Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Comparison of partial tuf gene sequences for the identification of lactobacilli

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.     

Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.     

Rapid PCR-based procedure to identify lactic acid bacteria: application to six common Lactobacillus species

The goal of this study was to develop a method allowing rapid identification of the lactic acid bacteria strains in use in the laboratory (Lactobacillus plantarum NCIMB8826; L. fermentum KLD; L. reuteri 100-23; L. salivarius UCC43321; L. paracasei LbTGS1.4; L. casei ATCC393), based on PCR amplification of 16S RNA coding sequences. First, specific forward oligonucleotides were designed in the variable regions of 16S RNA coding sequences of six Lactobacillus strains. The reverse oligonucleotide was designed in the region where the sequences were homologous for the six strains. The expected size of the amplification product was +/-1000 bp. The specificity of the method was tested on total chromosomal DNA. For five out of the six strains, the amplification of the fragment was strain-specific and the method was directly applicable to colonies. For the strain L. casei ATCC393, an additional argument to the classification of this bacteria in the paracasei group could be proposed. Validation of the developed method was performed by applying it to six Lactobacillus reference strains and to various species of bacteria.     

Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination

Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137(T) (= ATCC 393(T)). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (pi ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.     

Ribotyping ofLactobacillus casei group strains isolated from dairy products

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of L. casei and L. paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (= ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.     

Bacteriophages of lactobacilli

Lactobacilli are members of the bacterial flora of lactic starter cultures used to generate lactic acid fermentation in a number of animal or plant products used as human or animals foods. They can be affected by phage outbreaks, which can result in faulty and depreciated products. Two groups of phages specific of Lactobacillus casei have been thoroughly studied. 1. The first group is represented by phage PL-1. This phage behaves as lytic in its usual host L. casei ATCC 27092, but can lysogenize another strain, L. casei ATCC 334. Bacterial receptors of this phage are located in a cell-wall polysaccharide and rhamnose is the main component of the receptors. Ca2+ and adenosine triphosphate (ATP) are indispensable to ensure the injection of the phage DNA into the bacterial cell. The phage DNA is double-stranded, mostly linear, but with cohesive ends which enables it to be circularized. The vegetative growth of PL-1 proceeds according to the classical mode. Cell lysis is produced by an N-acetyl-muramidase at the end of vegetative growth. 2. The second group is represented by the temperate phage phi FSW of L. casei ATCC27139. It has been shown how virulent phages originate from this temperate phage in Japanese dairy plants. The lysogenic state of phi FSW can be altered either by point mutations or by the insertion of a mobile genetic element called ISL 1, which comes from the bacterial chromosome. This is the first transposable element that has been described in lactobacilli. Lysogeny appears to be widespread among lactobacilli since one study showed that 27% of 148 strains studied, representing 15 species, produced phage particles after induction by mitomycin C. Similarly, 23 out of 30 strains of Lactobacillus salivarius are lysogenic and produce, after induction by mitomycin C, temperate phages, killer particles, or defective phages. Temperate phages have also been found in 10 out of 105 strains of Lactobacillus bulgaricus or Lactobacillus lactis after induction by mitomycin C. Phages so far studied of the latter 2 and closely related lactobacilli, either temperate or isolated as lytic, may be divided into 4 unrelated groups called a, b, c and d. Most of these phages are found in group a and an unquestionable relationship has already been shown between lytic phages and temperate phages that belong to this group. Lytic phage LL-H of L. lactis LL 23, isolated in Finland, is one of the most representative of those of group a and has been extensively studied on the molecular level.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

干酪乳杆菌的近缘种及亚种部分看家基因的系统发育分析

【背景】16S rRNA基因序列分析已广泛应用于细菌的分类鉴定,但是存在一定局限性,而使用看家基因作为分子标记在近缘种及亚种间的系统发育分析中具有其独特的优势。【目的】研究16S rRNA、uvr C (核酸外切酶ABC,C亚基)和mur E (UDP-N-乙酰胞壁酰三肽合酶)基因序列对干酪乳杆菌的近缘种及亚种的区分能力。【方法】采用分离自传统发酵乳中的6株干酪乳杆菌为研究对象,选取uvr C和mur E基因片段,通过PCR扩增、测序,结合已公布的干酪乳杆菌的近缘种或亚种的相应序列计算遗传距离、构建系统发育树,并与16S rRNA基因序列分析技术进行比较。【结果】研究发现Lactobacilluscasei及相近种间的uvr C、mur E和联合基因(uvr C-mur E)构建的系统发育树拓扑结构与16S rRNA基因结果基本一致,区别在于相似性的不同,其分别为79.00%-99.16%、89.08%-99.20%、76.56%-99.69%和99.58%-100%。基于16S rRNA基因不能区分干酪乳杆菌的近缘种及亚种,而看家基因uvr C和mur E基因序列能够很好地区分干酪乳杆菌的近缘种及亚种,并且将uvr C和mur E基因串联使用后,试验菌株与参考菌株的分类关系更加清晰。【结论】联合基因(uvr C-mur E)可作为16SrRNA基因的辅助工具用于干酪乳杆菌的近缘种及亚种的快速准确鉴定。     

DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.     

Consensus-degenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS(-)) and EPS-producing (EPS(+)) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS(+) bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS(+) strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei.

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Genomic diversity of cultivable Lactobacillus populations residing in the neonatal and adult gastrointestinal tract

The objective of this study was to investigate the cultivable Lactobacillus population in adult and infant faecal material to identify strains shared across a number of individuals. A range of lactobacilli isolated on Lactobacillus-selective agar from faeces of 16 infants and 11 adults were genetically fingerprinted and further characterized by 16S rRNA gene sequencing. The relatedness of all the Lactobacillus strains isolated to known species was also determined both genetically and phenotypically. This study revealed that the human intestine is initially colonized by only a few (1-2) different cultivable strains whereas in adults the pattern becomes more complex with a higher diversity of strains. The adult samples contained three genetically distinct Lactobacillus strains in some cases, while infant samples generally harboured only one dominant Lactobacillus strain. Moreover, the species in general appeared to differ with Lactobacillus rhamnosus and Lactobacillus casei/paracasei found mainly in adults, whereas Lactobacillus gasseri and Lactobacillus salivarius were more commonly isolated in infant samples. The data reaffirm the differences in Lactobacillus populations both between individual subjects and between the infant and adult, with an overall change in the diversity and complexity from early stages of life to adulthood.     

Distribution of megaplasmids in Lactobacillus salivarius and other lactobacilli

The genome of Lactobacillus salivarius UCC118 includes a 242-kb megaplasmid, pMP118. We now show that 33 strains of L. salivarius isolated from humans and animals all harbor a megaplasmid, which hybridized with the repA and repE replication origin probes of pMP118. Linear megaplasmids that did not hybridize with the pMP118 repA probe were also found in some strains of L. salivarius, showing for the first time that a lactic acid bacterium has multiple megaplasmids. Phylogenetic analysis of the repE and groEL sequences of 28 L. salivarius strains suggested similar evolutionary paths for the chromosome and megaplasmid. Although the replication origin of circular megaplasmids in L. salivarius was highly conserved, genotypic and phenotypic comparisons revealed significant variation between megaplasmid-encoded traits. Furthermore, megaplasmids of sizes ranging from 120 kb to 490 kb were present in seven strains belonging to six other Lactobacillus species from among 91 strains and 47 species tested. The discovery of the widespread presence of megaplasmids in L. salivarius, and restricted carriage by other Lactobacillus species, provides an opportunity to study the contribution of large extrachromosomal replicons to the biology of Lactobacillus.     

Redox potential to discriminate among species of lactic acid bacteria

AIMS: To verify to what degree reducing capacity is a characterizing parameter of a species, and of the strains themselves within a given species, of lactic acid bacteria. METHODS AND RESULTS: Eighty-eight strains belonging to 10 species of lactic acid bacteria (LAB) isolated from traditional Italian cheeses were studied for their reduction activity: Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Streptococcus thermophilus, Lactococcus lactis ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus and Pediococcus pentosaceus. It was observed that the lactococci reached minimum redox potential before the lactobacilli. The reduction rate of Enterococcus spp. and L. lactis ssp. lactis was higher than that of the streptococci and Lactobacillus spp. All the P. pentosaceus strains had poor reduction activity compared with the other species. CONCLUSIONS: The evolution of the redox potential in milk over a time span of 24 h has been found to be a parameter that characterizes a species: the different courses corresponding to the species in question are clearly evident, and interesting differences can also be noted within the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: The reduction aptitude of strains might be used to select and adapt appropriate strains for use as starters for dairy products.     

Penicillin-binding proteins of various strains of Lactobacillus

Sensitivity of different species of Lactobacillus i.e. L. casei, L. plantarum, L. acidophillus, L. buchneri, L. jugurti and others to penicillins and cephalosporins of various generations was studied. Penicillin binding proteins (PBPs) of the Lactobacillus species were specified. It was shown that the number of PBPs depended on the Lactobacillus species. L. casei had the least number of PBPs (4) and L. brevis had the highest number of PBPs (11). Competition of 14C-benzylpenicillin with ampicillin, cefotaxime, ceftizoxime and cefoperazone for binding to separate PBPs in three strains of different Lactobacillus species was investigated.     

Identification and characterization of a strain-dependent cystathionine β/γ-lyase in Lactobacillus casei potentially involved in cysteine biosynthesis

The trans -sulfuration pathways allow the interconversion of cysteine and methionine with the intermediary formation of cystathionine and homocysteine. The genome database of Lactobacillus casei ATCC 334 provides evidence that this species cannot synthesize cysteine from methionine via the trans -sulfuration pathway. However, several L. casei strains use methionine as the sole sulfur source, which implies that these strains can convert methionine to cysteine. Cystathionine synthases and lyases play a crucial role in the trans -sulfuration pathway. By applying proteomic techniques, we have identified a protein in cell-free extracts of L. casei , which showed high homology to a gene product encoded in the genome of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactobacillus helveticus but not in the genome of L. casei ATCC 334. The presence of the gene was only found in strains able to grow on methionine as the sole sulfur source. Moreover, two gene variants were identified. Both gene variants were cloned and expressed heterologously in Escherichia coli . The recombinant enzymes exhibited cystathionine lyase activity in vitro and also cleaved cysteine, homocysteine and methionine releasing volatile sulfur compounds.     

Growth Stimulation of Lactobacillus Species by Lactic Streptococci

Cell extracts of Streptococcus species important in cheese starters stimulated the growth of Lactobacillus species common to Cheddar cheese. All Lactobacillus strains employed, with the exception of a strain of L. casei, were significantly stimulated by a strain of S. diacetilactis. L. casei was highly stimulated by both a strain of S. lactis and a strain of S. diacetilactis. The stimulant(s) was dialyzable and was partially inactivated by heat. The stimulatory principle was active at 10 C, indicating that the stimulatory effect may be influencing the growth of lactobacilli in Cheddar cheese during curing. Viable Streptococcus cells did not inhibit the growth of Lactobacillus species.     

Antibacterial polypeptides of Lactobacillus species

Twelve of 79 strains of the genus Lactobacillus , mainly isolated from plants or fermenting material, were found to inhibit at least one of the nine indicator strains of the species Lact. brevis, Pediococcus damnosus and Leucanostoc oenos . The antimicrobial activities from Lact. brevis B 37 and Lact. casei B 80 were caused by polypeptides detectable in the culture liquids. They are bacteriocins with a narrow antimicrobial spectrum. Brevicin 37 from Lact. brevis B 37 was active against many lactic acid bacteria and Nocardia corallina , whereas caseicin 80 from Lact. casei B 80 inhibits only one other strain of Lact. casei . Brevicin 37 is stable at 121C, caseicin 80 is inactivated above 60C, and both are inactivated under alkaline conditions.