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Effect of Nutritional Determinants and TonB on the Natural Transformation of Riemerella anatipestifer.

ABSTRACT: Riemerella anatipestifer is a gram-negative bacterium that is the first naturally competent bacterium identified in the family Flavobacteriaceae. However, the determinants that influence the natural transformation and the underlying mechanism remain unknown. In this study, we evaluated the effects of various nutritional factors of the GCB medium [glucose, L-glutamine, vitamin B1, Fe (NO₃)₃, NaCl, phosphate, and peptone], on the natural transformation of R. anatipestifer ATCC 11845. Among the assayed nutrients, peptone and phosphate affected the natural transformation of R. anatipestifer ATCC 11845, and the transformation frequency was significantly decreased when phosphate or peptone was removed from the GCB medium. When the iron chelator 2,2'-dipyridyl (Dip) was added, the transformation frequency was decreased by approximately 100-fold and restored gradually when Fe (NO₃)₃ was added, suggesting that the natural transformation of R. anatipestifer ATCC 11845 requires iron. Given the importance of TonB in nutrient transportation, we further identified whether TonB is involved in the natural transformation of R. anatipestifer ATCC 11845. Mutation of tonBA or tonBB, but not tbfA, was shown to inhibit the natural transformation of R. anatipestifer ATCC 11845 in the GCB medium. In parallel, it was shown that the tonBB mutant, but not the tonBA mutant, decreased iron acquisition in the GCB medium. This result suggested that the tonBB mutant affects the natural transformation frequency due to the deficiency of iron utilization.

SUBMITTER: Zhang L

PROVIDER: S-EPMC8383284 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Riemerella anatipestifer is a member of the family Flavobacteriaceae and a major causative agent of duck serositis. Little is known about its genetics and pathogenesis. Several bacteria are competent for natural transformation; however, whether R. anatipestifer is also competent for natural transformation has not been investigated. Here, we showed that R. anatipestifer strain ATCC 11845 can uptake the chromosomal DNA of R. anatipestifer strain RA-CH-1 in all growth phases. Subsequently, a natural transformation-based knockout method was established for R. anatipestifer ATCC 11845. Targeted mutagenesis gave transformation frequencies of ?10-5 transformants. Competition assay experiments showed that R. anatipestifer ATCC 11845 preferentially took up its own DNA rather than heterogeneous DNA, such as Escherichia coli DNA. Transformation was less efficient with the shuttle plasmid pLMF03 (transformation frequencies of ?10-9 transformants). However, the efficiency of transformation was increased approximately 100-fold using pLMF03 derivatives containing R. anatipestifer DNA fragments (transformation frequencies of ?10-7 transformants). Finally, we found that the R. anatipestifer RA-CH-1 strain was also naturally transformable, suggesting that natural competence is widely applicable for this species. The findings described here provide important tools for the genetic manipulation of R. anatipestiferIMPORTANCERiemerella anatipestifer is an important duck pathogen that belongs to the family Flavobacteriaceae At least 21 different serotypes have been identified. Genetic diversity has been demonstrated among these serotypes. The genetic and pathogenic mechanisms of R. anatipestifer remain largely unknown because no genetic tools are available for this bacterium. At present, natural transformation has been found in some bacteria but not in R. anatipestifer For the first time, we showed that natural transformation occurred in R. anatipestifer ATCC 11845 and R. anatipestifer RA-CH-1. Then, we established an easy gene knockout method in R. anatipestifer based on natural transformation. This information is important for further studies of the genetic diversity and pathogenesis in R. anatipestifer.

Project description:Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer, a Gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes hemoglobin-derived hemin as an iron source; however, the mechanism by which this bacterium acquires hemin from hemoglobin is largely unknown. Here, rhuA disruption was shown to impair iron utilization from duck hemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane hemin-binding protein, but it could not extract hemin from duck hemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A, and RhuAH149A lost hemin-binding ability, suggesting that amino acid sites at tyrosine 144 (Y144), Y177, and histidine 149 (H149) are crucial for hemin binding. Furthermore, rhuR, the gene adjacent to rhuA, encodes a TonB2-dependent hemin transporter. The function of rhuA in duck hemoglobin utilization was abolished in the rhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1 ΔrhuA rather than R. anatipestifer CH-1 ΔrhuR ΔrhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA hemin utilization locus exhibits no similarity to those of characterized hemin transport systems. Thus, this locus is a novel hemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum. IMPORTANCE In vertebrates, hemin from hemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain hemin from hemoglobin, but the mechanisms of hemin acquisition from hemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of hemin acquisition from mammalian hemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in hemin acquisition from duck hemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane hemin-binding protein, and RhuR was identified as a TonB2-dependent hemin transporter. Moreover, the function of RhuA in hemoglobin utilization is RhuR dependent and not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel hemin transportation system of Gram-negative bacteria. This study not only was important for understanding hemin uptake in R. anatipestifer but also enriched the knowledge about the hemin transportation pathway in Gram-negative bacteria.

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Effect of Nutritional Determinants and TonB on the Natural Transformation of Riemerella anatipestifer.

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