An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepl) and two genes coding for putative members of the ABC transporter family of proteins

Varmanen, Pekka; Rantanen, Terhi; Palva, Airi

doi:10.1099/13500872-142-12-3459

Cited by 34 publications

(34 citation statements)

References 44 publications

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“…Pro--NA has been shown to be efficiently hydrolyzed by PepI from L. helveticus and L. delbrueckii (13,31; this work). Significant activity was measured for Met--NA in mutant strains lacking both PepN and PepC (JLS244 and JLS247), which indicates the presence of at least one additional aminopeptidase.…”

Section: Discussionmentioning

confidence: 99%

Impaired Growth Rates in Milk of Lactobacillus helveticus Peptidase Mutants Can Be Overcome by Use of Amino Acid Supplements

Christensen¹,

Steele

2003

J Bacteriol

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To evaluate the contribution of intracellular peptidases to the growth of the 14-amino-acid (aa) auxotroph Lactobacillus helveticus CNRZ32, single-and multiple-peptidase-deletion mutants were constructed. Two broadspecificity aminopeptidases (PepC and PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) were inactivated through successive cycles of chromosomal gene replacement mutagenesis. The inactivation of all three peptidases in JLS247 (⌬pepC ⌬pepN ⌬pepX) did not affect the growth rate in amino acid-defined medium. However, the peptidase mutants generally had decreased specific growth rates when acquisition of amino acids required hydrolysis of the proteins in milk, the most significant result being a 73% increase in generation time for JLS247. The growth rate deficiencies in milk were overcome by amino acid supplements with some specificity to each of the peptidase mutants. For example, milk supplementation with Pro resulted in the most significant growth rate increase for ⌬pepX strains and a 7-aa supplement (Asn, Cys, Ile, Pro, Ser, Thr, and Val) resulted in a JLS247 growth rate indistinguishable from that of the wild type. Our results show that characterization of the activities of the broad-specificity aminopeptidases had little predictive value regarding the amino acid supplements found to enhance the milk growth rates of the peptidase mutant strains. These results represent the first determination of the physiological roles with respect to specific amino acid requirements for peptidase mutants grown in milk.

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Section: Discussionmentioning

confidence: 99%

Impaired Growth Rates in Milk of Lactobacillus helveticus Peptidase Mutants Can Be Overcome by Use of Amino Acid Supplements

Christensen¹,

Steele

2003

J Bacteriol

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“…The new plasmids, designated pKTH2172, pKTH2171, pKTH2150, and pKTH2170, respectively, were transferred into a fivefold peptidase-negative mutant strain, [XTOCN] Ϫ , of L. lactis MG1363 ( Table 1). The pepC and pepI genes reside in operon structures in L. helveticus (7,28). For this work, the pepC gene and pepI under the control of the pepX promoter (PpepXpepI) were isolated by PCR, transferred into pKTH2095, and cloned as plasmid constructs pKTH2179 and pKTH2175, respectively, directly into L. lactis.…”

Section: Resultsmentioning

confidence: 99%

Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

Luoma¹,

Peltoniemi²,

Joutsjoki³

et al. 2001

Appl Environ Microbiol

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For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.Lactic acid bacteria (LAB) play an important role in dairy fermentation processes and have a great influence on the quality and preservation of end products. The primary roles of LAB are to produce lactic acid from lactose, resulting in a pH decrease, and, by proteolysis, to liberate short peptides and free amino acids affecting the flavor and texture of dairy products.Since the concentration of free amino acids and small peptides is insufficient to support the growth of LAB to high cell densities in milk, these bacteria are dependent on a proteolytic system to liberate free amino acids from milk proteins. The proteolytic system of LAB consists of a cell envelope-associated proteinase, membrane-bound transport systems, and several cytoplasmic peptidase classes. The proteolytic system is particularly important in the development of flavor and texture of cheeses (9). Since Lactococcus strains, along with those of Lactobacillus, are widely used as starters in cheese manufacture, substantial effort has been directed in the last two decades toward elucidating the proteolytic mechanism of Lactococcus lactis. More recently, the proteolytic system of lactobacilli has also been extensively examined.Over 10 different peptidase types have been identified in various LAB strains, and a large number of peptidase genes have been cloned from different Lactococcus and Lactobacillus species and characterized (reviewed ...

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“…To address the possibility that a soluble transcription regulator controls slpD gene expression, the slpD promoter (P slpD ) region was fused with an L. helveticus pepI gene (38), chosen as the reporter gene because it encodes proline iminopeptidase (PepI) activity that can be easily assayed in lactobacilli. The P slpD -pepI fusion was cloned into low-copy-number plasmid vector pKTH2095 (27).…”

Section: Vol 184 2002 Isolation Of Three L Brevis S-layer Genes 6789mentioning

confidence: 99%

“…A transcriptional fusion between the slpD promoter region and the L. helveticus 53/7 proline iminopeptidase gene (pepI) (38) was constructed as follows. With the primer pair 1162 and 1163 (Table 1) the 255 bp upstream of the initiation codon of slpD was amplified by PCR from ATCC 14869 chromosomal DNA.…”

Section: Rt-pcrmentioning

confidence: 99%

Isolation of Three New Surface Layer Protein Genes ( slp ) from Lactobacillus brevis ATCC 14869 and Characterization of the Change in Their Expression under Aerated and Anaerobic Conditions

Jakava-Viljanen

Åvall-Jääskeläinen

Messner³

et al. 2002

J Bacteriol

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Two new surface layer (S-layer) proteins (SlpB and SlpD) were characterized, and three slp genes (slpB, slpC, and slpD) were isolated, sequenced, and studied for their expression in Lactobacillus brevis neotype strain ATCC 14869. Under different growth conditions, L. brevis strain 14869 was found to form two colony types, smooth (S) and rough (R), and to express the S-layer proteins differently. Under aerobic conditions R-colony type cells produced SlpB and SlpD proteins, whereas under anaerobic conditions S-colony type cells synthesized essentially only SlpB. Anaerobic and aerated cultivations of ATCC 14869 cells in rich medium also resulted in S-layer protein patterns similar to those of the S-and R-colony type cells, respectively. Electron microscopy suggested the presence of only a single S-layer with an oblique structure on the cells of both colony forms. The slpB and slpC genes were located adjacent to each other, whereas the slpD gene was not closely linked to the slpB-slpC gene region. Northern analyses confirmed that both slpB and slpD formed a monocistronic transcription unit and were effectively expressed, but slpD expression was induced under aerated conditions. slpC was a silent gene under the growth conditions tested. The amino acid contents of all the L. brevis ATCC 14869 S-layer proteins were typical of S-layer proteins, whereas their sequence similarities with other S-layer proteins were negligible. The interspecies identity of the L. brevis S-layer proteins was mainly restricted to the Nterminal regions of those proteins. Furthermore, Northern analyses, expression of a PepI reporter protein under the control of the slpD promoter, and quantitative real-time PCR analysis of slpD expression under aerated and anaerobic conditions suggested that, in L. brevis ATCC 14869, the variation of S-layer protein content involves activation of transcription by a soluble factor rather than DNA rearrangements that are typical for most of the S-layer phase variation mechanisms known.Surface layers (S layers) are monomolecular crystalline arrays of proteinaceous subunits present in almost all archaea and all major phylogenetic groups of bacteria (26,33). Most of the S layers are composed of subunits of a single protein or glycoprotein species capable of forming symmetrical arrays and covering the cell surface during all stages of growth. Slayer proteins commonly contain high numbers of acidic and hydrophobic amino acids but lack overall amino acid sequence homology with corresponding proteins from unrelated species. Moreover, a low pI is typical for S-layer proteins (31) except for those from different lactobacilli (5, 40) and Methanothermus fervidus (6). Diverse functions have been proposed for S layers, such as acting as molecular sieves, protective coats, molecular and ion traps, cell shape determinants, and promoters for cell adhesion and surface recognition (26). There is also increasing evidence that S-layer-carrying bacteria may use Slayer variation, by expressing alternative S-layer protein genes, for ada...

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An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepl) and two genes coding for putative members of the ABC transporter family of proteins

Cited by 34 publications

References 44 publications

Impaired Growth Rates in Milk of Lactobacillus helveticus Peptidase Mutants Can Be Overcome by Use of Amino Acid Supplements

Impaired Growth Rates in Milk of Lactobacillus helveticus Peptidase Mutants Can Be Overcome by Use of Amino Acid Supplements

Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

Isolation of Three New Surface Layer Protein Genes ( slp ) from Lactobacillus brevis ATCC 14869 and Characterization of the Change in Their Expression under Aerated and Anaerobic Conditions

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