The rsmS (ybaM) mutation causes bypass suppression of the RsmAB post-transcriptional virulence regulation system in enterobacterial phytopathogens.

Plant cell wall degrading enzymes (PCWDEs) are the primary virulence determinants of soft rotting bacteria such as the potato pathogen, Pectobacterium atrosepticum. The regulation of secondary metabolite (Rsm) system controls production of PCWDEs in response to changing nutrient conditions. This work identified a new suppressor of an rsmB mutation - ECA1172 or rsmS (rsmB suppressor). Mutants defective in rsmB (encoding a small regulatory RNA), show reduced elaboration of the quorum sensing molecule (N-3-oxohexanoyl-homoserine lactone; OHHL) and PCWDEs. However, OHHL and PCWDE production were partially restored in an rsmB, rsmS double mutant. Single rsmS mutants, overproduced PCWDEs and OHHL relative to wild type P. atrosepticum and exhibited hypervirulence in potato. RsmS overproduction also resulted in increased PCWDEs and OHHL. Homology searches revealed rsmS conservation across pathogens such as Escherichia coli (ybaM), Dickeya solani, Klebsiella pneumoniae and Shigella flexneri. An rsmS mutant of Pectobacterium carotovorum ATCC39048 showed bypass of rsmB-dependent repression of PCWDEs and OHHL production. P. carotovorum ATCC39048 produces the β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (a carbapenem). Production of the antibiotic was repressed in an rsmB mutant but partially restored in an rsmB, rsmS double mutant. This work highlights the importance of RsmS, as a conserved pleiotropic regulator of virulence and antibiotic biosynthesis.James Hutton Institut

Characterization of a novel virulence determinant in Erwinia carotovora subspecies atroseptica SCRI1043

This thesis is not available on this repository until the author agrees to make it public. If you are the author of this thesis and would like to make your work openly available, please contact us: [email protected] Library can supply a digital copy for private research purposes; interested parties should submit the request form here: http://www.lib.cam.ac.uk/collections/departments/digital-content-unit/ordering-imagesPlease note that print copies of theses may be available for consultation in the Cambridge University Library's Manuscript reading room. Admission details are at http://www.lib.cam.ac.uk/collections/departments/manuscripts-university-archivesThis study focused on the characterization of the gene rsmS in Erwinia carotovora subsp. atroseptica (Eca). Eca is a phytopathogenic Gram-negative bacteria, producing virulence factors, such as plant cell wall degrading enzymes {PCWDEs), which enable the initiation and maintenance of host plant invasion. rsmS was shown to encode a short polypeptide, and it has orthologues in many Gramnegative agriculturally and medically relevant bacteria. Plate and spectrophotometry-based assays and microarray studies showed that RsmS suppressed the transcription and activity of several PCWDEs, the transcription of type one, two and six secretion systems and of some of their substrates. RsmS also suppressed general virulence exhibited by Eca in potato tubers, and on the quorum sensing signaling molecule N-{3-oxohexanoylh-homoserine lactone. In many ways, RsmS acted in a similar fashion to RsmA, r a well characterized suppressor of virulence determinants (such as PCWDEs) and quorum sensing found in a wide variety of Gram-negative bacteria. RsmA activated rsmS transcription, and the transcription of both rsmS and rsmA was shown to be upregulated with increasing temperature. Thus, it is possible that RsmA exerts some of its inhibitory effects on virulence determinants through the positive regulation of RsmS. rsmS was shown to be transcribed from a promoter shared with its upstream neighbour, priC. It may also be transcribed from a promoter located within the priC open reading frame, but this result remains to be confirmed with future experiments. Although the genomic arrangement of priC and rsmS is conserved in almost all bacteria with an rsmS orthologue, no further regulatory connection was shown between their protein products. Interestingly, when rsmS was overexpressed without a 225 bp region located within priC, PCWDE activity was increased, in all mutant strains of Eca tested. The reason behind this dominant negative phenotype remains to be fully elucidated by future experiments. Both microarray and plasposon mutagenesis studies identified several genes coding for factors that are regulated by, or regulate, RsmS. It will require further analysis and experiments to unveil which one of these actually interacts with RsmS (directly or indirectly). Such genes include CRISPR-like genes, members of nitrogen metabolism pathways and membrane transport.