Overproduction and purification of the CcpA protein from Lactococcus lactis

In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xl1blue strain as a fusion with 6 x His tag. A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 microg/ml ampicillin at 37 degrees C and subsequently for 4 h after induction by IPTG. The procedure let us obtain 5 mg of homogenous CcpA protein. Glutaraldehyde crosslinking analysis indicated the formation of dimer or tetramer forms of the CcpA protein.     

In vitro DNA binding of purified CcpA protein from Lactococcus lactis IL1403

During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites. However, purified HPr-Ser-P protein from Bacillus subtilis was shown to slightly increase the DNA-binding capacity of the CcpA protein. It was also observed that CcpA bound to the cre box forms an apparently more stable complex than that resulting from unspecific binding. Competition gel retardation assay performed on DNA sequences from two PEP:PTS regions demonstrated that the ybhE, bglS, rheB, yebE, ptcB and yecA genes situated in these regions are most probably directly regulated by CcpA.     

Lactococcus lactis produces short-chain quinones that cross-feed Group B Streptococcus to activate respiration growth

Quinones are essential components of the respiration chain that shuttle electrons between oxidoreductases. We characterized the quinones synthesized by Lactococcus lactis , a fermenting bacterium that activates aerobic respiration when a haem source is provided. Two distinct subgroups were characterized: Menaquinones (MK) MK-8 to MK-10, considered as hallmarks of L. lactis , are produced throughout growth. MK-3 and demethylMK-3 [(D)MK-3] are newly identified and are present only late in growth. Production of (D)MK-3 was conditional on the carbon sugar and on the presence of carbon catabolite regulator gene ccpA . Electron flux driven by both (D)MK fractions was shared between the quinol oxidase and extracellular acceptors O₂, iron and, with remarkable efficiency, copper. Purified (D)MK-3, but not MK-8–10, complemented a menB defect in L. lactis . We previously showed that a respiratory metabolism is activated in Group B Streptococcus (GBS) by exogenous haem and MK, and that this activity is implicated in virulence. Here we show that growing lactococci donate (D)MK to GBS to activate respiration and stimulate growth of this opportunist pathogen. We propose that conditions favouring (D)MK production in dense microbial ecosystems, as present in the intestinal tract, could favour implantation of (D)MK-scavengers like GBS within the complex.     

Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.     

Roles of thioredoxin reductase during the aerobic life of Lactococcus lactis

Thiol-disulfide bond balance is generally maintained in bacteria by thioredoxin reductase-thioredoxin and/or glutathione-glutaredoxin systems. Some gram-positive bacteria, including Lactococcus lactis, do not produce glutathione, and the thioredoxin system is presumed to be essential. We constructed an L. lactis trxB1 mutant. The mutant was obtained under anaerobic conditions in the presence of dithiothreitol (DTT). Unexpectedly, the trxB1 mutant was viable without DTT and under aerated static conditions, thus disproving the essentiality of this system. Aerobic growth of the trxB1 mutant did not require glutathione, also ruling out the need for this redox maintenance system. Proteomic analyses showed that known oxidative stress defense proteins are induced in the trxB1 mutant. Two additional effects of trxB1 were not previously reported in other bacteria: (i) induction of proteins involved in fatty acid or menaquinone biosynthesis, indicating that membrane synthesis is part of the cellular response to a redox imbalance, and (ii) alteration of the isoforms of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GapB). We determined that the two GapB isoforms in L. lactis differed by the oxidation state of catalytic-site cysteine C152. Unexpectedly, a decrease specific to the oxidized, inactive form was observed in the trxB1 mutant, possibly because of proteolysis of oxidized GapB. This study showed that thioredoxin reductase is not essential in L. lactis and that its inactivation triggers induction of several mechanisms acting at the membrane and metabolic levels. The existence of a novel redox function that compensates for trxB1 deficiency is suggested.     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

pH influences growth and plasmid stability of recombinant Lactococcus lactis subsp. lactis

Continuous cultures of a recombinant Lactococcus lactis subsp. lactis strain were performed to display the effect of the fermentation pH on the specific growth rate and plasmid stability. The proportion of plasmid pIL252 bearing cells decreased exponentially with the number of generations. The influence of the pH on the rate of loss of plasmid pIL252 and on the specific growth rate of L. lactis IL2682 was described by second order polynomial equations. Optimal pH for the growth and plasmid stability were 6.39 and 6.41 respectively. © Rapid Science Ltd. 1998     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.     

A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation

Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride. Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB . GadC is homologous to putative glutamate-γ-aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane-spanning domains. GadB shows similarity to glutamate decarboxylases. A L . lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present. Expression of gadCB in L . lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression. Apparently, these genes encode a glutamate-dependent acid resistance mechanism of L . lactis that is optimally active under conditions in which it is needed to maintain viability. Immediately upstream of the chloride-dependent gadCB promoter P _gad , a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii . gadR expression is chloride and glutamate independent. A gadR mutant did not produce the 3 kb gadCB mRNA that is found in wild-type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.     

Generation of a membrane potential by Lactococcus lactis through aerobic electron transport

Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3',3'-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (Delta psi). Wild-type cells, grown aerobically in the presence of heme, generated a Delta psi even in the presence of the F(1)-F(o) ATPase inhibitor N,N'-dicyclohexylcarbodiimide, while a cytochrome bd-negative mutant strain (CydA Delta) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydA Delta cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the L. lactis ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.     

Lactococcus lactis and stress

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis.Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase.To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37°C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.     

Harnessing the respiration machinery for high-yield production of chemicals in metabolically engineered Lactococcus lactis

When modifying the metabolism of living organisms with the aim of achieving biosynthesis of useful compounds, it is essential to ensure that it is possible to achieve overall redox balance. We propose a generalized strategy for this, based on fine-tuning of respiration. The strategy was applied on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)-2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD⁺, and a high titer of 371 mM (32 g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361 mM (32 g/L) R-BDO with a yield of 81% or 365 mM (33 g/L) with a yield of 82%, respectively. These results demonstrate the great potential in using finely-tuned respiration machineries for bio-production.