RESUMO
The Tn5 insertion into the genome of Rhizobium leguminosarum bv viciae VF39, resulting in non-mucoid growth and formation of non-N2-fixing nodule-like structures on Vicia faba plants, was mapped within a 1.4-kb EcoRV-SacI fragment. Nucleotide sequence analysis revealed an ORF (pss4) of 263 amino acids (aa). Three transcription start points (tsp) were determined. Two of them were localized upstream from the first GTG codon; the third tsp was mapped in front of the second putative start codon (GTG) corresponding to Val64 of the Pss4 aa sequence. The expression of pss4 in a T7 RNA polymerase/promoter system produced a single approx. 29-kDa protein. Pss4 reveals similarity to several proteins involved in polysaccharide biosynthesis in various Rhizobium species. A nearly complete homology was found with PssA from Rl biovar phaseoli 8002 [Borthakur et al., Mol. Gen. Genet. 213 (1988) 155-162], except that Pss4 has an additional 63 aa on its N terminus.
Assuntos
Genes Bacterianos , Rhizobium leguminosarum/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Elementos de DNA Transponíveis/genética , DNA Bacteriano , Fabaceae/microbiologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plantas Medicinais , Polissacarídeos Bacterianos/biossíntese , RNA Mensageiro/química , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Rhizobium leguminosarum/fisiologia , Homologia de Sequência de Aminoácidos , Simbiose , Transcrição GênicaRESUMO
Employing heteroduplex and restriction analyses, two inverted copies of a 3.2.10(6) dalton transposable sequence, TnA, were found in RP4::TnA, a spontaneously arisen derivative of the plasmid RP4. Integration of the second copy of TnA causes loss of the conjugative properties of RP4. Both TnA sequences in RP4::TnA were localized and found to have opposite orientations. The DNA fragment corresponding to the individual transposon TnA was isolated after the endonuclease S1 digestion of RP4::TnA molecules annealed under conditions favoring intramolecular renaturation. The attempts to transform the cells of Escherichia coli QD5003, HB101[pCRI] and JC7623 with the isolated transposon were unsuccessful.
