Disruption of the carA gene in Pseudomonas syringae results in reduced fitness and alters motility

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 (δcarA) 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, δcarA 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. δcarA 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. Keywords: Pseudomonas syringae pv tomato, CarAB, P32, Virulence, Swarming, Biofilm formation Abbreviations: CFU, Colony forming units; CPSase, Carbamoylphosphate synthetase; FLOE, Fluorescently labeled oligonucleotide extension; G6PDH, Glucose-6-phosphate dehydrogenase; HR, Hypersensitive response; IVET, In vivo expression technology; KB, King's B; MG, Mannitol-glutamate; qPCR, Quantitative real-Time PCR; RACE, Rapid amplification of cDNA ends; T3SS, The type III secretions system; VBMM, Vogel-bonner minimal medium.