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Quantitative comparisons of bacterial population sizes in the field are greatly facilitated when each strain carries a unique selectable marker. This is particularly true when dispersal of bacteria between experimental plots in the field is expected or when one strain must be enumerated in the presence of a large excess of the other and measurement by difference or even by distinct colony morphology may give biased results. We are interested in comparisons of phyllosphere population sizes of mutants bearing defects in genes associated with pathogenicity to population sizes of wild-type Pseudomonas syringae pv. syringae (causal agent of bacterial brown spot disease) on snap bean (Phaseolus vulgaris L.) plants in the field (5, 8). The mutants carry selectable markers present on inserts that disrupt the genes of interest. For example, Tn5 mutants are resistant to kanamycin (Kan^r) due to the presence of the neomycin phosphotransferase gene (nptII) on the transposon. The issue that we faced in our studies was how to mark our wild-type strain in a way that would allow facile measurement of the population sizes to unambiguously differentiate them from those of mutant strains in field experiments.

One alternative is to use strains bearing spontaneous mutations that confer antibiotic resistance. These mutants are often easy to isolate, but they are less easy to characterize and may be less fit than the strain from which they were derived. In previous field experiments, we used a spontaneous nalidixic acid-resistant (Nal^r) mutant of P. syringae pv. syringae B728a as a surrogate wild-type strain (8). Although Nal^r B728a colonized bean leaves and caused disease in the field (8), we subsequently found that the derivative is less fit than the wild-type B728a. To better mark the wild-type strain, we sought to identify a site in the genome of B728a into which marker cassettes could be inserted in a site-directed manner without affecting fitness of the bacterium in the field. De Leij et al. (2) constructed derivatives of Pseudomonas fluorescens SBW25 with site-directed genomic insertions of marker genes (lacZY, aph-1 conferring Kan^r, and xylE) to examine the effects of gene insertions on the fitness of genetically modified bacteria. In their studies, however, bacterial fitness was assessed in greenhouse and growth chamber experiments, not under natural field conditions.

To identify a possible site for insertion of marker cassettes in the genome of B728a, we targeted a site (hereafter referred to as the landing site) located 51 bp upstream of the gacS open reading frame and within the roughly 235-bp intergenic region between the well-characterized regulatory gene gacS (global activator sensor kinase) (9) and cysM, a gene required for cysteine biosynthesis. The genes are transcribed in opposite directions away from the intergenic region (9). In P. syringae pv. syringae B728a, gacS is required for brown spot lesion formation; production of syringomycin, protease, N-acyl-L-homoserine lactone, and alginate; and a swarming behavior on soft agar (9, 10, 12, 13, 17). The landing site in the intergenic region was selected based on the finding that pEMH97 (9.7-kb HindIII fragment with gacS and cysM cloned in pLAFR3) containing a Tn3Gus insertion in the site retained the ability to restore the phenotype of the gacS mutant NPS3136 (gacS1::Tn5) to wild-type levels, whereas transposon insertions in gacS failed to do so (9). The objective of this study was to determine whether antibiotic resistance genes inserted into the landing site in the gacS-cysM intergenic region in the genome of B728a affect fitness of the bacterium in its natural habitat, leaves in the field.

Construction of site-directed marked derivatives. To introduce marker cassettes into the genome of B728a, we modified plasmid pEMH97 to contain a unique BamHI restriction sequence at the landing site. The restriction sequence was created by PCR using Pfu DNA polymerase (Stratagene, La Jolla, Calif). The modified plasmid pSSH-LS was digested with BamHI, and when necessary, the ends were made blunt with the Klenow fragment prior to ligation with the following marker cassettes: nptII (kanamycin resistance) (1), Spc interposon (spectinomycin resistance) (15), Cm interposon (chloramphenicol resistance) (3), and Gm^r (gentamycin resistance) (16). The markers were introduced into the chromosome of B728a by recombinational exchange.

Field experiments. Three field experiments were conducted to determine the relative fitness of the marked derivatives and B728a. The bacterial strains were inoculated onto snap bean seeds (cultivar Eagle; Asgrow Seed Co., Kalamazoo, Mich.) at the time of planting, and bacterial population sizes were monitored on germinating seeds and leaves of emergent plants as described previously (8). The treatments (i.e., bacterial strains) were in a randomized complete block design with three (1996 experiments) or eight (1997 experiment) blocks. Plot sizes were 8 by 8 m and 4 by 6 m in the 1996 and 1997 experiments, respectively. Bacterial population sizes were determined by dilution plating of individual leaf or seed homogenates as described previously (6, 8). The samples were plated on King's Medium B (11) containing rifampin (50 µg/ml) (KBR) supplemented with the appropriate antibiotics (kanamycin, 30 µg/ml; spectinomycin, 50 µg/ml; chloramphenicol, 30 µg/ml; gentamicin, 2 µg/ml; nalidixic acid, 50 µg/ml). Bacterial counts per sample were log₁₀ transformed before calculation of population statistics (7). Samples with no detectable colonies were assigned the limit of sensitivity of the plating assay (1.95 log CFU/sample for seeds collected immediately after planting; 2.57 log CFU/sample for all other samples). Evaluation of the relative fitness of the bacterial strains was based on the relative changes in population sizes over time. Treatment effects and treatment-by-time interactions were determined over all sampling time points using repeated-measures analysis of variance tests implemented with SAS Procedure Mixed (SAS Institute, Cary, N.C.) (14).

1996 experiments. In 1996, the relative fitnesses of B728a and the marked derivatives were compared in two adjacent experimental plots planted on 25 June. B728aTn3Gus, constructed by recombinational exchange of pEMH97::Tn3Gus (9) and therefore subjected to fewer molecular manipulations than the site-directed marked derivatives, was included as a control on the construction process. Additionally, the B728aTn3Gus construct provided a test of the effect of insert size on fitness. At roughly 7 kb, the Tn3Gus insert was over twice the size of any other insert tested.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   1996 field experiments. Mean population sizes of wild-type B728a and site-directed marked derivatives in association with field-grown bean plants are shown. The two experiments (A and B) were initiated on the same day in 1996. The bacterial strains were inoculated onto seeds immediately before planting. The sampling units collected were seeds at 0 days after planting (DAP); germinating seeds at 3 DAP; entire above-ground parts of emergent seedlings at 6 DAP; primary leaves at 10, 13, and 15 DAP; and leaflets from trifoliolate leaves at all other sampling times. Each datum point represents the mean log CFU/sample and standard error based on three replicate plots with six or eight individual samples per plot.

1997 experiment. To increase sensitivity in the detection of possible small differences in bacterial population sizes in an inherently variable system, we increased the number of blocks from three to eight in the 1997 experiment and monitored population sizes of the bacterial strains for a longer time (63 versus 31 days). Fewer subsamples were collected from each plot (eight in 1996 and five in 1997), and the plots were sampled less frequently. We included the Nal^r spontaneous mutant of B728a as a control strain with known decreased fitness relative to B728a.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   1997 field experiment. Mean population sizes of wild-type B728a, Nalr B728a, and site-directed marked derivatives in association with field-grown bean plants are shown. For clarity, the data are plotted in two groups. (A) B728a, Nalr B728a, and B728aCm; (B) B728a (same data as in panel A), B728aKan, B728aSpc, and B728aGm. The bacterial strains were inoculated onto seeds immediately before planting. The sampling units collected were seeds at 0 days after planting (DAP), germinating seeds at 3 DAP, entire above-ground parts of emergent seedlings at 6 DAP, primary leaves at 8 and 13 DAP, and leaflets from trifoliolate leaves at all other sampling times. Each datum point represents the mean log CFU/sample and standard error based on eight replicate plots with five individual samples per plot.