Genetics

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

Vector Inc group Selective markers Strains References Availability
Conjugation helper vectors
pRK2013 ColEI Km Methylosinus sp. 6 (Toukdarian and Lidstrom, 1984)
Methylomonas 16a (Sharpe et al., 2007)
pRK2073 Sm Methylomicrobium alcaliphilum 20Z (Mustakhimov et al., 2010)
strain S17-1 (tra genes in chromosome) Methylosinus trichosporium OB3b (Martin and Murrell, 1995)
Methylococcus capsulatus Bath (Stolyar et al., 1999)
Methylosinus trichosporium OB3b, Methylomicrobium album BG8 (Methylobacter albus BG8), Methylocystis parvus OBBP (Lloyd et al., 1999a; Lloyd et al., 1999b)
Methylomicrobium alcaliphilum 20Z (Mustakhimov et al., 2010)
Methylocella sylvestris SC2 (Theisen et al., 2005)
Replicating vectors
pVK100 Inc P Tc, Km Methylosinus trichosporium OB3b, Methylomicrobium album BG8 (Methylobacter albus BG8), Methylocystis parvus OBBP (Lloyd et al., 1999a; Lloyd et al., 1999b)
Methylosinus sporium (Ali et al., 2006)
pHM1 Inc Q Sm, Km Methylosinus trichosporium OB3b (Lloyd et al., 1999a)
pSRK-Km pBBR1 deriv Km Methylococcus capsulatus Bath (Welander and Summons, 2012)
Expression vectors
pGD500 IncP; reporter lacZ Tc Methylomicrobium album BG8 (Methylobacter albus BG8) (Chistoserdova et al., 1994) not available
pMHA203 IncP; reporter GFP; sigma54 promoter Km Methylocella silvestris BL2 (Theisen et al., 2005)
pBHR1 IncP; pCAT promoter Km Methylomonas 16a (Sharpe et al., 2007)
Promoter cloning vectors
pPROBE-NT derivative pBBR1; reporter GFP Km Methylococcus capsulatus Bath (Csaki et al., 2003)
pMHA200 IncP, ColE1; reporter GFP Km Methylocella silvestris BL2 (Theisen et al., 2005)
pMHA010 ColE1 (integrative);mob;  reporter GFP Gm Methylococcus capsulatus Bath (Ali and Murrell, 2009)
pMHA012 ColE1 (integrative);mob;  reporter GFP Km (Ali and Murrell, 2009)
pMHA020 ColE1 (integrative); reporter; reporter XylE Gm Methylococcus capsulatus Bath (Ali and Murrell, 2009)
pMHA030 ColE1 (integrative); mob;  reporter; reporter LacZ Gm Methylococcus capsulatus Bath (Ali and Murrell, 2009)
pMHA199 IncP, ColE1; mob; reporter XylE Km Methylococcus capsulatus Bath (Ali and Murrell, 2009)
pTSGex IncP, ColE1; mob; reporter GFP Km Methylomicrobium alcaliphilum 20Z (Mustakhimov et al., 2010)
Deletion (suicide) vectors
pBR325 oriV (ColE1) Ap Methylosinus sp. 6 (Toukdarian and Lidstrom, 1984)
pBR325mob oriV (ColE1)/mob Ap Methylosinus trichosporium OB3b (Martin and Murrell, 1995)
pAYC61 oriV (ColE1)/mob Ap, Tc Methylomicrobium album BG8 (Methylomonas albus BG8) (Berson and Lidstrom, 1997) not available; precursor to pCM184
Methylococcus capsulatus Bath (Stolyar et al., 1999)
pK18mob oriV (ColE1)/mob Km Methylosinus trichosporium OB3b (Stafford et al., 2003)
Methylosinus sporium (Ali et al., 2006)
pBR329mob oriV (ColE1)/mob Ap, Km Methylosinus trichosporium OB3b (Stafford et al., 2003)
pK18mobsacB oriV (ColE1)/mob w/sacB Km Methylococcus capsulatus Bath (Csaki et al., 2003)
Methylosinus trichosporium OB3b (Semrau et al., 2013)
pCM184 oriV (ColE1)/mob Ap, Tc Methylocella sylvestris (Chen et al., 2010) Addgene
Methylomicrobium alcaliphilum 20Z (Mustakhimov et al., 2010)
pUC18 Ap Methylocystis SC2 (Baani and Liesack, 2008)
pJQ200SK p15a w/sacB Gm Methylococcus capsulatus Bath (Welander and Summons, 2012)
pUTmini-Tn5km Mobilizable transposon vector for chromosomal insertion Km, Ap Methylosinus sp. B4S (Iguchi et al., 2013)
Marker exchange vectors
pGP704-sacB IncP (R6K) w/sacB as a selection Ap Methylomonas 16a (Sharpe et al., 2007)

 

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

Technique Strains References
Transformation Methylococcus capsulatus (Williams and Bainbridge, 1971)
Conjugation Methylosinus sp. 6 (Toukdarian and Lidstrom, 1984)
Methylococcus capsulatus Bath (Stolyar et al., 1999)
Methylosinus trichosporium OB3b (Lloyd et al., 1999a)
Methylomicrobium album BG8 (Methylomonas albus BG8) (Lloyd et al., 1999b)
Methylocystis parvus OBBP (Lloyd et al., 1999b)
Methylocella sylvestris BL2 (Theisen et al., 2005)
Methylomonas 16a (Sharpe et al., 2007)
Methylomicrobium alcaliphilum 20Z (Mustakhimov et al., 2010)
Electroporation Methylocystis SC2 (Baani and Liesack, 2008)
Methylocella silvestris BL2 (Crombie and Murrell, 2011)
Transposon mutagenesis Methylosinus sp. 6 (Toukdarian and Lidstrom, 1984)
Methylomonas 16a (Sharpe et al., 2007)

 

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

Alternate host Strain from which genes originated Genes expressed References
E. coli Methylococcus capsulatus Bath, Methylomicrobium album BG8 (Methylomonas albus BG8) mxaF (moxF) from T7 (Stephens et al., 1988)
E. coli Methylococcus capsulatus Bath glnA in pBR322 (Cardy and Murrell, 1990)
E. coli Methylococcus capsulatus Bath mmoB, mmoC from T7 (West et al., 1992)
E. coli Methylomonas sp. A4 mxaF (moxF) from T7 (Waechterbrulla et al., 1993)
E. coli Methylomicrobium album BG8 (Methylomonas albus BG8) mxaACKLD from T7 (Arps et al., 1995)
E. coli Methylococcus capsulatus Bath mopE from T7 (Fjellbirkeland et al., 2001)
E. coli Methylomicrobium alcaliphilum 20Z ectA (ectoine biosyn) from T7 (Reshetnikov et al., 2005)
E. coli Methylomicrobium alcaliphilum 20Z ectABC (ectoine biosyn) from T7 (Reshetnikov et al., 2006)
E. coli Methylomicrobium alcaliphilum 20Z pfp (phosphofructokinase) from T7 (Reshetnikov et al., 2008)
E. coli Methylomicrobium alcaliphilum 20Z ectR (regulator) from T7 (Mustakhimov et al., 2010)
E. coli Methylococcus capsulatus Bath fba from T7 (Rozova et al., 2010)
E. coli Methylosinus trichosporium OB3b fba from T7 (Rozova et al., 2012)
E. coli Methylomicrobium alcaliphilum 20Z ATP-dep fructokinase (But et al., 2012)
M. parvus OBBP Methylococcus capsulatus Bath Methylosinus trichosporium OB3b sMMO (Lloyd et al., 1999a)
M. album BG8 Methylococcus capsulatus Bath Methylosinus trichosporium OB3b sMMO (Lloyd et al., 1999a)