Stable Transformation of the Actinobacteria Frankia spp.

A stable and efficient plasmid transfer system was developed for nitrogen-fixing symbiotic actinobacteria of the genus Frankia, a key first step in developing a genetic system. Four derivatives of the broad-host-range cloning vector pBBR1MCS were successfully introduced into different Frankia strains by a filter mating with Escherichia coli strain BW29427. Initially, plasmid pHKT1 that expresses green fluorescent protein (GFP) was introduced into Frankia casuarinae strain CcI3 at a frequency of 4.0 × 10-3, resulting in transformants that were tetracycline resistant and exhibited GFP fluorescence. The presence of the plasmid was confirmed by molecular approaches, including visualization on agarose gel and PCR. Several other pBBR1MCS plasmids were also introduced into F. casuarinae strain CcI3 and other Frankia strains at frequencies ranging from 10-2 to 10-4, and the presence of the plasmids was confirmed by PCR. The plasmids were stably maintained for over 2 years and through passage in a plant host. As a proof of concept, a salt tolerance candidate gene from the highly salt-tolerant Frankia sp. strain CcI6 was cloned into pBBR1MCS-3. The resulting construct was introduced into the salt-sensitive F. casuarinae strain CcI3. Endpoint reverse transcriptase PCR (RT-PCR) showed that the gene was expressed in F. casuarinae strain CcI3. The expression provided an increased level of salt tolerance for the transformant. These results represent stable plasmid transfer and exogenous gene expression in Frankia spp., overcoming a major hurdle in the field. This step in the development of genetic tools in Frankia spp. will open up new avenues for research on actinorhizal symbiosis.IMPORTANCE The absence of genetic tools for Frankia research has been a major hindrance to the associated field of actinorhizal symbiosis and the use of the nitrogen-fixing actinobacteria. This study reports on the introduction of plasmids into Frankia spp. and their functional expression of green fluorescent protein and a cloned gene. As the first step in developing genetic tools, this technique opens up the field to a wide array of approaches in an organism with great importance to and potential in the environment.

Permanent Draft Genome Sequence for Frankia sp. Strain CeD, a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Casuarina equistifolia Grown in Senegal.

Frankiastrain CeD is a member ofFrankialineage Ib that is able to reinfect plants of theCasuarinafamilies. Here, we report a 5.0-Mbp draft genome sequence with a G+C content of 70.1% and 3,847 candidate protein-encoding genes.

Permanent Draft Genome Sequences for Two Variants of Frankia sp. Strain CpI1, the First Frankia Strain Isolated from Root Nodules of Comptonia peregrina.

Frankia stains CpI1-S and CpI1-P are members of Frankia lineage Ia that are able to reinfect plants of the Betulaceae and Myricaceae families. Here, we report two 7.6-Mbp draft genome sequences with 6,396 and 6,373 candidate protein-coding genes for CpI1-S and CpI1-P, respectively.

Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis.

UNLABELLED: The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. IMPORTANCE: The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont.

Draft Genome Sequence of Photorhabdus temperata Strain Meg1, an Entomopathogenic Bacterium Isolated from Heterorhabditis megidis Nematodes.

Photorhabdus temperata strain Meg1 is an entomopathogenic bacterium that forms a symbiotic association with Heterorhabditis nematodes. We report here a 4.9-Mbp draft genome sequence for P. temperata strain Meg1, with a G+C content of 43.18% and containing 4,340 candidate protein-coding genes.

Draft Genome Sequence of Frankia sp. Strain BMG5.23, a Salt-Tolerant Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Casuarina glauca Grown in Tunisia.

Nitrogen-fixing actinobacteria of the genus Frankia are symbionts of woody dicotyledonous plants termed actinorhizal plants. We report here a 5.27-Mbp draft genome sequence for Frankia sp. strain BMG5.23, a salt-tolerant nitrogen-fixing actinobacterium isolated from root nodules of Casuarina glauca collected in Tunisia.

Draft Genome Sequence of Frankia sp. Strain Thr, a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Casuarina cunninghamiana Grown in Egypt.

Nitrogen-fixing actinobacteria of the genus Frankia are symbionts of woody dicotyledonous plants termed actinorhizal plants. We report here a 5.3-Mbp draft genome sequence for Frankia sp. stain Thr, a nitrogen-fixing actinobacterium isolated from root nodules of Casuarina cunninghamiana collected in Egypt.

Draft Genome Sequence of Photorhabdus luminescens Strain BA1, an Entomopathogenic Bacterium Isolated from Nematodes Found in Egypt.

Photorhabdus luminescens strain BA1 is an entomopathogenic bacterium that forms a symbiotic association with Heterorhabditis nematodes. We report here a 5.0-Mbp draft genome sequence for P. luminscens strain BA1, with a G+C content of 42.46% and 4,250 candidate protein-coding genes.

Draft Genome Sequence of Frankia sp. Strain CcI6, a Salt-Tolerant Nitrogen-Fixing Actinobacterium Isolated from the Root Nodule of Casuarina cunninghamiana.

Members of the actinomycete genus Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a 5.57-Mbp draft genome sequence for Frankia sp. strain CcI6, a salt-tolerant nitrogen-fixing actinobacterium isolated from root nodules of Casurina cunninghamiana grown in Egyptian soils.