Transposon mutagenesis affecting thiosulfate oxidation in Bosea thiooxidans, a new chemolithoheterotrophic bacterium

Abstract

Transposon insertion mutagenesis was used to isolate mutants of Bosea thiooxidans which are impaired in thiosulfate oxidation. Suicide plasmid pSUP5011 was used to introduce the transposon Tn5 into B. thiooxidans via Escherichia coli S17.1-mediated conjugation. Neomycin-resistant transconjugants occurred at a frequency of 2.2 ؋ 10 ؊4 per donor. Transconjugants defective in thiosulfate oxidation were categorized into three classes on the basis of growth response, enzyme activities, and cytochrome patterns. Class I mutants were deficient in cytochrome c, and no thiosulfate oxidase activity was detected. Class II mutants retained the activities of key enzymes of thiosulfate metabolism, although at reduced levels. Mutants of this class grown on mixed-substrate agar plates deposited elemental sulfur on the colony surfaces. Class III mutants were unable to utilize thiosulfate, though they had normal levels of cytochrome c. The transposon insertions occurred at different chromosomal positions, as confirmed by Southern blotting of chromosomal DNA of mutants deficient in thiosulfate oxidation, a deficiency which resulted from single insertions of Tn5.Little is known about the structure, expression, and regulation of the genes associated with the oxidation of inorganic sulfur compounds of the obligately (17, 44) or facultatively (13,31,43) . These heterotrophs may benefit from sulfur oxidation in the detoxification of hydrogen peroxide (18,29). To date, preliminary work has been done to understand the genetical basis of sulfur metabolism by obligate chemolithoautotrophic (Thiobacillus ferrooxidans) (30) and facultatively chemolithoautotrophic (Thiobacillus versutus and Thiobacillus novellus) bacteria (9). However, little is known at the genetic level, and no genes involved in oxidation of sulfur have been identified or cloned (47-49). Until recently, no DNA probes for reduced inorganic sulfur oxidation genes existed (30). In addition, there is no defined mutant deficient in sulfur oxidation and an only rudimentary genetic system has been discussed for Thiobacillus novellus, Thiobacillus versutus (9), Paracoccus denitrificans GB17 (51), Thiosphaera pantotropha (5, 28), and a bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila (19). Genetic systems to study bacterial species capable of oxidizing inorganic sulfur compounds are only now being developed. Transposon mutagenesis of Bosea thiooxidans (8) was used to generate mutants impaired in thiosulfate oxidation.Transposon insertion mutagenesis has been applied to a wide range of gram-negative bacteria (5,16,34,35,45) for both strain construction and the analysis of mutant phenotypes, but this approach has not been exploited to study the mechanism of inorganic sulfur oxidation for chemolithoheterotrophs.B. thiooxidans BI-50 was selected for genetic studies of thiosulfate-oxidizing activity by transposon Tn5 insertion mutagenesis because (i) its level of spontaneous neomycin resistance above its base sensitivity level is very low; (ii) its generation ti...

Microorganisms Resistant to Free-Living Amoebae

“…The genus Bosea was described by Das et al (57) based on a single isolate, Bosea thiooxydans, recovered from agricultural field soil during a study of chemolithotrophic bacteria (56). Three additional species, B. eneae, B. vestrisii, and B. massiliensis (154), were recovered by amoebal coculture from hospital water supplies (150), demonstrating that some representatives of that genus are ARB commonly present in water.…”

Characterization of an rpoN mutant of Mesorhizobium ciceri

“…Digestions with restriction enzymes and DNA ligation were performed according to the manufacturers’ instructions (Promega, Inc. and New England Biochemical, Beverly, MA, USA). Southern blotting was performed by using a standard Southern blot protocol (Southern 1975) as described previously (Das and Mishra 1996).…”

Characterization of a symbiotically defective serine auxotroph ofMesorhizobium ciceri

“…DNA ligation was performed using Promega 2 Â rapid ligation buffer with 10 units of T4 DNA ligase (Promega, Inc.). Prehybridization and hybridization were performed as described earlier (Das & Mishra, 1996). The filter was hybridized with the probe DNA consisting of the Tn5mob (c. 5.4 kb) HindIII fragment obtained from pSUP5011.…”

“…The probe DNA was labelled using a DecalabelTM DNA labelling kit (MBI, Fermentas, Germany) with [a 32 P] dCTP (Sambrook et al, 1989). Prehybridization and hybridization were performed as described earlier (Das & Mishra, 1996). Prior to autoradiography, the blot was washed twice, each time with 2 Â SSC (standard saline citrate) containing 0.1% sodium dodecyl sulfate (SDS) for 20 min, after which an identical treatment with 0.2 Â SSC and 0.1 % SDS was performed at room temperature.…”