The global regulator GacS of a biocontrol bacterium Pseudomonas chlororaphis O6 regulates transcription from the rpoS gene encoding a stationary-phase sigma factor and affects survival in oxidative stress Beom Ryong Kang a , Baik Ho Cho a , Anne J. Anderson b , Young Cheol Kim a, * a Agricultural Plant Stress Research Center and Institute of Agriculture Science and Technology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, South Korea b Department of Biology, Utah State University, Logan, UT 84322-5305, USA Received 15 July 2003; received in revised form 29 September 2003; accepted 13 October 2003 Received by A.M. Campbell Abstract The global regulator, GacS (global activator for antibiotics and cyanide sensor kinase), of the rhizosphere bacterium Pseudomonas chlororaphis O6 (Pc O6) was required for increased resistance to hydrogen peroxide as cultures mature. Specific bands of peroxidase and catalase activity were absent in the stationary-phase cells of the Pc O6 gacS mutant, whereas a manganese superoxide dismutase (MnSOD) isozyme was expressed earlier and to a greater extent than in the wild-type. In the wild-type cell, transcript accumulation of rpoS was higher in late logarithmic (log)-phase cells than cells from mid log-phase or stationary-phase. Transcript abundance from rpoS was reduced in the gacS mutant throughout the growth phase compared to the wild-type expression. The sequence of a small RNA, rsmZ, found downstream of rpoS in other pseudomonads was lacking in Pc O6. This RNA is implicated in the control of genes activated by the GacS system. Thus, the mechanism by which GacS mediates the activation of genes under its control requires further investigation in Pc O6. D 2003 Elsevier B.V. All rights reserved. Keywords: Two-component sensor kinase; Stationary sigma factor RpoS; Catalase; Peroxidase; Superoxide dismutase; Regulatory RNA 1. Introduction Certain rhizobacteria stimulate plant growth, produce secondary metabolites antagonistic against soil-borne patho- gens, and induce plant disease resistance. Pseudomonas chlororaphis O6 (Pc O6) produces phenazines and hydro- gen cyanide that antagonize the growth of fungal pathogens (Radtke et al., 1994; Spencer et al., 2003). Colonization of roots by Pc O6 also induces systemic resistance against bacterial leaf pathogens (Spencer et al., 2003). Our studies are directed towards understanding the mechanisms by which the traits involved in the biocontrol potential of Pc O6 are regulated. In other biocontrol pseudomonads, the two-component GacS/GacA regulatory system controls the expression of secondary components involved in limiting pathogen per- formance (Heeb and Haas, 2001). GacS is a membrane- associated sensor kinase that becomes autophosphorylated through an as-yet unknown environmental stimulus. GacA is activated, by transfer of a phosphate residue from the GacS sensor kinase, to trigger the expression of downstream genes encoding beneficial traits. Enzymes and secondary metabolites controlled by the Gac system that have roles in biocontrol include phenazines, 2,4-diacylphloroglucinol, 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2003.10.004 Abbreviations: aa, amino acids; Ec, Escherichia coli; FeSOD, iron superoxide dismutase; GacA, a global activator of antibiotics and cyanide regulator; gacS and GacS, gene and protein encoding a global activator of antibiotics and cyanide sensor kinase; KB, King’s medium B; LB, Luria– Bertani; log, logarithmic; MnSOD, manganese superoxide dismutase; Pc O6, Pseudomonas chlororaphis O6; Pp, Pseudomonas putida; rpoS and RpoS, gene and protein encoding a stationary sigma factor; RT-PCR, reverse transcription polymerase chain reaction; rsmZ and RsmZ, gene and product encoding a regulatory RNA. * Corresponding author. Tel.: +82-62-530-2071; fax: +82-62-530- 0207. E-mail address: yckimyc@chonnam.ac.kr (Y.C. Kim). www.elsevier.com/locate/gene Gene 325 (2004) 137 – 143