Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

Chistoserdov, Andrei Y.; Tsygankov, Y. D.; Lidstrom, Mary E.

doi:10.1128/jb.173.18.5901-5908.1991

Cited by 30 publications

(28 citation statements)

References 55 publications

(64 reference statements)

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“…1). Eight of these polypeptides were present in pairs with a difference in mass of 1.5 to 2 kDa, as expected for unprocessed and mature forms of periplasmic polypeptides (10). These included a pair at 66 and 64.5 kDa, another at 37.5 and 35.5 kDa, a third at 17.5 and 15.5 kDa, and a fourth at 14.5 and 13 kDa.…”

Section: Resultsmentioning

confidence: 99%

“…Escherichia coli strains were grown at 37 or 30°C on Luria broth (25). Where appropriate, filter-sterilized antibiotic solutions were added to sterile medium in the following concentrations: ampicillin, 50 ,ug/ml; kanamycin, 50 ,ug/ml; rifamycin, 10 ,ug/ml; tetracycline, 10 ,ug/ml. When kanamycin and ampicillin were used together, the concentrations were 40 ,ug/ml each.…”

Section: Methodsmentioning

confidence: 99%

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Methanol oxidation genes in the marine methanotroph Methylomonas sp. strain A4

et al. 1993

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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 dehydrogenasespecific 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.Gram-negative methylotrophic bacteria oxidize methanol via a periplasmic methanol dehydrogenase, which contains the quinone cofactor pyrrolo-quinoline quinone and utilizes a soluble cytochrome c (called cytochrome CL) as an electron acceptor (5). This methanol oxidation system is complex, and more than 20 genes (mox genes) that are required for methanol dehydrogenase regulation, synthesis, and assembly and for synthesis of the cofactor pyrrolo-quinoline quinone (19) have been identified in the facultative methanol utilizer Methylobacterium exworquens AML.The structural genes for the methanol dehydrogenase alpha and beta subunits (moxF and moxI, respectively) have been cloned from the facultative methanol utilizers M. extorquens AM1 (28, 29), Methylobactenum organophilum XX (24), and Paracoccus denitrificans (16). In all three cases, the genes have been found to reside in a gene cluster together with the gene for cytochrome CL (moxG) and another gene of unknown function (mcxJ) (19). The order of these genes in all three bacteria is moxFJGI. Transcriptional start sites for this gene cluster have been mapped for the two Methylobacterium strains (4, 23), and a comparison of upstream sequences has identified a putative promoter sequence for these genes as follows: AAAGACA-18 bp-TAGAAA-4 to 5 bp-+1 (19).Although most of the genetic studies of ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Methanol oxidation genes in the marine methanotroph Methylomonas sp. strain A4

et al. 1993

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“…The transconjugants, however, are still defective in the ability to grow on C-2 compounds. We have been unable to comple- (2,9,18,22,29) carrying known genes associated with C-1 metabolism were introduced into the insertion mutants to determine whether they could complement the mutants. The clones tested were pAYC139 (mauBEDAC), pDN24 (moxAKLB), pDN202 (moxEQ), pM2 (ppc mc!…”

Section: Methodsmentioning

confidence: 99%

Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1

Chistoserdova

Lidstrom

1992

J Bacteriol

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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 cycle genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph that utilizes the serine cycle for C-1 assimilation (23). In the serine cycle, a C-1 unit is condensed with glycine to form serine, which is converted via hydroxypyruvate and D-glycerate into 3-phosphoglycerate (16). The net assimilation of C-1 compounds into cell constituents by this pathway requires the regeneration of one molecule of glycine for each molecule of 3-phosphoglycerate formed. The mechanism of synthesis of glycine from C-1 compounds remains unknown, but it is known that acetyl coenzyme A (acetyl-CoA) is a precursor (30). The use of labeled compounds and mutant studies have shown that the unknown route involved in the synthesis of glycine from acetyl-CoA is also essential for growth on ethanol, pyruvate, lactate, malonate, and hydroxybutyrate (6-8, 26, 27, 30). The M. extorquens AM1 methanol mutant 20BL was generated by nitrosoguanidine mutagenesis and lacks hydroxypyruvate reductase (HPR) (12). It does not grow on ethanol, but it grows normally on hydroxybutyrate (8,30). These data suggested that this key enzyme of the serine cycle might play a second role in the route for conversion of acetyl-CoA into glycine during growth on C-2 compounds (8) but not during growth on hydroxybutyrate (30). However, the genetic lesion in 20BL was not determined, and so it was not clear whether the phenotype observed was due to a single mutation or whether the structural gene for the serine cycle HPR was affected.To clarify this situation, it was necessary to construct a null serine cycle HPR mutant from the cloned structural gene and to determine its phenotype. In a recent paper, we described purification of the serine cycle HPR from M. extorquens AM1 and presented its N-terminal amino acid sequence (3). The present work was devoted to cloning of the gene encoding the serine cycle HPR and constructing an insertion HPR mutant of M. extorquens AM1 by gene replacement. * Corresponding author. MATERIALS AND METHODS...

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“…5) (1, 17). When these are expressed in E. coli, substantial processing occurs (2,6). Data from T7 expression experiments and immunoblotting experiments also suggest that the MADH large subunit has a leader sequence that is capable of being processed in E. coli cells (6).…”

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“…Plasmid pAYC152.1 was used for initial sequencing ( Fig. 1 and Table 1) (6), and the sequence data are shown in Fig. 2.…”

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The small-subunit polypeptide of methylamine dehydrogenase from Methylobacterium extorquens AM1 has an unusual leader sequence

Chistoserdov

Lidstrom

1991

J Bacteriol

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The nucleotide sequence for the N-terminal region of the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1 has revealed a leader sequence that is unusual in both its length and composition. Gene Table 1, and all Escherichia coli DHSa (F-recAl lacZYA r-m+ thi-J gyrA66 supE44 endAl 4801acZAM15) and E. coli CC118 (F-araD139 lacX74 phoA galE galK thi-J rpsE rpoB recAl) strains containing plasmids were grown on L medium (16) with the addition of the appropriate antibiotics (100 ,ug of ampicillin or kanamycin per ml or 20 ,ug of tetracycline per ml). Plasmid pAYC152.1 was used for initial sequencing ( Fig. 1 concern that the sequence might have been generated artifactually by rearrangements occurring during the subcloning either of the original 5.2-kb BamHI-HindIII insert in pAYC147 (5) or of the insert in pAYC152.1. Therefore, sequence data were generated independently from plasmid pAYC142, a clone containing mauA that was isolated directly from an independent partial clone library (5). An oligonucleotide (5'-GTTGTCCTGCGGCTTCCACTT-3') complementary to the region encoding the mature portion of the polypeptide was synthesized and used as a primer for sequencing of pAYC142. The sequence obtained was identical to the sequence shown in Fig. 2.To obtain further evidence that the start codons identified were translated, translational fusions of the mauA open reading frame with lacZ were generated (Fig. 1). These were constructed from plasmid pAYC150 (Table 1). Plasmid pAYC150a (Fig. 1) was generated by deleting an AccI-AccI fragment of pAYC150, treating the AccI recessed ends with Klenow fragment, and religating. This leads to the loss of approximately one-third of the mauA downstream sequence and brings in frame the sequences of this portion of mauA (mauA') and the part of lacZ in pUC19 (lacZ') (5,26

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Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

Cited by 30 publications

References 55 publications

Methanol oxidation genes in the marine methanotroph Methylomonas sp. strain A4

Methanol oxidation genes in the marine methanotroph Methylomonas sp. strain A4

Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1

The small-subunit polypeptide of methylamine dehydrogenase from Methylobacterium extorquens AM1 has an unusual leader sequence

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