Gene expression differences associated with intrinsic hindfoot muscle loss in the jerboa, Jaculus jaculus.

Vertebrate animals that run or jump across sparsely vegetated habitats, such as horses and jerboas, have reduced the number of distal limb bones, and many have lost most or all distal limb muscle. We previously showed that nascent muscles are present in the jerboa hindfoot at birth and that these myofibers are rapidly and completely lost soon after by a process that shares features with pathological skeletal muscle atrophy. Here, we apply an intra- and interspecies differential RNA-Seq approach, comparing jerboa and mouse muscles, to identify gene expression differences associated with the initiation and progression of jerboa hindfoot muscle loss. We show evidence for reduced hepatocyte growth factor and fibroblast growth factor signaling and an imbalance in nitric oxide signaling; all are pathways that are necessary for skeletal muscle development and regeneration. We also find evidence for phagosome formation, which hints at how myofibers may be removed by autophagy or by nonprofessional phagocytes without evidence for cell death or immune cell activation. Last, we show significant overlap between genes associated with jerboa hindfoot muscle loss and genes that are differentially expressed in a variety of human muscle pathologies and rodent models of muscle loss disorders. All together, these data provide molecular insight into the process of evolutionary and developmental muscle loss in jerboa hindfeet.

An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important gram-positive bacterium.

The gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. However, little is known about the bacterium's transcriptome architecture and mechanisms of posttranscriptional control. Here, we have applied transcription start site and termination mapping to generate a single-nucleotide-resolution RNA map of C. difficile 5' and 3' untranslated regions, operon structures, and noncoding regulators, including 42 sRNAs. Our results indicate functionality of many conserved riboswitches and predict cis-regulatory RNA elements upstream of multidrug resistance (MDR)-type ATP-binding cassette (ABC) transporters and transcriptional regulators. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, the functions of Hfq-based posttranscriptional regulatory networks in gram-positive pathogens remain controversial. Using Hfq immunoprecipitation followed by sequencing of bound RNA species (RIP-seq), we identify a large cohort of transcripts bound by Hfq and show that absence of Hfq affects transcript stabilities and steady-state levels. We demonstrate sRNA expression during intestinal colonization by C. difficile and identify infection-related signals impacting its expression. As a proof of concept, we show that the utilization of the abundant intestinal metabolite ethanolamine is regulated by the Hfq-dependent sRNA CDIF630nc_085. Overall, our study lays the foundation for understanding clostridial riboregulation with implications for the infection process and provides evidence for a global role of Hfq in posttranscriptional regulation in a gram-positive bacterium.

Defining the transcription landscape of the Gram-negative marine bacterium Vibrio harveyi.

Vibrio harveyi is a Gram-negative pathogenic bacterium ubiquitously present in natural aquatic systems. Although environmental adaptability in V. harveyi may be enabled by profound reprogramming of gene expression previously observed during responses to starvation, suboptimal temperatures and other stress factors, the key characteristics of V. harveyi transcripts and operons, such as their boundaries and size as well as location of small RNA genes, remain largely unknown. To reveal the main features of the V. harveyi transcriptome, total RNA of this organism was analyzed by differential RNA sequencing (dRNA-seq). Analysis of the dRNA-seq data made it possible to define the primary transcriptome of V. harveyi along with cis-acting regulatory elements (riboswitches and leader sequences) and new genes. The latter encode a number of putative polypeptides and new phylogenetically conserved antisense RNAs potentially involved in the post-transcriptional control of gene expression.

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea.

Prokaryotic genomes show a high level of information compaction often with different molecules transcribed from the same locus. Although antisense RNAs have been relatively well studied, RNAs in the same strand, internal RNAs (intraRNAs), are still poorly understood. The question of how common is the translation of overlapping reading frames remains open. We address this question in the model archaeon Halobacterium salinarum. In the present work we used differential RNA-seq (dRNA-seq) in H. salinarum NRC-1 to locate intraRNA signals in subsets of internal transcription start sites (iTSS) and establish the open reading frames associated to them (intraORFs). Using C-terminally flagged proteins, we experimentally observed isoforms accurately predicted by intraRNA translation for kef1, acs3 and orc4 genes. We also recovered from the literature and mass spectrometry databases several instances of protein isoforms consistent with intraRNA translation such as the gas vesicle protein gene gvpC1. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone dnaK and the aerobic respiration related cydA in both H. salinarum and Escherichia coli. Also, intraRNA translation evidence in H. salinarum, E. coli and yeast of a universal elongation factor (aEF-2, fusA and eEF-2) suggests that this is an ancient phenomenon present in all domains of life.

The Streptococcus suis transcriptional landscape reveals adaptation mechanisms in pig blood and cerebrospinal fluid.

Streptococcus suis (SS) is an important pathogen of pigs, and it is also recognized as a zoonotic agent for humans. SS infection may result in septicemia or meningitis in the host. However, little is known about genes that contribute to the virulence process and survival within host blood or cerebrospinal fluid (CSF). Small RNAs (sRNA) have emerged as key regulators of virulence in several bacteria, but they have not been investigated in SS. Here, using a differential RNA-sequencing approach and RNAs from SS strain P1/7 grown in rich medium, pig blood, or CSF, we present the SS genome-wide map of 793 transcriptional start sites and 370 operons. In addition to identifying 29 sRNAs, we show that five sRNA deletion mutants attenuate SS virulence in a zebrafish infection model. Homology searches revealed that 10 sRNAs were predicted to be present in other pathogenic Streptococcus species. Compared with wild-type strain P1/7, sRNAs rss03, rss05, and rss06 deletion mutants were significantly more sensitive to killing by pig blood. It is possible that rss06 contributes to SS virulence by indirectly activating expression of SSU0308, a virulence gene encoding a zinc-binding lipoprotein. In blood, genes involved in the synthesis of capsular polysaccharide (CPS) and subversion of host defenses were up-regulated. In contrast, in CSF, genes for CPS synthesis were down-regulated. Our study is the first analysis of SS sRNAs involved in virulence and has both improved our understanding of SS pathogenesis and increased the number of sRNAs known to play definitive roles in bacterial virulence.

Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori.

The global mapping of transcription boundaries is a key step in the elucidation of the full complement of transcriptional features of an organism. It facilitates the annotation of operons and untranslated regions as well as novel transcripts, including cis- and trans-encoded small RNAs (sRNAs). So called RNA sequencing (RNA-seq) based on deep sequencing of cDNAs has greatly facilitated transcript mapping with single nucleotide resolution. However, conventional RNA-seq approaches typically cannot distinguish between primary and processed transcripts. Here we describe the recently developed differential RNA-seq (dRNA-seq) approach, which facilitates the annotation of transcriptional start sites (TSS) based on deep sequencing of two differentially treated cDNA library pairs, with one library being enriched for primary transcripts. Using the human pathogen Helicobacter pylori as a model organism, we describe the application of dRNA-seq together with an automated TSS annotation approach for generation of a genome-wide TSS map in bacteria. Besides a description of transcriptome and regulatory features that can be identified by this approach, we discuss the impact of different library preparation protocols and sequencing platforms as well as manual and automated TSS annotation. Moreover, we have set up an easily accessible online browser for visualization of the H. pylori transcriptome data from this and our previous H. pylori dRNA-seq study.

Riboregulators and the role of Hfq in photosynthetic bacteria.

Anoxygenic and oxygenic bacteria directly convert solar energy into biomass using photosynthesis. The formation and composition of photosynthetic complexes has to be tightly controlled in response to environmental conditions, as exposure to sunlight can be harmful due to the generation of reactive oxygen species and the damaging effects of UV irradiation. Therefore, photosynthetic bacteria are exposed to a particular set of regulatory challenges in addition to those that also affect other bacteria, requiring sophisticated regulatory systems. Indeed, hundreds of potential regulatory RNAs have been identified in photosynthetic model bacteria as well as antisense RNAs (asRNAs) of up to several kb in length that protect certain mRNAs from degradation. The trans-acting small non-coding RNAs (sRNAs), PcrZ and PsrR1, control pigment and photosystem biogenesis in Rhodobacter sphaeroides and cyanobacteria, respectively. The asRNAs IsrR and As1_flv4 act as negative regulators and the asRNAs PsbA2R and PsbA3R as positive effectors of photosynthesis gene expression in Synechocystis 6803.

Transcriptome architecture of the three main lineages of agrobacteria.

Agrobacteria are a diverse, polyphyletic group of prokaryotes with multipartite genomes capable of transferring DNA into the genomes of host plants, making them an essential tool in plant biotechnology. Despite their utility in plant transformation, genome-wide transcriptional regulation is not well understood across the three main lineages of agrobacteria. Transcription start sites (TSSs) are a necessary component of gene expression and regulation. In this study, we used differential RNA-seq and a TSS identification algorithm optimized on manually annotated TSS, then validated with existing TSS to identify thousands of TSS with nucleotide resolution for representatives of each lineage. We extend upon the 356 TSSs previously reported in Agrobacterium fabrum C58 by identifying 1,916 TSSs. In addition, we completed genomes and phenotyping of Rhizobium rhizogenes C16/80 and Allorhizobium vitis T60/94, identifying 2,650 and 2,432 TSSs, respectively. Parameter optimization was crucial for an accurate, high-resolution view of genome and transcriptional dynamics, highlighting the importance of algorithm optimization in genome-wide TSS identification and genomics at large. The optimized algorithm reduced the number of TSSs identified internal and antisense to the coding sequence on average by 90.5% and 91.9%, respectively. Comparison of TSS conservation between orthologs of the three lineages revealed differences in cell cycle regulation of ctrA as well as divergence of transcriptional regulation of chemotaxis-related genes when grown in conditions that simulate the plant environment. These results provide a framework to elucidate the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. IMPORTANCE Transcription start sites (TSSs) are fundamental for understanding gene expression and regulation. Agrobacteria, a group of prokaryotes with the ability to transfer DNA into the genomes of host plants, are widely used in plant biotechnology. However, the genome-wide transcriptional regulation of agrobacteria is not well understood, especially in less-studied lineages. Differential RNA-seq and an optimized algorithm enabled identification of thousands of TSSs with nucleotide resolution for representatives of each lineage. The results of this study provide a framework for elucidating the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. The optimized algorithm also highlights the importance of parameter optimization in genome-wide TSS identification and genomics at large.

Cr(VI)-bioremediation mechanism of a novel strain Bacillus paramycoides Cr6 with the powerful ability to remove Cr(VI) from contaminated water.

This research provided an excellent novel hexavalent chromium (Cr(VI))-removal bacterium, Bacillus paramycoides Cr6, and investigated its removal mechanism from the perspective of molecular biology. Cr6 could resist up to 2500 mg/L Cr(VI), and the removal rate of 2000 mg/L Cr(VI) reached 67.3% under the optimal culture conditions of 220 r/min, pH 8 and 31 â„ƒ. When the initial concentration of Cr(VI) was 200 mg/L, Cr6 had a removal rate of 100% within 18 h. The differential transcriptome analysis identified two key structural genes named bcr005 and bcb765 of Cr6, which were upregulated by Cr(VI). Their functions were predicted and further confirmed by bioinformatic analyses and in vitro experiments. bcr005 encodes Cr(VI)-reductase BCR005, and bcb765 encodes Cr(VI)-binding protein BCB765. Real-time fluorescent quantitative PCRs were performed, and the data illustrated a parallel pathway (one is Cr(VI) reduction, and the other is Cr(VI) immobilisation) of Cr6 to remove Cr(VI), which relies on the synergistic expression of the genes bcr005 and bcb765 induced by different concentrations of Cr(VI). In summary, a deeper molecular mechanism of Cr(VI) microorganism removal was elaborated; Bacillus paramycoides Cr6 was an exceptional novel Cr(VI)-removed bacterial resource, while BCR005 and BCB765 were two new-found efficient enzymes that have potential practical applications for sustainable microbial remediation of Cr-contaminated water.

Sugarcane Genotypes with Contrasting Biological Nitrogen Fixation Efficiencies Differentially Modulate Nitrogen Metabolism, Auxin Signaling, and Microorganism Perception Pathways.

Sugarcane is an economically important crop that is used for the production of fuel ethanol. Diazotrophic bacteria have been isolated from sugarcane tissues, without causing visible plant anatomical changes or disease symptoms. These bacteria can be beneficial to the plant by promoting root growth and an increase in plant yield. Different rates of Biological Nitrogen Fixation (BNF) were observed in different genotypes. The aim of this work was to conduct a comprehensive molecular and physiological analysis of two model genotypes for contrasting BNF efficiency in order to unravel plant genes that are differentially regulated during a natural association with diazotrophic bacteria. A next-generation sequencing of RNA samples from the genotypes SP70-1143 (high-BNF) and Chunee (low-BNF) was performed. A differential transcriptome analysis showed that several pathways were differentially regulated among the two BNF-contrasting genotypes, including nitrogen metabolism, hormone regulation and bacteria recognition. Physiological analyses, such as nitrogenase and GS activity quantification, bacterial colonization, auxin response and root architecture evaluation, supported the transcriptome expression analyses. The differences observed between the genotypes may explain, at least in part, the differences in BNF contributions. Some of the identified genes might be involved in key regulatory processes for a beneficial association and could be further used as tools for obtaining more efficient BNF genotypes.

Genomic and Transcriptomic Investigation of the Physiological Response of the Methylotroph Bacillus methanolicus to 5-Aminovalerate.

The methylotrophic thermophile Bacillus methanolicus can utilize the non-food substrate methanol as its sole carbon and energy source. Metabolism of L-lysine, in particular its biosynthesis, has been studied to some detail, and methanol-based L-lysine production has been achieved. However, little is known about L-lysine degradation, which may proceed via 5-aminovalerate (5AVA), a non-proteinogenic ω-amino acid with applications in bioplastics. The physiological role of 5AVA and related compounds in the native methylotroph was unknown. Here, we showed that B. methanolicus exhibits low tolerance to 5AVA, but not to related short-chain (C4-C6) amino acids, diamines, and dicarboxylic acids. In order to gain insight into the physiological response of B. methanolicus to 5AVA, transcriptomic analyses by differential RNA-Seq in the presence and absence of 5AVA were performed. Besides genes of the general stress response, RNA levels of genes of histidine biosynthesis, and iron acquisition were increased in the presence of 5AVA, while an Rrf2 family transcriptional regulator gene showed reduced RNA levels. In order to test if mutations can overcome growth inhibition by 5AVA, adaptive laboratory evolution (ALE) was performed and two mutants-AVA6 and AVA10-with higher tolerance to 5AVA were selected. Genome sequencing revealed mutations in genes related to iron homeostasis, including the gene for an iron siderophore-binding protein. Overexpression of this mutant gene in the wild-type (WT) strain MGA3 improved 5AVA tolerance significantly at high Fe2+ supplementation. The combined ALE, omics, and genetics approach helped elucidate the physiological response of thermophilic B. methanolicus to 5AVA and will guide future strain development for 5AVA production from methanol.

Characterizing the Role of Exoribonucleases in the Control of Microbial Gene Expression: Differential RNA-Seq.

Differential RNA-Seq is a next-generation technology method to determine the significant transcriptomic differences between two and more samples. With this method it is possible to analyze the total RNA content of different samples making it the best global analysis method currently available to study the roles of exoribonucleases in the cell. These enzymes are responsible for the RNA processing and degradation in the cells and therefore affect the total RNA pool in ways not yet fully understood. In Escherichia coli there are three main degradative exoribonucleases RNase II, RNase R, and PNPase that degrade the RNA from the 3' to the 5'-end. These enzymes have several roles in the cell and even though they are degradative enzymes RNase II and PNPase can also protect some RNAs from degradation and PNPase can also act as an RNA polymerase under some conditions. The multiplicity of roles of these exoribonucleases leads to a very high number of transcripts that are affected by their absence in the cell. With the differential RNA-Seq it is possible to obtain a much deeper understanding of how these enzymes work and regulate the bacterial gene expression. In this chapter we have described a differential RNA-Seq data analysis protocol applied to the study of exoribonucleases. We also included the protocol for experimental validation of the RNA-Seq data using qPCR and motility assays. Although the methods described in this chapter were applied to the study of the exoribonucleases, they can also be used for other differential RNA-Seq studies.

Regulatory RNAs in photosynthetic cyanobacteria.

Regulatory RNAs play versatile roles in bacteria in the coordination of gene expression during various physiological processes, especially during stress adaptation. Photosynthetic bacteria use sunlight as their major energy source. Therefore, they are particularly vulnerable to the damaging effects of excess light or UV irradiation. In addition, like all bacteria, photosynthetic bacteria must adapt to limiting nutrient concentrations and abiotic and biotic stress factors. Transcriptome analyses have identified hundreds of potential regulatory small RNAs (sRNAs) in model cyanobacteria such as Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, and in environmentally relevant genera such as Trichodesmium, Synechococcus and Prochlorococcus. Some sRNAs have been shown to actually contain µORFs and encode short proteins. Examples include the 40-amino-acid product of the sml0013 gene, which encodes the NdhP subunit of the NDH1 complex. In contrast, the functional characterization of the non-coding sRNA PsrR1 revealed that the 131 nt long sRNA controls photosynthetic functions by targeting multiple mRNAs, providing a paradigm for sRNA functions in photosynthetic bacteria. We suggest that actuatons comprise a new class of genetic elements in which an sRNA gene is inserted upstream of a coding region to modify or enable transcription of that region.