Deciphering the Role of Multiple Thioredoxin Fold Proteins of Leptospirillum sp. in Oxidative Stress Tolerance.

Thioredoxin fold proteins (TFPs) form a family of diverse proteins involved in thiol/disulfide exchange in cells from all domains of life. Leptospirillum spp. are bioleaching bacteria naturally exposed to extreme conditions like acidic pH and high concentrations of metals that can contribute to the generation of reactive oxygen species (ROS) and consequently the induction of thiol oxidative damage. Bioinformatic studies have predicted 13 genes that encode for TFP proteins in Leptospirillum spp. We analyzed the participation of individual tfp genes from Leptospirillum sp. CF-1 in the response to oxidative conditions. Genomic context analysis predicted the involvement of these genes in the general thiol-reducing system, cofactor biosynthesis, carbon fixation, cytochrome c biogenesis, signal transduction, and pilus and fimbria assembly. All tfp genes identified were transcriptionally active, although they responded differentially to ferric sulfate and diamide stress. Some of these genes confer oxidative protection to a thioredoxin-deficient Escherichia coli strain by restoring the wild-type phenotype under oxidative stress conditions. These findings contribute to our understanding of the diversity and complexity of thiol/disulfide systems, and of adaptations that emerge in acidophilic microorganisms that allow them to thrive in highly oxidative environments. These findings also give new insights into the physiology of these microorganisms during industrial bioleaching operations.

A decade of metaproteomics: where we stand and what the future holds.

We are living through exciting times during which we are able to unravel the "microbial dark matter" in and around us through the application of high-resolution "meta-omics". Metaproteomics offers the ability to resolve the major catalytic units of microbial populations and thereby allows the establishment of genotype-phenotype linkages from in situ samples. A decade has passed since the term "metaproteomics" was first coined and corresponding analyses were carried out on mixed microbial communities. Since then metaproteomics has yielded many important insights into microbial ecosystem function in the various environmental settings where it has been applied. Although initial progress in analytical capacities and resulting numbers of proteins identified was extremely fast, this trend slowed rapidly. Here, we discuss several representative metaproteomic investigations of activated sludge, acid mine drainage biofilms, freshwater and seawater microbial communities, soil, and human gut microbiota. By using these case studies, we highlight current challenges and possible solutions for metaproteomics to realize its full potential, i.e. to enable conclusive links between microbial community composition, physiology, function, interactions, ecology, and evolution in situ.

Effects of Arsenite Resistance on the Growth and Functional Gene Expression of Leptospirillum ferriphilum and Acidithiobacillus thiooxidans in Pure Culture and Coculture.

The response of iron-oxidizing Leptospirillum ferriphilum YSK and sulfur-oxidizing Acidithiobacillus thiooxidans A01 to arsenite under pure culture and coculture was investigated based on biochemical characterization (concentration of iron ion and pH value) and related gene expression. L. ferriphilum YSK and At. thiooxidans A01 in pure culture could adapt up to 400 mM and 800 mM As(III) after domestication, respectively, although arsenite showed a negative effect on both strains. The coculture showed a stronger sulfur and ferrous ion oxidation activity when exposed to arsenite. In coculture, the pH value showed no significant difference when under 500 mM arsenite stress, and the cell number of At. thiooxidans was higher than that in pure culture benefiting from the interaction with L. ferriphilum. The expression profile showed that the arsenic efflux system in the coculture was more active than that in pure culture, indicating that there is a synergetic interaction between At. thiooxidans A01 and L. ferriphilum YSK. In addition, a model was proposed to illustrate the interaction between arsenite and the ars operon in L. ferriphilum YSK and At. thiooxidans A01. This study will facilitate the effective application of coculture in the bioleaching process by taking advantage of strain-strain communication and coordination.

Elevated temperature alters proteomic responses of individual organisms within a biofilm community.

Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. Here, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 °C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entire community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.

Systematics of leptospiraceae.

Leptospires are spirochetes that may be free-living saprophytes found in freshwater or may cause acute or chronic infection of animals. The family Leptospiraceae comprises three genera: Leptospira Leptospira Leptonema Leptonema, and Turneriella Turneriella. Within the genus Leptospira, three clades can be distinguished, of pathogens, nonpathogens, and an intermediate group. Leptospires are further divided into serovars; antigenically related serovars are clustered into serogroups for convenience.

Unifying vertical and nonvertical evolution: a stochastic ARG-based framework.

Evolutionary biologists have introduced numerous statistical approaches to explore nonvertical evolution, such as horizontal gene transfer, recombination, and genomic reassortment, through collections of Markov-dependent gene trees. These tree collections allow for inference of nonvertical evolution, but only indirectly, making findings difficult to interpret and models difficult to generalize. An alternative approach to explore nonvertical evolution relies on phylogenetic networks. These networks provide a framework to model nonvertical evolution but leave unanswered questions such as the statistical significance of specific nonvertical events. In this paper, we begin to correct the shortcomings of both approaches by introducing the "stochastic model for reassortment and transfer events" (SMARTIE) drawing upon ancestral recombination graphs (ARGs). ARGs are directed graphs that allow for formal probabilistic inference on vertical speciation events and nonvertical evolutionary events. We apply SMARTIE to phylogenetic data. Because of this, we can typically infer a single most probable ARG, avoiding coarse population dynamic summary statistics. In addition, a focus on phylogenetic data suggests novel probability distributions on ARGs. To make inference with our model, we develop a reversible jump Markov chain Monte Carlo sampler to approximate the posterior distribution of SMARTIE. Using the BEAST phylogenetic software as a foundation, the sampler employs a parallel computing approach that allows for inference on large-scale data sets. To demonstrate SMARTIE, we explore 2 separate phylogenetic applications, one involving pathogenic Leptospirochete and the other Saccharomyces.

Culturable and molecular phylogenetic diversity of microorganisms in an open-dumped, extremely acidic Pb/Zn mine tailings.

A combination of cultivation-based and molecular-based approaches was used to reveal the culturable and molecular diversity of the microbes inhabiting an open-dumped Pb/Zn mine tailings that was undergoing intensive acid generation (pH 1.9). Culturable bacteria found in the extremely acidic mine tailings were Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermotolerans and Acidiphilium cryptum, where the number of acidophilic heterotrophs was ten times higher than that of the iron- and sulfur-oxidizing bacteria. Cloning and phylogenetic analysis revealed that, in contrast to the adjacent AMD, the mine tailings possessed a low microbial diversity with archaeal sequence types dominating the 16S rRNA gene library. Of the 141 clones examined, 132 were represented by two sequence types phylogenetically affiliated with the iron-oxidizing archaea Ferroplasma acidiphilum and three belonged to two tentative groups within the Thermoplasma lineage so far represented by only a few environmental sequences. Six clones in the library were represented by the only bacterial sequence type and were closely related to the well-described iron-oxidizer L. ferriphilum. The significant differences in the prokaryotic community structures of the extremely acidic mine tailings and the AMD associated with it highlights the importance of studying the microbial communities that are more directly involved in the iron and sulfur cycles of mine tailings.

Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing.

The genus Leptospira is classified into 13 named species and 4 genomospecies based upon DNA-DNA reassociation studies. Phenotypic tests are unable to distinguish between species of Leptospira, and there is a need for a simplified molecular approach to the identification of leptospires. 16S rRNA gene sequences are potentially useful for species identification of Leptospira, but there are a large number of sequences of various lengths and quality in the public databases. 16S rRNA gene sequences of near full length and bidirectional high redundancy were determined for all type strains of the species of the Leptospiraceae. Three clades were identified within the genus Leptospira, composed of pathogenic species, nonpathogenic species, and another clade of undetermined pathogenicity with intermediate 16S rRNA gene sequence relatedness. All type strains could be identified by 16S rRNA gene sequences, but within both pathogenic and nonpathogenic clades as few as two or three base pairs separated some species. Sequences within the nonpathogenic clade were more similar, and in most cases < or =10 bp distinguished these species. These sequences provide a reference standard for identification of Leptospira species and confirm previously established relationships within the genus. 16S rRNA gene sequencing is a powerful method for identification in the clinical laboratory and offers a simplified approach to the identification of Leptospira species.

[Prevalence of the gene encoding the outer membrane lipoprotein LipL32 in leptospires of different taxons].

Primers flanking the fragment sized 677 bp have been constructed on the basis of nucleotide sequences of the gene encoding the outer membrane lipoprotein LipL32. PCR-analysis was used to reveal the prevalence of gene lipL32 among 73 Leptospiraceae family strains representing different genera and genomic species. The gene lipL32 appeared to be conservative across the pathogenic species. In contrast, it was not detected in the genome of nonpathogenic free-living leptospires. Thus the developed PCR test-system with primers LEP21/LEP22 may be efficiently used to differentiate these two distinct ecological groups of leptospires.

Reclassification of Leptospira parva Hovind-Hougen et al. 1982 as Turneriella parva gen. nov., comb. nov.

Analysis of the G+C content, DNA-DNA relatedness to other leptospires and 16S rRNA gene sequence of Leptospira parva showed that this species was not related to other Leptospira species. On the basis of these data, it is proposed that Leptospira parva should be transferred to the genus Turneriella as Turneriella parva gen. nov., comb. nov., with strain H(T) (=NCTC 11395(T)=ATCC BAA-1111(T)) as the type strain.

Iron meteorites can support the growth of acidophilic chemolithoautotrophic microorganisms.

Chemolithoautotrophy based on reduced inorganic minerals is considered a primitive energy transduction system. Evidence that a high number of meteorites crashed into the planet during the early period of Earth history led us to test the ability of iron-oxidizing bacteria to grow using iron meteorites as their source of energy. Here we report the growth of two acidophilic iron-oxidizing bacteria, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans, on a piece of the Toluca meteorite as the only source of energy. The alteration of the surface of the exposed piece of meteorite, the solubilization of its oxidized metal constituents, mainly ferric iron, and the formation of goethite precipitates all clearly indicate that iron-meteorite-based chemolithotrophic metabolism is viable.

Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies.

DNA relatedness was determined among 303 strains of Leptospira and Leptonema. Included in the analysis were reference strains from 228 well-characterized and recognized serovars. The study included 268 serovars from 29 named and one or more unnamed serogroups. The strains clustered into 17 DNA hybridization groups, representing 12 previously described species (292 strains) and five new genomospecies (11 strains). The largest groups included Leptospira interrogans (91 strains from 82 serovars), Leptospira santarosai (65 strains from 59 serovars), Leptospira borgpetersenii (49 strains from 43 serovars), Leptospira kirschneri (29 strains from 26 serovars) and Leptospira noguchii (20 strains from 20 serovars). The new genomospecies include Leptospira genomospecies 1 (two strains, serovars pinagchang and sichuan), Leptospira genomospecies 2 (six strains, serovars lushui, manhao 3, manzhuang, nanding, mengla and yunnan), Leptospira genomospecies 3 (one strain, serovar holland), Leptospira genomospecies 4 (one strain, serovar hualin) and Leptospira genomospecies 5 (one strain, serovar saopaulo). With the exception of Ballum, all serogroups with greater than one serovar studied were genetically heterogeneous. Phenotypic tests, including optimal growth temperature, lipase activity and growth inhibition by copper sulfate or 2,6-diaminopurine, were of little use in differentiating DNA relatedness groups. The name Leptospira alexanderi sp. nov. is proposed for Leptospira genomospecies 2 (type strain L 60T = ATCC 700520T, serovar manhao 3).

Real-time homogeneous assay of rapid cycle polymerase chain reaction product for identification of Leptonema illini.

Partial 16S rDNA sequences of eight Leptospira-like field isolates that reacted weakly or not at all to microscope agglutination test were found to be similar to the 16S rDNA sequence of the nonpathogen Leptonema illini-type strain 3055. Comparison of these sequences with those of Leptospira 16S rDNA sequences revealed a Leptonema species signature sequence for which a forward amplification primer was designed. This primer was used in conjunction with a bacterial-specific 16S rDNA universal reverse primer for developing a LightCycler-based rapid PCR protocol in which fluorescence emission due to the binding of SYBR green I dye to the amplified products was continuously monitored. A melting temperature (T(m)) determined from the melting curve of the amplified product immediately after PCR confirmed that the product was of Leptonema. The protocol for 24 samples consisting of 30 PCR cycles and melting curve acquisitions required 30 min to complete and agarose gel electrophoresis of the PCR products was not necessary. The method was specific as PCR products were detected from the seven Leptonema reference strains and the eight field isolates that had been previously verified as Leptonema by 16S rDNA sequencing, but not from the two representative strains from each of the eight Leptospira genospecies tested.

Rapid distinction between Leptonema and Leptospira by PCR amplification of 16S-23S ribosomal DNA spacer.

The PCR amplification of the genomic DNA of Leptonema illini strain 3055 using primers directed against conserved regions of the rRNA operon provided evidence that the 16S and 23S rRNA genes were linked via an intergenic spacer region. The sequencing of the intergenic spacer region indicated that it was 435 nucleotides in length and sequence similarity searches revealed that it bore no homology to any known sequences including tRNA available in databases. Further investigations using Southern blot hybridization revealed that there were two copies of these linked genes in the genome. However, similar PCR studies on a representative strain from each of the 23 serogroups of Leptospira interrogans, which are pathogenic, and eight strains from the 6 serogroups of Leptospira biflexa, which are non-pathogenic, revealed that the 16S and 23S rRNA genes were not linked.

A new serovar in the Grippotyphosa serogroup comprising leptospiral isolates from different regions.

Pulsed-field gel electrophoresis (PFGE) studies performed with leptospiral isolates led us to suspect the existence of a new serovar in the Grippotyphosa serogroup. The results obtained with reference serological techniques used in leptospiral identification, including cross-agglutination absorption and monoclonal antibody techniques, confirmed the existence of a new serovar exemplified by strain Dadas I. Four other isolates from different regions of the world were submitted for identification by PFGE and produced NotI restriction patterns similar to that of strain Dadas I. Our data demonstrate the power of PFGE for identifying leptospiral isolates. The name dadas is proposed for the new serovar.

The use of 16S rDNA sequence analysis to investigate the phylogeny of Leptospiraceae and related spirochaetes.

The 16S rDNA sequences from 15 Leptospiraceae were determined by automated PCR-directed cycle sequencing. Nucleotide comparisons, including those from published sequences for Leptospira canicola Moulton and Serpulina spp., were used to construct phylogenetic trees. Serpulina hyodysenteriae and S. innocens were related to each other but were distinct from the Leptospiraceae comprising Leptospira parva incertae sedis (Turneria parva H), Leptonema illini and Leptospira spp. The pathogenic and the saprophytic leptospires were distinct and separated from each other. Leptospira inadai occupied an intermediate position between the two forms. The pathogens formed three groups. Group I was represented by L. interrogans sensu stricto and L. kirschneri, Group II by L. weilii, L. borgpetersenii and L. santarosai, and Group III comprised L. noguchii and L. meyeri. The saprophytic species, L. wolbachii and L. biflexa sensu stricto shared about 99% sequence similarity. The freshwater isolates were distinct from the marine isolate L. biflexa sensu lato ancona Ancona Porto.

Characterization of Leptospiraceae by 16S DNA restriction fragment length polymorphisms.

Chromosomal DNA from 37 Leptospiracae representing genetic species, groups and reference strains together with five leptospire isolates and Escherichia coli were digested with the restriction endonucleases BamHI, ClaI and EcoRI. The Southern blots were hybridized with a biotinylated E. coli 1.5 kb 16S rDNA probe and gave 36 reproducible and unique patterns. With the exception of the type strain (Leptospira interrogans serovar icterohaemorrhagiae RGA) and neotype strain (serovar icterohaemorrhagiae Ictero no. 1) all the species and taxa examined could be differentiated from each other on the basis of their BamHI, ClaI and EcoRI restriction fragment length polymorphisms (RFLP). L. interrogans and L. borgpetersenii reference strains were heterogeneous, whereas Leptonema illini and L. parva incertae sedis were distinct and separate. Strains representing L. biflexa sensu lato presented divergent RFLP patterns. A porcine isolate was identified to be L. interrogans pomona Pomona.

Detection of Leptospiraceae by amplification of 16S ribosomal DNA.

The polymerase chain reaction (PCR) was developed to detect Leptospiraceae. Primers were used to amplify 1 631 base-pair (bp) 5'-region of 16S rDNA. Representative strains from the species, Leptospira interrogans sensu stricto, L. borgpetersenii, L. noguchii, L. santarosai, L. weilii, L. inadai, L. meyeri and the single member strain of Leptonema were amplified. In contrast, strains representing the saprophytic species. L. biflexa, L. wolbachii and L. parva were not amplified. There was no PCR product from 23 phylogenetically unrelated species of bacteria. As little as 10-1 pg of purified DNA and as few as 10-1 leptospires could be detected using the PCR analysis. Isolates of leptospires from clinical sources gave a positive PCR band, but those from surface waters did not.

Cloning, characterization and taxonomic significance of genes for the 5S ribosomal RNA of Leptonema illini strain 3055.

The genes encoding the 5S ribosomal RNA (rRNA) for Leptonema illini strain 3055 were isolated and sequenced. The 5S RNA molecule encoded was 117 nucleotides long. The genome of strain 3055 contained two genes for 5S rRNA that were located close together. The nucleotide sequences of the Leptonema illini genes exhibited less similarity to the rRNA gene of Leptospira interrogans strain Moulton and also to those of typical eubacterial genes than did the rRNA genes of other leptospires. However, the overall secondary structure of the 5S rRNA encoded exhibited a strong similarity to that of typical eubacterial 5S rRNA. Southern hybridization of the 5S rRNA gene probe with the genomic DNA of strain 965, which is currently classified as Leptospira biflexa, showed the latter to have close similarity to that of strain 3055. The physical map of strain 965 was quite similar to that of strain 3055 and was greatly different from that of any other strains of L. biflexa. In the organization of 5S rRNA genes, strain 965 is sufficiently different from other members of the genus Leptospira to be regarded as a member of the genus Leptonema.