Replication and active partition of integrative and conjugative elements (ICEs) of the SXT/R391 family : the line between ICEs and conjugative plasmids is getting thinner

Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs

The Family Frankiaceae

The family Frankiaceae, within the order Actinomycetales, contains bacteria isolated mainly from root nodules and occasionally from soil. Members of the genus Frankia have been found associated with the roots of 23 genera of dicots belonging to eight families. Historically, strains isolated in pure culture were grouped into two physiological categories, those that use carbohydrates and those that do not. Newer genomic information indicated that frankiae in general differ markedly in their complements of genes. Besides physiological grouping, these isolates were placed into four plant-compatibility groups (1-infective on Alnus and Myrica, 2-infective on Casuarina and Myrica, 3-infective on Elaeagnaceae and Myrica, 4-infective only on Elaeagnaceae). A 16S rRNA gene-based phylogenetic study, comprising non-isolated endophytes, yielded four clusters or clades, three of which are symbiotic (1-infective on Alnus and Casuarinaceae except Gymnostoma, 2-non-isolated strains in nodules of Rosaceae-Datisca-Coriaria-Rhamnaceae, 3-infective on Elaeagnaceae and Gymnostoma) and a fourth cluster that groups non-infective and non-effective strains. These groupings have been confirmed on the whole by analysis of other loci. DNA-DNA hybridization studies have yielded 12–15 genospecies, only one of which has been named, Frankia alni; one Candidatus Frankia datiscae was recently named to accommodate the genome of an endophyte in nodules of Datisca glomerata. The family Frankiaceae is close to Acidothermus, Cryptosporangium, Geodermatophilaceae (Geodermatophilus, Modestobacter, Blastococcus), Nakamurella, Sporichthya, and Fodinicola and was grouped into suborder Frankineae. A recent rearrangement has resulted in the elevation of suborder Frankineae to order Frankiales (Normand and Benson 2012b) containing families Acidothermaceae, Cryptosporangiaceae, Frankiaceae, Geodermatophilaceae, Nakamurellaceae, and Sporichthyaceae as well as the incertae sedis Fodinicola feengrottensis

Molecular Methods for Research on Actinorhiza

Actinorhizal root nodules result from the interaction between a nitrogen-fixing actinomycete from the genus Frankia and roots of dicotyledonous trees and shrubs belonging to 25 genera within 8 plant families. Most actinorhizal plants can reach high rates of nitrogen fixation comparable to those found in root nodule symbiosis of the legumes. As a consequence, these trees are able to grow in poor and disturbed soils and are important elements in plant communities worldwide. While the basic knowledge of these symbiotic associations is still poorly understood, actinorhizal symbioses emerged recently as original systems to explore developmental strategies to form nitrogen-fixing nodules. Many tools have been developed in recent years to explore the interaction between Frankia and actinorhizal plants including molecular biology, biochemistry, and genomics. However, technical difficulties are often encountered to explore these symbiotic interactions, mainly linked to the woody nature of the plant species and to the lack of genetic tools for their bacterial symbionts. In this chapter, we report an inventory of the main recent molecular tools and techniques developed for studying actinorhizae