|BUTCHER, BRONWYN - Cornell University|
|STOOS, KARI BROSSARD - Ithaca College|
Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/19/2016
Publication Date: 8/24/2016
Citation: Butcher, B.G., D'Amico, K.M., Stoos, K., Filiatrault, M.J. 2016. Disruption of the carA gene in Pseudomonas syringae alters motility and results in reduced fitness. BMC Microbiology. 16:194.
Interpretive Summary: Pathogenic bacteria must acquire nutrients from the host to grow and cause infection. To understand the mechanism by which a bacterial pathogen causes disease one must identify those gene products required for infection. Here we found that disruption of a gene (carA) in the plant pathogen Pseudomonas syringae results in reduced growth in minimal medium. We found that growth can be restored by the addition of the amino acid arginine and the pyrimidine uracil to the medium. We also found that deletion of carA results in reduced growth and reduced symptoms in infected tomato plants compared to the wild-type strain. Our results indicate that carA plays a critical role in the growth and fitness of Pseudomonas syringae in planta. Our data also suggests that the nutrient arginine is not present in sufficient amounts in the host and therefore targeting or modulating this pathway may represent a novel control method to combat disease caused by this plant pathogen.
Technical Abstract: Background: Pseudomonas syringae infects diverse plant species and is widely used in the study of effector function and the molecular basis of disease. Although the relationship between bacterial metabolism, nutrient acquisition and virulence has attracted increasing attention in bacterial pathology, there is limited knowledge regarding these studies in Pseudomonas syringae. The aim of this study was to investigate the function of the carA gene and the small RNA P32, and characterize the regulation of these transcripts. Results: Disruption of the carA gene (delta carA) which encodes the predicted small chain of carbamoylphosphate synthetase, resulted in arginine and pyrimidine auxotrophy in Pseudomonas syringae pv. tomato DC3000. Complementation with the wild type carA gene was able to restore growth to wild-type levels in minimal medium. Deletion of the small RNA P32, which resides immediately upstream of carA, did not result in arginine or pyrimidine auxotrophy. The expression of carA was influenced by the concentrations of both arginine and uracil in the medium. When tested for pathogenicity, delta carA showed reduced fitness in tomato as well as Arabidopsis when compared to the wild-type strain. In contrast, mutation of the region encoding P32 had minimal effect in planta. 'carA also exhibited reduced motility and increased biofilm formation, whereas disruption of P32 had no impact on motility or biofilm formation. Conclusions: Our data show that carA plays an important role in providing arginine and uracil for growth of the bacteria and also influences other factors that are potentially important for growth and survival during infection. Although we find that the small RNA P32 and carA are co-transcribed, P32 does not play a role in the phenotypes that carA is required for, such as motility, cell attachment, and virulence. Additionally, our data suggests that pyrimidines may be limited in the apoplastic space of the plant host tomato.