| Lignin containing oxo-alcohols or derivatives of benzene aromatic structural units of natural organic polymers is the world’s secondly most abundant reserves of organic compounds (cellulose occupys first). Lignin is a renewable source from nature and it can provide substantial aryl compounds. However, due to its complex structure, lignin is difficult to be degraded so far and that limits the value on applications. Guaiacyl structure (G unit) is a universal component of lignin which mainly exists in various types of lignin. The degradation mechanism of G unit basically represents all lignin pathways.Having research on G unit biodegradation not only brings great practical significance for lignin degradations but also plays an important role in pollution treatment by lignin-derived aromatic compounds (such as paper mill black liquor). Biodegradation of lignin compounds, such as ferulic acid (G unit) and its analogues-structural hydroxycinnamates (HCAs), has attracted great attentions so far. Therefore, we focused on the biodegradation of ferulic acid by Ralstonia solanacearum. Ralstonia solanacearum is a gram-negative bacterium which destroys the plant cell wall to get in touch with the xylem that leads to pathogenicity. According to bioinformatic annotation, there is a hypothesis that rsp0221-rsp0228 genes in Ralstonia solanacearum are corresponded to an island genes encoding enzymes required for hydroxycinnamates degradation to protocatechuate:rsp0221 is a two-component response regulator, rsp0222 and rsp0223 encode vanillate O-demethylase oxygenase subunit oxidoreductase protein and vanillate O-demethylase respectively, rsp0225 encodes p-hydroxycinnamoyl CoA hydratase, rsp0226 encodes vanillin dehydrogenase, rsp0227 encodes feruloyl-CoA synthase; Rsc1285 in R. solanacearum is a putative regulator on expression of type Ⅲ secretion system that may affect interaction between Ralstonia solanacearum and host plants. In this study, gene knockout and gene complemention techniques have been made to reveal the biodegradation of ferulic acid and analogues-structural hydroxycinnamates and function of genes in Ralstonia solanacearum.We have successfully constructed Δ rsp0221 mutant and Δ rsp0227mutant. High concentrations of Ferulic acid inhibited the growth of R. solanacearum and 0.5g/L Ferulic acid did not show effect on Ralstonia solanacearum. Ralstonia solanacearum could grow on ferulic acid as the sole carbon source. Δ rsp0227mutant failed to grow at the expense of HCAs but it growed well on HCAs catabolic intermediates as the sole carbon sources. Complemented rsp0227 could recover the growth of Δ rsp0227 mutant in HCAs as the sole carbon source. These results indicate that Rsp0227 is directly responsible for HCAs degradation. Δ rsp022/mutant grew as well as wild type in HCAs, suggesting that Rsp0221 is not directly related to ferulic acid and its structural analogues. In case of the ferulic acid degradation, ferulic acid could be degraded nearly 90% by wild type and Δ rsp0221mutant after one day. However, Δ rsp0227mutant couldn’t degrade ferulic acid.Furthermore, Type Ⅲ secretion system(T3SS) plays a crucial role in Ralstonia solanacearum invading into the xylem by injecting effectors. Therefore, we studied the effect of ferulic acid on the expression of type Ⅲ secretion system. It was found that addition of ferulic acid significantly promoted T3SS expression to much higher level(2-3 fold) whenever in wild type, Δ rsp022 Imatant or Δ rsp0227mutant, Indicating that ferulic acid might induce the process of cell wall decomposition by Ralstonia solanacearum.Expression of T3SS was significantly reduced by 1/2 in Δ rsc1285mutant. While complemented rsc1285 could recover the expression level of T3SS, suggesting that Rsc1285 is an important factor on T3SS. Ralstonia solanacearum T3SS is directly controlled by HrpB which is regulated by HrpG and PrhG.To our research, Rsc1285 controls the expression of hrpB and HrpB-regulating genes, but it is dispensable for the expression of hrpG and prhG. When it comes to plant infection, Δ rsc1285mutant grew as well as wild type in planta, indicationg that Rsc1285 has no effect on Ralstonia solanacearum invading host plants.The function of Rsc1285 in Ralstonia solanacearum remains to be revealed. |
PDF Full Text Request