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Type IV CRISPR-Cas are a distinct variety of highly derived CRISPR-Cas systems that appear to have evolved from type III systems through the loss of the target-cleaving nuclease and partial deterioration of the large subunit of the effector complex. All known type IV CRISPR-Cas systems are encoded on plasmids, integrative and conjugative elements (ICEs), or prophages, and are thought to contribute to competition between these elements, although the mechanistic details of their function remain unknown. There is a clear parallel between the compositions and likely origin of type IV and type I systems recruited by Tn7-like transposons and mediating RNA-guided transposition. We investigated the diversity and evolutionary relationships of type IV systems, with a focus on those in Acidithiobacillia, where this variety of CRISPR is particularly abundant and always found on ICEs. Our analysis revealed remarkable evolutionary plasticity of type IV CRISPR-Cas systems, with adaptation and ancillary genes originating from different ancestral CRISPR-Cas varieties, and extensive gene shuffling within the type IV loci. The adaptation module and the CRISPR array apparently were lost in the type IV ancestor but were subsequently recaptured by type IV systems on several independent occasions. We demonstrate a high level of heterogeneity among the repeats with type IV CRISPR arrays, which far exceed the heterogeneity of any other known CRISPR repeats and suggest a unique adaptation mechanism. The spacers in the type IV arrays, for which protospacers could be identified, match plasmid genes, in particular those encoding the conjugation apparatus components. Both the biochemical mechanism of type IV CRISPR-Cas function and their role in the competition among mobile genetic elements remain to be investigated.

Here, we report the draft sequence of Blautia luti strain DSM 14534T, originally isolated from human feces. This draft contains 74 contigs, comprising 3,718,760 bp with a G+C content of 42.87%. The annotated draft contains 3,338 coding sequences (CDSs) and 110 RNA genes.

Here, we report the draft sequence of Blautia luti strain DSM 14534 T , originally isolated from human feces. This draft contains 74 contigs, comprising 3,718,760 bp with a G+C content of 42.87%. The annotated draft contains 3,338 coding sequences (CDSs) and 110 RNA genes.

The family Acidiferrobacteraceae (order Acidiferrobacterales) currently contains Gram negative, neutrophilic sulfur oxidizers such as Sulfuricaulis and Sulfurifustis, as well as acidophilic iron and sulfur oxidizers belonging to the Acidiferrobacter genus. The diversity and taxonomy of the genus Acidiferrobacter has remained poorly explored. Although several metagenome and bioleaching studies have identified its presence worldwide, only two strains, namely Acidiferrobacter thiooxydans DSM 2932T, and Acidiferrobacter spp. SP3/III have been isolated and made publically available. Using 16S rRNA sequence data publically available for the Acidiferrobacteraceae, we herein shed light into the molecular taxonomy of this family. Results obtained support the presence of three clades Acidiferrobacter, Sulfuricaulis and Sulfurifustis. Genomic analyses of the genome sequences of A. thiooxydansT and Acidiferrobacter spp. SP3/III indicate that ANI relatedness between the SPIII/3 strain and A. thiooxydansT is below 95–96%, supporting the classification of strain SP3/III as a new species within this genus. In addition, approximately 70% of Acidiferrobacter sp. SPIII/3 predicted genes have a conserved ortholog in A. thiooxydans strains. A comparative analysis of iron, sulfur oxidation pathways, genome plasticity and cell-cell communication mechanisms of Acidiferrobacter spp. are also discussed.

Acidiphilium sp. DSM 27270 (Yenapatur) forms part of a microbial consortium isolated from copper mines in Chile, that is currently used in state of the art industrial-scale biotechnology. Its taxonomic assignment is still tentative and is metal resistance and homeostatic responses poorly characterized. Here we report the genomic taxonomy evaluation of the Yenapatur strain and the preliminary characterization of its metal resistance and homeostatic responses. The genome of Yenapatur was re-assembled, annotated and compared to other sequenced strains of the genus. Genomic signatures were derived to better define the taxonomy of the strain. The minimum inhibitory concentrations of diverse cations and anions (Cu (II), Fe (II), Mg (II), Mn (II), Zn (II), Al (III), As (III), Fe (III), chloride and nitrate) were determined. Known resistance determinants were profiled in the genome of Yenapatur and publically available sequenced Acidiphilium strains. Results are presented and discussed under the light of the operational conditions in which Yenapatur thrives.

Detailed descriptions of the consortia present in commercial mineral processing operations have emerged in recent years, improving our understanding of the biology and the ecology of bioleaching. In spite of this progress, one of the aspects of biomining microbial ecology that remains un-tackled is that of virus-host interactions. The effects of viruses on the dynamics of the bioleaching microbial consortia and their impact in metal recovery is presently unknown. To begin addressing this issue we asked a basic question: ¿Are there viruses in industrial bioleaching econiches? In this work, we answer that question experimentally, assessing the number and types of viral particles recovered in the leachates from different industrial settings, using epifluorescence and transmission electron microscopy. Findings emerging from this work point to an almost null presence of viral particles in the leachates from mineral processing operations, possibly due to structural stability issues of the particles in the extreme acidic and highly oxidant conditions favoured by their potential microbial hosts. In turn, DNA-loaded viral-size vesicles of presently unknown function are frequent and abundant in all samples analysed.

In this work we report the metagenome-derived draft genomic sequence of an enrichment culture dominated by A. ferrooxidans obtained from an airlift bioreactor inoculated with the microbial consortium recovered from the “Relave Viejo” tailing. The genome of this culture was assembled de-novo and by reference, generating a consensus assembly of 3.0 Mb. On the basis of 16S rRNA (100 % identity), average nucleotide identity analysis (99.33% identity) and in silico DNA-DNA hybridization against A. ferrooxidans ATCC 23270T (97.9%), the recovered genome is confirmed to pertain to A. ferrooxidans species. Comparative genomics results are presented to uncover the genetic traits of the variant surviving lime treatment and to further explore the genomic diversity of these model iron oxidizing species.

The family Acidiferrobacteraceae (order Acidiferrobacterales) currently contains three genera of chemolithoautotrophs: Sulfuricaulis (2016), Sulfurifustis (2015) and Acidiferrobacter (2011). While the two former are neutrophilic sulfur oxidizers isolated from lake sediments in Japan, the latter is an extremely acidophilic, moderately osmophilic, thermotolerant iron/sulfur oxidizer known to occur in macroscopic streamers in Rio Tinto, Spain and in acid waters worldwide. The type strains of both Sulfuricaulis limnicola (HA5T) and Sulfurifustis variabilis (skN76T) have been sequenced, and the draft genome of the ZJ isolate of Acidiferrobacter thiooxydans (MDCF01) has recently been deposited in public databases. Despite this fact, little evidence on the genomic diversity and evolution of this group has been presented so far. Using comparative genomic analyses and phylogenetic reconstruction strategies, we explored the evolutionary information contained in the available genome sequences to shed light on the taxonomic status of a novel isolate of the genus Acidiferrobacter (SP-III/3; DSM 27195).

Dispersal between genomes of certain mobile genetic elements and their gene cargo depends on conjugative type IV secretion systems. In this work, variants of these nanomachines, tra and trb, have been profiled in publicly available genomes of the genus Acidithiobacillus and in a set of relevant strains. Our analyses show that the trb system is of broad distribution, being present in most of the strains analyzed. In turn, the tra type is present in fewer strains of A. ferrooxidans, A. ferrivorans, A. ferriphilus and A. thiooxidans, and generally correlates with the presence of larger ICE in the respective genomes. Herein, sequence conservation, genomic context, integration site and synteny analyses are performed to infer functionality of the T4SS systems of the acidithiobacilli.

The genus Acidithiobacillus comprises several species of Gram-negative acidophilic bacteria that thrive in natural and man-made low pH environments in a variety of geo-climatic contexts. Beyond their fundamental interest as model extreme acidophiles, these bacteria are involved in the processing of minerals and the desulfurization of coal and natural gas, and are also sources of environmental pollution due to their generation of acid mine drainage and corrosion of cement and concrete structures. Acidithiobacillus spp. are therefore considered a biotechnologically relevant group of bacteria, and their identification and screening in natural and industrial environments is of great concern. Several molecular typing methodologies have been instrumental in improving knowledge of the inherent diversity of acidithiobacilli by providing information on the genetic subtypes sampled in public and private culture collections; more recently, they have provided specific insight into the diversity of acidithiobacilli present in industrial and natural environments. The aim of this review is to provide an overview of techniques used in molecular detection, identification and typing of Acidithiobacillus spp. These methods will be discussed in the context of their contribution to the general and specific understanding of the role of the acidithiobacilli in microbial ecology and industrial biotechnology. Emerging opportunities for industrial and environmental surveillance of acidithiobacilli using next-generation molecular typing methodologies are also reviewed.

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a woman’s reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive women’s health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 32 bacterial taxa of clinical importance, includingLactobacillus,Sneathia,Gardnerella, and 4 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, healthy ranges were determined in a group of 50 self-reported healthy women. The hrHPV portion of the test was evaluated against the Digene High-Risk HPV HC2 DNA test with vaginal samples obtained from 185 women. Results were concordant for 181/185 of the samples (overall agreement of 97.83%, Cohen’s kappa = 0.93), with sensitivity and specificity values of 94.74% and 98.64%, respectively. Two discrepancies were caused by the Digene assay’s known cross-reactivity with low-risk HPV types, while two additional samples were found to contain hrHPV not detected by Digene. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens.

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a woman´s reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive vaginal health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 31 bacterial taxa of clinical importance, including Lactobacillus, Sneathia, Gardnerella, and 3 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, reference ranges were determined in a group of 50 self-reported healthy women. The HPV sequencing portion of the test was evaluated against the digene High-Risk HPV HC2 DNA test. For hrHPV genotyping, agreement was 95.3% with a kappa of 0.804 (601 samples); after removal of samples in which the digene hrHPV probe showed cross-reactivity with lrHPV types, the sensitivity and specificity of the hrHPV genotyping assay were 94.5% and 96.6%, respectively, with a kappa of 0.841. For lrHPV genotyping, agreement was 93.9% with a kappa of 0.788 (148 samples), while sensitivity and specificity were 100% and 92.9%, respectively. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens. Fil: Bik, Elisabeth M.. Ubiome; Fil: Bird, Sara W.. Ubiome; Fil: Bustamante, Juan Pablo. Universidad Nacional de Entre Ríos. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática; Argentina Fil: Leon, Luis E.. Ubiome

The acidithiobacilli are sulfur-oxidizing acidophilic bacteria that thrive in both natural and anthropogenic low pH environments. They contribute to processes that lead to the generation of acid rock drainage in several different geoclimatic contexts, and their properties have long been harnessed for the biotechnological processing of minerals. Presently, the genus is composed of seven validated species, described between 1922 and 2015: Acidithiobacillus thiooxidans, A. ferrooxidans, A. albertensis, A. caldus, A. ferrivorans, A. ferridurans, and A. ferriphilus. However, a large number of Acidithiobacillus strains and sequence clones have been obtained from a variety of ecological niches over the years, and many isolates are thought to vary in phenotypic properties and cognate genetic traits. Moreover, many isolates remain unclassified and several conflicting specific assignments muddle the picture from an evolutionary standpoint. Here we revise the phylogenetic relationships within this species complex and determine the phylogenetic species boundaries using three different typing approaches with varying degrees of resolution: 16S rRNA gene-based ribotyping, oligotyping, and multi-locus sequencing analysis (MLSA). To this end, the 580 16S rRNA gene sequences affiliated to the Acidithiobacillus spp. were collected from public and private databases and subjected to a comprehensive phylogenetic analysis. Oligotyping was used to profile high-entropy nucleotide positions and resolve meaningful differences between closely related strains at the 16S rRNA gene level. Due to its greater discriminatory power, MLSA was used as a proxy for genome-wide divergence in a smaller but representative set of strains. Results obtained indicate that there is still considerable unexplored diversity within this genus. At least six new lineages or phylotypes, supported by the different methods used herein, are evident within the Acidithiobacillus species complex. Although the diagnostic characteristics of these subgroups of strains are as yet unresolved, correlations to specific metadata hint to the mechanisms behind econiche-driven divergence of some of the species/phylotypes identified. The emerging phylogenetic structure for the genus outlined in this study can be used to guide isolate selection for future population genomics and evolutionary studies in this important acidophile model.

Indexación: Web of Science; PubMed Leptospirillum ferriphilum Sp-Cl is a Gram negative, thermotolerant, curved, rod- shaped bacterium, isolated from an industrial bioleaching operation in northern Chile, where chalcocite is the major copper mineral and copper hydroxychloride atacamite is present in variable proportions in the ore. This strain has unique features as compared to the other members of the species, namely resistance to elevated concentrations of chloride, sulfate and metals. Basic microbiological features and genomic properties of this biotechnologically relevant strain are described in this work. The 2,475,669 bp draft genome is arranged into 74 scaffolds of 74 contigs. A total of 48 RNA genes and 2,834 protein coding genes were predicted from its annotation; 55 % of these were assigned a putative function. Release of the genome sequence of this strain will provide further understanding of the mechanisms used by acidophilic bacteria to endure high osmotic stress and high chloride levels and of the role of chloride-tolerant iron-oxidizers in industrial bioleaching operations. https://standardsingenomics.biomedcentral.com/articles/10.1186/s40793-016-0142-1

Bioleaching acidophiles inhabit environments with unusually high concentrations of iron that can potentially cause oxidative stress via the Fenton reaction in which dangerous reactive oxygen species (ROS) are generated. ROS can cause damage to proteins, nucleic acids, lipids and other macromolecules and thus have deleterious effects on cell growth and survival. Many of these microorganisms are chemolithotrophs with unusually high oxygen consumption rates that may exacerbate the problem of oxidative stress. Although some knowledge has been gained in recent years regarding the oxidative stress response in a few acidophiles, the general strategies used by them to face ROS challenges are still inadequately understood. Comparative genomics and multiple bioinformatic tools were used to explore 44 sequenced genomes of acidophilic bacteria and archaea in order to reconstruct their individual oxidative stress responses and to identify conserved strategies. The analyses revealed that acidophiles lack genes encoding typical oxidative stress response regulators and have an underrepresentation of classical ROS consumption enzymes (e.g. catalases) although they have a complete repertoire of repair systems for macromolecules (DNA, proteins and lipids). This suggests that stress mitigation is an active strategy in acidophiles confronting unavoidable ROS formation in their environment. Insights into the oxidative stress response in bioleaching acidophiles may contribute to a better understanding of the aspects that influence fitness of the microbial consortium driving bioleaching.

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation.

Previous physiological studies of the genus Ferroplasma have indicated that these microorganisms are capable of fixing CO2 in the presence of ferrous iron and low concentrations of yeast extract. Analysis of the gene complement of Ferroplasma acidarmanus fer1 and two partial genomes of Ferroplasma type I and II derived from the Iron Mountain acid mine drainage metagenome revealed the absence of several functional marker genes encoding key enzymes of three know alternative CO2 fixation routes present in archaea, i.e. the 3-hydroxypropionate cycle, the Ljungdahl–Wood pathway and the reverse TCA cycle. It is thus intriguing how these chemoautotrophic archaeal species deal with their requirements for carbon and suggests that they might have a distinct CO2 fixation route, as yet unreported. Using comparative genomics and metabolic reconstruction strategies, a putative pathway was detected for C1 fixation consisting of four main steps: 1) conversion of carbon monoxide to carbon dioxide with gain of energy and/or 2) reduction of carbon dioxide to formate, 3) incorporation of formate to tetrahydrofolate and 4) donation of the carbon moiety of tetrahydrofolate to glycine to produce serine. Steps 1 to 3 involve enzymes that correspond to some of the Ljungdahl–Wood pathway proteins, whereas step 4 resembles the well known “serine cycle”, utilized by methylotrophic microorganisms for formaldehyde fixation. Thus, this chimaeric pathway might represent the missing carbon fixation route in Ferroplasmatales. Herein, we discuss the implications of these findings in the context of central carbon metabolism requirements for biomass production in acidic environments.

“ Ferrovum myxofaciens ” is an iron-oxidizing betaproteobacterium with widespread distribution in acidic low-temperature environments, such as acid mine drainage streams. Here, we describe the genomic features of this novel acidophile and investigate the relevant metabolic pathways that enable its survival in these environments.

Background: Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome. Principal Findings: Comparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions. Significance: For many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche. © 2013 Acuña et al.

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation.

Background Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data. Results Here, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes map, rpoC, alaS and era results in improved interpretation of gene expression profiles in A. ferrooxidans. Conclusion This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems.

Development of reproducible genetic tools in the industrially important acidithiobacilli is urgently required. Inducible temperate phages which may be modified in vitro , propagated in suitable hosts, and used to transduce relevant genetic information to other strains and/or species are potentially valuable tools in this field of research. In order to address these current limitations, the genome sequence of an inducible temperate Myoviridae -like bacteriophage from the Acidithiobacillus caldus type strain was annotated and analyzed bioinformatically. Here, we announce the genome sequence of Aca ML1 and report major findings from its annotation.