We report the isolation, characterization, and development of a robust genetic system for a halophilic, Fe(II)-oxidizing bacterium isolated from a vertical borehole originating 714 m below the surface located in the Soudan Iron Mine in northern Minnesota, USA. Sequence analysis of the 16S rRNA gene places the isolate in the genus Marinobacter of the Gammaproteobacteria. The genome of the isolate was sequenced using a combination of short- and long-read technologies resulting in two contigs representing a 4.4 Mbp genome. Using genomic information, we used a suicide vector for targeted deletion of specific flagellin genes, resulting in a motility-deficient mutant. The motility mutant was successfully complemented by expression of the deleted genes in trans. Random mutagenesis using a transposon was also achieved. Capable of heterotrophic growth, this isolate represents a microaerophilic Fe(II)-oxidizing species for which a system for both directed and random mutagenesis has been established. Analysis of 16S rDNA suggests Marinobacter represents a major taxon in the mine, and genetic interrogation of this genus may offer insight into the structure of deep subsurface communities as well as an additional tool for analyzing nutrient and element cycling in the subsurface ecosystem.

Achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. Intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent A. xylosoxidans infection. Still, much of A. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. Here we demonstrate a promising genetic system for use in A. xylosoxidans ; generating a transposon mutant library which was then used to identify genes involved in biofilm development in vitro. We further described the effects of one of the genes found in the mutagenesis screen, encoding a putative enoyl-CoA hydratase, on biofilm structure and tolerance to antimicrobials. Through additional analysis, we find that a fatty acid signaling compound is essential to A. xylosoxidans biofilm ultrastructure and maintenance. This work describes methods for the genetic manipulation of A. xylosoxidans and demonstrated their use to improve our understanding of A. xylosoxidans pathophysiology.

Achromobacter xylosoxidans is associated with resilient nosocomial infections, with bacteraemia, pneumonia and chronic cystic fibrosis lung infection being the most common clinical presentations. Innate multi-drug resistance and a suite of virulence factors select for A. xylosoxidans infection during long-term antibiotic therapy, contributing to its persistence, treatment recalcitrance, association with poor clinical outcomes and emergence as a problematic pathogen. Horizontal gene transfer and maintenance of large genomes underpin the resilience and cosmopolitan lifestyle of A. xylosoxidans , and complicate its phylogenetic characterization.

Autotrophic bacteria utilizing Fe(II) as their energy and electron sources for growth affect multiple biogeochemical cycles. Some chemoheterotrophic bacteria have also been considered to exhibit an Fe(II) oxidation phenotype. For example, several Marinobacter strains have been reported to oxidize Fe(II) based on formation of oxidized iron bands in semi-solid gradient tubes that produce opposing concentration gradients of Fe(II) and oxygen. While gradient tubes are a simple and visually compelling method to test for Fe(II) oxidation, this method alone cannot confirm if, and to what extent, Fe(II) oxidation is linked to metabolism in chemoheterotrophic bacteria. Here we probe the possibility of protein-mediated and metabolic byproduct-mediated Fe(II) oxidation in Marinobacter subterrani JG233, a chemoheterotroph previously proposed to oxidize Fe(II). Results from conditional and mutant studies, along with measurements of Fe(II) oxidation rates suggest M. subterrani is unlikely to facilitate Fe(II) oxidation under microaerobic conditions. We conclude that the Fe(II) oxidation phenotype observed in gradient tubes inoculated with M. subterrani JG233 is a result of oligo-heterotrophic activity, shifting the location where oxygen dependent chemical Fe(II) oxidation occurs, rather than a biologically-mediated process. Importance Gradient tubes are the most commonly used method to isolate and identify neutrophilic Fe(II)-oxidizing bacteria. The formation of oxidized iron bands in gradient tubes provides a compelling assay to ascribe the ability to oxidize Fe(II) to autotrophic bacteria whose growth is dependent on Fe(II) oxidation. However, the physiological significance of Fe(II) oxidation in chemoheterotrophic bacteria is less well understood. Our work suggests that oligo-heterotrophic activity of certain bacteria may create a false-positive phenotype in gradient tubes by altering the location of the abiotic, oxygen-mediated oxidized iron band. Based on the results and analysis presented here, we caution against utilizing gradient tubes as the sole evidence for the capability of a strain to oxidize Fe(II) and that additional experiments are necessary to ascribe this phenotype to new isolates.

Achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. Intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent A. xylosoxidans infection. Still, much of A. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. Here we demonstrate a promising genetic system for use in A. xylosoxidans; generating a transposon mutant library which was then used to identify genes involved in biofilm development in vitro. We further described the effects of one of the genes found in the mutagenesis screen, encoding a putative enoyl-CoA hydratase, on biofilm structure and tolerance to antimicrobials. Through additional analysis, we find that a cis-2 fatty acid signaling compound is essential to A. xylosoxidans biofilm ultrastructure and maintenance. This work describes methods for the genetic manipulation of A. xylosoxidans and demonstrated their use to improve our understanding of A. xylosoxidans pathophysiology.