Transcriptional analysis and operon structure of the tagA?orf2?orf3?mop?tagD region on the Vibrio pathogenicity island in epidemic V. cholerae

Abstract

The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster. The VPI can excise from the chromosome and form extrachromosomal circular excision products. The VPI is 41.2-kb in size and encodes 29 potential proteins, several of which have no known function and whose regulation is not well understood. To determine the transcriptional organization of the tagA-orf2-orf3-mop-tagD region located at the 5'-(left) end of the VPI, we used reverse-transcriptase-PCR (RT-PCR), Northern blot analysis and DNA sequencing. RT-PCR primers were designed to transcribe and amplify regions spanning two or more open reading frames so as to establish the transcriptional organization. RT-PCR and Northern blot results demonstrated that the tagA-tagD region is transcribed as a polycistronic message and organized into several potential operons including tagA-orf2, orf3-mop, orf3-mop-tagD and tagD alone. Transcriptional lacZ fusions supported the existence of a promoter upstream of orf3 that was toxT-dependent. Interestingly, our data suggests that the orf3 promoter can drive the expression of either a long transcript (orf3-mop-tagD) or a short transcript (orf3-mop) without tagD. Our data also suggests that tagD can be expressed from two different promoters and that tagD is either transcribed alone or co-expressed with orf3-mop under certain conditions. These studies provide new insight into the genetic structure, transcriptional organization and regulation of a cluster of virulence genes on the VPI of epidemic V. cholerae.

Closely Related Vibrio alginolyticus Strains Encode an Identical Repertoire of Caudovirales-Like Regions and Filamentous Phages

“…The present study aimed to identify prophages and filamentous phages isolated from closely related V. alginolyticus strains, namely, the Kiel-alginolyticus ecotype [26], and to characterize their virulence potential. V. alginolyticus, a ubiquitous marine opportunistic pathogen can cause mass mortalities in shellfish, shrimp and fish, resulting in severe economic losses worldwide [27][28][29]. Additionally, wound infections and fatal septicemia in immunocompromised patients caused by V. alginolyticus have been reported in humans [30].…”

Protocol for Metatranscriptomic analysis of Intestinal Microbiota

“…This kit, tested on different bacterial species including Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Yersinia pestis, Bacillus anthracis Rhodobacter sphaeroides, has shown high efficiency (Applied Biosystems 2004, Xu Y, et al, 2003, Drysdale M, et al, 2004, Cheng HP, Yao SY. 2004, Zhang D. et al, 2004, Prithiviraj B. et al, 2005, Di Cello F, et al, 2005. The "MICROBExpress Bacterial mRNA Purification Kit" (Ambion, TX) was used to deplete the pool of 16S and 23S rRNA molecules present in the sample.…”