University of Groningen Lactococcus lactis bacteriophages: phage-host interaction and phage transduction Marcelli, Barbara

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Lactococcus lactis bacteriophages: phage-host interaction and phage transduction

Marcelli, Barbara

DOI:

10.33612/diss.118088081

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Publication date: 2020

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Marcelli, B. (2020). Lactococcus lactis bacteriophages: phage-host interaction and phage transduction. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.118088081

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Summary and general discussion

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Bacteriophage research started one hundred years ago when physician Frederick Twort and microbiologist Felix D’Herelle independently documented the appearance of “transparent spots” on their bacterial plates. These proved later on to be zones of bacterial lysis caused by the action of a type of viruses that infect bacterial cells: bacteriophages (or short: phages) (1, 2). Since then, the know-how of these bacterial viruses has made great progresses. Phages are now known to be the most abundant biological entities on the planet with an estimated total number of 1031_{. They can be found in all biological}

niches and it is believed that every bacterial cell can be infected by, at least, one type of phage (3). They play an important role in driving bacterial evolution, for example by performing transduction, a process of horizontal gene transfer (HGT) (4) and by constantly forcing their hosts to develop new and improved resistance mechanisms (5). Finally, bacteriophages are thought to be involved in determining oxygen production rates in ocean environments (6), in contributing to the toxicity of many bacterial species (7, 8), and in taking part in maintaining the microbial equilibrium in different biological niches (9, 10) .

Advances in next generation sequencing (NGS) techniques and the parallel decrease of the costs linked to this type of analysis make sequencing approaches increasingly more accessible to scientists worldwide. As a result, the number of whole bacteriophage genomes available to date in the NCBI database is nearly 9,300 which, compared with the same number registered in 2014 (1,500) (11), gives a good picture of the high rate at which new information is steadily made available to the scientific community. These advances have, in turn, led to a revision of bacteriophage taxonomy that is currently undertaken to better account for all the new phage genome sequences that are constantly released. It is foreseen that a genomic-based approach for classification will enable more accurate comparisons of bacteriophages that are at the moment considered to be distantly related (12, 13). Despite the significant progress obtained by bacteriophage research over the past decades, a lot still needs to be learned. For instance, the majority of the available bacteriophage genomes have not been experimentally evaluated, so that the function of most of the phage-encoded proteins is, nowadays, still unknown and waiting to be unraveled (14).

Bacteriophages infecting the Gram-positive lactic acid bacterium Lactococcus lactis are among the most thoroughly investigated, because of the detrimental effect that they can have on industrial dairy fermentation processes in which L. lactis is employed as a starter culture. Infection of starter cultures by bacteriophages is, in fact, one of the most common causes of fermentation failure and accounts for important economic losses in the dairy industry (15). To date, phages belonging to ten different species are known to infect L. lactis (16) with phages of three of the species, namely c2, 936 and P335, being the most commonly encountered in dairy environments (17, 18). Phage morphology, genome

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SUMMARY AND GENERAL DISCUSSION

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structure, host receptors and phage-host interaction have been thoroughly investigated in lactococcal phages of these three species (19–22). The information collected has proven extremely useful for the selection of the appropriate starter strains to be employed in industrial processes and in the establishment of strain rotation plans aimed at reducing bacteriophages spreading in production plants (23). Phages belonging to less commonly isolated species have also been shown to be able to colonize and spread in dairy plants (24–26) and, hence, research on every lactococcal bacteriophage species is significant both for the implications it can have for industrial fermentation engineering, and for the contribution it can give to bacteriophage-related data collection.

This thesis work was aimed at broadening the available knowledge on lactococcal bacteriophages of different species by analyzing a collection of phage isolates derived from dairy environments. The focus of this study was directed towards different aspects of bacteriophage biology: genome sequences, phage-host interaction, and phage-mediated horizontal gene transfer were investigated, and new insights were provided in all of these topics (Figure 1). Deep sequencing of the genomic DNA of all phage isolates enabled analyzing some of the most important genetic traits involved in phage-host interaction, which led to the identification of a new bacteriophage belonging to the rare lactococcal phage species 1706. We also unraveled the nature of the host receptor that this rare phage recognizes. Furthermore, the possible employment of bacteriophage transduction as a biotechnological tool for the improvement of metabolic activities of lactococcal starter strains was investigated. Finally, we here report, to the best of our knowledge, the first specific analysis of the establishment of a carrier state life cycle (CSLC) in L. lactis by a bacteriophage belonging to the c2 species, thus providing a new perspective on this poorly investigated phage replication mechanism.

Next generation sequencing (NGS) data analysis as an important approach in phage research

NGS (performed using the Illumina technology) data analysis played an essential role in the studies reported in this thesis.

In Chapter 2, a collection of dairy-isolated bacteriophages was studied in order to analyze the genomic determinants of phage-host recognition. First, the host range of all the phages was tested against a panel of L. lactis dairy isolates and laboratory strains. Second, the whole genome sequences of all phages and strains were determined. We could, thus, perform a comparative analysis of all the bacteriophages in our library and provide new insights into the correlation that exists between phage-host interaction phenotypic results and genomic characteristics of the lactococcal phages and their host strains. A closer analysis of bacteriophages belonging to the 936 species revealed that a total of four 936 bacteriophages in our library, namely MP111, MP118, MP144 and MP145,

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Figure 1. Schematic representation of the main topics studied in each chapter of this thesis and of their

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cannot be assigned with certainty to any of the known 936 bacteriophage subgroups. These subgroups have previously been proposed and are based on the correlation between phage receptor binding proteins (RBP) and the type of cell wall polysaccharide of their corresponding hosts (27). The aa sequences of the RBPs of the four phages mentioned above are very similar to each other and to those of other publicly available but unclassified 936 phages. However, they share low aa sequence similarity with the RBPs of previously classified 936 bacteriophages. These results indicate the possible existence of a new and yet uncharacterized 936-phage subgroup or, alternatively, the need to revise the criteria presently used to classify bacteriophages of this species. While investigating bacteriophages of the c2 species, our in silico analyses allowed firstly to divide these bacteriophages into the two known subspecies c2 and bIL67 (28), and the lactococcal strains into several groups on the basis of mutation patterns in the two protein receptors recognized by phages of the two subspecies: Pip and YjaE, respectively (28). The large data set derived from this analysis allowed confirming what is currently known regarding phage-host interaction in c2 phages and provided additional valuable in silico information for future studies of lactococcal bacteriophages. Secondly, we identified at least one exception to the current knowledge on phage-host interaction in c2 bacteriophages: the bIL67-type phage MP134 could only infect L. lactis MG1363, a strain that possesses a highly mutated YjaE receptor and a non-mutated Pip receptor, contrary to what would be expected. This result shows that the interaction of c2 bacteriophages with their hosts might be more intricate than currently known, a discovery that requires further investigation. Notwithstanding this, the outcome of this analysis could be interpreted in a different way if results of Chapter 5 of this thesis are also taken into consideration. In this chapter, a poorly investigated bacteriophage replication mechanism known as carrier state life cycle (CSLC) (29) was explored in L. lactis MG1363. Thorough analysis of the NGS data of the genome of this strain demonstrated that a CSLC bacteriophage (belonging to the c2 species) was present in a population of this strain. Our in silico and experimental data support the hypothesis that, upon infection of L. lactis MG1363 by phage CHPC966 (referred to as MP134 in chapter 2), the CSLC bacteriophage enters a more active lytic replication cycle producing a new phage progeny. Surprisingly, different single plaque-isolated CSLC phages derived from this infection event showed considerably different host ranges. Furthermore, a meticulous analysis of a selected group of these isolates demonstrated that the genomes of these phages are, in fact, not identical. To the best of our knowledge, this is the first reported study of a specific CSLC bacteriophage belonging to the c2 lactococcal phage species. Also, the data suggest that phages of this species might exploit this replication system as a mechanism for fast evolution and adaptation to new hosts. We hypothesize that CSLC represents an evolutionary strategy that could be employed by more bacteriophage species and that, in this regard, our work provides pioneering results for future investigation of this enigmatic bacteriophage life style. Finally, we believe that these results can help interpreting the before-mentioned result

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regarding the ability of phage MP134 to infect L. lactis MG1363, despite the fact that the

in silico analysis of this interaction predicted this to be not feasible. It is possible that the

plaques obtained during the host range assessment of phage MP134 were in fact caused by the newly discovered CSLC c2 bacteriophage residing inside the L. lactis MG1363 population. The existence of a CSLC phage in a population of this strain was, in fact, not known at the time when the Chapter 2 experiments were performed. This unexpected outcome highlights how important the characterization of new starter strains is in an industrial setting, and that deep sequencing of these strains might often or always be needed to reveal the possible presence of bacteriophages in a carrier state. Such studies should help identifying the culprits of fermentation failure and preventing infection outbreaks that are at the moment impossible to foresee.

Analyzing the least known phages

The specific species to which one of the bacteriophages of our library (named CHPC971) belongs, could be assessed after analyzing its complete genome sequence and comparing it with those of known bacteriophages registered in the NCBI. CHPC971 is only the 6th

reported isolate belonging to the rare lactococcal bacteriophage species 1706 (24, 30). A more detailed investigation on this bacteriophage is described in Chapter 3. We combined sugar competition assays, microscopy analysis and in silico investigation of phage-resistant derivatives of its original industrial host to prove that the receptor recognized by phage CHPC971 is a sugar molecule embedded in the cell wall polysaccharide pellicle that surrounds lactococcal cells. Furthermore, we were able to identify rhamnose as the main sugar involved in this specific phage-host interaction. We foresee this new data to be particularly useful for the optimization of those dairy fermentation processes that require using starter cultures prone to be infected by phages belonging to this rare species. Finally, a more in-depth analysis of the genome sequence of phage CHPC971 identified the candidate genes involved in host recognition, broadening the current information on this topic for the 1706 phages. It is noteworthy to mention that the genome of phage CHPC971 and of the five other 1706 isolates is composed, among others, of many genes with high similarity to genes of prophages of Firmicutes other than L. lactis such as Ruminococcus

torques and Clostridoum leptum. The confirmation that the phage CHLC971 genome also

has this peculiar composition is a further indication that 1706 phages might be derived from the acquisition, by an R. torques or C. leptum bacteriophage, of genes necessary to proliferate on lactococcal cells, as previously hypothesized (24). We believe that this peculiar genomic composition provides new confirmation that bacteriophage genomes evolve by acquisition of distinct functional modules (31).

Bacteriophages: not only enemies, but also exploitable biotechnological tools

With L. lactis playing such an indispensable role in industrial dairy fermentation it is not surprising that dairy companies are in constant need for new and reliable tools that can

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be used to improve, among others, the metabolic abilities of lactococcal starter strains. European Union legislation, however, forbids employing genetically modified organisms (GMOs) for the manufacture of foods (32). Therefore, all biotechnological tools routinely used in research laboratories to genetically modify bacterial strains cannot be utilized for the modification of any bacterial strain employed in food production. In view of this legislative impasse, we aimed at exploiting the phage-mediated horizontal gene transfer mechanism known as phage transduction for the mobilization of industrially relevant genetic traits among lactococcal strains. As phage transduction is a naturally occurring process, lactococcal strains obtained through this route should receive EU approval for use in industrial starter cultures. Starting from the bacteriophage collection already presented in Chapter 2, we identified three lytic bacteriophages as candidates for the establishment of a transduction protocol in L. lactis MG1363 and its derivative MG1614 as proof of concept that this method can be employed in industrial settings. Our choice was focused on strictly lytic bacteriophages because we aimed at establishing a tool that would rule out the possible integration, in the chromosomes of industrial strains, of intact or truncated phage genomes as that could lead to subsequent phage activation and consequent undesired bacterial lysis. The results in Chapter 4 prove that plasmid DNA can be successfully transferred by the three selected lytic phages. Several bottlenecks and restrictions can thwart successful transduction experiment: the length of the DNA fragment to be transferred cannot exceed the capacity of the phage capsid; the transduction frequency might depend on the sequence of the transferred DNA fragment; different bacteriophages could transduce the same DNA fragment at different frequencies. Our findings also indicate that DNA of chromosomal origin might prove more difficult to transfer using lytic phages. Even though chromosomal DNA transfer was not successfully achieved in our experimental settings, we cannot rule out that it can be attained after further testing of the bacteriophages selected here, or by different bacteriophages. In conclusion, the data reported in Chapter 2 demonstrate that phage transduction could, in principle, be used to transfer desirable genetic traits to relevant industrial lactococcal strains. However, it also highlights how laborious the setup of a proper protocol can prove to be. Despite the (majority of the) scientific community having often advocated the eventual approval of utilizing GMOs in European foods, this still seems to be difficult to achieve. I still believe that, although alternatives are possible and currently employed in industrial settings, the most rational foreseeable future should aim at overcoming this legislative obstacle.

Final remarks

In this thesis, various aspects of lactococcal bacteriophage biology have been investigated. The data presented provide new and useful information for future investigation not only on lactococcal bacteriophages, but on phages infecting Gram-positive bacteria in

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general. Although experimentally obtained results have been the basis of all the studies presented here, the analyses of the data extrapolated from full genome sequencing of both lactococcal bacteriophages and bacterial host strains allowed identifying candidate genes involved in host recognition, comparing bacteriophage genetic features and validating phenotypic evidence. An example of how powerful the combination of in silico and experimental approaches has been, is the identification of an CSLC bacteriophage that has gone undetected until now but is shown here to be present in the population of cells of many samples of the most studied lactococcal model strain, L. lactis MG1363. Analysis and careful interpretation of the data generated from deep sequencing of this lactococcal strain and of the phage progeny obtained during the experimental procedures, provided the data needed to confirm the very presence of the phage in the bacterial population and to prove the heterogeneity of the phage progeny.

Although it is clear that NGS represents an invaluable tool when combined with experimental approaches, an urgent need exists for the development of new bioinformatics tools that can deal with the rapidly increasing amounts of sequencing data that is continuously generated. This is especially true for bacteriophage research where a vast number of bacteriophage genes and genomes are now identified in metagenomics studies but cannot be validated due to the lack of information regarding their origin and potential host strains. This, in turn, is still making it more difficult to compare results from different studies and, undoubtedly, to discover new phage species and phage-host interaction mechanisms. In an era in which bacteriophage research is experiencing a renewed interest from the scientific community, we believe that this thesis work represents a prime example of how important the implementation of bacteriophage-related in silico data analysis tools will prove to be.

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