Transcriptional analysis of pqqD and study of the regulation of pyrroloquinoline quinone biosynthesis in Methylobacterium extorquens AM1

Methanol dehydrogenase, the enzyme that oxidizes methanol to formaldehyde in gram-negative methylotrophs, contains the prosthetic group pyrroloquinoline quinone (PQQ). To begin to analyze how the synthesis of PQQ is coordinated with the production of other methanol dehydrogenase components, the transcription of one of the key PQQ synthesis genes has been studied. This gene (pqqD) encodes a 29-amino- acid peptide that is thought to be the precursor for PQQ biosynthesis. A unique transcription start site was mapped to a guanidine nucleotide 95 bp upstream of the pqqD initiator codon. RNA blot analysis identified two transcripts, a major one of 240 bases encoding pqqD and a minor one of 1,300 bases encoding pqqD and the gene immediately downstream, pqqG. Both transcripts are present at similar levels in cells grown on methanol and on succinate, but the levels of PQQ are about fivefold higher in cells grown on methanol than in cells grown on succinate. These results suggest that PQQ production is regulated at a level different from the transcription of pqqD. The genes mxbM, mxbD, mxcQ, mxcE, and mxaB are required for transcription of the genes encoding the methanol dehydrogenase subunits and were assessed for their role in PQQ production. PQQ levels were measured in mutants defective in each of these regulatory genes and compared with levels of pqqD transcription, measured with a transcriptional fusion between the pqqD promoter and xylE. The results showed that only a subset of these regulatory genes (mxbM, mxbD, and mxaB) is required for transcription of pqqD, and only mxbM and mxbD mutants affected the final levels of PQQ significantly

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from Methylococcus capsulatus bath and Methylomonas albus BG8

An open-reading-frame fragment of a Methylobacterium sp. strain AM1 gene (moxF) encoding a portion of the methanol dehydrogenase structural protein has been used as a hybridization probe to detect similar sequences in a variety of methylotrophic bacteria. This hybridization was used to isolate clones containing putative moxF genes from two obligate methanotrophic bacteria, Methylococcus capsulatus Bath and Methylomonas albus BG8. The identity of these genes was confirmed in two ways. A T7 expression vector was used to produce methanol dehydrogenase protein in Escherichia coli from the cloned genes, and in each case the protein was identified by immunoblotting with antiserum against the Methylomonas albus methanol dehydrogenase. In addition, a moxF mutant of Methylobacterium strain AM1 was complemented to a methanol-positive phenotype that partially restored methanol dehydrogenase activity, using broad-host-range plasmids containing the moxF genes from each methanotroph. The partial complementation of a moxF mutant in a facultative serine pathway methanol utilizer by moxF genes from type I and type X obligate methane utilizers suggests broad functional conservation of the methanol oxidation system among gram-negative methylotrophs

Identification and nucleotide sequences of mxaA, mxaC, mxaK, mxaL, and mxaD genes from Methylobacterium extorquens AM1

The DNA sequence for a 4.4-kb HindIII-XhoI Methylobacterium extorquens AM1 DNA fragment that is known to contain three genes (mxaAKL) involved in incorporation of calcium into methanol dehydrogenase (I. W. Richardson and C. Anthony, Biochem. J. 287:709-7115, 1992) was determined. Five complete open reading frames and two partial open reading frames were found, suggesting that this region contains previously unidentified genes. A combination of sequence analysis, mutant complementation data, and gene expression studies showed that these genes correspond to mxaSACKLDorf1. Of the three previously unidentified genes (mxaC, mxaD, and orf1), mutant complementation studies showed that mxaC is required for methanol oxidation, while the function of the other two genes is still unknown

Cloning, Mutagenesis, and Physiological Effect of a Hydroxypyruvate Reductase Gene from Methylobacterium extorquens AM1

The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase

Genetic Engineering of a Radiation-Resistant Bacterium for Biodegradation of Mixed Wastes

The mixture of toxic chemicals, heavy metals, halogenated solvents and radionuclides in many DOE waste materials presents a challenging problem for separating the different species and disposing of individual contaminants. One approach for dealing with mixed wastes is to genetically engineer the radiation-resistant bacterium, Deinococcus radiodurans to survive in and detoxify DOE's mixed waste streams, and to develop process parameters for treating mixed wastes with such constructed strains. The goal for this project is to develop a suite of genetic tools for Deinococcus radiodurans and to use these tools to construct and test stable strains for detoxification of haloorganics in mixed wastes

Genetic Engineering of a Radiation-Resistant Bacterium for Biodegradation of Mixed Wastes

The mixture of toxic chemicals, heavy metals, halogenated solvents and radionuclides in many DOE waste materials presents a challenging problem for separating the different species and disposing of individual contaminants. One approach for dealing with mixed wastes is to genetically engineer the radiation-resistant bacterium, Deinococcus radiodurans to survive in and detoxify DOE's mixed waste streams, and to develop process parameters for treating mixed wastes with such constructed strains. The goal for this project is to develop a suite of genetic tools for Deinococcus radiodurans and to use these tools to construct and test stable strains for detoxification of haloorganics in mixed wastes

Genetic Engineering of a Radiation-Resistant Bacterium for Biodegradation of Mixed Wastes

The mixture of toxic chemicals, heavy metals, halogenated solvents and radionuclides in many DOE waste materials presents a challenging problem for separating the different species and disposing of individual contaminants. One approach for dealing with mixed wastes is to genetically engineer the radiation-resistant bacterium, Deinococcus radiodurans to survive in and detoxify DOE's mixed waste streams, and to develop process parameters for treating mixed wastes with such constructed strains. The goal for this project is to develop a suite of genetic tools for Deinococcus radiodurans and to use these tools to construct and test stable strains for detoxification of haloorganics in mixed wastes

Flux Analysis Uncovers Key Role of Functional Redundancy in Formaldehyde Metabolism

Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and (14)C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop

Methanol Oxidation Genes in the Marine Methanotroph Methylomonas sp. Strain A4

Methanol dehydrogenase has been purified from the type I marine methanotroph Methylomonas sp. strain A4 and found to be similar to other methanol dehydrogenase enzymes in subunit composition, molecular mass, and N-terminal sequence of the two subunits. A heterologous gene probe and a homologous oligonucleotide have been used to identify a DNA fragment from Methylomonas sp. strain A4 which contains moxF, the gene encoding the large subunit of methanol dehydrogenase. Protein expression experiments with Escherichia coli, immunoblotting of expression extracts, and partial DNA sequence determination have confirmed the presence of moxF on this DNA fragment. In addition, expression and immunoblot experiments have shown the presence of the genes for the small subunit of methanol dehydrogenase (moxI) and for the methanol dehydrogenase-specific cytochrome c (moxG). The moxG gene product has been shown to be cytochrome c552. The expression experiments have also shown that two other genes are present on this DNA fragment, and our evidence suggests that these are the homologs of moxJ and moxR, whose functions are unknown. Our data suggest that the order of these genes in Methylomonas sp. strain A4 is moxFJGIR, the same as in the facultative methylotrophs. The transcriptional start site for moxF was mapped. The sequence 5' to the transcriptional start does not resemble other promoter sequences, including the putative moxF promoter sequence of facultative methylotrophs. These results suggest that although the order of these genes and the N-terminal amino acid sequence of MoxF and MoxI are conserved between distantly related methylotrophs, the promoters for this gene cluster differ substantially