Michiel Wels

The lactic acid bacterium Lactococcus lactis is widely used for the fermentation of dairy products. Here, we present the draft genome sequences of 11 L. lactis subsp. cremoris strains isolated from different environments.

The ability of many bacteria to adapt to diverse environmental conditions is well known. Recent research has linked the process of bacterial adaptation to a niche to changes in the genome content and size, showing that many bacterial genomes reflect the constraints imposed by their habitat. However, some highly versatile bacteria are found in diverse niches that almost share nothing in common. Lactobacillus plantarum is a lactic acid bacterium that is found in a large variety of niches. With the aim of unravelling the link between genome evolution and ecological versatility of L. plantarum, we analysed the genomes of 54 L. plantarum strains isolated from different environments. Phylogenomic analyses coupled with the study of genetic functional divergence and gene-trait matching analysis revealed a mixed distribution of the strains, which was uncoupled from their environmental origin. Our findings demonstrate the high complexity of L. plantarum evolution, revealing the absence of specific genomic signatures marking adaptations of this species towards the diverse habitats it is associated with. This suggests fundamentally similar and parallel trends of genome evolution in L. plantarum, which occur in a manner that is apparently uncoupled from ecological constraint and reflects the nomadic lifestyle of this species.

The lactic acid bacterium Lactobacillus plantarum is intensively studied as a model probiotic species. Here, we present the draft genome sequence of the exopolysaccharide-producing strain SF2A35B.

The lactic acid bacterium Streptococcus thermophilus is widely used for the fermentation of dairy products. Here, we present the draft genome sequence of S. thermophilus C106 isolated from an artisanal cheese produced in the countryside of Ireland.

Background: To cope with environmental challenges bacteria possess sophisticated defense mechanisms that involve stress-induced adaptive responses. The canonical stress regulators CtsR and HrcA play a central role in the adaptations to a plethora of stresses in a variety of organisms. Here, we determined the CtsR and HrcA regulons of the lactic acid bacterium Lactobacillus plantarum WCFS1 grown under reference (28°C) and elevated (40°C) temperatures, using ctsR, hrcA, and ctsR-hrcA deletion mutants. Results: While the maximum specific growth rates of the mutants and the parental strain were similar at both temperatures (0.33 ± 0.02 h^-1 and 0.34 ± 0.03 h^-1, respectively), DNA microarray analyses revealed that the CtsR or HrcA deficient strains displayed altered transcription patterns of genes encoding functions involved in transport and binding of sugars and other compounds, primary metabolism, transcription regulation, capsular polysaccharide biosynthesis, as well as fatty acid metabolism. These transcriptional signatures enabled the refinement of the gene repertoire that is directly or indirectly controlled by CtsR and HrcA of L. plantarum. Deletion of both regulators, elicited transcriptional changes of a large variety of additional genes in a temperature-dependent manner, including genes encoding functions involved in cell-envelope remodeling. Moreover, phenotypic assays revealed that both transcription regulators contribute to regulation of resistance to hydrogen peroxide stress. The integration of these results allowed the reconstruction of CtsR and HrcA regulatory networks in L. plantarum, highlighting the significant intertwinement of class I and III stress regulons. Conclusions: Taken together, our results enabled the refinement of the CtsR and HrcA regulatory networks in L. plantarum, illustrating the complex nature of adaptive stress responses in this bacterium.

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.

Background: Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background. Results: Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type. Conclusions: The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.