Genetics

4. Genetics
4.1. Genetic Tools

A variety of genetic tools are available for methanotrophs and have been successfully applied for decades.  Replicating vectors are broad host-range vectors, most commonly of the IncP type, but other replicons have also been successful. The tools include vectors for general cloning, expression, promoter screening, chromosomal insertion, and gene deletion (see Table 4.1)

Table 4.1. Vectors Used in Methanotrophs

4.2 Gene Transfer

The most common mode of gene transfer for methanotrophs is conjugation, although electroporation has also been successful in Type II methanotrophs (Table 4.2). The one report of transformation (Williams and Bainbridge, 1971) has not been repeated.

4.2.1. Conjugation Protocols. Detailed protocols can be obtained in the references noted in Table 4.2. In general, conjugative plasmids are introduced into an E. coli donor strain by transformation.  It is common to use strain S17-1, which has transfer genes integrated into the chromosome.  Alternatively, triparental matings can be used with an E. coli donor strain, the recipient strain, and a helper strain containing a plasmid with transfer genes, such as pRK2013. Strains can be either grown on plates or in liquid cultures, then mixed, normally at a donor:recipient ratio of 1:2 to 1:5 and plated on a mating plate containing diluted rich medium plus the normal methanotroph medium, incubated on a substrate the methanotroph can use, normally methane. Alternatively, the mixture can be filtered and the mating is carried out on a filter placed on a mating plate. Mating time depends on the growth rate of the methanotroph, but 1-2 days is common.  Cells are then transferred to selective plates and allowed to grow out, compared to controls with E. coli not containing the donor plasmid.  Depending on the strain it can take 2-3 weeks for colonies to appear.  If an antibiotic-resistant methanotroph strain is not available, it is common to retain some amount of E. coli on the first transfer to selective plates and in this case, it is necessary to restreak single colonies to confirm they are the methanotroph containing the antibiotic resistance of the plasmid to be transferred.  Many methanotrophs are naturally resistant to 25 µg/ml naladixic acid, and if a nal-sensitive strain of E. coli is used, this can be used as a selective marker.

Table 4.2.  Gene Transfer Approaches Used in Methanotrophs

4.3.  Expression of Methanotroph Genes in Alternate Hosts

A number of methanotroph genes have been successful expressed in alternate hosts (Table 4.3).  However, the pMMO subunits are very toxic to E. coli and have not yet been successfully expressed, with the exception of engineered fragments of the soluble domain of the pmoB subunit (Balasubramanian et al., 2010). It is often difficult to clone these subunits in E. coli, and low copy, low expression vectors are sometimes needed. For the sMMO, neither methane oxidation nor propylene oxidation activity have been demonstrated for sMMO expressed in alternate hosts (Jahng et al., 1996; West et al., 1992), and it has been shown in E. coli that the hydroxylase component is inactive (West et al., 1992) However, active sMMO has been expressed in methanotroph hosts not containing sMMO (Lloyd et al., 1999a).

Table 4.3.  Expression of Methanotroph Genes in Alternate Hosts