Relation between cell-bound exopolysaccharide production via plasmid-encoded genes and rugose colony morphology in the probiotic Lactobacillus brevis KB290.

The probiotic Lactobacillus brevis KB290 is a natural producer of cell-bound exopolysaccharide (EPS), and the plasmid-encoded glycosyltransferase genes are responsible for this EPS production. KB290 forms unique rugose colonies inside an agar medium; this characteristic is useful for detecting and enumerating KB290 in the gut or feces. However, the genetic elements associated with this morphology remain unclear. Here, we aimed to investigate the relation between the plasmid eps genes and rugose colony morphology in KB290. The plasmid-cured mutants formed smooth colonies, and the rugose colony morphology was restored after complementation with the eps genes. The eps genes were successfully cloned and expressed in other L. brevis and L. plantarum strains. In these transformant strains, the presence of the EPS, consisting of glucose and N-acetylglucosamine, correlated with rugose colonies, indicating that EPS is responsible for rugose colony formation. To the best of our knowledge, this is the first report identifying the genetic factors influencing rugose colonies in Lactobacillus strains. This rugose colony formation may serve as a useful selective marker for KB290 in routine laboratory and research settings and can be used to detect the spontaneous loss of plasmids in this strain.

Activation of focal adhesion kinase in detached human epidermal cancer cells and their long-term survival might be associated with cell surface expression of laminin-5.

While normal epithelial cells are anchorage-dependent, cancer cells are anchorage-independent. To elucidate the mechanism underlying the anchorage-independence of cancer cells, we cultured detached cells in medium containing elastase. Detached human epidermal cancer cells (DJM-1) survived for at least 3 weeks and focal adhesion kinase was still phosphorylated. In contrast, most detached keratinocytes underwent rapid apoptosis and focal adhesion kinase was not phosphorylated while the cells were alive. Thus, discontinuation of the phosphorylation of focal adhesion kinase preceded cell death. Immunostaining showed laminin-5 expression on the surface of detached DJM-1 cells, but not on detached keratinocytes. Receptors for laminin-5 (i.e. integrins) were detected on both detached DJM-1 cells and keratinocytes. Laminins are secreted proteins, so we speculated that laminin-5 adhered to the surface of secreting DJM-1 cells via integrins and evoked activation of focal adhesion kinase, with the resultant signalling cascade promoting cellular survival. If this hypothesis is correct, cell surface expression of laminin-5 may be used to explain the characteristics of cancer, immortality, tumour formation, metastasis and angiogenesis.