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Scorpions represent an ancient lineage of arachnids that have radiated across the globe and are incredibly resilient-since some thrive in harsh environments and can exist on minimal and intermittent feedings. Given the emerging importance of microbiomes to an organism's health, it is intriguing to suggest that the long-term success of the scorpion bauplan may be linked to the microbiome. Little is known about scorpion microbiomes, and what is known, concentrates on the gut. The microbiome is not limited to the gut, rather it can be found within tissues, fluids and on external surfaces. We tested whether the scorpion telson, the venom-producing organ, of two species, Smeringurus mesaensis and Hadrurus arizonensis, contain bacteria. We isolated telson DNA from each species, amplified bacterial 16S rRNA genes, and identified the collection of bacteria present within each scorpion species. Our results show for the first time that telsons of non-buthid scorpion species do indeed contain bacteria. Interestingly, each scorpion species has a phylogenetically unique telson microbiome including Mollicutes symbionts. This study may change how we view scorpion biology and their venoms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Shimwell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The epigenetics of bacteria, and bacteria with a reduced genome in particular, is of great interest, but is still poorly understood. Mycoplasma gallisepticum, a representative of the class Mollicutes, is an excellent model of a minimal cell because of its reduced genome size, lack of a cell wall, and primitive cell organization. In this study we investigated DNA modifications of the model object Mycoplasma gallisepticum and their roles. We identified DNA modifications and methylation motifs in M. gallisepticum S6 at the genome level using single molecule real time (SMRT) sequencing. Only the ANCNNNNCCT methylation motif was found in the M. gallisepticum S6 genome. The studied bacteria have one functional system for DNA modifications, the Type I restriction-modification (RM) system, MgaS6I. We characterized its activity, affinity, protection and epigenetic functions. We demonstrated the protective effects of this RM system. A common epigenetic signal for bacteria is the m6A modification we found, which can cause changes in DNA-protein interactions and affect the cell phenotype. Native methylation sites are underrepresented in promoter regions and located only near the -35 box of the promoter, which does not have a significant effect on gene expression in mycoplasmas. To study the epigenetics effect of m6A for genome-reduced bacteria, we constructed a series of M. gallisepticum strains expressing EGFP under promoters with the methylation motifs in their different elements. We demonstrated that m6A modifications of the promoter located only in the -10-box affected gene expression and downregulated the expression of the corresponding gene., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Semashko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A Gram-stain-positive, non-spore-forming, rod-shaped, obligately anaerobic bacterium, designated strain BP52G T , was isolated from the hindgut of a Silver Drummer ( Kyphosus sydneyanus ) fish collected from the Hauraki Gulf, New Zealand. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate belonged to the family Erysipelotrichaceae in the phylum Firmicutes and was most closely related to Clostridium saccharogumia with 93.3 % sequence identity. Isolate BP52G T grew on agar medium containing mannitol as the sole carbon source. White, opaque and shiny colonies of the isolate measuring approximately 1 mm diameter grew within a week at 20-28 °C (optimum, 24 °C) and pH 6.9-8.5 (optimum, pH 7.8). BP52G T tolerated the addition of up to 1 % NaCl to the medium. Formate and acetate were the major fermentation products. The major cellular fatty acids were C 16 : 0 , C 16:1n-7t and C 18:1n-7t . The genome sequence of the isolate was determined. Its G+C content was 30.7 mol%, and the 72.65 % average nucleotide identity of the BP52G T genome to its closest neighbour with a completely sequenced genome ( Erysipelatoclostridium ramosum JCM 1298 T ) indicated low genomic relatedness. Based on the phenotypic and taxonomic characteristics observed in this study, a novel genus and species Tannockella kyphosi gen. nov., sp. nov. is proposed for isolate BP52G T (=NZRM 4757 T =JCM 34692 T ).

Plasmids are known to contain genes encoding for virulence factors and antibiotic resistance mechanisms. Their relevance in metagenomic data processing is steadily growing. However, with the increasing popularity and scale of metagenomics experiments, the number of reported plasmids is rapidly growing as well, amassing a considerable number of false positives due to undetected misassembles. Here, our previously published database PLSDB provides a reliable resource for researchers to quickly compare their sequences against selected and annotated previous findings. Within two years, the size of this resource has more than doubled from the initial 13,789 to now 34,513 entries over the course of eight regular data updates. For this update, we aggregated community feedback for major changes to the database featuring new analysis functionality as well as performance, quality, and accessibility improvements. New filtering steps, annotations, and preprocessing of existing records improve the quality of the provided data. Additionally, new features implemented in the web-server ease user interaction and allow for a deeper understanding of custom uploaded sequences, by visualizing similarity information. Lastly, an application programming interface was implemented along with a python library, to allow remote database queries in automated workflows. The latest release of PLSDB is freely accessible under https://www.ccb.uni-saarland.de/plsdb., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in Escherichia coli . DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique dus gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using in vitro activity tests as well as in vivo E. coli complementation assays with the enzyme from Mycoplasma capricolum (DusB MCap ), a model organism for the study of these parasitic bacteria, we show that, as expected for a DusB homolog, DusB MCap modifies U17 to D17 but also synthetizes D20/D20a combining therefore both E. coli DusA and DusB activities. Hence, this is the first case of a Dus enzyme able to modify up to three different sites as well as the first example of a tRNA-modifying enzyme that can modify bases present on the two opposite sides of an RNA-loop structure. Comparative analysis of the distribution of DusB homologs in Firmicutes revealed the existence of three DusB subgroups namely DusB1, DusB2 and DusB3. The first two subgroups were likely present in the Firmicute ancestor, and Mollicutes have retained DusB1 and lost DusB2. Altogether, our results suggest that the multisite specificity of the M. capricolum DusB enzyme could be an ancestral property.

Background: The gut microbiota is an emerging frontier in wildlife research and its importance to vertebrate health and physiology is becoming ever more apparent. Reptiles, in particular snakes, have not received the same attention given to other vertebrates and the composition of their wild gut microbiome remains understudied. The primary goal of this work was to describe the cloacal microbiota of two Colubrids, the Eastern Gartersnake (Thamnophis sirtalis sirtalis) and the Northern Watersnake (Nerodia sipedon sipedon), and if their cloacal microbiota differed as well as if it did between a wetland and upland population of the former species., Methods and Results: We utilized next-generation sequencing of cloacal swabs-a non-destructive proxy for the gut microbiota. The cloacal microbiome of Eastern Gartersnakes (N = 9) was like those of other snakes being comprised of Proteobacteria, Bacteroidetes, and Firmicutes, while that of Northern Watersnakes (N = 6) was dominated by Tenericutes. Seven microbial operational taxonomic units (OTUs), all members of Proteobacteria, were shared among all individuals and were indicative of a core microbiome in Eastern Gartersnakes, but these OTUs were not particularly relevant to Northern Watersnakes. The latter had greater OTU richness than did Eastern Gartersnakes, and habitat did not have any apparent effect on the microbial community composition in Eastern Gartersnakes., Conclusions: Our findings suggest host taxonomy to be a determining factor in the cloacal microbiota of snakes and that Tenericutes are associated with aquatic habitats. This is the first report to examine the cloacal microbiome of these species and provides a useful foundation for future work to build upon., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Traditional sampling methods for the study of poultry gut microbiota preclude longitudinal studies as they require euthanasia of birds for the collection of caecal and ileal contents. Some recent research has investigated alternative sampling methods to overcome this issue. The main goal of this study was to assess to what extent the microbial composition of non-invasive samples (excreta, litter and poultry dust) are representative of invasive samples (caecal and ileal contents). The microbiota of excreta, dust, litter, caecal and ileal contents (n = 110) was assessed using 16S ribosomal RNA gene amplicon sequencing. Of the operational taxonomic units (OTUs) detected in caecal contents, 99.7% were also detected in dust, 98.6% in litter and 100% in excreta. Of the OTUs detected in ileal contents, 99.8% were detected in dust, 99.3% in litter and 95.3% in excreta. Although the majority of the OTUs found in invasive samples were detected in non-invasive samples, the relative abundance of members of the microbial communities of these groups were different, as shown by beta diversity measures. Under the conditions of this study, correlation analysis showed that dust could be used as a proxy for ileal and caecal contents to detect the abundance of the phylum Firmicutes, and excreta as a proxy of caecal contents for the detection of Tenericutes. Similarly, litter could be used as a proxy for caecal contents to detect the abundance of Firmicutes and Tenericutes. However, none of the non-invasive samples could be used to infer the overall abundance of OTUs observed in invasive samples. In conclusion, non-invasive samples could be used to detect the presence and absence of the majority of the OTUs found in invasive samples, but could not accurately reflect the microbial community structure of invasive samples., Competing Interests: The authors declare no conflict of interest. Matthew Hilliar, who was a PhD candidate at University of New England at the time in which the study was carried out, is currently employed by Turosi. The funder provided support in the form of salaries for author MH during the write up of this study, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author are articulated in the ‘author contributions’ section. MH employment by Turosi after the current study was conducted does not alter our adherence to PLOS ONE policies on sharing data and materials.

CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Marine invertebrate microbiomes play important roles in diverse host and ecological processes. However, a mechanistic understanding of host-microbe interactions is currently available for a small number of model organisms. Here, an integrated taxonomic and functional analysis of the microbiome of the eastern oyster, Crassostrea virginica , was performed using 16S rRNA gene-based amplicon profiling, shotgun metagenomics, and genome-scale metabolic reconstruction. Relatively high variability of the microbiome was observed across individual oysters and among different tissue types. Specifically, a significantly higher alpha diversity was observed in the inner shell than in the gut, gill, mantle, and pallial fluid samples, and a distinct microbiome composition was revealed in the gut compared to other tissues examined in this study. Targeted metagenomic sequencing of the gut microbiota led to further characterization of a dominant bacterial taxon, the class Mollicutes , which was captured by the reconstruction of a metagenome-assembled genome (MAG). Genome-scale metabolic reconstruction of the oyster Mollicutes MAG revealed a reduced set of metabolic functions and a high reliance on the uptake of host-derived nutrients. A chitin degradation and an arginine deiminase pathway were unique to the MAG compared to closely related genomes of Mollicutes isolates, indicating distinct mechanisms of carbon and energy acquisition by the oyster-associated Mollicutes A systematic reanalysis of public eastern oyster-derived microbiome data revealed a high prevalence of the Mollicutes among adult oyster guts and a significantly lower relative abundance of the Mollicutes in oyster larvae and adult oyster biodeposits. IMPORTANCE Despite their biological and ecological significance, a mechanistic characterization of microbiome function is frequently missing from many nonmodel marine invertebrates. As an initial step toward filling this gap for the eastern oyster, Crassostrea virginica , this study provides an integrated taxonomic and functional analysis of the oyster microbiome using samples from a coastal salt pond in August 2017. The study identified high variability of the microbiome across tissue types and among individual oysters, with some dominant taxa showing higher relative abundance in specific tissues. A high prevalence of Mollicutes in the adult oyster gut was revealed by comparative analysis of the gut, biodeposit, and larva microbiomes. Phylogenomic analysis and metabolic reconstruction suggested the oyster-associated Mollicutes is closely related but functionally distinct from Mollicutes isolated from other marine invertebrates. To the best of our knowledge, this study represents the first metagenomics-derived functional inference of Mollicutes in the eastern oyster microbiome., (Copyright © 2021 Pimentel et al.)

Representatives of the Mollicutes class are the smallest, wall-less bacteria capable of independent reproduction. They are widespread in nature, most are commensals, and some are pathogens of humans, animals and plants. They are also the main contaminants of cell cultures and vaccine preparations. Despite limited biosynthetic capabilities, they are highly adaptable and capable of surviving under various stress and extreme conditions, including antimicrobial selective pressure. This review describes current understanding of antibiotic resistance (ABR) mechanisms in Mollicutes. Protective mechanisms in these bacteria include point mutations, which may include non-target genes, and unique gene exchange mechanisms, contributing to transfer of ABR genes. Better understanding of the mechanisms of emergence and dissemination of ABR in Mollicutes is crucial to control these hypermutable bacteria and prevent the occurrence of highly ABR strains., (Copyright © 2020 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

The study focuses on analysis of the compositional and diversity of bacteria in termite mound soils in comparison with the surrounding soils to verify the assertion that the high nutrient concentrations in termite mound soils influence a complex diversity of microorganisms. Here, whole DNA was extracted from soil samples collected from termite mounds and their surrounding soils which were 10 m apart and subsequently, sequenced using shotgun metagenomic approach. Our findings showed that both environments have several soil bacterial phyla in common. However, Proteobacteria and Actinobacteria significantly dominated the termite mound soils and the surrounding soils, respectively, with Tenericutes peculiar to only the termite mound soils. Furthermore, Bergeyella, Gloeothece, Thalassospira, and Glaciecola genera were exclusively identified in the termite mound soil samples. Diversity analysis showed that bacterial composition was different among the four sites (phyla level). This study also revealed a lot of unclassified groups of bacteria and this could point to the presence of potentially novel species. The differences observed in the bacterial structure and diversity from this study may be ascribed to variances in the physicochemical nature existing between the two environments. Mapping out schemes to culture these unclassified groups of bacteria discovered from this study would possibly set the platform for the discovery of novel bacteria for biotechnological applications.

Animals in captivity undergo a range of environmental changes from wild animals. An increasing number of studies show that captivity significantly affects the abundance and community structure of gut microbiota. The northern grass lizard (Takydromus septentrionalis) is an extensively studied lacertid lizard and has a distributional range covering the central and southeastern parts of China. Nonetheless, little is known about the gut microbiota of this species, which may play a certain role in nutrient and energy metabolism as well as immune homeostasis. Here, we examined the differences in the gut microbiota between two groups (wild and captive) of lizards through 16S rRNA sequencing using the Illumina HiSeq platform. The results demonstrated that the dominant microbial components in both groups consisted of Proteobacteria, Firmicutes, and Tenericutes. The two groups did not differ in the abundance of these three phyla. Citrobacter was the most dominant genus in wild lizards, while Morganella was the most dominant genus in captive lizards. Moreover, gene function predictions showed that genes at the KEGG pathway levels2 were more abundant in wild lizards than in captive lizards but, at the KEGG pathway levels1, the differences in gene abundances between wild and captive lizards were not significant. In summary, captivity exerted a significant impact on the gut microbial community structure and diversity in T. septentrionalis, and future work could usefully investigate the causes of these changes using a comparative approach., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The first comprehensive timetree is presented for phytoplasmas, a diverse group of obligate intracellular bacteria restricted to phloem sieve elements of vascular plants and tissues of their hemipteran insect vectors. Maximum likelihood-based phylogenetic analysis of DNA sequence data from the 16S rRNA and methionine aminopeptidase (map) genes yielded well resolved estimates of phylogenetic relationships among major phytoplasma lineages, 16Sr groups and known strains of phytoplasmas. Age estimates for divergences among two major lineages of Mollicutes based on a previous comprehensive bacterial timetree were used to calibrate an initial 16S timetree. A separate timetree was estimated based on the more rapidly-evolving map gene, with an internal calibration based on a recent divergence within two related 16Sr phytoplasma subgroups in group 16SrV thought to have been driven by the introduction of the North American leafhopper vector Scaphoideus titanus Ball into Europe during the early part of the 20th century. Combining the resulting divergence time estimates into a final 16S timetree suggests that evolutionary rates have remained relatively constant overall through the evolution of phytoplasmas and that the origin of this lineage, at ~641 million years ago (Ma), preceded the origin of land plants and hemipteran insects. Nevertheless, the crown group of phytoplasmas is estimated to have begun diversifying ~316 Ma, roughly coinciding with the origin of seed plants and Hemiptera. Some phytoplasma groups apparently associated with particular plant families or insect vector lineages generally arose more recently than their respective hosts and vectors, suggesting that vector-mediated host shifts have been an important mechanism in the evolutionary diversification of phytoplasmas. Further progress in understanding macroevolutionary patterns in phytoplasmas is hindered by large gaps in knowledge of the identity of competent vectors and lack of data on phytoplasma associations with non-economically important plants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The metabolic capacity, stress response and evolution of uncultured environmental Tenericutes have remained elusive, since previous studies have been largely focused on pathogenic species. In this study, we expanded analyses on Tenericutes lineages that inhabit various environments using a collection of 840 genomes., Results: Several environmental lineages were discovered inhabiting the human gut, ground water, bioreactors and hypersaline lake and spanning the Haloplasmatales and Mycoplasmatales orders. A phylogenomics analysis of Bacilli and Tenericutes genomes revealed that some uncultured Tenericutes are affiliated with novel clades in Bacilli, such as RF39, RFN20 and ML615. Erysipelotrichales and two major gut lineages, RF39 and RFN20, were found to be neighboring clades of Mycoplasmatales. We detected habitat-specific functional patterns between the pathogenic, gut and the environmental Tenericutes, where genes involved in carbohydrate storage, carbon fixation, mutation repair, environmental response and amino acid cleavage are overrepresented in the genomes of environmental lineages, perhaps as a result of environmental adaptation. We hypothesize that the two major gut lineages, namely RF39 and RFN20, are probably acetate and hydrogen producers. Furthermore, deteriorating capacity of bactoprenol synthesis for cell wall peptidoglycan precursors secretion is a potential adaptive strategy employed by these lineages in response to the gut environment., Conclusions: This study uncovers the characteristic functions of environmental Tenericutes and their relationships with Bacilli, which sheds new light onto the pathogenicity and evolutionary processes of Mycoplasmatales.

The C5-methylation of uracil to form 5-methyluracil (m 5 U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes , a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m 5 U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup use the ancestral tetrahydrofolate-dependent TrmFO enzyme to catalyze the formation of m 5 U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m 5 U1939 formation in 23S rRNA. RlmFO has replaced the S-adenosyl-L-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m 5 U-modifying enzymes that is highly dynamic and has undergone horizontal transfer.

The human gut microbiota is affected by genetic and environmental factors. It remains unclear how host genetic and environmental factors affect the composition and function of gut microbiota in populations living at high altitudes. We used a metagenome-wide analysis to investigate the gut microbiota composition in 15 native Tibetans and 12 Hans living on the Tibetan Plateau. The composition of gut microbiota differed significantly between these two groups ( P < 0.05). The Planctomycetes was the most abundant phyla both in native Tibetans and in Hans. Furthermore, the most relatively abundant phyla for native Tibetans were Bacteroidetes (15.66%), Firmicutes (11.10%), Proteobacteria (1.32%), Actinobacteria (1.10%), and Tenericutes (0.35%), while the most relatively abundant phyla for Hans were Bacteroidetes (16.28%), Firmicutes (8.41%), Proteobacteria (2.93%), Actinobacteria (0.49%), and Cyanobacteria (0.21%). The abundance of the majority of genera was significantly higher in Tibetans than in Hans ( P < 0.01). The number of microbial genes was 4.9 times higher in Tibetans than in Hans. The metabolic pathways and clusters of orthologous groups differed significantly between the two populations ( P < 0.05). The abundance of carbohydrate-active enzyme modules and antibiotic resistance genes was significantly lower in Tibetans compared to Hans ( P < 0.05). Our results suggest that different genetic factors (race) and environmental factors (diets and consumption of antibiotics) may play important roles in shaping the composition and function of gut microbiota in populations living at high altitudes., Competing Interests: All authors declare that they have no conflict of interest., (Copyright © 2020 Kang Li et al.)

Ureaplasma diversum is a member of the Mollicutes class responsible for urogenital tract infection in cattle and small ruminants. Studies indicate that the process of horizontal gene transfer, the exchange of genetic material among different species, has a crucial role in mollicute evolution, affecting the group's characteristic genomic reduction process and simplification of metabolic pathways. Using bioinformatics tools and the STRING database of known and predicted protein interactions, we constructed the protein-protein interaction network of U. diversum and compared it with the networks of other members of the Mollicutes class. We also investigated horizontal gene transfer events in subnetworks of interest involved in purine and pyrimidine metabolism and urease function, chosen because of their intrinsic importance for host colonization and virulence. We identified horizontal gene transfer events among Mollicutes and from Ureaplasma to Staphylococcus aureus and Corynebacterium , bacterial groups that colonize the urogenital niche. The overall tendency of genome reduction and simplification in the Mollicutes is echoed in their protein interaction networks, which tend to be more generalized and less selective. Our data suggest that the process was permitted (or enabled) by an increase in host dependence and the available gene repertoire in the urogenital tract shared via horizontal gene transfer.

Androctonus australis is one of the most ubiquitous and common scorpion species in desert and arid lands from North Africa to India and it has an important ecological role and social impact. The bacterial community associated to this arachnid is unknown and we aimed to dissect its species composition in the gut, gonads, and venom gland. A 16S rRNA gene culture-independent diversity analysis revealed, among six other taxonomic groups (Firmicutes, Betaproteobacteria, Gammaproteobacteria, Flavobacteria, Actinobacteria, and Cyanobacteria), a dominance of Mollicutes phylotypes recorded both in the digestive tract and the gonads. These related Mollicutes include two Spiroplasma phylotypes (12.5% of DGGE bands and 15% of clones), and a new Mycoplasma cluster (80% of clones) showing 16S rRNA sequence identities of 95 and 93% with Mollicutes detected in the Mexican scorpions Centruroides limpidus and Vaejovis smithi, respectively. Such scorpion-associated Mollicutes form a new lineage that share a distant ancestor with Mycoplasma hominis. The observed host specificity with the apparent phylogenetic divergence suggests a relatively long co-evolution of these symbionts with the scorpion hosts. From the ecological point of view, such association may play a beneficial role for the host fitness, especially during dormancy or molt periods., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The genus Mycoplasma, including species earlier classified in the genera Eperythrozoon and Haemobartonella, contains ~ 120 species and constitutes an extensively polyphyletic assemblage of bacteria within the phylum Tenericutes. Due to their small genome sizes and lack of unique characteristics, the relationships among the mycoplasmas/Tenericutes are not reliably discerned. Using genome sequences for 140 Tenericutes, their evolutionary relationships were examined using multiple independent approaches. Phylogenomic trees were constructed for 63 conserved proteins, 45 ribosomal proteins, three main subunits of RNA polymerase and 16S rRNA gene sequences. In all of these trees, Tenericutes species reliably grouped into four main clades designated as the "Acholeplasma", "Spiroplasma", "Pneumoniae" and "Hominis" clusters. These clades are also distinguished based on a similarity matrix constructed based on 16S rRNA gene sequences. Mycoplasma species were dispersed across 3 of these 4 clades highlighting their extensive polyphyly. In parallel, our comparative genomic analyses have identified > 100 conserved signature indels (CSIs) and 14 conserved signature proteins (CSPs), which are uniquely shared by the members of four identified clades, strongly supporting their monophyly and identifying them in molecular terms. Mycoplasma mycoides, the type species of the genus Mycoplasma, and a small number of other Mycoplasma species, formed a strongly supported clade within the "Spiroplasma" cluster. Nine CSIs and 14 CSPs reliably distinguish this clade from all other Mycoplasmatales species. The remainder of the Mycoplasmatales species are part of the "Pneumoniae" and "Hominis" clusters, which group together in phylogenetic trees. Here we are proposing that the order Mycoplasmatales should be emended to encompass only the Mycoplasma species within the "Spiroplasma" cluster and that a new order, Mycoplasmoidales ord. nov., should be created to encompass the other Mycoplasma species. The "Pneumoniae" and the "Hominis" clusters are proposed as two new families, Mycoplasmoidaceae fam. nov., which includes the genera Eperythrozoon, Ureaplasma, and the newly proposed genera Malacoplasma and Mycoplasmoides, and Metamycoplasmataceae fam. nov. to contain the newly proposed genera Metamycoplasma, Mycoplasmopsis, and Mesomycoplasma. The results presented here allow reliable discernment, both in phylogenetic and molecular terms, of the members of the two proposed families as well as different described genera within these families including members of the genus Eperythrozoon, which is comprised of uncultivable organisms. The taxonomic reclassifications proposed here, which more accurately portray the genetic diversity among the Tenericutes/Mycoplasma species, provide a new framework for understanding the biological and clinical aspects of these important microbes.

Background: The bacterial community present in the female lower genital tract plays an important role in maternal and neonatal health. Imbalances in this microbiota have been associated with negative reproductive outcomes, such as spontaneous preterm birth (sPTB), but the mechanisms underlying the association between a disturbed microbiota and sPTB remain poorly understood. An intrauterine infection ascending from the vagina is thought to be an important contributor to the onset of preterm labour. Our objective was to characterize the vaginal microbiota of pregnant women who had sPTB (n = 46) and compare to those of pregnant women who delivered at term (n = 170). Vaginal swabs were collected from women at 11-16 weeks of gestational age. Microbiota profiles were created by PCR amplification and pyrosequencing of the cpn60 universal target region., Results: Profiles clustered into seven community state types: I (Lactobacillus crispatus dominated), II (Lactobacillus gasseri dominated), III (Lactobacillus iners dominated), IVA (Gardnerella vaginalis subgroup B or mix of species), IVC (G. vaginalis subgroup A dominated), IVD (G. vaginalis subgroup C dominated) and V (Lactobacillus jensenii dominated). The microbiota of women who experienced preterm birth (< 37 weeks gestation) had higher richness and diversity and higher Mollicutes prevalence when compared to those of women who delivered at term. The two groups did not cluster according to CST, likely because CST assignment is driven in most cases by the dominance of one particular species, overwhelming the contributions of more rare taxa. In conclusion, we did not identify a specific microbial community structure that predicts sPTB, but differences in microbiota richness, diversity and Mollicutes prevalence were observed between groups., Conclusions: Although a causal relationship remains to be determined, our results confirm previous reports of an association between Mollicutes and sPTB and further suggest that a more diverse microbiome may be important in the pathogenesis of some cases.

The GroE chaperonin system, which comprises GroEL and GroES, assists protein folding in vivo and in vitro. It is conserved in all prokaryotes except in most, but not all, members of the class of mollicutes. In Escherichia coli, about 60 proteins were found to be obligatory clients of the GroE system. Here, we describe the properties of the homologs of these GroE clients in mollicutes and the evolution of chaperonins in this class of bacteria. Comparing the properties of these homologs in mollicutes with and without chaperonins enabled us to search for features correlated with the presence of GroE. Interestingly, no sequence-based features of proteins such as average length, amino acid composition and predicted folding/disorder propensity were found to be affected by the absence of GroE. Other properties such as genome size and number of proteins were also found to not differ between mollicute species with and without GroE. Our data suggest that two clades of mollicutes re-acquired the GroE system, thereby supporting the view that gaining the system occurred polyphyletically and not monophyletically, as previously debated. Our data also suggest that there might have been three isolated cases of lateral gene transfer from specific bacterial sources. Taken together, our data indicate that loss of GroE does not involve crossing a high evolutionary barrier and can be compensated for by a small number of changes within the few dozen client proteins.

Bacteria classified in Mycoplasma (M. bovis and M. bovigenitalium) and Ureaplasma (U. diversum) genera are associated with granular vulvovaginitis that affect heifers and cows at reproductive age. The traditional means for detection and speciation of mollicutes from clinical samples have been culture and serology. However, challenges experienced with these laboratory methods have hampered assessment of their impact in pathogenesis and epidemiology in cattle worldwide. The aim of this study was to develop a PCR strategy to detect and primarily discriminate between the main species of mollicutes associated with reproductive disorders of cattle in uncultured clinical samples. In order to amplify the 16S-23S rRNA internal transcribed spacer region of the genome, a consensual and species-specific nested-PCR assay was developed to identify and discriminate between main species of mollicutes. In addition, 31 vaginal swab samples from dairy and beef affected cows were investigated. This nested-PCR strategy was successfully employed in the diagnosis of single and mixed mollicute infections of diseased cows from cattle herds from Brazil. The developed system enabled the rapid and unambiguous identification of the main mollicute species known to be associated with this cattle reproductive disorder through differential amplification of partial fragments of the ITS region of mollicute genomes. The development of rapid and sensitive tools for mollicute detection and discrimination without the need for previous cultures or sequencing of PCR products is a high priority for accurate diagnosis in animal health. Therefore, the PCR strategy described herein may be helpful for diagnosis of this class of bacteria in genital swabs submitted to veterinary diagnostic laboratories, not demanding expertise in mycoplasma culture and identification., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bacterial interactions with plants and animals have been examined for many years; differently, only with the new millennium the study of bacterial-fungal interactions blossomed, becoming a new field of microbiology with relevance to microbial ecology, human health and biotechnology. Bacteria and fungi interact at different levels and bacterial endosymbionts, which dwell inside fungal cells, provide the most intimate example. Bacterial endosymbionts mostly occur in fungi of the phylum Mucoromycota and include Betaproteobacteria (Burkhoderia-related) and Mollicutes (Mycoplasma-related). Based on phylogenomics and estimations of divergence time, we hypothesized two different scenarios for the origin of these interactions (early vs late bacterial invasion). Sequencing of the genomes of fungal endobacteria revealed a significant reduction in genome size, particularly in endosymbionts of Glomeromycotina, as expected by their uncultivability and host dependency. Similar to endobacteria of insects, the endobacteria of fungi show a range of behaviours from mutualism to antagonism. Emerging results suggest that some benefits given by the endobacteria to their plant-associated fungal host may propagate to the interacting plant, giving rise to a three-level inter-domain interaction.

Background: Mollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes., Methods: A panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis., Results: Both assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10 -4 and 10 -5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well., Conclusions: These assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult.

The numbers of rectal sexually transmitted infections are on the rise especially among men who have sex with men. Males from men who have sex with men population are encouraged to send a rectal swab to the laboratory for sexually transmitted infection screening at their visit to the Genitourinary Medicine Clinic. In healthy asymptomatic males, the range of pathogens tested is limited therefore other pathogens may be left untreated allowing infections to persist among sexual partners. Molecular techniques have revolutionarised sexually transmitted infection testing enabling the detection of previously difficult-to-culture pathogens in extra-genital sites and have increased the evidence base for their clinical significance. The present study tests 107 rectal swabs from men who have sex with men negative for Chlamydia trachomatis and Neisseria gonorrhoeae against quantitative polymerase chain reaction (qPCR) assays targeting five common sexually transmitted bacteria which include Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum and Gardnerella vaginalis. The pathogenic role of these five bacteria in men who have sex with men is currently unknown. Amongst the 107 patients, a positive qPCR was obtained respectively for G. vaginalis 89 (83.2%); U. urealyticum 26 (24.3%); M. hominis 26 (24.3%); M. genitalium 10 (9.3%) and U. parvum 5 (4.7%). Bacterial loads in single and co-infections were compared for each organism. G. vaginalis and M. hominis loads were significantly ( p = 0.007 and p = 0.005, respectively) higher when co-infecting with at least one other organism. Amongst co-infections, the loads of each organism were assessed to determine possible synergies. G. vaginalis and M. hominis displayed a synergistic pattern ( r = 0.51; p = 0.02) which is in keeping with a similar synergy detected previously in the vagina of women with bacterial vaginosis. This study outlines that potential significant infections are being missed in men who have sex with men population; however, further research is warranted to confirm a pathogenesis in the rectal mucosa before routine screening can be introduced to clinical settings.

To better characterize murine intestinal microbiota, a large number (187) of Gram-positive-staining, rod- and coccoid-shaped, and facultatively or strictly anaerobic bacteria were isolated from small and large intestinal contents from mice. Based on 16S rRNA gene sequencing, a total 115 isolates formed three phylogenetically distinct clusters located within the family Erysipelotrichaceae. Group 1, as represented by strain NYU-BL-A3T, was most closely related to Allobaculum stercoricanis, with 16S rRNA gene sequence similarity values of 87.7 %. A second group, represented by NYU-BL-A4T, was most closely related to Faecalibaculum rodentium, with 86.6 % 16S rRNA gene sequence similarity. A third group had a nearly identical 16S rRNA gene sequence (99.9 %) compared with the recently described Faecalibaculum rodentium, also recovered from a laboratory mouse; however, this strain had a few differences in biochemical characteristics, which are detailed in an emended description. The predominant (>10 %) cellular fatty acids of strain NYU-BL-A3T were C16 : 0 and C18 : 0, and those of strain NYU-BL-A4T were C10 : 0, C16 : 0, C18 : 0 and C18 : 1ω9c. The two groups could also be distinguished by multiple biochemical reactions, with the group represented by NYU-BL-A4T being considerably more active. Based on phylogenetic, biochemical and chemotaxonomic criteria, two novel genera are proposed, Ileibacterium valens gen. nov., sp. nov. with NYU-BL-A3T (=ATCC TSD-63T=DSM 103668T) as the type strain and Dubosiella newyorkensis gen. nov., sp. nov. with NYU-BL-A4T (=ATCC TSD-64T=DSM 103457T) as the type strain.

Arbuscular mycorrhizal fungi (AMF, subphylum Glomeromycotina) are symbionts of most terrestrial plants. They commonly harbour endobacteria of a largely unknown biology, referred to as MRE (Mollicutes/mycoplasma-related endobacteria). Here, we propose to accommodate MRE in the novel genus 'Candidatus Moeniiplasma.' Phylogeny reconstructions based on the 16S rRNA gene sequences cluster 'Ca.Moeniiplasma' with representatives of the class Mollicutes, whereas phylogenies derived from amino acid sequences of 19 genes indicate that it is a discrete lineage sharing ancestry with the members of the family Mycoplasmataceae. Cells of 'Ca.Moeniiplasma' reside directly in the host cytoplasm and have not yet been cultivated. They are coccoid, ~500 nm in diameter, with an electron-dense layer outside the plasma membrane. However, the draft genomes of 'Ca.Moeniiplasma' suggest that this structure is not a Gram-positive cell wall. The evolution of 'Ca.Moeniiplasma' appears to be driven by an ultrarapid rate of mutation accumulation related to the loss of DNA repair mechanisms. Moreover, molecular evolution patterns suggest that, in addition to vertical transmission, 'Ca.Moeniiplasma' is able to transmit horizontally among distinct Glomeromycotina host lineages and exchange genes. On the basis of these unique lifestyle features, the new species 'Candidatus Moeniiplasma glomeromycotorum' is proposed.

Background: Bacteria of the class Mollicutes underwent extreme reduction of genomes and gene expression control systems. Only a few regulators are known to date. In this work, we describe a novel group of transcriptional regulators that are distributed within different Mollicutes and control the expression of restriction-modification systems (RM-systems)., Results: We performed cross-species search of putative regulators of RM-systems (C-proteins) and respective binding sites in Mollicutes. We identified a set of novel putative C-protein binding motifs distributed within Mollicutes. We studied the most frequent motif and respective C-protein on the model of Mycoplasma gallisepticum S6. We confirmed our prediction and identified key nucleotides important for C-protein binding. Further we identified novel target promoters of C-protein in M. gallisepticum., Conclusions: We found that C-protein of M. gallisepticum binds predicted conserved direct repeats of the (GTGTTAN 5 ) 2 motif. Apart from its own operon promoter, HsdC can bind to the promoters of the clpB chaperone gene and a tRNA cluster.

Tenericutes are a unique class of bacteria that lack a cell wall and are typically parasites or commensals of eukaryotic hosts. Environmental 16S rDNA surveys have identified a number of tenericute clades in diverse environments, introducing the possibility that these Tenericutes may represent non-host-associated, free-living microorganisms. Metagenomic sequencing of deep-sea methane seep sediments resulted in the assembly of two genomes from a Tenericutes-affiliated clade currently known as 'NB1-n' (SILVA taxonomy) or 'RF3' (Greengenes taxonomy). Metabolic reconstruction revealed that, like cultured members of the Mollicutes, these 'NB1-n' representatives lack a tricarboxylic acid cycle and instead use anaerobic fermentation of simple sugars for substrate level phosphorylation. Notably, the genomes also contained a number of unique metabolic features including hydrogenases and a simplified electron transport chain containing an RNF complex, cytochrome bd oxidase and complex I. On the basis of the metabolic potential predicted from the annotated genomes, we devised an anaerobic enrichment media that stimulated the growth of these Tenericutes at 10 °C, resulting in a mixed culture where these organisms represented ~60% of the total cells by targeted fluorescence in situ hybridization (FISH). Visual identification by FISH confirmed these organisms were not directly associated with Eukaryotes and electron cryomicroscopy of cells in the enrichment culture confirmed an ultrastructure consistent with the defining phenotypic property of Tenericutes, with a single membrane and no cell wall. On the basis of their unique gene content, phylogenetic placement and ultrastructure, we propose these organisms represent a novel class within the Tenericutes, and suggest the names Candidatus 'Izimaplasma sp. HR1' and Candidatus 'Izimaplasma sp. HR2' for the two genome representatives.

Mycoplasma bacteria are able to pass through sterilizing grade filters due to their small size and lack of a cell wall, making them a common contaminant of biopharmaceutical productions. The classical method for detecting Mycoplasma is described in the European Pharmacopeia (Ph.Eur) 2.6.7. The method takes 28 days to perform, due to the slow growing nature of some Mycoplasma species. The Ph.Eur has described Nucleic Acid Testing (NAT) as a rapid alternative to the classical method. Here we present the development of a quantitative polymerase chain reaction (qPCR) assay capable of unambiguous detection of Mycoplasma with high sensitivity and specificity. The broadness of detection and the specificity towards Mycoplasma has been investigated by in silico analysis of the primer sequences followed by testing on purified Mycoplasma DNA as well as DNA from closely related genera. The assay will in all probability detect at least 356 species and strains of Mycoplasma, Spiroplasma and Acholeplasma with high sensitivity. To our knowledge this assay has the most uniform amplification efficiency over the broadest range of species and it is extremely specific towards Mycoplasma. With appropriate validation, the assay can be applied as a powerful tool for rapid Mycoplasma detection in the biopharmaceutical industry., (Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Marine mammals play crucial ecological roles in the oceans, but little is known about their microbiotas. Here we study the bacterial communities in 337 samples from 5 body sites in 48 healthy dolphins and 18 healthy sea lions, as well as those of adjacent seawater and other hosts. The bacterial taxonomic compositions are distinct from those of other mammals, dietary fish and seawater, are highly diverse and vary according to body site and host species. Dolphins harbour 30 bacterial phyla, with 25 of them in the mouth, several abundant but poorly characterized Tenericutes species in gastric fluid and a surprisingly paucity of Bacteroidetes in distal gut. About 70% of near-full length bacterial 16S ribosomal RNA sequences from dolphins are unique. Host habitat, diet and phylogeny all contribute to variation in marine mammal distal gut microbiota composition. Our findings help elucidate the factors structuring marine mammal microbiotas and may enhance monitoring of marine mammal health.

Mycoplasmas are a large group of bacteria, sorted into different genera in the Mollicutes class, whose main characteristic in common, besides the small genome, is the absence of cell wall. They are considered cellular and molecular biology study models. We present an updated review of the molecular biology of these model microorganisms and the development of replicative vectors for the transformation of mycoplasmas. Synthetic biology studies inspired by these pioneering works became possible and won the attention of the mainstream media. For the first time, an artificial genome was synthesized (a minimal genome produced from consensus sequences obtained from mycoplasmas). For the first time, a functional artificial cell has been constructed by introducing a genome completely synthesized within a cell envelope of a mycoplasma obtained by transformation techniques. Therefore, this article offers an updated insight to the state of the art of these peculiar organisms' molecular biology.

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita.

The glacier ice worm, Mesenchytraeus solifugus, is a unique annelid, inhabiting only snow and ice in North American glaciers. Here, we analyzed the taxonomic composition of bacteria associated with M. solifugus based on the 16S rRNA gene. We analyzed four fixed-on-site and 10 starved ice worm individuals, along with glacier surface samples. In total, 1341 clones of 16S rRNA genes were analyzed for the ice worm samples, from which 65 bacterial phylotypes (99.0% cut-off) were identified. Of these, 35 phylotypes were closely related to sequences obtained from their habitat glacier and/or other components of cryosphere; whereas three dominant phylotypes were affiliated with animal-associated lineages of the class Mollicutes. Among the three, phylotype Ms-13 shared less than 89% similarity with database sequences and was closest to a gut symbiont of a terrestrial earthworm. Using fluorescence in situ hybridization, Ms-13 was located on the gut wall surface of the ice worms. We propose a novel genus and species, 'Candidatus Vermiplasma glacialis', for this bacterium. Our results raise the possibility that the ice worm has exploited indigenous glacier bacteria, while several symbiotic bacterial lineages have maintained their association with the ice worm during the course of adaptive evolution to the permanently cold environment., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches.

Lateral gene transfer (LGT)--which transfers DNA between two non-vertically related individuals belonging to the same or different species--is recognized as a major force in prokaryotic evolution, and evidence of its impact on eukaryotic evolution is ever increasing. LGT has attracted much public attention for its potential to transfer pathogenic elements and antibiotic resistance in bacteria, and to transfer pesticide resistance from genetically modified crops to other plants. In a wider perspective, there is a growing body of studies highlighting the role of LGT in enabling organisms to occupy new niches or adapt to environmental changes. The challenge LGT poses to the standard tree-based conception of evolution is also being debated. Studies of LGT have, however, been severely limited by a lack of computational tools. The best currently available LGT algorithms are parsimony-based phylogenetic methods, which require a pre-computed gene tree and cannot choose between sometimes wildly differing most parsimonious solutions. Moreover, in many studies, simple heuristics are applied that can only handle putative orthologs and completely disregard gene duplications (GDs). Consequently, proposed LGT among specific gene families, and the rate of LGT in general, remain debated. We present a Bayesian Markov-chain Monte Carlo-based method that integrates GD, gene loss, LGT, and sequence evolution, and apply the method in a genome-wide analysis of two groups of bacteria: Mollicutes and Cyanobacteria. Our analyses show that although the LGT rate between distant species is high, the net combined rate of duplication and close-species LGT is on average higher. We also show that the common practice of disregarding reconcilability in gene tree inference overestimates the number of LGT and duplication events.

The mreB gene family encodes actin-like proteins that determine cell shape by directing cell wall synthesis and often exists in one to three copies in the genomes of non-spherical bacteria. Intriguingly, while most wall-less bacteria do not have this gene, five to seven mreB homologs are found in Spiroplasma and Haloplasma, which are both characterized by cell contractility. To investigate the molecular evolution of this gene family in wall-less bacteria, we sampled the available genome sequences from these two genera and other related lineages for comparative analysis. The gene phylogenies indicated that the mreB homologs in Haloplasma are more closely related to those in Firmicutes, whereas those in Spiroplasma form a separate clade. This finding suggests that the gene family expansions in these two lineages are the results of independent ancient duplications. Moreover, the Spiroplasma mreB homologs can be classified into five clades, of which the genomic positions are largely conserved. The inference of gene gains and losses suggests that there has been an overall trend to retain only one homolog from each of the five mreB clades in the evolutionary history of Spiroplasma., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Nine species of uncultivable haemoplasmas and several Mycoplasma species were examined by partial sequencing of two protein-encoding housekeeping genes. Partial glyceraldehyde-3-phosphate dehydrogenase (gapA) and heat shock protein 70 (dnaK) gene sequences were determined for these Mollicute species; in total nine gapA sequences and ten dnaK sequences were obtained. Phylogenetic analyses of these sequences, along with those of a broad selection of Mollicute species downloaded from GenBank, for the individual genes, and for the gapA and dnaK concatenated data set, revealed a clear separation of the haemoplasmas from other species within the Mycoplasma genus; indeed the haemoplasmas resided within a single clade which was phylogenetically detached from the pneumoniae group of Mycoplasmas. This is the first report to examine the use of gapA and dnaK, as well as a concatenated data set, for phylogenetic analysis of the haemoplasmas and other Mollicute species. These results demonstrate a distinct phylogenetic separation between the haemoplasmas and Mycoplasmas that corresponds with the biological differences observed in these species, indicating that further evaluation of the haemoplasmas' relationship with the Mycoplasma genus is required to determine whether reclassification of the haemoplasmas is necessary., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiome. They are obligate biotrophs that colonize the roots of most land plants and enhance host nutrient acquisition. Many AMF themselves harbor endobacteria in their hyphae and spores. Two types of endobacteria are known in Glomeromycota: rod-shaped Gram-negative Candidatus Glomeribacter gigasporarum, CaGg, limited in distribution to members of the Gigasporaceae family, and coccoid Mollicutes-related endobacteria, Mre, widely distributed across different lineages of AMF. The goal of the present study is to investigate the patterns of distribution and coexistence of the two endosymbionts, CaGg and Mre, in spore samples of several strains of Gigaspora margarita. Based on previous observations, we hypothesized that some AMF could host populations of both endobacteria. To test this hypothesis, we performed an extensive investigation of both endosymbionts in G. margarita spores sampled from Cameroonian soils as well as in the Japanese G. margarita MAFF520054 isolate using different approaches (molecular phylotyping, electron microscopy, fluorescence in situ hybridization and quantitative real-time PCR). We found that a single AMF host can harbour both types of endobacteria, with Mre population being more abundant, variable and prone to recombination than the CaGg one. Both endosymbionts seem to retain their genetic and lifestyle peculiarities regardless of whether they colonize the host alone or together. These findings show for the first time that fungi support an intracellular bacterial microbiome, in which distinct types of endobacteria coexist in a single cell.

In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. Mollicutes microorganism contamination of tissue cultures is a major problem because it can determine various and severe alterations in cellular function. Thus methods able to detect and trace cell cultures with Mollicutes contamination are needed in the monitoring of cells grown under good manufacturing practice conditions, and cell lines in continuous culture must be tested at regular intervals. We here describe a multiplex quantitative polymerase chain reaction assay able to detect contaminant Mollicutes species in a single-tube reaction through analysis of 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes. The method shows a sensitivity, specificity, and robustness comparable with the culture and the indicator cell culture as required by the European Pharmacopoeia guidelines and was validated following International Conference on Harmonization guidelines and Food and Drug Administration requirements.

Many studies have explored the mechanisms involved in relative amino acid usage (RAAU) in a variety of prokaryotes and eukaryotes. A strong bias was observed in highly expressed genes (HEGs) of endosymbiotic bacteria. By means of correspondence analysis, we studied the major trends affecting internal variability of RAAU in Mollicutes. The principal trend is related to the usage of smaller, less aromatic, and GC-rich coded amino acids in HEGs. Given the nature of the genetic code, these properties are linked among them. Expectedly, we found a slow evolutionary rate of HEGs, which is likely driven by purifying selection. On the other hand, the rest of the genes accumulate rapid changes as a result of the extreme mutational bias toward A + T of the genomes and genetic drift, increasing internal variability. Amino acid changes across the phylogeny of the group were traced in order to estimate the mean molecular weight and aromaticity trends in each branch. Finally, we compared amino acid usage bias within and between Mollicutes and the free-living Firmicutes.

This study is the first to evaluate the occurrence of several Mollicutes species in Brazilian capuchin monkeys (Cebus spp.). Mollicutes were detected by culture and polymerase chain reaction (PCR) in samples of the oropharyngeal, conjuctiva, and genital mucosae of 58 monkeys. In the oropharynx, Mollicutes in general (generic PCR to the Class), and those of the genus Ureaplasma (genus PCR), were detected in 72.4% and 43.0% of the samples, respectively. The identified species in this site included: Mycoplasma arginini (43.1%), M. salivarium (41.4%), and M. pneumoniae (19.0%). Both Ureaplasma and Mycoplasma are genera of the order Mycoplasmatales. In the preputial/vaginal mucosa, PCR detected Mollicutes in general in 27.58% of the samples, the genus Ureaplasma in 32.7%, the species M. arginini in 8.6%, and Acholeplasma laidlawii of the order Acholeplasmatales in 1.7% In the conjunctiva, Mollicutes in general were detected in 29.3% of the samples, with 1.7% being identified as A. laidlawii. Culturing was difficult due to contamination, but two isolates were successfully obtained. The Mollicutes species of this study provided new insights into these bacteria in Brazilian Cebus. Studies are lacking of the actual risk of Mollicutes infection or the frequency at which primates serve as permanent or temporary reservoirs for Mollicutes. In the present study, the samples were collected from monkeys without clinical signs of infection. The mere presence of Mollicutes, particularly those also found in humans, nevertheless signals a need for studies to evaluate the impact of these microorganisms on the health of non-human primates (NHPs) and the possibility of cross-species transmission between NHPs and humans., (© 2013 Wiley Periodicals, Inc.)

The tree of life is paramount for achieving an integrated understanding of microbial evolution and the relationships between physiology, genealogy and genomics. It provides the framework for interpreting environmental sequence data, whether applied to microbial ecology or to human health. However, there remain many instances where there is ambiguity in our understanding of the phylogeny of major lineages, and/or confounding nomenclature. Here we apply recent genomic sequence data to examine the evolutionary history of members of the classes Mollicutes (phylum Tenericutes) and Erysipelotrichia (phylum Firmicutes). Consistent with previous analyses, we find evidence of a specific relationship between them in molecular phylogenies and signatures of the 16S rRNA, 23S rRNA, ribosomal proteins and aminoacyl-tRNA synthetase proteins. Furthermore, by mapping functions over the phylogenetic tree we find that the erysipelotrichia lineages are involved in various stages of genomic reduction, having lost (often repeatedly) a variety of metabolic functions and the ability to form endospores. Although molecular phylogeny has driven numerous taxonomic revisions, we find it puzzling that the most recent taxonomic revision of the phyla Firmicutes and Tenericutes has further separated them into distinct phyla, rather than reflecting their common roots.

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA(Arg)ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA(Arg)CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA(Arg)CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA(Arg) and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA(Arg)ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA(Arg)ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA(Arg)CCG and tadA, and then acquired a new tRNA(Arg)UCG. This permitted further tRNA(Arg)ACG mutations with tRNA(Arg)GCG or its disappearance, leaving a single tRNA(Arg)UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

Arbuscular mycorrhizal fungi (AMF) can host Gram-positive endobacteria (BLOs) in their cytoplasm. These have been identified as Mollicutes-related microbes based on an inventory of AMF spores from fungal collections. Bacteria-like organisms (BLOs) of unknown identity have also been reported in the cytoplasm of AMF associated with liverworts, the earliest-diverged extant lineage of land plants. A combination of morphological, molecular and phylogenetic analyses revealed that three samples of two liverwort species (Conocephalum conicum and Lunularia cruciata) growing spontaneously in a botanical garden harboured AMF belonging to Glomerales, and these, in turn, hosted coccoid BLOs. 16S rDNA sequences from these BLOs clustered with the Mollicutes sequences identified from the spore collections but revealed the presence of novel phylotypes. Electron microscopy and fluorescence in situ hybridization (FISH) confirmed the presence of BLOs inside the cytoplasm of AMF hyphae colonizing the liverwort thalli. The high genetic variability of BLOs in liverwort-AMF associations thriving in the same ecological niche raises questions about the mechanisms underlying such diversity., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Background Aims: The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions., Methods: We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing., Results: Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines., Conclusions: We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.

Trichomonas vaginalis can be naturally infected with intracellular Mycoplasma hominis. This bacterial infection may have implications for trichomonal virulence and disease pathogenesis. The objective of the study was to report the presence of M. hominis in Cuban T. vaginalis isolates and to describe the association between the phenotype M. hominis infected with RAPD genetic polymorphism of T. vaginalis. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 40 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with M. hominis. The trees drawn based on RAPD data showed no relations with metronidazole susceptibility and significantly association with the presence of M. hominis (P=0.043), which demonstrates the existence of concordance between the genetic relatedness and the presence of M. hominis in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the bacterial enters and/or survival., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Among all nucleic acid amplification technologies, Real-Time Transcription Mediated Amplification (Real-Time TMA) is an isothermal method that can amplify RNA targets a billion-fold in less than one hour's time. By using this method, a new assay was developed for detecting the presence of Mollicutes in mammalian cell cultures and biologics. Production of amplicons is monitored in real time by measuring continuously a fluorescence signal during the reaction. The shape of this signal curve is a sigmoid, where an initial baseline phase precedes an exponential phase ending with a maximum followed by a linear decreasing phase. The aim of this study was to develop a curve-analysis tool to unambiguously assign a Mollicutes positive or negative status to a biological sample. In this context, a statistical analysis of the data combined with the selection of the best predictors allowed the development of an algorithm which removes erroneous data and applies the best predictors to generate the Mollicutes status prediction. Our results demonstrate that this algorithm assigns positive/negative/invalid status coming from Real-Time TMA fluorescence signal analysis with a confidence (true predictions) in the results close to 100%., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Urogenital tract infections can be caused by a number of pathogens, some of which, like the obligate intracellular Chlamydia trachomatis, are difficult to culture, or the cell wall-less mollicutes, like M. hominis or Ureaplasma spp. Real-time PCR (qPCR) has become an important diagnostic tool as it enables not only the species-specific detection of the organism but also the quantification essential to define the etiological relevance of a facultative pathogenic bacterium. We developed a set of TaqMan qPCRs for the detection of the species M. genitalium and M. hominis (Mh/Mg-duplex qPCR), U. parvum and U. urealyticum (Uu/Up duplex-PCR), and C. trachomatis (CT-qPCR), and for typing of lymphogranuloma venereum-associated L-serovars of C. trachomatis (LGV-qPCR) as well as a sub-typing of L1, L2, and L3. In addition, the human gap-gene was amplified as quality control of the specimen, and a cryptic plasmid co-amplified in CT-qPCR as an inhibition control. The present protocol focuses on the step-by-step description for the establishment of these TaqMan multiplex qPCRs.

Background: Substitution matrices are key parameters for the alignment of two protein sequences, and consequently for most comparative genomics studies. The composition of biological sequences can vary importantly between species and groups of species, and classical matrices such as those in the BLOSUM series fail to accurately estimate alignment scores and statistical significance with sequences sharing marked compositional biases., Results: We present a general and simple methodology to build matrices that are especially fitted to the compositional bias of proteins. Our approach is inspired from the one used to build the BLOSUM matrices and is based on learning substitution and amino acid frequencies on real sequences with the corresponding compositional bias. We applied it to the large scale comparison of Mollicute AT-rich genomes. The new matrix, MOLLI60, was used to predict pairwise orthology relationships, as well as homolog families among 24 Mollicute genomes. We show that this new matrix enables to better discriminate between true and false orthologs and improves the clustering of homologous proteins, with respect to the use of the classical matrix BLOSUM62., Conclusions: We show in this paper that well-fitted matrices can improve the predictions of orthologous and homologous relationships among proteins with a similar compositional bias. With the ever-increasing number of sequenced genomes, our approach could prove valuable in numerous comparative studies focusing on atypical genomes., (© 2011 Lemaitre et al; licensee BioMed Central Ltd.)