Final Technical Report
P. fluorescens PfO-1 is a soil bacterium isolated by this laboratory from sandy loam soil (4). Because of the importance of adhesion for persistence in natural environments, we utilized adherence to sand as an assay to screen a library of PfO-1 mutants for defects in adhesion. Three adhesion defective mutants, PfO-5, PfO-10, and PfO-15 were recovered. PfO-5 and PfO-10 had different insertions in the same gene, which we called adnA, and also showed motility defects (3). PfO-15 was motile, but was hyper-flagellated. The insertion was in a different gene, adnB, which shows similarity to mot genes involved in flagella functions (Strain and Levy, unpublished). These early studies demonstrated the important but separable requirements for flagella and motility in adherence. In a field study, the adnA mutant PfO-5 was less able to persist than the wildtype PfO-1 and did not spread as fast or as far from the point of inoculation as did PfO-1 (7), linking adhesion and soil fitness. DNA sequencing revealed that AdnA shares 82% identity with the flagella regulator FleQ from P. aeruginosa (3). FleQ is required for adhesion of P. aeruginosa to respiratory mucin, which is important for pathogenesis (1, 2). Using a gene fusion approach, seven loci that are expressed in an AdnA-dependent manner were identified (8). The loci were called ''aba'', for affected by AdnA. We uncovered genes involved in motility, chemotaxis, LPS synthesis, and two genes of no known function. Four of the aba genes were not reported to be in the FleQ regulon (5). We recently began using the IVET (in vivo expression technology) promoter-trap to identify genes whose expression is upregulated in soil. We identified 22 sequences (termed iiv for induced in vivo) that are upregulated in sterile soil (9). Ten of these genes are similar to sequences present in genbank, and two sequences are classed as ''hypothetical''. We also found ten iiv genes that are antisense to known genes, providing new insight into genome organization (10). We have called these sequences ''cryptic'' because they were not detected during annotation of the P. fluorescens PfO-1 genome sequence. We have detected open reading frames for each of the 10 cryptic genes. To test the importance of soil-induced genes in survival or persistence, we constructed mutations in three iiv genes, and tested them in soil growth assays. Relative to the wildtype, these mutants had no growth defect in laboratory culture, but the mutations did increase the time needed to establish the population in sterile soil (9). These data provide support for our hypothesis that IVET will reveal genes involved in environmental (soil) survival. The gene iiv2 which has a colonization defect in sterile soil, has no similarity to known sequences and thus is a novel sequence with a role in soil colonization. In live soil microcosms, wildtype and iiv2 mutant bacteria were inoculated in a central ''core''. Persistence within the core and spread from the core were quantified, using a specific competitive PCR (6) approach which we developed and optimized. The Hv2 mutant showed reduced persistence within the microcosm core, and was impaired in its ability to spread out and colonize soil. These data further support the IVET approach for finding previously uncharacterized genes that are critical to fitness in soil. To directly confirm transcription, we have carried out RT-PCR analysis on cryptic iiv genes, and the sequences on the opposite DNA strand. Primers that permitted the amplification of a region internal to both cryptic and opposite gene were used. Reverse transcription used a single gene-specific primer, so that transcripts from both strands to be detected separately The RT-PCR experiments have confirmed expression of eight cryptic genes, and the genes encoded on the opposite strand The relative cryptic, opposite gene expression level varies between loci. For example, iiv14 is expressed more than its opposite gene, while both iiv5 and iiv23 appear to be expressed at lower levels than the opposite genes The iiv genes show increased expression in soil relative to laboratory media. We have begun to isolate mutants in which iiv promoter activity is increased to levels that can be detected in laboratory culture. We have successfully generated mutants that upregulate 15 different iiv genes, including five cryptic genes. In the case of the cryptic gene Hv14, de-repression resulted in an 18.3 fold increase, while for iiv23 the increase was 6 3 fold The mutation that led to de-repression of iiv23 was found to lie in the dsbD precursor gene Cloned dsbD precursor complemented the mutation, i e.repression of iiv23 was restored.
- Research Organization:
- Tufts University
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- FG02-97ER62493
- OSTI ID:
- 934437
- Report Number(s):
- DOE/ER62493-1 Final Report; TRN: US201022%%32
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
54 ENVIRONMENTAL SCIENCES
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
ADHESION
AMPLIFICATION
BACTERIA
DEFECTS
DNA
DNA SEQUENCING
GENES
IN VIVO
INOCULATION
LOAM
MICROCOSMS
MUTANTS
MUTATIONS
PATHOGENESIS
PRECURSOR
PROMOTERS
SOILS
SYNTHESIS
TRANSCRIPTION