AbstractsEngineering

Methylobacterium extorquens AM1 is a pink pigmented, facultative methylotroph that is capable of utilizing single-carbon compounds as the sole carbon and energy source. At least twenty-five genes are involved in the first step of methanol utilization, carried out by methanol dehydrogenase, in which methanol is oxidized to formaldehyde. In this project, the transcription of the methanol oxidation (Mox) system has been studied. The promoter region of mxaF, mxaW, pqqA and mxcQ has been defined through the measurement of catechol dioxygenase for cells containing constructs with different sizes of 5' deletion fragments fused to the xylE reporter gene. I have determined the size and location of transcripts using RT-PCR together with excluding the possibility of transcript overlap by screening the larger intergenic regions. No significant activity above the vector background level was observed. The results show that mxaFJGIRSACKLDEHB, pgqABC/DE , mxcQEtenA and the pggFG gene cluster from orf181 through the gene coding for a dioxygenase are each operons transcribed by a single promoter. The transcriptional initiation point of mxaW, mxbD, mxcQ and orf181 has been determined by primer extension. The alignment of the sequence upstream of the six transcriptional start points indicated conservation of -35 sequences, but more divergence of -10 sequences. In addition, a conserved multiple adenine plus one guanine sequence, termed the A-tract, was identified upstream of the -35 consensus region. In all cases, this region was found to be important for transcription of mox genes, and for mxaF, that of deleting or altering this sequence not only reduced transcription to the vector background, but mutants with the deletion or the altered sequence in the chromosome were not able to grow on methanol. Two new regulatory genes were identified. One, called tenA, was found downstream of mxcQE. The tenA mutant exhibits normal growth on methanol. However, analyses of promoter activity in this mutant demonstrated that it appears to mediate methanol induction of the Mox system. Transposon mutagenesis has led to the discovery of a negative regulator in the mxbDM-mxcQE positive amplification loop. The preliminary analysis the 5 ' UTR of mRNA in mxaF led to the hypothesis that two DNA sites are required for normal expression of mxaF in M. extroquens AM1.