Deoxyribonucleic Acid Homology Among Lactic Streptococci

A comparison was made by deoxyribonucleic acid homology of 45 strains of lactic streptococci, using two strains of Streptococcus cremoris and three strains of Streptococcus lactis as reference strains. All S. cremoris strains were grouped together by deoxyribonucleic acid homology. S. lactis strains formed a second group, except that three strains of S. lactis showed a high degree of homology with S. cremoris strains. The three Streptococcus diacetylactis strains could not be differentiated from S. lactis strains. In spite of these differences between S. lactis and S. cremoris strains, the majority of S. cremoris, S. lactis, and S. diacetylactis strains studied had at least 50% of their base sequences in common. In contrast, Streptococcus thermophilus strains generally showed little relationship with the other strains of lactic streptococci. The relevance of these findings to the selection of starter strains for cheese making is discussed.     

Potential of Lactic Streptococci to Produce Bacteriocin

A survey was made on the bacteriocin-producing potential of lactic streptococci. Bacteriocin-like activities were isolated and partially purified from about 5% of the 280 strains investigated. The frequency of production varied from about 1% in Streptococcus lactis subsp. diacetylactis to 9 and 7.5% in S. lactis and Streptococcus cremoris, respectively. Eight strains of S. cremoris produced bacteriocins which, on the basis of heat stability at different pH values and inhibitory spectrum, could be divided into four types. From 54 S. lactis strains, 5 strains produced inhibitory substances, namely, three nisin-like antibiotics and two different bacteriocins. Only 1 of 93 S. lactis subsp. diacetylactis strains produced a bacteriocin which was very similar to bacteriocins of type I in S. cremoris. All of the bacteriocins that were partially purified by ammonium sulfate precipitation showed very limited inhibitory spectra. Most of the lactic streptococci and a few members of the genera Clostridium, Leuconostoc, and Pediococcus were inhibited. None of the bacteriocins acted on gram-negative bacteria. The bacteriocinogenic strains were also characterized on the basis of plasmid content. All strains possessed between one and nine plasmids ranging from 1 to 50 megadaltons.     

Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.     

Taxonomic Structure and Monitoring of the Dominant Population of Lactic Acid Bacteria during Wheat Flour Sourdough Type I Propagation Using Lactobacillus sanfranciscensis Starters

The structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains of Lactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g⁻¹. Partial sequence analysis of the 16S rRNA and recA genes and PCR-denaturating gradient gel electrophoresis analysis using the rpoB gene allowed identification of Weissella confusa, Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rossiae, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Pediococcus pentosaceus, and Lactobacillus spp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain of L. sanfranciscensis was found in all the sourdoughs. Except for L. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes of L. sanfranciscensis and with W. confusa or L. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.     

Cooperation between Lactococcus lactis and Nonstarter Lactobacilli in the Formation of Cheese Aroma from Amino Acids

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of α-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced α-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

Stability of Lactic Acid Bacteria to Freezing as Related to Their Fatty Acid Composition

The viability of Streptococcus lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h was better preserved when the cells were grown in medium supplemented with oleic acid or Tween 80 (polyoxyethylene sorbitan monooleate). A pronounced change in the cellular fatty acid composition was noted when the bacteria were grown in the presence of Tween 80. In S. lactis the ratio of unsaturated to saturated fatty acids increased from 1.18 to 2.55 and in Lactobacillus sp. A-12 it increased from 0.85 to 1.67 when Tween 80 was added to the growth medium. The antibiotic cerulenin markedly inhibited the growth of lactic acid bacteria in tomato juice (TJ) medium but had almost no effect on the growth of the bacteria in TJ medium containing Tween 80 (or oleic acid). The antibiotic inhibited markedly the incorporation of [1-¹⁴C]acetate but had no inhibitory effect on the incorporation of exogenous [1-¹⁴C]oleate (or [1-¹⁴C]palmitate) into the lipid fractions of lactic acid bacteria. Thus, the fatty acid composition of lactic acid bacteria, inhibited by the antibiotic cerulenin, can be modulated by exogenously added oleic acid (or Tween 80) without the concurrent endogenous fatty acid synthesis from acetate. The data obtained suggest that cerulenin inhibits neither cyclopropane fatty acid synthesis nor elongation of fatty acid acyl intermediates. The radioactivity of cells grown in the presence of [1-¹⁴C]oleate and cerulenin was associated mainly with cyclopropane Δ19:0, 20:0 + 20:1, and 21:0 acids. As a consequence, cerulenin caused a decrease in the ratio of unsaturated to saturated fatty acids in lactic acid bacteria as compared with cells grown in TJ medium plus Tween 80 but without cerulenin. Cerulenin caused a decrease in the viability of S. lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h only when Tween 80 was present in the growth medium. We conclude that the sensitivity of lactic acid bacteria to damage from freezing can be correlated with specific alterations in the cellular fatty acids.     

Effects of Cultivation Conditions on Folate Production by Lactic Acid Bacteria

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.     

Characterization and Determination of Origin of Lactic Acid Bacteria from a Sorghum-Based Fermented Weaning Food by Analysis of Soluble Proteins and Amplified Fragment Length Polymorphism Fingerprinting

The group that includes the lactic acid bacteria is one of the most diverse groups of bacteria known, and these organisms have been characterized extensively by using different techniques. In this study, 180 lactic acid bacterial strains isolated from sorghum powder (44 strains) and from corresponding fermented (93 strains) and cooked fermented (43 strains) porridge samples that were prepared in 15 households were characterized by using biochemical and physiological methods, as well as by analyzing the electrophoretic profiles of total soluble proteins. A total of 58 of the 180 strains were Lactobacillus plantarum strains, 47 were Leuconostoc mesenteroides strains, 25 were Lactobacillus sake-Lactobacillus curvatus strains, 17 were Pediococcus pentosaceus strains, 13 were Pediococcus acidilactici strains, and 7 were Lactococcus lactis strains. L. plantarum and L. mesenteroides strains were the dominant strains during the fermentation process and were recovered from 87 and 73% of the households, respectively. The potential origins of these groups of lactic acid bacteria were assessed by amplified fragment length polymorphism fingerprint analysis.     

Effect of Fermentation Conditions on Growth of Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091 in Pure and Mixed Cultures

Two strains of mesophilic lactic acid bacteria, Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091, were grown in pure and mixed cultures in the presence or absence of citrate (15 mM) and at controlled (pH 6.5) or uncontrolled pH. Microbial cell densities at the end of growth, maximum growth rates, the pH decrease of the medium resulting from growth, and the corresponding acidification rates were determined to establish comparisons. The control of pH in pure cultures had no effect on L. lactis CNRZ 1091 populations. The final populations of S. cremoris AM2, however, were at least five times higher than when the pH was not controlled (4 × 10⁸ vs. 2 × 10⁹ CFU · ml⁻¹). The pH had no effect on the growth rate of either strain. That of S. cremoris AM2 (0.8 h⁻¹) was about twice that of L. lactis CNRZ 1091. When the pH fell below 5, the growth of both strains decreased or stopped altogether. Citrate had no effect on S. cremoris AM2, while final populations of L. lactis CNRZ 1091 were two to three times higher (3 × 10⁸ CFU · ml⁻¹); it had no effect on the maximum growth rates of the two strains. Citrate attenuated the pH decrease of the medium and reduced the maximum acidification rate of the culture by 50%, due to the growth of S. cremoris AM2. Acidification due to L. lactis CNRZ 1091, however, was very slight. Regardless of the conditions of pH and citrate, the total bacterial population in mixed culture was lower (by 39%) than that of the sum of each pure culture. Mixed culture improved the maximum growth rate of L. lactis CNRZ 1091 (0.6 h⁻¹) by 50%, while that of S. cremoris AM2 was unaffected. The acidification rate of the growth medium in mixed culture, affected by the presence of citrate, resulted from the development and activity of S. cremoris AM2.     

Synthesis of γ-Aminobutyric Acid by Lactic Acid Bacteria Isolated from a Variety of Italian Cheeses

The concentrations of γ-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg⁻¹. Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.     

Contribution of Lactococcus lactis Reducing Properties to the Downregulation of a Major Virulence Regulator in Staphylococcus aureus,the agr System

Staphylococcus aureus is a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol of S. aureus using lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability of Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence, agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties of L. lactis contribute to agr downregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% released agr downregulation in the presence of L. lactis. This downregulation still occurred in an S. aureus srrA mutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability of L. lactis reducing properties to interfere with the expression of S. aureus virulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.     

Behavior of Psychrotrophic Lactic Acid Bacteria Isolated from Spoiling Cooked Meat Products

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10°C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10°C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4°C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10⁸ CFU/g at 10°C after 7 to 12 days.     

Diversity analysis of dairy and non-dairy strains of Lactococcus lactis ssp. lactis by multilocus sequence analysis (MLSA)

The genetic diversity of 31 identified strains of Lactococcus lactis ssp. lactis isolated from different dairy and non-dairy sources were investigated at gene level using multilocus sequence analysis (MLSA) and PCR-RFLP based on the differences in four selected partial protein coding gene sequences: araT, encoding aromatic amino acid-specific aminotransferase; dtpT, encoding di/tri peptide transporter; yueF, encoding non-proteolytic protein, peptidase, M16 family; and pdhA, encoding pyruvate dehydrogenase E1 component α-subunit. A set of seven test strains from different isolation sources and one reference strain, L. lactis ssp. lactis NCDC 094, were analyzed by MLSA. The strains showed distinct diversity among themselves and exhibited a greater percent similarity with reference strains L. lactis ssp. lactis CV56 (CP002365.1), IL1403 (AE005176.1), and KF147 (CP001834.1) in comparison with L. lactis ssp. cremoris NZ9000 (CP002094.1), MG1363 (AM406671.1), and SK11 (CP00425.1). The MLSA revealed one distinct genomic lineage within strains exclusively of L. lactis ssp. lactis. This analysis also revealed no source-wise genetic relationship in the test strains analyzed. Further, PCR-RFLP of araT, dtpT, yueF and pdhA also characterized the single genomic lineage exclusively of L. lactis ssp. lactis within a total of 24 test strains.     

Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH

We describe the dynamics of changes in the intracellular pH (pH_i) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH_ex). Strains of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH_i within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH_ex from 7.0 to 5.0. Under these conditions, the pH_i of L. innocua remained neutral (between 7 and 8). In contrast, the pH_i values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH_ex was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pH_i at pH_ex 7.0, while different kinetics of pH_i regulation were observed in different species and also in different strains of S. thermophilus.     

Construction of Lactococcus lactis thyA-null using the Red recombination system

Lactococcus lactis—a food-grade nonpathogenic lactic acid bacterium—is used widely in the food industry. In this report, we describe an approach to construct deficient strains in L. lactis utilizing the λ-Red recombination system. Three kinds of recombinant proteins, λ exonuclease, β protein and γ protein, were induced by l-arabinose in L. lactis MG1363 harboring the plasmid pKD46. A chloramphenicol-resistant cassette was amplified from pGj103 containing homology arms of 50 nt to the thyA gene. The PCR-generated DNA fragment was then electroporated into L. lactis MG1363, which expressed the recombination proteins. ThyA-null strains resistant to chloramphenicol were obtained and their growth characteristics were analyzed in relation to thymidine requirement. The results revealed that the thyA gene in L. lactis MG1363 was successfully knocked out. This is the first time that the Red system has been used in a Gram-positive bacterium, and use of the techniques presented here should prompt rapid and efficient mutagenesis or modification of L. Lactis chromosomal genes.     

Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis

PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production.     

Improved acid-stress tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000 and <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 by overexpression of the anti-acid component <Emphasis Type="Italic">recT</Emphasis>

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.