Spontaneous deletion formation within the beta-galactosidase gene of Lactobacillus bulgaricus

Mollet, Beat; Delley, Michèle

doi:10.1128/jb.172.10.5670-5676.1990

Cited by 40 publications

(18 citation statements)

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“…L. delbrueckii subsp. bulgaricus NCDO1489 and mutants N310 and LFi5.F6 (spontaneous lacZ derivatives from NCDO1489) were from the Nestec collection (32), and strain CNRZ 397 was obtained from the Institut National de la Recherche Agronomique (Jouy en Josas, France). Lactococcus lactis subsp.…”

Section: Methodsmentioning

confidence: 99%

“…bulgaricus NCDO1489 were characterized by large chromosomal deletions of several kilobases including the lacS and lacZ genes, coding for a permease and ␤-galactosidase, respectively (11,32). We took advantage of the availability of some mutants with a proteinase-deficient phenotype to identify and localize the structural gene (prtB) coding for the cell surface proteinase.…”

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A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus

et al. 1996

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Investigation of the chromosomal region downstream of the lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus revealed the presence of a gene (prtB) encoding a proteinase of 1,946 residues with a predicted molecular mass of 212 kDa. The deduced amino acid sequence showed that PrtB proteinase displays significant homology with the N termini and catalytic domains of lactococcal PrtP cell surface proteinases and is probably synthesized as a preproprotein. However, the presence of a cysteine near the histidine of the PrtB active site suggests that PrtB belongs to the subfamily of cysteine subtilisins. The C-terminal region strongly differs from those of PrtP proteinases by having a high lysine content, an imperfect duplication of 41 residues, and a degenerated sequence compared with the consensus sequence for proteins anchoring in the cell walls of gram-positive bacteria. Finally, the product of the truncated prtM-like gene located immediately upstream of the prtB gene seems too short to be involved in the maturation of PrtB.Lactobacillus delbrueckii subsp. bulgaricus is one of the lactic acid bacteria used in industrial milk fermentation. The proteolytic system is essential to ensure rapid growth in milk and supply auxotrophic lactic acid bacteria with amino acids from caseins, the major milk proteins. This system is complex and has been extensively studied in lactococci (21, 35). The first step in milk casein degradation is performed by a cell surface proteinase, PrtP. Two types of PrtP enzymes have been distinguished on the basis of their substrate specificities. PI-type proteinases preferentially cleave ␤-casein, whereas PIII-type enzymes degrade ␣, ␤, and caseins (10). Lactococcal PrtPs are serine proteases and show extensive homology with subtilisins secreted by bacilli. Lactococcal proteinases are synthesized as inactive preproproteins. An N-terminal signal peptide of 33 residues is removed during translocation through the cytoplasmic membrane, and the C terminus remains anchored in the cell envelope. Then, a maturation process mediated by a membrane-located lipoprotein, PrtM, leads to the removal of the pro region (154 residues). The 33-kDa PrtM protein is encoded by a gene located immediately upstream and in the opposite direction of prtP (13, 49). Incubation of lactococci cells in a calcium-free buffer promotes self-digestion of the carboxy terminus of PrtP and its release into the extracellular medium.Lactobacilli have been investigated to a lesser extent but display a high cell surface proteinase activity with substrate specificity differing from that of lactococci (4). Cell surface proteinases have been purified from Lactobacillus paracasei subsp. paracasei NCDO151, L. helveticus CNRZ303, and L89, and L. delbrueckii subsp. bulgaricus CNRZ397 (24, 27, 33, 52). The sequence of the gene encoding proteinase from L. paracasei subsp. paracasei was determined; the deduced amino acid sequence shows 1,902 residues and a high degree of identity (96%) with lactococcal PrtP (15). The presence of a prtM gene ...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus

et al. 1996

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“…There is evidence that spontaneous mutations are influenced by DNA context (1,5,12,22,25,40). Interpreting the role of short sequence motifs, minor and extended direct repetitions, palindromes, and quasi-palindromes in predisposing particular nucleotides to mutate will be facilitated by analysis of many mutations, occurring in different genetic contexts.…”

Section: Vol 182 2000 Selection For Agrobacterial Dioxygenase Mutanmentioning

confidence: 99%

Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds in Agrobacterium tumefaciens : Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene

Parke

2000

J Bacteriol

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A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain of Agrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and ␤-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into the pcaB gene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described for Acinetobacter sp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker in pcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained with Acinetobacter sp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting the pcaHG genes of A. tumefaciens for spontaneous mutation. Sixteen strains carrying putative second-site mutations in pcaH or -G were subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (؊1) frameshifts, one (؉1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG from Pseudomonas putida and Acinetobacter sp. as well as of residues in other parts of the enzyme.

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“…In Lactobacillus bulgaricus a spontaneous insertion sequence (IS) element-mediated deletion of the lacZ gene altered lactose metabolism resulting in limited fermentation capacity. As a consequence, yoghurt, made with these altered L. bulgaricus, is not suffering from post-fermentation acidification during the shelf life period (Mollet and Delley, 1990). A gene inactivation method for several lactic acid bacteria has been described that is mediated via IS elements that may integrate into a gene, resulting in inhibition of function (Maguin et al 1996).…”

Section: Examples Of Lab Selected After Uncontrolled Genetic Alterationsmentioning

confidence: 99%

Safe use of genetically modified lactic acid bacteria in food. Bridging the gap between consumers, green groups, and industry

Sybesma¹,

Hugenholtz²,

Vos³

et al. 2006

Electron. J. Biotechnol.

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Spontaneous deletion formation within the beta-galactosidase gene of Lactobacillus bulgaricus

Cited by 40 publications

References 47 publications

A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus

A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus

Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds in Agrobacterium tumefaciens : Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene

Safe use of genetically modified lactic acid bacteria in food. Bridging the gap between consumers, green groups, and industry

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