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The gene designated pepR1, encoding a potential transcription regulator of Lactobacillus delbrueckii subsp. lactis DSM7290, was identified by sequence similarity of an open reading frame located upstream of the prolidase pepQ orientated in opposite direction. pepQ and pepR1 coding regions are spaced by 152 nucleotides. Upstream of the -35 region of pepQ, a 14-bp palindromic sequence, homologous to the catabolite responsive element, could be identified. The pepRl gene has the potential to encode a protein of 333 amino acids with a calculated molecular mass of 36955 Da and a calculated pl of 5.5. The deduced protein sequence shows significant identity to the catabolite control protein of Bacillus. Co-expression in Escherichia coli was studied with the pepR1-pepQ intergenic region fused to the promoterless beta-galactosidase reporter gene. The pepQ-beta-galactosidase hybrid displayed an enhanced expression in the presence of cloned pepR1.

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L-form strains of Proteus mirabilis and Escherichia coli lacking the cell wall represent an alternative prokaryotic cell system for the production of recombinant proteins (KLESSEN et al. 1988, LAPLACE et al. 1988a, 1989b). We could demonstrate that they are also able to synthesize the enzyme penicillin G acylase (PAC)1). PAC was processed and secreted into the medium by recombinant L-form strains. The synthesis of PAC was growth-associated and stably regulated. Expression, secretion, and processing were not temperature-dependent and occurred at 26 degrees C, 32 degrees C and even 37 degrees C. The expression vector pHC1 carried the pac gene under the control of the lac UV promotor and a kanamycin resistance gene. It could be maintained in L-form cells, showing low structural as well as segregational instability. The secretion of the biologically active enzyme into the medium indicated that the postranslational processing of the PAC molecule, including the excision of a 54 amino acid spacer peptide between the alpha and beta subunit, is not carried out in the periplasmic space, but occurs at the cytoplasmic membrane or autocatalytically.

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A gene (brnQ), encoding a carrier for branched-chain amino acids in Lactobacillus delbrückii subsp. lactis DSM7290 was cloned in the low-copy-number vector pLG339 by complementation of a transport-deficient Escherichia coli strain. The plasmid carrying the cloned gene restored growth of an E. coli strain mutated in 4 different branched-chain amino acid transport genes at low concentrations of isoleucine, and increased its sensitivity to valine. Transport assays showed that leucine, isoleucine and valine are transported by this carrier and that transport is driven by the proton motive force. Nucleotide sequence analysis revealed an open reading frame of 1338 bp encoding a hydrophobic protein of 446 amino acids with a calculated molecular mass of 47864 Daltons. The start site of brnQ transcription was determined by primer extension analysis using mRNA from Lactobacillus delbrückii subsp. lactis DSM7290. The hydropathy profile suggests the existence of at least 12 hydrophobic domains that probably form membrane-associated alpha-helices. Comparisons of the nucleotide sequence of brnQ from Lactobacillus delbrückii subsp. lactis DSM7290, the amino acid sequence of its product and the topology of the hydrophobic domains with those of the respective carrier genes and proteins of Salmonella typhimurium and Pseudomonas aeruginosa revealed extensive homology.

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Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N'-nitro-N-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl-beta-alanyl-L-proline (PhAc-beta Ala-Pro) phthalyl-L-leucine (Pht-Leu) or phthalylglycyl-L-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-beta Ala-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The Km of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45 degrees C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold.

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From a genomic library of Lactobacillus delbrueckii subsp. lactis (DSM7290) DNA, in the low-copy-number vector pLG339, a recombinant clone was selected, which complemented a mutation in the prolidase gene (pepQ) of Escherichia coli UK173. Nucleotide sequence analysis revealed an open reading frame of 1104 nucleotides corresponding to a protein of 368 amino acids with a calculated pI of 4.64 and a molecular mass of 41,087 Da. The start site of pepQ transcription was determined by primer extension analysis with mRNA prepared from L. delbrueckii. Based on homology of the gene product to various peptidases and on the substrate specificity determined, the peptidase was designated PepQ. The influence of various protease inhibitors and cations on peptidase activity indicated that PepQ is a metalloprotease. The absence of a membrane-spanning domain and a signal peptide sequence argues for a cytoplasmic localization of the enzyme.

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A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 DNA fragments from a Sau3A partial digestion in the low-copy-number vector pLG339, was used to screen Escherichia coli for the presence of peptidases. Using the chromogenic substrate leucine-beta-naphthylamide (Leu-NH-Nap) and E. coli strain CM89 lacking the corresponding enzyme activity in an enzymic plate assay, allowed the isolation of two peptidase genes; the newly described pepL and the recently cloned and sequenced pepN. Clones could be distinguished not only by the restriction pattern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu-NH-Nap. Three out of five clones were identified to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, together with 200 bp upstream of the proposed AUG initiation codon, was further subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids (34,541 Da). Searching the EMBL database revealed similarity to the prolinase of Lactobacillus helveticus (45.8% identity), to the iminopeptidases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active sites. The product encoded by the pepL gene was functional but could not be visualized on Coomassie-blue-stained polyacrylamide gels. High level expression of peptidase L in E. coli was achieved by placing the gene under the control of the T7 promoter.

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A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-beta-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50,909 Da. The enzyme is functional and extremely overexpressed in E. coli.

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Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity. The pure enzyme was stable at 4 degrees C or -20 degrees C and had a pH optimum at pH 9, and a pI of 4.7; the temperature optimum was at 37 degrees C. As the enzyme was activated by Co2+ and Zn2+, and deactivated by metal chelators, it appears to be a metallopeptidase. By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified. Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the alpha or beta position. Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed. Km values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.

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Cell extracts of Lactobacillus delbrueckii subsp. lactis DSM 7290 were found to exhibit unique peptolytic ability against unusual beta-alanyl-dipeptides. In order to clone the gene encoding this activity, designated pepV, a gene library of strain DSM 7290 genomic DNA, prepared in the low-copy-number plasmid pLG339, was screened for heterologous expression in Escherichia coli. Recombinant clones harbouring pepV were identified by their ability to allow the utilization of carnosine (beta-alanyl-histidine) as a source of histidine by the E. coli mutant strain UK197 (pepD, hisG). Complementation was observed in a colony harbouring a recombinant plasmid (pKV101), carrying pepV. A 2.4 kb fragment containing pepV was subcloned and its nucleotide sequence revealed an open reading frame (ORF) of 1413 nucleotides, corresponding to a protein with predicted molecular mass of 51998 Da. A single transcription initiation site 71 bp upstream of the ATG translational start codon was identified by primer extension. No significant homology was detected between pepV or its deduced amino acid sequence with any entry in the databases. The only similarity was found in a region conserved in the ArgE/DapE/CPG2/YscS family of proteins. This observation, and protease inhibitor studies, indicated that pepV is of the metalloprotease type. A second ORF present in the sequenced fragment showed extensive homology to a variety of amino acid permeases from E. coli and Saccharomyces cerevisiae.

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The transcriptional start points of ten Lactobacillus delbrückii ssp. lactis DSM7290 genes were determined by primer extension. The upstream located promoter regions, including potential -35 and -10 regions and the spacing between them were compared to the well-known Escherichia coli and Bacillus subtilis promoters. The Lb. delbrückii -35 consensus sequence (TTGACA) seems to be less conserved then the E. coli sequence. The nucleotides TGC were often found upstream of the -10 region (TATAAT). The most frequently observed spacing between the two core promoter regions was 17 nt and the main distance between the -10 region and the transcriptional start point was mostly determined to be 6 nt in contrast to 7 nt, as described for E. coli promoters. The preferred initiation nucleotides in Lb. delbrückii were shown to be definitely purines (A or G). The ribosome binding sites located downstream of the promoters revealed the consensus sequence 3'-UCCUCCU-5', being the predicted 3'-OH end of the Lactobacillus 16S rRNA with a high degree of homology to known 16S rRNAs.

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The gene for proline iminopeptidase from Lactobacillus delbrueckii subsp. lactis DSM 7290 coding for an enzyme that hydrolyses the synthetic substrate L-prolyl-beta-naphthylamide (Pro-beta NA) was cloned in Escherichia coli. An enzymic plate assay was used to screen for positive clones. The gene, designated pepI, was subcloned into vector pUC18 and sequenced. The nucleotide sequence revealed an 882 bp open reading frame encoding 294 amino acids, coding for an enzyme with a calculated molecular mass of 32883 Da. By cloning under control of the lac promoter the peptidase was highly expressed. Sequence analysis showed that pepI is of a new sequence type, distinct from all peptidases so far sequenced. Amino acid homology to the active site of a Pseudomonas putida esterase and inhibitor studies of the enzyme imply involvement of a serine residue in catalysis.

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Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties.

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Lactobacillus delbrückii ssp. lactis DSM7290 possesses an X-prolyl-dipeptidyl-aminopeptidase, designated PepX, which catalyses the hydrolytic removal of N-terminal dipeptidyl residues from peptides containing proline in the penultimate position. Using the specific substrate L-Ala-L-Pro-p-nitroanilide, PepX was purified by a four-step procedure including ammonium sulphate fractionation, hydrophobic interaction chromatography, ion exchange chromatography, and affinity chromatography. The N-terminus of the purified protein was sequenced. Screening of a gene library of chromosomal Lactobacillus delbrückii ssp. lactis DSM7290 DNA in the low-copy-number vector pLG339 resulted in the identification of the pepX gene in Escherichia coli using a specific plate assay with Gly-L-Pro-beta-naphthylamide as substrate. Nucleotide sequence analysis revealed an open reading frame of 2376 bp, coding for a protein of 792 amino acids with a molecular mass of 88449 Da.

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In cell extracts of Lactobacillus delbrückii ssp. lactis DSM7290 a peptidase with the ability to hydrolyse Phe-beta-naphthylamide (Phe-beta-NA) and His-beta-NA could be detected. Escherichia coli lacking the enzyme activity in an enzymic plate assay was used to screen high-copy-number and low-copy-number plasmid libraries of size-fractionated Lactobacillus DNA. Clones with the desired phenotype were detected, and the gene, designated pepN, was further subcloned and sequenced. A large open reading frame of 2529 nucleotides is predicted to encode a protein of 843 amino acids (95358 Da). Comparison of the pepN gene from Lb. delbrückii ssp. lactis DSM7290 indicates that it is homologous to genes of the family of Zn(2+)-metallohydrolases and PepN shows identity with the active centre Zn(2+)-binding motif of these enzymes. The substrate Lys-beta-NA is more effectively cleaved than Phe-beta-NA or His-beta-NA which were used for screening in E. coli. The cloned pepN gene was efficiently overexpressed in E. coli and subcloning of the gene in Lactobacillus casei resulted in a moderate overexpression of approximately 20-fold. The pepN gene product was purified from the pepN-deficient E. coli strain CM89, using the substrate Lys-p-nitroanilide (Lys-NH-Ph) in the assay procedure. In a four-step procedure including streptomycin sulfate precipitation, anion-exchange chromatography and gel filtration the peptidase was purified to electrophoretic homogeneity.

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Lactobacillus curvatus LTH683, a strain originally isolated from raw sausage, contains the single cryptic plasmid called pLC2. The sequence and genetic organization of the complete 2489-bp plasmid pLC2 was determined and used as the basis for construction of a series of vectors useful in Lactobacillus strains. The major parts of pLC2 nucleotide sequence could be aligned with other plasmids from gram-positive bacteria replicating by a rolling circle mechanism of replication (RCR). Direct evidence for a RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. Three protein-coding sequences could be predicted and the corresponding proteins were detected after in vitro transcription/translation of pLC2 plasmid DNA. ORFs 1 and 3 showed minor homologies to plasmids of gram-positive bacteria. The replication protein coded by ORF2 and its corresponding target sequence, the plus origin, were similar to replication regions of other gram-positive bacteria plasmids like pLS1, pWV01, and pE194. Upstream of the ori+ site, in a noncoding region, which was nonessential for replication, strong homology to other Lactobacillus plasmids like pC30i1, pLP1, pLJ1, and pLAB1000 could be detected. A palindromic sequence predicted to be the minus origin of replication was localized there. Small vectors (3213 bp) suitable for cloning in lactobacilli were constructed based on a 1635-bp DNA fragment of pLC2, containing the region necessary for replication, marked with the chloramphenicol resistance gene and a multiple cloning site.

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Lactococcus lactis ML3 possesses two different peptide transport systems of which the substrate size restriction and specificity have been determined. The first system is the earlier-described proton motive force-dependent di-tripeptide carrier (E. J. Smid, A. J. M. Driessen, and W. N. Konings, J. Bacteriol. 171:292-298, 1989). The second system is a metabolic energy-dependent oligopeptide transport system which transports peptides of four to at least six amino acid residues. The involvement of a specific oligopeptide transport system in the utilization of tetra-alanine and penta-alanine was established in a mutant of L. lactis MG1363 that was selected on the basis of resistance to toxic analogs of alanine and alanine-containing di- and tripeptides. This mutant is unable to transport alanine, dialanine, and trialanine but still shows uptake of tetra-alanine and penta-alanine. The oligopeptide transport system has a lower activity than the di-tripeptide transport system. Uptake of oligopeptides occurs in the absence of a proton motive force and is specifically inhibited by vanadate. The oligopeptide transport system is most likely driven by ATP or a related energy-rich, phosphorylated intermediate.

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A gene product with an apparent molecular mass of approximately 39,000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC. In this communication we report that the product was labelled with [3H]glycerol and [3H]palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase. The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys24 within the amino terminal region of EnvC, was replaced by tryptophane (Trp24). This protein was not labelled with [3H]glycerol. The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E. coli.

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A series of deletions removing progressively larger parts of the 5' flanking region of the Escherichia coli pepD gene was constructed. After fusing the resulting promoter fragments to the chromosomal malPQ operon, their activities were determined by assaying for amylomaltase, the product of the malQ gene. Transcription from the pepD promoter region in exponentially growing cells was estimated to be about 5 times less efficient than transcription from the induced lac promoter. Approximately 115 bp preceding the translation start site of the pepD gene are important for regular promoter functioning, whereas the more distal sequences could be deleted without any significant effects. In bacterial cultures containing limiting amounts of inorganic phosphate, the rate of de novo synthesis of peptidase D, simultaneously with the derepression of alkaline phosphatase, increased about fivefold as a consequence of phosphate starvation. This regulation was shown to occur at the transcriptional level by the use of chromosomal pepD promoter-malPQ fusions. The inducibility by phosphate limitation was conserved in all of the deletion clones in which the pepD promoter region was still functional. As demonstrated by the use of phoB, R, and M mutants, the modulation of pepD expression is independent of the genetic system controlling the pho regulon.

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