Quantification of gene expression in methanotrophs by competitive reverse transcription-polymerase chain reaction.
To improve the monitoring of methanotrophic activity, a competitive reverse transcription-polymerase chain reaction (RT-PCR) methodology was developed. Homologous internal RNA standards were created for mmoX and pmoA, genes encoding polypeptides of sMMO and pMMO, respectively. Using specific primer sets, expression of sMMO and pMMO could be quantified by means of competitive RT-PCR and capillary electrophoresis with uncoated bare-fused silica columns and UV detection. Using this technique, it was discovered that the amount of mRNA transcript for both mmoX and pmoA correlated well with whole-cell sMMO and pMMO activity respectively. A method for soil RNA extraction was also developed to utilize this RNA quantification technique for the monitoring of methanotrophic activity in situ. In a model soil slurry system with a background concentration of 2.9 micro M copper, it was found that only pmoA was transcribed by cells capable of expressing both forms of MMO. As pMMO and sMMO have very different substrate ranges and kinetics, this methodology may prove useful for optimizing in situ bioremediation by methanotrophs. Provided sufficient sequence information is available to create specific primer sets, these techniques can be applied for monitoring and measuring the activity of other microbial communities in situ.
Han
Jong-In
JI
Department of Civil and Environmental Engineering, The University of Michigan, 1351 Beal Avenue, Ann Arbor, Michigan 48109-2125, USA.
Semrau
Jeremy D
JD
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D008772:Methylococcaceae / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012988:Soil Microbiology; D013223:Statistics as Topic
D001426:Bacterial Proteins; D012329:RNA, Bacterial