The Expression of stlA in Photorhabdus luminescens Is Controlled by Nutrient Limitation

Abstract

Photorhabdus is a genus of Gram-negative entomopathogenic bacteria that also maintain a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus has an extensive secondary metabolism that is required for the interaction between the bacteria and the nematode. A major component of this secondary metabolism is a stilbene molecule, called ST. The first step in ST biosynthesis is the non-oxidative deamination of phenylalanine resulting in the production of cinnamic acid. This reaction is catalyzed by phenylalanine-ammonium lyase, an enzyme encoded by the stlA gene. In this study we show, using a stlA-gfp transcriptional fusion, that the expression of stlA is regulated by nutrient limitation through a regulatory network that involves at least 3 regulators. We show that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is absolutely required for stlA expression. We also show that stlA expression is modulated by σS and Lrp, regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria. This work is the first that describes pathway-specific regulation of secondary metabolism in Photorhabdus and, therefore, our study provides an initial insight into the complex regulatory network that controls secondary metabolism, and therefore mutualism, in this model organism.

Alarmone (p)ppGpp regulates the transition from pathogenicity to mutualism in Photorhabdus luminescens

“…The 1× modified M9 contains the same levels of tryptone and yeast extract as the LBP medium (1% (w/v) and 0.5 (w/v), respectively), while the 0.1× modified M9 contains only 10% of the tryptone and yeast extract levels of LBP. Again, growth and stlA promoter activity in the WT cultured in modified M9 confirmed the previously published results (Lango‐Scholey et al ., ); namely that (i) the growth rate in 0.1× modified M9 decreased much faster in comparison to 1× modified M9, confirming that cells were limited for carbon and/or nitrogen (Fig. C), and (ii) the reduced growth in 0.1× modified M9 and early entry into post‐exponential growth phase correlated with the kinetics of induction of the stlA promoter, as the expression of stlA ‐ gfp was found to occur at an earlier time‐point and at a lower cell density (Fig.…”

“…To further link the accumulation of (p)ppGpp to the production of ST we investigated the expression of stlA , catalyzing the first step in ST production, in the WT and the Δ relA Δ spoT strain. For this purpose, we constructed chromosomally located stlA ‐ gfp transcriptional fusions in the WT and the Δ relA Δ spoT strain, using a previously described mini‐Tn7 transposon strategy (Lango‐Scholey et al ., ). Both growth in LBP broth and stlA promoter activity were measured in the WT and this confirmed the previously published results (Lango‐Scholey et al ., ); namely that the activity of the stlA promoter increases during the post‐exponential growth phase (Fig.…”

“…Activity of the stlA promoter in WT and Δ relA Δ spoT in LBP medium. The fluorescence has been corrected for background, by subtracting the fluorescence of the promoter‐less gfp + control at each time‐point, followed by transformation into promoter activity using the equation [ΔGFP/Δt]/OD 600 , as described by (Lango‐Scholey et al ., ). C. Growth curves of WT and Δ relA Δ spoT in 1× or 0.1× modified M9 medium, respectively.…”

“…To investigate whether the accumulation of (p)ppGpp and stlA expression was linked to nutrient limitations during post‐exponential growth, we measured growth and stlA promoter activity in 1× and 0.1× modified M9 medium, respectively, as described previously (Lango‐Scholey et al ., ). The 1× modified M9 contains the same levels of tryptone and yeast extract as the LBP medium (1% (w/v) and 0.5 (w/v), respectively), while the 0.1× modified M9 contains only 10% of the tryptone and yeast extract levels of LBP.…”

“…Recent work has shown that secondary metabolism in Photorhabdus is associated with the mutualistic interaction between the bacterium and its nematode partner, and several global and pathway‐specific regulators involved in the coordination and regulation of secondary metabolism and mutualism have been identified (Watson et al ., ; Lango and Clarke, ; Joyce et al ., ; Easom and Clarke, ; Lango‐Scholey et al ., ). In this study we constructed a Δ relA and a Δ relA Δ spoT strain of P. luminescens TTO1 and confirmed that the Δ relA Δ spoT strain does not produce detectable levels of (p)ppGpp.…”