Identification of the cognate response regulator of the orphan histidine kinase OhkA involved in both secondary metabolism and morphological differentiation in Streptomyces coelicolor

Abstract

In the model actinomycete strain, Streptomyces coelicolor, an orphan histidine kinase (HK) named OhkA (encoded by SCO1596), which belongs to bacterial two-component regulatory systems (TCSs), has been identified as being involved in the regulation of both antibiotic biosynthesis and morphological development. However, its cognate response regulator (RR) remains unknown due to its isolated genetic location on the genome, which impedes the elucidation of the mechanism underlying OhkAmediated regulation. Here, we identified the orphan RR OrrA (encoded by SCO3008) as the cognate RR of OhkA according to mutant phenotypic changes, transcriptomics analysis, and bacterial two-hybrid experiment. Considering that the partner RR of the orphan HK is also orphan, a library of mutants with in-frame individual deletion of these functionally unknown orphan RRencoding genes were generated. Through phenotypic analysis, it was found that the ΔorrA mutant exhibited similar phenotypic changes as that of the ΔohkA mutant, showing increased production of actinorhodin (ACT) and undecylprodigiosin (RED), and pink colony surface. Further transcriptomics analysis showed these two mutants exhibited highly similar transcriptomics profiles. Finally, the direct interaction between OhkA and OrrA was revealed by bacterial two-hybrid system. The identification of the partner RR of OhkA lays a good foundation for an in-depth elucidation of the molecular mechanism underlying OhkA-mediated regulation of development and antibiotic biosynthesis in Streptomyces. Key points• OrrA was identified as the partner RR of the orphan histidine kinase OhkA.• The ΔorrA and ΔohkA mutants showed similar phenotype and transcriptomic profiling.• Specific interaction of OrrA and OhkA was revealed by bacterial two-hybrid system.

The regulatory genewblAis a target of the orphan response regulatorOrrAinStreptomyces coelicolor

“…1A and B) (Wang, 2019), and a quantitative assay indicated that Δ orrA produced extremely high amounts of both ACT and RED (Fig. 1C–E) (Wang, 2019), in agreement with previous reports (Xu et al ., 2017; Zheng et al ., 2021). Although the difference was not as striking as with ACT and RED, production of two other antibiotics, the calcium‐dependent antibiotic (CDA) (Fig.…”

“…5C), suggesting that sco1375 is not only regulated by OrrA but also by WblA. In addition, we also measured the expression of ohkA , an orphan sensor kinase gene that is the potential partner of OrrA (Zheng et al ., 2021). However, expression levels of ohkA were comparable in M145, Δ orrA and Δ wblA , suggesting that OrrA and WblA do not regulate ohkA , at least under the conditions tested.…”

“…In this study, we provide further evidence that the orphan response regulator OrrA is a pleiotropic regulator, strongly repressing the production of ACT and RED as reported (Xu et al ., 2017; Wang, 2019; Zheng et al ., 2021) and also exhibiting some repressive effects on the two antibiotics CDA and yCPK, in addition to impacting the development of S . coelicolor .…”

“…The impact of SCO3008 (OrrA) on antibiotic production was verified, and an effect on cellular development was revealed using a mutant strain with a deletion of sco3008 ( orrA ) (Wang, 2019). Recently, it was found that SCO3008 (OrrA) interacted and potentially paired with the orphan sensor kinase OhkA, and SCO3008 was named OrrA (for orphan response regulator A) in that report (Zheng et al ., 2021). Although a pleiotropic role in antibiotic production and cellular development has been demonstrated for OrrA (Xu et al ., 2017; Wang, 2019; Zheng et al ., 2021), the regulatory targets of OrrA were still unknown.…”

“…Recently, it was found that SCO3008 (OrrA) interacted and potentially paired with the orphan sensor kinase OhkA, and SCO3008 was named OrrA (for orphan response regulator A) in that report (Zheng et al ., 2021). Although a pleiotropic role in antibiotic production and cellular development has been demonstrated for OrrA (Xu et al ., 2017; Wang, 2019; Zheng et al ., 2021), the regulatory targets of OrrA were still unknown. In this study, we focused on deciphering the targets of OrrA, and we discovered that the regulatory gene wblA is a target of OrrA and that wblA may mediate some of the regulatory effects of OrrA.…”