SummaryGram-positive, nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of novel, safe production and delivery systems of heterologous proteins. Recombinant LAB strains were shown to elicit specific systemic and mucosal immune responses against selected antigens. For this reason, this group of bacteria is considered as a potential replacement of classical, often pathogenic, attenuated microbial carriers. Mucosal administration of recombinant LAB, especially via the best explored and universal oral route, offers many advantages in comparison to systemic inoculation, and is attractive from the immunological and practical point of view. Research aimed at designing efficient, mucosally applied vaccines in combination with improved immunization efficiency, monitoring of in vivo antigen production, determination of optimal dose for vaccination, strain selection and characterization is a priority in modern vaccinology. This paper summarizes and organizes the available knowledge on the application of LAB as live oral vaccine vectors. It constitutes a valuable source of general information for researchers interested in mucosal vaccine development and constructing LAB strains with vaccine potential.

Bacteriophage infection of Lactococcus species can cause serious disruption of dairy fermentation processes. The most common isolates from the dairy environment are Siphoviridae lytic 936-type phages. To gain specific knowledge about this group of phages in Polish dairies, we examined 90 isolates from 8 different locations. Based on restriction fragment length polymorphism analysis, coupled with physiological and molecular studies, the isolated phages were divided into 8 distinct groups. Whole-genome sequencing of single representatives from each phage group provided data about their biology and genetic composition. The phages present an overall conserved genome organization. High sequence homology to another Polish isolate, Lactococcus phage bIBB29, indicates their close phylogenetic relatedness to this strain. Such similarity may be suggestive of a general genome conservation among phages persisting in Polish dairies. Comparative genome analyses with other 936-type phages revealed several discriminative traits, including the presence and position of HNH endonuclease genes, varying number of orfs in the early gene region, and a putative TpeX gene. Interestingly, host range of the sequenced phages was restricted to L. lactis subsp. lactis biovar. diacetylactis strains. The results provide new data regarding phages present in the Polish dairy environment and permit analysis of their biology, genome composition and relatedness to other Lactococcus 936-type phages.

Lactococcus Ceduovirus (formerly c2virus) bacteriophages are among the three most prevalent phage types reported in dairy environments. Phages from this group conduct a strictly lytic lifestyle and cause substantial losses during milk fermentation processes, by infecting lactococcal host starter strains. Despite their deleterious activity, there are limited research data concerning Ceduovirus phages. To advance our knowledge on this specific phage group, we sequenced and performed a comparative analysis of 10 new Lactococcus lactis Ceduovirus phages isolated from distinct dairy environments. Host range studies allowed us to distinguish the differential patterns of infection of L. lactis cells for each phage, and revealed a broad host spectrum for most of them. We showed that 40% of the studied Ceduovirus phages can infect both cremoris and lactis strains. A preference to lyse strains with the C-type cell wall polysaccharide genotype was observed. Phage whole-genome sequencing revealed an average nucleotide identity above 80%, with distinct regions of divergence mapped to several locations. The comparative approach for analyzing genomic data and the phage lytic spectrum suggested that the amino acid sequence of the orf8-encoded putative tape measure protein correlates with host range. Phylogenetic studies revealed separation of the sequenced phages into two subgroups. Finally, we identified three types of phage origin of replication regions, and showed they are able to support plasmid replication without additional phage proteins.

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Here, we describe functional characterization of an early gene (gp46) product of a virulent Lactococcus lactis sk1-like phage, vB_Llc_bIBBF13 (abbr. F13). The GP46F13 protein carries a catalytically active RecA-like domain belonging to the P-loop NTPase superfamily. It also retains features characteristic for ATPases forming oligomers. In order to elucidate its detailed molecular function, we cloned and overexpressed the gp46 gene in Escherichia coli. Purified GP46F13 protein binds to DNA and exhibits DNA unwinding activity on branched substrates in the presence of adenosine triphosphate (ATP). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments demonstrate that GP46F13 forms oligomers, and further pull-down assays show that GP46F13 interacts with host proteins involved in replication (i.e., DnaK, DnaJ, topoisomerase I, and single-strand binding protein). Taking together the localization of the gene and the obtained results, GP46F13 is the first protein encoded in the early-expressed gene region with helicase activity that has been identified among lytic L. lactis phages up to date.