Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Cs1-4

Shicheng Chen,William J Hickey

doi:10.3389/fmicb.2011.00187

Abstract

The bacterium Delftia sp. Cs1-4 produces novel extracellular structures (nanopods) in conjunction with its growth on phenanthrene. While a full genome sequence is available for strain Cs1-4, genetic tools that could be applied to study phenanthrene degradation/nanopod production have not been reported. Thus, the objectives of this study were to establish such tools, and apply them for molecular analysis of nanopod formation or phenanthrene degradation. Three types of tools were developed or validated. First, we developed a new expression system based on a strong promoter controlling expression of a surface layer protein (NpdA) from Delftia sp. Cs1-4, which was ca. 2,500-fold stronger than the widely used lactose promoter. Second, the Cre-loxP system was validated for generation of markerless, in-frame, gene deletions, and for in-frame gene insertions. The gene deletion function was applied to examine potential roles in nanopod formation of three genes (omp32, lasI, and hcp), while the gene insertion function was used for reporter gene tagging of npdA. Lastly, pMiniHimar was modified to enhance gene recovery and mutant analysis in genome-wide transposon mutagenesis. Application of the latter to strain Cs1-4, revealed several new genes with potential roles in phenanthrene degradation or npdA expression. Collectively, the availability of these tools has opened new avenues of investigation in Delftia sp. Cs1-4 and other related genera/species with importance in environmental toxicology.

Highlights

Bacteria of the genus Delftia mediate a diversity of processes important in environmental toxicology, including xenobiotic biodegradation and biotransformation of heavy metals (Vacca et al, 2005; De Gusseme et al, 2010; Juarez-Jimenez et al, 2010; Leibeling et al, 2010; Paulin et al, 2010; Zhang et al, 2010; Morel et al, 2011; Yang et al, 2011)
Genome sequence data will be an essential resource for identification of functions in Delftia spp. that are key to these activities, and one recently completed genome is that of the phenanthrene degrader Delftia sp
Nanopods are tubular elements that contain outer membrane vesicles (OMV) within a sheath composed of a surface layer protein (SLP)

Summary

Introduction

Bacteria of the genus Delftia mediate a diversity of processes important in environmental toxicology, including xenobiotic biodegradation and biotransformation of heavy metals (Vacca et al, 2005; De Gusseme et al, 2010; Juarez-Jimenez et al, 2010; Leibeling et al, 2010; Paulin et al, 2010; Zhang et al, 2010; Morel et al, 2011; Yang et al, 2011). Nanopods are tubular elements that contain outer membrane vesicles (OMV) within a sheath composed of a surface layer protein (SLP). The latter was termed Nanopod protein A (NpdA), and mutants lacking this protein were unable to form nanopods. Proteomic analyses of nanopods revealed a variety of proteins that were associated with these structures, two being outer membrane protein 32 (Omp32) and hemolysin co-regulated protein (Hcp). These proteins were of interest as we hypothesized that they, along with NpdA, could have key roles in nanopod structure. We hypothesized that nanopod production may be regulated by quorum sensing

Objectives

Methods

Results

Discussion

Conclusion