D-alanylation of lipoteichoic acid: role of the D-alanyl carrier protein in acylation

The D-alanylation of membrane-associated lipoteichoic acid (LTA) in gram-positive organisms requires the D-alanine-D-alanyl carrier protein ligase (AMP) (Dcl) and the D-alanyl carrier protein (Dcp). The dlt operon encoding these proteins (dltA and dltC) also includes dltB and dltD. dltB encodes a putative transport system, while dltD encodes a protein which facilitates the binding of Dcp and Dcl for ligation with D-alanine and has thioesterase activity for mischarged D-alanyl-acyl carrier proteins (ACPs). In previous results it was shown that D-alanyl-Dcp donates its ester residue to membrane-associated LTA (M. P. Heaton and F. C. Neuhaus, J. Bacteriol. 176: 681-690, 1994). However, all efforts to identify an enzyme which catalyzes this D-alanylation process were unsuccessful. It was discovered that incubation of D-alanyl-Dcp in the presence of LTA resulted in the time-dependent hydrolysis of this D-alanyl thioester. D-Alanyl-ACP in the presence of LTA was not hydrolyzed. When Dcp was incubated with membrane-associated D-alanyl LTA, a time and concentration-dependent formation of D-alanyl-Dcp was found. The addition of NaCl to this reaction inhibited the formation of D-alanyl-Dcp and stimulated the hydrolysis of D-alanyl-Dcp. Since these reactions are specific for the carrier protein (Dcp), it is suggested that Dcp has a unique binding site which interacts with the poly(Gro-P) moiety of LTA. It is this specific interaction that provides the functional specificity for the D-alanylation process. The reversibility of this process provides a mechanism for the transacylation of the D-alanyl ester residues between LTA and wall teichoic acid.     

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens α-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P _nisA , Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.     

Sec-mediated secretion of bacteriocin enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

In Lactococcus lactis subsp. cremoris SK110 protein, instead of lipoteichoic acid, reacts with the group-N-specific antiserum

The reaction between cell-surface components, isolated from two Lactococcus lactis subsp. cremoris strains, with their Group-specific antiserum were studied. No reaction between purified lipoteichoic acid and the antiserum was observed. Both strains, however, did belong to the lactococci (Group-N streptococci), as was demonstrated by the positive reaction between the antiserum and an acid- (Lancefield) or alkaline-extract. Experiments with proteolytic enzymes demonstrated the involvement of protein in the antigenic material in the latter reaction.     

Biosynthesis of lipoteichoic acid in Lactobacillus rhamnosus: role of DltD in D-alanylation

The dlt operon (dltA to dltD) of Lactobacillus rhamnosus 7469 encodes four proteins responsible for the esterification of lipoteichoic acid (LTA) by D-alanine. These esters play an important role in controlling the net anionic charge of the poly (GroP) moiety of LTA. dltA and dltC encode the D-alanine-D-alanyl carrier protein ligase (Dcl) and D-alanyl carrier protein (Dcp), respectively. Whereas the functions of DltA and DltC are defined, the functions of DltB and DltD are unknown. To define the role of DltD, the gene was cloned and sequenced and a mutant was constructed by insertional mutagenesis of dltD from Lactobacillus casei 102S. Permeabilized cells of a dltD::erm mutant lacked the ability to incorporate D-alanine into LTA. This defect was complemented by the expression of DltD from pNZ123/dlt. In in vitro assays, DltD bound Dcp for ligation with D-alanine by Dcl in the presence of ATP. In contrast, the homologue of Dcp, the Escherichia coli acyl carrier protein (ACP), involved in fatty acid biosynthesis, was not bound to DltD and thus was not ligated with D-alanine. DltD also catalyzed the hydrolysis of the mischarged D-alanyl-ACP. The hydrophobic N-terminal sequence of DltD was required for anchoring the protein in the membrane. It is hypothesized that this membrane-associated DltD facilitates the binding of Dcp and Dcl for ligation of Dcp with D-alanine and that the resulting D-alanyl-Dcp is translocated to the primary site of D-alanylation.     

Functional analysis of D-alanylation of lipoteichoic acid in the probiotic strain Lactobacillus rhamnosus GG

Lipoteichoic acid (LTA) is a macroamphiphile molecule which performs several functions in gram-positive bacteria, such as maintenance of cell wall homeostasis. D-alanylation of LTA requires the proteins encoded by the dlt operon, and this process is directly related to the charge properties of this polymer strongly contributing to its function. The insertional inactivation of dltD of the probiotic strain Lactobacillus rhamnosus GG (ATCC 53103) resulted in the complete absence of D-alanyl esters in the LTA as confirmed by nuclear magnetic resonance analysis. This was reflected in modifications of the bacterial cell surface properties. The dltD strain showed 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of cationic peptides compared to wild-type results. However, in vitro experiments revealed no major differences for adhesion to human intestinal epithelial cells, biofilm formation, and immunomodulation. These properties are considered to be important for probiotics. The role of the dlt operon in lactobacilli is discussed in view of these results.     

Insertional mutagenesis to generate lantibiotic resistance in Lactococcus lactis

While the potential emergence of food spoilage and pathogenic bacteria with resistance to lantibiotics is a concern, the creation of derivatives of starter cultures and adjuncts that can grow in the presence of these antimicrobials may have applications in food fermentations. Here a bank of Lactococcus lactis IL1403 mutants was created and screened, and a number of novel genetic loci involved in lantibiotic resistance were identified.     

Signal peptide and propeptide optimization for heterologous protein secretion in Lactococcus lactis

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP(Nuc)) by that of L. lactis protein Usp45 (SP(Usp)) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP(Usp) than when directed by SP(Nuc). This SP(Usp) effect on Nuc secretion is not due to a better antifolding activity, since SP(Usp):Nuc precursor proteins display enzymatic activity in vitro, while SP(Nuc):Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP(Usp) and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.     

Eukaryotic membrane protein overproduction in Lactococcus lactis

Eukaryotic membrane proteins play many vital roles in the cell and are important drug targets. Approximately 25% of all genes identified in the genome are known to encode membrane proteins, but the vast majority have no assigned function. Although the generation of structures of soluble proteins has entered the high-throughput stage, for eukaryotic membrane proteins only a dozen high-resolution structures have been obtained so far. One major bottleneck for the functional and structural characterisation of membrane proteins is the overproduction of biologically active material. Recent advances in the development of the Lactococcus lactis expression system have opened the way for the high-throughput functional expression of eukaryotic membrane proteins.     

Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion

The use of Gram-positive bacteria for heterologous protein production proves to be a useful choice due to easy protein secretion and purification. The lactic acid bacterium Lactococcus lactis emerges as an attractive alternative to the Gram-positive model Bacillus subtilis. Here, we review recent work on the expression and secretion systems available for heterologous protein secretion in L. lactis, including promoters, signal peptides and mutant host strains known to overcome some bottlenecks of the process. Among the tools developed in our laboratory, inactivation of HtrA, the unique housekeeping protease at the cell surface, or complementation of the Sec machinery with B. subtilis SecDF accessory protein each result in the increase in heterologous protein yield. Furthermore, our lactococcal expression/secretion system, using both P(Zn)zitR, an expression cassette tightly controlled by environmental zinc, and a consensus signal peptide, SP(Exp4), allows efficient production and secretion of the staphylococcal nuclease, as evidenced by protein yields (protein amount/biomass) comparable to those obtained using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox we are developing should contribute to enlarge the use of L. lactis as a protein cell factory.     

A model system for the investigation of heterologous protein secretion pathways in Lactococcus lactis.

The capacity of recombinant strains of Lactococcus lactis to secrete a heterologous protein was investigated by constructing two expression-secretion vectors (pLET2 and pLET3) for use with a lactococcal gene expression system driven by the highly active T7 RNA polymerase. The vectors incorporated different lactococcal secretion leaders and translation initiation sequences. When tetanus toxin fragment C (TTFC) was used as a test protein, the quantities of TTFC produced by the pLET2-TTFC strain exceeded the rate of secretion of TTFC into the growth medium. However, nearly all of the soluble TTFC associated with the cell (3.4%) was translocated through the cell membrane. The pLET3-TTFC strain did not accumulate TTFC intracellularly and exhibited growth characteristics and viability identical to the growth characteristics and viability of the control strain. This strain secreted approximately 2.9 mg of TTFC per liter into the growth medium after 6 h of growth under test tube conditions. Our results indicate that L. lactis is capable of secreting substantial amounts of heterologous protein and also confirm the findings of other workers that the cell wall may serve as a functional barrier to the diffusion of some secreted proteins into the growth medium.     

Expression of green fluorescent protein in Lactococcus lactis

The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.     

Recognition of cyclonucleoside lesions by the Lactococcus lactis FPG protein

Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.     

Identification of a RecA-like protein in Lactococcus lactis

We have identified in Lactococcus lactis, an analogue of Escherichia coli RecA protein. Physiological responses such as ultraviolet (UV) and chemical mutagenesis and induction of prophage have been characterized and suggest the existence of RecA-like functions in this commercially important species. The putative RecA protein was detected at the position of an apparent molecular weight of 39 kDa by Western blot analysis by using antiserum against E coli RecA protein. In addition, the protein level is significantly increased after UV irradiation in a wild-type strain compared to the recombination deficient mutant strain.     

Autolysis of Lactococcus lactis is increased upon D-alanine depletion of peptidoglycan and lipoteichoic acids

Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when D-Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.     

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae,Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.     

Impact of osmotic stress on protein diffusion in Lactococcus lactis

We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram‐positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12‐transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ～ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram‐positive and Gram‐negative bacteria.     

Artificial signal peptide prediction by a hidden markov model to improve protein secretion via Lactococcus lactis bacteria

A hidden Markov model (HMM) has been utilized to predict and generate artificial secretory signal peptide sequences. The strength of signal peptides of proteins from different subcellular locations via Lactococcus lactis bacteria correlated with their HMM bit scores in the model. The results show that the HMM bit score +12 are determined as the threshold for discriminating secreteory signal sequences from the others. The model is used to generate artificial signal peptides with different bit scores for secretory proteins. The signal peptide with the maximum bit score strongly directs proteins secretion.