Showing papers in "Archives of Microbiology in 2010"

Production, purification and structural characterization of an exopolysaccharide produced by a probiotic Lactobacillus plantarum MTCC 9510

Abstract: Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and finds applications even in non-dairy foods and in therapeutics. Box-Behnken model of response surface methodology (RSM) was employed to formulate the production medium for exopolysaccharide (EPS). FT-IR spectral analysis of the purified EPS from Lactobacillus plantarum MTCC 9510 revealed prominent characteristic groups corresponding to polyhydric alcohols. The degradation temperature (Td) of the polysaccharide was found to be 260°C with the help of thermo gravimetric analysis (TGA). Structure elucidation of the EPS showed that it consists of a trisaccharide repeating unit of α-d-glucose, β-d-glucose and α-d-mannose.

How do bacteria sense and respond to low temperature

Abstract: Rigidification of the membrane appears to be the primary signal perceived by a bacterium when exposed to low temperature. The perception and transduction of the signal then occurs through a two-component signal transduction pathway consisting of a membrane-associated sensor and a cytoplasmic response regulator and as a consequence a set of cold-regulated genes are activated. In addition, changes in DNA topology due to change in temperature may also trigger cold-responsive mechanisms. Inducible proteins thus accumulated repair the damage caused by cold stress. For example, the fluidity of the rigidified membrane is restored by altering the levels of saturated and unsaturated fatty acids, by altering the fatty acid chain length, by changing the proportion of cis to trans fatty acids and by changing the proportion of anteiso to iso fatty acids. Bacteria could also achieve membrane fluidity changes by altering the protein content of the membrane and by altering the levels of the type of carotenoids synthesized. Changes in RNA secondary structure, changes in translation and alteration in protein conformation could also act as temperature sensors. This review highlights the various strategies by which bacteria senses low temperature signal and as to how it responds to the change.

Organic acid production and plant growth promotion as a function of phosphate solubilization by Acinetobacter rhizosphaerae strain BIHB 723 isolated from the cold deserts of the trans-Himalayas.

Abstract: An efficient phosphate-solubilizing plant growth-promoting Acinetobacter rhizosphaerae strain BIHB 723 exhibited significantly higher solubilization of tricalcium phosphate (TCP) than Udaipur rock phosphate (URP), Mussoorie rock phosphate (MRP) and North Carolina rock phosphate (NCRP). Qualitative and quantitative differences were discerned in the gluconic, oxalic, 2-keto gluconic, lactic, malic and formic acids during the solubilization of various inorganic phosphates by the strain. Gluconic acid was the main organic acid produced during phosphate solubilization. Formic acid production was restricted to TCP solubilization and oxalic acid production to the solubilization of MRP, URP and NCRP. A significant increase in plant height, shoot fresh weight, shoot dry weight, root length, root dry weight, and root, shoot and soil phosphorus (P) contents was recorded with the inoculated treatments over the uninoculated NP(0)K or NP(TCP)K treatments. Plant growth promotion as a function of phosphate solubilization suggested that the use of bacterial strain would be a beneficial addition to the agriculture practices in TCP-rich soils in reducing the application of phosphatic fertilizers.

Bacterial energy taxis: a global strategy?

Abstract: A functional energy metabolism is one of the most important requirements for survival of all kinds of organisms including bacteria. Therefore, many bacteria actively seek conditions of optimal metabolic activity, a behaviour which can be termed “energy taxis”. Motility, combined with the sensory perception of the internal energetic conditions, is prerequisite for tactic responses to different energy levels and metabolic yields. Diverse mechanisms of energy sensing and tactic response have evolved among various bacteria. Many of the known energy taxis sensors group among the methyl-accepting chemotaxis protein (MCP)-like sensors. This review summarizes recent advances in the field of energy taxis and explores the current concept that energy taxis is an important part of the bacterial behavioural repertoire in order to navigate towards more favourable metabolic niches and to survive in a specific habitat.

Isolation and characterization of the equol-producing bacterium Slackia sp. strain NATTS.

Abstract: Several kinds of carbohydrates such as sorbose, adonitol, and melezitose were found to enhance equol production from daidzein in an in vitro human fecal culture. Sorbose, one of the most effective carbohydrates, was used as a carbohydrate source for isolating the NATTS strain, which was a Gram-positive, non-spore-forming rod bacterium with high ability to convert daidzein to equol isolated from the 7th maintenance culture. The strain was found to belong to the genus Slackia family Coriobacteriaceae by 16S rRNA sequence-based analysis, and the prevalence of the Slackia sp. in Japanese adults was examined by reverse transcription-quantitative PCR (RT-qPCR), which was found to be 40% at a mean population level of 106 cells per gram of feces.

An overview of in silico protein function prediction.

Abstract: As the protein databases continue to expand at an exponential rate, fed by daily uploads from multiple large scale genomic and metagenomic projects, the problem of assigning a function to each new protein has become the focus of significant research interest in recent times. Herein, we review the most recent advances in the field of automated function prediction (AFP). We begin by defining what is meant by biological “function” and the means of describing such functions using standardised machine readable ontologies. We then focus on the various function-prediction programs available, both sequence and structure based, and outline their associated strengths and weaknesses. Finally, we conclude with a brief overview of the future challenges and outstanding questions in the field, which still remain unanswered.

The in vitro antibiofilm activity of selected marine bacterial culture supernatants against Vibrio spp.

Abstract: The aim of the work is to investigate the effect of marine bacterial culture supernatants on biofilm formation of Vibrio spp., a major menace in aquaculture industries. Vibrio spp. biofilm cause life-threatening infections in humans and animals. Forty-three marine bacterial culture supernatants were screened against the hydrophobicity index, initial attachment and biofilm formation in Vibrio spp. Twelve culture supernatants showed antibiofilm activity. The bacterial culture supernatants S8-07 (Bacillus pumilus) and S6-01 (B. indicus) inhibited the initial attachment, biofilm formation and dispersed the mature biofilm at 5% v/v concentration without inhibiting the growth. Analysis by light microscopy and confocal laser scanning microscopy showed that the architecture of the biofilm was destroyed by bacterial supernatants when compared to the control. The bacterial supernatants also reduce the surface hydrophobicity of Vibrio spp. which is one of the important requirements for biofilm formation. Further characterization of antibiofilm activity in S8-07 culture supernatant confirmed that it is an enzymatic activity and the size is more than 10 kDa and in S6-01, it is a heat-stable, non-protein compound. Furthermore, both the supernatants failed to show any biosurfactant activity. The culture supernatants of S8-07 and S6-01 with promising antibiofilm property have potential for application in medicine and marine aquaculture.

Effect of osmotic stress on plant growth promoting Pseudomonas spp.

Abstract: In this study we isolated and screened drought tolerant Pseudomonas isolates from arid and semi arid crop production systems of India. Five isolates could tolerate osmotic stress up to −0.73 MPa and possessed multiple PGP properties such as P-solubilization, production of phytohormones (IAA, GA and cytokinin), siderophores, ammonia and HCN however under osmotic stress expression of PGP traits was low compared to non-stressed conditions. The strains were identified as Pseudomonasentomophila, Pseudomonasstutzeri, Pseudomonas putida, Pseudomonas syringae and Pseudomonas monteilli respectively on the basis of 16S rRNA gene sequence analysis. Osmotic stress affected growth pattern of all the isolates as indicated by increased mean generation time. An increase level of intracellular free amino acids, proline, total soluble sugars and exopolysaccharides was observed under osmotic stress suggesting bacterial response to applied stress. Further, strains GAP-P45 and GRFHYTP52 showing higher levels of EPS and osmolytes (amino acids and proline) accumulation under stress as compared to non-stress conditions, also exhibited higher expression of PGP traits under stress indicating a relationship between stress response and expression of PGP traits. We conclude that isolation and screening of indigenous, stress adaptable strains possessing PGP traits can be a method for selection of efficient stress tolerant PGPR strains.

Advances in molecular detection of Aspergillus: an update

Abstract: Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a ‘gold standard’ for Aspergillus detection.

Saccharomyces cerevisiae strain UFMG 905 protects against bacterial translocation, preserves gut barrier integrity and stimulates the immune system in a murine intestinal obstruction model.

Abstract: Probiotic is a preparation containing microorganisms that confers beneficial effect to the host. This work assessed whether oral treatment with viable or heat-killed yeast Saccharomyces cerevisiae strain UFMG 905 prevents bacterial translocation (BT), intestinal barrier integrity, and stimulates the immunity, in a murine intestinal obstruction (IO) model. Four groups of mice were used: mice undergoing only laparotomy (CTL), undergoing intestinal obstruction (IO) and undergoing intestinal obstruction after previous treatment with viable or heat-killed yeast. BT, determined as uptake of 99mTc-E. coli in blood, mesenteric lymph nodes, liver, spleen and lungs, was significantly higher in IO group than in CTL group. Treatments with both yeasts reduced BT in blood and all organs investigated. The treatment with both yeasts also reduced intestinal permeability as determined by blood uptake of 99mTc-DTPA. Immunological data demonstrated that both treatments were able to significantly increase IL-10 levels, but only viable yeast had the same effect on sIgA levels. Intestinal lesions were more severe in IO group when compared to CTL and yeasts groups. Concluding, both viable and heat-killed cells of yeast prevent BT, probably by immunomodulation and by maintaining gut barrier integrity. Only the stimulation of IgA production seems to depend on the yeast viability.

Oxidative stress protection and the repair response to hydrogen peroxide in the hyperthermophilic archaeon Pyrococcus furiosus and in related species

Abstract: Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100 degrees C. Addition of H(2)O(2) (0.5 mM) to a growing culture resulted in the cessation of growth with a 2-h lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function, while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H(2)O(2) were not more resistant to the subsequent addition of H(2)O(2) (0.5-5.0 mM). Consequently, there is little if any induced protective response to peroxide. The organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin, and alkyl hydroperoxide reductase. Related hyperthermophiles contain homologs of the proteins involved in the constitutive protective mechanism but these organisms were more sensitive to peroxide than P. furiosus and lack several of its peroxide-responsive ORFs.

Environmental and genetic factors that contribute to Escherichia coli K-12 biofilm formation

Abstract: Biofilms are communities of bacteria whose formation on surfaces requires a large portion of the bacteria’s transcriptional network. To identify environmental conditions and transcriptional regulators that contribute to sensing these conditions, we used a high-throughput approach to monitor biofilm biomass produced by an isogenic set of Escherichia coli K-12 strains grown under combinations of environmental conditions. Of the environmental combinations, growth in tryptic soy broth at 37°C supported the most biofilm production. To analyze the complex relationships between the diverse cell-surface organelles, transcriptional regulators, and metabolic enzymes represented by the tested mutant set, we used a novel vector-item pattern-mining algorithm. The algorithm related biofilm amounts to the functional annotations of each mutated protein. The pattern with the best statistical significance was the gene ontology ‘pyruvate catabolic process,’ which is associated with enzymes of acetate metabolism. Phenotype microarray experiments illustrated that carbon sources that are metabolized to acetyl-coenzyme A, acetyl phosphate, and acetate are particularly supportive of biofilm formation. Scanning electron microscopy revealed structural differences between mutants that lack acetate metabolism enzymes and their parent and confirmed the quantitative differences. We conclude that acetate metabolism functions as a metabolic sensor, transmitting changes in environmental conditions to biofilm biomass and structure.

Effects of the antibiotic growth promoters flavomycin and florfenicol on the autochthonous intestinal microbiota of hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂)

Abstract: The 16S rDNA PCR-DGGE and rpoB quantitative PCR (RQ-PCR) techniques were used to evaluate the effects of dietary flavomycin and florfenicol on the autochthonous intestinal microbiota of hybrid tilapia. The fish were fed four diets: control, dietary flavomycin, florfenicol and their combination. After 8 weeks of feeding, 6 fish from each cage were randomly chosen for the analysis. The total number of intestinal bacteria was determined by RQ-PCR. The results showed that dietary antibiotics significantly influenced the intestinal microbiota and dramatically reduced the intensity of total intestinal bacterial counts. The intensity of some phylotypes (EU563257, EU563262 and EU563255) were reduced to non-detectable levels by both dietary antibiotics, while supplementation of florfenicol to the diet also reduced the intensity of the phylotypes EU563242 and EU563262, uncultured Mycobacterium sp.-like, uncultured Cyanobacterium-like and uncultured Cyanobacterium (EU563246). Dietary flavomycin only reduced the OTU intensity of one phylotype, identified as a member of the phylum Fusobacteria. The antibiotic combination only reduced the phylotypes EU563242 and EU563262. Based on our results, we conclude that the reduced effect of florfenicol on intestinal microbiota was stronger than that of flavomycin, and when flavomycin and florfenicol were added in combination, the effect of florfenicol overshadowed that of flavomycin.

Antibacterial mechanism of soybean isoflavone on Staphylococcus aureus.

Abstract: Effects of different flavonoids on various bacterial strains have been extensively reported; however, the mechanism(s) of their action on bacterial cells remain largely elusive. In this study, the antibacterial mechanism of soybean isoflavone (SI) on Staphylococcus aureus is systematically investigated using 4′6-diamidino-2-phenylindole (DAPI) staining, pBR322DNA decatenation experiment mediated by topoisomerase and agarose gel electrophoresis for direct decatenation. The results of fluorescence microscopy and fluorescence spectrophotometer indicated that DAPI was integrated in Staphylococcus aureus. Additionally, the quantity of both DNA and RNA reduced to 66.47 and 60.18%, respectively, after treated with SI for 28 h. Effects of SI on topoisomerase I and II were also investigated. SI completely inhibited the pBR322DNA unwinding mediated by topoisomerase I and topoisomerase II at the concentration of 6.4 mg/ml and could denature the plasmid DNA at the concentration of 12.8 mg/ml. These results indicate that topoisomerase I and II are the most important targets by SI to restrain bacterial cell division.

Bacteriophages and cancer

Abstract: Bacteriophages can be used effectively to cure bacterial infections. They are known to be active against bacteria but inactive against eukaryotic cells. Nevertheless, novel observations suggest that phages are not neutral for higher organisms. They can affect physiological and immunological processes which may be crucial to their expected positive effects in therapies. Bacteriophages are a very differentiated group of viruses and at least some of them can influence cancer processes. Phages may also affect the immunological system. In general, they activate the immunological response, for example cytokine secretion. They can also switch the tumor microenvironment to one advantageous for anticancer treatment. On the other hand, bacteriophages are used as a platform for foreign peptides that may induce anticancer effects. As bacterial debris can interfere with bacteriophage activity, phage purification is significant for the final effect of a phage preparation. In this review, results of the influence of bacteriophages on cancer processes are presented which have implications for the perspective application of phage therapy in patients with cancer and the general understanding of the role of bacteriophages in the human organism.

Ergot alkaloid biosynthesis in Aspergillus fumigatus : conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH

Abstract: Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His6-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD+ resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K M values for chanoclavine-I and NAD+ were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s−1.

On the photosynthetic properties of marine bacterium COL2P belonging to Roseobacter clade.

Abstract: Aerobic anoxygenic phototrophs (AAPs) are prokaryotic microorganisms capable of harvesting light using bacteriochlorophyll-based reaction centres Marine AAP communities are generally dominated by species belonging to the Roseobacter clade For this reason, we used marine Roseobacter-related strain COL2P as a model organism to characterize its photosynthetic apparatus, level of pigmentation and expression of photosynthetic complexes This strain contained functional photosynthetic reaction centres with bacteriochlorophyll a and spheroidenone as the main light-harvesting pigments, but the expression of the photosynthetic apparatus was significantly reduced when compared to truly photoautotrophic species Moreover, the absence of peripheral light-harvesting complexes largely reduced its light-harvesting capacity The size of the photosynthetic unit was limited to 354 ± 10 BChl a molecules supplemented by the same number of spheroidenone molecules The contribution of oxidative phosphorylation and photophosphorylation was analysed by respiration and fluorometric measurements Our results indicate that even with a such reduced photosynthetic apparatus, photophosphorylation provides up to three times higher electron fluxes than aerobic respiration These results suggest that light-derived energy can provide a substantial fraction of COL2P metabolic needs

Inhibition of Vibrio cholerae biofilm by AiiA enzyme produced from Bacillus spp.

Abstract: Vibrio cholerae is the causative agent of water-borne diarrheal disease, cholera The formation of biofilm favors survival and persistence of V cholerae in the aquatic environment and also inside the host AHL lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus spp, blocks quorum sensing in Gram-negative bacteria by hydrolyzing N-acyl-homoserine lactones (AHLs) In the present investigation, AiiA-mediated inhibition of V cholerae biofilm was studied Two novel alleles of aiiA-encoding genes from Bacillus spp were expressed in E coli, and the results demonstrated that AiiA enzyme is a potent inhibitor of V cholerae biofilm

Unicellular cyanobacteria with a new mode of life: the lack of photosynthetic oxygen evolution allows nitrogen fixation to proceed.

Abstract: Some unicellular N2-fixing cyanobacteria have recently been found to lack a functional photosystem II of photosynthesis. Such organisms, provisionally termed UCYN-A, of the oceanic picoplanktion are major contributors to the global marine N-input by N2-fixation. Since their photosystem II is inactive, they can perform N2-fixation during the day. UCYN-A organisms cannot be cultivated as yet. Their genomic analysis indicates that they lack genes coding for enzymes of the Calvin cycle, the tricarboxylic acid cycle and for the biosynthesis of several amino acids. The carbon source in the ocean that allows them to thrive in such high abundance has not been identified. Their genomic analysis implies that they metabolize organic carbon by a new mode of life. These unicellular N2-fixing cyanobacteria of the oceanic picoplankton are evolutionarily related to spheroid bodies present in diatoms of the family Epithemiaceae, such as Rhopalodia gibba. More recently, spheroid bodies were ultimately proven to be related to cyanobacteria and to express nitrogenase. They have been reported to be completely inactive in all photosynthetic reactions despite the presence of thylakoids. Sequence data show that R. gibba and its spheroid bodies are an evolutionarily young symbiosis that might serve as a model system to unravel early events in the evolution of chloroplasts. The cell metabolism of UCYN-A and the spheroid bodies may be related to that of the acetate photoassimilating green alga Chlamydobotrys.

Environmental Escherichia coli occur as natural plant growth-promoting soil bacterium

Abstract: Currently, it is presumed that Escherichia coli is not a normal inhabitant of the soil. Soilborne E. coli strains were isolated from broad range of 7 geoclimatic zones of India, indicating that E. coli can survive and thrive under different extreme soil conditions. Diversity among E. coli strains from widely separated geographic regions using enterobacterial repetitive intergenic consensus (ERIC)-PCR did not reveal any relationships between the genotypes and the source of isolation. Inoculation of maize (Zea mays cv. Arkil) seeds with E. coli NBRIAR3 (NBRIAR3) significantly enhanced (P < 0.05) plant growth and nutrient uptake, when compared with uninoculated control. Presence or absence of NBRIAR3 did not affect significantly (P < 0.05) diversity indexes, using substrate utilization patterns on the Biolog Eco plates. Clone libraries based on 16S rRNA gene from rhizosphere of maize plants demonstrated rather similar phylotype diversity from the uninoculated control and NBRIAR3-treated rhizosphere soil, which further indicated that NBRIAR3 did not exert a major influence on the overall bacterial diversity. The methodological approach described in this study supports the idea that E. coli should be treated as native soil bacterium instead of as an “indicator” of the possible presence of other fecal coliform bacteria.

Characterization of an O-desmethylangolensin-producing bacterium isolated from human feces

Abstract: A bacterium that converted daidzein to O-desmethylangolensin was isolated from the feces of healthy humans. It was an obligately anaerobic, nonsporeforming, nonmotile and Gram-positive rod. The isolate used glucose, sucrose, raffinose, maltose, and fructose as carbon sources. It did not hydrolyze gelatin, esculin, or starch. The strain was urease, acid phosphatase, and arginine dihydrolase positive. It was catalase, oxidase, H(2)S, and indole negative. The major products of glucose fermentation were butyrate and lactate. Its mol% G+C was 51.2. The major cellular fatty acids were C(16:0) DMA, C(16:0), and C(16:0) aldehyde. The structural type of cell wall peptidoglycan was suggested to be A1gamma. The isolate was susceptible to beta-lactam, cefem, and macrolide antibiotics and resistant to aminoglycoside and quinolone antibiotics. The bacterium was related to Eubacterium ramulus ATCC29099(T), Eubacterium rectale ATCC33656(T), and species of the genus Roseburia, but the highest 16S rRNA gene similarity to these described species was only 94.4%, consistent with its being classified as a novel genus. Based on the above, the isolate, named strain SY8519, was identified as belonging to a novel genus in the Clostridium rRNA cluster XIVa.

Culturable endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane and its non-transgenic isolines

Abstract: The diversity of endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane plants and its isoline was evaluated by cultivation followed by amplified rDNA restriction analysis (ARDRA) of randomly selected strains. Transgenic and non-transgenic cultivars and their crop management (herbicide application or manual weed control) were used to assess the possible non-target effects of genetically modified sugarcane on the fungal endophytic community. A total of 14 ARDRA haplotypes were identified in the endophytic community of sugarcane. Internal transcribed spacer (ITS) sequencing revealed a rich community represented by 12 different families from the Ascomycota phylum. Some isolates had a high sequence similarity with genera that are common endophytes in tropical climates, such as Cladosporium, Epicoccum, Fusarium, Guignardia, Pestalotiopsis and Xylaria. Analysis of molecular variance indicated that fluctuations in fungal population were related to both transgenic plants and herbicide application. While herbicide applications quickly induced transient changes in the fungal community, transgenic plants induced slower changes that were maintained over time. These results represent the first draft on composition of endophytic filamentous fungi associated with sugarcane plants. They are an important step in understanding the possible effects of transgenic plants and their crop management on the fungal endophytic community.

Inhibitory activity against the fish pathogen Lactococcus garvieae produced by Lactococcus lactis TW34, a lactic acid bacterium isolated from the intestinal tract of a Patagonian fish

Abstract: After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis In addition, TW34 showed to be sensitive to different antibiotics The production of the inhibitory agent against L garvieae was growth associated, and it was significantly influenced by the incubation temperature The optimal temperature for the antimicrobial production was as low as 15°C Both acidification and hydrogen peroxide production were ruled out as the source of inhibition In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin The bacteriocin was highly thermostable (121°C for 15 min) and active between pH 3 and 11 It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C Our results suggest that the strain L lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish

Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes.

Abstract: Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.

Reduced expression of virulence factors in multidrug-resistant Pseudomonas aeruginosa strains

Abstract: MDR Pseudomonas aeruginosa strains are isolated from clinical specimens with increasing frequency. It seems that acquiring genes which determine antibiotic resistance usually comes at a biological cost of impaired bacterial physiology. There is no information on investigations comparing phenotypic differences in MDR and MDS P. aeruginosa strains in literature. The study included 150 clinical P. aeruginosa isolates (75 classified as MDS and 75 as MDR). PFGE analysis revealed five pairs of identical isolates in the group of MDR strains and the results obtained for these strains were not included in the statistical analyses. MDR strains adhered to polystyrene to a lesser extent than MDS strains. The growth rate in the liquid medium was significantly lower for MDR strains. Detectable amounts of alginate were present in the culture supernatants of seven MDS and six MDR strains. The MDR P. aeruginosa strains which were investigated produced significantly lower amounts of extracellular material binding Congo Red, lower lipolytic, elastase, LasA protease, phospholipase C activity and pyocyanin quantity in culture supernatants when compared with MDS strains. No significant differences were observed between MDR and MDS strains in proteolytic activity. In conclusion, the MDR P. aeruginosa strains have impaired virulence when compared to MDS strains.

Molecular characterization and isolation of cytochrome P450 genes from the filamentous fungus Aspergillus oryzae.

Abstract: We explored the molecular diversity of cytochrome P450 genes in the filamentous fungus Aspergillus oryzae using bioinformatic and experimental approaches. Based on bioinformatic annotation, we found 155 putative genes of cytochromes P450 in the whole genome sequence; however, 13 of 155 appeared to be pseudogenes due to sequence deletions and/or inframe stop codon(s). There are 87 families of A. oryzae cytochromes P450 (AoCYPs), indicating considerable phylogenetic diversity. To characterize A. oryzae AoCYPs, we attempted to isolate cDNAs using RT-PCR and determined their transcriptional capabilities. To date, we have confirmed gene expression of 133 AoCYPs and cloned 121 AoCYPs as full-length cDNAs encoding a mature open reading frame. Using experimentally deduced sequences and intron-exon organization, we analyzed AoCYPs phylogenetically. We also identified intronic consensus sequences in AoCYPs genes. The experimentally validated exonic and intronic sequences will be a powerful advantage in identification and characterization of novel P450s from various ascomycetous fungi.

Molecular characteristics of community-associated methicillin-resistant Staphylococcus aureus strains for clinical medicine.

Abstract: Infections caused by methicillin-resistant S. aureus strains are mainly associated with a hospital setting. However, nowadays, the MRSA infections of non-hospitalized patients are observed more frequently. In order to distinguish them from hospital-associated methicillin-resistant S. aureus (HA-MRSA) strains, given them the name of community-associated methicillin-resistant S. aureus (CA-MRSA). CA-MRSA strains most commonly cause skin infections, but may lead to more severe diseases, and consequently the patient’s death. The molecular markers of CA-MRSA strains are the presence of accessory gene regulator (agr) of group I or III, staphylococcal cassette chromosome mec (SCCmec) type IV, V or VII and genes encoding for Panton–Valentine leukocidin (PVL). In addition, CA-MRSA strains show resistance to β-lactam antibiotics. Studies on the genetic elements of CA-MRSA strains have a key role in the unambiguous identification of strains, monitoring of infections, improving the treatment, work on new antimicrobial agents and understanding the evolution of these pathogens.

Mutations of ferric uptake regulator (fur) impair iron homeostasis, growth, oxidative stress survival, and virulence of Xanthomonas campestris pv. campestris.

Abstract: Iron is essential in numerous cellular functions. Intracellular iron homeostasis must be maintained for cell survival and protection against iron's toxic effects. Here, we characterize the roles of Xanthomonas campestris pv. campestris (Xcc) fur, which encodes an iron sensor and a transcriptional regulator that acts in iron homeostasis, oxidative stress, and virulence. Herein, we isolated spontaneous Xcc fur mutants that had high intracellular iron concentrations due to constitutively high siderophore levels and increased expression of iron transport genes. These mutants also had reduced aerobic plating efficiency and resistance to peroxide killing. Moreover, one fur mutant was attenuated on a host plant, thus indicating that fur has important roles in the virulence of X. campestris pv. campestris.

Extracellular monoenzyme deglycosylation system of 7-O-linked flavonoid β-rutinosides and its disaccharide transglycosylation activity from Stilbella fimetaria

Abstract: We screened for microorganisms able to use flavonoids as a carbon source; and one isolate, nominated Stilbella fimetaria SES201, was found to possess a disaccharide-specific hydrolase. It was a cell-bound ectoenzyme that was released to the medium during conidiogenesis. The enzyme was shown to cleave the flavonoid hesperidin (hesperetin 7-O-α-rhamnopyranosyl-β-glucopyranoside) into rutinose (α-rhamnopyranosyl-β-glucopyranose) and hesperetin. Since only intracellular traces of monoglycosidase activities (β-glucosidase, α-rhamnosidase) were produced, the disaccharidase α-rhamnosyl-β-glucosidase was the main system utilized by the microorganism for hesperidin hydrolysis. The enzyme was a glycoprotein with a molecular weight of 42224 Da and isoelectric point of 5.7. Even when maximum activity was found at 70°C, it was active at temperatures as low as 5°C, consistent with the psychrotolerant character of S. fimetaria. Substrate preference studies indicated that the enzyme exhibits high specificity toward 7-O-linked flavonoid β-rutinosides. It did not act on flavonoid 3-O-β-rutinoside and 7-O-β-neohesperidosides, neither monoglycosylated substrates. In an aqueous medium, the α-rhamnosyl-β-glucosidase was also able to transfer rutinose to other acceptors besides water, indicating its potential as biocatalyst for organic synthesis. The monoenzyme strategy of S. fimetaria SES201, as well as the enzyme substrate preference for 7-O-β-flavonoid rutinosides, is unique characteristics among the microbial flavonoid deglycosylation systems reported.

S-Adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces

Abstract: The production of antibiotics in different Streptomyces strains has been reported to be stimulated by the external addition of S-adenosylmethionine (SAM) and by overexpression of the SAM synthetase gene metK. We investigated the influence of SAM addition, and of the expression of SAM biosynthetic genes, on the production of the aminocoumarin antibiotic novobiocin in the heterologous producer strain Streptomyces coelicolor M512 (nov-BG1). External addition of SAM did not influence novobiocin accumulation. However, overexpression of a SAM synthase gene stimulated novobiocin formation, concomitant with an increase of the intracellular SAM concentration. Streptomyces genomes contain orthologs of all genes required for the SAM cycle known from mammals. In contrast, most other bacteria use a different cycle for SAM regeneration. Three secondary metabolic gene clusters, coding for the biosynthesis of structurally very different antibiotics in different Streptomyces strains, were found to contain an operon comprising all five putative genes of the SAM cycle. We cloned one of these operons into an expression plasmid, under control of a strong constitutive promoter. However, transformation of the heterologous novobiocin producer strain with this plasmid did not stimulate novobiocin production, but rather showed a detrimental effect on cell viability in the stationary phase and strongly reduced novobiocin accumulation.