Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.

Intercellular mobility and homing of an archaeal rDNA intron confers a selective advantage over intron- cells of Sulfolobus acidocaldarius.

Some intron-containing rRNA genes of archaea encode homing-type endonucleases, which facilitate intron insertion at homologous sites in intron- alleles. These archaeal rRNA genes, in contrast to their eukaryotic counterparts, are present in single copies per cell, which precludes intron homing within one cell. However, given the highly conserved nature of the sequences flanking the intron, homing may occur in intron- rRNA genes of other archaeal cells. To test whether this occurs, the intron-containing 23S rRNA gene of the archaeal hyperthermophile Desulfurococcus mobilis, carried on nonreplicating bacterial vectors, was electroporated into an intron- culture of Sulfolobus acidocaldarius. PCR experiments demonstrated that the intron underwent homing and spread through the culture. By using a double drug-resistant mutant of S. acidocaldarius, it was shown that spreading resulted partly from a selective advantage of intron+ cells and partly from intercellular mobility of the intron and homing.

Dominance of one bacterial phylotype at a Mid-Atlantic Ridge hydrothermal vent site.

Microbial community structure in natural environments has remained largely unexplored yet is generally considered to be complex. It is shown here that in a Mid-Atlantic Ridge hydrothermal vent habitat, where food webs depend on prokaryotic primary production, the surface microbial community consists largely of only one bacterial phylogenetic type (phylotype) as indicated by the dominance of a single 16S rRNA sequence. The main part of its population occurs as an ectosymbiont on the dominant animals, the shrimp Rimicaris exoculata, where it grows as a monoculture within the carapace and on the extremities. However, the same bacteria are also the major microbial component of the free-living substrate community. Phylogenetically, this type forms a distinct branch within the epsilon-Proteobacteria. This is different from all previously studied chemoautotrophic endo- and ectosymbioses from hydrothermal vents and other sulfidic habitats in which all the bacterial members cluster within the gamma-Proteobacteria.

Impacto da diversidade bacteriana sob a degradação clorotalonil no solo manejado com biochar

A diversidade microbiana é geralmente considerada por seu papel nos principais processos do ecossistema, tais como a decomposição da matéria orgânica e ciclos biogeoquímicos. No entanto, informações sobre o impacto da diversidade em funções menores, como degradação de xenobióticos são escassas. Nós estudamos a partir da abordagem da \'diluição para extinção\', o papel da diversidade sobre a capacidade da comunidade microbiana em degradar o fungicida clorotalonil (organoclorado). Também estudamos o comportamento da comunidade bacteriana após aplicação do pesticida no solo com e sem biochar. A diversidade microbiana do solo natural foi alterada artificialmente por diluição, constituindo um gradiente de diversidade (SN > 10-1 > 10-3 > 10-6), seguido pela inoculação em amostras de solo estéril e posterior reestruturação (15 dias). Após a reestruturação da comunidade, as amostras foram manejadas com biochar (1% m/m) e tratadas com a dose de campo do CHT. O comportamento da comunidade bacteriana foi estudo por PCR-DGGE e qPCR do gene 16S rDNA através de um experimento com molécula fria (não radiomarcada). Enquanto a capacidade de degradação do CHT foi estudada por radiorespirometria (14C-CHT). Inicialmente, a comunidade de bactérias foi influenciada pelo gradiente de diversidade obtido por diluição. A separação dos grupos bacterianos se mostrou bastante similar nos três primeiros períodos pré-aplicação do CHT (SN > 10-1 - 10-3 > 10-6), enquanto que no período de 15 dias, a dinâmica de grupos foi alterada (SN > 10-1 > 10-3 - 10-6). O fungicida e o biochar não exerceram efeitos na comunidade bacteriana no tempo zero (imediatamente após a aplicação), a modificação no perfil da comunidade foi atribuído à diluição. Nos períodos de 21 e 42 dias, o perfil comunidade bacteriana apresentou forte modificação. Os grupos bacterianos se mostraram mais dispersos quando considerado somente o CHT. Embora, a análise de ANOSIM indicou não haver diferença nas amostras com e sem biochar, sugerindo que o clorotalonil foi quem mais contribuiu na dispersão dos grupos bacterianos. No período de 42 d, a comunidade apresentou resposta positiva, sendo observado aumentos no número de bandas e no índice de Shannon em todos tratamentos. Isto possivelmente, devido a menor concentração do fungicida disponível na solução do solo, diminuindo assim, os efeitos deletérios sobre a comunidade. Os dados de qPCR não apresentaram alteração no número de copias do gene 16S rDNA em todos os tratamentos. A remoção da diversidade impactou fortemente a capacidade da comunidade bacteriana de degradar o clorotalonil. Apesar da capacidade de degradar não ter sido perdida, a mínima alteração na diversidade promoveu elevada redução na taxa de mineralização do CHT. A dissipação do CHT se mostrou rápida (D50 < 1 dia) em todos os tratamentos, além disso, a formação de 14C-resíduos não extraíveis foi constituiu um dos principais mecanismos de dissipação do CHT. A partir da degradação do fungicida, foram detectados três metabólitos. Conclui-se que a modificação por diluição da diversidade bacteriana promoveu impacto negativo na mineralização do clorotalonil. E que a formação de resíduos não extraíveis consistiu no principal mecanismo de dissipação do CHT em ambos solos.

Table 2: Bacterial diversity in Ny-Ålesund, Spitzbergen

We describe the antibiotic resistance profiling of bacterial isolates collected from Ny-Alesund, Arctic, as part of the Indian Arctic Summer Expedition 2009. It was interesting to note that the bacterial isolates collected from the Arctic showed multidrug resistance. 32% of the isolates were found to be multi- drug resistant with several combinations of antibiotics. The 16S rRNA sequencing results shows a diverse group of bacteria belonging to Phyla Proteobacteria, Actinobacteria and Bacteriodetes and their relatedness was studied by phylogenetic analysis. While analysing the plasmid profiling, the most resistant two strains of Pseudomonas migulae showed multiple plasmids of varying sizes ~5.2-5.3 kb and ~9.5 kb. The extent and frequency of multidrug resistance in the polar bacteria deserves close monitoring and efforts to understand the various molecular mechanisms of drug resistance and control the spread of antibiotic resistance in polar environment is called for.

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation

The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNAand differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.

Australian microhylid frogs (Cophixalus and Austrochaperina): phylogeny, taxonomy, calls, distributions and breeding biology

Despite a considerable surge in herpetological research in Australia over the last couple of decades the Australian microhylid frogs (Cophixalus and Austrochaperina) remain relatively poorly known. Herein I present the results of extensive fieldwork and molecular, morphological and call analysis with the aim of resolving taxonomy, call variation and distributions, and increasing our understanding of breeding biology. Analysis of 943 base pairs of mitochondrial 16S rRNA and 12S rRNA provides a well supported phylogeny that is largely consistent with current taxonomy. Levels of divergence between species are substantial and significant phylogeographic structuring is evident in C. ornatus, C. neglectus and C. aenigma, sp. nov. The description of C. concinnus was based on a mixed collection of two species from Thornton Peak and a new species is described to resolve this. C. aenigma, sp. nov., is described from high-elevation (>750 m) rainforest across the Carbine, Thornton, Finnigan and Bakers Blue Mountain uplands, north-east Queensland. C. concinnus is redescribed as a highly distinct species restricted to rainforest and boulder fields at the summit of Thornton Peak (>1100 m). Despite protection in Daintree National Park in the Wet Tropics World Heritage Area, predictions of the impact of global warming suggest C. concinnus to be of very high conservation concern ( Critically Endangered, IUCN criteria). The mating call of two species ( C. mcdonaldi and C. exiguus) is described for the first time and high levels of call variation within C. ornatus, C. neglectus, C. hosmeri, C. aenigma and Austrochaperina fryi are presented. Such variation is often attributable to genetically divergent lineages, altitudinal variation and courtship; however, in some instances ( particularly within C. hosmeri) the source or function of highly distinct calls at a site remains obscure. Molecular, morphological and call analyses allow the clarification of species distributions, especially in the northern mountains of the Wet Tropics. Notes are presented on the breeding biology of C. aenigma, C. bombiens, C. concinnus, C. exiguus, C. infacetus, C. mcdonaldi, C. monticola, C. neglectus, C. ornatus and C. saxatilis, which are largely consistent with previous accounts: small terrestrial clutches usually attended by a male. Courtship behaviour in C. ornatus is described and the first records of multiple clutching in Australian microhylids are presented (for C. ornatus and C. infacetus).

The influence of substrate kinetics on the microbial community structure in granular anaerobic biomass

The development of a strong, active granular sludge bed is necessary for optimal operation of upflow anaerobic sludge blanket reactors. The microbial and mechanical structure of the granules may have a strong influence on desirable properties such as growth rate, settling velocity and shear strength. Theories have been proposed for granule microbial structure based on the relative kinetics of substrate degradation, but contradict some observations from both modelling and microscopic studies. In this paper, the structures of four granule types were examined from full-scale UASB reactors, treating wastewater from a cannery, a slaughterhouse, and two breweries. Microbial structure was determined using fluorescence in situ hybridisation probing with 16S rRNA-directed oligonucleotide probes, and superficial structure and microbial density (volume occupied by cells and microbial debris) assessed using scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The granules were also modelled using a distributed parameter biofilm model, with a previously published biochemical model structure, biofilm modelling approach, and model parameters. The model results reflected the trophic structures observed, indicating that the structures were possibly determined by kinetics. Of particular interest were results from simulations of the protein grown granules, which were predicted to have slow growth rates, low microbial density, and no trophic layers, the last two of which were reflected by microscopic observations. The primary cause of this structure, as assessed by modelling, was the particulate nature of the wastewater, and the slow rate of particulate hydrolysis, rather than the presence of proteins in the wastewater. Because solids hydrolysis was rate limiting, soluble substrate concentrations were very low (below Monod half saturation concentration), which caused low growth rates. (C) 2003 Elsevier Ltd. All rights reserved.

Dynamics of a temperature-related coral disease outbreak

During the austral summer of 2001/2002, a coral epizootic occurred almost simultaneously with a bleaching event on the fringing reefs of Magnetic Island (Great Barrier Reef region), Australia. This resulted in a 3- to 4-fold increase in the mean percentage of partial mortality rate in a population of the hard coral Montipora aequituberculata. The putative disease state, ‘atramentous necrosis’, was observed on both bleached and normally-pigmented M. aequituberculata, and presented blackened lesions that spread within days across the colony surface and throughout the population. Diseased portions of the corals were only visible for 3 to 4 wk, with diseased tissues becoming covered in sediment and algae, which rapidly obscured evidence of the outbreak. Diseased colonies were again observed in the summer of 2002/2003 after being absent over the 2002 winter. Analysis of when diseased and bleached corals were first observed, and when and where the mortality occurred on individual colonies, indicated virtually all the mortality over the summer could be attributed to the disease and not to the bleaching. Fluorescence in situ hybridisation (FISH) techniques and cloning, and analysis of the 16S rRNA genes from diseased coral tissue, identified a mixed microbial assemblage in the diseased tissues particularly within the Alphaproteobacteria, Firmicutes and Bacteroidetes. While it is not possible in this study to distinguish between a disease-causing microbial community versus secondary invaders, the bacterial 16S rDNA sequences identified within the blackened lesions demonstrated high similarity to sequences from black band disease and white plague infected corals, suggesting either common aetiological agents or development of a bacterial community that is specific to degrading coral tissues. Temperature-induced coral disease outbreaks, with the potential for elevated levels of mortality, may represent an added problem for corals during the warmer summer months and an added dimension to predicted increases in water temperature from climate change.

Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches

Diverse ketosynthase (KS) genes were retrieved from the microbial community associated with the Great Barrier Reef sponge Pseudoceratina clavata. Bacterial isolation and metagenomic approaches were employed. Phylogenetic analysis of 16S rRNA of culturable sponge-associated bacterial communities comprised eight groups over four phyla. Ten KS domains were amplified from four genera of isolates and phylogenetics demonstrated that these KS domains were located in three clusters (actinobacterial, cyanobacterial and trans-AT type). Metagenomic DNA of the sponge microbial community was extracted to explore community KS genes by two approaches: direct amplification of KS domains and construction of fosmid libraries for KS domain screening. Five KS domains were retrieved from polymerase chain reaction (PCR) amplification using sponge metagenome DNA as template and five fosmid clones containing KS domains found using multiplex PCR screening. Analysis of selected polyketide synthase (PKS) from one fosmid showed that the PKS consists of two modules. Open reading frames located up- and downstream of the PKS displayed similarity with membrane synthesis-related proteins such as cardiolipin synthase. Metagenome approaches did not detect KS domains found in sponge isolates. All KS domains from both metagenome approaches formed a single cluster with KS domains originating from metagenomes derived from other sponge species from other geographical regions.

Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus)

The ornate tropical rock lobster, Panulirus ornatus has substantial potential as an aquaculture species though disease outbreaks during the animal's extended larval lifecycle are major constraints for success. In order to effectively address such disease-related issues, an improved understanding of the composition and dynamics of the microbial communities in the larval rearing tanks is required. This study used flow cytometry and molecular microbial techniques (clone libraries and denaturing gradient gel electrophoresis (DGGE)) to quantify and characterise the microbial community of the water column in the early stages (developmental stage I-II) of a P. ornatus larval rearing system. DGGE analysis of a 5000 L larval rearing trial demonstrated a dynamic microbial community with distinct changes in the community structure after initial stocking (day I to day 2) and from day 4 to day 5, after which the structure was relatively stable. Flow cytometry analysis of water samples taken over the duration of the trial demonstrated a major increase in bacterial load leading up to and peaking on the first day of the initial larval moult (day 7), before markedly decreasing prior to when > 50% of larvae moulted (day 9). A clone library of a day 10 water sample taken following a mass larval mortality event reflected high microbial diversity confirmed by statistical analysis indices. Sequences retrieved from both clone library and DGGE analyses were dominated by gamma- and alpha-Proteobacteria affiliated organisms with additional sequences affiliated with beta- and epsilon-Proteobacteria, Bacteroidetes, Cytophagales and Chlamydiales groups. Vibrio affiliated species were commonly retrieved in the clone library, though absent from DGGE analysis.

Detection of bacterial DNA in cardiac vegetations by PCR after the completion of antimicrobial treatment for endorcarditis

PCR with broad-range primers for prokaryotic 16S rRNA genes was used to identify bacterial DNA in tissue from patients undergoing valve replacements following a previous episode of infective endocarditis (IF). Of eight valves investigated, bacterial DNA was detected in three from patients for whom IE had been treated by antibiotic therapy 5, 12 and 18 months previously. The demonstration of bacterial DNA within resected heart valves suggests either recurrence of infection, treatment failure or the persistence of bacterial debris within the cardiac vegetation. There may also be implications for routine use of PCR in the diagnosis of infection. © 2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases.

Evaluation of PCR in the molecular diagnosis of endocarditis

Objective. Infective endocarditis (IE) is diagnosed by the Duke criteria, which can be inconclusive particularly when blood cultures are negative. This study investigated the application of polymerase chain reaction (PCR) to identify bacterial DNA in excised valvular tissue, and its role in establishing the diagnosis of IE. Methods. Ninety-eight patients undergoing valve replacement surgery were studied. Twenty-eight patients were confirmed as definite for endocarditis by the Duke criteria; nine were considered as possible and 61 had no known or previous microbial infection of the endocardium. A broad-range PCR technique was used to amplify prokaryotic 16S rRNA genes present within homogenised heart valve tissue. Subsequent DNA sequencing of the PCR amplicon allowed identification of the infecting microorganism. Results. PCR results demonstrated the presence of bacterial DNA in the heart valves obtained from 14 out of 20 (70%) definite IE patients with positive blood cultures preoperatively. The causative microorganism for one patient with definite culture negative endocarditis was identified by PCR. Two out of nine (22%) of the valves from possible endocarditis patients also had bacterial DNA present converting them into the definite criteria whereas in the valves of seven out of nine (78%) of these patients no bacterial DNA was detected. Conclusion. The application of PCR to the explanted valves in patients with possible or confirmed diagnosis can augment the Duke criteria thereby improving post-surgical antimicrobial therapeutic options. © 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Diversity of Vancomycin Genes in Sub-tropical Soils

With the increased antibiotic exposure from anthropogenic sources, soil microbes are an ever-increasing ecological pool of resistant bacteria. This is the case with bacterial resistance to vancomycin through transfer of van-resistance genes by transposons. Studies show that bacterial species other than enteroccoci harbor genetic-like elements such as the Tn1546 transposon containing vancomycin-resistant genes. Overuse and misuse of antibiotics in hospital settings and agricultural practices have led to an increase in transferability of vancomycin-resistant genes among microbes. The objective of this project is to analyze the diversity of these genes found in the soil microbes from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine the degree of resistance. Results showed that all bacterial isolates were resistant to penicillin at the 10 µg concentration and most were susceptible to varying vancomycin concentrations (10 µg, 20 µg, and 30 µg). A 1465 bp fragment was amplified from the 16S rDNA gene using 27F and 1492R universal primers from the multi-antibiotic resistant bacteria and sequenced to identify the isolates. Three Gram-negative bacteria genera were identified with the closest phylogenetic match to: Pseudomonas sp., Stenotrophomonas sp., Xanthomonas sp., as well as two Gram-positive bacteria genera: Bacillus sp. and Brevibacillus sp. The isolates’ vanA and vanB genes were amplified using the respective primers. Ongoing work is underway to sequence and compare these known van resistant genes, with the goal of revealing intrinsic vancomycin resistance present in soil bacteria.

Preliminary identification of dehalogenation specificities exhibited by dehalorespiring bacteria from a marine microbial community

The ability of a previously PCB-enriched microbial culture from Venice Lagoon marine sediments to dechlorinate pentachlorophenol (PCP) and 2,3,5-trichlorophenol (2,3,5-TCP) was confirmed under anaerobic conditions in microcosms consisting of site water and sediment. Dechlorination activities against Aroclor 1254 PCB mixture were also confirmed as control. Pentachlorophenol was degraded to 2,4,6-TCP (75.92±0.85 mol%), 3,5-DCP (6.40±0.75 mol%), and phenol (15.40±0.87 mol%). From the distribution of the different dechlorination products accumulated in the PCP-spiked cultures over time, two dechlorination pathways for PCP were proposed: (i) PCP to 2,3,4,6-TeCP, then to 2,4,6-TCP through the removal of both meta double-flanked chlorine substituents (main pathway); (ii) alternately, PCP to 2,3,5,6-TeCP, 2,3,5-TCP, 3,5-DCP, then phenol, through the removal of the para double-flanked chlorine, followed by ortho single-flanked chlorines, and finally meta unflanked chlorines (minor pathway). Removal of meta double-flanked chlorines is thus preferred over all other substituents. 2,3,5-TCP, that completely lacks double-flanked chlorines, was degraded to 3,5-DCP through removal of the ortho single-flanked chlorine, with a 99.6% reduction in initial concentration of 2,3,5-TCP by week 14. 16S rRNA PCR-DGGE using Chloroflexi-specific primers revealed a different role of the two microorganisms VLD-1 and VLD-2, previously identified as dechlorinators in the Aroclor 1254 PCB-enriched community, in the dehalogenation of chlorophenols. VLD-1 was observed both in PCP- and TCP-dechlorinating communities, whereas VLD-2 only in TCP-dechlorinating communities. This indicates that VLD-1 and VLD-2 may both dechlorinate ortho single-flanked chlorines, but only VLD-1 is able to remove double-flanked meta or para chlorines.