RESUMO
We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.
Assuntos
Escherichia coli/genética , Klebsiella/genética , Quinolonas , Clonagem Molecular , DNA/análise , Escherichia coli/metabolismo , Cofator PQQ , Mapeamento por Restrição , Transformação BacterianaRESUMO
A generally applicable method is described for reintroduction of mutant plasmid-borne alleles to the chromosome of Klebsiella pneumoniae using bacteriophage lambda. We used this method to make stable chromosomal transposon insertions in genes for biosynthesis of pyrroloquinoline quinone in K. pneumoniae.
Assuntos
Cromossomos Bacterianos/metabolismo , Elementos de DNA Transponíveis , Klebsiella pneumoniae/genética , Plasmídeos , Quinolonas/metabolismo , Alelos , Bacteriófago lambda/genética , Coenzimas/metabolismo , Mutação , Cofator PQQ , Mapeamento por RestriçãoRESUMO
A 6940 bp Klebsiella pneumoniae chromosomal DNA fragment, containing genes involved in pyrroloquinoline quinone (PQQ) biosynthesis, was sequenced. Six open reading frames, pqqA, pqqB, pqqC, pqqD, pqqE and pqqF were identified in the pqq operon, which coded for polypeptides of 2764 (23 amino acids), 33,464, 28,986, 10,436, 42,881 and 83,616 Da, respectively. The transcription startpoint was mapped by primer extension analysis, upstream of pqqA, and promoter boxes could be identified. The gene products of pqqB, pqqC, pqqE and pqqF were detected in maxi-cells and the molecular weights of the proteins corresponded with the molecular weights deduced from the nucleotide sequence. The gene products of pqqA, pqqB, pqqC, pqqD and pqqE show 49%-64% identity in amino acid sequence with those of pqqIV, pqqV, pqqI, pqqII and pqqIII respectively in the cloned pqq cluster of Acinetobacter calcoaceticus. The 84 kDa protein encoded by pqqF, which is not present in the cloned pqq cluster of A. calcoaceticus but which is essential for PQQ biosynthesis in K. pneumoniae and Escherichia coli, seems to belong to a family of proteases.
Assuntos
Klebsiella pneumoniae/genética , Óperon , Sequência de Aminoácidos , Sequência de Bases , Coenzimas/metabolismo , DNA Bacteriano/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Família Multigênica , Mutação , Fases de Leitura Aberta , Cofator PQQ , Plasmídeos , Regiões Promotoras Genéticas , Quinolonas/metabolismo , RNA Mensageiro/genética , Mapeamento por Restrição , Alinhamento de Sequência , Transcrição GênicaRESUMO
In Klebsiella pneumoniae, six genes, constituting the pqqABCDEF operon, which are required for the synthesis of the cofactor pyrroloquinoline quinone (PQQ) have been identified. The role of each of these K. pneumoniae Pqq proteins was examined by expression of the cloned pqq genes in Escherichia coli, which cannot synthesize PQQ. All six pqq genes were required for PQQ biosynthesis and excretion into the medium in sufficient amounts to allow growth of E. coli on glucose via the PQQ-dependent glucose dehydrogenase. Mutants lacking the PqqB or PqqF protein synthesized small amounts of PQQ, however. PQQ synthesis was also studied in cell extracts. Extracts made from cells containing all Pqq proteins contained PQQ. Lack of each of the Pqq proteins except PqqB resulted in the absence of PQQ. Extracts lacking PqqB synthesized PQQ slowly. Complementation studies with extracts containing different Pqq proteins showed that an extract lacking PqqC synthesized an intermediate which was also detected in the culture medium of pqqC mutants. It is proposed that PqqC catalyzes the last step in PQQ biosynthesis. Studies with cells lacking PqqB suggest that the same intermediate might be accumulated in these mutants. By using pqq-lacZ protein fusions, it was shown that the expression of the putative precursor of PQQ, the small PqqA polypeptide, was much higher than that of the other Pqq proteins. Synthesis of PQQ most likely requires molecular oxygen, since PQQ was not synthesized under anaerobic conditions, although the pqq genes were expressed.