Recognition of Cyclonucleoside Lesions by the Lactococcus lactis FPG Protein

Abstract

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.     

Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been implicated as a host receptor of (certain) 936-type phages, is specified by a large gene cluster, which, among different lactococcal strains, contains highly conserved regions as well as regions of diversity. The regions of diversity within this cluster on the genomes of lactococcal strains MG1363, SK11, IL1403, KF147, CV56, and UC509.9 were used for the development of a multiplex PCR system to identify the pellicle genotype of lactococcal strains used in this study. The resulting comparative analysis revealed an apparent correlation between the pellicle genotype of a given host strain and the host range of tested 936-type phages. Such a correlation would allow prediction of the intrinsic 936-type phage sensitivity of a particular lactococcal strain and substantiates the notion that the lactococcal pellicle polysaccharide represents the receptor for (certain) 936-type phages while also partially explaining the molecular reasons behind the observed narrow host range of such phages.     

Functional Display of a Heterologous Protein on the Surface of Lactococcus lactis by Means of the Cell Wall Anchor of Staphylococcus aureus Protein A

In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S. aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L. lactis. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface. L. lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.     

Expression of a Lactococcus lactis Phage Resistance Mechanism by Streptococcus thermophilus

The 7.8-kb lactococcal plasmid pSRQ700 encodes the LlaII restriction/modification system which recognizes and cleaves the sequence 3(prm1)-GATC-5(prm1). When the plasmid pSRQ700 is introduced into a phage-sensitive Lactococcus lactis strain, strong phage resistance is conferred by the LlaII system. In this report, we show that pSRQ700 cannot replicate in Streptococcus thermophilus. However, if cloned into the vector pNZ123, the native LlaII system is expressed and strong phage resistance is conferred to various industrial S. thermophilus strains. Resistance against phages isolated from yogurt and mozzarella wheys was observed. To our knowledge, this is the first report of increased phage resistance in S. thermophilus.     

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.     

乳酸乳球菌作为基因工程受体菌研究进展

乳酸乳球菌(Lactococcus lactis)是一种"公认安全"的革兰氏阳性细菌,广泛存在于人、畜的肠道中并发挥许多重要的生理功能。由于它兼具安全性与益生性,近几年来研究者们开始关注用乳酸乳球菌作为受体菌来表达外源蛋白。随着生物技术的发展,人们对乳酸乳球菌基因表达及调控过程的认识不断深入并构建了一系列表达,成功地表达了许多外源蛋白,初步展示出良好的应用前景。主要对近年来国内外将乳酸乳球菌作为外源蛋白表达受体菌方面的研究进展做简要综述。     

Crystal Structure of ORF12 from Lactococcus lactis Phage p2 Identifies a Tape Measure Protein Chaperone

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is detected in the second half of the lytic cycle, indicating that it is a late-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-Å resolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface. Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice was identified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeated motif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex between ORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fitted exquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act as a chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of the lactococcal siphophage p2.During industrial milk fermentation, Lactococcus lactis cells are added to transform milk into an array of fermented products such as cheese. However, this manufacturing process may be impaired by lytic phages present in the factory environment as well as in the milk itself (30). Due to the destructive effects of phage infections on bacterial fermentation, much effort has been undertaken to isolate and study the biodiversity of these bacteriophages. Lactococcal bacteriophages belong to at least 10 different genetically distinct species of double-stranded DNA viruses (9). Of them, three lactococcal phage species, all belonging to the Siphoviridae family, are the major source of problems in milk fermentation, namely, the 936, P335, and c2 species (7, 28, 29). Furthermore, members of the 936 species are by far responsible for the majority of infections (50 to 80%) (1, 24, 41). Numerous phages of the 936 species have been isolated, and several have been characterized at the genome level (25). However, little is known concerning their molecular mechanisms of infection, although we recently solved the structure of the receptor-binding protein (RBP) of our model 936-like phage, namely, the virulent phage p2 (38, 43), and of phages belonging to the P335 species (27, 34, 37, 38).As with all viruses, bacteriophage genomes are quite compact, leaving little room for noncoding sequences (4). In fact, phage genes are disposed in an operon-type organization (4), and the order of genes corresponds to the different phases of the infection cycle. Moreover, genes are often in clusters (referred to as modules), with gene products from adjacent genes generally found to interact with each other. Interestingly, phage genome organization, including individual gene order, is often conserved within a given species, particularly within the Siphoviridae family. In the case of L. lactis virulent phages belonging to the 936 or P335 species, this principle applies particularly to the morphogenesis gene module, which includes all the genes coding for the phage structural protein genes. For the tail assembly, a module comprises a set of genes between the portal protein, which is connecting the tail to the capsid, and the RBP, which is located at the tip of the tail and is involved in host recognition (39, 43).The characterization of tail assembly genes of lactococcal phages has been more extensive for temperate siphophages belonging to the P335 species (27, 34, 37, 38). Because of the similarities in genome organization, the findings in this phage species can, in some cases, be used as clues toward understanding the morphology of 936-like phages. For the temperate phage Tuc2009 (P335 species), all structural proteins required for tail and baseplate assembly have been identified (27, 34, 37, 38). Genes located between those coding for the tape measure protein (TMP) and BppL (RBP) were identified as corresponding to components of the baseplate structure, located at the tail distal end. Furthermore, a gene coding for the major tail protein (MTP) was also identified at a position upstream from tmp. Between the genes coding for the MTP and those coding for the TMP in Tuc2009 are two gene products identified as gpG and gpGT, which are not present in the phage particle. These two proteins were named based on their likely role analogous to the tail assembly proteins present in coliphage lambda, a model virus belonging to the Siphoviridae family (21, 27, 47). gpGT has an essential role in lambda tail assembly, acting prior to tail shaft assembly, while the role of gpG in tail assembly is not known (21). Both gpG and gpGT are also absent from mature lambda virions (21). It has been argued that they may act as assembly chaperones (47).A close examination of 936 genomes indicates the presence of two genes coding for gpG and gpGT-like proteins. Analysis of the phage p2 genome, closely related to that of lactococcal phage sk1 (6), revealed that the putative tail assembly proteins could correspond to gene products ORF12 and ORF13. These two genes are followed by the TMP gene corresponding to orf14, other genes coding for other structural proteins, and the RBP gene orf18. During our ongoing investigation of the structure of phage p2, we report here the cloning, expression, and crystal structure of ORF12 in order to decipher its role in the tail assembly process.     

Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.     

Molecular Recognition by a Polymorphic Cell Surface Receptor Governs Cooperative Behaviors in Bacteria

Cell-cell recognition is a fundamental process that allows cells to coordinate multicellular behaviors. Some microbes, such as myxobacteria, build multicellular fruiting bodies from free-living cells. However, how bacterial cells recognize each other by contact is poorly understood. Here we show that myxobacteria engage in recognition through interactions between TraA cell surface receptors, which leads to the fusion and exchange of outer membrane (OM) components. OM exchange is shown to be selective among 17 environmental isolates, as exchange partners parsed into five major recognition groups. TraA is the determinant of molecular specificity because: (i) exchange partners correlated with sequence conservation within its polymorphic PA14-like domain and (ii) traA allele replacements predictably changed partner specificity. Swapping traA alleles also reprogrammed social interactions among strains, including the regulation of motility and conferred immunity from inter-strain killing. We suggest that TraA helps guide the transition of single cells into a coherent bacterial community, by a proposed mechanism that is analogous to mitochondrial fusion and fission cycling that mixes contents to establish a homogenous population. In evolutionary terms, traA functions as a rare greenbeard gene that recognizes others that bear the same allele to confer beneficial treatment.     

RNA Recognition by the MS2 Phage Coat Protein

The interaction of MS2 coat protein and its translational operator hairpin is a very well-characterized RNA–protein complex. The recent high-resolution cocrystal structure successfully explains many biochemical experiments measuring the affinity of the protein–RNA interaction for mutant proteins and chemically modified RNAs. However, an analysis of a tight binding variant of the RNA suggests that the conformation of the free RNA is also an important determinant of the affinity.     

Molecular characterization of a phage-encoded resistance system in Lactococcus lactis

A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009 protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.     

Molecular Characterization of a Phage-Encoded Resistance System in Lactococcus lactis

A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep₂₀₀₉, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep₂₀₀₉ protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.     

Molecular Differentiation of Lactococcus lactis Subspecies lactis and cremoris Strains by Ribotyping and Site Specific-PCR

Twenty-five strains of Lactococcus lactis subspecies lactis and subspecies cremoris obtained from dairy industry and environmental collections were examined by 16S RNA automated ribotyping profiles and site-specific PCR (S-PCR). By automated ribotyping, the majority of strains were classified in accordance with phenotypic characterization, with the exception of one lactis (220) and two cremoris (BO32 and 140) strains. A complete differentiation of subspecies lactis and cremoris in agreement with conventional phenotypic methods was achieved by S-PCR with a set of site-specific primer pairs (PR1, RM4, and F3) designed particularly from a deletion region found in subspecies cremoris, but not in lactis. Therefore, S-PCR with primers (PR1, RM4, and F3) is a rapid and very sensitive method for the distinction of lactis and cremoris subspecies in dairy production. Received: 19 June 2000 / Accepted: 17 July 2000     

Rapid Multiplex Detection and Differentiation of Listeria Cells by Use of Fluorescent Phage Endolysin Cell Wall Binding Domains

The genus Listeria comprises food-borne pathogens associated with severe infections and a high mortality rate. Endolysins from bacteriophages infecting Listeria are promising tools for both their detection and control. These proteins feature a modular organization, consisting of an N-terminal enzymatically active domain (EAD), which contributes lytic activity, and a C-terminal cell wall binding domain (CBD), which targets the lysin to its substrate. Sequence comparison among 12 different endolysins revealed high diversity among the enzyme''s functional domains and allowed classification of their CBDs into two major groups and five subclasses. This diversity is reflected in various binding properties, as determined by cell wall binding assays using CBDs fused to fluorescent marker proteins. Although some proteins exhibited a broad binding range and recognize Listeria strains representing all serovars, others target specific serovars only. The CBDs also differed with respect to the number and distribution of ligands recognized on the cells, as well as their binding affinities. Surface plasmon resonance analysis revealed equilibrium affinities in the pico- to nanomolar ranges for all proteins except CBD006, which is due to an internal truncation. Rapid multiplexed detection and differentiation of Listeria strains in mixed bacterial cultures was possible by combining CBDs of different binding specificities with fluorescent markers of various colors. In addition, cells of different Listeria strains could be recovered from artificially contaminated milk or cheese by CBD-based magnetic separation by using broad-range CBDP40 and subsequently identified after incubation with two differently colored CBD fusion proteins of higher specificity.Listeria belong to the low G+C Gram-positive bacteria; are ubiquitously present in nature; and can be isolated from many sources such as soil, water, sewage effluents, and the feces of humans and animals (52). Within the genus Listeria, six species are recognized (Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi), in addition to the recently proposed new species L. marthii (12) and L. rocourtiae (24). Five major serovar (sv.) groups exist (1/2, 3, 4, 6, and 7), and at least 16 subserovars can be distinguished. Variations are mainly due to the structure and composition of cell wall-associated carbohydrates, wall teichoic acid (WTA), and lipoteichoic acid (LTA). However, serotyping does not necessarily correlate with the species (7, 8). Listeria monocytogenes is an opportunistic, intracellular pathogen causing an infection termed listeriosis and is exclusively transmitted via contaminated food such as raw meat, milk products, fish products, and vegetables. Listeriosis is a serious infection primarily affecting immunocompromised patients, pregnant women, the elderly, and newborns and is characterized by a high mortality rate up to >40% (5). Strains belonging to sv. groups 1/2 and 4b have been responsible for the majority of Listeria infections in humans (52).Based on their host specificity, bacteriophages are useful tools for bacterial detection and differentiation (reviewed in references 40 and 43). To date, many phages infecting Listeria cells have been isolated. All of them are strictly genus specific. The few known virulent (obligately lytic) phages have a very broad host range (19, 29), whereas the majority are temperate and are restricted to a limited number of host strains within the individual serovar groups (32). Various applications based on Listeria phages have been developed, including phage typing (27) and the detection of viable Listeria cells by a recombinant luciferase reporter phage (31).Bacteriophage endolysins are peptidoglycan hydrolases that mediate lysis of the host cell at the end of the lytic multiplication cycle. These enzymes represent powerful tools with many applications in molecular biology, biotechnology, and medicine (26). Listeria phage endolysins show a domain organization and belong to category 1 of modular enzymes, in which catalysis and substrate specificity are clearly separated (18). They feature an N-terminal enzymatically active domain (EAD) and a C-terminal cell wall binding domain (CBD) (30, 33). The EAD determines the catalytic activity of the enzyme, and the CBD is responsible for targeting the protein to the bacterial cell wall. Surprisingly, the CBDs feature high binding specificity; although they are able to lyse all Listeria cell walls, the individual endolysins display highest activity against cells or cell walls from specific serovars. Besides recognition specificity, the CBDs feature very strong, saturation-dependent binding to listerial cell walls, with equilibrium constants in the nanomolar ranges (30). Because of the absence of an outer membrane in Gram-positive bacteria, the cell wall can also be accessed from outside, enabling the CBD to attach to its ligand. These properties can be harnessed for rapid and efficient labeling and immobilization of bacterial cells (23, 30). While CBD118 recognizes cells of Listeria sv. groups 1/2 and 3 and predominantly binds at the polar and septal regions of these cells, CBD500 and CBD PSA (21, 30) exhibit binding over the entire cell surfaces of strains, belonging to sv. groups 4, 5, and 6. Although the ligands recognized by the various CBDs have not been conclusively identified, we have strong evidence for an involvement of cell wall-associated carbohydrates in recognition and binding. Furthermore, the proteins retain their lectinlike binding function in complex matrices and environments, such as infected eukaryotic cells (15) and homogenized food and enrichment cultures (23).Fluorescent proteins (FP) such as the green fluorescent protein are very popular tools in molecular biology, medicine, and cell biology, based on their wide compatibility, lack of toxicity, incredible stability, and the fact that they do not require any cofactors other than oxygen for chromophore formation. Several FP derivatives with shifted spectral characteristics such as blue, cyan, and yellow fluorescent proteins have been developed (reviewed in references 45 and 49), which are useful for simultaneous staining applications. In addition to the green fluorescent protein (GFP) variants, several red fluorescent proteins have been described (14, 34). The RedStar protein (RS) (20) was derived from dsRed and features rapid development of high fluorescence intensity and a reduced tendency for oligomerization.The aim of the present study was to develop a comprehensive toolbox consisting of different combinations of Listeria phage CBDs and FPs. Toward this goal, we first established a classification system for CBDs from all known Listeria phage endolysins, and characterized representative CBDs from each class regarding their binding range binding affinity, and spatial distribution and density of ligands on the cell surface. We then used fluorescent proteins for the construction of differently tagged reporter-CBDs and demonstrate the suitability of using these proteins in a single and simple assay for simultaneous detection and differentiation of Listeria strains in mixed cultures. We also provide proof of concept for application of this technique for differential staining and identification of different Listeria strains after recovery from contaminated food by magnetic separation with CBD-coated paramagnetic beads (CBD-MS).     

Bacteriophage T4D Receptor and the Escherichia coli Cell Wall Structure: Binding of Endotoxin-Like Particles to the Cell Wall

A variety of degradative treatments have been used to investigate the nature of the structure and components of the cell walls of Escherichia coli B. The binding and localization of the endotoxin-like particles found on the cell walls were of special interest because some of them are associated with the site where the inner tail tube of bacteriophage T4D penetrates the cell wall. Modified cell walls were obtained by heating a suspension of bacterial cells originally in 0.1 M phosphate, pH 7.0, after the addition of 12.5 M NaOH to a final concentration of 0.25 M. With regard to the endotoxin-like particles, it was found that: (i) at least part of them still remained bound to the modified cell wall after the alkali treatment; (ii) the subsequent incubation of alkali-treated cell walls with lysozyme destroyed the bacterial form and released a complex of endotoxin-like particles together with a fibrous material; (iii) on the other hand, treatment with 45% phenol at 70°C removed the endotoxin-like particles from the surface of the alkali-treated cell walls, but most of the fibrous material was left on the cell wall; and (iv) incubation of alkali-treated cell walls with 5 mM ethylenediaminetetraacetic acid at 20°C also removed the endotoxin-like particles, but did not disrupt the rodlike bacterial form. However, if the ethylenediaminetetraacetic acid treatment was performed at 55°C, the bacterium-like form was destroyed. These differential sensitivities to ethylenediaminetetraacetic acid suggested that loosely bound divalent metal ions normally hold these endotoxin-like particles on the cell wall surface, but that probably more tightly bound metal ions are involved in the determination of cell shape. Analysis of the protein components of the alkalitreated cell walls showed that only one protein was present in significant amounts, and this protein had an electrophoretic mobility similar to that of the Braun lipoprotein. This protein was released from the alkali-treated cell walls upon heating with 2% sodium dodecyl sulfate at 100°C. Phospholipids were also absent from this structure. The distribution of the remaining cell wall components on the alkali-treated cell walls is discussed.     

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.