Bakteriális gének szerepe a szimbiotikus gümő inváziójában = The role of bacterial genes in the invasion of the symbiotic nodule

Hoszú távú célunk, hogy megértsük a szimbiotikus nitrogénkötő gümő kifejlődését, és azonosítsuk az invázióban fontos bakteriális géneket. E pályázatban feladatunk volt, hogy (i) elemezzük azon rkp géneket, melyek a Sinorhizobium meliloti 41 törzs specifikus KR5 kapszuláris poliszacharidjának bioszintézisében vesznek részt, (ii) meghatározzuk pontos funkcióját a pha géneknek, melyekről feltételeztük, hogy a pH-homeosztázisban fontosak. Fágreceptorban hibás baktérium és host range fág mutánsok molekuláris genetikai elemzése révén kimutattuk, hogy a 16-3 fág receptorának fontos alkotója az RkpM fehérje, és azonosítottuk a fág h génjét, mely a farki rost fehérjét kódolja. A KR5 antigénről kimutattuk, hogy csak a szimbiotikus gümő inváziójakor fontos, de nem alkotója a fágreceptornak. Befejeztük a DNS-szekvencia meghatározást az rkp-3 régióban, és vizsgáltuk az azonosított gének szerepét. Egyes és kettős rkpY mutánsok segítségével, amelyekben a második mutáció különböző rkp génekben volt kimutattuk, hogy (i) az rkpY mutánsok által termelt poliszacharid nem prekurzora a KR5 antigénnek, és hogy (ii) az rkpR gén ennek az új poliszacharidnak a bioszintézisében játszik szerepet. A pha gének szerepét kifordított membrán vezikulákon végzett fiziológiai kísérletekben vizsgáltuk. Kimutattuk, hogy a K+ adás proton kiáramlást eredményez, ami alátámasztja korábbi feltételezésünket, hogy e gének egy K+/H+ antiportert kódolnak. | Our long term goal is to understand the development of nitrogen fixing symbiotic nodule and to identify bacterial genes involved in the invasion. In this project we focus on (i) the analysis of the rkp genes involved in the biosynthesis of the strain specific KR5 polysaccharide of Sinorhizobium meliloti 41, and (ii) analysis of function of pha gene clusters presumed to be important in pH-homeostasis. The molecular genetic analysis of phage receptor bacterial and host-range phage mutants resulted in the identification of the RkpM protein as an essential part of the 16-3 phage receptor, and in the identification of h gene of the phage encoding the tail-fiber protein. The KR5 antigen were shown only to be important in the invasion of symbiotic nodule but not in the structure of phage receptor. DNA-sequence analysis of the rkp-3 region was completed and the role of the identified genes were examined. Using single and double rkpY mutants, where the second mutations were in different rkp genes, it was shown that (i) the polysaccharide produced by the rkpY mutant was not a precursor of the KR5 antigen, (ii) the rkpR gene is involved in the synthesis of this new polysaccharide. The role of the pha genes were examined on everted membrane vesicles. It was shown that addition of K+ results in proton efflux supporting our hypothesis that pha genes encode for a K+/H+ antiporter

Identification of Tail Genes in the Temperate Phage 16-3 of Sinorhizobium meliloti 41 ▿

Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor

Genetic Analysis of the rkp-3 Gene Region in Sinorhizobium meliloti 41: rkpY Directs Capsular Polysaccharide Synthesis to K R 5 Antigen Production

International audienc

The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure

The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the K(R)5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpl-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the K(R)5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago saliva) symbiosis

H Protein of Bacteriophage 16-3 and RkpM Protein of Sinorhizobium meliloti 41 Are Involved in Phage Adsorption

The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition

Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an α-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C–βII isoenzyme involved in human insulin signaling