RESUMO
A mutational and structural analysis of Rhizobium loti nodulation genes in strains NZP2037 and NZP2213 was carried out. Unlike the case with other Rhizobium strains examined to date, nodB was found on an operon separate from nodACIJ. Sequence analysis of the nodACIJ and nodB operon regions confirm that R. loti common nod genes have a gene organization different from that of other Rhizobium spp. At least 4 copies of nodD-like sequences were identified in R. loti. The complete nucleotide sequence of one of these, nodD3, was determined. A new host-specific nod gene, nolL, was identified adjacent to nodD3. NolL shares homology with NodX and other O-acetyl transferases. Mutational analysis of the nod regions of strains NZP2037 and NZP2213 showed that nodD3, nodI, nodJ, and nolL were all essential for R. loti strains to effectively nodulate the extended host Lotus pedunculatus, but were not necessary for effective nodulation of the less restrictive host, Lotus corniculatus. Both nodD3 and nolL were essential for R. loti strains to nodulate Leucaena leucocephala.
Assuntos
Genes Bacterianos , Rhizobium/genética , Aciltransferases/genética , Amidoidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , Primers do DNA/genética , DNA Bacteriano/genética , Fabaceae/microbiologia , Rearranjo Gênico , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Fixação de Nitrogênio/genética , Óperon , Plantas Medicinais , Homologia de Sequência de Aminoácidos , SimbioseRESUMO
A broad-host-range bidirectional promoter reporter vector, pSPV4, has been constructed to analyse the activity of cloned divergent regulatory regions. Plasmid pSPV4 contains a pair of divergent promoterless reporter genes (lacZ and gusA) that are bisected by an extensive multiple cloning site. [corrected] The transcriptional fusion vector pSPV4 has a distinct advantage over unidirectional promoter probe vectors in that it can determine the activity of a cloned bidirectional regulatory region simultaneously in both directions using the same set of cells. The relative activity of each of the divergent promoters, and hence the reporter genes, is therefore not a function of the particular growth state of the culture. To demonstrate the utility of pSPV4, the promoter activity of two Rhizobium loti nod regulatory regions were examined. Although the nod gene organisation in this species is unusual, the promoter activity of these two divergent nod regulatory regions is consistent with the conventional mode of nod expression (constitutive towards the regulatory nodD gene and inducible in the divergent nod gene direction). The construction of the bidirectional reporter vector, pSPV4, involved two intermediary plasmids, pSPV1 and pSPV2. Both of these constructs will also be useful for other researchers, especially given the trend towards the utilisation of gusA as a reporter gene.
Assuntos
Aciltransferases , Amidoidrolases , Genes Bacterianos , Vetores Genéticos , Fixação de Nitrogênio/genética , Regiões Promotoras Genéticas , Rhizobium/genética , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Expressão Gênica , Genes Reporter , Dados de Sequência MolecularRESUMO
The efficiency of plating of phages derived by ultraviolet induction of, or by lytic growth on, certain strains of Streptococcus cremoris was found to vary by as much as 10 depending on the lactose concentration of the medium in which the indicator bacteria were grown and the length of time the stationary-phase indicator cultures were aged. This effect was noted only when the culture was used as an indicator for phages that had previously grown on an apparently unrelated strain of bacteria. Conditions of culturing and aging had no detectable effect upon the ability of a strain to serve as an indicator for phage that had previously been cultured on the same strain. These observations suggest the presence of some kind of physiologically labile restriction system in strains of S. cremoris. The implications of this finding for increasing the sensitivity of the host range test in determining phage susceptibility, whether from induced lysates, whey, or lytic phage stocks, are discussed. It is recommended that, for all such testing, the concentration of lactose in buffered media be increased to such levels as required to obtain a final pH similar to that of a freshly coagulated milk culture, namely, below pH 5.0.
