Construction and Environmental Release of a Sinorhizobium meliloti Strain Genetically Modified To Be More Competitive for Alfalfa Nodulation

Abstract

؊ mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492-498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.

Brucella BioR Regulator Defines a Complex Regulatory Mechanism for Bacterial Biotin Metabolism

“…There, promoter sequences of bioBbme (396 bp), bioYbme (372 bp), and bioRbme (377 bp) were synthesized by integrated DNA technology (IDT) and encoded by the following three plasmids: pIDT-PbioBbme, pIDT-PbioYbme, and pIDT-PbioRbme (Table 1). Subsequently, the promoter regions were cut from the pIDT vector with SmaI and BamHI and cloned into pRG970, a low-copy-number lacZ reporter vector (42,43), via the same cuts, yielding pRG-PbioBbme, pRGPbioYbme, and pRG-PbioRbme, respectively ( Table 1). All the required recombinant plasmids were verified by either PCR detection, digestion of restriction enzymes, or direct DNA sequencing.…”

Oxygen Reactivity of PutA from Helicobacter Species and Proline-Linked Oxidative Stress

“…Glutamate that is formed by the oxidation of proline eventually enters the tricarboxylic acid cycle via ␣-ketoglutarate. Proline catabolism has been shown to be an important energy pathway in various gramnegative bacteria such as Bradyrhizobium japonicum, Sinorhizobium meliloti, and Helicobacter pylori, with proline serving as a preferred respiratory substrate in various environments (12,25,38,58,59).…”

Survival of Genetically Modified and Self-Cloned Strains of Commercial Baker's Yeast in Simulated Natural Environments: Environmental Risk Assessment

“…In contrast to only a few studies on genetically engineered yeasts, many studies on genetically engineered bacteria have been reported, such as Pseudomonas strains used for bioremediation and lactic acid bacteria used for probiotics (1,7,19,21). Specific methods for the detection of genetically engineered bacteria have been reported previously (11,26,27,30).…”