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Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated in vivo with MS2-tagged trans -sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia Sinorhizobium meliloti . The three proteomes were rather distinct, with that of EcpR1 particularly enriched in cell cycle-related enzymes, whilst sharing several transcription/translation-related proteins recurrently identified associated with sRNAs. Strikingly, MetK, the synthetase of the major methyl donor S-adenosylmethionine, was reliably recovered as a binding partner of the three sRNAs, which reciprocally co-immunoprecipitated with a FLAG-tagged MetK variant. Induced (over)expression of the trans -sRNAs and MetK depletion did not influence canonical riboregulatory traits, `for example, protein titration or sRNA stability, respectively. An in vitro filter assay confirmed binding of AbcR2, NfeR1 and EcpR1 to MetK and further revealed interaction of the protein with other non-coding and coding transcripts but not with the 5S rRNA. These findings uncover a broad specificity for RNA binding as an unprecedented feature of this housekeeping prokaryotic enzyme.

Bacterial small-RNA (sRNA) sequences are functional RNAs, which play an important role in regulating the expression of a diverse class of genes. It is thus critical to identify such sRNA sequences and their probable mRNA targets. Here, we discuss new procedures to identify and characterize sRNA and their targets via the introduction of an integrated online platform 'PresRAT'. PresRAT uses the primary and secondary structural attributes of sRNA sequences to predict sRNA from a given sequence or bacterial genome. PresRAT also finds probable target mRNAs of sRNA sequences from a given bacterial chromosome and further concentrates on the identification of the probable sRNA-mRNA binding regions. Using PresRAT, we have identified a total of 66,209 potential sRNA sequences from 292 bacterial genomes and 2247 potential targets from 13 bacterial genomes. We have also implemented a protocol to build and refine 3D models of sRNA and sRNA-mRNA duplex regions and generated 3D models of 50 known sRNAs and 81 sRNA-mRNA duplexes using this platform. Along with the server part, PresRAT also contains a database section, which enlists the predicted sRNA sequences, sRNA targets, and their corresponding 3D models with structural dynamics information.

The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa , including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser., (Copyright © 2021 Gerovac et al.)

In the past, the potato plant microbiota and rhizosphere have been studied in detail to improve plant growth and fitness. However, less is known about the postharvest potato tuber microbiome and its role in storage stability. The storage stability of potatoes depends on genotype and storage conditions, but the soil in which tubers were grown could also play a role. To understand the ecology and functional role of the postharvest potato microbiota, we planted four potato varieties in five soil types and monitored them until the tubers started sprouting. During storage, the bacterial community of tubers was analysed by next-generation sequencing of the 16S rRNA gene amplicons. The potato tubers exhibited soil-dependent differences in sprouting behaviour. The statistical analysis revealed a strong shift of the tuber-associated bacterial community from harvest to dormancy break. By combining indicator species analysis and a correlation matrix, we predicted associations between members of the bacterial community and tuber sprouting behaviour. Based on this, we identified Flavobacterium sp. isolates, which were able to influence sprouting behaviour by inhibiting potato bud outgrowth.

Rfam is a database of RNA families where each of the 3444 families is represented by a multiple sequence alignment of known RNA sequences and a covariance model that can be used to search for additional members of the family. Recent developments have involved expert collaborations to improve the quality and coverage of Rfam data, focusing on microRNAs, viral and bacterial RNAs. We have completed the first phase of synchronising microRNA families in Rfam and miRBase, creating 356 new Rfam families and updating 40. We established a procedure for comprehensive annotation of viral RNA families starting with Flavivirus and Coronaviridae RNAs. We have also increased the coverage of bacterial and metagenome-based RNA families from the ZWD database. These developments have enabled a significant growth of the database, with the addition of 759 new families in Rfam 14. To facilitate further community contribution to Rfam, expert users are now able to build and submit new families using the newly developed Rfam Cloud family curation system. New Rfam website features include a new sequence similarity search powered by RNAcentral, as well as search and visualisation of families with pseudoknots. Rfam is freely available at https://rfam.org., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pseudomonas putida is a micro-organism with great potential for industry due to its stress-endurance traits and easy manipulation of the metabolism. However, optimization is still required to improve production yields. In the last years, manipulation of bacterial small non-coding RNAs (ncRNAs) has been recognized as an effective tool to improve the production of industrial compounds. So far, very few ncRNAs are annotated in P. putida beyond the generally conserved. In the present study, P. putida was cultivated in a two-compartment scale-down bioreactor that simulates large-scale industrial bioreactors. We performed RNA-Seq of samples collected at distinct locations and time-points to predict novel and potentially important ncRNAs for the adaptation of P. putida to bioreactor stress conditions. Instead of using a purely genomic approach, we have rather identified regions of putative ncRNAs with high expression levels using two different programs (Artemis and sRNA detect). Only the regions identified with both approaches were considered for further analysis and, in total, 725 novel ncRNAs were predicted. We also found that their expression was not constant throughout the bioreactor, showing different patterns of expression with time and position. This is the first work focusing on the ncRNAs whose expression is triggered in a bioreactor environment. This information is of great importance for industry, since it provides possible targets to engineer more effective P. putida strains for large-scale production.

Small non-coding RNAs (sRNAs) regulate numerous cellular processes in all domains of life. Several approaches have been developed to identify them from RNA-seq data, which are efficient for eukaryotic sRNAs but remain inaccurate for the longer and highly structured bacterial sRNAs. We present APERO, a new algorithm to detect small transcripts from paired-end bacterial RNA-seq data. In contrast to previous approaches that start from the read coverage distribution, APERO analyzes boundaries of individual sequenced fragments to infer the 5' and 3' ends of all transcripts. Since sRNAs are about the same size as individual fragments (50-350 nucleotides), this algorithm provides a significantly higher accuracy and robustness, e.g., with respect to spontaneous internal breaking sites. To demonstrate this improvement, we develop a comparative assessment on datasets from Escherichia coli and Salmonella enterica, based on experimentally validated sRNAs. We also identify the small transcript repertoire of Dickeya dadantii including putative intergenic RNAs, 5' UTR or 3' UTR-derived RNA products and antisense RNAs. Comparisons to annotations as well as RACE-PCR experimental data confirm the precision of the detected transcripts. Altogether, APERO outperforms all existing methods in terms of sRNA detection and boundary precision, which is crucial for comprehensive genome annotations. It is freely available as an open source R package on https://github.com/Simon-Leonard/APERO., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Streptococcal species constitute a large group of commensal and pathogenic Gram-positive bacteria that exist in a wide variety of habitats. The family of small RNAs is typically ranged in size from 50 to 300 nucleotides, and acts as regulators in bacteria. The last decade has witnessed the increasing findings of small RNAs (sRNAs), which play important regulatory roles in the variety of biological processes in streptococci. In this review, we summarized the recent achievements in the identification of streptococcal sRNAs, mainly in Streptococcus pyogenes and Streptococcus pneumoniae . In addition, we particularly focused on the functions that sRNAs exert in the regulatory networks of both phenotypical traits and pathogenicity. The fact that sRNAs act as a critical fine-tuning regulator of streptococci may not only reveal in-depth mechanisms of bacterial post-transcriptional regulations in response to environmental perturbance, but also provide promising approaches to the better management of streptococcal infections.

A precise understanding of the genomic organization into transcriptional units and their regulation is essential for our comprehension of opportunistic human pathogens and how they cause disease. Using single-molecule real-time (PacBio) sequencing we unambiguously determined the genome sequence of Streptococcus pneumoniae strain D39 and revealed several inversions previously undetected by short-read sequencing. Significantly, a chromosomal inversion results in antigenic variation of PhtD, an important surface-exposed virulence factor. We generated a new genome annotation using automated tools, followed by manual curation, reflecting the current knowledge in the field. By combining sequence-driven terminator prediction, deep paired-end transcriptome sequencing and enrichment of primary transcripts by Cappable-Seq, we mapped 1015 transcriptional start sites and 748 termination sites. We show that the pneumococcal transcriptional landscape is complex and includes many secondary, antisense and internal promoters. Using this new genomic map, we identified several new small RNAs (sRNAs), RNA switches (including sixteen previously misidentified as sRNAs), and antisense RNAs. In total, we annotated 89 new protein-encoding genes, 34 sRNAs and 165 pseudogenes, bringing the S. pneumoniae D39 repertoire to 2146 genetic elements. We report operon structures and observed that 9% of operons are leaderless. The genome data are accessible in an online resource called PneumoBrowse (https://veeninglab.com/pneumobrowse) providing one of the most complete inventories of a bacterial genome to date. PneumoBrowse will accelerate pneumococcal research and the development of new prevention and treatment strategies.

The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway. This allows in turn the formation of catalysts by an anabolic process, eventually leading to the accumulation of ribozymes. These results demonstrate that rudimentary self-reproducing RNA systems based on recombination possess an inherent capacity to assimilate an expanded repertoire of chemical resources and suggest that coupled catabolism and anabolism could have arisen at a very early stage in primordial living systems.

Bacteria are an exceedingly diverse group of organisms whose molecular exploration is experiencing a renaissance. While the classical view of bacterial gene expression was relatively simple, the emerging view is more complex, encompassing extensive post-transcriptional control involving riboswitches, RNA thermometers, and regulatory small RNAs (sRNAs) associated with the RNA-binding proteins CsrA, Hfq, and ProQ, as well as CRISPR/Cas systems that are programmed by RNAs. Moreover, increasing interest in members of the human microbiota and environmental microbial communities has highlighted the importance of understudied bacterial species with largely unknown transcriptome structures and RNA-based control mechanisms. Collectively, this creates a need for global RNA biology approaches that can rapidly and comprehensively analyze the RNA composition of a bacterium of interest. We review such approaches with a focus on RNA-seq as a versatile tool to investigate the different layers of gene expression in which RNA is made, processed, regulated, modified, translated, and turned over., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Small RNAs (sRNAs) in bacteria have emerged as key players in transcriptional and post-transcriptional regulation of gene expression. Here, we present a statistical analysis of different sequence- and structure-related features of bacterial sRNAs to identify the descriptors that could discriminate sRNAs from other bacterial RNAs. We investigated a comprehensive and heterogeneous collection of 816 sRNAs, identified by northern blotting across 33 bacterial species and compared their various features with other classes of bacterial RNAs, such as tRNAs, rRNAs and mRNAs. We observed that sRNAs differed significantly from the rest with respect to G+C composition, normalized minimum free energy of folding, motif frequency and several RNA-folding parameters like base-pairing propensity, Shannon entropy and base-pair distance. Based on the selected features, we developed a predictive model using Random Forests (RF) method to classify the above four classes of RNAs. Our model displayed an overall predictive accuracy of 89.5%. These findings would help to differentiate bacterial sRNAs from other RNAs and further promote prediction of novel sRNAs in different bacterial species.

Public aquaria offer numerous educational opportunities for visitors while touch-tank exhibits offer guests the ability to directly interact with marine life via physical contact. Despite the popularity of touch-tanks, there is a paucity of research about animal health in these exhibits and, in particular, there is little research on the microbial communities in these highly interactive exhibits. Microbial community structure can have implications for both host health and habitat function. To better understand the microbiome of a touch-tank we used high-throughput sequencing of the 16S rRNA gene to analyze the microbial community on the dorsal and ventral surfaces of cow-nose rays (Rhinoptera bonasus) as well as their environment in a frequently visited touch-tank exhibit at the New England Aquarium. Our analyses revealed a distinct microbial community associated with the skin of the ray that had lower diversity than the surrounding habitat. The ray skin was dominated by three orders: Burkholderiales (∼55%), Flavobacteriales (∼19%), and Pseudomonadales (∼12%), taxonomic groups commonly associated with other fish species. Our results provide a survey of ray-associated bacterial communities in a touch-tank environment, thereby laying the foundation for future studies examining the role of potential challenges to ray microbiota and their associated health., (© 2017 Wiley Periodicals, Inc.)

Unlabelled: Bacteria use multidrug efflux pumps to export drugs and toxic compounds out of the cell. One of the most important efflux pumps in Escherichia coli is the AcrAB-TolC system. Small regulatory RNAs (sRNAs) are known to be major posttranscriptional regulators that can enhance or repress translation by binding to the 5' untranslated region (UTR) of mRNA targets with the help of a chaperone protein, Hfq. In this study, we investigated the expression of acrA, acrB, and tolC translational fusions using 27 Hfq-dependent sRNAs overexpressed from plasmids. No significant sRNA regulation of acrA or acrB was detected. SdsR (also known as RyeB), an abundant and well-conserved stationary-phase sRNA, was found to repress the expression of tolC, the gene encoding the outer membrane protein of many multidrug resistance efflux pumps. This repression was shown to be by direct base pairing occurring upstream from the ribosomal binding site. SdsR overexpression and its regulation of tolC were found to reduce resistance to novobiocin and crystal violet. Our results suggest that additional targets for SdsR exist that contribute to increased antibiotic sensitivity and reduced biofilm formation. In an effort to identify phenotypes associated with single-copy SdsR and its regulation of tolC, the effect of a deletion of sdsR or mutations in tolC that should block SdsR pairing were investigated using a Biolog phenotypic microarray. However, no significant phenotypes were identified. Therefore, SdsR appears to modulate rather than act as a major regulator of its targets., Importance: AcrAB-TolC is a major efflux pump present in E. coli and Gram-negative bacteria used to export toxic compounds; the pump confers resistance to many antibiotics of unrelated classes. In this study, we found that SdsR, a small RNA expressed in stationary phase, repressed the expression of tolC, resulting in increased sensitivity to some antibiotics. This extends the findings of previous studies showing that sRNAs contribute to the regulation of many outer membrane proteins; manipulating or enhancing their action might help in sensitizing bacteria to antibiotics., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: The aim of the present study was to evaluate the prevalence of haemoplasma infection in cats in Portugal and to assess risk factors for infection., Methods: Real-time polymerase chain reaction techniques were used to assess 236 urban and rural cats from central and southern Portugal., Results: The overall prevalence of haemoplasma in the target population was 27.1% (64/236), with individual species' prevalences as follows: 17.8% (42/236) 'Candidatus Mycoplasma haemominutum' (CMhm), 14.4% (34/236) Mycoplasma haemofelis (Mhf) and only 5.9% (14/236) 'Candidatus Mycoplasma turicensis' (CMt). Multiple infections were detected in 8.1% (19/236) of the samples, with triple and double infections with Mhf and CMhm being most commonly detected (5.9% [14/236] of cats). Haemoplasma infection was significantly higher in shelter cats (P = 0.015) than in cats with other lifestyles (eg, free-roaming/house pet/blood donors). Haemoplasma prevalence was also higher in cats with feline immunodeficiency virus infection (FIV; P = 0.011). Although sex was not significantly associated with haemoplasma infection (P = 0.050), CMt was predominantly found in males (P = 0.032). Also, the presence of haemoplasma multiple infections was statistically associated with being in a shelter (P = 0.021), male (P = 0.057) and with FIV co-infection (P = 0.004). No evidence of an association between haemoplasma infection and geographical location, age or feline leukaemia virus co-infection was found., Conclusions and Relevance: The results obtained in our study are consistent with the documented worldwide prevalence of feline haemoplasma infections, suggesting that the three main feline haemoplasma species are common in Portugal., (© ISFM and AAFP 2014.)

Noncoding RNAs are integral to a wide range of biological processes, including translation, gene regulation, host-pathogen interactions and environmental sensing. While genomics is now a mature field, our capacity to identify noncoding RNA elements in bacterial and archaeal genomes is hampered by the difficulty of de novo identification. The emergence of new technologies for characterizing transcriptome outputs, notably RNA-seq, are improving noncoding RNA identification and expression quantification. However, a major challenge is to robustly distinguish functional outputs from transcriptional noise. To establish whether annotation of existing transcriptome data has effectively captured all functional outputs, we analysed over 400 publicly available RNA-seq datasets spanning 37 different Archaea and Bacteria. Using comparative tools, we identify close to a thousand highly-expressed candidate noncoding RNAs. However, our analyses reveal that capacity to identify noncoding RNA outputs is strongly dependent on phylogenetic sampling. Surprisingly, and in stark contrast to protein-coding genes, the phylogenetic window for effective use of comparative methods is perversely narrow: aggregating public datasets only produced one phylogenetic cluster where these tools could be used to robustly separate unannotated noncoding RNAs from a null hypothesis of transcriptional noise. Our results show that for the full potential of transcriptomics data to be realized, a change in experimental design is paramount: effective transcriptomics requires phylogeny-aware sampling.

Listeria monocytogenes is a Gram-positive human-pathogen bacterium that served as an experimental model for investigating fundamental processes of adaptive immunity and virulence. Recent novel technologies allowed the identification of several hundred non-coding RNAs (ncRNAs) in the Listeria genome and provided insight into an unexpected complex transcriptional machinery. In this review, we discuss ncRNAs that are encoded on the opposite strand of the target gene and are therefore termed antisense RNAs (asRNAs). We highlight mechanistic and functional concepts of asRNAs in L. monocytogenes and put these in context of asRNAs in other bacteria. Understanding asRNAs will further broaden our knowledge of RNA-mediated gene regulation and may provide targets for diagnostic and antimicrobial development.

6S RNA is a highly abundant small non-coding RNA widely spread among diverse bacterial groups. By competing with DNA promoters for binding to RNA polymerase (RNAP), the RNA regulates transcription on a global scale. RNAP produces small product RNAs derived from 6S RNA as template, which rearranges the 6S RNA structure leading to dissociation of 6S RNA:RNAP complexes. Although 6S RNA has been experimentally analysed in detail for some species, such as Escherichia coli and Bacillus subtilis, and was computationally predicted in many diverse bacteria, a complete and up-to-date overview of the distribution among all bacteria is missing. In this study we searched with new methods for 6S RNA genes in all currently available bacterial genomes. We ended up with a set of 1,750 6S RNA genes, of which 1,367 are novel and bona fide, distributed among 1,610 bacteria, and had a few tentative candidates among the remaining 510 assembled bacterial genomes accessible. We were able to confirm two tentative candidates by Northern blot analysis. We extended 6S RNA genes of the Flavobacteriia significantly in length compared to the present Rfam entry. We describe multiple homologs of 6S RNAs (including split 6S RNA genes) and performed a detailed synteny analysis.

The genus Xanthomonas comprises a large group of plant-pathogenic bacteria. The infection and bacterial multiplication in the plant tissue depends on the type III secretion system and other virulence determinants. Recent studies revealed that bacterial virulence is also controlled at the post-transcriptional level by small non-coding RNAs (sRNAs). In this review, we highlight our current knowledge about sRNAs and RNA-binding proteins in Xanthomonas species.

Small RNAs (sRNAs) constitute a large and heterogeneous class of bacterial gene expression regulators. Much like eukaryotic microRNAs, these sRNAs typically target multiple mRNAs through short seed pairing, thereby acting as global posttranscriptional regulators. In some bacteria, evidence for hundreds to possibly more than 1,000 different sRNAs has been obtained by transcriptome sequencing. However, the experimental identification of possible targets and, therefore, their confirmation as functional regulators of gene expression has remained laborious. Here, we present a strategy that integrates phylogenetic information to predict sRNA targets at the genomic scale and reconstructs regulatory networks upon functional enrichment and network analysis (CopraRNA, for Comparative Prediction Algorithm for sRNA Targets). Furthermore, CopraRNA precisely predicts the sRNA domains for target recognition and interaction. When applied to several model sRNAs, CopraRNA revealed additional targets and functions for the sRNAs CyaR, FnrS, RybB, RyhB, SgrS, and Spot42. Moreover, the mRNAs gdhA, lrp, marA, nagZ, ptsI, sdhA, and yobF-cspC were suggested as regulatory hubs targeted by up to seven different sRNAs. The verification of many previously undetected targets by CopraRNA, even for extensively investigated sRNAs, demonstrates its advantages and shows that CopraRNA-based analyses can compete with experimental target prediction approaches. A Web interface allows high-confidence target prediction and efficient classification of bacterial sRNAs.

Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein-binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap.

The biochemical pattern of Cowan and Steel (BPCS) was compared with a commercial biochemical strip for the identification of Corynebacterium pseudotuberculosis isolated from small ruminants. On 16S rRNA gene sequencing, 40/78 coryneform isolates from the lymph nodes of sheep and goats with lesions resembling caseous lymphadenitis were identified as C. pseudotuberculosis. The sensitivities of the BPCS and the commercial biochemical strip relative to 16S rRNA sequencing were 80% and 85%, and their specificities were 92.1% and 94.7%, respectively; the level of agreement between the BPCS and the commercial biochemical strip was high (κ=0.82). Likelihood ratios for positive and negative results were 10.0 and 0.22 for the BPCS, and 16.0 and 0.16 for the commercial biochemical strip, respectively. These results indicate that the BPCS and the commercial biochemical strip are both useful for identifying C. pseudotuberculosis in veterinary microbiology laboratories., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In Escherichia coli, 12 distinct RNA structures within the transcripts encoding ribosomal proteins interact with specific ribosomal proteins to allow autogenous regulation of expression from large multi-gene operons, thus coordinating ribosomal protein biosynthesis across multiple operons. However, these RNA structures are typically not represented in the RNA Families Database or annotated in genomic sequences databases, and their phylogenetic distribution is largely unknown. To investigate the extent to which these RNA structures are conserved across eubacterial phyla, we created multiple sequence alignments representing 10 of these messenger RNA (mRNA) structures in E. coli. We find that while three RNA structures are widely distributed across many phyla of bacteria, seven of the RNAs are narrowly distributed to a few orders of Gammaproteobacteria. To experimentally validate our computational predictions, we biochemically confirmed dual L1-binding sites identified in many Firmicute species. This work reveals that RNA-based regulation of ribosomal protein biosynthesis is used in nearly all eubacterial phyla, but the specific RNA structures that regulate ribosomal protein biosynthesis in E. coli are narrowly distributed. These results highlight the limits of our knowledge regarding ribosomal protein biosynthesis regulation outside of E. coli, and the potential for alternative RNA structures responsible for regulating ribosomal proteins in other eubacteria.

Bacteria live in many dynamic environments with alternating cycles of feast or famine that have resulted in the evolution of mechanisms to quickly alter their metabolic capabilities. Such alterations often involve complex regulatory networks that modulate expression of genes involved in nutrient uptake and metabolism. A great number of protein regulators of metabolism have been characterized in depth. However, our ever-increasing understanding of the roles played by RNA regulators has revealed far greater complexity to regulation of metabolism in bacteria. Here, we review the mechanisms and functions of selected bacterial RNA regulators and discuss their importance in modulating nutrient uptake as well as primary and secondary metabolic pathways.

A previously healthy Chinese male returned from working in the Malaysian jungle with a fever. A blood culture grew Gram-negative bacilli that were initially identified as Burkholderia cepacia by the VITEK 2 system but were subsequently found to be Burkholderia pseudomallei by partial sequencing of the 16S rRNA gene. The identification of B. pseudomallei using commercially available automated systems is problematic and clinicians in non-endemic areas should be aware of the possibility of melioidosis in patients with a relevant travel history and blood cultures growing Burkholderia spp.

Endosporin is used in veterinary for the prophylaxis and treatment of disbacteriosis, intestinal infections, festering wounds and postpartum pyoinflammatory complications in agricultural animals. The probiotic is based on two Bacillus strains which inhibit growth of a broad spectrum of pathogenic microorganisms and synthesise proteolytic enzymes and other biologically active secondary metabolites, particularly - polysaccharides. The activity of these two strains was supplementary. For the species identification of these strains, sequences of 16S rRNA genes and fatty acid content of cell walls were analysed. It was found that the both strains belong to B. velezensis. Limitations of application of 16S rRNA sequences for identification of closely related species are discussed in the paper. A method of 16S rRNA sequence profiling by polymorphic nucleotides was proposed. It was also shown that usefulness of Bacillus strains in probiotics is mostly based on their unique strain specific properties rather than on general species characteristics.

Trimethoprim/sulfamethoxazole is widely used in the treatment of infectious diseases caused by bacterial pathogens in aquaculture. However, the practice of antibiotic administration can promote the emergence of resistant strains of bacteria and result in a wane in efficacy over time. The objective of this study was to assess the effect of oral treatment with trimethoprim/sulfamethoxazole on the gastrointestinal (GI) microbiota of healthy gibel carp and those affected with bacterial enteritis. By using denaturing gradient gel electrophoresis (DGGE), the changes in the predominant bacterial communities were directly depicted for the first time. The main findings were (1) Actinobacteria, Firmicutes and Proteobacteria were the predominant phyla in the healthy gibel carp intestine; (2) administration of antibiotics had a more profound impact on the intestinal microflora of healthy fish than of the diseased ones; and (3) Enterobacteriaceae might be one of the major drug-resistant bacteria in the gibel carp intestine. This study provides an insight into the effect of antibiotic treatment on the establishment and colonization of fish GI microbiota and speculates on some possible drug-resistant bacteria.

Using dietary fibre to control childhood diarrhoea has rarely been discussed. However, dietary fibre is being proposed to prevent diarrhoea in piglets. The present study aimed to study the effects of introducing fibre in the post-weaning piglet diet and its particle size on the intestinal ecosystem before and after an experimental infection with Escherichia coli. A total of thirty-six post-weaning piglets were assigned to four experimental diets: a negative control (NC) diet, the same diet with 4 % wheat bran coarse (WBc) particle size or finely milled (WBF) and a positive control (PC) diet with an antibiotic. On day 9, animals were challenged with E. coli. Faecal and digesta samples were obtained before and after the experimental infection and changes in the microbial ecosystem were measured. Animals fed the WBc and the PC diets showed a significant reduction in the faecal score compared with the NC diet. The inclusion of WBc in the diet increased total volatile fatty acid concentration, reduced Bacteroidetes in the faeces before and after the experimental infection compared with the NC diet and increased Firmicutes at the end of the experiment. Based on the results, diarrhoea scours and the composition of the pig gut microbial community are modified by the inclusion of a relatively small amount of wheat bran in the diet, being the physical presentation of the fibre a determinant of that difference.

Background: Three species of feline haemoplasma are recognised: Mycoplasma haemofelis (Mhf), 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus Mycoplasma turicensis (CMt). This study compared a reverse line blot hybridization (RLB) assay for simultaneous detection of Mhf, CMhm with three separate quantitative real-time polymerase chain reaction (qPCR) assays used for diagnosis of Mhf, CMhm and CMt. The RLB and qPCR assays were applied to DNA extracted from blood samples collected from 154 cats from Trinidad and Tobago., Results: CMhm and Mhf DNA were detected using both RLB and qPCR. CMt DNA was detected by qPCR only. Comparing RLB and qPCR for the detection of CMhm DNA, 40 (26.3%) and 48 (31.6%) cats, respectively, were positive. The difference was more marked for Mhf, with RLB detecting a total of only 11 (7.2%) positive cats whereas qPCR detected 41 (27.0%) positive cats. Using qPCR as a gold standard, haemoplasma infected cats were more likely to be retrovirus positive (OR = 5.68, P = 0.02) and older (median age 5.5 years), than non-infected cats. In addition, CMhm positive cats were more likely to be male (OR = 3.4, P = 0.04)., Conclusions: Overall the qPCR was more sensitive than RLB. In addition, age (median 5.5 years) and retrovirus positivity were risk factors for infection with the feline haemoplasmas in this study population. Further studies on feline haemoplasma infections in cats are needed to determine the significance of detecting small amounts of haemoplasma DNA, feline retrovirus infection and other associated risk factors on the clinical manifestation of disease.

Toxin-antitoxin systems are widespread in bacteria and archaea. They perform diverse functional roles, including the generation of persistence, maintenance of genetic loci and resistance to bacteriophages through abortive infection. Toxin-antitoxin systems have been divided into three types, depending on the nature of the interacting macromolecules. The recently discovered Type III toxin-antitoxin systems encode protein toxins that are inhibited by pseudoknots of antitoxic RNA, encoded by short tandem repeats upstream of the toxin gene. Recent studies have identified the range of Type I and Type II systems within current sequence databases. Here, structure-based homology searches were combined with iterative protein sequence comparisons to obtain a current picture of the prevalence of Type III systems. Three independent Type III families were identified, according to toxin sequence similarity. The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria. Functional assays confirmed that representatives from all three families act as toxin-antitoxin loci within Escherichia coli and at least two of the families confer resistance to bacteriophages. This study shows that active Type III toxin-antitoxin systems are far more diverse than previously known, and suggests that more remain to be identified.

Background: The rhesus monkey is an important animal model to study human vaginal health to which lactic acid bacteria play a significant role. However, the vaginal lactic acid bacterial species richness and relative abundance in rhesus monkeys is largely unknown., Methods: Vaginal swab samples were aseptically obtained from 200 reproductive-aged female rhesus monkeys. Following Rogosa agar plating, single bacterial colonies representing different morphotypes were isolated and analyzed for whole-cell protein profile, species-specific polymerase chain reaction, and 16S rRNA gene sequence., Results:   A total of 510 Lactobacillus strains of 17 species and one Pediococcus acidilactici were identified. The most abundant species was Lactobacillus reuteri, which colonized the vaginas of 86% monkeys. Lactobacillus johnsonii was the second most abundant species, which colonized 36% of monkeys. The majority of monkeys were colonized by multiple Lactobacillus species., Conclusions: The vaginas of rhesus monkeys are frequently colonized by multiple Lactobacillus species, dominated by L. reuteri., (© 2012 John Wiley & Sons A/S.)

Aim: To evaluate the microbial ecology of the coronal and apical segments of infected root canal systems using a complete sampling technique and next-generation sequencing., Methodology: The roots of 23 extracted teeth with apical periodontitis were sectioned in half, horizontally, and cryo-pulverized. Bacterial communities were profiled using tagged 454 pyrosequencing of the 16S rDNA hypervariable V5-V6 region., Results: The sequences were classified into 606 taxa (species or higher taxon), representing 24 bacterial phyla or candidate divisions and one archaeal phylum. Proteobacteria were more abundant in the apical samples (P < 0.05), whilst Actinobacteria were in significantly higher proportions in the coronal samples. The apical samples harboured statistically significantly more taxa than the coronal samples (P = 0.01) and showed a higher microbial diversity. Several taxa belonging to fastidious obligate anaerobes were significantly more abundant in the apical segments of the roots compared with their coronal counterparts., Conclusions: Endodontic infections are more complex than reported previously. The apical part of the root canal system drives the selection of a more diverse and more anaerobic community than the coronal part. The presence of a distinct ecological niche in the apical region explains the difficulty of eradication of the infection and emphasizes the need for new treatment approaches to be developed., (© 2012 International Endodontic Journal.)

Upon starvation, the bacterium Bacillus subtilis enters the process of sporulation, lasting several hours and culminating in formation of a spore, the most resilient cell type known. We show that a few days following sporulation, the RNA profile of spores is highly dynamic. In aging spores incubated at high temperatures, RNA content is globally decreased by degradation over several days. This degradation might be a strategy utilized by the spore to facilitate its dormancy. However, spores kept at low temperature exhibit a different RNA profile with evidence supporting transcription. Further, we demonstrate that germination is affected by spore age, incubation temperature, and RNA state, implying that spores can acquire dissimilar characteristics at a time they are considered dormant. We propose that, in contrast to current thinking, entering dormancy lasts a few days, during which spores are affected by the environment and undergo corresponding molecular changes influencing their emergence from quiescence., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nucleic acid phylogenetic profiling (NAPP) classifies coding and non-coding sequences in a genome according to their pattern of conservation across other genomes. This procedure efficiently distinguishes clusters of functional non-coding elements in bacteria, particularly small RNAs and cis-regulatory RNAs, from other conserved sequences. In contrast to other non-coding RNA detection pipelines, NAPP does not require the presence of conserved RNA secondary structure and therefore is likely to identify previously undetected RNA genes or elements. Furthermore, as NAPP clusters contain both coding and non-coding sequences with similar occurrence profiles, they can be analyzed under a functional perspective. We recently improved the NAPP pipeline and applied it to a collection of 949 bacterial and 68 archaeal species. The database and web interface available at http://napp.u-psud.fr/ enable detailed analysis of NAPP clusters enriched in non-coding RNAs, graphical display of phylogenetic profiles, visualization of predicted RNAs in their genome context and extraction of predicted RNAs for use with genome browsers or other software.

Only few small RNAs (sRNAs) have been characterized in Mycobacterium tuberculosis and their role in regulatory networks is still poorly understood. Here we report a genome-wide characterization of sRNAs in M. tuberculosis integrating experimental and computational analyses. Global RNA-seq analysis of exponentially growing cultures of M. tuberculosis H37Rv had previously identified 1373 sRNA species. In the present report we show that 258 (19%) of these were also identified by microarray expression. This set included 22 intergenic sRNAs, 84 sRNAs mapping within 5'/3' UTRs, and 152 antisense sRNAs. Analysis of promoter and terminator consensus sequences identified sigma A promoter consensus sequences for 121 sRNAs (47%), terminator consensus motifs for 22 sRNAs (8.5%), and both motifs for 35 sRNAs (14%). Additionally, 20/23 candidates were visualized by Northern blot analysis and 5' end mapping by primer extension confirmed the RNA-seq data. We also used a computational approach utilizing functional enrichment to identify the pathways targeted by sRNA regulation. We found that antisense sRNAs preferentially regulated transcription of membrane-bound proteins. Genes putatively regulated by novel cis-encoded sRNAs were enriched for two-component systems and for functional pathways involved in hydrogen transport on the membrane.

Background: Salmonella enterica serotype Typhi is the cause of typhoid fever. It is a human-restricted pathogen, and few data exist on S. Typhi gene expression in humans., Methodology/principal Findings: We applied an RNA capture and amplification technique, Selective Capture of Transcribed Sequences (SCOTS), and microarray hybridization to identify S. Typhi transcripts expressed in the blood of five humans infected with S. Typhi in Bangladesh. In total, we detected the expression of mRNAs for 2,046 S. Typhi genes (44% of the S. Typhi genome) in human blood; expression of 912 genes was detected in all 5 patients, and expression of 1,100 genes was detected in 4 or more patients. Identified transcripts were associated with the virulence-associated PhoP regulon, Salmonella pathogenicity islands, the use of alternative carbon and energy sources, synthesis and transport of iron, thiamine, and biotin, and resistance to antimicrobial peptides and oxidative stress. The most highly represented group were genes currently annotated as encoding proteins designated as hypothetical, unknown, or unclassified. Of the 2,046 detected transcripts, 1,320 (29% of the S. Typhi genome) had significantly different levels of detection in human blood compared to in vitro cultures; detection of 141 transcripts was significantly different in all 5 patients, and detection of 331 transcripts varied in at least 4 patients. These mRNAs encode proteins of unknown function, those involved in energy metabolism, transport and binding, cell envelope, cellular processes, and pathogenesis. We confirmed increased expression of a subset of identified mRNAs by quantitative-PCR., Conclusions/significance: We report the first characterization of bacterial transcriptional profiles in the blood of patients with typhoid fever. S. Typhi is an important global pathogen whose restricted host range has greatly inhibited laboratory studies. Our results suggest that S. Typhi uses a largely uncharacterized genetic repertoire to survive within cells and utilize alternate energy sources during infection.

sRNA-Xcc1 is a trans-acting sRNA recently identified from the plant pathogenic bacterium Xanthomonas campestris pathovar campestris (Xcc). Here, the phylogenetic distribution, predicted secondary structure and regulation of expression of sRNA-Xcc1 were analyzed. The analysis showed (1) a total 81 sRNA-Xcc1 homologs that are found in some bacterial strains that are taxonomically unrelated, belonging to the α-, β-, γ- and δ-proteobacteria (2) that some sRNA-Xcc1 homologs are located in a plasmid-borne transposon or near a transposase coding gene, (3) that sRNA-Xcc1 is encoded by a integron gene cassette in Xcc and sRNA-Xcc1 homologs occur in integron gene cassettes of some uncultured bacteria and (4) that sRNA-Xcc1 homologs have a highly conserved sequence motif and a stable consensus secondary structure. These findings strongly support the idea that sRNA-Xcc1 represents a novel family of sRNAs which may be originally captured by integrons from natural environments and then spread among different bacterial species via horizontal gene transfer, possibly by means of transposons and plasmids. The expression analysis results demonstrated that the transcription of sRNA-Xcc1 is under the positive control of the key virulence regulators HrpG and HrpX, indicating that sRNA-Xcc1 may be involved in the virulence regulation of Xcc.

An expanding number of metabolite-binding riboswitch classes are being discovered in the noncoding portions of bacterial genomes. Findings over the last decade indicate that bacteria commonly use these RNA genetic elements as regulators of metabolic pathways and as mediators of changes in cell physiology. Some riboswitches are surprisingly complex, and they rival protein factors in their structural and functional sophistication. Each new riboswitch discovery expands our knowledge of the biochemical capabilities of RNA, and some give rise to new questions that require additional study to be addressed. Some of the greatest prospects for riboswitch research and some of the more interesting mysteries are discussed in this review., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Regulatory small RNAs (sRNAs), also known as non-coding RNA, are not translated into proteins and widespread in prokaryotes and eukaryotes. sRNAs involve in multiple fundamental cellular events. They are emerging regulatory elements that are gaining momentum. Knowledge of sRNA largely originates from eukaryotes. Prokaryotic sRNAs, particularly those of pathogen are only recently explored. The main types, function, and methodology to predict pathogen sRNAs are summarized in this review. Special focus is sRNAs regulating pathogen gene expression, particularly that of Mycobacterium tuberculosis, which is hitherto the most successful pathogen afflicting mankind., (Copyright © 2010 Wiley-Liss, Inc.)

Background: The interest in non-coding RNAs (ncRNAs) constantly rose during the past few years because of the wide spectrum of biological processes in which they are involved. This led to the discovery of numerous ncRNA genes across many species. However, for most organisms the non-coding transcriptome still remains unexplored to a great extent. Various experimental techniques for the identification of ncRNA transcripts are available, but as these methods are costly and time-consuming, there is a need for computational methods that allow the detection of functional RNAs in complete genomes in order to suggest elements for further experiments. Several programs for the genome-wide prediction of functional RNAs have been developed but most of them predict a genomic locus with no indication whether the element is transcribed or not., Results: We present NOCORNAc, a program for the genome-wide prediction of ncRNA transcripts in bacteria. NOCORNAc incorporates various procedures for the detection of transcriptional features which are then integrated with functional ncRNA loci to determine the transcript coordinates. We applied RNAz and NOCORNAc to the genome of Streptomyces coelicolor and detected more than 800 putative ncRNA transcripts most of them located antisense to protein-coding regions. Using a custom design microarray we profiled the expression of about 400 of these elements and found more than 300 to be transcribed, 38 of them are predicted novel ncRNA genes in intergenic regions. The expression patterns of many ncRNAs are similarly complex as those of the protein-coding genes, in particular many antisense ncRNAs show a high expression correlation with their protein-coding partner., Conclusions: We have developed NOCORNAc, a framework that facilitates the automated characterization of functional ncRNAs. NOCORNAc increases the confidence of predicted ncRNA loci, especially if they contain transcribed ncRNAs. NOCORNAc is not restricted to intergenic regions, but it is applicable to the prediction of ncRNA transcripts in whole microbial genomes. The software as well as a user guide and example data is available at http://www.zbit.uni-tuebingen.de/pas/nocornac.htm.

The bacterial RNA network includes most of the same components found in eukaryotes, and many of the interactions that under lie transcription, RNA processing and stability, translation, and protein secretion are conserved. The major difference is that all of these functions take place in a single cellular compartment. In spite of the absence of membrane-bound organelles, or in some cases because of it, key components of the RNA network are localized to specific subcellular spaces or structures to ensure proper processing and regulation. This chapter focuses on what is known about subcellular localization of the bacterial RNA network and what insights localization provides to regulation of the RNA infrastructure of the cell.

Exploitation of microbial wealth, of which almost 95% or more is still unexplored, is a growing need. The taxonomic placements of a new isolate based on phenotypic characteristics are now being supported by information preserved in the 16S rRNA gene. However, the analysis of 16S rDNA sequences retrieved from metagenome, by the available bioinformatics tools, is subject to limitations. In this study, the occurrences of nucleotide features in 16S rDNA sequences have been used to ascertain the taxonomic placement of organisms. The tetra- and penta-nucleotide features were extracted from the training data set of the 16S rDNA sequence, and was subjected to an artificial neural network (ANN) based tool known as self-organizing map (SOM), which helped in visualization of unsupervised classification. For selection of significant features, principal component analysis (PCA) or curvilinear component analysis (CCA) was applied. The SOM along with these techniques could discriminate the sample sequences with more than 90% accuracy, highlighting the relevance of features. To ascertain the confidence level in the developed classification approach, the test data set was specifically evaluated for Thiobacillus, with Acidiphilium, Paracocus and Starkeya, which are taxonomically reassigned. The evaluation proved the excellent generalization capability of the developed tool. The topology of genera in SOM supported the conventional chemo-biochemical classification reported in the Bergey manual.

This is the first report, to our knowledge, of two temporally and geographically related nosocomial Asaia lannensis infections in a paediatric setting. Two patients with idiopathic dilated cardiomyopathy awaiting cardiac transplantation developed bacteraemia during their hospital stay. The physical location of both patients, the temporal association of infections, as well as the isolation of two identical pathogens suggested a nosocomial transmission. Commonly used identification methods and instruments failed to identify the isolated pathogens and only 16S rRNA gene sequencing provided definitive identification. These isolates of A. lannensis showed an unfavourable susceptibility pattern including resistance to carbapenems, all beta-lactam agents and fluoroquinolones.

Small, hydrophobic proteins whose synthesis is repressed by small RNAs (sRNAs), denoted type I toxin-antitoxin modules, were first discovered on plasmids where they regulate plasmid stability, but were subsequently found on a few bacterial chromosomes. We used exhaustive PSI-BLAST and TBLASTN searches across 774 bacterial genomes to identify homologs of known type I toxins. These searches substantially expanded the collection of predicted type I toxins, revealed homology of the Ldr and Fst toxins, and suggested that type I toxin-antitoxin loci are not spread by horizontal gene transfer. To discover novel type I toxin-antitoxin systems, we developed a set of search parameters based on characteristics of known loci including the presence of tandem repeats and clusters of charged and bulky amino acids at the C-termini of short proteins containing predicted transmembrane regions. We detected sRNAs for three predicted toxins from enterohemorrhagic Escherichia coli and Bacillus subtilis, and showed that two of the respective proteins indeed are toxic when overexpressed. We also demonstrated that the local free-energy minima of RNA folding can be used to detect the positions of the sRNA genes. Our results suggest that type I toxin-antitoxin modules are much more widely distributed among bacteria than previously appreciated.

Background: Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. The severity of these infections is highly variable and depends on multiple parameters including the genome content of the bacterium as well as the condition of the infected host. Clinically and epidemiologically, S. aureus shows a particular capacity to survive and adapt to drastic environmental changes including the presence of numerous antimicrobial agents. Mechanisms triggering this adaptation remain largely unknown despite important research efforts. Most studies evaluating gene content have so far neglected to analyze the so-called intergenic regions as well as potential antisense RNA molecules., Principal Findings: Using high-throughput sequencing technology, we performed an inventory of the whole transcriptome of S. aureus strain N315. In addition to the annotated transcription units, we identified more than 195 small transcribed regions, in the chromosome and the plasmid of S. aureus strain N315. The coding strand of each transcript was identified and structural analysis enabled classification of all discovered transcripts. RNA purified at four time-points during the growth phase of the bacterium allowed us to define the temporal expression of such transcripts. A selection of 26 transcripts of interest dispersed along the intergenic regions was assessed for expression changes in the presence of various stress conditions including pH, temperature, oxidative shocks and growth in a stringent medium. Most of these transcripts showed expression patterns specific for the defined stress conditions that we tested., Conclusions: These RNA molecules potentially represent important effectors of S. aureus adaptation and more generally could support some of the epidemiological characteristics of the bacterium.

The lone star tick, Amblyomma americanum (Acari: Ixodidae), is commonly reported from people and animals throughout the eastern U.S. and is associated with transmission of a number of emerging diseases. To better define the microbial communities within lone star ticks, 16S rRNA gene based analysis using bacteria-wide primers, followed by sequencing of individual clones (n = 449) was used to identify the most common bacterial operational taxonomic units (OTUs) present within colony-reared and wild A. americanum. The colony-reared ticks contained primarily sequence affiliated with members of the genus Coxiella (89%; 81/91), common endosymbionts of ticks, and Brevibacterium (11%; 10/91). Similarly, analysis of clones from unfed wild lone star ticks revealed that 96.7% (89/92) of all the OTUs identified were affiliated with Coxiella-like endosymbionts, as compared with only 5.1-11.7% (5/98-9/77) of those identified from wild lone star ticks after feeding. In contrast, the proportion of OTUs identified as Rickettsia sp. in wild-caught ticks increased from 2.2% (2/92) before feeding to as high as 46.8% (36/77) after feeding, and all Rickettsia spp. sequences recovered were most similar to those described from the spotted fever group Rickettsia, specifically R. amblyommii and R. massiliae. Additional characterization of the Rickettsiales tick community by polymerase chain reaction, cloning, and sequencing of 17 kDa and gltA genes confirmed these initial findings and suggested that novel Rickettsia spp. are likely present in these ticks. These data provide insight into the overall, as well as the rickettsial community of wild lone star ticks and may ultimately aid in identification of novel pathogens transmitted by A. americanum.

Identification and characterization of functional elements in the noncoding regions of genomes is an elusive and time-consuming activity whose output does not keep up with the pace of genome sequencing. Hundreds of bacterial genomes lay unexploited in terms of noncoding sequence analysis, although they may conceal a wide diversity of novel RNA genes, riboswitches, or other regulatory elements. We describe a strategy that exploits the entirety of available bacterial genomes to classify all noncoding elements of a selected reference species in a single pass. This method clusters noncoding elements based on their profile of presence among species. Most noncoding RNAs (ncRNAs) display specific signatures that enable their grouping in distinct clusters, away from sequence conservation noise and other elements such as promoters. We submitted 24 ncRNA candidates from Staphylococcus aureus to experimental validation and confirmed the presence of seven novel small RNAs or riboswitches. Besides offering a powerful method for de novo ncRNA identification, the analysis of phylogenetic profiles opens a new path toward the identification of functional relationships between co-evolving coding and noncoding elements.

The impact of a moderate consumption of an instant coffee on the general composition of the human intestinal bacterial population was assessed in this study. Sixteen (16) healthy adult volunteers consumed a daily dose of 3 cups of coffee during 3 weeks. Faecal samples were collected before and after the consumption of coffee, and the impact of the ingestion of the product on the intestinal bacteria as well as the quantification of specific bacterial groups was assessed using nucleic acid-based methods. Although faecal profiles of the dominant microbiota were not significantly affected after the consumption of the coffee (Dice's similarity index=92%, n=16), the population of Bifidobacterium spp. increased after the 3-week test period (P=0.02). Moreover, in some subjects, there was a specific increase in the metabolic activity of Bifidobacterium spp. Our results show that the consumption of the coffee preparation resulting from water co-extraction of green and roasted coffee beans produce an increase in the metabolic activity and/or numbers of the Bifidobacterium spp. population, a bacterial group of reputed beneficial effects, without major impact on the dominant microbiota.

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (10(1) to 10(6)) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortutitum, M. scrofalaceum, M. intracellular, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR.

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed.