Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore

Abstract

In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.

A mutant in one of two exbD loci of a TonB system in Flavobacterium psychrophilum shows attenuated virulence and confers protection against cold water disease

“…Up-and downregulation of extracellular enzymes by iron limitation has been described for different bacteria (Tokuda et al, 1998;Enard & Expert, 2000;Maunsell et al, 2006). Although in some cases this regulation is part of a more complex pathway related to virulence (Beare et al, 2003), the function of this association remains unclear.…”

Characterisation of the thermostable protease AprX in strains of Pseudomonas fluorescens and impact on the shelf-life of dairy products: preliminary results

“…The AprX protein and its encoding gene have been studied extensively (Liu et al , 2007; Maunsell et al , 2006; Nicodeme et al ., 2005; Woods et al ., 2001; Liao and McCallus, 1998). Differences in AprX enzyme activities were highlighted, but the identification of the most proteolytic strains was not completely elucidated (Dufour et al ., 2008; Marchand et al , 2009a, 2009b).…”

Cell-surface signaling inPseudomonas: stress responses, iron transport, and pathogenicity

“…However, there are no evident Fur or IS boxes in the aprA promoter region. The expression of the aprA gene in P. fluorescens is also repressed by iron (plausibly through Fur) and positively regulated by σ PbrA (the σ PvdS homolog of P. fluorescens ) (Maunsell et al ., ). These findings suggest that iron‐ and σ PvdS ‐mediated control of AprA production could be a conserved trait of fluorescent pseudomonads.…”