Horizontal Gene Transfer of Genes Encoding Copper-Containing Membrane-Bound Monooxygenase (CuMMO) and Soluble Di-iron Monooxygenase (SDIMO) in Ethane- and Propane-Oxidizing Rhodococcus Bacteria.

The families of copper-containing membrane-bound monooxygenases (CuMMOs) and soluble di-iron monooxygenases (SDIMOs) are involved not only in methane oxidation but also in short-chain alkane oxidation. Here, we describe Rhodococcus sp. strain ZPP, a bacterium able to grow with ethane or propane as the sole carbon and energy source, and report on the horizontal gene transfer (HGT) of actinobacterial hydrocarbon monooxygenases (HMOs) of the CuMMO family and the sMMO (soluble methane monooxygenase)-like SDIMO in the genus Rhodococcus. The key function of HMO in strain ZPP for propane oxidation was verified by allylthiourea inhibition. The HMO genes (designated hmoCAB) and those encoding sMMO-like SDIMO (designated smoXYB1C1Z) are located on a linear megaplasmid (pRZP1) of strain ZPP. Comparative genomic analysis of similar plasmids indicated the mobility of these plasmids within the genus Rhodococcus. The plasmid pRZP1 in strain ZPP could be conjugatively transferred to a recipient Rhodococcus erythropolis strain in a mating experiment and showed similar ethane- and propane-consuming activities. Finally, our findings demonstrate that the horizontal transfer of plasmid-based CuMMO and SDIMO genes confers the ability to use ethane and propane on the recipient. IMPORTANCE CuMMOs and SDIMOs initiate the aerobic oxidation of alkanes in bacteria. Here, the supposition that horizontally transferred plasmid-based CuMMO and SDIMO genes confer on the recipient similar abilities to use ethane and propane was proposed and confirmed in Rhodococcus. This study is a living example of HGT of CuMMOs and SDIMOs and outlines the plasmid-borne properties responsible for gaseous alkane degradation. Our results indicate that plasmids can support the rapid evolution of enzyme-mediated biogeochemical processes.

Horizontal gene transfer of three co-inherited methane monooxygenase systems gave rise to methanotrophy in the Proteobacteria.

The critical role that bacterial methanotrophs have in regulating the environmental concentrations of the potent greenhouse gas, methane, under aerobic conditions is dependent on monooxygenase enzymes which oxidise the substrate as both a carbon and energy source. Despite the importance of these organisms, the evolutionary origins of aerobic methane oxidation capability and its relationship to proteobacterial evolution is not well understood. Here we investigated the phylogenetic relationship of proteobacterial methanotrophs with related, non-methanotrophic bacteria using 16S rRNA and the evolution of two forms of methane monooxygenase: membrane bound (pMMO and pXMO) and cytoplasmic (sMMO). Through analysis we have concluded that extant proteobacterial methanotrophs evolved from up to five ancestral species, and that all three methane monooxygenase systems, pMMO, pXMO and sMMO, were likely present in the ancestral species (although pXMO and sMMO are not present in most of the present day methanotrophs). Here we propose that the three monooxygenase systems entered the ancestral species by horizontal gene transfer, with these likely to have pre-existing physiological and metabolic attributes that supported conversion to methanotrophy. Further, we suggest that prior to these enzyme systems developing methane oxidation capabilities, the membrane-bound and cytoplasmic monooxygenases were already both functionally and phylogenetically associated. These results not only suggest that sMMO and pXMO have a far greater role in methanotrophic evolution than previously understood but also implies that the co-inheritance of membrane bound and cytoplasmic monooxygenases have roles additional to that of supporting methanotrophy.

Tagged Highly Degenerate Primer (THDP)-PCR for Community Analysis of Methane- and Ammonia-oxidizing Bacteria Based on Copper-containing Membrane-bound Monooxygenases (CuMMO).

We describe a two-step PCR strategy using tagged highly degenerate primer (THDP-PCR) targeting copper-containing membrane-bound monooxygenases (CuMMO) genes for community analysis of methane- or ammonia-oxidizing bacteria. This strategy consists of a primary CuMMO gene-specific PCR followed by a secondary PCR with a tag as a single primer. This strategy remarkably increases the divergence of CuMMO gene amplicons while maintaining PCR efficiency without obvious amplification bias. This THDP-PCR strategy can be extended to other functional gene-based community analysis with design of new highly degenerate primer covering target functional gene sequences.

An improved protocol with a highly degenerate primer targeting copper-containing membrane-bound monooxygenase genes for community analysis of methane- and ammonia-oxidizing bacteria.

The copper-containing membrane-bound monooxygenase (CuMMO) family comprises key enzymes for methane or ammonia oxidation: particulate methane monooxygenase (PMMO) and ammonia monooxygenase (AMO). To comprehensively amplify CuMMO genes, a two-step PCR strategy was developed using a newly designed tagged highly degenerate primer (THDP; degeneracy = 4608). Designated THDP-PCR, the technique consists of primary CuMMO gene-specific PCR followed by secondary PCR with a tag as a single primer. No significant bias in THDP-PCR amplification was found using various combinations of template mixtures of pmoA and amoA genes, which encode key subunits of the pMMO and AMO enzymes, respectively, from different microbes. THDP-PCR was successfully applied to nine different environmental samples and revealed relatively high contents of complete ammonia oxidation (Comammox)-related bacteria and a novel group of the CuMMO family. The levels of freshwater cluster methanotrophs obtained by THDP-PCR were much higher than those obtained by conventional methanotroph-specific PCR. The THDP-PCR strategy developed in this study can be extended to other functional gene-based community analyses, particularly when the target gene sequences lack regions of high consensus for primer design.