An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by -1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family. In L. sanfranciscensis type strain DSM 20451T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.

Lactobacillus sanfranciscensis CB1, an important sourdough lactic acid bacterium, can withstand low pH after initial exposure to sublethal acidic conditions. The sensitivity to low pH varied according to the type of acid used. Treatment of LB: sanfranciscensis CB1 with chloramphenicol during acid adaptation almost completely eliminated the protective effect, suggesting that induction of protein synthesis was required for the acid-tolerance response. Two constitutively acid-tolerant mutants, CB1-5R and CB1-7R, were isolated using natural selection techniques after sequential exposure to lactic acid (pH 3.2). Two-dimensional gel electrophoresis analysis of protein expression by non-adapted, acid-adapted and acid-tolerant mutant cells of LB: sanfranciscensis showed changes in the levels of 63 proteins. While some of the modifications were common to the acid-adapted and acid-tolerant mutant cells, several differences, especially regarding the induced proteins, were determined. The two mutants showed a very similar level of protein expression. Antibodies were used to identify heat-shock proteins DnaJ, DnaK, GroES and GrpE. Only GrpE showed an increased level of expression in the acid-adapted and acid-tolerant mutants as compared with non-adapted cells. The N-terminal sequence was determined for two proteins, one induced in both the acid-adapted and mutant cells and the other showing the highest induction factor of those proteins specifically induced in the acid-adapted cells. This second protein has 60% identity with the N-terminal portion of YhaH, a transmembrane protein of Bacillus subtilis, which has 54 and 47% homology with stress proteins identified in Listeria monocytogenes and Bacillus halodurans. The constitutively acid-tolerant mutants showed other different phenotypic features compared to the parental strain: (i) the aminopeptidase activity of CB1-5R decreased and that of CB1-7R markedly increased, especially in acid conditions; (ii) the growth in culture medium at 10 degrees C and in the presence of 5% NaCl was greater (the same was found for acid-adapted cells); and (iii) the acidification rate during sourdough fermentation in acid conditions was faster and greater.

The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451(T) on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451(T)DeltagshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451(T)DeltagshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 microM to 10.5 microM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451(T)DeltagshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 microM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451(T)DeltagshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.

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.

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.

Protein hydrolysis and amino acid metabolism contribute to the beneficial effects of sourdough fermentation on bread quality. In this work, genes of Lactobacillus sanfranciscensis strain DSM 20451 involved in peptide uptake and hydrolysis were identified and their expression during growth in sourdough was determined. Screening of the L. sanfranciscensis genome with degenerate primers targeting prt and analysis of proteolytic activity in vitro provided no indication for proteolytic activity. Proteolysis in aseptic doughs and sourdoughs fermented with L. sanfranciscensis was inhibited upon the addition of an aspartic protease inhibitor. These results indicate that proteolysis was not linked to the presence of L. sanfranciscensis DSM 20451 and that this strain does not harbor a proteinase. Genes encoding the peptide transport systems Opp and DtpT and the intracellular peptidases PepT, PepR, PepC, PepN, and PepX were identified. Both peptide uptake systems and the genes pepN, pepX, pepC, and pepT were expressed by L. sanfranciscensis growing exponentially in sourdough, whereas pepX was not transcribed. The regulation of the expression of Opp, DtpT, and PepT during growth of L. sanfranciscensis in sourdough was investigated. Expression of Opp and DtpT was reduced approximately 17-fold when the peptide supply in dough was increased. The expression of PepT was dependent on the peptide supply to a lesser extent. Thus, the accumulation of amino nitrogen by L. sanfranciscensis in dough is attributable to peptide hydrolysis rather than proteolysis and amino acid metabolism by L. sanfranciscensis during growth in sourdough is limited by the peptide availability.

Exopolysaccharides (EPS) produced in situ by sourdough lactobacilli affect rheological properties of dough as well as bread quality and may serve as prebiotics. The aim of this study was to characterize EPS-formation by Lactobacillus sanfranciscensis TMW 1.392 at the molecular level. A levansucrase gene from L. sanfranciscensis TMW 1.392 encompassing 2,300 bp was sequenced. This levansucrase is predicted to be a cell-wall associated protein of 879 amino acids with a relative molecular weight (M(R)) of 90,000. The levansucrase gene was heterologously expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme exhibited transferase and hydrolase activities and produced glucose, fructose, 1-kestose and levan from sucrose; truncation of the N-terminal domain did not affect catalytic activity. Kestose formation was enhanced relative to fructose and levan formation by low temperature or high sucrose levels. During growth in wheat doughs, strain TMW 1.392 utilized sucrose to form fructose, 1-kestose, and fructan, whereas a levansucrase deletion mutant, L. sanfranciscensis TMW 1392Deltalev, lost the ability to hydrolyze sucrose, and did not produce fructan or 1-kestose. These results indicate that, in L. sanfranciscensis TMW 1.392, sucrose metabolism and formation of fructan and 1-kestose is dependent on the activity of a single enzyme, levansucrase.

A reporter system was developed to study gene expression in Lactobacillus sanfranciscensis. It was based on the Escherichia coli/Lactobacillus shuttle vector pLP3537 and the melA gene encoding alpha-galactosidase originating from Lactobacillus plantarum. melA was functionally expressed in E. coli and L. sanfranciscensis, and activity was easily monitored in vivo as well as in vitro by applying an optimized enzyme assay. The reporter system was validated by demonstrating the induction of the dnaK operon of L. sanfranciscensis by NaCl stress. The complete operon, which was composed of hrcA, grpE, dnaK, and dnaJ, was sequenced. A 299-bp sequence upstream of this operon, including a putative sigmaA-type promoter and a single conserved Controlling Inverted Repeat of Chaperone Expression element, was amplified. This amplicon was cloned directly upstream of melA. Both reporter enzyme activity and Northern hybridization analyses of dnaK and melA revealed a transcriptional induction, reaching its maximum when the culture was exposed to 0.75 M NaCl.

The effect of sublethal hydrostatic pressure on the transcriptome of Lactobacillus sanfranciscensis was determined using a shot-gun-microarray. Among the 750 spots that passed quality analysis 42 genes were induced, while six were repressed when cells were incubated at 45 MPa for 30 min. The nature of genes and their differential expression clearly indicate cellular efforts to counteract a decrease in translational capacity. The majority of high pressure affected genes were found to encode either translation factors (EF-G, EF-TU), ribosomal proteins (S2, L6, L11), genes changing translational accuracy or molecular chaperones (GroEL, ClpL). These data agree with previously reported effects observed in in vitro studies as well as with physiological and proteomic data. This study provides in vivo evidence to identify ribosomes and impaired translation among primary targets for high pressure treatment. The observed induction of heat as well as cold shock genes (e.g. hsp60, gyrA) may be explained as a result of high pressure affected protein synthesis.

Lactobacillus sanfranciscensis is the key bacterium in traditional sourdough fermentation. The molecular background of its oxygen tolerance was investigated by comparison of wild type and NADH-oxidase (Nox) knock out mutants. The nox gene of L. sanfranciscensis DSM20451(T) coding for a NADH-oxidase (Nox) was inactivated by single crossover integration to yield strain L. sanfranciscensis DSM20451�Gnox. By inactivation of the native NADH-oxidase gene, it was ensured that besides fructose, O(2) can react as an electron acceptor. In aerated cultures the mutant strain was only able to grow in MRS media supplemented with fructose as electron acceptor, whereas the wild type strain showed a fructose independent growth response. The use of oxygen as an external electron acceptor enables L. sanfranciscensis to shift from acetyl-phosphate into the acetate branch and gain an additionally ATP, while the reduced cofactors were regenerated by Nox-activity. In aerated cultures the wild type strain formed a fermentation ratio of lactate to acetate of 1.09 in MRS supplemented with fructose after 24 h of fermentation, while the mutant strain formed a fermentation ratio of 3.05. Additionally, L. sanfranciscensis showed manganese-dependent growth response in aerated cultures, the final OD and growth velocity was increased in media supplemented with manganese. The expression of two predicted Mn(2+)/Fe(2+) transporters MntH1 and MntH2 in L. sanfranciscensis DSM20451(T) was verified by amplification of a 318 bp fragment of MntH1 and a 239 bp fragment of MntH2 from cDNA library obtained from aerobically, exponentially growing cells of L. sanfranciscensis DSM20451(T) in MRS. Moreover, the mutant strain DSM20451�Gnox was more sensitive to the superoxide generating agent paraquat and showed inhibition of growth on diamide-treated MRS-plates without fructose supplementation.

Lactobacillus sanfranciscensis is a lactic acid bacterium that characterizes the sourdough environment. The genetic differences of 24 strains isolated in different years from sourdoughs, mostly collected in Italy, were examined and compared by PFGE and multilocus sequence typing (MLST). The MLST scheme, based on the analysis of six housekeeping genes (gdh, gyrA, mapA, nox, pgmA and pta) was developed for this study. PFGE with the restriction enzyme ApaI proved to have higher discriminatory power, since it revealed 22 different pulsotypes, while 19 sequence types were recognized through MLST analysis. Notably, restriction profiles generated from three isolates collected from the same firm but in three consecutive years clustered in a single pulsotype and showed the same sequence type, emphasizing the fact that the main factors affecting the dominance of a strain are correlated with processing conditions and the manufacturing environment rather than the geographical area. All results indicated a limited recombination among genes and the presence of a clonal population in L. sanfranciscensis. The MLST scheme proposed in this work can be considered a useful tool for characterization of isolates and for in-depth examination of the strain diversity and evolution of this species.

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.

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.

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.

Lactobacillus sanfranciscensis is the predominant lactic acid bacteria (LAB) species in Chinese traditional sourdoughs and conduces to the flavor and rheology properties of Chinese steamed bread, a staple food originated in China over 1500years ago. The aim of this study is to describe the intraspecific diversity of 98 L. sanfranciscensis isolates from 11 Chinese traditional sourdoughs in different regions by multilocus sequence typing (MLST) and multiplex random amplified polymorphic DNA-polymerase chain reaction (multiplex RAPD-PCR). MLST scheme was reduced from six gene fragments (gdh, gyrA, mapA, nox, pgmA and pta) to five (gdh, gyrA, mapA, nox and pta) since the fragment of pgmA displayed only one allele. 10 different sequence types (STs) were revealed by MLST and 6 of them containing 79.8% of the isolates were classified into one clonal complex, demonstrating a close relationship among them. The multiplex-RAPD analysis was performed by employing the combined primers OPL-05+RD1 and divided the 98 L. sanfranciscensis isolates into 6 types with the similarity level of 70%. According to the result, it seems that the genotypic variations of L. sanfranciscensis strains showed by MLST have no relations to geographical origin. MLST seems to have a higher discriminatory power compared with multiplex-RAPD since it produced more groups, but multiplex-RAPD could help to distinguish some strains in the same ST. Hence, an optimal genotypic characterization of L. sanfranciscensis was obtained under the combination of MLST and multiplex-RAPD analysis, targeting different genetic variations.