Novel type I restriction specificities through domain shuffling of HsdS subunits in Lactococcus lactis.
ABSTRACT This study identifies a natural system in Lactococcus lactis, in which a restriction modification specificity subunit resident on a 6159 bp plasmid (pAH33) alters the specificity of a functional R/M mechanism encoded by a 20.3 kb plasmid, pAH82. The new specificity was identified after phenotypic and molecular analysis of a 26.5 kb co-integrate plasmid (pAH90), which was detected after bacteriophage challenge of the parent strain. Analysis of the regions involved in the co-integration revealed that two novel hybrid hsdS genes had been formed during the co-integration event. The HsdS chimeras had interchanged the C- and N-terminal variable domains of the parent subunits, generating two new restriction specificities. Comparison of the parent hsdS genes with other type I specificity determinants revealed that the region of the hsdS genes responsible for the co-integration event is highly conserved among lactococcal type I hsdS determinants. Thus, as hsdS determinants are widespread in the genus Lactococcus, new restriction specificities may evolve rapidly after homologous recombination between these genes. This study demonstrates that, similar to previous observations in Gram-negative bacteria, a Gram-positive bacterium can acquire novel restriction specificities naturally through domain shuffling of resident HsdS subunits.
Full-textDOI: · Available from: Aidan Coffey, May 28, 2015
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ABSTRACT: Lactococcus lactis subsp. cremoris strains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin, L. lactis strains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extra-chromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids of L. lactis UC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped and genes that are typically associated with lactococcal plasmids identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance of L. lactis UC509.9. AbiB-escape mutants were generated for phage sk1, which were found to carry mutations in orf6 encoding the major capsid protein of this phage. In addition, a comparative analysis of all currently available lactococcal plasmid sequences was performed, revealing previously unknown, common genetic dairy accessories.Applied and Environmental Microbiology 05/2014; 80(14). DOI:10.1128/AEM.01070-14 · 3.95 Impact Factor
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ABSTRACT: Attack on the Host Bacterial Genome: Type II RM SystemsWorks have demonstrated that RM systems also act as a watchdog maintaining epigenetic order in cells (Fig. 5). Alteration in the epigenetic status might lead to double-strand breakage of the self genome by the restriction enzyme,34,68,77,78 which can result in cell death or genome rearrangements. This may eliminate unstable cells and maintain the epigenome status.Figure 5.Host attack by restriction systems in conflict with epigenetic systems. A) Postsegregational killing by Type II systems. When a resident RM gene complex is replaced by a competitor genetic element, a decrease in the modification enzyme level exposes newly (more...)Some Type II RM systems cause chromosomal cleavage of their host cells when their genes are eliminated, for example, by a competitor genetic element (Figs. 1D and 5A).68 When an RM system is stably maintained in a cell, the restriction enzyme does not cleave the genomic DNA because of protection through epigenetic methylation by the cognate methyltransferases. However, when the RM system is lost, the concentration of the restriction and modification enzymes is decreased through cell division,79 resulting in undermethylated sites on newly replicated chromosomes.80 The remaining restriction enzyme molecules cleave the unmethylated recognition sequence and cause cell death. The net result is survival of cells that were not invaded by the competitor. This process is called "postsegregational killing" or "genetic addiction".24 The capability of an RM system in forcing maintenance on its host can become stronger by a mutation in its methyltransferase.81 Figure 1E visualizes the effect of postsegregational killing during the formation of bacterial colony. An unstable plasmid in the bacterial cell is lost during colony formation and leads to formation of papillae (Fig. 1E, left). However, when the EcoRI RM system is present on the plasmid, no papillae are formed because the plasmid-free cells are killed (Fig. 1E, right).Postsegregational killing occurs because of a conflict between the RM system (or the plasmid) and the host bacteria and is an example of intragenomic conflicts. A theoretical work demonstrated that starting from very few copies, a postsegregational killing gene can increase in a population in the presence of a spatial structure (Fig. 1F (ii)). In the absence of the spatial structure, the gene is quickly lost unless it is abundant at the beginning (Fig. 1F (i)).82 Post-segregational cell killing by one RM system is inhibited by the presence of another RM system recognizing the same DNA sequence, because the M protein of the latter system protects the genome from cleavage by the R protein of the former system.83 This indicates the presence of competition for recognition sequences between RM systems. Thus, a recognition sequence of RM systems defines an incompatibility group. This competition explains the individual specificity and collective diversity in RM systems' sequence recognition. The competition may be one-sided when the recognition sequence of one RM is included in the recognition sequence of the other RM.84 Another incompatibility relationship between RM systems is found in a regulatory protein that delays expression of the R protein upon entry of an RM system into a new host bacterial cell.85 Recent studies revealed a common pathway of stress-induced cell death in bacteria.86,87 Transcriptome analysis during postsegregational killing by a Type II RM system revealed its similarity to killing by several bacteriocidal antibiotics.78 Thus, RM systems switch on the death pathway intrinsic to the host bacterial cells. Gene products that program bacterial cell death, such as the restriction enzymes discussed here, are likely to work upstream of the common cell death pathway.
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ABSTRACT: Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. However, S. macedonicus is closely related to commensal opportunistic pathogens of the Streptococcus bovis/Streptococcus equinus complex. We analyzed the pSMA198 plasmid isolated from the dairy strain Streptococcus macedonicus ACA-DC 198 in order to provide novel clues about the main ecological niche of this bacterium. pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and to the best of our knowledge it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 sequence revealed a high degree of similarity with plasmids isolated from Lactococcus lactis strains deriving from milk or its products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Additionally, cloning of the pSMA198 ori in L. lactis revealed a 100% stability of replication over 100 generations. Both pSMA198 and the chromosome of S. macedonicus exhibit a high percentage of potential pseudogenes, indicating that they have co-evolved under the same gene decay processes. We identified chromosomal regions in S. macedonicus that may have originated from pSMA198, also supporting a long co-existence of the two replicons. pSMA198 was also found in divergent biotypes of S. macedonicus and in strains isolated from dispersed geographic locations (e.g. Greece and Switzerland) showing that pSMA198's acquisition is not a recent event. Here we propose that S. macedonicus acquired plasmid pSMA198 from L. lactis via an ancestral genetic exchange event that took place most probably in milk or dairy products. We provide important evidence that point towards the dairy origin of this species.PLoS ONE 01/2015; 10(1):e0116337. DOI:10.1371/journal.pone.0116337 · 3.53 Impact Factor