Resistance to the Bacteriocin Lcn972 Deciphered by Genome Sequencing.

In view of the current threat of antibiotic resistance, new antimicrobials with low risk of resistance development are demanded. Lcn972 is a lactococcal bacteriocin that inhibits septum formation by binding to the cell wall precursor lipid II in Lactococcus. It has a species-specific spectrum of activity, making Lcn972 an attractive template to develop or improve existing antibiotics. The aim of this work was to identify mutations present in the Lcn972-resistant clone Lactococcus cremoris D1-20, previously evolved from the sensitive strain L. cremoris MG1614. Whole-genome sequencing and comparison over the reference genome L. cremoris MG1363 identified several unexpected mutations in the parental strain MG1614, likely selected during in-house propagation. In the Lcn972R clone, two previously identified mutations were mapped and confirmed. Additionally, another transposition event deregulating cellobiose uptake was identified along with three point mutations of unknown consequences for Lcn972 resistance. Two new independent evolution experiments exposing L. cremoris MG1614 to Lcn972 revealed transposition of IS981 into the LLMG_RS12285 locus as the predominant mutation selected by Lcn972. This event occurs early during evolution and was found in 100% of the evolved clones, while other mutations were not selected. Therefore, activation of LLMG_RS12285 coding for a putative anti-ECF (extra-cytoplasmic function) sigma factor is regarded as the main Lcn972 resistance factor in L. cremoris MG1614.

Cell wall modifications that alter the exolytic activity of lactococcal phage endolysins have little impact on phage growth.

Bacteriophages are a nuisance in the production of fermented dairy products driven by starter bacteria and strategies to reduce the risk of phage infection are permanently sought. Bearing in mind that the bacterial cell wall plays a pivotal role in host recognition and lysis, our goal was to elucidate to which extent modifications in the cell wall may alter endolysin activity and influence the outcome of phage infection in Lactococcus. Three lactococcal endolysins with distinct catalytic domains (CHAP, amidase and lysozyme) from phages 1,358, p2 and c2 respectively, were purified and their exolytic activity was tested against lactococcal mutants either overexpressing or lacking genes involved in the cell envelope stress (CES) response or in modifying peptidoglycan (PG) composition. After recombinant production in E. coli, Lys1358 (CHAP) and LysC2 (muramidase) were able to lyse lactococcal cells in turbidity reduction assays, but no activity of LysP2 was detected. The degree of PG acetylation, namely C6-O-acetylation and de-N-acetylation influenced the exolytic activity, being LysC2 more active against cells depleted of the PG deacetylase PgdA and the O-acetyl transferase OatA. On the contrary, both endolysins showed reduced activity on cells with an induced CES response. By measuring several growth parameters of phage c2 on these lactococcal mutants (lytic score, efficiency of plaquing, plaque size and one-step curves), a direct link between the exolytic activity of its endolysin and phage performance could not be stablished.

Bacteriocin-phage interaction (BaPI): Phage predation of Lactococcus in the presence of bacteriocins.

Bacteriophages infecting dairy starter bacteria are a leading cause of milk fermentation failure and strategies to reduce the risk of phage infection in dairy settings are demanded. Along with dairy starters, bacteriocin producers (protective cultures) or the direct addition of bacteriocins as biopreservatives may be applied in food to extend shelf-life. In this work, we have studied the progress of infection of Lactococcus cremoris MG1363 by the phage sk1, in the presence of three bacteriocins with different modes of action: nisin, lactococcin A (LcnA), and lactococcin 972 (Lcn972). We aimed to reveal putative bacteriocin-phage interactions (BaPI) that could be detrimental and increase the risk of fermentation failure due to phages. Based on infections in broth and solid media, a synergistic effect was observed with Lcn972. This positive sk1-Lcn972 interaction could be correlated with an increased burst size. sk1-Lcn972 BaPI occurred independently of a functional SOS and cell envelope stress response but was lost in the absence of the major autolysin AcmA. Furthermore, BaPI was not exclusive to the sk1-Lcn972 pairing and could be observed with other phages and lactococcal strains. Therefore, bacteriocins may facilitate phage predation of dairy lactococci and their use should be carefully evaluated.

Combined use of bacteriocins and bacteriophages as food biopreservatives. A review.

Throughout history, humans have consistently developed strategies to prevent food-associated illnesses. However, despite our multiple technological advances, food safety is still an issue of concern. Moreover, there is a demand for gaining access to less processed and naturally preserved food. Food biopreservation, understood as the use of natural antimicrobials already present in food with a long history of safe consumption, is seen as a plausible strategy to reduce the intensity of current preservation technologies (e.g., presence of chemically synthesized food preservatives). In that sense, the combined use of several antimicrobial strategies, known as hurdle technology, has been often chosen as a means to improve the efficacy of food biopreservation. This review intends to summarize the most recent examples of the combined use of bacteriocins and bacteriophages to extend food shelf-life and reduce the risks associated with the presence of foodborne bacteria along the food chain. However, while the efficacy of bacteriocins has been extensively documented, bacteriophages have only started to be assessed as potential food biopreservatives more recently. Within this context, we would like to consider whether these two types of natural antimicrobials would help each other to overcome bottlenecks in food biopreservation.

Resident TP712 Prophage of Lactococcus lactis Strain MG1363 Provides Extra Holin Functions to the P335 Phage CAP for Effective Host Lysis.

Prophages are widely present in Lactococcus lactis, a lactic acid bacterium (LAB) that plays a key role in dairy fermentations. L. lactis MG1363 is a laboratory strain used worldwide as a model LAB. Initially regarded as plasmid and prophage free, MG1363 carries two complete prophages, TP712 and MG-3. Only TP712 seems to be inducible but unable to lyse the host. Several so-called TP712 lysogens able to lyse upon prophage induction were reported in the past, but the reason for their lytic phenotype remained unknown. In this work, we describe CAP, a new P335 prophage detected in the "lytic TP712 lysogens" which had remained unnoticed. CAP is able to be excised after mitomycin C treatment, along with TP712, and able to infect L. lactis MG1363-like strains but not the lytic TP712 lysogens. Both phages cooperate for efficient host lysis. While the expression in trans of the CAP lytic genes was sufficient to trigger cell lysis, this process was boosted when the resident TP712 prophage was concomitantly induced. Introduction of mutations into the TP712 lytic genes revealed that its holin but not its endolysin plays a major role. Accordingly, it is shown that the lytic activity of the recombinant CAP endolysin relies on membrane depolarization. Revisiting the seminal work that generated the extensively used L. lactis MG1363 strain led us to conclude that the CAP phage was originally present in its ancestor, L. lactis NCDO712, and our results solved long-standing mysteries around the MG1363 resident prophage TP712 reported in the "presequencing" era. IMPORTANCE Prophages are bacterial viruses that integrate into the chromosomes of bacteria until an environmental trigger induces their lytic cycle, ending with lysis of the host. Prophages present in dairy starters can compromise milk fermentation and represent a serious threat in dairy plants. In this work, we discovered that two temperate phages, TP712 and CAP, infecting the laboratory strain Lactococcus lactis MG1363 join forces to lyse the host. Based on the in vitro lytic activity of the LysCAP endolysin, in combination with mutated versions of TP712 lacking either its holin or endolysin, we conclude that this cooperation relies on the combined activity of the holins of both phages that boost the activity of LysCAP. The presence of an additional prophage explains the lytic phenotype of the lysogens formerly thought to be single TP712 lysogens that had remained a mystery for many years.

Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in Lactococcus lactis.

Resistance against antimicrobial peptides (AMPs) is often mediated by detoxification modules that rely on sensing the AMP through a BceAB-like ATP-binding cassette (ABC) transporter that subsequently activates a cognate two-component system (TCS) to mount the cell response. Here, the Lactococcus lactis ABC transporter YsaDCB is shown to constitute, together with TCS-G, a detoxification module that protects L. lactis against bacitracin and the bacteriocin Lcn972, both AMPs that inhibit cell wall biosynthesis. Initially, increased expression of ysaDCB was detected by RT-qPCR in three L. lactis resistant to Lcn972, two of which were also resistant to bacitracin. These mutants shared, among others, single-point mutations in ysaB coding for the putative Bce-like permease. These results led us to investigate the function of YsaDCB ABC-transporter and study the impact of these mutations. Expression in trans of ysaDCB in L. lactis NZ9000, a strain that lacks a functional detoxification module, enhanced resistance to both AMPs, demonstrating its role as a resistance factor in L. lactis. When the three different ysaB alleles from the mutants were expressed, all of them outperformed the wild-type transporter in resistance against Lcn972 but not against bacitracin, suggesting a distinct mode of protection against each AMP. Moreover, P ysaD promoter fusions, designed to measure the activation of the detoxification module, revealed that the ysaB mutations unlock transcriptional control by TCS-G, resulting in constitutive expression of the ysaDCB operon. Finally, deletion of ysaD was also performed to get an insight into the function of this gene. ysaD encodes a secreted peptide and is part of the ysaDCB operon. YsaD appears to modulate signal relay between the ABC transporter and TCS-G, based on the different response of the P ysaD promoter fusions when it is not present. Altogether, the results underscore the unique features of this lactococcal detoxification module that warrant further research to advance in our overall understanding of these important resistance factors in bacteria.

Insight into the Lytic Functions of the Lactococcal Prophage TP712.

The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria.

Adaptive Evolution of Industrial Lactococcus lactis Under Cell Envelope Stress Provides Phenotypic Diversity.

Lactococcus lactis is widely used as a starter in the manufacture of cheese and fermented milk. Its main role is the production of lactic acid, but also contributes to the sensory attributes of cheese. Unfortunately, the diversity of suitable strains to be commercialized as dairy starters is limited. In this work, we have applied adaptive evolution under cell envelope stress (AE-CES) as means to provide evolved L. lactis strains with distinct physiological and metabolic traits. A total of seven strains, three of industrial origin and four wild nisin Z-producing L. lactis, were exposed to subinhibitory concentrations of Lcn972, a bacteriocin that triggers the cell envelope stress response in L. lactis. Stable Lcn972 resistant (Lcn972R) mutants were obtained from all of them and two mutants per strain were further characterized. Minimal inhibitory Lcn972 concentrations increased from 4- to 32-fold compared to their parental strains and the Lcn972R mutants retained similar growth parameters in broth. All the mutants acidified milk to a pH below 5.3 with the exception of one that lost the lactose plasmid during adaptation and was unable to grow in milk, and two others with slower acidification rates in milk. While in general phage susceptibility was unaltered, six mutants derived from three nisin Z producers became more sensitive to phage attack. Loss of a putative plasmid-encoded anti-phage mechanism appeared to be the reason for phage susceptibility. Otherwise, nisin production in milk was not compromised. Different inter- and intra-strain-dependent phenotypes were observed encompassing changes in cell surface hydrophobicity and in their autolytic profile with Lcn972R mutants being, generally, less autolytic. Resistance to other antimicrobials revealed cross-protection mainly to cell wall-active antimicrobials such as lysozyme, bacitracin, and vancomycin. Finally, distinct and shared non-synonymous mutations were detected in the draft genome of the Lcn972R mutants. Depending on the parental strain, mutations were found in genes involved in stress response, detoxification modules, cell envelope biogenesis and/or nucleotide metabolism. As a whole, the results emphasize the different strategies by which each strain becomes resistant to Lcn972 and supports the feasibility of AE-CES as a novel platform to introduce diversity within industrial L. lactis dairy starters.

Bacteriophages in the Dairy Environment: From Enemies to Allies.

The history of dairy farming goes back thousands of years, evolving from a traditional small-scale production to the industrialized manufacturing of fermented dairy products. Commercialization of milk and its derived products has been very important not only as a source of nourishment but also as an economic resource. However, the dairy industry has encountered several problems that have to be overcome to ensure the quality and safety of the final products, as well as to avoid economic losses. Within this context, it is interesting to highlight the role played by bacteriophages, or phages, viruses that infect bacteria. Indeed, bacteriophages were originally regarded as a nuisance, being responsible for fermentation failure and economic losses when infecting lactic acid bacteria, but are now considered promising antimicrobials to fight milk-borne pathogens without contributing to the increase in antibiotic resistance.

Reduced Binding of the Endolysin LysTP712 to Lactococcus lactis ΔftsH Contributes to Phage Resistance.

Absence of the membrane protease FtsH in Lactococcus lactis hinders release of the bacteriophage TP712. In this work we have analyzed the mechanism responsible for the non-lytic phenotype of L. lactis ΔftsH after phage infection. The lytic cassette of TP712 contains a putative antiholin-pinholin system and a modular endolysin (LysTP712). Inducible expression of the holin gene demonstrated the presence of a dual start motif which is functional in both wildtype and L. lactis ΔftsH cells. Moreover, simulating holin activity with ionophores accelerated lysis of wildtype cells but not L. lactis ΔftsH cells, suggesting inhibition of the endolysin rather than a role of FtsH in holin activation. However, zymograms revealed the synthesis of an active endolysin in both wildtype and L. lactis ΔftsH TP712 lysogens. A reporter protein was generated by fusing the cell wall binding domain of LysTP712 to the fluorescent mCherry protein. Binding of this reporter protein took place at the septa of both wildtype and L. lactis ΔftsH cells as shown by fluorescence microscopy. Nonetheless, fluorescence spectroscopy demonstrated that mutant cells bound 40% less protein. In conclusion, the non-lytic phenotype of L. lactis ΔftsH is not due to direct action of the FtsH protease on the phage lytic proteins but rather to a putative function of FtsH in modulating the architecture of the L. lactis cell envelope that results in a lower affinity of the phage endolysin to its substrate.

Modulation of Lactobacillus casei bacteriophage A2 lytic/lysogenic cycles by binding of Gp25 to the early lytic mRNA.

The genetic switch of Lactobacillus casei bacteriophage A2 is regulated by the CI protein, which represses the early lytic promoter PR and Cro that abolishes expression from the lysogenic promoter PL . Lysogens contain equivalent cI and cro-gp25 mRNA concentrations, i.e., CI only partially represses P(R), predicting a lytic cycle dominance. However, A2 generates stable lysogens. This may be due to Gp25 binding to the cro-gp25 mRNA between the ribosomal binding site and the cro start codon, which abolishes its translation. Upon lytic cycle induction, CI is partially degraded, cro-gp25 mRNA levels increase, and Cro accumulates, launching viral progeny production. The concomitant concentration increase of Gp25 restricts cro mRNA translation, which, together with the low but detectable levels of CI late during the lytic cycle, promotes reentry of part of the cell population into the lysogenic cycle, thus explaining the low proportion of L. casei lysogens that become lysed (∼ 1%). A2 shares its genetic switch structure with many other Firmicutes phages. The data presented may constitute a model of how these phages make the decision for lysis versus lysogeny.

Surface glycosaminoglycans protect eukaryotic cells against membrane-driven peptide bacteriocins.

Enzymatic elimination of surface glycosaminoglycans or inhibition of their sulfation provokes sensitizing of HT-29 and HeLa cells toward the peptide bacteriocins nisin A, plantaricin C, and pediocin PA-1/AcH. The effect can be partially reversed by heparin, which also lowers the susceptibility of Lactococcus lactis to nisin A. These data indicate that the negative charge of the glycosaminoglycan sulfate residues binds the positively charged bacteriocins, thus protecting eukaryotic cells from plasma membrane damage.

Differential expression of cro, the lysogenic cycle repressor determinant of bacteriophage A2, in Lactobacillus casei and Escherichia coli.

Expression of bacteriophage A2-encoded cro in Escherichia coli gives rise to two co-linear polypeptides, Cro and Cro*, which were proposed to form a regulatory tandem to modulate the frequency with which the phage would choose between the lytic and the lysogenic cycles. In this communication, it is reported that Cro is the canonical product of the gene cro while Cro* results from a -1 ribosome frameshift during translation and is twelve amino acids shorter than Cro. However, frameshifting was not observed during phage development in Lactobacillus casei. Furthermore, wild type phages and cro-frameshifting negative mutants present the same phenotype, thus corroborating that only the canonical form of Cro is needed to produce a viable phage progeny.

Surface glycosaminoglycans mediate adherence between HeLa cells and Lactobacillus salivarius Lv72.

BACKGROUND: The adhesion of lactobacilli to the vaginal surface is of paramount importance to develop their probiotic functions. For this reason, the role of HeLa cell surface proteoglycans in the attachment of Lactobacillus salivarius Lv72, a mutualistic strain of vaginal origin, was investigated. RESULTS: Incubation of cultures with a variety of glycosaminoglycans (chondroitin sulfate A and C, heparin and heparan sulfate) resulted in marked binding interference. However, no single glycosaminoglycan was able to completely abolish cell binding, the sum of all having an additive effect that suggests cooperation between them and recognition of specific adhesins on the bacterial surface. In contrast, chondroitin sulfate B enhanced cell to cell attachment, showing the relevance of the stereochemistry of the uronic acid and the sulfation pattern on binding. Elimination of the HeLa surface glycosaminoglycans with lyases also resulted in severe adherence impairment. Advantage was taken of the Lactobacillus-glycosaminoglycans interaction to identify an adhesin from the bacterial surface. This protein, identify as a soluble binding protein of an ABC transporter system (OppA) by MALDI-TOF/(MS), was overproduced in Escherichia coli, purified and shown to interfere with L. salivarius Lv72 adhesion to HeLa cells. CONCLUSIONS: These data suggest that glycosaminoglycans play a fundamental role in attachment of mutualistic bacteria to the epithelium that lines the cavities where the normal microbiota thrives, OppA being a bacterial adhesin involved in the process.

Induction, structural characterization, and genome sequence of Lv1, a prophage from a human vaginal Lactobacillus jensenii strain.

The prophage Lv1, harbored by a vaginal Lactobacillus jensenii isolate, was induced by several different anticancer, antimicrobial, and antiseptic agents, suggesting that they contribute to the adverse vaginal effects associated with their therapeutic use. Of special interest with respect to its novelty was the inducing effect of nonoxynol-9, a non-ionic detergent commonly used as a spermicide. The Lv1 genome consists of a 38,934-bp dsDNA molecule with cohesive ends, in which 48 ORFs were recognized, and is organized into functional modules. Lv1 belongs to the family Siphoviridae and, more precisely, to the proposed Sfi21-like genus. The capsid-tail junction of the Lv1 virions is fragile such that most particles become disrupted, suggesting that the virus is defective and thus unable to generate fertile progeny. However, genome analysis did not provide evidence of the defective nature of the prophage, other than the finding that its genome is shorter than those of other, related, phages. Further analysis indicated that prophage Lv1 suffered deletions in its right half to the extent that it no longer fulfill the minimum packaging limits, thereby generating the observed unstable particles.

Induction, structural characterization, and genome sequence of Lv1, a prophage from a human vaginal Lactobacillus jensenii strain

The prophage Lv1, harbored by a vaginal Lactobacillus jensenii isolate, was induced by several different anticancer, antimicrobial, and antiseptic agents, suggesting that they contribute to the adverse vaginal effects associated with their therapeutic use. Of special interest with respect to its novelty was the inducing effect of nonoxynol-9, a non-ionic detergent commonly used as a spermicide. The Lv1 genome consists of a 38,934-bp dsDNA molecule with cohesive ends, in which 48 ORFs were recognized, and is organized into functional modules. Lv1 belongs to the family Siphoviridae and, more precisely, to the proposed Sfi21-like genus. The capsid-tail junction of the Lv1 virions is fragile such that most particles become disrupted, suggesting that the virus is defective and thus unable to generate fertile progeny. However, genome analysis did not provide evidence of the defective nature of the prophage, other than the finding that its genome is shorter than those of other, related, phages. Further analysis indicated that prophage Lv1 suffered deletions in its right half to the extent that it no longer fulfill the minimum packaging limits, thereby generating the observed unstable particles (AU)

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Bacteriophage induction versus vaginal homeostasis: role of H(2)O(2) in the selection of Lactobacillus defective prophages.

Vaginal disorders associated with systemic chemotherapy arise by direct inhibition of the resident microbiota (dominated by lactobacilli) or, possibly, by induction of prophages harbored in their genomes, leading to cell lysis. In the present study, proficient Lactobacillus phages could not be isolated from vaginal exudates. However, lysogeny appeared to be widespread, although about half of the strains harbored prophage sequences that were not responsive to SOS activation. In other cases, prophage induction was achieved, but viable phages were not generated, despite the fact that the induced supernatants of some strains were bactericidal. In one case, this activity was accompanied by the production of a bacteriophage subsequently identified as a member of the family Siphoviridae (isometric capsid and long non-contractile tail). Most of the lactobacilli tested generated hydrogen peroxide, which acted as an inducer of the SOS response, suggesting that H2O2 selects for strains that harbor SOS-insensitive, defective prophages, which are thus unable to promote vaginal lactobacilli phage-induced lysis.

Bacteriophage induction versus vaginal homeostasis: role of H2O2 in the selection of Lactobacillus defective prophages

Vaginal disorders associated with systemic chemotherapy arise by direct inhibition of the resident microbiota (dominated by lactobacilli) or, possibly, by induction of prophages harbored in their genomes, leading to cell lysis. In the present study, proficient Lactobacillus phages could not be isolated from vaginal exudates. However, lysogeny appeared to be widespread, although about half of the strains harbored prophage sequences that were not responsive to SOS activation. In other cases, prophage induction was achieved, but viable phages were not generated, despite the fact that the induced supernatants of some strains were bactericidal. In one case, this activity was accompanied by the production of a bacteriophage subsequently identified as a member of the family Siphoviridae (isometric capsid and long non-contractile tail). Most of the lactobacilli tested generated hydrogen peroxide, which acted as an inducer of the SOS response, suggesting that H2O2 selects for strains that harbor SOS-insensitive, defective prophages, which are thus unable to promote vaginal lactobacilli phage-induced lysis (AU)

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A new eremophilanolide from Senecio sinuatus Gilib.

From the hexane extracts of Senecio sinuatus roots, the new 3beta-angeloyloxy-6beta-hydroxyeremophil-1(10)-en-8beta,12-olide (3), along with the known compounds 3beta-angeloyloxy-6beta-hydroxyeremophil-1(10)-ene (1), 3beta-senecioyloxy-6beta-hydroxyeremophil-1(10)-ene (2), and 3beta-angeloyloxy-6beta,8alpha-dihydroxyeremophil-1(10)-en-8beta,12-olide (4), were isolated. Complete 1H and 13C NMR chemical shift assignments of 1-4 were achieved using one- and two-dimensional NMR techniques, including gHMQC and gHMBC experiments. A Monte Carlo search, followed by B3LYP/6-31G*DFT calculation, provided the theoretical conformations of the eremophilane rings, which were in agreement with results derived from 1H-1H NMR coupling constant analysis, and confirmed by NOESY experiments.