Transcriptome analysis of Pseudomonas syringae identifies new genes, noncoding RNAs, and antisense activity.
To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illumina's high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoN-dependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation.
Filiatrault
Melanie J
MJ
USDA Agricultural Research Service, Plant-Microbe Interactions Research Unit, Cornell University, Plant Science Bldg., Room 334, Ithaca, NY 14853, USA. melanie.filiatrault@ars.usda.gov
Stodghill
Paul V
PV
Bronstein
Philip A
PA
Moll
Simon
S
Lindeberg
Magdalen
M
Grills
George
G
Schweitzer
Peter
P
Wang
Wei
W
Schroth
Gary P
GP
Luo
Shujun
S
Khrebtukova
Irina
I
Yang
Yong
Y
Thannhauser
Theodore
T
Butcher
Bronwyn G
BG
Cartinhour
Samuel
S
Schneider
David J
DJ
D002942:Circular Dichroism; D019295:Computational Biology; D020869:Gene Expression Profiling; D016680:Genome, Bacterial / Q000235:genetics; D008957:Models, Genetic; D021141:Nucleic Acid Amplification Techniques; D011401:Promoter Regions, Genetic / Q000235:genetics; D044224:Pseudomonas syringae / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics*; D022661:RNA, Untranslated / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000235:genetics
D016372:RNA, Antisense; D022661:RNA, Untranslated