Transcriptome Analysis of
            <i>Pseudomonas syringae</i>
            Identifies New Genes, Noncoding RNAs, and Antisense Activity

Filiatrault, Melanie J.; Stodghill, Paul; Bronstein, Philip A.; Moll, Simon; Lindeberg, Magdalen; Grills, George S.; Schweitzer, Peter A.; Wang, Wei; Schroth, Gary P.; Luo, Shujun; Khrebtukova, Irina; Yang, Yong; Thannhauser, Theodore W.; Butcher, Bronwyn G.; Cartinhour, Samuel; Schneider, David J.

doi:10.1128/jb.01445-09

Cited by 117 publications

(137 citation statements)

References 88 publications

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“…Second, in our profiles we experienced fluctuating sequence read coverage, and at times the read profiles were fragmented by gaps without read coverage even within regions with high read counts. Other RNAseq-based transcriptome studies have also reported fluctuating read coverage (15,23,59), which may be caused by stochastic artifacts introduced during sample preparation or be due to possible degradation of RNA (85). During the cDNA library preparation, sequencing biases may be introduced due to different efficiencies in fragmentation, reverse transcription, and amplification, all of which can be affected by sequence composition as well as the level of transcripts.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Transcriptome Analysis of the Periodontopathogenic Bacterium Porphyromonas gingivalis W83

Høvik

Yu²,

Olsen

et al. 2012

J Bacteriol

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High-density tiling microarray and RNA sequencing technologies were used to analyze the transcriptome of the periodontopathogenic bacterium Porphyromonas gingivalis. The compiled P. gingivalis transcriptome profiles were based on total RNA samples isolated from three different laboratory culturing conditions, and the strand-specific transcription profiles generated covered the entire genome, including both protein coding and noncoding regions. The transcription profiles revealed various operon structures, 5=-and 3=-end untranslated regions (UTRs), differential expression patterns, and many novel, not-yetannotated transcripts within intergenic and antisense regions. Further transcriptome analysis identified the majority of the genes as being expressed within operons and most 5= and 3= ends to be protruding UTRs, of which several 3= UTRs were extended to overlap genes carried on the opposite/antisense strand. Extensive antisense RNAs were detected opposite most insertion sequence (IS) elements. Pairwise comparative analyses were also performed among transcriptome profiles of the three culture conditions, and differentially expressed genes and metabolic pathways were identified. With the growing realization that noncoding RNAs play important biological functions, the discovery of novel RNAs and the comprehensive transcriptome profiles compiled in this study may provide a foundation to further understand the gene regulation and virulence mechanisms in P. gingivalis. The transcriptome profiles can be viewed at and downloaded from the Microbial Transcriptome Database website, http://bioinformatics.forsyth.org/mtd.

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Section: Discussionmentioning

confidence: 99%

Comprehensive Transcriptome Analysis of the Periodontopathogenic Bacterium Porphyromonas gingivalis W83

Høvik

Yu²,

Olsen

et al. 2012

J Bacteriol

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“…The transcription start point (+) for each transcriptional group in the dar gene cluster was determined using the 59 rapid amplification of cDNA ends (RACE) method (Carrió n et al, 2012;Filiatrault et al, 2010;Maruyama et al, 1995). The synthesis of single-stranded cDNA was performed using total DNA-free RNA obtained from a P. chlororaphis PCL1606 culture growing in TPG medium for 24 h at 25 uC as described previously.…”

Section: Methodsmentioning

confidence: 99%

darR and darS are regulatory genes that modulate 2-hexyl, 5-propyl resorcinol transcription in Pseudomonas chlororaphis PCL1606

et al. 2014

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Pseudomonas chlororaphis PCL1606 synthesizes the antifungal antibiotic 2-hexyl, 5-propyl resorcinol (HPR), which is crucial for the biocontrol of fungal soil-borne pathogens. The genetic basis for HPR production lies in the dar genes, which are directly involved in the biosynthesis of HPR. In the present study, we elucidated the genetic features of the dar genes. Reverse transcription PCR experiments revealed an independent organization of the dar genes, except for darBC, which was transcribed as a polycistronic mRNA. In silico analysis of each gene revealed putative promoters and terminator sequences, validating the proposed gene arrangement. Moreover, experiments utilizing 59 rapid amplification of cDNA ends were used to determine the transcriptional initiation sites for the darA, darBC, darS and darR gene promoters, and subsequently to confirm the functionality of these regions. The results of quantitative real-time PCR experiments indicated that biosynthetic dar genes were not only modulated through the global regulator gacS, but also through darS and darR. The interplay between darS and darR revealed transcriptional cross-inhibition. However, these results also showed that other regulatory parameters play a role in HPR production, such as the environmental conditions and additional regulatory genes. INTRODUCTIONPseudomonas chlororaphis PCL1606, isolated from the rhizospheres of healthy avocado trees, has been used in previous studies as an antagonist bacterial strain against different soilborne phytopathogenic fungi, including Rosellinia necatrix, the causal agent of avocado white root rot, and Fusarium oxysporum f. sp. radicis-lycopersici, the causal agent of tomato crown and foot rot (Cazorla et al., 2006). The antifungal antibiotic 2-hexyl, 5-propyl resorcinol (HPR) is a major factor in the antagonistic phenotype of this rhizobacterium (Cazorla et al., 2006), and recent studies have demonstrated a crucial role for this antibiotic in the biocontrol of R. necatrix and F. oxysporum (Calderó n et al., 2013; González-Sánchez et al., 2010).HPR was first detected in an unidentified Pseudomonas sp., and exhibited moderate antifungal and antibacterial properties . The genetic basis for HPR production was first examined in P. chlororaphis subsp. auranthiaca BL915. In this strain, the genes responsible for HPR production are located in a cluster containing three presumptive biosynthetic genes (darA, darB and darC), followed by two independent sequences homologous to the regulatory genes from the AraC/XylS family (darS and darR; Nowak- Thompson et al., 2003). This dar cluster has recently been identified in P. chlororaphis PCL1606 (Calderó n et al., 2013). The construction of insertion mutants for each homologous dar gene in P. chlororaphis PCL1606 and the corresponding complementary derivative strains restoring HPR production revealed a key role for darA and darB genes in both HPR biosynthesis and the antagonistic phenotype (Calderó n et al., 2013). A minor role in HPR biosynthesis has been proposed for darC, as de...

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“…In the past 10 years, the development of new approaches based on high-resolution tiling arrays and RNA deep sequencing (RNA-seq) has uncovered that a significant proportion (depending on the study, varying between 3% and >50%) of protein coding genes are also transcribed from the reverse complementary strand (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In most cases, overlapping transcription generates a noncoding antisense transcript whose size can vary between various tens of nucleotides (cisencoded small RNAs) to thousands of nucleotides (antisense RNAs).…”

mentioning

confidence: 99%

Genome-wide antisense transcription drives mRNA processing in bacteria

Lasa

Toledo‐Arana

Dobin

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

225

277

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RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5′ and 3′ untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels.antisense RNA | overlapping transcription | RNA processing | posttranscriptional regulation | microRNA F or many years, the catalog of transcripts (transcriptome) produced by bacterial cells was limited to the transcription products of known annotated genes (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). In the past 10 years, the development of new approaches based on high-resolution tiling arrays and RNA deep sequencing (RNA-seq) has uncovered that a significant proportion (depending on the study, varying between 3% and >50%) of protein coding genes are also transcribed from the reverse complementary strand (1-17). In most cases, overlapping transcription generates a noncoding antisense transcript whose size can vary between various tens of nucleotides (cisencoded small RNAs) to thousands of nucleotides (antisense RNAs). The antisense transcript can cover the 5′ end, 3′ end, middle, entire gene, or even various contiguous genes. Alternatively, overlapping transcription can also be due to the overlap between long 5′ or 3′ UTRs of mRNAs transcribed in the opposite direction. Independent of the mechanism by which it is generated, overlapping transcription has been proposed to affect the expression of the target gene at different levels [for review, see Thomason and Storz (18)]. These mechanisms include: (i) the overlapped transcript affects the stability of the target RNA by either promoting (RNA degradation) or blocking (RNA stabilization) cleavage by endoribonucleases or exoribonucleases; (ii) the overlapped transcript induces a change in the structure of the mRNA that affects transcription termination (transcription attenuation); (iii) the overlapped transcript prevents RNA polymerase from binding or extending the transcript encoded in the opposite strand (transcription interference); and (iv) the overl...

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Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity

Cited by 117 publications

References 88 publications

Comprehensive Transcriptome Analysis of the Periodontopathogenic Bacterium Porphyromonas gingivalis W83

Comprehensive Transcriptome Analysis of the Periodontopathogenic Bacterium Porphyromonas gingivalis W83

darR and darS are regulatory genes that modulate 2-hexyl, 5-propyl resorcinol transcription in Pseudomonas chlororaphis PCL1606

Genome-wide antisense transcription drives mRNA processing in bacteria

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