Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes

Abstract

Chloromethane gas is produced naturally in the phyllosphere, the compartment defined as the aboveground parts of vegetation, which hosts a rich bacterial flora. Chloromethane may serve as a growth substrate for specialized aerobic methylotrophic bacteria, which have been isolated from soil and water environments, and use cmu genes for chloromethane utilization. Evidence for the presence of chloromethane-degrading bacteria on the leaf surfaces of Arabidopsis thaliana was obtained by specific quantitative PCR of the cmuA gene encoding the two-domain methyltransferase corrinoid protein of chloromethane dehalogenase. Bacterial strains were isolated on a solid mineral medium with chloromethane as the sole carbon source from liquid mineral medium enrichment cultures inoculated with leaves of A. thaliana. Restriction analysis-based genotyping of cmuA PCR products was used to evaluate the diversity of chloromethane-degrading bacteria during enrichment and after strain isolation. The isolates obtained, affiliated to the genus Hyphomicrobium based on their 16S rRNA gene sequence and the presence of characteristic hyphae, dehalogenate chloromethane, and grow in a liquid culture with chloromethane as the sole carbon and energy source. The cmu genes of these isolates were analysed using new PCR primers, and their sequences were compared with those of previously reported aerobic chloromethane-degrading strains. The three isolates featured a colinear cmuBCA gene arrangement similar to that of all previously characterized strains, except Methylobacterium extorquens CM4 of known genome sequence.

Toward a List of Molecules as Potential Biosignature Gases for the Search for Life on Exoplanets and Applications to Terrestrial Biochemistry

“…Although the biosynthetic pathways are well understood (Rhew et al, 2003;Roje, 2006), the exact ecological role and physiological function of methyl chloride remain to be determined, despite earlier suggestions that methyl chloride is produced as a toxicant to deter predators and/or suppress competitors (Hartmans et al, 1986;Manley, 2002). Methyl chloride is widely produced by many plant and algal species (Wuosmaa and Hager, 1990;Nadalig et al, 2011;Rhew et al, 2014) and also by fungi and marine bacteria (Tait and Moore, 1995;Khalil and Rasmussen, 1999).…”

Fluorescence-Based Bacterial Bioreporter for Specific Detection of Methyl Halide Emissions in the Environment

“…Quantitative PCR (qPCR) measurements on cDNA preparations were done with 96-well reaction PCR plates by using a GeneAmp 5700 sequence detection system (Applied Biosystems). Primers cmuA802F (5=-TTCAACGGCGAYATGTATCCYGG-3=) (37) and cmuA968R (5=-CCR CCRTTRTAVCCVACYTC-3=) (30) were used for amplification of the cmuA gene, and amplification of the 16S rRNA gene rrnA was performed with primers BACT1369F and PROK1492R (38). PCRs were carried out with a final volume of 20 l, using 4 l of a cDNA preparation (diluted with ultrapure molecular biology-grade water; Sigma) with a 3 M final concentration of each primer and commercial 1ϫ SYBR green PCR mix (Eurogentec).…”

“…Insights into the biological transformation of methyl halides have become available from studies on the physiology and genetics of bacteria that can degrade methyl halides and utilize chloromethane as the only source of carbon and energy for growth, which have been isolated from various environments, including soils (22)(23)(24)(25), sludge (26)(27)(28), seawater (29), and the phyllosphere (30). The biochemistry and genetics of chloromethane degradation have been elucidated in detail for Methylobacterium extorquens CM4 (31-34), a strain isolated from soil of a petrochemical factory in Tatarstan (23), and the complete genome sequence of this strain was determined and analyzed (35,36).…”

Culture-dependent and culture-independent methods reveal diverse methylotrophic communities in terrestrial environments

“…Using cultivation-dependent and -independent methods, methylotrophs in the plant phyllosphere have indeed been shown to degrade methanol and methyl chloride, thereby acting as plant-associated sinks for these compounds (Gogleva et al 2010;Knief et al 2010;Nadalig et al 2011). Terrestrial one-carbon compound utilization may also be important from an agricultural point of view as microbial degradation of DMS and DMSO in soil may increase the amount of inorganic sulfur available for plants (Kertesz and Mirleau 2004).…”