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The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.IMPORTANCEIn the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities., Competing Interests: P.P.D.W., I.M.H.V.R., and N.N.M.E.V.P. work for dsm-firmenich.

Until the late 2000s, lactococci substantially contributed to the discovery of various plasmid-borne phage defence systems, rendering these bacteria an excellent antiphage discovery resource. Recently, there has been a resurgence of interest in identifying novel antiphage systems in lactic acid bacteria owing to recent reports of so-called 'defence islands' in diverse bacterial genera. Here, 321 plasmid sequences from 53 lactococcal strains were scrutinized for the presence of antiphage systems. Systematic evaluation of 198 candidates facilitated the discovery of seven not previously described antiphage systems, as well as five systems, of which homologues had been described in other bacteria. All described systems confer resistance against the most prevalent lactococcal phages, and act post phage DNA injection, while all except one behave like abortive infection systems. Structure and domain predictions provided insights into their mechanism of action and allow grouping of several genetically distinct systems. Although rare within our plasmid collection, homologues of the seven novel systems appear to be widespread among bacteria. This study highlights plasmids as a rich repository of as yet undiscovered antiphage systems., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by traAd and trsAd , respectively. Despite their significant sequence divergence, TraAd and TrsAd represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in traAd or trsAd , respectively, were shown to act as efficient donors provided the corresponding recipient expresses either traAd or trsAd , thus demonstrating in trans reciprocal complementarity of these proteins in conjugation systems., Competing Interests: PdeW, IvR & NvP are employed by the company dsm-firmenich. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 The Author(s).)

Lactococcal conjugative plasmids are poorly characterized compared to those harbored by numerous other Gram-positive bacteria, despite their significance in dairy fermentations and starter culture development. Furthermore, the transcriptional landscape of these lactococcal conjugation systems and their regulation have not been studied in any detail. Lactococcal plasmids pNP40 and pUC11B possess two genetically distinct and prevalent conjugation systems. Here, we describe the detailed transcriptional analysis of the pNP40 and pUC11B conjugation-associated gene clusters, revealing three and five promoters, respectively, for which the corresponding transcriptional start sites were identified. Regulation of several of these promoters, and therefore conjugation, is shown to involve the individual or concerted activities of the corresponding relaxase and transcriptional repressor(s) encoded by each conjugative plasmid. This work highlights how the conjugative potential of these systems may be unlocked, with significant implications for the starter culture and food fermentation industry., Competing Interests: PdeW, IvR & NvP are employed by the company dsm-firmenich. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 The Authors. Published by Elsevier B.V.)

Bacteriophages (or phages) represent one of the most persistent threats to food fermentations, particularly large-scale commercial dairy fermentations. Phages infecting lactic acid bacteria (LAB) that are used as starter cultures in dairy fermentations are well studied, and in recent years there have been significant advances in defining the driving forces of LAB-phage coevolution. The means by which different starter bacterial species defend themselves against phage predation and the chromosomal or plasmid location of the genes encoding these defense mechanisms have dictated the technological approaches for the development of robust starter cultures. In this review, we highlight recent advances in defining phage-host interactions and how phage resistance occurs in different bacterial species. Furthermore, we discuss how these insights continue to transform the dairy fermentation industry and how they also are anticipated to guide food fermentations involving plant-based alternatives in the future.

Competence refers to the specialized physiological state in which bacteria undergo transformation through the internalization of exogenous DNA in a controlled and genetically encoded process that leads to genotypic and, in many cases, phenotypic changes. Natural transformation was first described in Streptococcus pneumoniae and has since been demonstrated in numerous species, including Bacillus subtilis and Neisseria gonorrhoeae . Homologs of the genes encoding the DNA uptake machinery for natural transformation have been reported to be present in several lactic acid bacteria, including Lactobacillus spp., Streptococcus thermophilus , and Lactococcus spp. In this review, we collate current knowledge of the phenomenon of natural transformation in Gram-positive bacteria. Furthermore, we describe the mechanism of competence development and its regulation in model bacterial species. We highlight the importance and opportunities for the application of these findings in the context of bacterial starter cultures associated with food fermentations as well as current limitations in this area of research.

In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a laborious manual process, Bionano Genomics platforms now offer high-throughput, automated optical mapping based on chips packed with nanochannels through which unwound DNA is guided and the fluorescent DNA backbone and specific restriction sites are recorded. Although the raw image data obtained is of high quality, the processing and assembly software accompanying the platforms is closed source and does not seem to make full use of data, labeling approximately half of the measured signals as unusable. Here we introduce two new software tools, independent of Bionano Genomics software, to extract and process molecules from raw images (OptiScan) and to perform molecule-to-molecule and molecule-to-reference alignments using a novel signal-based approach (OptiMap). We demonstrate that the molecules detected by OptiScan can yield better assemblies, and that the approach taken by OptiMap results in higher use of molecules from the raw data. These tools lay the foundation for a suite of open-source methods to process and analyze high-throughput optical mapping data. The Python implementations of the OptiTools are publicly available through http://www.bif.wur.nl/., Competing Interests: No authors have competing interests.

Co-cultivation of brewers' yeast (Saccharomyces cerevisiae) with Cyberlindnera fabianii makes it possible to steer aroma and alcohol levels by changing the inoculation ratio of the two yeasts. A dynamic model was developed based on mono-culture performance of brewers' yeast and C. fabianii in controlled bioreactors with aerated wort as medium, describing growth rate, carbohydrate utilization, ethanol production, maintenance, oxygen consumption and ergosterol biosynthesis/use for cell membrane synthesis (the last one only for brewers' yeast). The parameters were estimated by fitting models to experimental data of both mono-cultivations. To predict the fermentation outcome of brewers' yeast and C. fabianii in co-cultivation, the two models were combined and the same parameter settings were used. The co-cultivation model was experimentally validated for the inoculum ratios 1:10 and 1:100 brewers' yeast over C. fabianii. The use of predictive modelling supported the hypothesis that performance of brewers' yeast in co-cultivation is inhibited by oxygen depletion which is required for the biosynthesis of ergosterol. This dynamic modelling approach and the parameters involved may also be used to predict the performance of brewers' yeast in the co-cultivation with other yeast species and to give guidance to optimize the fermentation outcome., (Copyright © 2019. Published by Elsevier Ltd.)

Esters are essential for the flavor and aroma of fermented products, and are mainly produced by alcohol acyl transferases (AATs). A recently discovered AAT family named Eat (Ethanol acetyltransferase) contributes to ethyl acetate synthesis in yeast. However, its effect on the synthesis of other esters is unknown. In this study, the role of the Eat family in ester synthesis was compared to that of other Saccharomyces cerevisiae AATs (Atf1p, Atf2p, Eht1p, and Eeb1p) in silico and in vivo . A genomic study in a collection of industrial S. cerevisiae strains showed that variation of the primary sequence of the AATs did not correlate with ester production. Fifteen members of the EAT family from nine yeast species were overexpressed in S. cerevisiae CEN.PK2-1D and were able to increase the production of acetate and propanoate esters. The role of Eat1p was then studied in more detail in S. cerevisiae CEN.PK2-1D by deleting EAT1 in various combinations with other known S. cerevisiae AATs. Between 6 and 11 esters were produced under three cultivation conditions. Contrary to our expectations, a strain where all known AATs were disrupted could still produce, e.g., ethyl acetate and isoamyl acetate. This study has expanded our understanding of ester synthesis in yeast but also showed that some unknown ester-producing mechanisms still exist.

Increasing interest in new beer types has stimulated the search for approaches to extend the metabolic variation of brewers' yeast. Therefore, we tested two approaches using non-conventional yeast to create a beer with lower ethanol content and a complex aroma bouquet. First, the mono-culture performance was monitored of 49 wild yeast isolates of Saccharomyces cerevisiae (16 strains), Cyberlindnera fabianii (9 strains) and Pichia kudriavzevii (24 strains). Interestingly, both C. fabianii and P. kudriavzevii isolates produced relatively more esters compared with S. cerevisiae isolates, despite their limited fermentation capacity. Next, one representative strain of each species (Sc131, Cf65 and Pk129) was applied as co-culture with brewers' yeast (ratio 1:1). Co-cultures with Cf65 and Pk129 resulted in a beer with lower alcohol content (3.5, 3.8 compared with 4.2% v/v) and relatively more esters. At higher inoculum ratios of Cf65 over brewers' yeast, growth inhibition of brewers' yeast was observed, most likely caused by competition for oxygen between brewers' yeast and Cf65 resulting in a reduced level of ethanol and altered aroma profiles. With this study, we demonstrate the feasibility of using non-conventional yeast species in co-cultivation with traditional brewers' yeast to tailor aroma profiles as well as the final ethanol content of beer., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)