The ATP-dependent RNA helicase HrpB plays an important role in motility and biofilm formation in Xanthomonas citri subsp. citri

Abstract

BackgroundRNA helicases are enzymes that catalyze the separation of double-stranded RNA (dsRNA) using the free energy of ATP binding and hydrolysis. DEAD/DEAH families participate in many different aspects of RNA metabolism, including RNA synthesis, RNA folding, RNA-RNA interactions, RNA localization and RNA degradation. Several important bacterial DEAD/DEAH-box RNA helicases have been extensively studied. In this study, we characterize the ATP-dependent RNA helicase encoded by the hrpB (XAC0293) gene using deletion and genetic complementation assays. We provide insights into the function of the hrpB gene in Xanthomonas citri subsp. citri by investigating the roles of hrpB in biofilm formation on abiotic surfaces and host leaves, cell motility, host virulence of the citrus canker bacterium and growth in planta.ResultsThe hrpB gene is highly conserved in the sequenced strains of Xanthomonas. Mutation of the hrpB gene (∆hrpB) resulted in a significant reduction in biofilms on abiotic surfaces and host leaves. ∆hrpB also exhibited increased cell dispersion on solid medium plates. ∆hrpB showed reduced adhesion on biotic and abiotic surfaces and delayed development in disease symptoms when sprayed on susceptible citrus leaves. Quantitative reverse transcription-PCR assays indicated that deletion of hrpB reduced the expression of four type IV pili genes. The transcriptional start site of fimA (XAC3241) was determined using rapid amplification of 5′-cDNA Ends (5′RACE). Based on the results of fimA mRNA structure predictions, the fimA 5′ UTR may contain three different loops. HrpB may be involved in alterations to the structure of fimA mRNA that promote the stability of fimA RNA.ConclusionsOur data show that hrpB is involved in adherence of Xanthomonas citri subsp. citri to different surfaces. In addition, to the best of our knowledge, this is the first time that a DEAH RNA helicase has been implicated in the regulation of type IV pili in Xanthomonas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0655-1) contains supplementary material, which is available to authorized users.

Transcriptome profiling ofVariovorax paradoxusEPS under different growth conditions reveals regulatory and structural novelty in biofilm formation

“…The most specifically highly upregulated gene in our analysis was Varpa_1640 (Table 1, green ), which was not identified in our previous biofilm mutant screen. This protein is predicted to be a DEAD-box RNA helicase, a widespread family of proteins recently shown to play a role in biofilm formation [26]. We hypothesize that this protein is active in RNA turnover in V. paradoxus EPS based on the KEGG predicted RNA degradation pathway [27] (Figure 6A).…”

“…The PNPase, enolase, and RNAses are all highly expressed in biofilm ( Supplemental Data S1, S2 ), leading to the hypothesis that differential expression of Varpa_1640 is a mechanism for regulating RNA stability and drastically altering the gene expression profile. A relationship between a DEAD-box helicase and biofilm growth was recently described in the plant pathogen Xanthomonas citri [26], based on mutations in the HrpB helicase resulting in reduced expression of Type IV pili. Intriguingly, our prior work indicated that Type IV pili are critical for the switch between biofilm and swarming phenotypes in V. paradoxus EPS [15], suggesting a possible link between RNA stability, pilus formation, and attachment phenotypes.…”

The ecnA antitoxin is not only important for human pathogens: Evidences of its role in the plant pathogen Xathomonas citri subsp. citri

“…Motility assays were performed as previously described (40). Bacteria were grown overnight in NBY medium, and 3 µL of bacterial culture (OD600 0.3) was then spotted onto a plate containing SB medium plus 0.5 % (wt/v) agar (Difco, Franklin Lakes, NJ) (41) for the sliding motility tests or NYGB medium 0.25 % (wt/v) agar (42) for the swimming motility tests.…”

Transcriptome profiling of Variovorax paradoxus EPS under different growth conditions reveals regulatory and structural novelty in biofilm formation

“…The most specifically highly upregulated gene in our analysis was Varpa_1640 (Table 1, green), which was not identified in our previous biofilm mutant screen. This protein is predicted to be a DEAD-box RNA helicase, a widespread family of proteins recently shown to play a role in biofilm formation [33]. We hypothesize that this protein is active in RNA turnover in V. paradoxus EPS based on the KEGG predicted RNA degradation pathway [34] (Fig.…”

“…The PNPase, enolase and RNAses are all highly expressed in biofilm (Supplementary Data S1 and S2), leading to the hypothesis that differential expression of Varpa_1640 is a mechanism for regulating RNA stability and drastically altering the gene expression profile. A relationship between a DEAD-box helicase and biofilm growth was recently described in the plant pathogen Xanthomonas citri [33], based on mutations in the HrpB helicase resulting in reduced expression of type-IV pili. Intriguingly, our prior work indicated that type-IV pili are critical for the switch between biofilm and swarming phenotypes in V. paradoxus EPS [22], suggesting a possible link between RNA stability, pilus formation and attachment phenotypes.…”