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

Abstract

BackgroundPseudomonas 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.ResultsDisruption 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0819-z) contains supplementary material, which is available to authorized users.

Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis

“…It has been observed that carA plays an important role in virulence in other bacterial species, such as Pseudomonas syringae ( 46 ), Escherichia coli ( 47 ), Xanthomonas citri ( 48 ) and Francisella tularensis ( 49 ). Similar to the genomic organization characterized in B. subtilis , several pyr genes ( pyR , pyrP , pyrB , pyrC , carA , carB , pyrF and pyrE ) of S. aureus are located on an operon and transcribed from a single promoter ( Figure 3B ).…”

Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism

“…Genes adjacent (3′ end) to the short 5′UTRs identified in B. cenocepacia include homologues of genes known to harbour cis-regulatory structures in other bacterial species, e.g. ribonuclease E 34 (BCAL2888), a proline-betaine transporter 35 (BCAL1252), ribosomal proteins 36 (BCAL0115, BCAL2091, BCAL2765, BCAL2714, BCAL3348), carA 37 (BCAL1260) and tRNA-synthetases 36 (BCAL3373, BCAL3436). Attenuation, observed for many short 5′UTRs, is indicative of a cis-regulatory function.…”

Efficacy of Antimicrobial Peptide DP7, Designed by Machine-Learning Method, Against Methicillin-Resistant Staphylococcus aureus

“…Specifically, carAB , a component of the pyrimidine pathway, was downregulated by 17- and 10-fold relative to the control. As a part of the arginine and pyrimidine pathways, the carAB gene encodes the enzyme carbamoylphosphate synthetase (CPS), which catalyzes the synthesis of carbamoylphosphate (Piette et al, 1984; Martinussen and Hammer, 1998; Nicoloff et al, 2001; Butcher et al, 2016). Carbamoylphosphate is a common intermediate in arginine and pyrimidine biosynthesis (Werner et al, 1985).…”

“…Although the exact regulated mode of carAB in S. aureus is still unclear, similar studies performed on other gram-negative microbes suggest that arginine is likely involved in the regulation (Sekowska et al, 2001). Regulation of the carAB operon in P. aeruginosa is controlled by arginine and pyrimidines at the transcriptional level, possibly through an attenuation mechanism (Lu et al, 1997; Butcher et al, 2016). Further studies on E. coli identified an arginine box located upstream of the control region of carAB that served as the initiation site, but which was blocked upon arginine recognition (Piette et al, 1984; Crowell et al, 1987; Alwan et al, 2017).…”