The effect of zinc limitation on the transcriptome of Pseudomonas protegens Pf-5 Chee Kent Lim, 1 Karl A. Hassan, 1 Anahit Penesyan, 1 Joyce E. Loper 2 and Ian T. Paulsen 1 * 1 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia. 2 USDA-ARS Horticultural Crops Research Laboratory and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA. Summary Zinc is an important nutrient but can be lacking in some soil environments, influencing the physiology of soil-dwelling bacteria. Hence, we studied the global effect of zinc limitation on the transcriptome of the rhizosphere biocontrol strain Pseudomonas prote- gens Pf-5 (formerly Pseudomonas fluorescens). We observed that the expression of the putative zinc uptake regulator (Zur) gene was upregulated, and we mapped putative Zur binding sites in the Pf-5 genome using bioinformatic approaches. In line with the need to regulate intracellular zinc concentrations, an array of potential zinc transporter genes was found to be zinc-regulated. To adapt to low-zinc conditions, a gene cluster encoding non-zinc-requiring paralogues of zinc-dependent proteins was also significantly upregulated. Similarly, transcription of genes encod- ing non-zinc-requiring paralogues of ribosomal pro- teins L31 and L36 was increased by zinc limitation. A strong transcriptional downregulation of the putative copper chaperone gene (copZ) was also observed, suggesting interplay between zinc and copper home- ostasis. Importantly, zinc also affected biocontrol attributes in Pf-5, most notably reducing the expres- sion of the gene cluster responsible for biosynthesis of the antibiotic 2,4-diacetylphloroglucinol (DAPG) under zinc limitation. This study clearly defines changes to the molecular physiology of Pf-5 that enable it to survive under zinc limitation. Introduction Pseudomonas protegens Pf-5 (previously called Pseu- domonas fluorescens Pf-5) (Ramette et al., 2011) is a soil-inhabiting biocontrol bacterium that can suppress a wide variety of plant pathogenic bacteria, fungi and oomyc- etes (Howell and Stipanovic, 1979; 1980; Loper et al., 2007). Pf-5 suppresses the growth of these pathogens primarily via the secretion of a range of secondary metabo- lites such as hydrogen cyanide, pyoluteorin, pyrrolnitrin, rhizoxin analogues and 2,4-diacetylphloroglucinol (DAPG) (Howell and Stipanovic, 1979; 1980; Nowak-Thompson et al., 1994; Whistler et al., 1998; Loper et al., 2008). Genome sequencing further highlighted the biocontrol properties of Pf-5, revealing that a large proportion of its genetic information is dedicated to biocontrol functions, such as the production of secondary metabolites, a number of which were previously unknown (Paulsen et al., 2005). The concentrations of micronutrients vary considerably within the soil environments that may be inhabited by P. protegens Pf-5 and related biocontrol organisms. Nonetheless, micronutrients are essential to most organ- isms and are likely to have profound effects on the physi- ology of soil-dwelling microbes. Zinc is considered an essential micronutrient mainly due to its unique chemical properties. Because zinc does not undergo redox reac- tions and can act as a Lewis acid or an electrophile, it serves as a cofactor for many proteins, mediating distinct catalytic reactions in a large number of enzymes (Andreini et al., 2006; Haas et al., 2009). For example, it has been estimated that zinc may act as a cofactor within approxi- mately 5% of the proteins found in bacteria (Berg and Shi, 1996; Andreini et al., 2006), including ribosomal proteins, DNA and RNA polymerases (Blaby-Haas et al., 2011) and DNA primases (Pan and Wigley, 2000), which serve in the essential housekeeping functions of DNA, RNA and protein synthesis. Additionally, zinc is a cofactor for a range of accessory proteins, such as some b-lactamases (Andreini et al., 2006) that may be essential under par- ticular stress conditions. In line with its importance in the cell, zinc ions have been shown to accumulate to the same levels as iron and calcium in some bacterial cells (Outten and O’Halloran, 2001). Although it is important for bacterial cells to accumulate a sufficient quantity of zinc to fulfil the range of essential cellular reactions, zinc in excess can be toxic as it can compete with other metals for binding sites in proteins (Loisel et al., 2008; Blaby-Haas et al., 2011). Therefore, bacteria must employ measures to maintain a fine zinc Received 4 April, 2012; revised 9 July, 2012; accepted 21 July, 2012. *For correspondence. E-mail ian.paulsen@mq.edu.au; Tel. (+61) 2 9850 8152; Fax (+61) 2 9850 8313. Environmental Microbiology (2012) doi:10.1111/j.1462-2920.2012.02849.x © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd