize hybrids with different genotypes.
129 citations
TL;DR: It is postulate that the putative vpT6SS systems contribute to adhesion of V. parahaemolyticus to host cells.
Abstract: Analysis of the genome sequence of Vibrio parahaemolyticus reveals two IcmF family genes in putative type VI secretion system (vpT6SS) clusters in chromosomes 1 (icmF1) and 2 (icmF2). The icmF1 gene is present in majority of clinical isolates (87.5 %), but has a low fraction (25.0 %) in environmental isolates. However, icmF2 is contained in all strains of both clinical and environmental sources. Deletion of either icmF1 or hcp1 significantly reduced bacterial adhesion to Caco-2 cells or HeLa monolayers. However, the ΔicmF2 and Δhcp2 mutants showed decreased adhesion only to HeLa monolayers. Western blot analysis showed that Hcp2 was present both in the supernatant and pellet samples in the wild-type strain, but only in the pellet of the ΔicmF2 mutant, indicating that Hcp2 is a translocon of T6SS2. Although vpT6SS1 might be functional in cellular adhesion, the putative translocon Hcp1 was not detectable. Quantitative PCR revealed 10-fold and 17-fold less transcripts of hcp1 and icmF1 mRNA than those of hcp2 and icmF2 accordingly. Thus, we postulate that the putative vpT6SS systems contribute to adhesion of V. parahaemolyticus to host cells.
107 citations
TL;DR: A phylogenetic comparison of nitrogen fixation gene (nifH) is presented with the aim of elucidating the processes underlying the evolutionary history of this catalytic ability among actinobacteria.
Abstract: It was assumed for a long time that the ability to catalyze atmospheric nitrogen (diazotrophy) has a narrow distribution among actinobacteria being limited to the genus Frankia. Recently, the number of nitrogen fixation (nifH) genes identified in other non-Frankia actinobacteria has dramatically increased and has opened investigation on the origin and emergence of diazotrophy among actinobacteria. During the last decade, Mycobacterium flavum, Corynebacterium autotrophicum and a fluorescent Arthrobacter sp. have been reported to have nitrogenase activity, but these studies have not been further verified. Additional reports of nitrogen fixation by Agromyces, Microbacterium, Corynebacterium and Micromonospora isolated from root nodules of leguminous and actinorhizal plants have increased. For several actinobacteria, nitrogen fixation was demonstrated by the ability to grow on nitrogen-free medium, acetylene reduction activity, 15N isotope dilution analysis and identification of a nifH gene via PCR amplification. Moreover, the analyses of draft genome sequences of actinobacteria including Slackia exigua, Rothia mucilaginosa and Gordonibacter pamelaeae have also revealed the presence of nifH-like sequences. Whether these nifH sequences are associated with effective nitrogen fixation in these actinobacteria taxa has not yet been demonstrated. These genes may be vertically or horizontally transferred and be silent sequences. These ideas merit further investigation. This minireview presents a phylogenetic comparison of nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of this catalytic ability among actinobacteria.
90 citations
TL;DR: Evaluation of the antifungal activity of the isolated lipopeptides showed that fengycins are the most active ones, the first report of an endophytic Bacillus subtilis producing all three major families oflipopeptide antibiotics containing a very heterogeneous mixture of homologues.
Abstract: In a previous study (Malfanova et al. in Microbial Biotech 4:523–532, 2011), we described the isolation and partial characterization of the biocontrol endophytic bacterium B. subtilis HC8. Using thin-layer chromatography, we have detected several bioactive antifungal compounds in the methanolic extract from the acid-precipitated supernatant of HC8. In the present study, we have further analyzed this methanolic extract using liquid chromatography-mass spectrometry. Based on the comparison of retention times and molecular masses with those of known antifungal compounds, we identified three families of lipopeptide antibiotics. These include four iturins A having fatty acyl chain lengths of C14 to C17, eight fengycins A (from C14 to C18 and from C15 to C17 containing a double bond in the acyl chain), four fengycins B (C15 to C18), and five surfactins (C12 to C16). Evaluation of the antifungal activity of the isolated lipopeptides showed that fengycins are the most active ones. To our knowledge, this is the first report of an endophytic Bacillus subtilis producing all three major families of lipopeptide antibiotics containing a very heterogeneous mixture of homologues. The questions remain open which of these lipopeptides (1) are being produced during interaction with the plant and (2) are contributing to the biocontrol activity of HC8.
85 citations
TL;DR: It is demonstrated that R. tropici EPS chemical composition and yield differ when grown with different substrates in a defined minimal medium in batch culture, and it is found that optimizing the C/N ratio has a greater impact upon R. Tropici EPS production than upon other rhizobia strains.
Abstract: Rhizobium tropici, a legume-symbiont soil bacterium, is known for its copious production of exopolysaccharide (EPS) Many aspects of this organism’s growth and EPS production, however, remain uncharacterized, including the influence of environment and culturing conditions upon EPS Here, we demonstrate that R tropici EPS chemical composition and yield differ when grown with different substrates in a defined minimal medium in batch culture Exopolysaccharide was quantified from R tropici grown using arabinose, glucose, sucrose, mannitol, fructose, or glutamate as a sole carbon source All tested substrates produced plenteous amounts of exopolysaccharide material Variations in pH and carbon-to-nitrogen (C/N) ratio also resulted in assorted cell growth and exopolysaccharide production differences We found that optimizing the C/N ratio has a greater impact upon R tropici EPS production than upon R tropici growth A maximum EPS yield of 408 g/L was realized under optimized conditions, which is large even in comparison with other known rhizobia We provide evidence that the chemical composition of R tropici EPS can vary with changes to the growth environment The composition of glucose-grown EPS contained rhamnose-linked residues that were not present in arabinose-grown EPS
79 citations
TL;DR: Increased understanding of the molecular and functional diversity of P450s in this fungus will facilitate comprehension of metabolic diversity in basidiomycetes and has future biotechnology applications.
Abstract: We explored the molecular diversity and functional capabilities of cytochrome P450 monooxygenases (P450s) from the brown-rot basidiomycete Postia placenta. Using bioinformatic and experimental data, we found 250 genes of P450s in the whole genome, including 60 putative allelic variants. Phylogenetic analysis revealed the presence of 42 families, including 18 novel families. Comparative phylogenetic analysis of P450s from P. placenta and the white-rot basidiomycete Phanerochaete chrysosporium suggested that vigorous gene duplication and molecular evolution occurred after speciation of basidiomycetes. Among the 250 gene models, 184 were isolated as full-length cDNA and transformed into Saccharomyces cerevisiae to construct a functional library in which recombinant P450s were co-expressed with yeast NADPH-P450 oxidoreductase. Using this library, the catalytic potentials of P450s against a wide variety of compounds were investigated. A functionomic survey allowed the discovery of novel catalytic properties of P. placenta P450s. The phylogenetic diversity of the CYP53 family in P. placenta was clear, and CYP53D2 is capable of converting stilbene derivatives. This is the first report of this peculiar function of the CYP53 family. Our increased understanding of the molecular and functional diversity of P450s in this fungus will facilitate comprehension of metabolic diversity in basidiomycetes and has future biotechnology applications.
75 citations
TL;DR: The results indicate that the ability to oxidize arsenite is widespread in various unusual taxa, and molecular methods for their detection require further improvement.
Abstract: In the present study cultivation-dependent and molecular methods were applied in combination to investigate the arsenite-oxidizing communities in enrichment cultures from arsenic and lead smelter-impacted soils with respect to both 16S rRNA and arsenite oxidase gene diversity. Enrichments with arsenite as the only electron donor resulted in completely different communities than enrichments with yeast extract and the simultaneous presence of arsenite. The lithoautotrophic community appeared to be dominated by Ferrimicrobium-related Actinobacteria, unusual Acidobacteria, Myxobacteria, and α-Proteobacteria but the heterotrophic community comprised many Dokdonella-related γ-Proteobacteria. Gene sequences of clones encoding arsenite oxidase from the enrichment for lithoautotrophs belonged to three major clusters with sequences from non-cultivated microorganisms. So, primers used to detect arsenite oxidase genes could amplify the genes from many α-, β- and γ-Proteobacteria, but not from various strains of the other phyla present in the enrichment for lithotrophs. This was also observed for the isolates where arsenite oxidase genes from new proteobacterial isolates of the genera Burkholderia, Bosea, Alcaligenes, Bradyrhizobium and Methylobacterium could be amplified but the genes of the new Rhodococcus isolate S43 could not. The results indicate that the ability to oxidize arsenite is widespread in various unusual taxa, and molecular methods for their detection require further improvement.
65 citations
TL;DR: Based on phylogenetic and phenotypic studies, the isolate merits recognition as a member of a novel genus and species, for which the name Acetatifactor muris is proposed.
Abstract: We used selective agar media for culturing bacteria from the caecum of mice fed a high calorie diet. In addition to the isolation of Enterobacteriaceae growing on a medium containing cholesterol and bile salts, we focused on the characterization of strain CT-m2T, which, based on 16S rDNA analysis, did not appear to correspond to any currently described organisms. The isolate belongs to the Clostridium cluster XIV and is most closely related to members of the Lachnospiraceae, including the genera Anaerostipes, Blautia, Butyrivibrio, Clostridium, Coprococcus, Eubacterium, Robinsoniella, Roseburia, Ruminococcus and Syntrophococcus (≤90 % similarity). Strain CT-m2T is a non-motile Gram-positive rod that does not form spores and has a G + C content of DNA of 48.5 %. Cells grow under strictly anoxic conditions (100 % N2) and produce acetate and butyrate after growth in reduced WCA broth. In contrast to related species, the new bacterium does not metabolize glucose and is positive for phenylalanine arylamidase, and its major cellular fatty acid is C14:0. Based on phylogenetic and phenotypic studies, the isolate merits recognition as a member of a novel genus and species, for which the name Acetatifactor muris is proposed. The type strain is CT-m2T (= DSM 23669T = ATCC BAA-2170T).
64 citations
TL;DR: Microbial diversity within formation water and oil from two compartments in Bokor oil reservoir from a Malaysian petroleum oil field was examined, suggesting that primers may play an important role in determining which taxa would be detected.
Abstract: Microbial diversity within formation water and oil from two compartments in Bokor oil reservoir from a Malaysian petroleum oil field was examined. A total of 1,056 16S rRNA gene clones were screened from each location by amplified ribosomal DNA restriction analysis. All samples were dominated by clones affiliated with Marinobacter, some novel Deferribacteraceae genera and various clones allied to the Methanococci. In addition, either Marinobacterium- or Pseudomonas-like operational taxonomic units were detected from either compartment. A systematic comparison with the existing pertinent studies was undertaken by analysing the microbial amplicons detected and the PCR primers used. The analyses demonstrated that bacterial communities were site specific, while Archaea co-occurred more frequently. Amplicons related to Marinobacter, Marinobacterium and Pseudomonas were detected in a number of the studies examined, suggesting they may be ubiquitous members in oil reservoirs. Further analysis of primers used in those studies suggested that most primer pairs had fairly broad but low matches across the bacterial and archaeal domains, while a minority had selective matches to certain taxa or low matches to all the microbial taxa tested. Thus, it indicated that primers may play an important role in determining which taxa would be detected.
54 citations
TL;DR: The characterization of Est6 revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5.
Abstract: A deep-sea sediment metagenomic library was constructed and screened for lipolytic enzymes by activity-based approach. Nine novel lipolytic enzymes were identified, and the amino acid sequences shared 56% to 84% identity to other lipolytic enzymes in the database. Phylogenetic analysis showed that these enzymes belonged to family IV lipolytic enzymes. One of the lipolytic enzymes, Est6, was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form. The recombinant protein was purified by Ni-nitrilotriacetic affinity chromatography column and characterized using p-nitrophenyl esters with various chain lengths. The est6 gene consisted of 909 bp that encoded 302 amino acid residues. Est6 was most similar to a lipolytic enzyme from uncultured bacterium (ACL67845, 61% identity) isolated from the South China Sea marine sediment metagenome. The characterization of Est6 revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5.
54 citations
TL;DR: Kuwaiti habitats with two-decade history of oil pollution were surveyed for their inhabitant oil-utilizing bacterioflora and it was concluded that the indigenous microflora could be involved in bioremediation programs without bioaugmentation or nitrogen fertilization.
Abstract: Kuwaiti habitats with two-decade history of oil pollution were surveyed for their inhabitant oil-utilizing bacterioflora. Seawater samples from six sites along the Kuwaiti coasts of the Arabian Gulf and desert soil samples collected from seven sites all over the country harbored oil-utilizing bacteria whose numbers made up 0.0001–0.01% of the total, direct, microscopic counts. The indigenous bacterioflora in various sites were affiliated to many species. This was true when counting was made on nitrogen-containing and nitrogen-free media. Seawater samples harbored species belonging predominantly to the Gammaproteobacteria and desert soil samples contained predominantly Actinobacteria. Bacterial species that grew on the nitrogen-free medium and that represented a considerable proportion of the total in all individual bacterial consortia were diazotrophic. They gave positive acetylene-reduction test and possessed the nifH genes in their genomes. Individual representative species could utilize a wide range of aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy. Quantitative determination showed that the individual species consumed crude oil, n-octadecane and phenanthrene, in batch cultures. It was concluded that the indigenous microflora could be involved in bioremediation programs without bioaugmentation or nitrogen fertilization. Irrigation would be the most important practice in bioremediation of the polluted soil desert areas.
TL;DR: Increased salinities caused decreased nitrogenase activity and were accompanied by a lower proportion of cyanobacterial nifH transcripts, which was strongest in mats higher up in the littoral zone.
Abstract: Microbial mats are often found in intertidal areas experiencing a large range of salinities. This study investigated the effect of changing salinities on nitrogenase activity and on the composition of the active diazotrophic community (nifH transcript libraries) of three types of microbial mats situated along a littoral gradient. All three mat types exhibited highest nitrogenase activity at salinities close to ambient seawater or lower. The response to lower or higher salinity was strongest in mats higher up in the littoral zone. Changes in nitrogenase activity as the result of exposure to different salinities were accompanied by changes in the active diazotrophic community. The two stations higher up in the littoral zone showed nifH expression by Cyanobacteria (Oscillatoriales and Chroococcales) and Proteobacteria (Gammaproteobacteria and Deltaproteobacteria). At these stations, a decrease in the relative contribution of Cyanobacteria to the nifH transcript libraries was observed at increasing salinity coinciding with a decrease in nitrogenase activity. The station at the low water mark showed low cyanobacterial contribution to nifH transcript libraries at all salinities but an increase in deltaproteobacterial nifH transcripts under hypersaline conditions. In conclusion, increased salinities caused decreased nitrogenase activity and were accompanied by a lower proportion of cyanobacterial nifH transcripts.
TL;DR: This is the first study that the Aquimarina species possesses both direct and indirect algicidal activities, and should be classified as representing a novel species, for which the name A. salinaria sp.
Abstract: A bacterial strain designated antisso-27T, previously isolated from saltpan in Taiwan while screening for bacteria for algicidal activity, was characterized using the polyphasic taxonomic approach. Strain antisso-27T was Gram-negative, aerobic, brownish yellow colored, rod-shaped, non-flagellated and non-gliding. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain antisso-27T belonged to the genus Aquimarina within the family Flavobacteriaceae with relatively low sequence similarities of 94.0–96.6% to other valid Aquimarina spp. It contained iso-C17:0 3-OH, iso-C15:0, iso-C16:0, iso-C15:1 and iso-C15:0 3-OH as the main fatty acids and contained a menaquinone with six isoprene units (MK-6) as the major isoprenoid quinone. Major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, an uncharacterized aminolipid and five uncharacterized phospholipids. Strain antisso-27T employed direct mode of algicidal lysis to Chlorella vulgaris strain 211-31; nevertheless, it released an algicidal substance against M. aeruginosa strain MTY01. This is the first study that the Aquimarina species possesses both direct and indirect algicidal activities. On the basis of the phylogenetic and phenotypic data, strain antisso-27T should be classified as representing a novel species, for which the name A. salinaria sp. nov. is proposed. The type strain is A. salinaria antisso-27T (= BCRC 80080T = LMG 25375T).
TL;DR: The HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalliporphyrin toxicity by binding an excess of these compounds.
Abstract: Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P. gingivalis during planktonic growth, biofilm formation, epithelial cell adhesion and invasion, and employed hmuY, hmuR and hmuY-hmuR mutants to assess the involvement of HmuY and HmuR proteins in the acquisition of metalloporphyrins. Iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) supported planktonic growth of P. gingivalis cells, biofilm accumulation, as well as survival, adhesion and invasion of HeLa cells in a way analogous to protoheme. In contrast, cobalt(III), gallium(III) and copper(II) protoporphyrin IX exhibited antimicrobial activity against P. gingivalis, and thus represent potentially useful antibacterial compounds with which to target P. gingivalis. P. gingivalishmuY, hmuR and hmuY-hmuR mutants showed decreased growth and infection of epithelial cells in the presence of all metalloporphyrins examined. In conclusion, the HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalloporphyrin toxicity by binding an excess of these compounds.
TL;DR: It is suggested that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.
Abstract: Multi- and pan-antibiotic-resistant bacteria area major health challenge in hospital settings. Furthermore,when susceptible bacteria establish surface-attached biofilm populations, they become recalcitrant to antimicrobial therapy. Therefore, there is a need for novel antimicrobials that are effective against multi-drug-resistant and surface-attached bacteria. A screen to identify prokaryote-derived antimicrobials from a panel of over 100 bacterial strains was performed. One compound isolated from Citrobacter freundii exhibited antimicrobial activity against a wide range of Gram-negative bacteria and was effective against biofilms. Random transposon mutagenesis was performed to find mutants unable to produce the antimicrobial compound.Transposons mapped to a bacteriocin gene located on a small plasmid capable of replication in Escherichia coli. The plasmid was sequenced and found to be highly similar to a previously described colicinogenic plasmid.Expression of the predicted bacteriocin immunity gene conferred bacteriocin immunity to E. coli. The predicted bacteriocin gene, colA-43864, expressed in E. coli was sufficient to generate anti-microbial activity, and purified recombinant ColA-43864 was highly effective in killing E. coli, Citrobacter species, and Klebsiella pneumoniae cells in a planktonic and biofilm state. This study suggests that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.
TL;DR: It is proposed that the nif gene clusters remained stable despite genetic variations occurring in the Frankia genomes.
Abstract: The nucleotide sequence of a 20.5-kb genomic region harboring nif genes was determined and analyzed. The fragment was obtained from Frankia sp. EuIK1 strain, an indigenous symbiont of Elaeagnus umbellata. A total of 20 ORFs including 12 nif genes were identified and subjected to comparative analysis with the genome sequences of 3 Frankia strains representing diverse host plant specificities. The nucleotide and deduced amino acid sequences showed highest levels of identity with orthologous genes from an Elaeagnus-infecting strain. The gene organization patterns around the nif gene clusters were well conserved among all 4 Frankia strains. However, characteristic features appeared in the location of the nifV gene for each Frankia strain, depending on the type of host plant. Sequence analysis was performed to determine the transcription units and suggested that there could be an independent operon starting from the nifW gene in the EuIK strain. Considering the organization patterns and their total extensions on the genome, we propose that the nif gene clusters remained stable despite genetic variations occurring in the Frankia genomes.
TL;DR: The main sporulation factors responsible for heat resistance in Geobacillus stearothermophilus, a useful biological indicator for steam sterilization, were evaluated and the model obtained accurately describes positive pH and negative manganese sulfate concentration influence on spore heat resistance.
Abstract: Biological indicators are important tools in infection control via sterilization process monitoring. The use of a standardized spore crop with a well-defined heat resistance will guarantee the quality of a biological indicator. Ambient factors during sporulation can affect spore characteristics and properties, including heat resistance. The aim of this study is to evaluate the main sporulation factors responsible for heat resistance in Geobacillus stearothermophilus, a useful biological indicator for steam sterilization. A sequence of a three-step optimization of variables (initial pH, nutrient concentration, tryptone, peptone, beef extract, yeast extract, manganese sulfate, magnesium sulfate, calcium chloride and potassium phosphate) was carried out to screen those that have a significant influence on heat resistance of produced spores. The variable exerting greatest influence on G. stearothermophilus heat resistance during sporulation was found to be the initial pH. Lower nutrient concentration and alkaline pH around 8.5 tended to enhance decimal reduction time at 121 °C (D121°C). A central composite design enabled a fourfold enhancement in heat resistance, and the model obtained accurately describes positive pH and negative manganese sulfate concentration influence on spore heat resistance.
TL;DR: In this paper, the expression of a hemolysin coregulated protein (Hcp1) was found to be strictly regulated in Vibrio alginolyticus.
Abstract: Type VI secretion system (T6SS) is a highly conserved bacterial protein secretion system and is precisely regulated in Gram-negative pathogens. In Vibrio alginolyticus, an important fish pathogen, two complete T6SS gene clusters (T6SSVA1 and T6SSVA2) were identified. In this study, expression of a hemolysin coregulated protein (Hcp1), which is one of the hallmarks of T6SS, was found to be strictly regulated in this bacterium. We showed that the expression of Hcp1 was growth phase-dependent and the production of Hcp1 reached a maximum in the exponential phase. The expression of Hcp1 was positively and negatively regulated by quorum sensing regulators LuxO and LuxR, respectively. In addition, we observed that Hcp1 expression required the alternative sigma factor RpoN and the enhancer-binding protein VasH, which is encoded in T6SSVA1 gene cluster. Moreover, LuxR, RpoN, and VasH could positively regulate the expression of other T6SS genes. Taken together, we demonstrated that the expression of T6SS in V. alginolyticus was under the regulation of quorum sensing and alternative sigma factor.
TL;DR: It is concluded that the electron transport chain in Z. mobilis contains at least two electron pathways to oxygen and that one of its functions might be to prevent endogenous oxidative stress.
Abstract: The ethanol-producing bacterium Zymomonas mobilis is of great interest from a bioenergetic perspective because, although it has a very high respiratory capacity, the respiratory system does not appear to be primarily required for energy conservation. To investigate the regulation of respiratory genes and function of electron transport branches in Z. mobilis, several mutants of the common wild-type strain Zm6 (ATCC 29191) were constructed and analyzed. Mutant strains with a chloramphenicol-resistance determinant inserted in the genes encoding the cytochrome b subunit of the bc (1) complex (Zm6-cytB), subunit II of the cytochrome bd terminal oxidase (Zm6-cydB), and in the catalase gene (Zm6-kat) were constructed. The cytB and cydB mutants had low respiration capacity when cultivated anaerobically. Zm6-cydB lacked the cytochrome d absorbance at 630 nm, while Zm6-cytB had very low spectral signals of all cytochromes and low catalase activity. However, under aerobic growth conditions, the respiration capacity of the mutant cells was comparable to that of the parent strain. The catalase mutation did not affect aerobic growth, but rendered cells sensitive to hydrogen peroxide. Cytochrome c peroxidase activity could not be detected. An upregulation of several thiol-dependent oxidative stress-protective systems was observed in an aerobically growing ndh mutant deficient in type II NADH dehydrogenase (Zm6-ndh). It is concluded that the electron transport chain in Z. mobilis contains at least two electron pathways to oxygen and that one of its functions might be to prevent endogenous oxidative stress.
TL;DR: The unusual case that two S-layer glycoproteins are co-assembled into a single S- layer is demonstrated, which might impact the pathogenicity of this bacterium.
Abstract: The Gram-negative oral pathogen Tannerella forsythia is decorated with a 2D crystalline surface (S-) layer, with two different S-layer glycoprotein species being present. Prompted by the predicted virulence potential of the S-layer, this study focused on the analysis of the arrangement of the individual S-layer glycoproteins by a combination of microscopic, genetic, and biochemical analyses. The two S-layer genes are transcribed into mRNA and expressed into protein in equal amounts. The S-layer was investigated on intact bacterial cells by transmission electron microscopy, by immune fluorescence microscopy, and by atomic force microscopy. The analyses of wild-type cells revealed a distinct square S-layer lattice with an overall lattice constant of 10.1 ± 0.7 nm. In contrast, a blurred lattice with a lattice constant of 9.0 nm was found on S-layer single-mutant cells. This together with in vitro self-assembly studies using purified (glyco)protein species indicated their increased structural flexibility after self-assembly and/or impaired self-assembly capability. In conjunction with TEM analyses of thin-sectioned cells, this study demonstrates the unusual case that two S-layer glycoproteins are co-assembled into a single S-layer. Additionally, flagella and pilus-like structures were observed on T. forsythia cells, which might impact the pathogenicity of this bacterium.
TL;DR: Proteins that are secreted specifically by hypervirulent strains were identified, mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins.
Abstract: Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.
TL;DR: This work corroborates the association of Rhodopirellula spp.
Abstract: The increasing ecological significance of Planctomycetes and the still limited knowledge of this group prompted us to obtain cultured isolates from the sediment of a treatment water recycling tank of a marine fish farm. Presence of strains from this group was assessed in the sediments and water column of the tank. Eleven isolates were obtained from the sediment sample by exploiting Planctomycetes natural resistance to several antibiotics and their capacity to degrade organic matter. Based on morphological characteristics and resistance to antibiotics, Planctomycetes were identified. Their phylogenetic affiliation was confirmed by the sequence analysis of the 16S rRNA gene that revealed the presence of a group of 6 isolates closely related to Rhodopirellula baltica and a cluster of 5 isolates with 97.7–97.9 % of similarity to this species, which probably are a different species of Rhodopirellula. ERIC-PCR profiles showed a higher discrimination within the two groups and allowed the identification of nine different genotypes within the isolated strains. This work corroborates the association of Rhodopirellula spp. with fish farm environments.
TL;DR: Results indicate that TdrA is an outer membrane receptor and a protective immunogen that is likely to be involved in iron acquisition and, as a result, required for optimal bacterial virulence.
Abstract: Pseudomonas fluorescens is a Gram-negative bacterium and a common aquaculture pathogen. In this study, we identified from a pathogenic P. fluorescens strain a TonB-dependent outer membrane receptor, TdrA, as a secreted protein and examined its function and vaccine potential. TdrA is composed of 746 residues and possesses conserved structural domains of TonB-dependent outer membrane receptors. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tdrA was upregulated under conditions of iron starvation and during infection of host cells. Consistently, iron depletion induced increased production of TdrA protein in the outer membrane. Compared to the wild type, a tdrA-knock out mutant (1) was unable to grow in the absence of iron, (2) exhibited drastically attenuated overall bacterial virulence, and (3) was impaired in the ability to establish lethal infection in host tissues. Purified recombinant TdrA (rTdrA), when used as a subunit vaccine to immunize flounder, was able to induce strong protective immunity, including production of serum-specific antibodies that resulted in effective protection against lethal-dose P. fluorescens challenge. Together, these results indicate that TdrA is an outer membrane receptor and a protective immunogen that is likely to be involved in iron acquisition and, as a result, required for optimal bacterial virulence.
TL;DR: The high roughage diet was found to cause more methane emissions for either maintenance or ad-lib group, but the total methanogenic abundance was not influenced by roughage proportion and showed no significant difference between groups.
Abstract: This study aims to investigate the influence of diet roughage proportion on the methanogenic communities from the rumen and fecal samples in Altay local sheep native to Xinjiang and better understand the association of methanogenic diversity or abundance with methane emissions of the ruminants. In this study, the high roughage diet was found to cause more methane emissions for either maintenance or ad-lib group, but the total methanogenic abundance was not influenced by roughage proportion and showed no significant difference between groups. Furthermore, the denaturing gradient gel electrophoresis was conducted to reveal the difference in methanogenic diversity. Phylogenetic analysis showed that the sequences obtained were divided into three groups, affiliated to the genus of Methanobrevibacter, Methanocorpusculum and an unidentified methanogenic-like group. Of these sequences, the predominant diversity from the genus of Methanobrevibacter and the unidentified methanogenic-like archaeons in the rumen was found to be significantly induced by the high roughage diet, implying that the variation of diversity at the species or strain level might have an effect on methane emissions from the rumen. Further analysis showed that five methangenic sequences from the rumen were possibly associated with the differential methane emissions.
TL;DR: The real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification.
Abstract: A species-specific method to detect and quantify Planktothrix agardhii was developed by combining the SYBR Green I real-time polymerase chain reaction technique with a simplified DNA extraction procedure for standard curve preparation. Newly designed PCR primers were used to amplify a specific fragment within the rpoC1 gene. Since this gene exists in single copy in the genome, it allows the direct achievement of cell concentrations. The cell concentration determined by real-time PCR showed a linear correlation with the cell concentration determined from direct microscopic counts. The detection limit for cell quantification of the method was 8 cells μL−1, corresponding to 32 cells per reaction. Furthermore, the real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification of the potentially toxic cyanobacterium P. agardhii.
TL;DR: Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source and the AMP-forming acetyl-CoA synthetase gene of Kuenenia stuttgartiensis was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates.
Abstract: Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.
TL;DR: The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores, and mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides.
Abstract: Bacillus amyloliquefaciens LBM 5006 produces an antimicrobial factor active against Paenibacillus larvae, a major honeybee pathogen. The antagonistic effect and the mode of action of the antimicrobial factor were investigated. The antibacterial activity was produced starting at mid-logarithmic growth phase, reaching its maximum during the stationary phase. Exposure of cell suspensions of P. larvae to this antimicrobial resulted in loss of cell viability and reduction in optical density associated with cell lysis. Scanning electron microscopy showed damaged cell envelope and loss of protoplasmic material. The antimicrobial factor was stable for up to 80°C, but it was sensitive to proteinase K and trypsin. Mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides. The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores. This is the first report on iturin activity against P. larvae. This antimicrobial presents potential for use in the control of American foulbrood disease.
TL;DR: The data indicate that reduced phosphorus oxidizing bacteria are abundant in the environment and provide strong evidence for the importance of bacterial P oxidation in nature.
Abstract: Concentrations of hypophosphite and phosphite oxidizing bacteria were found to be high, relative to bacterial concentrations growing on phosphate, in sediment and soil during winter and summer seasons from 12 common terrestrial and aquatic sites using a most probable number method. The percent of total culturable bacterial concentrations that could use these reduced phosphorus compounds as a sole source of phosphorus were as follows: hypophosphite, 7–100%; phosphite, 10–67%; aminoethylphosphonate, 34–270%. The average MPN/g (±SEM) was as follows: phosphate, 6.19 × 106 (±2.40 × 106); hypophosphite, 2.61 × 106 (±1.35 × 106) phosphite, 1.91 × 106 (±1.02 × 106); aminoethylphosphonate, 3.90 × 106 (± 1.95 × 106). Relatively high concentrations of reduced phosphorus oxidizing bacteria were found in both pristine sites and sites with urban and agricultural disturbance. Concentrations of reduced phosphorus oxidizing bacteria in anoxic sediments and soil were equivalent. Our data indicate that reduced phosphorus oxidizing bacteria are abundant in the environment and provide strong evidence for the importance of bacterial P oxidation in nature.
TL;DR: Within the nanchangmycin gene cluster (nan), it was identified that two SARP-family regulatory genes, nanR1 and nanR2, were both required to activate the transcription of all nan polyketide genes and led to a threefold increase in NAN production.
Abstract: The nanchangmycin (NAN) produced by Streptomyces nanchangensis NS3226 is a polyether antibiotic resembling monensin in their gene clusters and the chemical structures. They can inhibit gram-positive bacteria and be a growth promoter for ruminants. Within the nanchangmycin gene cluster (nan), we identified that two SARP-family regulatory genes, nanR1 and nanR2, were both required to activate the transcription of all nan polyketide genes. Overexpression of NanR1 and NanR2 in wild-type increase NAN yields by at least three folds. Bioinformatic analysis of the immediate upstream DNA sequence of each nan gene and quantitative real-time RT-PCR analysis of the nan operons identified five putative SARP binding sites. Moreover, deletion of an AraC-family repressor gene nanR4 increased expression of NanR1 and R2 and led to a threefold increase in NAN production.
TL;DR: The fish infection results indicated that mutation of luxT led to marginal attenuation in the virulence of V. alginolyticus, suggesting that LuxT might play a role in the fine-tuning of the virulent via QS in V.Alginolyticsus.
Abstract: Vibrio alginolyticus, an opportunistic pathogen that causes vibriosis in miscellaneous fish species, has brought about serious economic damage to the mariculture industry in South China. The mechanism of virulence regulation in V. alginolyticus is yet not known except a Vibrio harveyi-like quorum sensing (QS) system that is established to manipulate the expression of diverse genes including those encoding virulence determinants. In this study, a new TetR family QS regulator, luxT, was identified and characterized in V. alginolyticus. The transcription of luxT gene was cell density dependent and was positively regulated by LuxU, an established QS component relaying the signal from three paralleled QS regulatory systems in V. harveyi. In addition, luxT positively regulated both luxO at transcriptional level and luxR at post-transcriptional level, which is thoroughly different from the established QS regulation mode in V. harveyi and Vibrio vulnificus. The mutant of luxT deletion produced markedly decreased total extracellular proteases and reduced motility ability compared to the wild type and the complemented strain luxT+. The fish infection results indicated that mutation of luxT led to marginal attenuation in the virulence of V. alginolyticus, suggesting that LuxT might play a role in the fine-tuning of the virulence via QS in V. alginolyticus.