References

Higashi S, Abe M (1979) Appl. Environ. Microbiol. 39, 297-301 Lebunh et al. (2000) J. Syst. Evol. Microbiol. 50, 2207-2223

DEVELOPING GENETIC TOOLS FOR FRANKIA, THE BACTERIAL PARTNER OF THE ACTINORHIZAL SYMBIOSIS

Dept of Microbiology, Univ. of New Hampshire, Durham, NH 03824-2617, USA

Genetic analysis of Frankia is in its infancy. There are no known systems for gene transfer. Reliable standardized mutagenesis protocols have not yet been firmly established. One major drawback for genetic studies on Frankia is the lack of genetic markers. To identify potential genetic markers, twelve strains of Frankia were screened for resistance to antimetabolites, antibiotics and heavy metals by the use of a growth inhibition assay. Several strains had distinctive patterns of resistance that are potentially useful as genetic markers. The physical properties of the Frankia genome are being investigated by the use of PFGE. To develop a collection of genetically marked Frankia strains, chemical and physical mutagens were used to generate axotrophic, antimetabolite-resistant and antibiotic-resistant mutants. In the case of strain Eullc, EMS induced tetracycline-resistant and lincomycin-resistant mutants occurred at frequencies of 3.2 x 10"3 and 4.7 x 10"4, respectively. With strain Cel. 17, EMS induced lincomycin-resistant and 5-fluorouracil-resistant mutants occurred at frequencies of 1 x 10"5 and 4 x 10~5, respectively. The conjugative transposon Tn916 was successfully introduced into Frankia by filter mating with Enterococcus faecalis as the donor. These transconjugates were stable and have been maintained in culture. PCR and DNA hybridization experiments confirmed the insertion of the tetM gene. This is the first successful transfer and expression of foreign DNA into Frankia.

GENOMIC ANALYSIS AND GENETIC STUDY OF PHOSPHATE REGULATION GENES (pho REGULONS) IN SINORHIZOBIUM MELILOTI 1021

Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1 Canada

In bacteria, the transport and assimilation of phosphorus compounds from the environment is regulated by the PhoR-PhoB two component system. PhoR is a transmembrane sensor histidine kinase and PhoB is a transcriptional activator. In phosphorus limiting conditions PhoR activates PhoB by phosphorylation. The Pho regulon consistes of genes whose expression is regulated by PhoB. Phosphorylated PhoB binds to an 18 base pair (pho Box) containing two 7 bp repeats of 5'—CT{T/G}TCAT—3', which are present in the promoter region of phosphate regulated genes.

In S. meliloti we have previously identified two phosphate transport systems. The PhoCDET system is an ABC-type high affinity phosphate transport system. The OrfA-Pit system is a low affinity system which is expressed in cells growing in excess phosphate but repressed in cells growing under conditions of Pi limitation. We have cloned a 7.5 kb Hindlll gene fragment which includes the phoR-pstS-pstC-pstA-pstB and partial phoU genes. PstSCAB forms another ABC type phosphate transporter in E. coli. By Tn5-B20 and lacZ-aacCl cassette gene disruptions/fusions, we mutated the pstA, pstB andphoR genes respectively. We found: (a) pstA-pstB-phoU-phoB are in one operon, (b) pstB expression is not regulated by the media phosphate concentration and is independent of phoB, (c) in free-living cells, pstB mutants behave in the same manner as phoU or phoB regulatory mutants, i.e. they exhibit an alkaline phosphatase negative phenotype, (d) in plant tests, a pstB mutant had normal nitrogen fixation ability and like phoB mutations, the pstB mutation suppressed the Fix" phenotype of phoCDET mutants, (e) phoB expression is neither regulated by phosphate concentration nor does its expression appear to be autoregulated. Sequence analysis showed that there is a putative pho box upstream of pstS genes.

We collected all the available pho boxes from S. meliloti and E. coli, then built a matrix with the frequency of the four nucleotides at each position of the pho box and generated the S. meliloti pho box consensus motif CTGTCAT AAAT CTGTCAT. Employing this matrix we analyzed the complete S. meliloti genome sequence. Approximately 110 putative pho boxes were found to be located around different promoter regions: 71 pho boxes belong to known genes, and 40 to unknown genes. To further analyze the Pho regulon, we cloned about 20 genes or promoters including nadE2, Smc01907, Smb20427, Smc00801, sra, rhbF, CRP, recF, katA, ppk, afuA which contain putative pho boxes and are studying their promoter activity in low and high phosphate condition.

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