Abstract
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.
Highlights
Ubiquitous and abundant in marine environments, the genus Marinobacter contains a metabolic diversity and environmental versatility that has gained recent attention as a prospective biocatalyst for wax ester synthesis (Holtzapple and Schmidt-Dannert, 2007; Lenneman et al, 2013) and remediation of hydrocarbon contaminated environments (Strong et al, 2013; Fathepure, 2014)
A bacterium belonging to the genus Marinobacter, designated isolate JG233, was isolated from a downwardly oriented borehole located on the lowest level of the mine, approximately 714 m below the surface
Anaerobic and Fe(II)-rich water started welling from the deeper of these boreholes. The effluent of these boreholes is ideal for microaerophilic Fe(II)-oxidizing bacteria as oxygen diffusion forms opposing Fe(II) and oxygen gradients
Summary
Ubiquitous and abundant in marine environments, the genus Marinobacter contains a metabolic diversity and environmental versatility that has gained recent attention as a prospective biocatalyst for wax ester synthesis (Holtzapple and Schmidt-Dannert, 2007; Lenneman et al, 2013) and remediation of hydrocarbon contaminated environments (Strong et al, 2013; Fathepure, 2014). A bacterium belonging to the genus Marinobacter, designated isolate JG233, was isolated from a downwardly oriented borehole located on the lowest level of the mine, approximately 714 m below the surface. Heterotrophic cultivation estimates concentration of Marinobacter to be approximately 105 CFU/mL from the site where JG233 was isolated (data not shown). These findings are further supported by cultivation-independent analysis where Marinobacter species constituted a significant fraction of 16S rDNA sequences from several sites within the mine (unpublished work). The presence of Marinobacter suggests the genus is well suited to survival in the high salinity and Fe(II) found in the mine, and as such is likely to influence nutrient and geochemical cycling
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