The SmpB-tmRNA tagging system plays important roles in Streptomyces coelicolor growth and development.

The ssrA gene encodes tmRNA that, together with a specialized tmRNA-binding protein, SmpB, forms part of a ribonucleoprotein complex, provides a template for the resumption of translation elongation, subsequent termination and recycling of stalled ribosomes. In addition, the mRNA-like domain of tmRNA encodes a peptide that tags polypeptides derived from stalled ribosomes for degradation. Streptomyces are unique bacteria that undergo a developmental cycle culminating at sporulation that is at least partly controlled at the level of translation elongation by the abundance of a rare tRNA that decodes UUA codons found in a relatively small number of open reading frames prompting us to examine the role of tmRNA in S. coelicolor. Using a temperature sensitive replicon, we found that the ssrA gene could be disrupted only in cells with an extra-copy wild type gene but not in wild type cells or cells with an extra-copy mutant tmRNA (tmRNA(DD)) encoding a degradation-resistant tag. A cosmid-based gene replacement method that does not include a high temperature step enabled us to disrupt both the ssrA and smpB genes separately and at the same time suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Indeed, mutant cells show growth and sporulation defects at high temperature and under optimal culture conditions. Interestingly, even though these defects can be completely restored by wild type genes, the DeltassrA strain was only partially corrected by tmRNA(DD). In addition, wildtype tmRNA can restore the hygromycin-resistance to DeltassrA cells while tmRNA(DD) failed to do so suggesting that degradation of aberrant peptides is important for antibiotic resistance. Overall, these results suggest that the tmRNA tagging system plays important roles during Streptomyces growth and sporulation under both normal and stress conditions.

Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development.

The cadA gene in Dictyostelium encodes the Ca2+-dependent cell adhesion molecule DdCAD-1, which is expressed soon after the initiation of development. To investigate the biological role of DdCAD-1, the cadA gene was disrupted by homologous recombination. The cadA-null cells showed a 50% reduction in EDTA-sensitive cell adhesion. The remaining EDTA-sensitive adhesion sites were resistant to dissociation by anti-DdCAD-1 antibody, suggesting that they were distinct adhesion sites. Cells that lacked DdCAD-1 were able to complete development and form fruiting bodies. However, they displayed abnormal slug morphology and culmination was delayed by approximately 6 hours. The yield of spores was reduced by approximately 50%. The proportion of prestalk cells in cadA(-) slugs showed a 2.5-fold increase over the parental strain. When cadA(-) cells were transfected with pcotB::GFP to label prespore cells, aberrant cell-sorting patterns in slugs became apparent. When mutant prestalk cells were mixed with wild-type prespore cells, mutant prestalk cells were unable to return to the anterior position of chimeric slugs, suggesting defects in the sorting mechanism. The wild-type phenotype was restored when cadA(-) cells were transfected with a cadA-expression vector. These results indicate that, in addition to cell-cell adhesion, DdCAD-1 plays a role in cell type proportioning and pattern formation.