BCRC Strain Collection Catalog & Shopping Cart

  Home / BCRC Content / 12936 / 

Return

  Research Article

The information shown in this page was generated using the cross-referenced linkage within public domain database between their strains and BCRC related strains. Usually the information provided from public domain databases varies with diffent confidences and errors, BCRC provides the related information here at best effort, but BCRC doesn't take the responsibility about the correctness of the information provided here.

1. Kochhar  S, Hottinger  H, Chuard  N, Taylor  PG, Atkinson  T, Scawen  MD, Nicholls  DJ,     ( 1992 )

Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli.

European journal of biochemistry 208 (3)
PMID : 1396685  :   DOI  :   10.1111/j.1432-1033.1992.tb17250.x    
Abstract >>
NAD(+)-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220-215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499-8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (greater than 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60-66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. alpha 2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.
KeywordMeSH Terms
Lactate Dehydrogenases
2. Fortina  MG, Ricci  G, Mora  D, Guglielmetti  S, Manachini  PL,     ( 2003 )

Unusual organization for lactose and galactose gene clusters in Lactobacillus helveticus.

Applied and environmental microbiology 69 (6)
PMID : 12788721  :   DOI  :   10.1128/aem.69.6.3238-3243.2003     PMC  :   PMC161534    
Abstract >>
The nucleotide sequences of the Lactobacillus helveticus lactose utilization genes were determined, and these genes were located and oriented relative to one another. The lacLM genes (encoding the beta-galactosidase protein) were in a divergent orientation compared to lacR (regulatory gene) and lacS (lactose transporter). Downstream from lacM was an open reading frame (galE) encoding a UDP-galactose 4 epimerase, and the open reading frame had the same orientation as lacM. The lacR gene was separated from the downstream lacS gene by 2.0 kb of DNA containing several open reading frames that were derived from fragmentation of another permease gene (lacS'). Northern blot analysis revealed that lacL, lacM, and galE made up an operon that was transcribed in the presence of lactose from an upstream lacL promoter. The inducible genes lacL and lacM were regulated at the transcriptional level by the LacR repressor. In the presence of glucose and galactose galE was transcribed from its promoter, suggesting that the corresponding enzyme can be expressed constitutively. Lactose transport was inducible by addition of lactose to the growth medium.
KeywordMeSH Terms
Multigene Family
3. Chen  YS, Christensen  JE, Broadbent  JR, Steele  JL,     ( 2003 )

Identification and characterization of Lactobacillus helveticus PepO2, an endopeptidase with post-proline specificity.

Applied and environmental microbiology 69 (2)
PMID : 12571057  :   DOI  :   10.1128/aem.69.2.1276-1282.2003     PMC  :   PMC143593    
Abstract >>
A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-beta-casein-(f203-209)-rho-nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that L. helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L. helveticus PepO, Lactococcus lactis PepO2, L. lactis PepO, and Lactobacillus rhamnosus PepO, respectively. Northern hybridization results indicated that the transcript containing pepO2 was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L. helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as beta-casein(f193-209).
KeywordMeSH Terms
Bacterial Proteins
Metalloendopeptidases
4. Chavagnat  F, Haueter  M, Jimeno  J, Casey  MG,     ( 2002 )

Comparison of partial tuf gene sequences for the identification of lactobacilli.

FEMS microbiology letters 217 (2)
PMID : 12480101  :   DOI  :   10.1111/j.1574-6968.2002.tb11472.x    
Abstract >>
Comparative analysis of partial tuf sequences was evaluated for the identification and differentiation of lactobacilli. Comparison of the amino acid sequences allowed differentiation between species and also between the subspecies of Lactobacillus delbrueckii. The nucleotide sequence comparison allowed differentiation between other subspecies and between some strains. Lactobacilli from several collections and isolates from dairy samples were clearly identified by comparison of short tuf sequences with those of the type strains. In evaluating the taxonomy of the Lactobacillus casei-related taxa, different tuf amino acid signatures are in favour of a classification into three distinct species. The type strain designation for the L. casei species is discussed.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
5. Kaminski  PA,     ( 2002 )

Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus.

The Journal of biological chemistry 277 (17)
PMID : 11836245  :   DOI  :   10.1074/jbc.M111995200    
Abstract >>
Lactobacillus helveticus contains two types of N-deoxyribosyltransferases: DRTase I catalyzes the transfer of 2'-deoxyribose between purine bases exclusively whereas DRTase II is able to transfer the 2'-deoxyribose between two pyrimidine or between pyrimidine and purine bases. An Escherichia coli strain, auxotrophic for guanine and unable to use deoxyguanosine as source of guanine, was constructed to clone the corresponding genes. By screening a genomic bank for the production of guanine, the L. helveticus ptd and ntd genes coding for DRTase I and II, respectively, were isolated. Although the two genes have no sequence similarity, the two deduced polypeptides display 25.6% identity, with most of the residues involved in substrate binding and the active site nucleophile Glu-98 being conserved. Overexpression and purification of the two proteins shows that DRTase I is specific for purines with a preference for deoxyinosine (dI) > deoxyadenosine > deoxyguanosine as donor substrates whereas DRTase II has a strong preference for pyrimidines as donor substrates and purines as base acceptors. Purine analogues were substrates as acceptor bases for both enzymes. Comparison of DRTase I and DRTase II activities with dI as donor or hypoxanthine as acceptor and colocalization of the ptd and add genes suggest a specific role for DRTase I in the metabolism of dI.
KeywordMeSH Terms
6. Okuyama  K, Noguchi  T,     ( 2000 )

Molecular cloning and expression of the nucleoside deoxyribosyltransferase-II gene from Lactobacillus helveticus.

Bioscience, biotechnology, and biochemistry 64 (10)
PMID : 11129605  :   DOI  :   10.1271/bbb.64.2243    
Abstract >>
Nucleoside deoxyribosyltransferase-II, which catalyzes transfer of glycosyl residues from a donor deoxynucleoside to an acceptor base, was purified from Lactobacillus helveticus and its gene was cloned. Analysis of the nucleotide sequence showed the presence of a 474-nucleotide open reading frame encoding a protein of 158 amino acids with a molecular weight of 18,317. The active enzyme can be produced in large quantities in E. coli cells using the cloned gene.
KeywordMeSH Terms
7. Callegari  ML, Ventura  M,     ( 2000 )

S-layer gene as a molecular marker for identification of Lactobacillus helveticus.

FEMS microbiology letters 189 (2)
PMID : 10930751  :   DOI  :   10.1111/j.1574-6968.2000.tb09243.x    
Abstract >>
The paper describes two methods that allow rapid and accurate identification of Lactobacillus helveticus strains based on the nucleotide sequence of the gene coding for the surface layer (S-layer) protein. The first method is based on a polymerase chain reaction amplification using primers targeting a specific fragment of the S-layer gene. The second method involved a portion of the S-layer gene of L. helveticus as a probe in a hybridisation test. The specificity of these protocols was checked for DNA samples isolated from various Lactobacillus strains.
KeywordMeSH Terms
Genes, Bacterial
8. Parkin  KL, Fenster  KM,     ( 2000 )

Characterization of an arylesterase from Lactobacillus helveticus CNRZ32.

Journal of applied microbiology 88 (4)
PMID : 10792515  :  
Abstract >>
An esterase gene (estA) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The estA gene consisted of a 558 bp open reading frame encoding a putative peptide of 21.3 kDa. Protein sequence homology searches using BLAST revealed that EstA had low amino acid sequence identity with the serine-dependent arylesterases TesI (24%) and EtpA (26%) from Escherichia coli and Vibrio mimicus, respectively. A recombinant EstA fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstA revealed that it was a serine-dependent enzyme having a monomeric Mr of 22.6-25.1 kDa. Optimum temperature, NaCl concentration and pH for EstA activity were determined to be 35-40 degrees C, 3.5% NaCl and 7.5-8.0, respectively. EstA had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). EstA hydrolysed a variety of ester compounds and preferred those with substituted phenyl alcohol and short-chain fatty acid groups. Site-directed mutagenesis suggested that the S10 and H164 residues were essential for EstA activity.
KeywordMeSH Terms
Bacterial Proteins
9. Savijoki  K,     ( 2000 )

Purification and molecular characterization of a tripeptidase (PepT) from Lactobacillus helveticus.

Applied and environmental microbiology 66 (2)
PMID : 10653753  :   DOI  :   10.1128/aem.66.2.794-800.2000     PMC  :   PMC91898    
Abstract >>
A tripeptidase (PepT) from a thermophilic dairy starter strain of Lactobacillus helveticus was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase with a molecular mass of 150 kDa. PepT exhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly (K(m), 2.6 mM; V(max), 80.2 micromol. min(-1). microg(-1)). Some of the hydrophobic dipeptides were slowly hydrolyzed, distinguishing the Lactobacillus PepT from its counterpart in mesophilic Lactococcus lactis. No activity against tetrapeptides or amino acid p-nitroanilide derivatives was observed. The pepT gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 encoded a 47-kDa PepT protein which exhibited 53% identity with the PepT from L. lactis. The mRNA analyses indicated that pepT conforms a novel operon structure with an ORF1 located upstream. Several putative -35/-10 regions preceded the operon, but only one transcription start site located downstream of the first putative -10 region was identified. An inverted repeat structure with DeltaG of -64.8 kJ/mol was found downstream of the PepT-encoding region.
KeywordMeSH Terms
Aminopeptidases
10. Mileski  GJ, Weimer  BC, Steele  JL,     ( 1999 )

Genetic characterization of a cell envelope-associated proteinase from Lactobacillus helveticus CNRZ32.

Journal of bacteriology 181 (15)
PMID : 10419958  :   PMC  :   PMC103591    
Abstract >>
A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. The prtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that prtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of alpha(s1)-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.
KeywordMeSH Terms
Bacterial Proteins
11. Scheirlinck  I, Van der Meulen  R, Van Schoor  A, Vancanneyt  M, De Vuyst  L, Vandamme  P, Huys  G,     ( 2007 )

Influence of geographical origin and flour type on diversity of lactic acid bacteria in traditional Belgian sourdoughs.

Applied and environmental microbiology 73 (19)
PMID : 17675431  :   DOI  :   10.1128/AEM.00894-07     PMC  :   PMC2075033    
Abstract >>
A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.
KeywordMeSH Terms
Bacterial Typing Techniques
Fermentation
Genetic Variation
12. Mozzi  F, Vaningelgem  F, Hébert  EM, Van der Meulen  R, Foulquié Moreno  MR, Font de Valdez  G, De Vuyst  L,     ( 2006 )

Diversity of heteropolysaccharide-producing lactic acid bacterium strains and their biopolymers.

Applied and environmental microbiology 72 (6)
PMID : 16751563  :   DOI  :   10.1128/AEM.02780-05     PMC  :   PMC1489642    
Abstract >>
Thirty-one lactic acid bacterial strains from different species were evaluated for exopolysaccharide (EPS) production in milk. Thermophilic strains produced more EPS than mesophilic ones, but EPS yields were generally low. Ropiness or capsular polysaccharide formation was strain dependent. Six strains produced high-molecular-mass EPS. Polymers were classified into nine groups on the basis of their monomer composition. EPS from Enterococcus strains were isolated and characterized.
KeywordMeSH Terms
13. Naser  SM, Hagen  KE, Vancanneyt  M, Cleenwerck  I, Swings  J, Tompkins  TA,     ( 2006 )

Lactobacillus suntoryeus Cachat and Priest 2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen 1919) Bergey et al. 1925 (Approved Lists 1980).

International journal of systematic and evolutionary microbiology 56 (Pt 2)
PMID : 16449439  :   DOI  :   10.1099/ijs.0.64001-0     DOI  :   10.1099/ijs.0.64001-0    
Abstract >>
Strain R0052, isolated from a North American dairy starter culture, was initially identified as Lactobacillus acidophilus based on phenotypic analyses. However, upon sequencing the 16S rRNA gene, it became clear that the isolate was very highly related to Lactobacillus suntoryeus, Lactobacillus helveticus and Lactobacillus gallinarum, as similarities ranging from 99.3 to 99.8 % were observed. As an initial screening test to investigate the relatedness of strain R0052 and reference strains of L. suntoryeus, L. helveticus and L. gallinarum, the partial sequences for the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), the translational elongation factor Tu (tuf), a surface-layer protein (slp) and the Hsp60 chaperonins (groEL) were determined and they revealed high relatedness between all of the strains. The determination of the 16S-23S rRNA internally transcribed spacer (ITS) sequences revealed 98.3-100% similarity between L. suntoryeus and L. helveticus strains. SDS-PAGE of whole-cell proteins did not distinguish between these species. Fluorescent amplified fragment length polymorphism (FAFLP) could distinguish between these taxa, but they still constituted a single cluster within the L. acidophilus group. Finally, DNA-DNA hybridization experiments between strain R0052 and the type strains of L. helveticus and L. suntoryeus yielded reassociation values above 70% and confirmed that these names are synonyms.
KeywordMeSH Terms
14. Duwat  P, Ehrlich  SD, Gruss  A,     ( 1992 )

A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction.

Journal of bacteriology 174 (15)
PMID : 1629178  :   DOI  :   10.1128/jb.174.15.5171-5175.1992     PMC  :   PMC206342    
Abstract >>
An internal fragment of the recA gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction. The internal 348- or 360-bp recA DNA segments from Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesanteroides, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus salivarus subsp. thermophilus were amplified, cloned, and sequenced. The G + C contents of the DNA from these species range from 28 to 52%. The sequences of the bacterial recA genes show strong relatedness. This method is particularly useful for the recovery of the recA genes of gram-positive bacteria and avoids the difficulties of using a genetic complementation test for cloning.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
Polymerase Chain Reaction
15. Callanan  MJ, Beresford  TP, Ross  RP,     ( 2005 )

Genetic diversity in the lactose operons of Lactobacillus helveticus strains and its relationship to the role of these strains as commercial starter cultures.

Applied and environmental microbiology 71 (3)
PMID : 15746373  :   DOI  :   10.1128/AEM.71.3.1655-1658.2005     PMC  :   PMC1065143    
Abstract >>
Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the beta-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced beta-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed beta-galactosidase in the absence of lactose.
KeywordMeSH Terms
Lac Operon
16. Cappa  F, Cattivelli  D, Cocconcelli  PS,     ( 2005 )

The uvrA gene is involved in oxidative and acid stress responses in Lactobacillus helveticus CNBL1156.

Research in microbiology 156 (10)
PMID : 16125908  :   DOI  :   10.1016/j.resmic.2005.06.003    
Abstract >>
The uvrA gene of Lactobacillus helveticus CNBL1156 coding for subunit A of the excinuclease ABC complex involved in the nucleotide excision repair mechanism was identified. Analysis of the uvrA locus revealed the presence of three open reading frames, merR, sat and uvrA, which coded respectively for a MerR-like regulatory protein, a putative protein with homology to streptothricin acetyl transferase and for a UvrA protein. RNA analysis by northern blotting and RT-PCR showed that sat and uvrA were transcriptionally coupled. UvrA from L. helveticus contained the conserved domains of bacterial excinuclease A, as well as the two ATP binding sites and the zinc binding domains. The transcriptional activity of uvrA indicated that this gene was activated by exposure to UV radiation and oxidative stress. In addition, we observed that the expression of uvrA was inducible by pH; moreover, the role of UvrA in protection against stress was confirmed by acid adaptation experiments. Pretreatment of cells at pH 5 conferred resistance to H2O2, suggesting a specific adaptive response to pH-induced DNA damage. The results from this study indicate that UvrA contributes to acid and oxidative tolerance in L. helveticus, and suggest that it plays a role in survival at low pH under normal conditions.
KeywordMeSH Terms
Heat-Shock Response
Oxidative Stress
17. Ventura  M, Canchaya  C, van Sinderen  D, Fitzgerald  GF, Zink  R,     ( 2004 )

Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny.

Applied and environmental microbiology 70 (5)
PMID : 15128574  :   DOI  :   10.1128/aem.70.5.3110-3121.2004     PMC  :   PMC404453    
Abstract >>
The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F(1)F(0)-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.
KeywordMeSH Terms
Operon
18. Anand  R, Kaminski  PA, Ealick  SE,     ( 2004 )

Structures of purine 2'-deoxyribosyltransferase, substrate complexes, and the ribosylated enzyme intermediate at 2.0 A resolution.

Biochemistry 43 (9)
PMID : 14992575  :   DOI  :   10.1021/bi035723k    
Abstract >>
The structure of class I N-deoxyribosyltransferase from Lactobacillus helveticus was determined by X-ray crystallography. Unlike class II N-deoxyribosyltransferases, which accept either purine or pyrimidine deoxynucleosides, class I enzymes are specific for purines as both the donor and acceptor base. Both class I and class II enzymes are highly specific for deoxynucleosides. The class I structure reveals similarities with the previously determined class II enzyme from Lactobacillus leichmanni [Armstrong, S. A., Cook, W. J., Short, S. A., and Ealick, S. E. (1996) Structure 4, 97-107]. The specificity of the class I enzyme for purine deoxynucleosides can be traced to a loop (residues 48-62), which shields the active site in the class II enzyme. In the class I enzyme, the purine base itself shields the active site from the solvent, while the smaller pyrimidine base cannot. The structure of the enzyme with a bound ribonucleoside shows that the nucleophilic oxygen atom of Glu101 hydrogen bonds to the O2' atom, rendering it unreactive and thus explaining the specificity for 2'-deoxynucleosides. The structure of a ribosylated enzyme intermediate reveals movements that occur during cleavage of the N-glycosidic bond. The structures of complexes with substrates and substrate analogues show that the purine base can bind in several different orientations, thus explaining the ability of the enzyme to catalyze alternate deoxyribosylation at the N3 or N7 position.
KeywordMeSH Terms
19. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836     DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
Databases, Nucleic Acid
Evolution, Molecular
Internet
20. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836     DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
Databases, Nucleic Acid
Evolution, Molecular
Internet
21. Ventura  M, Canchaya  C, Meylan  V, Klaenhammer  TR, Zink  R,     ( 2003 )

Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification.

Applied and environmental microbiology 69 (11)
PMID : 14602655  :   DOI  :   10.1128/aem.69.11.6908-6922.2003     PMC  :   PMC262312    
Abstract >>
We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus.
KeywordMeSH Terms
22. Cousin  S, Gulat-Okalla  ML, Motreff  L, Gouyette  C, Bouchier  C, Clermont  D, Bizet  C,     ( 2012 )

Lactobacillus gigeriorum sp. nov., isolated from chicken crop.

International journal of systematic and evolutionary microbiology 62 (Pt 2)
PMID : 21421927  :   DOI  :   10.1099/ijs.0.028217-0    
Abstract >>
In the early 1980s, a facultatively anaerobic, non-motile, short rod, designated 202(T), was isolated from a chicken crop and identified as a homofermentative lactic acid bacterium. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain was affiliated with the genus Lactobacillus, clustering within the Lactobacillus acidophilus-delbrueckii group. In this analysis, strain 202(T) appeared to be most closely related to the type strains of Lactobacillus intestinalis and Lactobacillus amylolyticus, with gene sequence similarities of 96.1 and 96.2 %, respectively. Strain 202(T) was found to differ from these two species, however, when investigated by multilocus sequence analysis, and it also differed in terms of some of its metabolic properties. On the basis of these observations, strain 202(T) is considered to represent a novel species in the genus Lactobacillus, for which the name Lactobacillus gigeriorum sp. nov. is proposed; the type strain is 202(T) (= CRBIP 24.85(T) = DSM 23908(T)).
KeywordMeSH Terms
23. Mollet  B, Pilloud  N,     ( 1991 )

Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes.

Journal of bacteriology 173 (14)
PMID : 2066342  :   DOI  :   10.1128/jb.173.14.4464-4473.1991     PMC  :   PMC208110    
Abstract >>
By complementing appropriate gal lesions in Escherichia coli K802, we were able to isolate the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes of Lactobacillus helveticus. Tn10 transposon mutagenesis, together with in vivo complementation analysis and in vitro enzyme activity measurements, allowed us to map these two genes. The DNA sequences of the genes and the flanking regions were determined. These revealed that the two genes are organized in the order galK-galT in an operonlike structure. In an in vitro transcription-translation assay, the galK and galT gene products were identified as 44- and 53-kDa proteins, respectively, data which corresponded well with the DNA sequencing data. The deduced amino acid sequence of the galK gene product showed significant homologies to other prokaryotic and eukaryotic galactokinase sequences, whereas galactose-1-phosphate uridyl transferase did not show any sequence similarities to other known proteins. This observation, together with a comparison of known gal operon structures, suggested that the L. helveticus operon developed independently to a translational expression unit having a different gene order than that in E. coli, Streptococcus lividans, or Saccharomyces cerevisiae. DNA sequencing of the flanking regions revealed an open reading frame downstream of the galKT operon. It was tentatively identified as galM (mutarotase) on the basis of the significant amino acid sequence homology with the corresponding Streptococcus thermophilus gene.
KeywordMeSH Terms
Genes, Bacterial
24. Hagen  KE, Tramp  CA, Altermann  E, Welker  DL, Tompkins  TA,     ( 2010 )

Sequence analysis of plasmid pIR52-1 from Lactobacillus helveticus R0052 and investigation of its origin of replication.

Plasmid 63 (2)
PMID : 20051251  :   DOI  :   10.1016/j.plasmid.2009.12.004    
Abstract >>
Lactobacillus helveticus R0052 is a bacterium used in commercial probiotic preparations. R0052 contains a small, cryptic plasmid comprised of eight open reading frames, four of which encode proteins of unknown function. Based on the sequence of the replication initiation protein RepA, pIR52-1 is a member of the recently described RepA_N family of Gram-positive theta-replicating plasmids. The repA gene of pIR52-1 is the minimal origin of replication for L. helveticus and other Lactobacillus hosts. Additionally, pIR52-1 belongs to a subgroup of the RepA_N plasmid family which have RepA proteins of high amino acid identity and a conserved, non-coding element upstream of repA which, in pIR52-1, is responsible for the control of plasmid copy number and contributes to plasmid maintenance.
KeywordMeSH Terms
Sequence Analysis, DNA
25. Scheirlinck  I, Van der Meulen  R, Van Schoor  A, Vancanneyt  M, De Vuyst  L, Vandamme  P, Huys  G,     ( 2008 )

Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting.

Applied and environmental microbiology 74 (8)
PMID : 18310426  :   DOI  :   10.1128/AEM.02771-07     PMC  :   PMC2293155    
Abstract >>
A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.
KeywordMeSH Terms
Biodiversity
Food Microbiology
26. Naser  SM, Dawyndt  P, Hoste  B, Gevers  D, Vandemeulebroecke  K, Cleenwerck  I, Vancanneyt  M, Swings  J,     ( 2007 )

Identification of lactobacilli by pheS and rpoA gene sequence analyses.

International journal of systematic and evolutionary microbiology 57 (Pt 12)
PMID : 18048724  :   DOI  :   10.1099/ijs.0.64711-0    
Abstract >>
The aim of this study was to evaluate the use of the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial gene sequences for species identification of members of the genus Lactobacillus. Two hundred and one strains representing the 98 species and 17 subspecies were examined. The pheS gene sequence analysis provided an interspecies gap, which in most cases exceeded 10 % divergence, and an intraspecies variation of up to 3 %. The rpoA gene sequences revealed a somewhat lower resolution, with an interspecies gap normally exceeding 5 % and an intraspecies variation of up to 2 %. The combined use of pheS and rpoA gene sequences offers a reliable identification system for nearly all species of the genus Lactobacillus. The pheS and rpoA gene sequences provide a powerful tool for the detection of potential novel Lactobacillus species and synonymous taxa. In conclusion, the pheS and rpoA gene sequences can be used as alternative genomic markers to 16S rRNA gene sequences and have a higher discriminatory power for reliable identification of species of the genus Lactobacillus.
KeywordMeSH Terms
27. Blaiotta  G, Fusco  V, Ercolini  D, Aponte  M, Pepe  O, Villani  F,     ( 2008 )

Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation.

Applied and environmental microbiology 74 (1)
PMID : 17993558  :   DOI  :   10.1128/AEM.01711-07     PMC  :   PMC2223197    
Abstract >>
A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.
KeywordMeSH Terms
Polymorphism, Restriction Fragment Length
28. Takiguchi  R, Hashiba  H, Aoyama  K, Ishii  S,     ( 1989 )

Complete nucleotide sequence and characterization of a cryptic plasmid from Lactobacillus helveticus subsp. jugurti.

Applied and environmental microbiology 55 (6)
PMID : 2764571  :   PMC  :   PMC202923    
Abstract >>
A small cryptic plasmid, pLJ1, was isolated from Lactobacillus helveticus subsp. jugurti and was cloned into Escherichia coli HB101 by using pBR329 as a vector. Plasmid pLJ1 was 3,292 base pairs long and had single restriction endonuclease sites for PvuII, KpnI, AvaII, Acci, HindIII, and EcoRI. In a maxicell system, pLJ1 produced a protein of about 41 kilodaltons.
KeywordMeSH Terms
Plasmids
29. Sun  Z, Harris  HM, McCann  A, Guo  C, Argimón  S, Zhang  W, Yang  X, Jeffery  IB, Cooney  JC, Kagawa  TF, Liu  W, Song  Y, Salvetti  E, Wrobel  A, Rasinkangas  P, Parkhill  J, Rea  MC, O'Sullivan  O, Ritari  J, Douillard  FP, Paul Ross  R, Yang  R, Briner  AE, Felis  GE, de Vos  WM, Barrangou  R, Klaenhammer  TR, Caufield  PW, Cui  Y, Zhang  H, O'Toole  PW,     ( 2015 )

Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera.

Nature communications 6 (N/A)
PMID : 26415554  :   DOI  :   10.1038/ncomms9322     PMC  :   PMC4667430    
Abstract >>
Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.
KeywordMeSH Terms
Phylogeny
30. Wakai  T, Yamamoto  N,     ( 2013 )

A novel branched chain amino acids responsive transcriptional regulator, BCARR, negatively acts on the proteolytic system in Lactobacillus helveticus.

PloS one 8 (10)
PMID : 24146802  :   DOI  :   10.1371/journal.pone.0075976     PMC  :   PMC3795697    
Abstract >>
Transcriptional negative regulation of the proteolytic system of Lactobacillus helveticus CM4 in response to amino acids seems to be very important for the control of antihypertensive peptide production; however, it remains poorly understood. A 26-kDa protein with N-terminal cystathionine �]-synthase domains (CBS domain protein), which seems to be involved in the regulatory system, was purified by using a DNA-sepharose bound 300-bp DNA fragment corresponding to the upstream regions of the six proteolytic genes that are down-regulated by amino acids. The CBS domain protein bound to a DNA fragment corresponding to the region upstream of the pepV gene in response to branched chain amino acids (BCAAs). The expression of the pepV gene in Escherichia coli grown in BCAA-enriched medium was repressed when the CBS domain protein was co-expressed. These results reveal that the CBS domain protein acts as a novel type of BCAA-responsive transcriptional regulator (BCARR) in L. helveticus. From comparative analysis of the promoter regions of the six proteolysis genes, a palindromic AT-rich motif, 5'-AAAAANNCTWTTATT-3', was predicted as the consensus DNA motif for the BCARR protein binding. Footprint analysis using the pepV promotor region and gel shift analyses with the corresponding short DNA fragments strongly suggested that the BCARR protein binds adjacent to the pepV promoter region and affects the transcription level of the pepV gene in the presence of BCAAs. Homology search analysis of the C-terminal region of the BCARR protein suggested the existence of a unique �]�\�]�]�\�] fold structure that has been reported in a variety of ACT (aspartate kinase-chorismate mutase-tyrA) domain proteins for sensing amino acids. These results also suggest that the sensing of BCAAs by the ACT domain might promote the binding of the BCARR to DNA sequences upstream of proteolysis genes, which affects the gene expression of the proteolytic system in L. helveticus.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
31. Stressler  T, Eisele  T, Schlayer  M, Lutz-Wahl  S, Fischer  L,     ( 2013 )

Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis.

PloS one 8 (7)
PMID : 23894590  :   DOI  :   10.1371/journal.pone.0070055     PMC  :   PMC3716637    
Abstract >>
The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 ?kat(H-Ala-Pro-pNA) L(-1), which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 ?kat(H-Ala-pNA) L(-1). After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn(2+)-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co(2+)>Zn(2+)>Mn(2+)>Ca(2+)>Mg(2+). PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50�XC and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.
KeywordMeSH Terms
32. Huang  CH, Chang  MT, Huang  MC, Wang  LT, Huang  L, Lee  FL,     ( 2012 )

Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene.

Journal of the science of food and agriculture 92 (13)
PMID : 22555934  :   DOI  :   10.1002/jsfa.5692    
Abstract >>
To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing.
KeywordMeSH Terms
Base Sequence
Genotype
Phylogeny
33. Joerger  MC, Klaenhammer  TR,     ( 1990 )

Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J.

Journal of bacteriology 172 (11)
PMID : 2228964  :   DOI  :   10.1128/jb.172.11.6339-6347.1990     PMC  :   PMC526818    
Abstract >>
Lactobacillus helveticus 481 produces a 37-kDa bacteriocin called helveticin J. Libraries of chromosomal DNA from L. helveticus were prepared in lambda gt11 and probed for phage-producing fusion proteins that could react with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologous inserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clones specifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not produce helveticin J or strains producing unrelated bacteriocins. HJ1 and HJ4 lysogens produced beta-galactosidase fusion proteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) was isolated by screening a library of L. helveticus chromosomal DNA in lambda EMBL3 with the insert DNA from phage HJ4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' end of another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticus DNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from the termination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end of another ORF (ORF4) was found. A Bg/II fragment containing ORF2 and ORF3 was cloned into pGK12, and the recombinant plasmid, pTRK135, was transformed into Lactobacillus acidophilus via electroporation. Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an acitivity spectrum that was the same as that of helveticin J.
KeywordMeSH Terms
Bacteriocins
Genes, Bacterial
34.     ( 1997 )

Cloning and functional expression in Escherichia coli of the gene encoding the di- and tripeptide transport protein of Lactobacillus helveticus.

Applied and environmental microbiology 63 (6)
PMID : 9172341  :   PMC  :   PMC168514    
Abstract >>
The gene encoding the di- and tripeptide transport protein (DtpT) of Lactobacillus helveticus (DtpTLH) was cloned with the aid of the inverse PCR technique and used to complement the dipeptide transport-deficient and proline-auxotrophic Escherichia coli E1772. Functional expression of the peptide transporter was shown by the uptake of prolyl-[14C] alanine in whole cells and membrane vesicles. Peptide transport via DtpT in membrane vesicles is driven by the proton motive force. The system has specificity for di- and tripeptides but not for amino acids or tetrapeptides. The dtpTLH gene consists of 1,491 bp, which translates into a 497-amino-acid polypeptide. DtpTLH shows 34% identity to the di- and tripeptide transport protein of Lactococcus lactis and is also homologous to various peptide transporters of eukaryotic origin, but the similarity between these proteins is confined mainly to the N-terminal halves.
KeywordMeSH Terms
Genes, Bacterial
Membrane Transport Proteins
35.     ( 1997 )

Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme.

Applied and environmental microbiology 63 (7)
PMID : 9212432  :   PMC  :   PMC168581    
Abstract >>
The Lactobacillus helveticus L-(+)-lactate dehydrogenase (L-LDH) gene (ldhL) was isolated from a lambda library. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 323 amino acids (35.3 kDa). The deduced sequence of the 35-kDa protein revealed a relatively high degree of identity with other lactobacillar L-LDHs. The highest identity (80.2%) was observed with the Lactobacillus casei L-LDH. The sizes and 5' end analyses of ldhL transcripts showed that the ldhL gene is a monocistronic transcriptional unit. The expression of ldhL, studied as a function of growth, revealed a high expression level at the logarithmic phase of growth. The ldhL gene is preceded by two putative -10 regions, but no corresponding -35 regions could be identified. By primer extension analysis, the ldhL transcripts were confirmed to be derived from the -10 region closest to the initiation codon. However, upstream of these regions additional putative -10/-35 regions could be found. The L-LDH was overexpressed in Escherichia coli and purified to homogeneity by two chromatographic steps. The purified L-LDH was shown to be a nonaliosteric enzyme, and amino acid residues involved in allosteric regulation were not conserved in L. helveticus L-LDH. However, a slight enhancement of enzyme activity was observed in the presence of fructose 1,6-diphosphate, particularly at neutral pH. A detailed enzymatic characterization of L-LDH was performed. The optimal reaction velocity was at pH 5.0, where the kinetic parameters K(m), and Kcat for pyruvate were 0.25 mM and 643 S-1, respectively.
KeywordMeSH Terms
36.     ( 1997 )

Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32.

Journal of bacteriology 179 (8)
PMID : 9098049  :   DOI  :   10.1128/jb.179.8.2529-2533.1997     PMC  :   PMC179000    
Abstract >>
An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa. Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp. lactis DSM7290, L. helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp. cremoris AM2. A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively. PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected.
KeywordMeSH Terms
37.     ( 1996 )

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

Microbiology (Reading, England) 142 (Pt 12) (N/A)
PMID : 9004508  :   DOI  :   10.1099/13500872-142-12-3459    
Abstract >>
A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.
KeywordMeSH Terms
Operon
Promoter Regions, Genetic
38.     ( 1996 )

Characterization of a prolinase gene and its product and an adjacent ABC transporter gene from Lactobacillus helveticus.

Microbiology (Reading, England) 142 (Pt 4) (N/A)
PMID : 8936308  :   DOI  :   10.1099/00221287-142-4-809    
Abstract >>
A prolinase (pepR) gene was cloned from an industrial Lactobacillus helveticus strain (53/7). Three clones, hybridizing with a gene probe specific for a peptidase shown to have activity against di- and tripeptides, were detected from a L. helveticus genomic library constructed in Escherichia coli. None of the three clones, however, showed enzyme activity against the di- or tripeptide substrates tested. One of the clones, carrying a vector with a 5.5 kb insert, was further characterized by DNA sequencing. The sequence analysis revealed the presence of two ORFs, ORF1 and ORF2 of 912 and 1602 bp, respectively. ORF2, located upstream of and in the opposite orientation to ORF1, had a promoter region overlapping that of ORF1. ORF1 had the capacity to encode a 35083 Da protein. When amplified by PCR, ORF1 with its control regions specified a 35 kDa protein in E. coli that was able to hydrolyse dipeptides, with highest activity against Pro-Leu, whereas from the tripeptides tested, only Leu-Leu-Leu was slowly degraded. By the substrate-specificity profile and protein homologies, the 35 kDa protein was identified as a prolinase. The activity of the cloned prolinase was inhibited by p-hydroxymercuribenzoate. Northern and primer-extension analyses of ORF1 revealed a 1.25 kb transcript and two adjacent transcription start sites, respectively, thus confirming the DNA sequence data. ORF2 had encoding capacity for a 59.5 kDa protein that showed significant homology to several members of the family of ABC transporters. Determination of the mRNA levels at different growth phases revealed that the pepR gene and ORF2 are transcribed in L. helveticus at the exponential and stationary phases of growth, respectively. Furthermore, two ORF2 deletion constructs, carrying the intact pepR gene, showed that this upstream operon adversely affected PepR activity in E. coli, which explains the enzymic inactivity of the original clones.
KeywordMeSH Terms
Genes, Bacterial
39.     ( 1996 )

DNA sequence analysis, expression, distribution, and physiological role of the Xaa-prolyldipeptidyl aminopeptidase gene from Lactobacillus helveticus CNRZ32.

Applied microbiology and biotechnology 44 (6)
PMID : 8867635  :  
Abstract >>
Lactobacillus helveticus CNRZ32 possesses an Xaa-prolyldipeptidyl aminopeptidase (PepX), which releases amino-terminal dipeptides from peptides containing proline residues in the penultimate position. The PepX gene, designated pepX, from Lb. helveticus CNRZ32 was sequenced. Analysis of the sequence identified a putative 2379-bp pepX open-reading frame, which encodes a polypeptide of 793 amino acid residues with a deduced molecular mass of 88,111 Da. The gene shows significant sequence identity with sequenced pepX genes from lactic acid bacteria. The product of the gene contains a motif that is almost identical with the active-site motif of the serine-dependent PepX from lactococci. The introduction of pepX into Lactococcus lactis LM0230 on either pGK12 (a low-copy-number plasmid vector) or pIL253 (a high-copy-number plasmid vector) did not result in a significant increase in PepX activity, while the introduction of pepX into CNRZ32 on pGK12 resulted in a four-fold increase in PepX activity. Southern hybridization experiments revealed that the pepX gene from CNRZ32 is well conserved in lactobacilli, pediococci and streptococci. The physiological role of PepX during growth in lactobacillus MRS (a rich medium containing protein hydrolysates along with other ingredients) and milk was examined by comparing growth of CNRZ32 and a CNRZ32 PepX-negative derivative. No difference in growth rate or acid production was observed between CNRZ32 and its PepX-negative derivative in MRS. However, the CNRZ32 PepX-negative derivative grew in milk at a reduced specific growth rate when compared to wild-type CNRZ32. Introduction of the cloned PepX determinant into the CNRZ32 PepX-negative derivative resulted in a construct with a specific growth rate similar to that of wild-type CNRZ32.
KeywordMeSH Terms
40.     ( 1996 )

Molecular characterization, over-expression and purification of a novel dipeptidase from Lactobacillus helveticus.

Applied microbiology and biotechnology 45 (5)
PMID : 8766699  :  
Abstract >>
A dipeptidase gene (pepD) from an industrial Lactobacillus helveticus strain was isolated by colony hybridization. An open reading frame (ORF) of 1422 base pairs (bp) with a coding capacity for a 53.5-kDa protein (PepD) was identified. The ORF was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -51.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 53.5-kDa protein revealed no marked homologies when compared to the data bases of EMBL and SWISS-PROT. The 5'end of the 1.6-kb pepD transcript was determined both by a conventional primer extension method and using an automated sequencer. pepD was found to be maximally expressed at late exponential growth. The pepD gene was cloned into an expression vector to over-produce PepD in Escherichia coli JM105. Purification of PepD to homogeneity was achieved using three chromatographic steps. PepD was able to hydrolyze a number of dipeptides with the exception of those containing a proline residue. Optimal PepD activity was observed at pH 6.0 and 55 degrees C. The enzyme was inhibited by p-hydroxymercuribenzoate and reactivated by dithiothreitol whereas ethylenediaminetetraacetate had no inhibitory effect on PepD. The enzymatic properties of PepD suggest that it represents a novel dipeptidase type among lactic acid bacteria.
KeywordMeSH Terms
Genes, Bacterial
41.     ( 1996 )

Sequencing, distribution, and inactivation of the dipeptidase A gene (pepDA) from Lactobacillus helveticus CNRZ32.

Journal of bacteriology 178 (3)
PMID : 8550503  :   DOI  :   10.1128/jb.178.3.701-704.1996     PMC  :   PMC177715    
Abstract >>
Previously, the gene for a general dipeptidase (pepDA) was isolated from a gene bank of Lactobacillus helveticus CNRZ32. The pepDA gene consists of a 1,422-bp open reading frame which could encode a polypeptide of 53.5 kDa. No significant identity was found between the deduced amino acid sequence of the pepDA product and the sequence for other polypeptides reported in GenBank. Southern hybridization studies with a pepDA probe indicated that the nucleotide sequence for pepDA is not well conserved among a variety of lactic acid bacteria. Growth studies indicated that a pepDA deletion had no detectable effect on growth rate or acid production by L. helveticus CNRZ32 in milk. Furthermore, no difference in total cellular dipeptidase activity was detected between the mutant and wild-type strains during logarithmic growth in MRS medium.
KeywordMeSH Terms
Genes, Bacterial
42.     ( 1995 )

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus.

Microbiology (Reading, England) 141 (Pt 12) (N/A)
PMID : 8574400  :   DOI  :   10.1099/13500872-141-12-3067    
Abstract >>
The X-prolyl dipeptidyl aminopeptidase gene (pepX) of an industrially used Lactobacillus helveticus strain has been detected by nucleic acid hybridization, cloned, characterized and sequenced. One ORF of 2379 bp with coding capacity for a 90.6 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with delta G of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 90.6 kDa protein showed 49.3, 49.4 and 77.7% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lactobacillus delbrueckii subsp. lactis, respectively. Northern blotting revealed a 2.6 kb transcript and one transcription start site was identified via primer extension analysis using an A.L.F. sequencer. In a bioreactor study, the expression of pepX in Lb. helveticus was studied as a function of growth. Transcription of pepX was typical of exponential growth phase expression. The pepX gene has been cloned into pKK223-3 and expressed at a high level in Escherichia coli JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6.5 and 45 degrees C. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu2+, Cd2+ and Zn2+. EDTA and 1,10-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding DTT, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.
KeywordMeSH Terms
Genes, Bacterial
43.     ( 1994 )

Characterization of the Lactobacillus helveticus CNRZ32 pepC gene.

Applied and environmental microbiology 60 (1)
PMID : 8117086  :   PMC  :   PMC201308    
Abstract >>
Sequence analysis of the aminopeptidase C gene (pepC) from Lactobacillus helveticus CNRZ32 identified a 1,332-nucleotide open reading frame coding for a polypeptide with motifs characteristic of cysteine proteinases. Homology to the pepC gene appears to be widely distributed among lactic acid bacteria.
KeywordMeSH Terms
Genes, Bacterial
44.     ( 1994 )

Characterization of IS1201, an insertion sequence isolated from Lactobacillus helveticus.

Gene 145 (1)
PMID : 8045427  :   DOI  :   10.1016/0378-1119(94)90325-5    
Abstract >>
IS1201, a 1387-bp insertion sequence isolated from Lactobacillus helveticus, was identified by its nucleotide (nt) sequence. It carries a single open reading frame encoding a 369-amino-acid protein, which shares homology with transposases found in a class of related IS, including ISRm3 from Rhizobium meliloti, IS256 from Staphylococcus aureus, IS6120 from Mycobacterium smegmatis, IS1081 from M. bovis, IST2 from Thiobacillus ferroxidans and IS406 from Pseudomonas cepacia. IS1201 has terminal inverted repeats of 24 bp in length and a target site duplication of 8 bp. Its copy number ranges from 3 to about 16 per L. helveticus genome. No homology was found between the nt sequence of IS1201 and those of the other bacterial IS from the same class. These results, together with previous observations [de los Reyes-Gavil?n et al., Appl. Environ. Microbiol., 58 (1992) 3429-3432], confirm that IS1201 can be used as a specific DNA probe for the identification of L. helveticus strains.
KeywordMeSH Terms
DNA Transposable Elements
45.     ( 1994 )

Nucleotide sequence and distribution of the pepPN gene from Lactobacillus helveticus CNRZ32.

FEMS microbiology letters 119 (1��2��)
PMID : 8039668  :   DOI  :   10.1111/j.1574-6968.1994.tb06864.x    
Abstract >>
The Lactobacillus helveticus CNRZ32 gene encoding a di-/tri- pepidase with prolinase activity (pepPN) was sequenced. An open reading frame of 912 base pairs was identified corresponding to a peptide with a molecular mass of 35.04 kDa. Southern hybridization indicated that the gene sequence is well conserved in strains of lactobacilli and pediococci.
KeywordMeSH Terms
Genes, Bacterial
46. Zwahlen  MC, Mollet  B,     ( 1994 )

ISL2, a new mobile genetic element in Lactobacillus helveticus.

Molecular & general genetics : MGG 245 (3)
PMID : 7816043  :   DOI  :   10.1007/bf00290113    
Abstract >>
Spontaneous, phenotypically stable mutations at the beta-galactosidase locus (lacL-lacM) in Lactobacillus helveticus were identified and analyzed. We found that a significant number of mutations were caused by integration of a new IS element, ISL2, into these lac genes. ISL2 is 858 bp long, flanked by 16-bp perfect inverted repeats and generates 3-bp target duplications upon insertion. It contains one open reading frame, which shows significant homology (40.1% identity) to the putative transposase of IS702 from Cyanobacterium calothrix. ISL2 is present in 4-21 copies in the L. helveticus genome, but it is not found in other lactic acid bacteria. Its divergence in copy number and genomic locations in different L. helveticus strains makes it useful as a tool for strain identification by genetic fingerprinting.
KeywordMeSH Terms
DNA Transposable Elements
Genes, Bacterial
47. Varmanen  P, Vesanto  E, Steele  JL, Palva  A,     ( 1994 )

Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus.

FEMS microbiology letters 124 (3)
PMID : 7851738  :   DOI  :   10.1111/j.1574-6968.1994.tb07302.x    
Abstract >>
An aminopeptidase N (pepN) gene was detected by DNA hybridization from an industrially important Lactobacillus helveticus strain using part of the L. helveticus CNRZ32 pepN gene as the probe. One of five hybridization positive clones was characterized in more detail. A subcloned 3.7 kb fragment, positive in hybridization and encoding aminopeptidase activity, was sequenced and analyzed. Only one open reading frame (ORF) of 2532 base pairs with a coding capacity for a 95.9 kDa protein could be found. The deduced amino acid sequence of the 95.9 kDa protein showed homology to PepN proteins from other lactic acid bacteria and carried the conserved catalytic and zinc binding sites of the neutral zinc metallo-peptidase family confirming the identity of the pepN gene. A 2.75 kb transcript and two transcription start sites were identified with mRNA analyses. Expression of pepN in L. helveticus, studied as the function of growth, revealed a high level of pepN transcripts throughout the growth, in contrast to the steady state levels of other peptidase mRNAs from L. helveticus analyzed in our laboratory.
KeywordMeSH Terms
48. Pridmore  D, Stefanova  T, Mollet  B,     ( 1994 )

Cryptic plasmids from Lactobacillus helveticus and their evolutionary relationship.

FEMS microbiology letters 124 (3)
PMID : 7851737  :   DOI  :   10.1111/j.1574-6968.1994.tb07300.x    
Abstract >>
Three different cryptic plasmids from Lactobacillus helveticus have been identified and their DNA sequences determined. Analysis and comparisons of their primary structures revealed stretches of DNA with considerable homology. Thus, large portions of the plasmid non-coding sequences were conserved at 80-90% identity between the different plasmids identified so far in L. helveticus. Nevertheless, different plasmids found in a same host strain utilise different genes of replication, probably acquired during evolution from different replicons from Gram-positive bacterial origins. A remnant structure of such a possible genetic integration of a foreign replication gene into one of the plasmids of L. helveticus was identified.
KeywordMeSH Terms
49. Christensen  JE, Lin  DL, Palva  A, Steele  JL,     ( 1995 )

Sequence analysis, distribution and expression of an aminopeptidase N-encoding gene from Lactobacillus helveticus CNRZ32.

Gene 155 (1)
PMID : 7698673  :   DOI  :   10.1016/0378-1119(94)00924-h    
Abstract >>
Lactobacillus (Lb.) helveticus CNRZ32 possesses a 97-kDa metalloenzyme with aminopeptidase activity (PepN; EC 3.4.11.2). A 3.8-kb fragment encoding PepN was cloned into pIL253 and designated pSUW34. Transformation of lactococcus (Lc.) lactis LM0230 with pSUW34 resulted in > 180-fold increase in general aminopeptidase (AP) activity using L-lysine-p-nitroanilide. Southern hybridization was conducted to determine the distribution of homology to the CNRZ32 pepN gene among lactic-acid bacteria (LAB). Hybridization was observed with strains of lactobacilli, pediococci, leuconostoc, streptococci and lactococci. The pepN gene was sequenced and found to encode a protein containing 844 amino acid (aa) residues. A comparison of Lb. helveticus CNRZ32 pepN to Lb. delbrueckii ssp. lactis DSM7290 pepN indicated 69.5% nucleotide (nt) identity and 71.8% aa identity, while comparison to pepN from Lc. lactis ssp. cremoris MG1363 indicated 61.1% nt identity and 49.2% aa identity. Alignment of peptidase aa sequences of LAB, Escherichia coli, yeast and mammalian origin display homology in the zinc-binding domain, as well as a conserved region upstream from the putative active site.
KeywordMeSH Terms
Aminopeptidases
50. Bhowmik  T, Steele  JL,     ( 1994 )

Cloning, characterization and insertional inactivation of the Lactobacillus helveticus D(-) lactate dehydrogenase gene.

Applied microbiology and biotechnology 41 (4)
PMID : 7765104  :  
Abstract >>
A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD+ oxidoreductase, EC 1.1.1.28] from Lactobacillus helveticus CNRZ32 was identified from a genomic library by complementation of Escherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kb DraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-)LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci or Lactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative of L. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant.
KeywordMeSH Terms
51.     ( 1994 )

Characterization and expression of the Lactobacillus helveticus pepC gene encoding a general aminopeptidase.

European journal of biochemistry 224 (3)
PMID : 7925424  :   DOI  :   10.1111/j.1432-1033.1994.00991.x    
Abstract >>
An aminopeptidase C gene (pepC) was detected by nucleic acid hybridization from an industrially important Lactobacillus helveticus strain. Three hybridization positive clones were isolated from a gene library of this L. helveticus strain, and one of them was characterized in more detail. Deletion mapping localized the hybridization positivity into a 2.8-kb fragment, which also encoded aminopeptidase activity. This fragment was sequenced and two open reading frames (ORF1 and 2) of 1347 and 840 base pairs were identified. The ORF1 was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -49.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of ORF1, with an encoding capacity for a 51.4-kDa protein, was shown to share 48.3% and 98.0% identities with the PepC proteins from Lactococcus lactis and L. helveticus CNRZ32, respectively, thus confirming that ORF1 codes for an aminopeptidase C. mRNA size analyses revealed 1.7-kb and 2.7-kb transcripts in Northern blot with the pepC-specific probe. A further analysis with the pepC- and ORF2-specific probes showed that downstream ORF2 is co-transcribed with the pepC gene at the exponential phase of growth whereas, at the stationary growth phase, transcripts derived from the pepC promoter were below the detection limit, and the ORF2 was expressed by its own promoter. The 5' end mapping of the pepC transcripts with primer extension revealed one transcription start site suggesting a new position for the pepC promoter region when compared to that predicted for the L. helveticus CNRZ32 pepC gene. Expression of pepC was also studied in L. helveticus as the function of growth in a bioreactor study. Transcription of pepC was typical to exponential growth phase expression. The level of total thiol-aminopeptidase activity, however, remained nearly constant throughout the stationary growth phase.
KeywordMeSH Terms
Genes, Bacterial
52. Schmid  M, Muri  J, Melidis  D, Varadarajan  AR, Somerville  V, Wicki  A, Moser  A, Bourqui  M, Wenzel  C, Eugster-Meier  E, Frey  JE, Irmler  S, Ahrens  CH,     ( 2018 )

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level.

Frontiers in microbiology 9 (N/A)
PMID : 29441050  :   DOI  :   10.3389/fmicb.2018.00063     PMC  :   PMC5797582    
Abstract >>
Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus-to our knowledge-identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories "cell wall/membrane biogenesis" and "defense mechanisms" were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.
KeywordMeSH Terms
CRISPR/Cas
PacBio
comparative genomics
dairy industry
metagenomics
natural whey starter cultures
strain-specific genes
whole genome sequencing
CRISPR/Cas
PacBio
comparative genomics
dairy industry
metagenomics
natural whey starter cultures
strain-specific genes
whole genome sequencing
53. Moser  A, Wüthrich  D, Bruggmann  R, Eugster-Meier  E, Meile  L, Irmler  S,     ( 2017 )

Amplicon Sequencing of the slpH Locus Permits Culture-Independent Strain Typing of Lactobacillus helveticus in Dairy Products.

Frontiers in microbiology 8 (N/A)
PMID : 28775722  :   DOI  :   10.3389/fmicb.2017.01380     PMC  :   PMC5517455    
Abstract >>
The advent of massive parallel sequencing technologies has opened up possibilities for the study of the bacterial diversity of ecosystems without the need for enrichment or single strain isolation. By exploiting 78 genome data-sets from Lactobacillus helveticus strains, we found that the slpH locus that encodes a putative surface layer protein displays sufficient genetic heterogeneity to be a suitable target for strain typing. Based on high-throughput slpH gene sequencing and the detection of single-base DNA sequence variations, we established a culture-independent method to assess the biodiversity of the L. helveticus strains present in fermented dairy food. When we applied the method to study the L. helveticus strain composition in 15 natural whey cultures (NWCs) that were collected at different Gruy?re, a protected designation of origin (PDO) production facilities, we detected a total of 10 sequence types (STs). In addition, we monitored the development of a three-strain mix in raclette cheese for 17 weeks.
KeywordMeSH Terms
Lactobacillus helveticus
natural whey culture
population composition
semiconductor sequencing
strain typing
Lactobacillus helveticus
natural whey culture
population composition
semiconductor sequencing
strain typing
54.     ( 2013 )

Comparative analysis of proteolytic enzymes need for processing of antihypertensive peptides between Lactobacillus helveticus CM4 and DPC4571.

Journal of bioscience and bioengineering 115 (3)
PMID : 23182500  :   DOI  :   10.1016/j.jbiosc.2012.09.020    
Abstract >>
To understand high amount of production and detailed processing of antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in Lactobacillus helveticus CM4 fermented milk, whole genome sequence of the CM4 strain was completed and compared to previously reported whole genome sequence of L. helveticus DPC4571. It revealed 2,028,493 bp of DNA sequence and encoding of 2174 open reading frames in the whole genome sequence with the highest homology to the genome sequence of L. helveticus DPC 4571. Comparative analysis focused on proteolytic enzymes between CM4 and DPC4571 strains revealed existence of 23 kinds of identical intracellular peptidase genes in both strains but no prtY type proteinase gene in DPC4571. Immunoblotting analysis with an antibody raised against the PrtY proteinase showed existence of the 45 kDa PrtY protein in CM4 but not in DPC4571 in the cell extracts. The cell wall-associated proteinase activity was higher in the CM4 than that in the DPC4571 throughout all fermentation period, and the amounts of VPP and IPP in CM4 and DPC4571 fermented milk were correlated with the proteinase activity on the cell wall. Moreover, slight difference of the �]-casein hydrolysates by cell wall-associated extracellular proteinases between CM4 and DPC4571 cells was detected by a MALDI-TOF/TOF analysis. These results suggest that the extracellular proteinase activity might affect on the productivity of VPP and IPP in L. helveticus fermented milk and some peptidases might play important role in following precise processing to release VPP and IPP.
KeywordMeSH Terms
55.     ( 2013 )

S-layer protein mediates the stimulatory effect of Lactobacillus helveticus MIMLh5 on innate immunity.

Applied and environmental microbiology 79 (4)
PMID : 23220964  :   DOI  :   10.1128/AEM.03056-12     PMC  :   PMC3568609    
Abstract >>
The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-�eB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the host's defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention.
KeywordMeSH Terms
Immunity, Innate
56.     ( 1998 )

Characterization of the Lactobacillus helveticus groESL operon.

Research in microbiology 149 (4)
PMID : 9766226  :  
Abstract >>
This study utilized inverse polymerase chain reactions to characterize a 2.7-kb region of the Lactobacillus helveticus LH212 chromosome that included two complete and one truncated open reading frames (ORFs). Protein homology searches showed that the first two ORFs encoded homologs to the universally conserved heat shock proteins GroES and GroEL. Amino acids encoded by the 5' end of the truncated ORF that was downstream of groEL showed good homology to the amino terminal end of the Streptococcus pneumoniae DNA mismatch repair enzyme HexA. Nucleotide sequence analysis identified a putative transcriptional promoter upstream of groES that was comprised of -35 and -10 hexamers flanked upstream and downstream by copies of the conserved Gram-positive heat shock gene regulatory sequence, CIRCE. A large inverted repeat that may function as a rho-independent transcriptional terminator was located between groEL and the third ORF. Northern hybridization of an LH212 groEL gene fragment to RNA isolated from cells that had been heat shocked at 52 degrees C for 0, 5, 10 or 15 min detected a 2.2-kb transcript in each of the cell preparations. Densitometry showed the concentration of this mRNA species was approximately 4-fold higher after heat shock for 5 or 10 min and 3-fold higher after 15 min of heat shock.
KeywordMeSH Terms
Operon
57.     ( 1998 )

Molecular characterization of a stress-inducible gene from Lactobacillus helveticus.

Journal of bacteriology 180 (23)
PMID : 9829922  :   PMC  :   PMC107698    
Abstract >>
A gene (htrA) coding for a stress-inducible HtrA-like protein from Lactobacillus helveticus CNRZ32 was cloned, sequenced, and characterized. The deduced amino acid sequence of the gene exhibited 30% identity with the HtrA protein from Escherichia coli; the putative catalytic triad and a PDZ domain that characterize the HtrA family of known bacterial serine proteases were also found in the sequence. Expression of the L. helveticus htrA gene in a variety of stress conditions was analyzed at the transcriptional level. The strongest induction, resulting in over an eightfold increase in the htrA transcription level, was found in growing CNRZ32 cells exposed to 4% (wt/vol) NaCl. Enhanced htrA mRNA expression was also seen in CNRZ32 cells after exposure to puromycin, ethanol, or heat. The reporter gene gusA was integrated in the Lactobacillus chromosome downstream of the htrA promoter by a double-crossover event which also interrupted the wild-type gene. The expression of gusA in the stress conditions tested was similar to that of htrA itself. In addition, the presence of an intact htrA gene facilitated growth under heat stress but not under salt stress.
KeywordMeSH Terms
Genes, Bacterial
Heat-Shock Proteins
Periplasmic Proteins
58.     ( 1998 )

Genetic characterization and physiological role of endopeptidase O from Lactobacillus helveticus CNRZ32.

Applied and environmental microbiology 64 (9)
PMID : 9726890  :   PMC  :   PMC106740    
Abstract >>
A previously identified insert expressing an endopeptidase from a Lactobacillus helveticus CNRZ32 genomic library was characterized. Nucleotide sequence analysis revealed an open reading frame of 1,941 bp encoding a putative protein of 71.2 kDa which contained a zinc-protease motif. Protein homology searches revealed that this enzyme has 40% similarity with endopeptidase O (PepO) from Lactococcus lactis P8-2-47. Northern hybridization revealed that pepO is monocistronic and is expressed throughout the growth phase. CNRZ32 derivatives lacking PepO activity were constructed via gene replacement. Enzyme assays revealed that the PepO mutant had significantly reduced endopeptidase activity when compared to CNRZ32 with two of the three substrates examined. Growth studies indicated that PepO has no detectable effect on growth rate or acid production by Lactobacillus helveticus CNRZ32 in amino acid defined or skim milk medium.
KeywordMeSH Terms
Bacterial Proteins
59.     ( 1998 )

Conservation of the major cold shock protein in lactic acid bacteria.

Current microbiology 37 (5)
PMID : 9767713  :  
Abstract >>
Primers designed from consensus regions of the major cold shock gene of different bacterial species were used in PCR amplification of Lactic Acid Bacteria (LAB). An appropriately-sized PCR product was obtained from Lactococcus lactis subsp. lactis LL43-1 and MG1363; Lactococcus lactis subsp. cremoris LC10-1, LC11-1, and LC12-1; Streptococcus thermophilus ST1-1; Enterococcus faecalis EF1-1; Lactobacillus acidophilus LA1-1; Lactobacillus helveticus LH1-1; Pediococcus pentosaceus PP1-1; and Bifidobacterium animalis BA1-1. The PCR products were cloned and sequenced. The deduced amino acid sequences displayed high sequence similarity with the major cold shock proteins of Escherichia coli and Bacillus subtilis and the human Y-box factor. The amino acid residues of the cold shock domain implicated in nucleic acid binding in several unrelated species were also highly conserved in the LAB strains. It is possible, therefore, that this protein in LAB may also act as a transcriptional enhancer to other cold shock genes and/or act as an RNA chaperone unwinding tightly folded RNA molecules.
KeywordMeSH Terms
Cold Temperature

331, Shih-Pin Rd., Hsinchu 30062, Taiwan

Phone: +886-3-5223191

E-mail: bcrcweb@firdi.org.tw

web maintainance: +886-3-5223191 ext 593

Copyright © 2018.BCRC All rights reserved.The duplication or use of information and data such as texts or images or any linkage the website at the "bcrc.firdi.org.tw" is only permitted with the indication of the source or with prior approval by the BCRC(Bioresource Collection and Research Center).