Competitive control of endoglucanase gene engXCA expression in the plant pathogen Xanthomonas campestris by the global transcriptional regulators HpaR1 and Clp

Abstract

Transcriptional regulators are key players in pathways that allow bacteria to alter gene expression in response to environmental conditions. However, work to understand how such transcriptional regulatory networks interact in bacterial plant pathogens is limited. Here, in the phytopathogen Xanthomonas campestris, we demonstrate that the global transcriptional regulator HpaR1 influences many of the same genes as another global regulator Clp, including the engXCA gene that encodes extracellular endoglucanase. We demonstrate that HpaR1 facilitates the binding of RNA polymerase to the engXCA promoter. In addition, we show that HpaR1 binds directly to the engXCA promoter. Furthermore, our in vitro tests characterize two binding sites for Clp within the engXCA promoter. Interestingly, one of these sites overlaps with the HpaR1 binding site. Mobility shift assays reveal that HpaR1 has greater affinity for binding to the engXCA promoter. This observation is supported by promoter activity assays, which show that the engXCA expression level is lower when both HpaR1 and Clp are present together, rather than alone. The data also reveal that HpaR1 and Clp activate engXCA gene expression by binding directly to its promoter. This transcriptional activation is modulated as both regulators compete to bind to overlapping sites on the engXCA promoter. Bioinformatics analysis suggests that this mechanism may be used broadly in Xanthomonas campestris pv. campestris (Xcc) and is probably widespread in Xanthomonads and, potentially, other bacteria. Taken together, these data support a novel mechanism of competitive activation by two global regulators of virulence gene expression in Xcc which is probably widespread in Xanthomonads and, potentially, other bacteria.

Flp, a Fis‐like protein, contributes to the regulation of type III secretion and virulence processes in the phytopathogen Xanthomonas campestris pv. campestris

“…(B) In vitro transcription experiment using Flp protein (final density arranged from 0, 0.1, 0.20 and 0.5 µM) in the transcription system. A template DNA fragment containing the clp promoter (Liu et al ., ) and a 121‐bp hrpX fragment extending from +1 to +121 relative to the TIS were used as controls.…”

“…ChIP assay was performed as previously described with minor modifications (Liu et al , ). In brief, a strain producing an Flp protein fused with 3 × Flag‐tag (3 × Flag::Flp) at the N‐terminus of Flp was first constructed.…”

Punicalagin: a monomer with anti-Eimeria tenella effect from fruit peel of Punica granatum L.

“…Library construction and Illumina sequencing was performed by Novogene China. An RNA-seq analysis was performed according to the protocol recommended by the manufacturer (Illumina Inc., San Diego, CA ) ( Qiao et al, 2016 ; Liu et al, 2019 ).…”

Xanthomonas campestris VemR enhances the transcription of the T3SS key regulator HrpX via physical interaction with HrpG

“…The two fragments were joined using overlap extension PCR, and the resulting recombinant fragment was cloned into the suicide plasmid pK18 mobsacB (Schäfer et al, 1994 ). The resulting recombined plasmid named pK vemR :: flag (Table S1 ) was introduced into Xcc strain 8004 by conjugation, and the transconjugants chromosomally encoding VemR::3 × FLAG protein were screened and confirmed by the procedure described previously (Liu et al, 2019 ). The obtained variant strain was named 8004/VemR::3 × FLAG (Table S1 ).…”