Abstract
The GntR family transcription regulator HpaR1 identified from Xanthomonas campestris pv. campestris has been previously shown to positively regulate the genes responsible for hypersensitive reaction and pathogenicity and to autorepress its own expression. Here, we demonstrated that HpaR1 is a global regulator that positively regulates diverse biological processes, including xanthan polysaccharide production, extracellular enzyme activity, cell motility and tolerance to various stresses. To investigate the regulatory mechanisms of HpaR1, we began with xanthan polysaccharide production, which is governed by a cluster of gum genes. These are directed by the gumB promoter. Disruption of HpaR1 significantly reduced gumB transcription and an electrophoretic mobility shift assay demonstrated that HpaR1 interacts directly with gumB promoter. DNase I footprint analysis revealed that HpaR1 and RNA polymerase were bound to the sequences extending from −21 to +10 and −41 to +29 relative to the transcription initiation site of gumB, respectively. Furthermore, in vitro transcription assays showed that HpaR1 facilitated the binding of RNA polymerase to gumB promoter, leading to an enhancement of its transcription. These results suggest that HpaR1 regulates gumB transcription via a mechanism similar but different to what was found, until now, to only be used by some MerR family transcription activators.
Highlights
Pectate lyase (U) 0.68 ± 0.04a 0.32 ± 0.03b 0.65 ± 0.06a a key regulator for zinc homeostasis, which positively affects the expression of the hrp cluster via HrpX13
To further investigate whether HpaR1 is involved in other cellular processes, we examined the activity of extracellular enzymes, xanthan production, biofilm dispersal, cell motility and stress tolerance of the hpaR1 mutant, which all contribute to the full virulence of Xcc
The result displayed that similar to the wild type, the mutant did not form aggregates. This indicates that HpaR1 regulates the production of extracellular enzymes and polysaccharides, some of which can influence aggregative behaviour[18], mutation of hpaR1 did not lead to an aggregated mode of growth. These results reveal that Xcc HpaR1 is a global regulator that regulates diverse cellular processes, including the production of extracellular enzymes and xanthan, and cell motility, as well as tolerance to various stresses
Summary
Pectate lyase (U) 0.68 ± 0.04a 0.32 ± 0.03b 0.65 ± 0.06a a key regulator for zinc homeostasis, which positively affects the expression of the hrp cluster via HrpX13. HpaR1 is a GntR family transcriptional regulator, which may regulate indirectly the expression of hrpG14. GntR family members belong to the helix-turn-helix group of bacterial transcriptional regulators. GntR members possess a conserved N-terminal winged helix-turn-helix DNA-binding domain and a diverse C-terminal signalling domain. We found that HpaR1 is a global regulator of diverse Xcc cellular processes. We describe how HpaR1 is involved in the regulation of extracellular enzyme and xanthan production, cell motility, and the tolerance of Xcc to various stresses. We demonstrate that HpaR1 positively regulates xanthan production by binding to the gumB promoter region to facilitate the interaction between RNA polymerase and the promoter, leading to a transcriptional enhancement of the gum cluster of genes
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