Regulation of indigoidine production in <i>Vogesella indigofera</i> by a novel two-component system

Indigoidine is a blue pigment synthesized by several bacteria, including <i>Vogesella indigofera</i>. Industrial production of indigoidine has been a research focus, but less is known about why bacteria make this pigment or how its biosynthesis is regulated. We isolated <i>V. indigofera</i> strain OSW_575 and investigated the basis for its indigoidine production using genomic and genetic approaches. Mutation of the indigoidine synthase gene <i>igiD</i> eliminated pigment production, and complementation restored it. A transposon mutagenesis screen uncovered 34 mutations across 20 genes that affect pigment production, including some involved in metabolism, translation, protein homeostasis, and regulation. Three chaperones that combat misfolded proteins, <i>dnaK</i>, <i>dnaJ</i>, and <i>grpE</i>, were required for indigoidine production, while mutations affecting the <i>clpAP</i> proteasome resulted in hyperpigmentation. These results are consistent with prior studies and suggest a role for the protein homeostasis system in regulating indigoidine. We also found that the alternative sigma factor <i>rpoN</i> contributes to indigoidine production. Finally, one transposon mutation affected a predicted sensor histidine kinase, which we dub <i>tciK</i>. Our genetic characterization of <i>tciK</i> and its cognate response regulator <i>tciR</i> suggests that they function in the same pathway to regulate indigoidine. TciR is of interest due to its non-canonical domain architecture that combines an N-terminal REC domain and C-terminal RsbW-like anti-sigma factor domain. Further investigation of this system may reveal novel regulatory mechanisms. To our knowledge, this is the first study to employ genetic tools in <i>V. indigofera</i>, and we propose it as a useful experimental system for studying the regulation and function of indigoidine.IMPORTANCEDespite being known to science for more than a century, <i>Vogesella indigofera</i> has been the focus of few studies. We isolated a strain of this bacterium, sequenced its genome, and investigated which genes contribute to its production of the blue pigment indigoidine. We found that mutations in genes involved in metabolism, protein homeostasis, and regulation can affect pigment production. One locus required for indigoidine production encodes a novel two-component regulation system. We conducted a preliminary genetic characterization of this system, which includes a non-canonical response regulator. Based on the results, we propose our strain as a model organism for studying indigoidine production and regulation.