Background: Although posttranscriptional modification of mitochondrial (mt) transcripts plays key roles in completion of the coding information and in the expression of mtDNA-encoded genes, there is little experimental evidence on the polyadenylation status and the location of mt gene poly(A) sites for non-human mammals. Results: Poly(A)-enriched RNA-Seq reads collected for two wild-caught bank voles (Clethrionomys glareolus) were mapped to the complete mitochondrial genome of that species. Transcript polyadenylation was detected as unmapped adenine residues at the ends of the mapped reads. Where the tRNA punctuation model applied, there was the expected polyadenylation, except for the nad5 transcript, whose polyadenylated 3′ end is at an intergenic sequence/cytochrome b boundary. As in human, two pairs of bank vole genes, nad4l/nad4 and atp8/atp6, are expressed from bicistronic transcripts. TAA stop codons of four bank vole protein-coding genes (nad1, atp6, cox3 and nad4) are incompletely encoded in the DNA and are completed by polyadenylation. This is three genes (nad2, nad3 and cob) less than in human. The bank vole nad2 gene encodes a full stop codon (TAA in one vole and TAG in the other), which is followed by a 2 bp UTR and the gene conforms to the tRNA punctuation model. In contrast, the annotations of the reference mouse and some other rodent mt genomes in GenBank include complete TAG stop codons in both nad1 and nad2, which overlap downstream trnI and trnW, respectively. Thus the RNA-Seq data of bank voles provides a model for stop codons of mt-encoded genes in mammals comparable to humans, but at odds with some of the interpretation based purely on genomic data in mouse and other rodents. Conclusions: This work demonstrates how RNA-Seq data were useful to recover mtDNA transcriptome data in a non-model rodent and to shed more light on mammalian mtDNA transcriptome and post-transcriptional modification. Even though gene content and organisation of mtDNA are strongly conserved among mammals, annotations that neglect the transcriptome may be prone to errors in relation to the stop codons.

Background: Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs. Results: Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and % predicted D L CO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples. Conclusions: We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and D L CO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.

Background: Trichomes, developing from the epidermis of nearly all terrestrial plants, provide good structural resistance against insect herbivores and an excellent model for studying the molecular mechanisms underlying cell fate determination. Regulation of trichomes in Rosids has been well characterized. However, little is known about the cell proliferation molecular processes during multicellular trichome formation in Asterids. Results: In this study, we identified two point mutations in a novel allele (Wo v ) at Wo locus. Ectopic expression of Wo v in tobacco and potato induces much more trichome formation than wild type. To gain new insights into the underlying mechanisms during the processes of these trichomes formation, we compared the gene expression profiles between Wo v transgenic and wild-type tobacco by RNA-seq analysis. A total of 544 co-DEGs were detected between transgenic and wild-type tobacco. Functional assignments of the co-DEGs indicated that 33 reliable pathways are altered in transgenic tobacco plants. The most noticeable pathways are fatty acid metabolism, amino acid biosynthesis and metabolism, and plant hormone signal transduction. Results suggest that these enhanced processes are critical for the cell proliferation during multicellular trichome formation in transgenic plants. In addition, the transcriptional levels of homologues of trichome regulators in Rosids were not significantly changed, whereas homologues of genes (Wo and SlCycB2) in Asterids were significantly upregulated in Wo v transgenic tobacco plants. Conclusions: This study presents a global picture of the gene expression changes induced by Wo v - gene in tobacco. And the results provided us new insight into the molecular processes controlling multicellular formation in tobacco. Furthermore, we inferred that trichomes in solanaceous species might share a common network.

Background: The migratory locust, Locusta migratoria manilensis, is an immensely destructive agricultural pest that forms a devastating and voracious gregarious phase. The fungal insect pathogen, Metarhizium acridum, is a specialized locust pathogen that has been used as a potent mycoinsecticide for locust control. Little, however, is known about locust immune tissue, i.e. fat body and hemocyte, responses to challenge by this fungus. Methods: RNA-seq (RNA sequencing) technology were applied to comparatively examine the different roles of locust fat body and hemocytes, the two major contributors to the insect immune response, in defense against M. acridum. According to the sequence identity to homologies of other species explored immune response genes, immune related unigenes were screened in all transcriptome wide range from locust and the differential expressed genes were identified in these two tissues, respectively. Results: Analysis of differentially expressed locust genes revealed 4660 and 138 up-regulated, and 1647 and 23 down-regulated transcripts in the fat body and hemocytes, respectively after inoculation with M. acridum spores. GO (Gene Ontology) enrichment analysis showed membrane biogenesis related proteins and effector proteins significantly differentially expressed in hemocytes, while the expression of energy metabolism and development related transcripts were enriched in the fat body after fungal infection. A total of 470 immune related unigenes were identified, including members of the three major insect immune pathways, i.e. Toll, Imd (immune deficiency) and JAK/STAT (janus kinase/signal transduction and activator of transcription). Of these, 58 and three were differentially expressed in the insect fat body or hemocytes after infection, respectively. Of differential expressed transcripts post challenge, 43 were found in both the fat body and hemocytes, including the LmLys4 lysozyme, representing a microbial cell wall targeting enzyme. Conclusions: These data indicate that locust fat body and hemocytes adopt different strategies in response to M. acridum infection. Fat body gene expression after M. acridum challenge appears to function mainly through activation of innate immune related genes, energy metabolism and development related genes. Hemocyte responses attempt to limit fungal infection primarily through regulation of membrane related genes and activation of cellular immune responses and release of humoral immune factors.

Background: N-ethyl-N-nitrosourea (ENU) mutagen has become the method of choice for inducing random mutations for forward genetics applications. However, distinguishing induced mutations from sequencing errors or sporadic mutations is difficult, which has hampered surveys of potential biases in the methodology in the past. Addressing this issue, we created a large cohort of mice with biological replicates enabling the confident calling of induced mutations, which in turn allowed us to conduct a comprehensive analysis of potential biases in mutation properties and genomic location. Results: In the exome sequencing data we observe the known preference of ENU to cause A : T ⇒ G : C transitions in longer genes. Mutations were frequently clustered and inherited in blocks hampering attempts to pinpoint individual causative mutations by genome analysis only. Furthermore, ENU mutations were biased towards areas in the genome that are accessible in testis, potentially limiting the scope of forward genetic approaches to only 1–10 % of the genome. Conclusion: ENU provides a powerful tool for exploring the genome-phenome relationship, however forward genetic applications that require the mutation to be passed on through the germ line may be limited to explore only genes that are accessible in testis.

Background: Alcohol abuse and alcoholism are significant public health problems, but the genetic basis for individual variation in alcohol sensitivity remains poorly understood. Drosophila melanogaster presents a powerful model system for dissecting the genetic underpinnings that determine individual variation in alcohol-related phenotypes. We performed genome wide association analyses for alcohol sensitivity using the sequenced, inbred lines of the D. melanogaster Genetic Reference Panel (DGRP) together with extreme QTL mapping in an advanced intercross population derived from sensitive and resistant DGRP lines. Results: The DGRP harbors substantial genetic variation for alcohol sensitivity and tolerance. We identified 247 candidate genes affecting alcohol sensitivity in the DGRP or the DGRP-derived advanced intercross population, some of which met a Bonferroni-corrected significance threshold, while others occurred among the top candidate genes associated with variation in alcohol sensitivity in multiple analyses. Among these were candidate genes associated with development and function of the nervous system, including several genes in the Dopamine decarboxylase (Ddc) cluster involved in catecholamine synthesis. We found that 58 of these genes formed a genetic interaction network. We verified candidate genes using mutational analysis, targeted gene disruption through RNAi knock-down and transcriptional profiling. Two-thirds of the candidate genes have been implicated in previous Drosophila, mouse and human studies of alcohol-related phenotypes. Conclusions: Individual variation in alcohol sensitivity in Drosophila is highly polygenic and in part determined by variation in evolutionarily conserved signaling pathways that are associated with catecholamine neurotransmitter biosynthesis and early development of the nervous system.

Background: Transcription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes—five—and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants. Methods: The three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.). Results: Phenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions. Conclusions: We report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene—nodD4—might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresses.

Background: Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. Methods: The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Results: Here we describe a novel Type II-C CRISPR and its associated genes—cas1, cas2, and cas9—in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Conclusions: Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.

Background: Perchlorate is a widely distributed anion that is toxic to humans, but serves as a valuable electron acceptor for several lineages of bacteria. The ability to utilize perchlorate is conferred by a horizontally transferred piece of DNA called the perchlorate reduction genomic island (PRI). Methods: We compared genomes of perchlorate reducers using phylogenomics, SNP mapping, and differences in genomic architecture to interrogate the evolutionary history of perchlorate respiration. Results: Here we report on the PRI of 13 genomes of perchlorate-reducing bacteria from four different classes of Phylum Proteobacteria (the Alpha-, Beta-, Gamma- and Epsilonproteobacteria). Among the different phylogenetic classes, the island varies considerably in genetic content as well as in its putative mechanism and location of integration. However, the islands of the densely sampled genera Azospira and Magnetospirillum have striking nucleotide identity despite divergent genomes, implying horizontal transfer and positive selection within narrow phylogenetic taxa. We also assess the phylogenetic origin of accessory genes in the various incarnations of the island, which can be traced to chromosomal paralogs from phylogenetically similar organisms. Conclusion: These observations suggest a complex phylogenetic history where the island is rarely transferred at the class level but undergoes frequent and continuous transfer within narrow phylogenetic groups. This restricted transfer is seen directly by the independent integration of near-identical islands within a genus and indirectly due to the acquisition of lineage-specific accessory genes. The genomic reversibility of perchlorate reduction may present a unique equilibrium for a metabolism that confers a competitive advantage only in the presence of an electron acceptor, which although widely distributed, is generally present at low concentrations in nature.

Background: Methicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms. Methods: USA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus™ Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®). Results: Comparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001). Conclusions: This sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300.