<p>Abstract</p> <p>Background</p> <p>Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from <it>Mycobacterium tuberculosis</it>, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms.</p> <p>Results</p> <p>We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from <it>Listeria monocytogenes </it>and <it>Leishmania major</it>, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXG<b>KD</b>R) are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides.</p> <p>Conclusion</p> <p>This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases and they have no homologues in humans. This study provides a foundation for examining the biological role of this new family of phosphatases and their potential as pharmaceutical targets against infectious diseases.</p>

Two-year cycle spruce beetle (Dendroctonus rufipennis [Kirby]) adults were collected from emergence traps installed on two types of hosts (stumps and windfalls) in two adjacent timber harvesting areas in central British Columbia over a 5-year period to determine the incidence of associated fungi, bacteria, yeasts, nematodes, and mites. There was no difference in either the mean size or the female ratio of spruce beetles between host types or areas. Yeasts and bacteria were the most common associates of the spruce beetle and were isolated from the majority of beetles, regardless of host type. Pesotum sp. A, a blue-stain fungus, was the most common filamentous species and was isolated from 63% of the 221 beetles sampled. This fungus appears to be closely associated with the spruce beetle regardless of host type. At least 25 taxa of other filamentous fungi (OFF) were also isolated from beetles. Most of the OFF were common, wind-dispersed species, prevalent in the environment, e.g., Penicillium and Cladosporium spp., and are likely only incidental associates of the spruce beetle. The OFF were more likely to be isolated from beetles emerging from windfalls than from stumps. This may have been due to higher moisture in windfalls, which promoted the growth of some OFF. The incidence of mites and nematodes associated with the spruce beetle was relatively low; however, only those observed on the exoskeleton were recorded. The association of microorganisms with the spruce beetle did not vary between the two timber harvesting areas sampled in this study. Despite differences in the two types of host material, the spruce beetle is able to maintain a consistent association with yeasts, bacteria, and Pesotum sp. A.

Bacteria, fungi and parasites are in constant contact with the insect gut environment and can influence different aspects of the host gut physiology. Usually, some of these microorganisms develop and survive in the digestive tract. Therefore, the gut environment must be able to tolerate certain populations of these organisms for the establishment of interactions between non-pathogenic bacteria, parasites and the gut. This review provides a brief overview of the biological and molecular mechanisms that microorganisms use to interact with the gut epithelia in mosquitoes and speculates on their significances for the development of bacteria and Trypanosoma cruzi in the guts of triatomines.

Amoebae-resistant microorganisms exhibit a specific lifestyle. Unlike allopatric specialized intracellular pathogens, they have not specialized because they infect the amoebae via amoebal attack and present a sympatric lifestyle with species from different phyla. In this review, we compare the genomes from bacteria (Legionella pneumophila, Legionella drancourtii, Candidatus'Protochlamydia amoebophila,'Rickettsia bellii, Candidatus'Amoebophilus asiaticus') and a virus (mimivirus) that multiply naturally in amoebae. The objective is to highlight the genomic traits characterizing these microorganisms and their niche by comparison with other specialized pathogens. The genome of intra-amoebal microorganisms is significantly larger than that of their relatives, contradicting the genome reduction theory mostly accepted for intracellular pathogens. This is probably due to the fact that they are not specialized and therefore maintain their genome size. Moreover, the presence of many horizontally transferred genes and mobilomes in their genomes suggests that these microorganisms acquired genetic material from their neighbors and amoebal host, thus increasing their genome size. Important features involved in gene transfer and pathogenicity were thus acquired. These characteristics suggest that amoebae constitute a gene melting pot, allowing diverse microorganisms to evolve by the same pathway characterized by gene acquisition, and then either adapt to the intra-amoebal lifestyle or create new pathogens.

A rapid and cost-effective technique for identification of microorganisms was explored using fluorescence microscopy and image analysis, and classification was done with trained neural network. The microorganisms used in this study are Bacillus thuringiensis (C399), Escherichia coli K12 (ATCC 10798), Lactobacillus brevis (LJH240), Listeria innocua (C366), and Staphylococcus epidermis (LJH343). After staining the microorganisms with fluorescent dyes [diamidino-2-phenyl-indole and acridine orange (AO)], images of the microorganisms were captured using a digital camera attached to a light microscope. Geometrical, optical, and textural features were extracted from the images using image analysis. Parameters extracted from images of microorganisms stained with AO gave better results for classification of the microorganisms. From these parameters, the best identification parameters that could classify the microorganisms with higher accuracy were selected using a probabilistic neural network (PNN). PNN was then used to classify the microorganisms with a 100% accuracy using nine identification parameters. These parameters are: 45° run length non-uniformity, width, shape factor, horizontal run length non-uniformity, mean gray level intensity, ten percentile values of the gray level histogram, 99 percentile values of the gray level histogram, sum entropy, and entropy. When the five microorganisms were mixed together then, also the PNN could classify the microorganisms with 100% accuracy using these nine parameters.

To verify the dominance of microorganisms in wastewater biological treatment, PCR-DGGE (denaturing gradient gel electrophoresis) was performed as a supplementary support method for screening of the dominant microorganisms from activated sludge. Results suggest that the dominant microorganisms in activated sludge are primarily responsible for strengthening its effectiveness as a biological treatment system, followed by the non-main dominant microorganisms, whereas the non-dominant microorganisms showed no effects. The degree of microbial abundance present on the profile of PCR-DGGE was in line with the treatment efficiency of augmented activated sludge with isolated cultures, suggesting that PCR-DGGE can be used as an effective supplementary method for verifying culturable dominant microorganisms in activated sludge of coking wastewater.

The effects of high concentrations of copper (Cu) and zinc (Zn) in a soil treated with biosolids previously spiked with these metals on poplar (Populus deltoides×yunnanensis) were investigated in a pot trial. The total soil metal concentrations in the treatments were 12, 46, 137, and 226mg Cu/kg and 25, 141, 433, and 686mg Zn/kg. Copper accumulation was lower in poplar leaves than Zn and the maximum bioconcentration factor was 0.8 for Cu and 10 for Zn. Copper was not found to be toxic to plants at any level of application or to mycorrhiza up to 137mg/kg, but it was found to be toxic to soil microorganisms at all levels of Cu addition. Copper application increased mycorrhiza colonisation up to 137mg Cu/kg and root dry matter at 226mg Cu/kg, but had no effect on leaf dry matter. Increasing Zn rate decreased all plant and soil parameters. Lower percentages of Cu in the soil exchangeable fraction, and a lower Cu²⁺ concentrations in soil solution relative to Zn indicated lower bioavailability of Cu. Dehydrogenase activity was reduced by 50% at total solution-phase Cu and Zn concentrations of 0.1 and 27mg/L, respectively, and solid-phase exchangeable Cu and Zn concentrations of 5 and 169mg/kg, respectively.

The surfaces of massive corals of the genus Favia from Eilat, Red Sea, and from Heron Island, Great Barrier Reef, are covered by a layer of eukaryotic microorganisms. These microorganisms are embedded in the coral mucus and tissue. In the Gulf of Eilat, the prevalence of corals covered by patches of eukaryotic microorganisms was positively correlated with a decrease in water temperatures (from 25-28 °C in the summer to 20-23 °C in winter). Comparisons carried out using transmission and scanning electron microscopy showed morphological similarities between the microorganisms from the two geographically distant reefs. The microorganisms found on and in the tissues were approximately 5-15 μm in diameter, surrounded by scales in their cell wall, contained a nucleus, and included unique auto-florescent coccoid bodies of approximately 1 μm. Such morphological characters suggested that these microorganisms are stramenopile protists and in particular thraustochytrids. Molecular analysis, carried out using specific primers for stramenopile 18S rRNA genes, revealed that 90% (111/123) of the clones in the gene libraries were from the Thraustochytriidae. The dominant genera in this family were Aplanochytrium sp., Thraustochytrium sp., and Labyrinthuloides sp. Ten stramenopile strains were isolated and cultured from the corals. Some strains showed ≥97% similarity to clones derived from libraries of mucus-associated microorganisms retrieved directly from these corals. Fatty acid characterization of one of the prevalent strains revealed a high percentage of polyunsaturated fatty acids, including omega-3. The possible association of these stramenopiles in the coral holobiont appeared to be a positive one.

The Petit-Saut ecosystem is a hydroelectric reservoir covering 365km² of flooded tropical forest. This reservoir and the Sinnamary Estuary downstream of the dam are subject to significant mercury methylation. The mercury methylation potential of plankton and biofilm microorganisms/components from different depths in the anoxic reservoir water column and from two different sites along the estuary was assessed. For this, reservoir water and samples of epiphytic biofilms from the trunk of a submerged tree in the anoxic water column and from submerged branches in the estuary were batch-incubated from 1h to 3months with a nominal 1000ng/L spike of Hg(II) chloride enriched in ¹⁹⁹Hg. Methylation rates were determined for different reservoir and estuarine communities under natural nutrient (reservoir water, estuary freshwater) and artificial nutrient (culture medium) conditions. Methylation rates in reservoir water incubations were the highest with plankton microorganisms sampled at −9.5m depth (0.5%/d) without addition of biofilm components. Mercury methylation rates of incubated biofilm components were strongly enhanced by nutrient addition. The results suggested that plankton microorganisms strongly contribute to the total Hg methylation in the Petit-Saut reservoir and in the Sinnamary Estuary. Moreover, specific methylation efficiencies (%Me¹⁹⁹Hgnet/cell) suggested that plankton microorganisms could be more efficient methylating actors than biofilm consortia and that their methylation efficiency may be reduced in the presence of biofilm components. Extrapolation to the reservoir scale of the experimentally determined preliminary methylation efficiencies suggested that plankton microorganisms in the anoxic water column could produce up to 27mol MeHg/year. Taking into account that (i) demethylation probably occurs in the reservoir and (ii) that the presence of biofilm components may limit the methylation efficiency of plankton microorganisms, this result is highly consistent with the annual net MeHg production estimated from mass balances (8.1mol MeHg/year, Muresan et al., 2008a).