( 1992 )
Purification and some properties of acidocin 8912, a novel bacteriocin produced by Lactobacillus acidophilus TK8912.
PMID : 1368836 :
Acidocin 8912, a bacteriocin produced by Lactobacillus acidophilus TK8912, was purified by ammonium sulfate fractionation and successive chromatographies on CM-cellulose, Sephadex G-50, Sephadex G-25, and reversed-phase HPLC on Aquapore RP-300. The purified acidocin 8912 migrated as a single band on SDS-PAGE. The molecular weight was estimated to be 5200 by SDS-PAGE, and 5400 by HPLC gel filtration on TSKgel G3000PWXL. Both the amino acid composition and the N-terminal amino acid sequence analysis indicated that acidocin 8912 was a peptide composed of presumably 50 amino acids containing a Lys residue at the N-terminus. The purified acidocin 8912 showed a bactericidal effect on sensitive cells but not a bacteriolytic effect.
( 2003 )
Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus.
PMID : 12847288 : DOI : 10.1073/pnas.1332765100 PMC : PMC166420
Lactobacillus acidophilus is a probiotic organism that displays the ability to use prebiotic compounds such as fructooligosaccharides (FOS), which stimulate the growth of beneficial commensals in the gastrointestinal tract. However, little is known about the mechanisms and genes involved in FOS utilization by Lactobacillus species. Analysis of the L. acidophilus NCFM genome revealed an msm locus composed of a transcriptional regulator of the LacI family, a four-component ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose phosphorylase. Transcriptional analysis of this operon demonstrated that gene expression was induced by sucrose and FOS but not by glucose or fructose, suggesting some specificity for nonreadily fermentable sugars. Additionally, expression was repressed by glucose but not by fructose, suggesting catabolite repression via two cre-like sequences identified in the promoter-operator region. Insertional inactivation of the genes encoding the ABC transporter substrate-binding protein and the fructosidase reduced the ability of the mutants to grow on FOS. Comparative analysis of gene architecture within this cluster revealed a high degree of synteny with operons in Streptococcus mutans and Streptococcus pneumoniae. However, the association between a fructosidase and an ABC transporter is unusual and may be specific to L. acidophilus. This is a description of a previously undescribed gene locus involved in transport and catabolism of FOS compounds, which can promote competition of beneficial microorganisms in the human gastrointestinal tract.
( 1999 )
Identification of the pH-inducible, proton-translocating F1F0-ATPase (atpBEFHAGDC) operon of Lactobacillus acidophilus by differential display: gene structure, cloning and characterization.
PMID : 10510230 : DOI : 10.1046/j.1365-2958.1999.01557.x
The influence of low pH on inducible gene expression in Lactobacillus acidophilus was investigated by the use of differential display. Logarithmic phase cultures were exposed to pH 3.5 for various intervals, and RNA was isolated and reverse transcribed. The resultant cDNAs were subjected to PCR and the products were resolved by electrophoresis. Several cDNA products were induced after exposure to pH 3.5. One of these products, a 0.7 kb fragment, showed sequence similarity to bacterial atpBEF genes of the atp operon, whose genes encode the various subunits of the F1F0-ATPase. With the 0.7 kb differential display product as a probe, hybridizations with total RNA from untreated and acid-treated L. acidophilus verified the acid inducibility of this operon. The increase in atp mRNA induced by low pH was accompanied by an increase in the activity of the enzyme in membrane extracts. The full-length atp operon was sequenced, and its genes were in the order of atpBEFHAGDC, coding for the a, c, b, delta, alpha, gamma, beta and epsilon subunits respectively. The operon contained no i gene, but was preceded by a 122 bp intergenic space, which contained putative extended -10 and -35 promoter regions. Primer extension analysis of RNA from cultures that were shifted from pH 5.6 to pH 3. 5, and held for 0, 30 or 45 min, revealed that the transcriptional start site did not change position as a function of culture pH or time after exposure to pH 3.5. The primary structure and genetic organization indicated that the H+-ATPase of L. acidophilus is a typical F1F0-type ATPase. The similarity to streptococcal ATPases and the acid inducibility of this operon suggest that it may function in the ATP-dependent extrusion of protons and maintenance of cytoplasmic pH. Finally, the use of differential display RT-PCR was an effective approach to identify genes in L. acidophilus induced by an environmental stimulus.
( 2007 )
Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22.
PMID : 17227458 : DOI : 10.1111/j.1574-6968.2006.00614.x
Beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04+/-0.26 mM, 28.8+/-0.2 micromol D-glucose released per min per mg protein, and 0.73+/-0.07 mM, 361+/-12 micromol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7+/-3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. Beta-galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.
( 2005 )
Extracellular homopolysaccharides and oligosaccharides from intestinal lactobacilli.
PMID : 16108811 : DOI : 10.1111/j.1365-2672.2005.02638.x
To characterize lactobacilli isolated from the intestines of ducks or pigs with respect to the production of extracellular homopolysaccharides (HoPS) and oligosaccharides. Lactobacillus strains of duck or pig origin were screened for HoPS synthesis and >25% of the isolates produced fructans or glucans from sucrose. Glucan-forming strains were found within the species Lactobacillus reuteri and Lactobacillus animalis and fructan-forming strains were found within Lactobacillus mucosae, Lactobacillus crispatus and Lactobacillus acidophilus. The glucan-forming strains of L. reuteri but not L. animalis produced glucose-oligosaccharides in additon to the respective polymers, and two fructan-forming strains of L. acidophilus produced kestose. Genes coding for glycosyltransferases were detected by PCR and partially characterized by sequence analysis. A large proportion of lactobacilli from intestinal habitats produce HoPS from sucrose and polysaccharide formation is generally associated with the formation of glucose- and fructose oligosaccharides. The characterization of the metabolic potential of intestinal lactobacilli contributes to the understanding of the molecular basis of autochthony in intestinal habitats. Moreover, this is the first report of glucose-oligosaccharide production during growth of lactobacilli, and one novel fructosyltransferase and one novel glucansucrase were partially characterized on the genetic level.
( 2004 )
Identification and phenotypic characterization of the cell-division protein CdpA.
PMID : 15527978 : DOI : 10.1016/j.gene.2004.08.004
Analysis of the automated computer annotation of the early draft phase genome of Lactobacillus acidophilus NCFM revealed the previously discovered S-layer gene slpA and an additional partial ORF with weak similarities to S-layer proteins. The entire gene was sequenced to reveal a 1799-bp gene coding for 599 amino acids with a calculated molecular mass of 64.8 kDa. No transcription or translation signals could be determined in close proximity to the 5'-region. However, a strong putative terminator with a free energy of -16.84 kcal/mol was identified directly downstream of the gene. A PSI-Blast analysis showed similarities to members of S-layer proteins, cell-wall associated proteinases and hexosyl-transferases. Calculation of an unrooted phylogenetic tree with other examples of S-layer proteins and proteinases placed the deduced protein separately from both groups. A derivative of L. acidophilus NCFM was constructed by targeted integration into the gene. SDS-PAGE analysis of non-covalently linked proteins of the cell wall of the mutant, compared to the wild type, revealed the loss of a cell-surface protein. Phenotypic analyses of the mutant revealed significant changes in cell morphology, altered responses to various environmental stresses, and lowered cell adhesion. Based on the in silico and functional analyses, we ascertained that this protein plays a role in cell-wall processing during the growth and cell-cell separation and designated the gene as cell-division protein, cdpA.
( 2004 )
Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance.
PMID : 15345415 : DOI : 10.1128/AEM.70.9.5315-5322.2004 PMC : PMC520879
Amino acid decarboxylation-antiporter reactions are one of the most important systems for maintaining intracellular pH between physiological limits under acid stress. We analyzed the Lactobacillus acidophilus NCFM complete genome sequence and selected four open reading frames with similarities to genes involved with decarboxylation reactions involved in acid tolerance in several microorganisms. Putative genes encoding an ornithine decarboxylase, an amino acid permease, a glutamate gamma-aminobutyrate antiporter, and a transcriptional regulator were disrupted by insertional inactivation. The ability of L. acidophilus to survive low-pH conditions, such as those encountered in the stomach or fermented dairy foods, was investigated and compared to the abilities of early- and late-stationary-phase cells of the mutants by challenging them with a variety of acidic conditions. All of the integrants were more sensitive to low pH than the parental strain. Interestingly, each integrant also exhibited an adaptive acid response during logarithmic growth, indicating that multiple mechanisms are present and orchestrated in L. acidophilus in response to acid challenge.
van Sinderen D,
( 2004 )
Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny.
PMID : 15128574 : DOI : 10.1128/aem.70.5.3110-3121.2004 PMC : PMC404453
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.
( 2012 )
Lactobacillus gigeriorum sp. nov., isolated from chicken crop.
PMID : 21421927 : DOI : 10.1099/ijs.0.028217-0
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)).
( 2010 )
Characterization of a novel Lactobacillus species closely related to Lactobacillus johnsonii using a combination of molecular and comparative genomics methods.
PMID : 20849602 : DOI : 10.1186/1471-2164-11-504 PMC : PMC2997000
Comparative genomic hybridization (CGH) constitutes a powerful tool for identification and characterization of bacterial strains. In this study we have applied this technique for the characterization of a number of Lactobacillus strains isolated from the intestinal content of rats fed with a diet supplemented with sorbitol. Phylogenetic analysis based on 16S rRNA gene, recA, pheS, pyrG and tuf sequences identified five bacterial strains isolated from the intestinal content of rats as belonging to the recently described Lactobacillus taiwanensis species. DNA-DNA hybridization experiments confirmed that these five strains are distinct but closely related to Lactobacillus johnsonii and Lactobacillus gasseri. A whole genome DNA microarray designed for the probiotic L. johnsonii strain NCC533 was used for CGH analysis of L. johnsonii ATCC 33200T, L. johnsonii BL261, L. gasseri ATCC 33323T and L. taiwanensis BL263. In these experiments, the fluorescence ratio distributions obtained with L. taiwanensis and L. gasseri showed characteristic inter-species profiles. The percentage of conserved L. johnsonii NCC533 genes was about 83% in the L. johnsonii strains comparisons and decreased to 51% and 47% for L. taiwanensis and L. gasseri, respectively. These results confirmed the separate status of L. taiwanensis from L. johnsonii at the level of species, and also that L. taiwanensis is closer to L. johnsonii than L. gasseri is to L. johnsonii. Conventional taxonomic analyses and microarray-based CGH analysis have been used for the identification and characterization of the newly species L. taiwanensis. The microarray-based CGH technology has been shown as a remarkable tool for the identification and fine discrimination between phylogenetically close species, and additionally provided insight into the adaptation of the strain L. taiwanensis BL263 to its ecological niche.
( 2010 )
Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus.
PMID : 20461509 : DOI : 10.1007/s00253-010-2634-z
While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from alpha-linoleic acid by the enzyme linoleate isomerase (LI, EC 18.104.22.168) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers.
( 2010 )
Heterologous expression of glycoside hydrolase family 2 and 42 �]-galactosidases of lactic acid bacteria in Lactococcus lactis.
PMID : 20822875 : DOI : 10.1016/j.syapm.2010.07.002
This study characterized a glycoside hydrolase family 42 (GH42) �]-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 �]-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 �]-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 �]-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 �]-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 �]-galactosidases formed structurally similar GOS with varying preferences. The diversity of lactic acid bacteria �]-galactosidase activity in L. lactis CCE can be exploited in future nutritional or therapeutic applications.
( 2007 )
Identification of lactobacilli by pheS and rpoA gene sequence analyses.
PMID : 18048724 : DOI : 10.1099/ijs.0.64711-0
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.
( 2007 )
Oxalate consumption by lactobacilli: evaluation of oxalyl-CoA decarboxylase and formyl-CoA transferase activity in Lactobacillus acidophilus.
PMID : 17953571 : DOI : 10.1111/j.1365-2672.2007.03388.x
This study was undertaken to evaluate the oxalate-degrading activity in several Lactobacillus species widely used in probiotic dairy and pharmaceutical preparations. Functional characterization of oxalyl-CoA decarboxylase and formyl-CoA transferase in Lactobacillus acidophilus was performed in order to assess the possible contribution of Lactobacillus in regulating the intestinal oxalate homeostasis. In order to determine the oxalate-degrading ability in 60 Lactobacillus strains belonging to 12 species, a screening was carried out by using an enzymatic assay. A high variability in the oxalate-degrading capacity was found in the different species. Strains of Lact. acidophilus and Lactobacillus gasseri showed the highest oxalate-degrading activity. Oxalyl-CoA decarboxylase and formyl-CoA transferase genes from Lact. acidophilus LA14 were cloned and sequenced. The activity of the recombinant enzymes was assessed by capillary electrophoresis. Strains of Lactobacillus with a high oxalate-degrading activity were identified. The function and significance of Lact. acidophilus LA14 oxalyl-CoA decarboxylase and formyl-CoA transferase in oxalate catabolism were demonstrated. These results suggest the potential use of Lactobacillus strains for the degradation of oxalate in the human gut. Identification of probiotic strains with oxalate-degrading activity can offer the opportunity to provide this capacity to individuals suffering from an increased body burden of oxalate and oxalate-associated disorders.
( 2008 )
Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation.
PMID : 17993558 : DOI : 10.1128/AEM.01711-07 PMC : PMC2223197
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.
van Belkum MJ,
( 2015 )
Solution structure of acidocin B, a circular bacteriocin produced by Lactobacillus acidophilus M46.
PMID : 25681186 : DOI : 10.1128/AEM.04265-14 PMC : PMC4375312
Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four �\-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ?7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four �\-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.
( 2015 )
A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus.
PMID : 25966711 : DOI : 10.1194/jlr.M059444 PMC : PMC4479338
Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the �G12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.
( 2013 )
Strain-specific detection of orally administered canine jejunum-dominated Lactobacillus acidophilus LAB20 in dog faeces by real-time PCR targeted to the novel surface layer protein.
PMID : 23758090 : DOI : 10.1111/lam.12117
Lactobacillus acidophilus LAB20 has potential to be a probiotic strain because it can be present at high numbers in the jejunum of dog. To specifically detect LAB20 from dog faecal samples, a real-time PCR protocol was developed targeting the novel surface (S) layer protein gene of LAB20. The presence of S-layer protein was verified by N-terminal sequencing of the approximately 50-kDa major band from SDS-PAGE gel. The corresponding S-layer gene was amplified by inverse PCR using homology to known S-layers and sequenced. This novel S-layer protein has low sequence similarity to other S-layer proteins in the N-terminal region (32-211 aa, 7-39%). This enabled designing strain-specific PCR primers. The primer set was utilized to study intestinal persistence of LAB20 in dog that was fed with LAB20 fermented milk for 5 days. The results showed that LAB20 can be detected from dog faecal sample after 6 weeks with 10(4�P53) DNA copies g(-1) postadministration. It suggested that LAB20 could be a good candidate to study the mechanism behind its persistence and dominance in dog intestine and maybe utilize it as a probiotic for canine. A real-time PCR method was developed to detect Lactobacillus acidophilus LAB20, a strain that was previously found dominant in canine gastrointestinal (GI) tract. The quantitative detection was based on targeting to variation region of a novel S-layer protein found in LAB20, allowing to specifically enumerate LAB20 from dog faeces. The results showed that the real-time PCR method was sensitive enough to be used in later intervention studies. Interestingly, LAB20 was found to persist in dog GI tract for 6 weeks. Therefore, LAB20 could be a good candidate to study its colonization and potentially utilize as a canine probiotic.
( 2013 )
Development of a tiered multilocus sequence typing scheme for members of the Lactobacillus acidophilus complex.
PMID : 24038697 : DOI : 10.1128/AEM.02257-13 PMC : PMC3837765
Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.
( 2012 )
Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene.
PMID : 22555934 : DOI : 10.1002/jsfa.5692
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.
( 1997 )
Identification of the replication region of the Lactobacillus acidophilus plasmid pLA106.
PMID : 9084151 : DOI : 10.1111/j.1574-6968.1997.tb10292.x
The complete nucleotide sequence of plasmid pLA106 (2862 bp) from Lactobacillus acidophilus TK8912 was determined. Based on this sequence, the location of the genes for mobilization-plasmid recombination protein (mob), replication origin (ori), transcriptional repressor protein (repA) and replication initiation protein (repB) were predicted. Deletion analysis showed that the 1.4-kb PstI-EcoRV fragment carrying the ori, repA and repB genes is able to replicate in Lactobacillus and Escherichia coli cells. The plasmid pLA106 appears to have most features of the pLS1/pE194 plasmid family.
( 1996 )
Isolation, partial characterization, and mode of action of Acidocin J1132, a two-component bacteriocin produced by Lactobacillus acidophilus JCM 1132.
PMID : 8975617 : PMC : PMC167854
Lactobacillus acidophilus JCM 1132 produces a heat-stable, two-component bacteriocin designated acidocin J1132 that has a narrow inhibitory spectrum. Maximum production of acidocin J1132 in MRS broth was detected at pH 5.0. Acidocin J1132 was purified by ammonium sulfate precipitation and sequential cation exchange and reversed-phase chromatographies. Acidocin J1132 activity was associated with two components, termed alpha and beta. On the basis of N-terminal amino acid sequencing and the molecular masses of the alpha and beta components, it is interpreted that the compounds differ by an additional glycine residue in the beta component. Both alpha and beta had inhibitory activity, and an increase in activity by the complementary action of the two components was observed. Acidocin J1132 is bactericidal and dissipates the membrane potential and the pH gradient in sensitive cells, which affect such proton motive force-dependent processes as amino acid transport. Acidocin J1132 also caused efflux of preaccumulated amino acid taken up via a unidirectional ATP-driven transport system. Secondary structure prediction revealed the presence of an amphiphilic alpha-helix region that could form hydrophilic pores. These results suggest that acidocin J1132 is a pore-forming bacteriocin that creates cell membrane channels through the "barrel-stave" mechanism.
( 1995 )
Identification, cloning, and nucleotide sequence of a silent S-layer protein gene of Lactobacillus acidophilus ATCC 4356 which has extensive similarity with the S-layer protein gene of this species.
PMID : 8522531 : DOI : 10.1128/jb.177.24.7222-7230.1995 PMC : PMC177603
The bacterial S-layer forms a regular structure, composed of a monolayer of one (glyco)protein, on the surfaces of many prokaryotic species. S-layers are reported to fulfil different functions, such as attachment structures for extracellular enzymes and major virulence determinants for pathogenic species. Lactobacillus acidophilus ATCC 4356, which originates from the human pharynx, possesses such an S-layer. No function has yet been assigned to the S-layer of this species. Besides the structural gene (slpA) for the S-layer protein (S-protein) which constitutes this S-layer, we have identified a silent gene (slpB), which is almost identical to slpA in two regions. From the deduced amino acid sequence, it appears that the mature SB-protein (44,884 Da) is 53% similar to the SA-protein (43,636 Da) in the N-terminal and middle parts of the proteins. The C-terminal parts of the two proteins are identical except for one amino acid residue. The physical properties of the deduced S-proteins are virtually the same. Northern (RNA) blot analysis shows that only the slpA gene is expressed in wild-type cells, in line with the results from sequencing and primer extension analyses, which reveal that only the slpA gene harbors a promoter, which is located immediately upstream of the region where the two genes are identical. The occurrence of in vivo chromosomal recombination between the two S-protein-encoding genes will be described elsewhere.
( 1995 )
Identification of the replication region of Lactobacillus acidophilus plasmid pLA103.
PMID : 8566697 : DOI : 10.1111/j.1574-6968.1995.tb07872.x
The structure of the region necessary for replication of the plasmid pLA103 from Lactobacillus acidophilus TK8912 has been characterized. Sequence analysis revealed that the replication region contained an open reading frame (OrfA) encoding a 282-amino acid peptide preceded by a 22-bp tandem repeat sequence region. The predicted OrfA protein showed homology to the replication protein of a plasmid from Pediococcus halophilus. The plasmid containing the repeat sequence region preceding OrfA was able to replicate in the Lactobacillus host when provided with OrfA in trans, suggesting that the repeat sequence region contains the origin sequence essential for the pLA103 replication.
( 1993 )
Molecular analysis of the lactacin F operon.
PMID : 8285694 : PMC : PMC182548
Lactacin F is a nonlantibiotic, heat-stable, peptide bacteriocin produced by Lactobacillus johnsonii VPI11088. Molecular analysis of the lactacin F DNA region characterized a small operon that codes for three open reading frames, designated lafA, lafX, and ORFZ. The peptide encoded by lafA, the lactacin F structural gene, was compared with various peptide bacteriocins from lactic acid bacteria, and similarities were identified in the amino and carboxy termini of the propeptides. Site-directed mutagenesis of the LafA precursor at the two glycine residues in positions -1 and -2 defined an essential motif for processing of mature lactacin F. The involvement of the peptides encoded by lafX and ORFZ in bacteriocin expression was investigated by subcloning various fragments from the lactacin F region into the shuttle vector pGKV210. In addition to lafA, expression of lafX is essential to lactacin F activity. The lactacin F operon resembles the genetic organization of lactococcin M. Although no function has been assigned to ORFZ by genetic analysis, both peptide Z and the lactococcin M immunity protein are predicted to be integral membrane proteins with four putative transmembrane segments. Lactacin F activity, defined by bactericidal action on Lactobacillus delbrueckii, is dependent on the expression of two genes, lafA and lafX.
( 1995 )
Cloning and nucleotide sequence of the gene for acidocin 8912, a bacteriocin from Lactobacillus acidophilus TK8912.
PMID : 8554765 :
Acidocin 8912 is a bacteriocin produced by Lactobacillus acidophilus TK8912. The acidocin 8912 structural gene, acdT, was cloned and determined. It was located on the 14-kb plasmid pL103 and encoded a 46 amino acid precursor including a 20 amino acid N-terminal extension. The precursor sequence of the acdT gene shows a conservation of the general structural characteristics of the bacteriocin precursors from some lactic acid bacteria.
( 1994 )
Heterodimeric deoxynucleoside kinases of Lactobacillus acidophilus R-26: functional assignment of subunits using limited proteolysis controlled by end-product inhibitors.
PMID : 8172906 : DOI : 10.1021/bi00183a041
Heterodimeric quaternary structures for two enzyme complexes from Lactobacillus acidophilus R-26 exhibiting deoxycytidine kinase/deoxyadenosine kinase (I) and deoxyguanosine kinase/deoxyadenosine kinase(II) activities have been proven by the following steps: (1) separation of each complex into two components on SDS-PAGE at pH 6.6; (2) N-terminal amino acid sequencing of each component; (3) functional assignment of each component by differential limited proteolysis. The third step was facilitated by the finding that the binding of a specific end-product inhibitor dNTP, to each kinase active site makes the corresponding kinase subunit resistant to trypsin, while leaving the heterologous kinase subunit susceptible to proteolysis. Analysis on SDS-PAGE has revealed only two fragments (15.8 and 11.0 kDa) following proteolysis of dCyd kinase/dAdo kinase (I) with trypsin in the presence of dATP. This may indicate that the kinase polypeptide chain (27.2 kDa) not protected by dNTP is cut by trypsin at a single specific site, with concomitant loss of activity. Thus, this work presents a unique approach to the clarification of structure and function of enzymes composed of heterologous subunits.
( 1994 )
Identification and purification of a protein that induces production of the Lactobacillus acidophilus bacteriocin lactacin B.
PMID : 7986029 : PMC : PMC201849
Lactacin B is a heat-stable bacteriocin produced by Lactobacillus acidophilus N2 that is active against closely related lactobacilli, including Lactobacillus delbrueckii subsp. lactis (formerly Lactobacillus leichmannii) ATCC 4797. Pure producer cultures propagated in MRS broth (initial pH 6.5) contain no lactacin B; it is detected only in cultures maintained at pH 5.0 to 6.0 and produced optimally at pH 6.0 S. F. Barefoot and T. R. Klaenhammer, Antimicrob. Agents Chemother. 26:328-334, 1984). Associative growth of producer and indicator, L. delbrueckii subsp. lactis ATCC 4797, resulted in production of an inhibitor identical to lactacin B. Associative growth increased lactacin B production from nondetectable levels (< 100 activity units [AU]/ml) to between 3,200 and 6,400 AU/ml in MRS broth (initial pH 6.5) and resulted in early but equal production of lactacin B (approximately 25,600 AU/ml) in broth maintained at pH 6.0. Indicator cells, but not spent culture filtrates, induced lactacin B production. Indicator cells disrupted by a French pressure cell yielded cell-free filtrates containing inducing activity. Chromatofocusing and gel filtration high-performance liquid chromatography of cell-free filtrates yielded a protein with a pI of 4.1 and a molecular size of approximately 58 kDa that induced lactacin B production. Analytical isoelectric focusing yielded a single protein band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels contained a 28-kDa protein suggesting a two-subunit structure. Protein sequencing identified an N-terminal serine and 18 additional amino acids. To our knowledge, there are not previous descriptions of proteins that induce bacteriocin production in lactic acid bacteria.
( 1995 )
Isolation and characterization of acidocin A and cloning of the bacteriocin gene from Lactobacillus acidophilus.
PMID : 7793908 : PMC : PMC167361
Acidocin A, a bacteriocin produced by Lactobacillus acidophilus TK9201, is active against closely related lactic acid bacteria and food-borne pathogens including Listeria monocytogenes. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential ion-exchange and reversed-phase chromatographies. The molecular mass was determined by high-performance liquid chromatography gel filtration to be 6,500 Da. The sequence of the first 16 amino acids of the N terminus was determined, and oligonucleotide probes based on this sequence were constructed to detect the acidocin A structural gene acdA. The probes hybridized to the 4.5-kb EcoRI fragment of a 45-kb plasmid, pLA9201, present in L. acidophilus TK9201, and the hybridizing region was further localized to the 0.9-kb KpnI-XbaI fragment. Analysis of the nucleotide sequence of this fragment revealed that acidocin A was synthesized as an 81-amino-acid precursor including a 23-amino-acid N-terminal extension. An additional open reading frame (ORF2) encoding a 55-amino-acid polypeptide was found downstream of and in the same operon as acdA. Transformants containing this ORF2 became resistant to acidocin A, suggesting that ORF2 encodes an immunity function for acidocin A. The 7.2-kb SacI-XbaI fragment containing the upstream region of acdA of pLA9201 was necessary for acidocin A expression in the acidocin A-deficient mutant, L. acidophilus TK9201-1, and other Lactobacillus strains.
de Berardinis V,
( 2017 )
Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis.
PMID : 28581482 : DOI : 10.1038/nchembio.2397
Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.
( 1988 )
Amino-terminal nucleotide-binding sequences of a Lactobacillus deoxynucleoside kinase complex isolated by novel affinity chromatography.
PMID : 2851331 : DOI : 10.1021/bi00423a021
A highly efficient new affinity medium for deoxycytidine kinase, deoxycytidine 5'-tetraphosphate-Sepharose (dCp4-Sepharose), has been constructed. A dCp4-Sepharose column effects a one-step, 19,000-fold, purification to homogeneity of dCyd kinase from the ammonium sulfate fraction of Lactobacillus acidophilus R-26 extract, with 60% recovery. dCTP, a potent end-product inhibitor, is used as an eluent, and it also stabilizes the extremely labile purified enzyme. A noncompeting deoxyadenosine kinase activity accompanies the deoxycytidine kinase activity eluted. Native polyacrylamide gel electrophoresis shows a single protein band, which coincides with both deoxycytidine kinase and deoxyadenosine kinase activities at several gel concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide band of 26,000 daltons. Since the native enzyme is known to have an Mr of 50,000, it appears that the enzyme is composed of two subunits of similar size. Sequence analysis of the intact protein from the N-terminus reveals but a single amino acid species per residue up to the 17th residue; at the 18th, 21st, 26th, and 27th residue positions of the sequence, however, there appear to be two different amino acids in almost equal amounts. This may indicate that the enzyme is composed of two nonidentical subunits having the same amino acid sequence near the N-terminus. Residues 6-13 contain the highly conserved Gly-X-X-Gly-X-Gly-Lys sequence found at the active sites of kinases and other nucleotide-binding proteins.
( 2013 )
Improved membrane protein expression in Lactococcus lactis by fusion to Mistic.
PMID : 23519161 : DOI : 10.1099/mic.0.066621-0
Difficulty overexpressing (eukaryotic) membrane proteins is generally considered as the major impediment in their structural and functional research. Lactococcus lactis possesses many properties ideal for membrane protein expression. In order to investigate membrane protein expression in L. lactis, we created a novel expression system by introducing Mistic, a short peptide previously identified in Bacillus subtilis, into L. lactis. The potential of this system was demonstrated in the overexpression of a eukaryotic membrane protein (pkjDes4) and a prokaryotic membrane protein (pkjLi), a newly isolated linoleate isomerase from Lactobacillus acidophilus. The expression levels reached up to 4.4 % and 45.2 % for pkjDes4 and pkjLi, respectively, which represented an exceptionally robust ability to overproduce membrane proteins. Moreover, the expressed pkjLi was functional, with its catalysing nature characterized for the first time in this species. Up to 0.852 mg ml(-1) conjugated linoleic acid was obtained during the linoleic acid conversion catalysed by the recombinant lactococcal strains. In summary, we established a membrane protein expression system in L. lactis and examined its functionality. Our results demonstrate that the Mistic chaperoning strategy can be efficiently applied to L. lactis hosts and show its extraordinary capacity to facilitate the high-yield production of intractable membrane proteins.
( 1998 )
Conservation of the major cold shock protein in lactic acid bacteria.
PMID : 9767713 :
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.