Characterization of two prevalent yet distinct conjugation systems among lactococci

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Ortiz Charneco, Guillermo
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University College Cork
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Lactic acid bacteria are Gram-positive, non-motile, lactic acid-producing microorganisms, with Lactococcus lactis and Lactococcus cremoris representing species that are extensively applied as starter bacteria in dairy fermentations. Starter culture providers continuously aim to expand their culture catalogues and enhance their existing cultures to maintain pace with the ever-changing consumer demand for products with diverse organoleptic attributes. Plasmids present in L. lactis and L. cremoris species harbor genes associated with many important technological traits, such as bacteriophage resistance, lactose metabolism and exopolysaccharide (EPS) production. There are practical limitations associated with the isolation of new strains, while there are regulatory hurdles associated with the application of genetically-modified organisms (GMOs). Consequently, the enhancement of existing starter strains through natural gene transfer methods including conjugation can be a highly advantageous approach, though with its challenges as (plasmid) conjugation efficiencies between lactococcal strains reported in literature have been very low. This thesis describes the optimization and characterization of two plasmid-encoded conjugation systems that are commonly found in L. lactis and L. cremoris strains. Chapter II details an optimized protocol for conjugation with the lactococcal conjugative plasmid pNP40, which represents a highly prevalent conjugation system among lactococci. A combined approach of in silico and mutational analyses facilitated the functional assignment of many conjugation-associated genes including a transcriptional repressor of this system. Furthermore, certain non-conjugative plasmids were observed to co-mobilise together with the conjugative plasmid. Chapter III details the mutational and functional analysis of the pUC11B conjugation gene cluster, which was demonstrated to be capable of self-transfer using the optimized conjugation protocol described in Chapter II. This conjugation system, along with those encoded by plasmids pMRC01 and pAF22, represents the second most prevalent type of conjugation system among lactococcal plasmids in public databases. Within the conjugation gene cluster, a transcriptional repressor and an anti-restriction modification (RM) system were identified. Interestingly, the anti-RM system promotes more efficient conjugation of this plasmid into recipient strains with Type II or Type III RM systems. Chapter IV focuses on the transcriptional analysis of the conjugation gene clusters of pNP40 and pUC11B, revealing the presence of three and five promoters, respectively, as well as their precise transcriptional start sites. Moreover, regulation of several of these promoters appears to be mediated by the individual or concerted action of the corresponding relaxase and transcriptional repressor associated with each conjugation system. In Chapter V, pNP40- and pUC11B-mediated mobilisation of smaller, non-conjugative plasmids present in the same donor strains is reported. The origin of transfer (oriT) sequences of pNP40 and pUC11B were determined. Sequences similar to these oriT sequences present in other, non-conjugatable plasmids had been reported to be required for their mobilisation. Moreover, the relaxases of both conjugation systems presented specific nickase activity to their respective oriT-containing sequences. Finally, the presence of additional copies of the auxiliary gene mobC in the same donor strain was shown to confer a high-frequency mobilisation phenotype. Chapter VI details the identification and characterization of surface adhesins encoded within the two prevalent lactococcal conjugation gene clusters. The surface adhesion functions of both pNP40 and pUC11B conjugation systems appear to be interchangeable between these two otherwise divergent systems. Additionally, overexpression of these proteins promoted significant cell clumping phenotypes, reminiscent of their cell adhesin function. In summary, the thesis provides a wide range of novel findings which facilitated comprehensive insights into the functionality, prevalence and diversity of lactococcal conjugation systems and the means by which conjugation is regulated. Understanding these systems and the enhancement of conjugation processes is invaluable to the rapid development of robust and technologically desirable starter cultures for sustainable food production systems.
Conjugation , Lactococci , Mobilisation , Transcription , Regulation , Adhesion , Plasmid , Anti-restriction
Ortiz Charneco, G. 2022. Characterization of two prevalent yet distinct conjugation systems among lactococci. PhD Thesis, University College Cork.