PCR analysis

The polymerase chain reaction (PCR) (Mullis and Faloona, 1987) was used in an attempt to amplify genes from AO22, E9 and VUN 10010 corresponding to the ones previously reported to be involved in heavy metal resistance and PAH degradation (outlined in Chapter 1) and characterisation of the isolates to a species level. For this purpose it was first necessary to design oligonucleotide primers corresponding to published genes. The following sections outline the design of primers and the setting up of PCR.

2.8.1 Criteria for design of oligonucleotide primers

As outlined by Sambrook et al., (1989), the following criteria were considered:

• The length of each primer was at least 18 bases to allow for specific binding to the template.

• The primers contained no self-complementary regions and are not complementary to each other either

• The base composition chosen such that the primer pairs had similar annealing temperatures.

Where possible, primers were designed to amplify overlapping regions of the operon of interest (i.e. mer, pbr). In some cases, a number of related genes were aligned and areas of relative homology selected for primer design (i.e. pah genes) internally to the individual genes. It was sometimes necessary (i.e. for mer and pah genes) to design degenerate primers (more than one nucleotide in a particular position). These are represented by the following: R=A+G, Y=C+T, M=A+C, W=A+T, V=G+A+C. All primers were commercially synthesised by Sigma Genosys (Castle Hill, NSW), with cartridge purification. All primers were obtained as a dried powder and were

73 resuspended in sterile dH2O at a concentration of 2 µg/µL, prior to dilution at appropriate concentrations for PCR and sequencing (refer to Sections 2.8.2and 2.11.1).

2.8.1.1 Primer design for the amplification of mercury resistance genes

Primers were designed to determine the presence of the mer operon in Alcaligenes sp. AO22, A. woluwensis E9 and Consortium VUN 10010, using a number of reported mer

gene sequences. The majority of the primers were designed based on the P. aeruginosa

Tn501mer operon (Z00027) which carries the archetypal merRTPADE genes (Misra et al., 1984; Brown et al., 1986; Barrineau et al., 1984) (Section 1.4). Additional primers were designed based on the alignment of the genes from plasmid pDU1358 of Serratia marcescens (M24940) (Nucifora et al., 1989a)and the Tn21 operon of Shigella flexneri

(AP000342) (Barrineau et al., 1984; Misra et al., 1984; Misra et al., 1985), to address possible heterogeneity of the genes in our isolates, compared to these ones. The primer pair mercP-cA, which corresponds to the merP, merC and merA genes, was designed based on the plasmid NR1 sequence (NM1MER) (Holt et al., 1999). Details of the mer

operons are outlined in Section 1.4. Figure 2.2 provides an overview of the primer locations relating to the merRTPADEorf-2 genes. Further details for each primer are provided in Table 2.2.

2.8.1.2 Primer design for the amplification of lead resistance genes

Primers for the detection of lead resistance (pbr) genes were designed based on the only published lead resistance operon to date, from Cupriavidus metallidurans CH34 (X71400) (Borremans et al., 2001) (Section 1.5). Figure 2.5provides an overview of the primer locations and further details for each primer are provided in Table 2.2.

2.8.1.3 Primer design for the amplification of cadmium resistance genes

Primers for the detection of the cadmium resistance gene (cadA) and regulatory gene (cadC) were designed based on the pI258 plasmid of Staphylococcus aureus

an overview of the primer locations and further details for each primer are provided in Table 2.2.

2.8.1.4 Primer design for the amplification of genes relating to PAH degradation

Unlike the mer and pbr primer pairs, which were designed to span two or more genes, the primers targeting PAH degradation genes were all designed to amplify individual genes, due to the wide inter-species variation of operon structures (see Section 1.7). The primers were designed based on the regions of highest homology in a number of PAH degradation genes. The genes used were from the operons of Ralstonia sp. U2 (AF036940) (Fuenmayor et al., 1998; Zhou et al., 2001), Pseudomonas stutzeri

(AF039533) (Bosch et al., 1999a), P. putida (PSENAPDOXA) (Simon et al., 1993),

Pseudomonas fluorescens (AF004283) (unpublished), Commonas testosteroni

(AF252550) (Moser and Stahl, 2001) and P. aeruginosa (PSEORF1). Figures 2.7-2.13 provide overviews of the primer locations relating to PAH degradation. Further details of primers are provided inTable 2.2.

2.8.2 PCR conditions

PCR mixes were set up by adding: 1 μg genomic DNA template (prepared as described in Section 2.4), 5 μL 5X buffer, 2 μL 50 mM MgCl2, 1 μL dNTPs (1 0 mM mix of

dATP, dCTP, dGTP, dTTP), 3.5 μL of each forward and reverse primer (0.1 μg/μL), and 0.5 μL DNA polymerase enzyme (5 U/ μL) and dH2O to a total volume of 50 μL. AmpliTaq® DNA polymerase (Perkin Elmer) and Taq DNA polymerase (Gibco BRL Life Technologies) enzymes were used for standard PCRs while Expand Long Template PCR system (Roche) was used longer fragments (>1000 bp) and Platinum® Taq DNA polymerase (Invitrogen) was used for reactions that required high levels of sensitivity. Negative control PCRs were set up as above, without the DNA template. PCRs were

Figure 2.2 Location of primers designed to amplify the merRTPADEorf-2 genes. This figure indicates the location of and expected size of amplified products for each primer pair. These primers were designed based on the mer operons of Tn501 (Z00027), Tn21

Figure 2.3 Location of primers designed to amplify the merC gene. This figure indicates the location of and expected size of amplified products for each primer pair. These primers were designed based on the mer operon of Tn21 (AP000342).

Figure 2.4 Location of primers designed to amplify the merB gene. This figure indicates the location of and expected size of amplified products for each primer pair. These primers were designed based on the mer operon of pDU1358 (M24940). The additional primer, mer9 was designed to further amplify the 3’ section of this gene beyond mer24.

77 run in the PTC-100 or PTC-200 Peltier Thermal Cycler (MJ Research) using the following typical cycles: an initial denaturation step at 95oC for 5 minutes followed by 35 cycles of: 94oC for 90 seconds (denaturation); 55-58oC for 1 minute (annealing); 70oC for 3 minutes (elongation); followed by a final elongation step of 70oC for 7 minutes. All amplification reactions were electrophoresed as described in Section 2.7.