Identification and Characterization of Novel Genetic Markers Associated with Biological Control Activities in Bacillus subtilis
-Raghavendra Joshi and Brian B. McSpadden Gardener Department of Plant Pathology, The Ohio State University, OARDC, 1680 Madison Ave., Wooster 44691.
Accepted for publication 3 October 2005.
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RESULTS
Characterization of strain-specific DNA fragments. In order to identify DNA markers for biological control in B. subtilis, SSH was conducted to identify sequences present in the genomes of strains GB03 and QST713 but absent in the genome of strain 168. In all, 149 subtracted fragments (74 from GB03 and 75 from QST713) were cloned and analyzed. Fragment lengths varied from 180 to 1,520 bp, with a median fragment size of 560 bp. Se- quence analyses revealed that 80 of the subtracted fragments (36 from GB03 and 44 from QST713) had significant nucleotide homology (E value <1E-4) to previously identified Bacillus sequences. The sequences of these fragments can be found in GenBank under accession numbers DQ011366-401 (GB03) and DQ0113402-446 (QST713) in the same order as presented in Tables 1 and 2, respectively. In several instances, clones from both strains GB03 and QST713 were identified that had similarity to gene fragments from homologous operons. These included genes for the biosynthesis of cyclic lipopeptides (bam/bmy, fen, and srf ) and the cell wall component teichuronic acid (tua). Subtracted fragments matching the yndJ gene from B. subtilis subsp. amyloliquefaciens strain FZB42 also were recovered from both strains GB03 and QST713. Additionally, multiple sporulation gene fragments (spo) were found in both subtracted libraries.
DISCUSSION:
We characterized over 60,000 bp of genomic sequence obtained from strains of B. subtilis used in two different biocontrol prod- ucts. Nearly half of the 149 sequences we analyzed were sufficiently different from those presently in GenBank that no functional assignments could be made (data not shown). We expect that many of those unique sequences represent noncoding regions which are likely to be strain specific and therefore of limited value for population studies. Nonetheless, further characterization of these unique sequences is warranted because some may encode novel genes, an unknown fraction of which may be involved in biological control. Of the 80 Bacillus-like sequences we obtained, 65 were associated with genes predicted to encode a variety of metabolic functions. Much research has gone into establishing the function of B. subtilis genes, but such work has yet to reveal the function of all of the ORFs identified in strain 168 (48). In addition, their potential roles in bacterial fitness in the natural environment and biocontrol activities in a managed environment remain highly speculative. Nonetheless, several of these gene fragments identified present intriguing new avenues for investigating the nature of antibiotic-mediated biocontrol. For example, dppC is pre- dicted to code for dipeptide transporters and the homologs identi-
Nearly half of the 149 sequences we analyzed were sufficiently different from those presently in GenBank that no functional assignments could be made
Identification of subtracted genome fragments obtained from Bacillus subtilis QST713 [AGRAQUEST]
Bacillus amyloliquefaciens- RESULTS
Subtracted fragments matching the yndJ gene from B. subtilis subsp. amyloliquefaciens strain FZB42 also were recovered from both strains GB03 and QST713.
Bacillus cereus - RESULTS
.....with the exception that one of two faint bands amplified from most B. cereus strains comigrated with the ituC product amplified from the positive control strain, QST713.
...And, interestingly enough, only some of the B. cereus isolates and B. pumilus GB34 were maximally antagonistic to Phytophthora sojae in these assays, though QST713 did show a marginal degree of inhibition in all assays (i.e., average score of 1.0).
Bacillus licheniformis (page 7)
...However, similar types of associations were observed in other Bacillus spp. As a group, all of the B. pumilus and B. licheniformis isolates scored (weakly) positive for one or more gene markers and were more inhibitory to R. solani (P = 0.01) compared with those strains lacking the scored markers. Additionally, the two B. pumilus isolates that were scored weakly positive for the bmy gene (i.e., 1.1a2 and 2.5a) were significantly more inhibitory to Pythium ultimum than the other isolates lacking all of the markers (P = 0.04). However, presence of the amplifiable markers was not necessarily a good predictor of biocontrol capacity in B. pumilus because the commercialized biocontrol strain GB34 was scored negative for all of the amplifiable targets, yet displayed in vitro in- hibition capacities similar to those of the commercialized B. subtilis strains GB03, MBI600, and QST713.
