Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu

Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique Il-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria, It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions.

Investigation of candidate division TM7, a recently recognized major lineage of the domain bacteria with no known pure-culture representatives

A molecular approach was used to investigate a recently described candidate division of the domain Bacteria, TM7, currently known only from environmental 16S ribosomal DNA sequence data, A number of TM7-specific primers and probes were designed and evaluated. Fluorescence in situ hybridization (FISH) of a laboratory scale bioreactor using two independent TM7-specific probes revealed a conspicuous sheathed-filament morphotype, fortuitously enriched in the reactor. Morphologically, the filament matched the description of the Eikelboom morphotype 0041-0675 widely associated with bulking problems in activated-sludge wastewater treatment systems. Transmission electron microscopy of the bioreactor sludge demonstrated that the sheathed-filament morphotype had a typical gram-positive cell envelope ultrastructure. Therefore, TM7 is only the third bacterial lineage recognized to have gram-positive representatives. TM7-specific FISH analysis of two full-scale wastewater treatment plant sludges, including the one used to seed the laboratory scale reactor, indicated the presence of a number of morphotypes, including sheathed filaments. TM7-specific PCR clone libraries prepared from the two full-scale sludges yielded 23 novel TM7 sequences. Three subdivisions could be defined based on these data and publicly available sequences. Environmental sequence data and TM7-specific FISH analysis indicate that members of the TM7 division are present in a variety of terrestrial, aquatic, and clinical habitats. A highly atypical base substitution (Escherichia coli position 912; C to U) for bacterial 16S rRNAs was present in almost all TM7 sequences, suggesting that TM7 bacteria, like Archaea, may be streptomycin resistant at the ribosome level.

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae, The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented, A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria, Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group, Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species, A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.

Isolation of Gemmata-like and Isosphaera-like planctomycete bacteria from soil and freshwater

New cultured strains of the planctomycete division (order Planctomycetales) of the domain Bacteria related to species in the genera Gemmata and Isosphaera were isolated from soil, freshwater, and a laboratory ampicillin solution. Phylogenetic analysis of the 16S rRNA gene from eight representative isolates showed that all the isolates were members of the planctomycete division. Six isolates clustered with Gemmata obscuriglobus and related strains, while two isolates clustered with Isosphaera pallida. A double-membrane-bounded nucleoid was observed in Gemmata-related isolates but not in Isosphaera-related isolates, consistent with the ultrastructures of existing species of each genus. Two isolates from this study represent the first planctomycetes successfully cultivated from soil.

16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the Green Non-Sulfur bacteria.

Microorganisms play an important role in the biogeochemistry of the ocean surface layer, but spatial and temporal structures in the distributions of specific bacterioplankton species are largely unexplored, with the exceptions of those organisms that can be detected by either autofluorescence or culture methods. The use of rRNA genes as genetic markers provides a tool by which patterns in the growth, distribution, and activity of abundant bacterioplankton species can be studied regardless of the ease with which they can be cultured. Here we report an unusual cluster of related 16S rRNA genes (SAR202, SAR263, SAR279, SAR287, SAR293, SAR307) cloned from seawater collected at 250 m in the Sargasso Sea in August 1991, when the water column was highly stratified and the deep chlorophyll maximum was located at a depth of 120 m. Phylogenetic analysis and an unusual 15-bp deletion confirmed that the genes were related to the Green Non-Sulfur phylum of the domain Bacteria. This is the first evidence that representatives of this phylum occur in the open ocean. Oligonucleotide probes were used to examine the distribution of the SAR202 gene cluster in vertical profiles (0-250 m) from the Atlantic and Pacific Oceans, and in discrete (monthly) time series (O and 200 m) (over 30 consecutive months in the Western Sargasso Sea. The data provide robust statistical support for the conclusion that the SAR202 gene cluster is proportionately most abundant at the lower boundary of the deep chlorophyll maximum (P = 2.33 x 10(-5)). These results suggest that previously unsuspected stratification of microbial populations may be a significant factor in the ecology of the ocean surface layer.

Degradation of detergent (linear alkylbenzene sulfonate) in an anaerobic stirred sequencing-batch reactor containing granular biomass

This study aimed to determine the efficiency of an anaerobic stirred sequencing-batch reactor containing granular biomass for the degradation of linear alkylbenzene sulfonate (LAS), a surfactant present in household detergent. The bioreactor was monitored for LAS concentrations in the influent, effluent and sludge, pH, chemical oxygen demand, bicarbonate alkalinity, total solids, and volatile solids. The degradation of LAS was found to be higher in the absence of co-substrates (53%) than in their presence (24-37%). Using the polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE), we identified populations of microorganisms from the Bacteria and Archaea domains. Among the bacteria, we identified uncultivated populations of Arcanobacterium spp. (94%) and Opitutus spp. (96%). Among the Archaea, we identified Methanospirillum spp. (90%), Methanosaeta spp. (98%), and Methanobacterium spp. (96%). The presence of methanogenic microorganisms shows that LAS did not inhibit anaerobic digestion. Sampling at the last stage of reactor operation recovered 61 clones belonging to the domain bacteria. These represented a variety of phyla: 34% shared significant homology with Bacteroidetes, 18% with Proteobacteria, 11% with Verrucomicrobia, 8% with Fibrobacteres, 2% with Acidobacteria, 3% with Chlorobi and Firmicutes, and 1% with Acidobacteres and Chloroflexi. A small fraction of the clones (13%) were not related to any phylum. Published by Elsevier Ltd.

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

Intracellular compartmentation in planctomycetes

The phylum Planctomycetes of the domain Bacteria consists of budding, peptidoglycan-less organisms important for understanding the origins of complex cell organization. Their significance for cell biology lies in their possession of intracellular membrane compartmentation. All planctomycetes share a unique cell plan, in which the cell cytoplasm is divided into compartments by one or more membranes, including a major cell compartment containing the nucleoid. Of special significance is Gemmata obscuriglobus, in which the nucleoid is enveloped in two membranes to form a nuclear body that is analogous to the structure of a eukaryotic nucleus. Planctomycete compartmentation may have functional physiological roles, as in the case of anaerobic ammonium-oxidizing anammox planctomycetes, in which the anammoxosome harbors specialized enzymes and is wrapped in an envelope possessing unique ladderane lipids. Organisms in phyla other than the phylum Planctomycetes may possess compartmentation similar to that of some planctomycetes, as in the case of members of the phylum Poribacteria from marine sponges.

Culturable bacterial symbionts isolated from two distinct sponge species (Pseudoceratina clavata and Rhabdastrella globostellata) from the Great Barrier Reef display similar phylogenetic diversity

The diversity of the culturable microbial communities was examined in two sponge species-Pseudoceratina clavata and Rhabdastrella globostellata. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis. The bacterial community structures represented in both sponges were found to be similar at the phylum level by the same four phyla in this study and also at a finer scale at the species level in both Firmicutes and Alphaproteobacteria. The majority of the Alphaproteobacteria isolates were most closely related to isolates from other sponge species including alpha proteobacterium NW001 sp. and alpha proteobacterium MBIC3368. Members of the low %G + C gram-positive (phylum Firmicutes), high %G + C gram-positive (phylum Actinobacteria), and Cytophaga-Flavobacterium-Bacteroides (phylum Bacteroidetes) phyla of domain Bacteria were also represented in both sponges. In terms of culturable organisms, taxonomic diversity of the microbial community in the two sponge species displays similar structure at phylum level. Within phyla, isolates often belonged to the same genus-level monophyletic group. Community structure and taxonomic composition in the two sponge species P. clavata and Rha. globostellata share significant features with those of other sponge species including those from widely separated geographical and climatic regions of the sea.

Metagenoma de comunidades microbianas expostas à radiação natural e a metais

Many species have specialized to live in the most varied existing environments showing the remarkable adaptability of the microbial world the most diverse physicochemical conditions. Environments exposed to natural radiation and metals are scarce around the world, presenting a microbiota still unknown. With a total number estimated between 4 and 6 x 1030 microrganisms on earth, they constitute an enormous biological and genetic pool to be explored. Metagenomic approach independent of cultivation, provides a new form to access to the potential genomic environmental samples becoming a powerful tool for the elucidation of ecological functions, metabolic profiles, as well as to identify new biomolecules. In this context, the genetic material of environmental soil and water samples from Açude Boqueirao Parelhas-RN, under the influence of natural radiation and the presence of metals, was extracted, pirosequencing and the generated sequences were analyzed by bioinformatics programs (MG-RAST and STAMP). Taxonomic comparative profiles of both samples showed high abundance of Domain Bacteria, followed by a small portion attributable to Eucaryota Domains, Archaea and Viruses. Proteobacteria, Actinobacteria and Bacterioidetes phyla showed the greater dominance in both samples. Important genera and species associated with resistance to various stressors found in region were observed. Sequences related to oxidative and heat stress, DNA replication and repair, and resistance to toxic compounds were observed, suggesting a significant relationship between the microbiota and their metabolic profile, influenced by regional environmental variables. The results of this study add valuable and unpublished data on the composition of microbial communities in these regions

Hydrochemical and microbiological properties of groundwater from pristine aquifer sampled at the Lower Rhine Embayment

The hydrochemistry and the microbial diversity of a pristine aquifer system near Garzweiler, Germany next to the open-pit lignite mine Garzweiler 1, were characterized. Hydrogeochemical and isotopic data indicate a recent activity of sulfate-reducing bacteria in the Tertiary marine sands. The community structure in the aquifer was studied by fluorescence in situ hybridization (FISH). Up to 7.3 x 10**5 cells/ml were detected by DAPIstaining. Bacteria (identified by the probe EUB338) were dominant, representing 51.9% of the total cell number (DAPI). Another 25.7% of total cell were affiliated with the domain Archaea as identified by the probe ARCH915. Within the domain Bacteria, the beta-Proteobacteria were most abundant (21.0% of total cell counts). Using genusspecific probes for sulfate-reducing bacteria (SRB), 2.5% of the total cells were identified as members of the genus Desulfotomaculum. This reflects the predominant role these microorganisms have been found to play in sulfatereducing zones of aquifers at other sites. Previously, all SRB cultured from this site were from the spore-forming genera Desulfotomaculum and Desulfosporosinus. Samples were taken after pumping for >= 40 min and after parameters such as temperature, pH, redox potential, oxygen and conductivity of the groundwater had remained stable for >= 15 min due to recharge of aquifer water. Hybridization and microscopy counts of hybridized and 4',6'-diamidino-2-phenylindole (DAPI)- stained cells were performed as described in Snaidr et al., (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Means were calculated from 10 to 20 randomly chosen fields on each filter section, corresponding to 800-1000 DAPI stained cells. Counting results were always corrected by subtracting signals observed with the probe NON338. Formamide concentrations and oligonucleotide probes used please see further details.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Endophytic bacteria in long-term in vitro cultivated axenic pineapple microplants revealed by PCR-DGGE

In vitro propagated plants are believed to be free of microbes. However, after 5 years of in vitro culture of pineapple plants, without evidence of microbial contamination, the use of culture-independent molecular approach [classifying heterogeneous nucleic acids amplified via universal and specific 16S rRNA gene by polymerase chain reaction (PCR)], and further analysis by denaturing gradient gel electrophoresis (DGGE) revealed endophytic bacteria in roots, young and mature leaves of such plants. The amplification of 16S rRNA gene (Bacteria domain) with the exclusion of the plant chloroplast DNA interference, confirmed the presence of bacterial DNA, from endophytic microorganisms within microplant tissues. PCR-DGGE analysis revealed clear differences on bacterial communities depending on plant organ. Group-specific DGGE analyses also indicated differences in the structures of Actinobacteria, Alphaproteobacteria and Betaproteobacteria communities in each part of plants. The results suggest the occurrence of a succession of bacterial communities colonizing actively the microplants organs. This study is the first report that brings together evidences that pineapple microplants, previously considered axenic, harbor an endophytic bacterial community encompassing members of Actinobacteria, Alphaproteobacteria and Betaproteobacteria group which is responsive to differences in organs due to plant development.