Bacteria adapt themselves to host environments by altering the pattern of gene expression. The promoter-recognizing subunit σ of bacterial RNA polymerase plays a major role in the selection of genes to be transcribed. Among seven σ factors of Escherichia coli, σ38 is responsible for the transcription of genes in the stationary phase and under stressful conditions. We found a transient increase of σ38 when E. coli was injected into the hemocoel of Drosophila melanogaster. The loss of σ38 made E. coli rapidly eliminated in flies, and flies infected with σ38-lacking E. coli stayed alive longer than those infected with the parental strain. This was also observed in fly lines defective in humoral immune responses, but not in flies in which phagocytosis was impaired. The lack of σ38 did not influence the susceptibility of E. coli to phagocytosis, but made them vulnerable to killing after engulfment. The changes caused by the loss of σ38 were recovered by the forced expression of σ38-encoding rpoS as well as σ38-regulated katE and katG coding for enzymes that detoxify reactive oxygen species. These results collectively suggested that σ38 contributes to the prolonged survival of E. coli in Drosophila by inducing the production of enzymes that protect bacteria from killing in phagocytes. Considering the similarity in the mechanism of innate immunity against invading bacteria between fruit flies and humans, the products of these genes could be new targets for the development of more effective antibacterial remedies.
An RNA chaperone of Escherichia coli, called host factor required for phage Qβ RNA replication (Hfq), forms a complex with small noncoding RNAs to facilitate their binding to target mRNA for the alteration of translation efficiency and stability. Although the role of Hfq in the virulence and drug resistance of bacteria has been suggested, how this RNA chaperone controls the infectious state remains unknown. In the present study, we addressed this issue using Drosophila melanogaster as a host for bacterial infection. In an assay for abdominal infection using adult flies, an E. coli strain with mutation in hfq was eliminated earlier, whereas flies survived longer compared with infection with a parental strain. The same was true with flies deficient in humoral responses, but the mutant phenotypes were not observed when a fly line with impaired hemocyte phagocytosis was infected. The results from an assay for phagocytosis in vitro revealed that Hfq inhibits the killing of E. coli by Drosophila phagocytes after engulfment. Furthermore, Hfq seemed to exert this action partly through enhancing the expression of σ38, a stress-responsive σ factor that was previously shown to be involved in the inhibition of phagocytic killing of E. coli, by a posttranscriptional mechanism. Our study indicates that the RNA chaperone Hfq contributes to the persistent infection of E. coli by maintaining the expression of bacterial genes, including one coding for σ38, that help bacteria evade host immunity.
Bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a destructive disease in rice fields. Can Tho is one of the most important rice-growing areas in the Mekong Delta, which is vulnerable to climate change, making the disease more damaging in this region. Deployment of resistance genes is considered an economic and eco-friendly approach to control the disease. However, Xoo exists in different races with diverse reactions on different resistance genes. Thus, for effective management of BB, it is essential to understand the diversity of contemporary Xoo population to deploy appropriate resistance genes in rice fields. This study aims at assessing the Xoo population diversity (race composition) in rice fields of Can Tho using pathogenicity reactions on the near-isogenic lines (pathotypes) in combination with insertion sequence-PCR technique using J3 primer (genotypes). Among 132 isolates obtained from BB-infected leaf samples collected from six rice-growing areas of Can Tho, 126 isolates were identified as Xoo using PCR with the specific primers XOO290F/R. The contemporary Xoo population in Can Tho was composed of four races including two classic standard races (5 and 7) and two newly emerged ones (5* and 5**) of which races 5 and 5* were the most predominant. Seven haplotypes were identified in the four races and haplotypes I and III were predominant, accounting for 50.79% and 40.48%, respectively. The combination of the pathotypic and genotypic analyses showed genetic variations in races 5 and 5*. These results could be used for deployment of appropriate BB resistance cultivars in rice fields of Can Tho.
Eugenol oxidase (EUGO), a member of the vanillyl alcohol oxidase family, catalyzes the oxidative reaction of vanillyl alcohol to vanillin. This compound is responsible for the vanilla aroma and is widely used as a flavoring agent in food, cosmetics, and pharmaceuticals. Previously, EUGO was cloned and expressed in E. coli TOP10, and purified by anion-exchange chromatography with Q-Sepharose resin but the purification factor was low. To improve the efficiency of the EUGO purification, in this study, we cloned eugo gene into pET-28a vector and expressed it in E. coli Tunetta. The SDS-PAGE analysis of protein extracts obtained from E. coli expressing EUGO under different induction conditions showed that EUGO was expressed mostly in the soluble fraction at 6 hours after induction with 0.1 mM IPTG at 25oC. EUGO was purified by immobilized−metal affinity chromatography with Ni2+-NTA agarose and the in vitro enzymatic activity was characterized. The specific activity of purified EUGO was nearly 4-fold higher than that of the crude enzyme sample. In particular, the enzyme preparation produced by the purification method based on Ni-NTA affinity in this study was 2,5-fold more pure than that produced by Q-sepharose purification method described previously.
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