Multiple competitive PCR-DGGE as a tool for quantifying and profiling defined mixed cultures of lactic acid bacteria during production of probiotics from complex polysaccharides.

AIMS: To apply a denaturing gradient gel electrophoretic (DGGE) method to quantify and profile individual strains during a mixed culture fermentation. METHODS AND RESULTS: DNA was extracted during the culture of lactic acid bacteria (LAB) and amplified in a multiple competitive PCR (cPCR) using general primers targeting 16S rDNA and DNA from Streptococcus salivarius as competitive DNA. Subsequently the 200-kb amplified fragments were separated by DGGE. The method was validated in pure cultures and used to profile a mixture of three LAB grown on glucose, soluble starch and glycogen from mussel processing waste. The inclusion of a starch- and glycogen-degrading strain (Lactobacillus plantarum) and a weakly amylotic nisin-resistant strain (Lact. paracasei) allowed proliferation of the nisin producing Lactococcus lactis which in itself is unable to grow on complex carbohydrates. cPCR-DGGE permitted the monitoring of a different strain succession on the different carbohydrates, related to amylolytic activity and substrate consumption, acid production and nisin production. CONCLUSIONS: cPCR-DGGE is a useful tool for profiling defined mixed cultures of bacteria and hence allows their interaction to be studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Provided validation of the method for each specific case, it may be appropriate to monitor and control the reproducibility of any defined mixed culture of bacteria, with the advantage that an increase in the strain numbers to be monitored does not yield an increase in the labour of the procedure.

Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021.

Sinorhizobium meliloti is an alpha-proteobacterium that forms agronomically important N(2)-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid/peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.

The composite genome of the legume symbiont Sinorhizobium meliloti.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Dynamics of the microbial community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rRNA hybridization.

The microbial community developing during the spontaneous fermentation of sour cassava starch was investigated by cultivation-independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rDNA followed by sequencing of the most intense bands showed that the dominant organisms were all lactic acid bacteria (LAB), mainly close relatives of Bifidobacterium minimum, Lactococcus lactis, Streptococcus sp., Enterococcus saccharolyticus and Lactobacillus plantarum., Close relatives of Lb. panis, Leuconostoc mesenteroides and Ln. citreum were also found. A complementary analysis using hybridization of 16S rRNA with phylogenetic probes was necessary to detect the presence of the recently discovered species Lb. manihotivorans. Although it represented up to 13% of the total lactic acid bacteria of sour cassava starch, this species could not be detected by DGGE as the PCR product migrated to the same position as Lc. lactis. In addition, it was shown that a strong pH decrease in the time course of fermentation was most probably responsible for the competitive selection of acid-resistant LAB vs. both homo and heterofermentative acid-sensitive LAB.

Microbial community dynamics during production of the Mexican fermented maize dough pozol.

The dynamics of the microbial community responsible for the traditional fermentation of maize in the production of Mexican pozol was investigated by using a polyphasic approach combining (i) microbial enumerations with culture media, (ii) denaturing gradient gel electrophoresis (DGGE) fingerprinting of total community DNA with bacterial and eukaryotic primers and sequencing of partial 16S ribosomal DNA (rDNA) genes, (iii) quantification of rRNAs from dominant microbial taxa by using phylogenetic oligonucleotide probes, and (iv) analysis of sugars and fermentation products. A Streptococcus species dominated the fermentation and accounted for between 25 and 75% of the total flora throughout the process. Results also showed that the initial epiphytic aerobic microflora was replaced in the first 2 days by heterofermentative lactic acid bacteria (LAB), including a close relative of Lactobacillus fermentum, producing lactic acid and ethanol; this heterolactic flora was then progressively replaced by homofermentative LAB (mainly close relatives of L. plantarum, L. casei, and L. delbrueckii) which continued acidification of the maize dough. At the same time, a very diverse community of yeasts and fungi developed, mainly at the periphery of the dough. The analysis of the DGGE patterns obtained with bacterial and eukaryotic primers targeting the 16S and 18S rDNA genes clearly demonstrated that there was a major shift in the community structure after 24 h and that high biodiversity-according to the Shannon-Weaver index-was maintained throughout the process. These results proved that a relatively high number of species, at least six to eight, are needed to perform this traditional lactic acid fermentation. The presence of Bifidobacterium, Enterococcus, and enterobacteria suggests a fecal origin of some important pozol microorganisms. Overall, the results obtained with different culture-dependent or -independent techniques clearly confirmed the importance of developing a polyphasic approach to study the ecology of fermented foods.

Molecular diversity of lactic acid bacteria from cassava sour starch (Colombia).

Lactic acid bacteria and more particularly lactobacilli and Leuconostoc, are widely found in a wide variety of traditional fermented foods of tropical countries, made with cereals, tubers, meat or fish. These products represent a source of bacterial diversity that cannot be accurately analysed using classical phenotypic and biochemical tests. In the present work, the identification and the molecular diversity of lactic acid bacteria isolated from cassava sour starch fermentation were assessed by using a combination of complementary molecular methods: Randomly Amplified Polymorphic DNA fingerprinting (RAPD), plasmid profiling, hybridization using rRNA phylogenetic probes and partial 16S rDNA sequencing. The results revealed a large diversity of bacterial species (Lb. manihotivorans, Lb. plantarum, Lb. casei, Lb. hilgardii, Lb. buchneri, Lb. fermentum, Ln. mesenteroides and Pediococcus sp.). However, the most frequently isolated species were Lb. plantarum and Lb. manihotivorans. The RAPD analysis revealed a large molecular diversity between Lb. manihotivorans or Lb. plantarum strains. These results, observed on a rather limited number of samples, reveal that significant bacterial diversity is generated in traditional cassava sour starch fermentations. We propose that the presence of the amylolytic Lb. manihotivorans strains could have a role in sour starch processing.

Design and evaluation of a Lactobacillus manihotivorans species-specific rRNA-targeted hybridization probe and its application to the study of sour cassava fermentation.

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.

Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations.

The distribution of microorganisms in pozol balls, a fermented maize dough, was investigated by a polyphasic approach in which we used both culture-dependent and culture-independent methods, including microbial enumeration, fermentation product analysis, quantification of microbial taxa with 16S rRNA-targeted oligonucleotide probes, determination of microbial fingerprints by denaturing gradient gel electrophoresis (DGGE), and 16S ribosomal DNA gene sequencing. Our results demonstrate that DGGE fingerprinting and rRNA quantification should allow workers to precisely and rapidly characterize the microbial assemblage in a spontaneous lactic acid fermented food. Lactic acid bacteria (LAB) accounted for 90 to 97% of the total active microflora; no streptococci were isolated, although members of the genus Streptococcus accounted for 25 to 50% of the microflora. Lactobacillus plantarum and Lactobacillus fermentum, together with members of the genera Leuconostoc and Weissella, were the other dominant organisms. The overall activity was more important at the periphery of a ball, where eucaryotes, enterobacteria, and bacterial exopolysacharide producers developed. Our results also showed that the metabolism of heterofermentative LAB was influenced in situ by the distribution of the LAB in the pozol ball, whereas homolactic fermentation was controlled primarily by sugar limitation. We propose that starch is first degraded by amylases from LAB and that the resulting sugars, together with the lactate produced, allow a secondary flora to develop in the presence of oxygen. Our results strongly suggest that cultivation-independent methods should be used to study traditional fermented foods.

Culture-independent quantification of physiologically-active microbial groups in fermented foods using rRNA-targeted oligonucleotide probes: application to pozol, a Mexican lactic acid fermented maize dough.

Nine phylogenetic oligonucleotide probes were used to describe at the genus level the microbial community responsible for the spontaneous fermentation of maize, leading to the production of Mexican pozol. Ribosomal RNAs of specific groups and genera, in particular, lactic acid bacteria, were quantified using a culture-independent approach. In the early stage of the fermentation, Lactococcus and Leuconostoc appeared to be the dominant genera. A contrario, these represented minor genera at the end of the fermentation when Lactobacillus dominated the process. In addition, eukaryotes seemed to play a significant role throughout the fermentation and enterobacteria could be detected by this method.

Recovery of total microbial RNA from lactic acid fermented foods with a high starch content.

An optimized procedure for the recovery of RNA from micro-organisms involved in the fermentation of starchy foods (mainly hard-to-lyse lactic acid bacteria) is reported. Critical steps for the extraction were: cell recovery by differential centrifugation; cell wall digestion with both mutanolysin and lysozyme; and CTAB treatment for the elimination of starch. Digestion of starch with alpha-amylase did not improve extraction yields. The method yielded high amounts of RNA from pozol, a Mexican maize-based fermented food, and was found to extract total RNA efficiently from all the micro-organisms potentially present in these ecosystems. Both rRNA and mRNA recovered were of high quality and suitable for hybridization studies.

Repression of phenol catabolism by organic acids in Ralstonia eutropha.

During batch growth of Ralstonia eutropha (previously named Alcaligenes eutrophus) on phenol in the presence of acetate, acetate was found to be the preferred substrate; this organic acid was rapidly metabolized, and the specific rate of phenol consumption was considerably decreased, although phenol consumption was not abolished. This decrease corresponded to a drop in phenol hydroxylase and catechol-2,3-dioxygenase specific activities, and the synthesis of the latter was repressed at the transcriptional level. Studies with a mutant not able to consume acetate indicated that the organic acid itself triggers the repression. Other organic acids were also found to repress phenol degradation. One of these, benzoate, was found to completely block the catabolism of phenol (diauxic growth). A mutant unable to metabolize benzoate was also unable to develop on benzoate-phenol mixtures, indicating that the organic acid rather than a metabolite involved in benzoate degradation was responsible for the repression observed.

Benzoate degradation via the ortho pathway in Alcaligenes eutrophus is perturbed by succinate.

During batch growth of Alcaligenes eutrophus on benzoate-plus-succinate mixtures, substrates were simultaneously metabolized, leading to a higher specific growth rate (mu = 0.56 h-1) than when a single substrate was used (mu = 0.51 h-1 for benzoate alone and 0.44 h-1 for succinate alone), without adversely affecting the growth yield (0.57 Cmol/Cmol). Flux distribution analysis revealed that succinate dehydrogenase most probably controls the rate of total succinate consumption (the maximum flux being 9.7 mmol.g-1.h-1). It is postulated that the relative consumption rate of each substrate is in part related to modified levels of gene expression but to a large extent is dependent upon the presence of succinate, end product of the beta-ketoadipate pathway. Indeed, the in vitro beta-ketoadipate-succinyl coenzyme A transferase activity was seen to be inhibited by succinate, a coproduct of the reaction.

Microbiological and biochemical characterization of cassava retting, a traditional lactic Acid fermentation for foo-foo (cassava flour) production.

The overall kinetics of retting, a spontaneous fermentation of cassava roots performed in central Africa, was investigated in terms of microbial-population evolution and biochemical and physicochemical parameters. During the traditional process, endogenous cyanogens were almost totally degraded, plant cell walls were lysed by the simultaneous action of pectin methylesterase and pectate lyase, and organic acids (C(inf2) to C(inf4)) were produced. Most microorganisms identified were found to be facultative anaerobes which used the sugars (sucrose, glucose, and fructose) present in the roots as carbon sources. After 24 h of retting, the fermentation reached an equilibrium that was reproducible in all the spontaneous fermentations studied. Lactic acid bacteria were largely predominant (over 99% of the total flora after 48 h) and governed the fermentation. The epiphytic flora was first replaced by Lactococcus lactis, then by Leuconostoc mesenteroides, and finally, at the end of the process, by Lactobacillus plantarum. These organisms produced ethanol and high concentrations of lactate, which strongly acidified the retting juice. In addition, the rapid decrease in partial oxygen pressure rendered the process anaerobic. Strict anaerobes, such as Clostridium spp., developed and produced the volatile fatty acids (mainly butyrate) responsible, together with lactate, for the typical flavor of retted cassava. Yeasts (mostly Candida spp.) did not seem to play a significant role in the process, but their increasing numbers in the last stage of the process might influence the flavor and the preservation of the end products.

Flux limitations in the ortho pathway of benzoate degradation of Alcaligenes eutrophus: metabolite overflow and induction of the meta pathway at high substrate concentrations.

The growth behaviour of Alcaligenes eutrophus using various concentrations of benzoate was investigated. In batch culture, growth was exponential and growth rate (mu) and yields (Y) were high [mu = 0.51 h-1 and Yx/benzoate = 0.56 mol carbon (mol carbon)-1] when low concentrations of benzoate (< 5 mM) were used. These kinetic parameters were close to the maxima determined in a benzoate-limited chemostat [mu(max) = 0.55 h-1 and YX/benzoatemax = 0.57 mol carbon (mol carbon)-1] and the part of the energy for maintenance was limited (mATP = 4.3 +/- 2.2 mmol ATP g-1 h-1). When higher concentrations of benzoate were used (up to 40 mM), several metabolic limitations appeared. The specific rate of benzoate consumption was not altered, whereas growth was inhibited [Ki(benzoate) approximately 27 mM]. Furthermore, high concentrations of catechol together with some 1,2-dihydro-1,2-dihydroxybenzoate (DHB) transiently accumulated in the medium. The accumulation of catechol was attributed to limiting flux through catechol 1,2-dioxygenase estimated to be 5.2 mmol g-1 h-1, whereas that of DHB was provoked by an imbalance in the NADH/NAD+ intracellular content. The direct consequence of DHB accumulation was the induction of the meta pathway for the degradation of catechol, and this pathway contributed up to 20% of the total flux of catechol to the central metabolism. Finally, when very high concentrations of benzoate were used (55 mM), both growth and the specific rate of benzoate degradation were diminished due to a strong decrease in benzoate 1,2-dioxygenase specific activity.

Acetate utilization is inhibited by benzoate in Alcaligenes eutrophus: evidence for transcriptional control of the expression of acoE coding for acetyl coenzyme A synthetase.

During batch growth of Alcaligenes eutrophus on benzoate-acetate mixtures, benzoate was the preferred substrate, with acetate consumption being delayed until the rate of benzoate consumption had diminished. This effect was attributed to a transcriptional control of the synthesis of acetyl coenzyme A (acetyl-CoA) synthetase, an enzyme necessary for the entry of acetate into the central metabolic pathways, rather than to a biochemical modulation of the activity of this enzyme. Analysis of a 2.4-kb mRNA transcript hybridizing with the A. eutrophus acoE gene confirmed this repression effect. In a benzoate-limited chemostat culture, derepression was observed, with no increase in the level of expression following an acetate pulse. Benzoate itself was not the signal triggering the repression of acetyl-CoA synthetase. This role was played by catechol, which transiently accumulated in the medium when high specific rates of benzoate consumption were reached. The lack of rapid inactivation of the functional acetyl-CoA synthetase after synthesis has been stopped enables A. eutrophus to retain the capacity to metabolize acetate for prolonged periods while conserving minimal protein expenditure.

Origin of enzymes involved in detoxification and root softening during cassava retting.

The origin of root softening during cassava retting was investigated in a natural retting and in a sterile fermentation. Softening only occurred in the natural retting. Although high activities of endogenous pectin methyl esterase were found in cassava extracts from both fermentations, the depolymerizing enzymes polygalacturonase, active at low pH, and pectate lyase were only found in the non-sterile retting. No cellulase or xylanase activity was observed. The role of pectinases in the softening of cassava roots was confirmedin vitro using commericial enzymes. Root softening is therefore due to the combined action of endogenous pectin methyl esterase and exogenous bacterial depolymerizing enzymes. Detoxification occurred in both fermentations, confirming that the linamarase responsible for the destruction of cassava cyanide glycosides was mainly endogenous, even though microbial ß-glucosidases may help in the detoxication.