Nutritional requirements and media development for Lactococcus lactis IL1403

Lactic acid bacteria are extensively used in food technology and for the production of various compounds, but they are fastidious in nutrient requirements. In order to elucidate the role of each component precisely, defined multicomponent media are required. This study focuses on determining nutrient auxotrophies and minimizing media components (amino acids, vitamins, metal ions, buffers and additional compounds) for the cultivation of Lactococcus lactis subsp. lactis IL1403, using microtitre plates and test tubes. It was shown that glutamine and asparagine were the most important media components for achieving higher biomass yields while the branched-chain amino acids were necessary to increase specific growth rate. The amino acid and glucose ratio was reduced to achieve minimal residual concentration of amino acids in the medium after the growth of cells, whereas the specific growth rate and biomass yield of cells were not considerably affected. As the percentage of each consumed amino acid compared to initial amount is larger than measurement error, these optimized media are important for achieving more precise data about amino acid utilization and metabolism.     

Influence of Carbohydrate Starvation and Arginine on Culturability and Amino Acid Utilization of Lactococcus lactis subsp. lactis

Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.     

Identification of Lactococcus lactis Genes Required for Bacteriophage Adsorption

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that 645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and 645 that showed no homology in the C-terminal part.     

pSEUDO, a Genetic Integration Standard for Lactococcus lactis

Plasmid pSEUDO and derivatives were used to show that llmg_pseudo_10 in Lactococcus lactis MG1363 and its homologous locus in L. lactis IL1403 are suitable for chromosomal integrations. L. lactis MG1363 and IL1403 nisin-induced controlled expression (NICE) system derivatives (JP9000 and IL9000) and two general stress reporter strains (NZ9000::PhrcA-GFP and NZ9000::PgroES-GFP) enabling in vivo noninvasive monitoring of cellular fitness were constructed.     

Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.     

Cloning,Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.     

Adsorption of bisphenol A by lactic acid bacteria,Lactococcus, strains

Ten strains of the genus Lactococcus were examined for their ability to remove bisphenol A [2, 2-bis(4-hydroxyphenyl)propane; BPA], which is known as an endocrine disrupter. Nine strains of the lactococci tested could remove BPA from media during growth, although the removal ratio was below 9%. When BPA was incubated with lyophilized cells of lactococci for 1 h, the concentration of BPA in the media was decreased by 9–62%. Especially, the highest removal ratio of BPA was observed for Lactococcus lactis subsp. lactis 712. The lactococci could adsorb BPA but not degrade it, because the lactococci maintained the ability to remove BPA from the medium after autoclaving. When the lyophilized cells of L. lactis subsp. lactis 712 were also incubated with six analogues of BPA, they effectively adsorbed hydrophobic compounds such as 2, 2′-diphenylpropane and bisphenol A dimethylether. The BPA-adsorbing ability of lactococci could be due to the hydrophobic binding effect. The removal ratio of BPA by L. lactis subsp. lactis 712 was increased after treatment with sodium dodecyl sulfate and decreased after digestion with trypsin. These results suggest that the hydrophobic proteins on cell surface may be involved in the BPA-adsorbing ability of lactococci.     

Using highly efficient nonlinear experimental design methods for optimization of Lactococcus lactis fermentation in chemically defined media

Optimization of fermentation media and processes is a difficult task due to the potential for high dimensionality and nonlinearity. Here we develop and evaluate variations on two novel and highly efficient methods for experimental fermentation optimization. The first approach is based on using a truncated genetic algorithm with a developing neural network model to choose the best experiments to run. The second approach uses information theory, along with Bayesian regularized neural network models, for experiment selection. To evaluate these methods experimentally, we used them to develop a new chemically defined medium for Lactococcus lactis IL1403, along with an optimal temperature and initial pH, to achieve maximum cell growth. The media consisted of 19 defined components or groups of components. The optimization results show that the maximum cell growth from the optimal process of each novel method is generally comparable to or higher than that achieved using a traditional statistical experimental design method, but these optima are reached in about half of the experiments (73–94 vs. 161, depending on the variants of methods). The optimal chemically defined media developed in this work are rich media that can support high cell density growth 3.5–4 times higher than the best reported synthetic medium and 72% higher than a commonly used complex medium (M17) at optimization scale. The best chemically defined medium found using the method was evaluated and compared with other defined or complex media at flask‐ and fermentor‐scales. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media

An efficient method for genetic transformation of lactococci by electroporation is presented. Highly competent lactococci for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers. These cells could be stored at −85°C without loss of competence. With Lactococcus lactis subsp. cremoris BC101, a transformation frequency of 5.7 × 10⁷ transformants per μg of pIL253 DNA was obtained, which represents 5% of the surviving cells. All the lactococcal strains tested could be transformed by the present method.     

Reduced Lysis upon Growth of Lactococcus lactis on Galactose Is a Consequence of Decreased Binding of the Autolysin AcmA

When Lactococcus lactis subsp. lactis IL1403 or L. lactis subsp. cremoris MG1363 is grown in a medium with galactose as the carbon source, the culture lyses to a lesser extent in stationary phase than when the bacteria are grown in a medium containing glucose. Expression of AcmA, the major autolysin of L. lactis, is not influenced by the carbon source. Binding studies with a fusion protein consisting of the MSA2 protein of Plasmodium falciparum and the C-terminal peptidoglycan-binding domain of AcmA revealed that cell walls of cells from both subspecies grown on galactose bind less AcmA than cell walls of cells grown on glucose. Cells grown on glucose or galactose and treated with trichloroacetic acid prior to AcmA binding bind similar amounts of AcmA. Analysis of the composition of the lipoteichoic acids (LTAs) of L. lactis IL1403 cells grown on glucose or galactose showed that the LTA composition is influenced by the carbon source: cells grown on galactose contain LTA with less galactose than cells grown on glucose. In conclusion, growth of L. lactis on galactose changes the LTA composition in the cell wall in such a way that less AcmA is able to bind to the peptidoglycan, resulting in a decrease in autolysis.     

Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

     

A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation

A mutant of fast milk-coagulating (Fmc⁺) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc⁻) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc⁺ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.     

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese.     

Starvation-Induced Stress Resistance in Lactococcus lactis subsp. lactis IL1403

Carbohydrate-starved cultures of Lactococcus lactis subsp. lactis IL1403 showed enhanced resistance to heat, ethanol, acid, osmotic, and oxidative stresses. This cross-protection seems to be established progressively during the transitional growth phase, with maximum resistance occurring when cells enter the stationary phase. Chloramphenicol or rifamycin treatment does not abolish the development of a tolerant cell state but, on the contrary, seems to provoke this response in L. lactis subsp. lactis.     

A dynamic,genome-scale flux model of Lactococcus lactis to increase specific recombinant protein expression

Recently, lactic acid bacteria (LAB) have attracted a great deal of interest because of their potential to serve as oral delivery vehicles for recombinant protein vaccines. An important limitation to their use is the typically low level of heterologous expression obtained in LAB. To address this, a dynamic flux balance analysis (DFBA) model was used to identify gene targets for increasing specific expression of Green Fluorescent Protein (GFP), a model heterologous protein, in Lactococcus lactis IL1403. Two strains, each targeting one of the top model-identified genes, were constructed and tested in vivo. Data show that both strains, by a conservative estimate, achieved 15% higher GFP per cell than the control strain, a qualitative confirmation of the model predictions. A genome-scale DFBA model for L. lactis growing on M17 medium is presented along with the procedure for screening gene targets and a powerful method for visualizing fluxes in genome-scale metabolic networks.     

Identification and Characterization of Lactococcal-Prophage-Carried Superinfection Exclusion Genes

Superinfection exclusion (Sie) proteins are prophage-encoded phage resistance systems. In this study, genes encoding Sie systems were identified on the genomes of Lactococcus lactis subsp. cremoris MG1363 and L. lactis subsp. lactis IL1403. These Sie systems are genetically distinct and yet were shown to act specifically against a particular subset of the 936 phage group. Each of the systems allows normal phage adsorption while affecting plasmid transduction and intracellular phage DNA replication, which points to the blocking of phage DNA injection as their common mode of action. Sie-specifying genes found on the MG1363 prophages are also present in various lactococcal strains, whereas the prophage-encoded Sie systems of IL1403 do not appear to be as widely disseminated.     

Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis

The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lactis subsp. lactis (M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the pepXP gene. After inactivation of the gene in L. lactis subsp. lactis MG1363, which is Lac^- and Prt^-, the Lac⁺ Prt⁺ determinants were transferred by conjugation by using L. lactis subsp. lactis 712 as the donor. Since growth of the transconjugants relative to the PepXP⁺ strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of pepXP, was not required for PepXP activity in L. lactis. A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and pepXP mutants. Therefore, altered expression of the pepXP-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products.     

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.     

Projector: automatic contig mapping for gap closure purposes

Projector was designed for automatic positioning of contigs from an unfinished prokaryotic genome onto a template genome of a closely related strain or species. Projector mapped 84 contigs of Lactococcus lactis MG1363 (corresponding to 81% of the assembly nucleotides) against the genome of L.lactis IL1403. Ninety three percent of subsequent gap closure PCRs were successful. Moreover, a significant improvement in the N50 and N80 values (describing the assembly quality) was observed after the use of Projector. Because increasing numbers of bacterial genomes are being sequenced, Projector provides an efficient method to close a significant number of remaining gaps in the late stages of a genome sequencing project.