ABSTRACT
Streptococcus pneumoniae two-component regulatory systems (TCSs) are important systems that perceive and respond to various host environmental stimuli. In this study, we have explored the role of TCS09 on gene expression and phenotypic alterations in S. pneumoniae D39. Our comparative transcriptomic analyses identified 67 differently expressed genes in total. Among those, agaR and the aga operon involved in galactose metabolism showed the highest changes. Intriguingly, the encapsulated and nonencapsulated hk09-mutants showed significant growth defects under nutrient-defined conditions, in particular with galactose as a carbon source. Phenotypic analyses revealed alterations in the morphology of the nonencapsulated hk09- and tcs09-mutants, whereas the encapsulated hk09- and tcs09-mutants produced higher amounts of capsule. Interestingly, the encapsulated D39∆hk09 showed only the opaque colony morphology, while the D39∆rr09- and D39∆tcs09-mutants had a higher proportion of transparent variants. The phenotypic variations of D39ΔcpsΔhk09 and D39ΔcpsΔtcs09 are in accordance with their higher numbers of outer membrane vesicles, higher sensitivity against Triton X-100 induced autolysis, and lower resistance against oxidative stress. In conclusion, these results indicate the importance of TCS09 for pneumococcal metabolic fitness and resistance against oxidative stress by regulating the carbohydrate metabolism and thereby, most likely indirectly, the cell wall integrity and amount of capsular polysaccharide.
ABSTRACT
Differential editing of transcripts from the Newcastle disease virus (NDV) phosphoprotein gene results in mRNAs capable of encoding the phosphoprotein (P), the V protein, and the W protein which share a common N-terminus but specify different C-termini. Whereas the expression and viral incorporation of the P - and V proteins by NDV has been documented, evidence for the existence of a W protein was lacking. To analyze expression of the NDV W protein, two peptides encompassing predicted antigenic sites of the unique C-terminal W protein amino acid sequence of NDV Clone 30 were used for the generation of W-specific rabbit antisera. One of them detected plasmid-expressed W protein and identified W protein after infection by indirect immunofluorescence and Western blot analyses. W protein was absent in cells infected by a newly generated recombinant NDV lacking W protein expression. Furthermore, Western blot and mass spectrometric analyses indicated the incorporation of W protein into viral particles. Confocal microscopic analyses of infected cells revealed nuclear accumulation of W protein that could be attributed to a bipartite nuclear localization sequence (NLS) within its unique C-terminal part. Redistribution of the W protein to the cytoplasm within transfected cells confirmed functionality of the NLS after mutation of its two basic clusters. This finding was additionally corroborated in cells infected with a recombinant virus expressing the mutated W protein.
