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.     

Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.     

Autolysis of Lactococcus lactis Is Influenced by Proteolysis

The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in an L. lactis acmA deletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.     

利用乳酸乳球菌AcmA表面展示b-1, 3-1, 4-葡聚糖酶

采用PCR扩增乳酸乳球菌(Lactococcus lactis)MB191菌株的全长肽聚糖水解酶基因acmA, 通过C-末端融合构建了与绿色荧光基因gfp的融合基因acmA-gfp, 再连接于表达载体pMG36k上后得到可组成型表达AcmA-GFP融合蛋白的重组质粒pMB137, 然后将该质粒电转化导入到乳酸乳球菌AS1.2829中获得重组菌MB137。经SDS-PAGE检测, 重组菌MB137可表达预期的分子量约74 kD的蛋白质。Western blotting、细胞分级分离组分的荧光活性测定和特异GFP二抗标记的流式细胞仪检测证实GFP被成功锚定在重组菌细胞表面, 被锚定蛋白约占总表达融合蛋白的35%。进一步通过从枯草芽胞杆菌BF7658基因组中扩增去信号肽序列的b-1, 3-1, 4葡聚糖酶基因gls, 来取代pMB137中的gfp, 得到携带融合基因acmA-gls的重组质粒pMB138, 经导入到乳酸乳球菌AS1.2829后得到重组菌MB138, 其全细胞b-1, 3-1, 4-葡聚糖水解酶的活性约为12 U/mL菌液, 明显高于对照菌株。     

AcmD,a Homolog of the Major Autolysin AcmA of Lactococcus lactis,Binds to the Cell Wall and Contributes to Cell Separation and Autolysis

Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under standard laboratory conditions AcmD was mainly secreted into the culture supernatant. An L. lactis acmAacmD double mutant formed longer chains than the acmA single mutant, indicating that AcmD contributes to cell separation. This phenotype could be complemented by plasmid-encoded expression of AcmD in the double mutant. No clear difference in cellular lysis and protein secretion was observed between both mutants. Nevertheless, overexpression of AcmD resulted in increased autolysis when AcmA was present (as in the wild type strain) or when AcmA was added to the culture medium of an AcmA-minus strain. Possibly, AcmD is mainly active within the cell wall, at places where proper conditions are present for its binding and catalytic activity. Various fusion proteins carrying either the three LysM repeats of AcmA or AcmD were used to study and compare their cell wall binding characteristics. Whereas binding of the LysM domain of AcmA took place at pHs ranging from 4 to 8, LysM domain of AcmD seems to bind strongest at pH 4.     

利用乳酸乳球菌AcmA表面展示β-1,3-1,4-葡聚糖酶

采用PCR扩增乳酸乳球(Lactococcus lactis)MBl91菌株的全长肽聚糖水解酶基因acmA,通过C-末端融合构建了与绿色荧光基因gfp的融合基因acmA-gfp,再连接于表达载体pMG36k上后得到可组成型表达AcmA-GFP融合蛋白的重组质粒pMB137,然后将该质粒电转化导入到乳酸乳球菌AS1.2829中获得重组菌MB137.经SDS-PAGE检测.重组菌MB137可表达预期的分子量约74 kD的蛋白质.Western blotting、细胞分级分离组分的荧光活性测定和特异GFP 二抗标记的流式细胞仪检测证实GFP被成功锚定在重组茵细胞表面,被锚定蛋白约占总表达融合蛋白的35%.进一步通过从枯草芽胞杆菌BF7658基因组中扩增去信号肽序列的β-1,3-1,4葡聚糖酶基因gls,来取代pMB137中的gfp,得到携带融合基因acmA-gls的重组质粒pMB138,经导入到乳酸乳球茵AS1.2829后得到重组菌MB138,其全细胞β-1,3-1,4-葡聚糖水解酶的活性约为12 U/mL茵液,明显高于对照茵株.     

AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning

AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N-terminal active site domain and a C-terminal peptidoglycan-binding domain. The active site domain is homologous to that of muramidase-2 of Enterococcus hirae, however, RP-HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N-acetylglucosaminidase. In the C-terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall-hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning.     

Positive role of peptidoglycan breaks in lactococcal biofilm formation

Bacterial attachment to solid matrices depends on adhesive molecules present on the cell surface. Here we establish a positive correlation between peptidoglycan (PG) breaks, rather than particular molecules, and biofilm-forming capacity in the Gram-positive bacterium Lactococcus lactis. The L. lactis acmA strain, which is defective in PG hydrolase, adhered less efficiently than the wild-type (wt) strain to different solid surfaces and was unable to form biofilms. These phenotypes were abolished by addition of lysozyme, a PG hydrolytic enzyme. Thus, the presence of PG breaks introduced by PG hydrolase, and not the AcmA protein itself, appears to be responsible for biofilm formation. Two different genetic screens confirmed the importance of PG breaks in L. lactis biofilm formation. Using the chain-forming ability of the acmA strain as a phenotypic indicator of PG integrity, we selected for insertional mutants generating short chains. Five independent mutants were all mapped to ponA, which encodes the PG synthesis enzyme PBP1A. Double acmA ponA mutants displayed increased adhesion and biofilm-forming capacity. Direct selection for strains with increased biofilm-forming capacity resulted in the isolation of another five mutations in ponA. Based on these results, we conclude that PG breaks are important for both adhesion and biofilm formation in L. lactis.     

A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis.

A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis (J. W. Sanders, G. Venema, J. Kok, and K. Leenhouts, Mol. Gen. Genet., in press) was exploited for the inducible expression of homologous and heterologous genes. An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR. The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of the lactococcal bacteriophage r1t, and the autolysin gene of L. lactis, acmA. Basal activity of Pgad resulted in a low level of expression of all three proteins. Growth in the presence of 0.5 M NaCl of a strain containing the gadC::lacZ fusion resulted in a 1,500-fold increase of beta-galactosidase activity. The background activity levels of LytPR and AcmA had no deleterious effects on cell growth, but induction of lysin expression by addition of 0.5 M NaCl resulted in inhibition of growth. Lysis was monitored by following the release of the cytoplasmic marker enzyme PepX. Released PepX activity was maximal at 1 day after induction of lytPR expression with 0.1 M NaCl. Induction of acmA expression resulted in slower release of PepX from the cells. The presence of the inducing agent NaCl resulted in the stabilization of osmotically fragile cells.     

Characterization of the Highly Autolytic Lactococcus lactis subsp. cremoris Strains CO and 2250

Two highly autolytic Lactococcus lactis subsp. cremoris strains (CO and 2250) were selected and analyzed for their autolytic properties. Both strains showed maximum lysis when grown in M17 broth containing a limiting concentration of glucose (0.4 to 0.5%) as the carbohydrate source. Lysis did not vary greatly with pH or temperature but was reduced when strains were grown on lactose or galactose. Growth in M17 containing excess glucose (1%) prevented autolysis, although rapid lysis of L. lactis subsp. cremoris CO did occur in the presence of 1% glucose if sodium fluoride (an inhibitor of glycolysis) was added to the medium. Maximum cell lysis in a buffer system was observed early in the stationary phase, and for CO, two pH optima were observed for log-phase and stationary-phase cells (6.5 and 8.5, respectively). Autolysins were extracted from the cell wall fraction of each strain by using either 4% sodium dodecyl sulfate (SDS), 6 M guanidine hydrochloride, or 4 M lithium chloride, and their activities were analyzed by renaturing SDS-polyacrylamide gel electrophoresis on gels containing Micrococcus luteus or L. lactis subsp. cremoris CO cells as the substrate. More than one lytic band was observed on each substrate, with the major band having an apparent molecular mass of 48 kDa for CO. Each lytic band was present throughout growth and lysis. These results suggest that at least two different autolytic enzymes are present in the autolytic L. lactis subsp. cremoris strains. The presence of the lactococcal cell wall hydrolase gene, acmA (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrikman, J. Bacteriol. 177:1554-1563, 1995), in strains 2250 and CO was confirmed by Southern hybridization. Analysis of an acmA deletion mutant of 2250 confirmed that the gene was involved in cell separation and had a role in cell lysis.     

Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum

A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.     

Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis.

Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms. The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cremoris MG1363 by complementation of an Escherichia coli mutant. The gene was sequenced, and the putative amino acid sequence was deduced. The promoter was mapped by both primer extension and analysis of beta-galactosidase expressed from strains carrying fusion between upp promoter fragments and the lacLM gene. The results showed that the upp gene was expressed from its own promoter. After in vitro construction of an internal deletion, a upp mutant was constructed by a double-crossover event. This implicated the utilization of a plasmid with a thermosensitive origin of replication and a new and easy way to screen for double crossover events in both gram-positive and gram-negative bacterial strains. The phenotype of the uracil phosphoribosyltransferase-deficient strain was established. Surprisingly, the upp strain is resistant only to very low concentrations of 5-fluorouracil. Secondary mutants in thymidine phosphorylase and thymidine kinase were isolated by selection for resistance to high concentrations of 5-fluorouracil.     

Expression and excretion of human pancreatic secretory trypsin inhibitor in lipoprotein-deletion mutant of Escherichia coli

We constructed a gene coding for the 56-amino acid human pancreatic secretory trypsin inhibitor (PSTI), and ligated it on a plasmid downstream from the trp promoter and the signal peptide sequence of alkaline phosphatase. The resulting plasmid was transfected into a lipoprotein deletion mutant (Escherichia coli JE5505) and the plasmid-carrying cells were induced with 3-indoleacrylic acid. A considerable amount (50 micrograms/ml culture) of the mature PSTI protein was detected in the culture supernatant. The excreted PSTI was identical to the natural PSTI protein with respect to the trypsin-inhibiting activity, the N-terminal and the C-terminal amino acid sequences and the amino acid composition.     

Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain

The gene encoding Mur1, a Streptococcus thermophilus peptidoglycan hydrolase, was cloned by homology with acmA, the Lactococcus lactis major autolysin gene. Mur1 is a 24.7-kDa protein endowed with a putative signal peptide. Sequence analysis evidenced that Mur1 encompasses exactly the AcmA region containing the catalytic domain, but lacks the one containing amino acid repeats involved in cell wall binding. Mur1 appears to be expressed and cell-associated in S. thermophilus, as revealed by immunoblot analysis. These results suggest that the cell wall attachment mode of Mur1 differs from that of most peptidoglycan hydrolases described so far.     

Cell immobilization studies using a cellulose-binding domain fused to PrtP in Lactococcus lactis

The cellulose-binding domain (CBD) of XylA was fused with PrtP, NisP and AcmA anchors derived from Lactococcus lactis under P45 promoter and Usp45 secretion signal. The fusion construct with the anchor PrtP (334 aa) was shown to exhibit the most efficient immobilization. The CBD-PrtP construct on the other hand was not efficiently attached to the cell wall and as such was found mainly in the supernatant. Results also showed that expression of the CBD-NisP anchor fusion led to a similar result. This raised the question if more efficient binding of the anchor to the cell wall by sortase could enhance the efficiency of cell immobilization to the cellulosic material. However, expressing sortase with the CBD-PrtP fusion did not improve the immobilization of the cells to cellulose.     

Tripeptidase gene (pepT) of Lactococcus lactis: molecular cloning and nucleotide sequencing of pepT and construction of a chromosomal deletion mutant.

The gene encoding a tripeptidase (pepT) of Lactococcus lactis subsp. cremoris (formerly subsp. lactis) MG1363 was cloned from a genomic library in pUC19 and subsequently sequenced. The tripeptidase of L. lactis was shown to be homologous to PepT of Salmonella typhimurium with 47.4% identity in the deduced amino acid sequences. L. lactis PepT was enzymatically active in Escherichia coli and allowed growth of a peptidase-negative leucine-auxotrophic E. coli strain by liberation of Leu from a tripeptide. Using a two-step integration-excision system, a pepT-negative mutant of L. lactis was constructed. No differences between the growth of the mutant and that of the wild-type strain in milk or in chemically defined medium with casein as the sole source of essential amino acids were observed.     

利用N-乙酰葡糖胺糖苷酶在乳酸乳球菌表面展示超氧化物歧化酶

分别将乳酸乳球菌(Lactococcus lactis)N-乙酰葡糖胺糖苷酶基因(acmA)的信号肽序列(ss)、C-末端结构域(cA)及全长基因,与来自大肠杆菌(Escherichiacoli)的超氧化物歧化酶(SOD)基因sod构建成融合基因ss-cA-sod和acmA-sod,并连接于表达载体pMG36K,然后导入乳酸乳球菌ATCC11454菌株,获得了能在细胞表面展示SOD的重组工程菌MB193和MB194。经SDS-PAGE验证,重组菌MB193和MB194可分别表达产生分子量约为46和64kD的融合酶蛋白cA-SOD和AcmA-SOD。通过黄嘌呤氧化酶法测定MB193和MB194菌株的全细胞Mn-SOD酶活力分别为(2.63±0.51)U/mL和(3.51±0.64)U/mL,明显高于仅在细胞内表达产生SOD的对照重组菌MB192的酶活性(1.53±0.38)U/mL,且表达融合酶AcmA-SOD的重组菌MB194具有最大的表面展示效率(56.4%)。     

Effect of the deletion of the σ32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli . The P1 promoter of topA has previously been shown to be a σ³²-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is >10-fold more sensitive to heat treatment (52°C) than the wild type. After brief treatment at 42°C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52°C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42°C (displayed on SDS–polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.     

Regulation of the ribonucleotide reductases in Lactococcus lactis subsp. cremoris

Lactococcus lactis has two essential ribonucleotide reductases for DNA biosynthesis and repair which are affected in the presence or absence of oxygen. Expression of glutaredoxin like protein (NrdH), the hydrogen donor for ribonucleotide reductase, was found to be regulated by the FNR like proteins (FlpA and FlpB). Proteomics study demonstrated that expression level of NrdH significantly decreased in the flpA and flpAB deletion mutants. The nrdH gene is located in an nrdHIEF operon and encoding the NrdEF ribonucleotide reductase, which is active under aerobic and anaerobic conditions. Regulation of expression of the nrdHIEF operons was investigated using beta-galactosidase as a reporter gene. The 588 bp fragment containing the nrdH promoter and gene cloned into the pORI vector immediately upstream of a promoterless lacZ gene. Constructed plasmid was transferred into wild type (MG1363), single mutant (flpA orflpB) and double mutant (flpAB). Aerobically, nrdH promoter activity is 15-fold higher than anaerobic expression.