Preliminary validation of microarray results using reverse transcription quantitative PCR

An attempt was made to validate the microarray results using reverse transcription quantitative PCR (RT-qPCR) with a SYBR®Green-based approach using the Applied Biosystems 7000 Real-Time PCR System (Applied Biosystems). Genes selected for the RT-qPCR were the 16S rRNA gene, RNA polymerase encoding gene (rpoB) (Msil3868),mmoX(Msil1262 ),glcB/Malate synthase (Msil2501),

icl/isocitrate lyase (Msil3157), fur/ferric uptake regulator (Msil1272) and glycine dehydrogenase (Msil1215). Detailed procedures of the RT-qPCR are described in Chapter 2insection 2.4.4. Synthesis of cDNA from RNA extracted from methane or acetate grownMethylocella silvestriswere confirmed by amplificationmmoX and 16S rRNA genes from the cDNA by RT-PCR (Figure 6.9).

Figure 6.9. Amplification of 16S rRNA gene (Lane 1 to 6) andmmoX gene (Lane 7 to 12) from cDNA generated from RNA extracted from Methylocella silvestris

grown either on methane or acetate. Lane 1 and lane7 = cDNA (methane), lane 2 and lane 8 = RT negative (methane), Lane 3 and lane 9 = cDNA (acetate), Lane 4 and lane 10 = RT negative (acetate), Lane 5 and lane 11 = PCR positive control (Methylocella silvestris genomic DNA), Lane 6 and lane 12 = Negative control (Non-template control), M=1 kbp DNA ladder.

However, when the RT-qPCR was carried out with the cDNA, the dissociation curve analysis indicated the formation of non-specific amplicons at the end of the assay. Further attempts were not made in this regard due to time limitations.

6.3 Discussion

The use of high quality RNA is the first step in obtaining meaningful gene expression data. The quality of the RNA used in this study for the microarray experiments were judged by determining the RNA integrity number (RIN) that reflects the integrity of the RNA (Fleigeet al., 2006). Total RNA extracted using the hot phenol-chloroform method as described by Gilbertet al.(2000) yielded high

quality RNA as revealed by analyses using the 2100 Bioanalyzer (Agilent Technologies, USA).

The technical and biological replicates of RNA used in the microarray experiment did not have any major effect on the findings of the microarray results as shown by the normalized signal intensity values of probes generated from the different replicates (Figure 6.5) and the PCA analysis (Figure 6.6). However, to make the microarray results more meaningful, only these data obtained from three biological replicates were included in the final analyses. Cluster analyses of the data obtained from the microarray experiments were carried out based upon biological replicates (Figure 6.7). Cluster analysis is traditionally used in phylogenetic research and has also been adopted for microarray analysis. It is an also an easy way to find out which genes are differentially transcribed under different growth conditions. The results of the cluster analysis indicated that genes found to be highly upregulated during growth on methane were found to be highly downregulated during growth on acetate, and vice versa, as expected.

To provide global information on gene expression during growth on C1or C2

compounds, microarray experiments were carried out by comparing methane (C1

compound) with acetate (C2compound) growth conditions. During growth on

methane, more genes (n=169; 4.25% of the total genome) were found to be upregulated compared to the cells grown on acetate (n=85; 2.14% of the total

genome).As expected, all the genes of the sMMO operons, both the structural genes

e.g., mmoXYBZDCand the regulatory genese.g., mmoR, orf2andmmoG,were

The fold changes in the expression of sMMO gene clusters as observed in the microarray experiment are present inFigure 6.10.

Figure 6.10. The sMMO operon and related genes and their fold changes as observed during growth of Methylocella silvestris on methane in the microarray experiment.

ThemmoXYBZDCgene cluster encodes different subunits of the hydroxylase and associated protein of sMMO, whilemmoR, orf2andmmoGencode the regulatory proteins required for the transcription of sMMO (Please seeChapter 1 section 1.4.2 for details about the genes located in the sMMO gene cluster and their functions). Upregulation of all of the genes of the sMMO operon inMethylocella silvestris

during growth on methane is in agreement with the earlier observation of Theisenet al.(2005). These authors detected the expression of transcript of themmoXYBZDC

operon by RT-PCR with cDNA generated from RNA extracted fromMethylocella silvestrisgrown on methane. These authors also demonstrated that acetate represses the transcription of sMMO, which is in agreement with the microarray results observed here. In addition, the RT-PCR result carried out here also indicated that

acetate significantly repressed the transcription ofmmoX(Figure 6.9) in

Methylocella silvestris.

It is important to note that although all the genes of the sMMO operon are under the control of the same promoter, their transcription level varied from40 to 1000 fold (Figure 6.10) during growth on methane. It is not unexpected and a similar

phenomenon has been observed at the transcription and translation level of the

aceBAKoperon inEscherichia coliduring growth on acetate (Chunget al., 1993).

aceA, aceBandaceKencode isocitrate lyase, malate synthase and isocitrate dehydrogenase kinase/phosphatase respectively (Brice & Kornberg, 1968) . These three genes are also under the control of the same promoter, however, their

transcription and translation patterns are highly variable during growth on acetate and is related to premature transcriptional termination (Chunget al., 1993).

It is very interesting to note that two other genes located immediately downstream

(3′) of the sMMO operon, e.g.,Msil1271 and Msil1272 were also upregulated during growth on methane (> 1000 fold) (Figure 6.10). Msil1271 encodes a hypothetical protein with no significant identity to any characterized genes, while Msil1272 is annotated in the genome as a ferric uptake regulator (Fur). Fur is a global

transcriptional regulator involved in the regulation of genes responsible for iron acquisition (Hantke, 2001; Rodriguez & Smith, 2003). Iron is an essential

component of the α subunit of sMMO since iron is an essential component of the

diiron active site of sMMO (Green & Dalton, 1985; Foxet al., 1989). Iron is also required for the activity of formate dehydrogenases (Laukelet al., 2003) and the

required during growth on methane for the metabolism of the oxidation products arising from methane (Zhanget al., 2005). It may be possible that during growth on methane,Methylocella silvestrisupregulates the transcription offurso that Fur can in turn induce the transcription of other genes required for the uptake of iron from the medium for the activity of sMMO. For example, two other genes Msil0825 and Msil0826 that encode ExbD and ExbB respectively were found to be upregulated ( 2 to 3 fold) during growth on methane. ExbD and ExbB are membrane-bound transport proteins possibly involved in iron acquisition. InXanthomonas campestris

pv.campestrisExb B and ExbD are essential for ferric uptake (Wiggerichet al., 1997). InEscherichia coli, ExbB and ExbD are required for the transport of siderophores (Ahmeret al., 1995). InMethylocella silvestris, Msil0825 and

Msil0826 gene products could also be involved in iron acquisition. In addition, Fur may have some regulatory effect on the transcription of these genes.

During the methane oxidation process, methanol produced from the methane by the methane monooxygenase is converted into formaldehyde by the methanol

dehydrogenase (Antony, 1982) (seeChapter1,section 1.3, figure 1.4 for methane oxidation pathway). Methanol dehydrogenase is a pyrroloquinoline quinone (PQQ)- linked enzyme encoded by themxagene clusteri.e., mxaFJGIRSACKLDEH. Unusually none of these genes were found to be significantly upregulated during growth on methane, except for two genes Msil1739 and Msil2260 (2 to 160 fold) that are involved in PQQ biosynthesis process. PQQ acts as a prosthetic group for several bacterial enzymes, including methanol dehydrogenase (Anthony, 1982). It was surprising not to see the upregulation of other genes of themxagene cluster during growth on methane, since methanol dehydrogenase is the second key enzyme