The 3,308,274-bp sequence of the chromosome of Lactobacillus plantarum strain WCFS1, a single colony isolate of strain NCIMB8826 that was originally isolated from human saliva, has been determined, and contains 3,052 predicted protein-encoding genes. Putative biological functions could be assigned to 2,120 (70%) of the predicted proteins. Consistent with the classification of L. plantarum as a facultative heterofermentative lactic acid bacterium, the genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, all of which appear to belong to the class of potentially highly expressed genes in this organism, as was evident from the codon-adaptation index of individual genes. Moreover, L. plantarum encodes a large pyruvate-dissipating potential, leading to various end-products of fermentation. L. plantarum is a species that is encountered in many different environmental niches, and this flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 25 complete PTS sugar transport systems. Moreover, the chromosome encodes >200 extracellular proteins, many of which are predicted to be bound to the cell envelope. A large proportion of the genes encoding sugar transport and utilization, as well as genes encoding extracellular functions, appear to be clustered in a 600-kb region near the origin of replication. Many of these genes display deviation of nucleotide composition, consistent with a foreign origin. These findings suggest that these genes, which provide an important part of the interaction of L. plantarum with its environment, form a lifestyle adaptation region in the chromosome.

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

Lactococcus lactisstrains are known to carry plasmids encoding industrially important traits.L. lactissubsp.cremorisSK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologousrepB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside onL. lactisSK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP,pepO,pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while thepabAandpabBgenes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependentd-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted α-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these “adaptation” genes have not been described before onL. lactisplasmids. Moreover, several genes were identified for the first time inL. lactis, possibly reflecting horizontal gene transfer.

Background: Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action.Results: TheLactobacillus plantarumWCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-Jandcps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-Iandcps3A-J). We constructedL. plantarumWCFS1 gene deletion mutants that lack individual (Δcps1A-I,Δcps2A-J, Δcps3A-Jand Δcps4A-J) or combinations ofcpsclusters (Δcps1A-3Jand Δcps1A-3I, Δcps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. Thecpscluster deletions influenced the expression of variable gene sets in the individualcpscluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants incpscluster 1 and 2, as well as between mutant incpsclusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness ofcps1A-Iandcps3A-J, all clusters are active and functional inL. plantarum. The Δcps1A-Istrain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies ofcps2A-J,cps3A-Jorcps4A-Jproduced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of thecpscluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-κB activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to aL. plantarumwild-type derivative, TLR2 activation remained unaffected by the Δcps1A-Iand Δcps3A-Jmutants but appeared slightly increased after stimulation with the Δcps2A-Jand Δcps4A-Jmutants, while the Δcps1A-3Jand Δcps1A-3J,Δcps4A-Jmutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling.Conclusions: Our study reveals that modulation of surface glycan characteristics inL. plantarumhighlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently completecpsclusters (cps2A-Jandcps4A-J) contributed individually to this shielding, the removal of allcpsclusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis inL. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis , while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis . The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum . Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.