Integrative plasmid vector for constructing single-copy reporter systems to study gene regulation in Rhizobium meliloti and related species

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available.

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Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome

Halmillawewa

Restrepo-Córdoba

Perry

et al. 2015

Mol Genet Genomics

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The ntrPR Operon of Sinorhizobium meliloti Is Organized and Functions as a Toxin-Antitoxin Module

Bodogai¹,

Ferenczi²,

Bashtovyy³

et al. 2006

MPMI

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The chromosomal ntrPR operon of Sinorhizobium meliloti encodes a protein pair that forms a toxin-antitoxin (TA) module, the first characterized functional TA system in Rhizobiaceae. Similarly to other bacterial TA systems, the toxin gene ntrR is preceded by and partially overlaps with the antitoxin gene ntrP. Based on protein homologies, the ntrPR operon belongs to the vapBC family of TA systems. The operon is negatively autoregulated by the NtrPNtrR complex. Promoter binding by NtrP is weak; stable complex formation also requires the presence of NtrR. The N-terminal part of NtrP is responsible for the interaction with promoter DNA, whereas the C-terminal part is required for protein-protein interactions. In the promoter region, a direct repeat sequence was identified as the binding site of the NtrPNtrR complex. NtrR expression resulted in the inhibition of cell growth and colony formation; this effect was counteracted by the presence of the antitoxin NtrP. These results and our earlier observations demonstrating a less effective downregulation of a wide range of symbiotic and metabolic functions in the ntrR mutant under microoxic conditions and an increased symbiotic efficiency with the host plant alfalfa suggest that the ntrPR module contributes to adjusting metabolic levels under symbiosis and other stressful conditions.

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A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16‐3 is functional and convertible to suppressor tRNA

Blaha

Semsey

Ferenczi

et al. 2004

Molecular Microbiology

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SummarySeveral temperate bacteriophage utilize chromosomal sequences encoding putative tRNA genes for phage attachment. However, whether these sequences belong to genes which are functional as tRNA is generally not known. In this article, we demonstrate that the attachment site of temperate phage 16-3 ( attB ) nests within an active proline tRNA gene in Rhizobium meliloti 41. A loss-of-function mutation in this tRNA gene leads to significant delay in switching from lag to exponential growth phase. We converted the putative Rhizobium gene to an active amber suppressor gene which suppressed amber mutant alleles of genes of 16-3 phage and of Escherichia coli origin in R. meliloti 41 and in Agrobacterium tumefaciens GV2260. Upon lysogenization of R. meliloti by phage 16-3 , the proline tRNA gene retained its structural and functional integrity. Aspects of the co-evolution of a temperate phage and its bacterium host is discussed. The side product of this work, i.e. construction of amber suppressor tRNA genes in Rhizobium and Agrobacterium , for the first time widens the options of genetic study.

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Integrative plasmid vector for constructing single-copy reporter systems to study gene regulation in Rhizobium meliloti and related species

Cited by 6 publications

References 19 publications

Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome

Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome

The ntrPR Operon of Sinorhizobium meliloti Is Organized and Functions as a Toxin-Antitoxin Module

A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16‐3 is functional and convertible to suppressor tRNA

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