Showing papers in "Fems Microbiology Letters in 2011"

Purpureocillium, a new genus for the medically important Paecilomyces lilacinus

Abstract: Paecilomyces lilacinus was described more than a century ago and is a commonly occurring fungus in soil. However, in the last decade this fungus has been increasingly found as the causal agent of infections in man and other vertebrates. Most cases of disease are described from patients with compromised immune systems or intraocular lens implants. In this study, we compared clinical isolates with strains isolated from soil, insects and nematodes using 18S rRNA gene, internal transcribed spacer (ITS) and partial translation elongation factor 1-α (TEF) sequences. Our data show that P. lilacinus is not related to Paecilomyces, represented by the well-known thermophilic and often pathogenic Paecilomyces variotii. The new genus name Purpureocillium is proposed for P. lilacinus and the new combination Purpureocillium lilacinum is made here. Furthermore, the examined Purpureocillium lilacinum isolated grouped in two clades based on ITS and partial TEF sequences. The ITS and TEF sequences of the Purpureocillium lilacinum isolates used for biocontrol of nematode pests are identical to those causing infections in (immunocompromised) humans. The use of high concentrations of Purpureocillium lilacinum spores for biocontrol poses a health risk in immunocompromised humans and more research is needed to determine the pathogenicity factors of Purpureocillium lilacinum.

Ploidy in cyanobacteria

Abstract: A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. Synechococcus PCC 7942 and Synechococcus WH7803 were found to contain 3-4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, Synechocystis PCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the 'Kazusa' strain more than 20 years ago. The results reveal that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.

Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes

Abstract: Lipoteichoic acid (LTA) is a zwitterionic polymer found in the cell wall of many Gram-positive bacteria. A widespread and one of the best-studied forms of LTA consists of a polyglycerolphosphate (PGP) chain that is tethered to the membrane via a glycolipid anchor. In this review, we will summarize our current understanding of the enzymes involved in glycolipid and PGP backbone synthesis in a variety of different Gram-positive bacteria. The recent identification of key LTA synthesis proteins allowed the construction and analysis of mutant strains with defined defects in glycolipid or backbone synthesis. Using these strains, new information on the functions of LTA for bacterial growth, physiology and during developmental processes was gained and will be discussed. Furthermore, we will reintroduce the idea that LTA remains in close proximity to the bacterial membrane for its function during bacterial growth rather than as a surface-exposed structure.

Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures.

Abstract: The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications.

Adherence and motility characteristics of clinical Acinetobacter baumannii isolates

Abstract: Acinetobacter baumannii continues to be a major health problem especially in hospital settings. Herein, features that may play a role in persistence and disease potential were investigated in a collection of clinical A. baumannii strains from Australia. Twitching motility was found to be a common trait in A. baumannii international clone I strains and in abundant biofilm formers, whereas swarming motility was only observed in isolates not classified within the international clone lineages. Bioinformatic analysis of the type IV fimbriae revealed a correlation between PilA sequence homology and motility. A high level of variability in adherence to both abiotic surfaces and epithelial cells was found. We report for the first time the motility characteristics of a large number of A. baumannii isolates and present a direct comparison of A. baumannii binding to nasopharyngeal and lung epithelial cells.

From Saccharomyces cerevisiae to Candida glabrata in a few easy steps: important adaptations for an opportunistic pathogen

Abstract: The opportunistic human fungal pathogen Candida glabrata is closely related to Saccharomyces cerevisiae, yet it has evolved to survive within mammalian hosts. Which traits help C. glabrata to adapt to this different environment? Which specific responses are crucial for its survival in the host? The main differences seem to include an extended repertoire of adhesin genes, high drug resistance, an enhanced ability to sustain prolonged starvation and adaptations of the transcriptional wiring of key stress response genes. Here, we discuss the properties of C. glabrata with a focus on the differences to related fungi.

High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain.

Abstract: Lacticin 3147 is a two-peptide broad spectrum lantibiotic produced by Lactococcus lactis DPC3147 shown to inhibit a number of clinically relevant Gram-positive pathogens. Initially isolated from an Irish kefir grain, lacticin 3147 is one of the most extensively studied lantibiotics to date. In this study, the bacterial diversity of the Irish kefir grain from which L. lactis DPC3147 was originally isolated was for the first time investigated using a high-throughput parallel sequencing strategy. A total of 17 416 unique V4 variable regions of the 16S rRNA gene were analysed from both the kefir starter grain and its derivative kefir-fermented milk. Firmicutes (which includes the lactic acid bacteria) was the dominant phylum accounting for > 92% of sequences. Within the Firmicutes, dramatic differences in abundance were observed when the starter grain and kefir milk fermentate were compared. The kefir grain-associated bacterial community was largely composed of the Lactobacillaceae family while Streptococcaceae (primarily Lactococcus spp.) was the dominant family within the kefir milk fermentate. Sequencing data confirmed previous findings that the microbiota of kefir milk and the starter grain are quite different while at the same time, establishing that the microbial diversity of the starter grain is not uniform with a greater level of diversity associated with the interior kefir starter grain compared with the exterior.

Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624

Abstract: Aflatoxin B(1) (AFB(1) ) is a potent mycotoxin with mutagenic, carcinogenic, teratogenic, hepatotoxic, and immunosuppressive properties. In order to develop a bioremediation system for AFB(1) -contaminated foods by white-rot fungi or ligninolytic enzymes, AFB(1) was treated with manganese peroxidase (MnP) from the white-rot fungus Phanerochaete sordida YK-624. AFB(1) was eliminated by MnP. The maximum elimination (86.0%) of AFB(1) was observed after 48 h in a reaction mixture containing 5 nkat of MnP. The addition of Tween 80 enhanced AFB(1) elimination. The elimination of AFB(1) by MnP considerably reduced its mutagenic activity in an umu test, and the treatment of AFB(1) by 20 nkat MnP reduced the mutagenic activity by 69.2%. (1) H-NMR and HR-ESI-MS analysis suggested that AFB(1) is first oxidized to AFB(1) -8,9-epoxide by MnP and then hydrolyzed to AFB(1) -8,9-dihydrodiol. This is the first report that MnP can effectively remove the mutagenic activity of AFB(1) by converting it into AFB(1) -8,9-dihydrodiol.

Aspergillus biofilms: clinical and industrial significance.

Abstract: The biofilm phenotype is an increasingly important concept in mycological research. Recently, there has been a developing interest in whether Aspergillus species are truly able to form biofilms or not. Industrial mycologists have long been aware of biofilms and their benefit in fermentation processes, whereas clinically their role is uncertain. This review provides an update on the impact that Aspergillus biofilms have medically and industrially, and will discuss biofilm development, and our current understanding of its molecular basis. The role of exopolymeric substance and how this substance relates to antimicrobial recalcitrance will also be discussed.

Lactic acid bacteria fermentation of human milk oligosaccharide components, human milk oligosaccharides and galactooligosaccharides

Abstract: Human milk contains about 7% lactose and 1% human milk oligosaccharides (HMOs) consisting of lactose with linked fucose, N -acetylglucosamine and sialic acid. In infant formula, galactooligosaccharides (GOSs) are added to replace HMOs. This study investigated the ability of six strains of lactic acid bacteria (LAB), Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Streptococcus thermophilus and Leuconostoc mesenteroides subsp. cremoris , to digest HMO components, defined HMOs, and GOSs. All strains grew on lactose and glucose. N -acetylglucosamine utilization varied between strains and was maximal in L. plantarum ; fucose utilization was low or absent in all strains. Both hetero- and homofermentative LAB utilized N -acetylglucosamine via the Embden–Meyerhof pathway. Lactobacillus acidophilus and L. plantarum were the most versatile in hydrolysing pNP analogues and the only strains releasing mono- and disaccharides from defined HMOs. Whole cells of all six LAB hydrolysed oNP-galactoside and pNP-galactoside indicating β-galactosidase activity. High β-galactosidase activity of L. reuteri, L. fermentum, S. thermophilus and L. mesenteroides subsp. cremoris whole cells correlated to lactose and GOS hydrolysis. Hydrolysis of lactose and GOSs by heterologously expressed β-galactosidases confirmed that LAB β-galactosidases are involved in GOS digestion. In summary, the strains of LAB used were not capable of utilizing complex HMOs but metabolized HMO components and GOSs.

Tight control of mycotoxin biosynthesis gene expression in Aspergillus flavus by temperature as revealed by RNA-Seq

Abstract: To better understand the effect of temperature on mycotoxin biosynthesis, RNA-Seq technology was used to profile the Aspergillus flavus transcriptome under different temperature conditions. This approach allowed us to quantify transcript abundance for over 80% of fungal genes including 1153 genes that were differentially expressed at 30 and 37 °C. Eleven of the 55 secondary metabolite clusters were upregulated at the lower temperature, including aflatoxin biosynthesis genes, which were among the most highly upexpressed genes. On average, transcript abundance for the 30 aflatoxin biosynthesis genes was 3300 times greater at 30 °C as compared with 37 °C. The results are consistent with the view that high temperature negatively affects aflatoxin production by turning down transcription of the two key transcriptional regulators, aflR and aflS. Subtle changes in the expression levels of aflS to aflR appear to control transcription activation of the aflatoxin cluster.

Quorum‐sensing autoinducer molecules produced by members of a multispecies biofilm promote horizontal gene transfer to Vibrio cholerae

Abstract: Vibrio cholerae, the causative agent of cholera and a natural inhabitant of aquatic environments, regulates numerous behaviors using a quorum-sensing (QS) system conserved among many members of the marine genus Vibrio. The Vibrio QS response is mediated by two extracellular autoinducer (AI) molecules: CAI-I, which is produced only by Vibrios, and AI-2, which is produced by many bacteria. In marine biofilms on chitinous surfaces, QS-proficient V. cholerae become naturally competent to take up extracellular DNA. Because the direct role of AIs in this environmental behavior had not been determined, we sought to define the contribution of CAI-1 and AI-2 in controlling transcription of the competence gene, comEA, and in DNA uptake. In this study we demonstrated that comEA transcription and the horizontal acquisition of DNA by V. cholerae are induced in response to purified CAI-1 and AI-2, and also by autoinducers derived from other Vibrios co-cultured with V. cholerae within a mixed-species biofilm. These results suggest that autoinducer communication within a consortium may promote DNA exchange among Vibrios, perhaps contributing to the evolution of these bacterial pathogens.

A genome-wide polyketide synthase deletion library uncovers novel genetic links to polyketides and meroterpenoids in Aspergillus nidulans.

Abstract: Fungi possess an advanced secondary metabolism that is regulated and coordinated in a complex manner depending on environmental challenges. To understand this complexity, a holistic approach is necessary. We initiated such an analysis in the important model fungus Aspergillus nidulans by systematically deleting all 32 individual genes encoding polyketide synthases. Wild-type and all mutant strains were challenged on different complex media to provoke induction of the secondary metabolism. Screening of the mutant library revealed direct genetic links to two austinol meroterpenoids and expanded the current understanding of the biosynthetic pathways leading to arugosins and violaceols. We expect that the library will be an important resource towards a systemic understanding of polyketide production in A. nidulans.

Facultative methanotrophy: false leads, true results, and suggestions for future research

Abstract: Methanotrophs are a group of phylogenetically diverse microorganisms characterized by their ability to utilize methane as their sole source of carbon and energy Early studies suggested that growth on methane could be stimulated with the addition of some small organic acids, but initial efforts to find facultative methanotrophs, ie, methanotrophs able to utilize compounds with carbon-carbon bonds as sole growth substrates were inconclusive Recently, however, facultative methanotrophs in the genera Methylocella, Methylocapsa, and Methylocystis have been reported that can grow on acetate, as well as on larger organic acids or ethanol for some species All identified facultative methanotrophs group within the Alphaproteobacteria and utilize the serine cycle for carbon assimilation from formaldehyde It is possible that facultative methanotrophs are able to convert acetate into intermediates of the serine cycle (eg malate and glyoxylate), because a variety of acetate assimilation pathways convert acetate into these compounds (eg the glyoxylate shunt of the tricarboxylic acid cycle, the ethylmalonyl-CoA pathway, the citramalate cycle, and the methylaspartate cycle) In this review, we summarize the history of facultative methanotrophy, describe scenarios for the basis of facultative methanotrophy, and pose several topics for future research in this area

Model of the molecular basis for hydroxylamine oxidation and nitrous oxide production in methanotrophic bacteria

Abstract: Many methane-oxidizing bacteria (MOB) have been shown to aerobically oxidize ammonia and hydroxylamine (NH(2)OH) to produce nitrite and nitrous oxide (N(2)O). Genome sequences of alphaproteobacterial, gammaproteobacterial, and verrucomicrobial methanotrophs revealed the presence of haoAB, cytL, cytS, nirS or nirK, and norCB genes that may be responsible for N(2)O production, and additional haoAB genes were sequenced from two strains of Methylomicrobium album. The haoAB genes of M. album ATCC 33003 were inducible by ammonia and NH(2)OH, similar to haoAB induction by ammonia in Methylococcus capsulatus Bath. Increased expression of genes encoding nitric oxide reductase (cNOR; norCB) was measured upon exposure of M. capsulatus Bath to NaNO(2) and NO-releasing sodium nitroprusside. Only incubations of M. capsulatus Bath with methane, ammonia, and nitrite produced N(2)O. The data suggest a possible pathway of nitrite reduction to NO by reversely operating NH(2)OH oxidoreductase and NO reduction to N(2)O by cNOR independently or in conjunction with ammonia-induced enzymes (i.e. HAO or cytochrome c'-β). Results of this study show that MOB likely have diverse mechanisms for nitrogen oxide metabolism and detoxification of NH(2)OH that involve conventional and unconventional enzymes.

An endophytic/pathogenic Phoma sp. from creosote bush producing biologically active volatile compounds having fuel potential.

Abstract: A Phoma sp. was isolated and characterized as endophytic and as a pathogen of Larrea tridentata (creosote bush) growing in the desert region of southern Utah, USA. This fungus produces a unique mixture of volatile organic compounds (VOCs), including a series of sesquiterpenoids, some alcohols and several reduced naphthalene derivatives. Trans-caryophyllene, a product in the fungal VOCs, was also noted in the VOCs of this pungent plant. The gases of Phoma sp. possess antifungal properties and is markedly similar to that of a methanolic extract of the host plant. Some of the test organisms with the greatest sensitivity to the Phoma sp. VOCs were Verticillium, Ceratocystis, Cercospora and Sclerotinia while those being the least sensitive were Trichoderma, Colletotrichum and Aspergillus. We discuss the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship with implications for utilization as an energy source.

The activity of bacteriocins from Carnobacterium maltaromaticum UAL307 against Gram-negative bacteria in combination with EDTA treatment

Abstract: Bacteriocins from Gram-positive bacteria are potent antimicrobial peptides that inhibit pathogenic and food-spoilage bacteria. They are usually ineffective against Gram-negative bacteria because they cannot penetrate the outer membrane (OM). Disruption of the OM of some Gram-negative bacteria was reported to sensitize them to certain bacteriocins. This study evaluates the activity of three purified bacteriocins [carnocyclin A (CclA), carnobacteriocin BM1 (CbnBM1) and piscicolin 126 (PisA)] produced by Carnobacterium maltaromaticum UAL307, which has been approved for preservation of food in United States and Canada, against three Gram-negative bacteria (Escherichia coli DH5α, Pseudomonas aeruginosa ATCC 14207 and Salmonella Typhimurium ATCC 23564). Their efficacy is compared with bacteriocins of other classes: the lantibiotics nisin A (positive control) and gallidermin, and the cyclic peptide subtilosin A (SubA). In combination with EDTA, CclA inhibited both E. coli and Pseudomonas. PisA inhibited Pseudomonas, but CbnBM1 showed weak activity toward Pseudomonas. In comparison, nisin and gallidermin inhibited the growth of all three strains, whereas SubA was active against E. coli and Pseudomonas only at high concentrations. The results reveal that UAL307 bacteriocins can inhibit Gram-negative bacteria if the OM is weakened, and that the different classes of bacteriocins in this study exert unique modes of action toward such bacteria.

Genome sequence of Staphylococcus lugdunensis N920143 allows identification of putative colonization and virulence factors

Abstract: Staphylococcus lugdunensis is an opportunistic pathogen related to Staphylococcus aureus and Staphylococcus epidermidis. The genome sequence of S. lugdunensis strain N920143 has been compared with other staphylococci, and genes were identified that could promote survival of S. lugdunensis on human skin and pathogenesis of infections. Staphylococcus lugdunensis lacks virulence factors that characterize S. aureus and harbours a smaller number of genes encoding surface proteins. It is the only staphylococcal species other than S. aureus that possesses a locus encoding iron-regulated surface determinant (Isd) proteins involved in iron acquisition from haemoglobin.

Maintaining network security: how macromolecular structures cross the peptidoglycan layer.

Abstract: Peptidoglycan plays a vital role in bacterial physiology, maintaining cell shape and resisting cellular lysis from high internal turgor pressures. Its integrity is carefully maintained by controlled remodeling during growth and division by the coordinated activities of penicillin-binding proteins, lytic transglycosylases, and N-acetylmuramyl-l-alanine amidases. However, its small pore size (∼2 nm) and covalently closed structure make it a formidable barrier to the assembly of large macromolecular cell-envelope-spanning complexes involved in motility and secretion. Here, we review the strategies used by Gram-negative bacteria to assemble such macromolecular complexes across the peptidoglycan layer, while preserving its essential structural role. In addition, we discuss evidence that suggests that peptidoglycan can be integrated into cell-envelope-spanning complexes as a structural and functional extension of their architecture.

Isolation and identification of soil bacteria growing at the expense of arbuscular mycorrhizal fungi

Abstract: Soil-microorganism symbioses are of fundamental importance for plant adaptation to the environment. Research in microbial ecology has revealed that some soil bacteria are associated with arbuscular mycorrhizal fungi (AMF). However, these interactions may be much more complex than originally thought. To assess the type of bacteria associated with AMF, we initially isolated spores of Glomus irregulare from an Agrostis stolonifera rhizosphere. The spores were washed with sterile water and plated onto G. irregulare mycelium growing in vitro in a root-free compartment of bicompartmented Petri dishes. We hypothesized that this system should select for bacteria closely associated with the fungus because the only nutrients available to the bacteria were those derived from the hyphae. Twenty-nine bacterial colonies growing on the AMF hyphae were subcultured and identified using 16S rRNA gene sequences. All bacterial isolates showed high sequence identity to Bacillus cereus, Bacillus megaterium, Bacillus simplex, Kocuria rhizophila, Microbacterium ginsengisoli, Sphingomonas sp. and Variovorax paradoxus. We also assessed bacterial diversity on the surface of spores by PCR-denaturating gradient gel electrophoresis. Finally, we used live cellular imaging to show that the bacteria isolated can grow on the surface of hyphae with different growing patterns in contrast to Escherichia coli as a control.

Quantification of butyryl CoA:acetate CoA-transferase genes reveals different butyrate production capacity in individuals according to diet and age

Abstract: The gastrointestinal microbiota produces short-chain fatty acids, especially butyrate, which affect colonic health, immune function and epigenetic regulation. To assess the effects of nutrition and aging on the production of butyrate, the butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium clusters lV and XlVa, the main butyrate producers, were analysed. Faecal samples of young healthy omnivores (24 ± 2.5 years), vegetarians (26 ± 5 years) and elderly (86 ± 8 years) omnivores were evaluated. Diet and lifestyle were assessed in questionnaire-based interviews. The elderly had significantly fewer copies of the butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014), while vegetarians showed the highest number of copies (P=0.048). The thermal denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting curve related to Roseburia/Eubacterium rectale spp. was significantly more variable in the vegetarians than in the elderly. The Clostridium cluster XIVa was more abundant in vegetarians (P=0.049) and in omnivores (P<0.01) than in the elderly group. Gastrointestinal microbiota of the elderly is characterized by decreased butyrate production capacity, reflecting increased risk of degenerative diseases. These results suggest that the butyryl-CoA:acetate CoA-transferase gene is a valuable marker for gastrointestinal microbiota function.

Reduced virulence is an important characteristic of biofilm infection of Streptococcus suis.

Abstract: Streptococcus suis 2 (SS2) is a zoonotic pathogen that can participate in biofilm formation to survive in hostile environments. In this study, virulent SS2 strains HA9801 and ZY05719 displayed increased biofilm formation compared with SS2 avirulent strain T15. In addition, a 58% reduction in adherence to HEp-2 cells was observed for HA9801 biofilm cells, compared with HA9801 planktonic cells. The 50% lethal dose (LD(50) ) of biofilm cells was 40-fold greater than that of planktonic cells. Quantification of expression levels of known virulence genes by real-time PCR revealed that the transcription levels of the gdh, cps2 and mrp genes in biofilm cells were downregulated, while the sly and gapdh genes were upregulated. HA9801 biofilm and planktonic vaccines provided 60% and 46% protection, respectively, when challenged with 50 times the LD(50) of the HA9801 strain. These results suggest a possible connection between virulence and the ability of biofilm formation; cell adhesion, transcription levels and virulence properties are different between biofilm cells and planktonic cells. Furthermore, this work offers a novel insight into bacterium infection mechanisms, which suggests that a virulent strain may be able to decrease its virulence by forming a biofilm so that it can achieve persistent infection in vivo.

Flagella and pili are both necessary for efficient attachment of Methanococcus maripaludis to surfaces

Abstract: Methanococcus maripaludis has two surface appendages, namely flagella and pili. Flagella have been shown to be required for swimming, but no specific role has been assigned as yet to pili. In this report, wild-type M. maripaludis cells are compared with mutants lacking either pili or flagella or both surface appendages in their ability to attach to a variety of surfaces including nickel, gold and molybdenum grids as well as glass, silicon and mica. Wild-type cells attached to varying degrees to all surfaces tested, except mica, via their flagella as observed by scanning electron microscopy. Large cables of flagella were found to leave the cell and to be unwound on the surface. In addition, such cables were often found to connect cells. In contrast, cells lacking either flagella or pili or both surface appendages were unable to attach efficiently to any surfaces. This indicates a second role for flagella in addition to swimming in M. maripaludis, as well as a first role for pili in this organism, namely in surface attachment.

Metabolism of organochlorine pesticide heptachlor and its metabolite heptachlor epoxide by white rot fungi, belonging to genus Phlebia.

Abstract: White rot fungi of the genus Phlebia have demonstrated a high capacity to degrade organic pollutants, including polychlorinated dibenzo-p-dioxins and polychlorinated biphenyls. In this study, we evaluated the ability of 18 white rot fungi species of genus Phlebia to degrade heptachlor and heptachlor epoxide, and described the metabolic pathways by selected white rot fungi. Phlebia tremellosa, Phlebia brevispora and Phlebia acanthocystis removed about 71%, 74% and 90% of heptachlor, respectively, after 14 days of incubation. A large amount of heptachlor epoxide and a small amount of 1-hydroxychlordene and 1-hydroxy-2,3-epoxychlordene were detected as metabolic products of heptachlor from most fungal cultures. The screening of heptachlor epoxide-degrading fungi revealed that several fungi are capable of degrading heptachlor epoxide, which is a recalcitrant metabolite of heptachlor. Phlebia acanthocystis, P. brevispora, Phlebia lindtneri and Phlebia aurea removed about 16%, 16%, 22% and 25% of heptachlor epoxide, respectively, after 14 days of incubation. Heptachlor diol and 1-hydroxy-2,3-epoxychlordene were produced in these fungal cultures as metabolites, suggesting that the hydrolysis and hydroxylation reaction occur in the epoxide ring and in position 1 of heptachlor epoxide, respectively.

Visible light during mycelial growth and conidiation of Metarhizium robertsii produces conidia with increased stress tolerance.

Abstract: Light conditions during mycelial growth are known to influence fungi in many ways. The effect of visible-light exposure during mycelial growth was investigated on conidial tolerance to UVB irradiation and wet heat of Metarhizium robertsii, an insect-pathogenic fungus. Two nutrient media and two light regimens were compared. Conidia were produced on (A) potato dextrose agar plus yeast extract medium (PDAY) (A1) under dark conditions or (A2) under continuous visible light (provided by two fluorescent lamps with intensity 5.4 W m−2). For comparison, the fungus was also produced on (B) minimal medium (MM) under continuous-dark incubation, which is known to produce conidia with increased tolerance to heat and UVB radiation. The UVB tolerances of conidia produced on PDAY under continuous visible light were twofold higher than conidia produced on PDAY medium under dark conditions, and this elevated UVB tolerance was similar to that of conidia produced on MM in the dark. The heat tolerance of conidia produced under continuous light was, however, similar to that of conidia produced on MM or PDAY in the dark. Conidial yield on PDAY medium was equivalent when the fungus was grown either under continuous-dark or under continuous-light conditions.

Anti‐Candida effect of bacillomycin D‐like lipopeptides from Bacillus subtilis B38

Abstract: Bacillus subtilis B38, isolated from soil, showed antimicrobial activity against human pathogenic Candida albicans species. Specific PCR primers revealed the presence of the bamC gene, which is involved in the biosynthesis of bacillomycin D. Three anti-Candida compounds designated a(1) , a(2) and a(3) were purified from culture supernatant and identified using matrix-assisted laser desorption/ionization time-of-flight MS as analogues of bacillomycin D-like lipopeptides of 14, 15 and 16 carbon fatty acid long chains, respectively. The compound a(3) displayed the strongest fungicidal activity against pathogenic C. albicans strains. It was even more active than amphotericin B with a lethal concentration of 59.07 vs. 135.26 μM of the antimycotic drug against the pathogenic strain C. albicans sp. 311 isolated from finger nail. Only moderate or weak anti-Candida activity was recorded for a(1) and a(2) compounds. Furthermore, a(3) showed the highest hemolytic activity, reaching 50% hemolysis at 22.14 μM, whereas a(1) and a(2) displayed a limited hemolysis at 68.26 and 37.41 μM, respectively. These findings suggest that the acyl chain length of bacillomycin D-like lipopeptides plays a major role in hemolytic and antifungal activities.

Phylogenetic diversity and dietary association of rumen Treponema revealed using group‐specific 16S rRNA gene‐based analysis

Abstract: Treponema spp. are a commonly detected bacterial group in the rumen that are involved in the degradation of soluble fibers. In this study, a ruminal Treponema group-specific PCR primer targeting the 16S rRNA gene was designed and used to assess the phylogenetic diversity and diet association of this group in sheep rumen. Total DNA was extracted from rumen digesta of three sheep fed a diet based on alfalfa/orchardgrass hay or concentrate. The real-time PCR quantification indicated that the relative abundance of the Treponema group in the total rumen bacteria was as high as 1.05%, while the known species Treponema bryantii accounted for only 0.02%. Fingerprints of the Treponema community determined by 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE) analysis tended to differ among the diets. Principal component analysis of the DGGE profiles distinguished those Treponema associated with either the hay or the concentrate diets. Analysis of a Treponema 16S rRNA gene clone library showed phylogenetically distinct operational taxonomic units for a specific dietary condition, and significant (P=0.001) differences in community composition were observed among clone libraries constructed from each dietary regimen. The majority of clones (75.4%) had <97% sequence similarity with known Treponema. These results suggest the predominance of uncultured Treponema that appear to have distinct members related to the digestion of either hay or concentrate diet.

Biogenesis of the cyanobacterial thylakoid membrane system--an update.

Abstract: Current molecular analyses suggest that initial steps of the biogenesis of cyanobacterial photosystems progress in a membrane subfraction representing a biosynthetic center with contact to both plasma and thylakoid membranes. This special membrane fraction is defined by the presence of the photosystem II assembly factor PratA. The proposed model suggests that both biogenesis of protein complexes and insertion of chlorophyll molecules into the photosystems occur in this intermediate membrane system.

Transcriptional repressor Rex is involved in regulation of oxidative stress response and biofilm formation by Streptococcus mutans.

Abstract: The transcriptional repressor Rex has been implicated in the regulation of energy metabolism and fermentative growth in response to redox potential. Streptococcus mutans, the primary causative agent of human dental caries, possesses a gene that encodes a protein with high similarity to members of the Rex family of proteins. In this study, we showed that Rex-deficiency compromised the ability of S. mutans to cope with oxidative stress and to form biofilms. The Rex-deficient mutant also accumulated less biofilm after 3days than the wild-type strain, especially when grown in sucrose-containing medium, but produced more extracellular glucans than the parental strain. Rex-deficiency caused substantial alterations in gene transcription, including those involved in heterofermentative metabolism, NAD 1 regeneration and oxidative stress. Among the upregulated genes was gtfC, which encodes glucosyltransferase C, an enzyme primarily responsible for synthesis of water-insoluble glucans. These results reveal that Rex plays an important role in oxidative stress responses and biofilm formation by S. mutans.

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples.

Abstract: Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.