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Applied and Environmental Microbiology, June 2008, p. 3644-3651, Vol. 74, No. 12
0099-2240/08/$08.00+0     doi:10.1128/AEM.00429-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Increased Fitness of Pseudomonas fluorescens Pf0-1 Leucine Auxotrophs in Soil

Wook Kim and Stuart B. Levy^*

Center for Adaptation Genetics and Drug Resistance, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111

Received 15 February 2008/ Accepted 9 April 2008

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The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-encoding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology has previously identified 22 discrete genetic loci in Pseudomonas fluorescens Pf0-1 that were specifically activated in the soil environment, of which 10 were present in an antisense orientation relative to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs, iiv19 and leuA2, in soil. Although iiv19 was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter the ability of P. fluorescens to colonize or survive in soil. Surprisingly, the absence of the leuA2 gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the iiv19 gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage of the bacterium in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of Pseudomonas spp. in the lungs of cystic fibrosis patients.

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Sterile liquid media have long served as, and remain, the preferred conduit for studying gene expression in bacteria. Consequently, most current knowledge has come from the perspective of free-living planktonic populations in shaking flasks and test tubes. However, it is widely acknowledged that bacteria in nature rarely exist solely in the planktonic form but rather as constituents of structurally organized communities.

Escherichia coli K-12 has served as the primary model in almost all fundamental aspects of microbiology. One may argue that E. coli K-12 has been passaged and studied in shaken broth more often than any other bacterium. Since the initial annotation of the complete genome of K-12 a decade ago, the percentage of identified chromosomal gene products without a function assignment has decreased from approximately 33 percent to 10 percent (28). Although the proportion of genes of unknown function will continue to shrink over time, the functions of some of the remaining genes may have to be elucidated outside the flask. Gene assignment in newly sequenced genomes is an automated process that is largely based on sequence features derived from previously annotated genomes, a process innately biased toward genes identified under laboratory conditions. Understandably, the proportion of genes without a function assignment is markedly higher in other genomes, especially those that are remotely related to E. coli K-12.

Pseudomonas fluorescens is a versatile organism that is readily isolated from soil, in bulk and from the rhizosphere. P. fluorescens Pf0-1 has long served as a model organism in our ongoing interests in understanding the genetic bases of bacterial proliferation and persistence in soil (7, 9-11, 20, 29). Our experimental systems utilize the same batch of soil from which P. fluorescens Pf0-1 was originally isolated (9), presenting an exceptional opportunity to study the organism as close to its natural habitat as possible in the laboratory. Application of in vivo expression technology (IVET) identified a set of 22 discrete loci in the genome of P. fluorescens Pf0-1 that were induced specifically in soil (33). This set of iiv (induced in vivo) genes included the so-called "cryptic genes" whose sequences did not share any homology to known genes while overlapping those previously annotated on the opposite DNA strand (34). Overlapping structures as such appear frequently in viral genomes but have been rarely reported in bacteria (31, 34).

One of the obvious challenges in studying the individual function of overlapping genes is that the introduction of a mutation in one also affects the other, especially when one member of the pair is cryptic in nature. We sought to examine the individual roles of one of the previously identified overlapping pairs of genes, iiv19 and leuA2, directly in the soil environment. This gene pair was chosen for study because the function of the leuA2 gene product had already been described in great detail. Here, we report an unforeseen soil-specific fitness advantage resulting from a mutation in the leuA2 locus that is independent of its cryptic iiv19 counterpart.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains and culture conditions.
Pseudomonas fluorescens Pf0-1 was isolated from loam soil (9). For competition experiments, strains were labeled and verified by using the mini-Tn7 system as previously described (44), with kanamycin resistance via plasmid pHRB2 (24) or streptomycin resistance (33a) via a derivative of the plasmid pUC18T-miniTn7T (8) harboring the cassette from pHRP315 (26). Deletion mutants of Pf0-1 were constructed as already described (33). Splicing by overlap extension-PCR (SOE-PCR) (16) was used to create the deletion constructs with the suicide plasmid pSR47s (22). Pf0-119 is a construct with a complete deletion of the leuA2-iiv19 overlapping locus, created by the SOE-PCR primers 19D5f (5'-GCGAAGTGCTGCATTACG) and 19D5r (5'-CTCTCACACCCTCATTCAGGGAACGATCAGGCGCCTC) for the 5' end and 19D3f (5'-GAGGCGCCTGATCGTTCCCTGAATGAGGGTGTGAGAG) and 19D3r (5'-AGCAACCTGCTGCTGGAC) for the 3' end. Pf0-1leuB is a construct with a complete deletion of the leuB locus, created by the SOE-PCR primers leuB5f (5'-TTGAGCAACGTGTCCACG) and leuB5r (5'-GCTTTCCTCGTGTCTTCAGGCTTTCCTCGTGTCTTCAG) for the 5' end and leuB3f (5'-CTGAAGACACGAGGAAAGCTAATCTCTCGGGCCCGCTG) and leuB3r (5'-ATGGCGCTCATCCACTCG) for the 3' end. Each deletion construct was cloned into pGEM-T Easy (Promega) then subcloned into the NotI site in pSR47s. Deletions were confirmed by PCR using primers iiv19A (5'-TTGCCAAACGTGAGGAGACG) and iiv19B (5'-GCCGCAACCTCAATTTCAGT) for the iiv19-leuA2 locus and primers leuB1 (5'-GTCGCCTCGTTGGTGAAAG) and leuB2 (5'-TTCGGTCGGGTAAGCGTC) for the leuB locus. Strain Pf0-119PEleuA is Pf0-119 complemented with a hybrid leuA gene comprising the leuA2 promoter from Pf0-1 fused to a promoterless leuA open reading frame (ORF) from E. coli MG1655 by SOE-PCR: primers PleuAF (5'-AGTCCTCGAGTTCTTCAATGGCAACACCG) and PleuAR (5'-GAAAATAATGACTTGCTGGCTCATCGGAAAATTCCTTG) amplified the Pf0-1 promoter, and primers EleuAF (5'-CAAGGAATTTTCCGATGAGCCAGCAAGTCATTATTTTC) and EleuAR (5'-AGTCGGATCCCGGCAATACGGCAATATG) amplified the leuA ORF from MG1655. The hybrid construct was cloned into pGEM-T Easy, then subcloned into the ApaI and BamHI sites of the mini-Tn7 plasmid pHRB2 (24), and introduced into Pf0-119 by conjugation. All routine cloning was done with E. coli 10B (Invitrogen), and E. coli S17.1 pir (35) was used as the donor strain in conjugations. All E. coli strains were grown in LB (Invitrogen) at 37°C.

P. fluorescens strains were grown in Pseudomonas minimal medium (PMM) (18) with shaking at 250 rpm at 30°C. We assessed the growth of strains individually or in competition with another strain in 2.5 ml PMM inoculated with ca. 10⁴ CFU. Each culture was serially diluted 16 h later and enumerated on PMM solidified with 1.5% agar. Leucine, isoleucine, valine, and histidine were used as supplements at 200 µM each, and ampicillin, kanamycin, and streptomycin were each added at 50 µg/ml when required. All amino acids and antibiotics were purchased from Sigma.

Reverse transcription-PCR (RT-PCR) and 5' RACE.
RNA was purified from Pf0-1 grown in PMM by using the RNeasy minikit (Qiagen). Each sample was treated with DNase I (Qiagen) on-column and later with RQ1 DNase I (Promega) off-column. Each sample was then repurified using the RNeasy minikit. cDNA was synthesized from 0.5 µg RNA by using the Superscript III kit (Invitrogen), primer 19RT-F (5'-ACCTGGCTGAATTCGATCTG) for leuA2, and primer 19RT-R2 (5'-AACGTCGACCTCGTGACTG) for iiv19. PCR was performed using the cDNA as templates and 19RT-F and 19RT-R2 as primers. cDNA generated by use of 19RT-R2 was used as the template for the 5' rapid amplification of cDNA ends (RACE) system (Invitrogen) with primers 19GSP2 (5'-CGAAGAGTGCAACCAGATC) and 19GSP3 (5'-GGTGCACACAGCGTTCTC). 5' RACE products from three discrete cDNA preparations were cloned into pGEM-T Easy (Promega) and sequenced using the T7 and SP6 primers at the Tufts University Core Facility (Boston, MA).

Soil assays.
Gamma-irradiated loam soil (11) was inoculated and sampled as previously described (33) with slight modifications. Strains were grown in PMM overnight and serially diluted in distilled water to ca. 10⁴ CFU/ml, and 1 ml was mixed with 5 g of soil. This resulted in ca. 50% water-holding capacity. The initial population density (CFU/g soil) was determined by sampling 0.5 g of soil at 30 minutes following inoculation. Each soil sample was vortexed for 30 seconds in 1 ml distilled water, briefly rested to settle large particles, and then serially diluted and enumerated on PMM solidified with 1.5% agar. Soil was supplemented with leucine, isoleucine, valine, or histidine by the addition of 20 µl of 1 mM stock solution during bacterial inoculation or as noted otherwise. Competition experiments were carried out in the same manner except that both strains at 0.5 ml each were mixed together prior to being mixed with soil and enumerated using PMM supplemented with the appropriate antibiotic. Competitive index values were calculated as the output ratio of the mutant population density to the wild-type population density divided by the input ratio of the mutant population density to the wild-type population density as previously described (41).

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Molecular analyses of the iiv19-leuA2 locus.
The leuA2 gene specifies the 2-isopropylmalate synthase, the first specific enzyme in the leucine biosynthesis pathway (38). In P. fluorescens Pf0-1, antisense to leuA2 resides a cryptic promoter termed iiv19, whose activity was sufficient for detection by IVET in soil but not in minimal medium (PMM) (33). To determine whether iiv19 is indeed capable of transcribing a message, RT-PCR was carried out using strand-specific oligonucleotides. Products from both strands were detected only when reverse transcriptase was supplied (Fig. 1A), confirming that each of the two genes—iiv19 and leuA2— is actively transcribed. Furthermore, the transcription initiation site was mapped to be 373 bp downstream from the 3' end of the leuA2 gene on the opposite strand by 5' RACE (Fig. 1B). BPROM (http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) and NNPP (http://www.fruitfly.org/seq_tools/promoter.html) analyses of the immediate upstream region did not reveal recognizable ⁷⁰-dependent promoter sequences, and the termination site of the transcript remains elusive, as 3' RACE attempts did not yield conclusive results.

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FIG. 1. Transcription and organization of iiv19 and leuA2 genes. (A) Results of RT-PCR using gene-specific primers; "+" and "–" indicate the presence and absence of reverse transcriptase in the reaction, respectively. (B) Schematic of the overlapping genetic organization of iiv19 and leuA2. The numbers indicate the nucleotide positions of the leuA2 ORF (bp 1 to 1680). The transcript start site for iiv19 (bp 1307 relative to the start site of the leuA2 ORF) was mapped by 5' RACE. The 3' end of iiv19 is stretched out to the first translational stop site, but the precise location of the 3' terminus of the iiv19 transcript is unknown. The horizontal bar (bp 843 to 1232) represents the RT-PCR product indicated in panel A.

Since iiv19 and leuA2 directly overlap one another (Fig. 1B), transcription of the leuA2 gene should have a direct influence on iiv19 expression, and vice versa, through DNA template competition. The leuA gene of E. coli is strongly repressed in the presence of exogenous leucine by a regulatory mechanism known as transcription attenuation (23). Although the iiv19 transcript can be detected by RT-PCR from Pf0-1 grown in PMM (Fig. 1A), its expression is below the threshold level of the IVET screen (33). We reasoned that the addition of leucine to PMM would reduce leuA2 transcription, which in turn may allow iiv19 to be transcribed at a higher rate. Northern analyses were carried out using RNA isolated from Pf0-1 grown in PMM and the rich King's B medium supplemented with leucine at various concentrations ranging between 0 to 1 mM. We did not observe any notable differences in the leuA2 transcript levels up to the highest level of leucine supplied, and iiv19 transcripts were not detected in any of the samples (data not shown). These results indicated that expression of the leuA2 gene in Pf0-1 was not regulated by leucine. In agreement with these observations, a recent bioinformatics study reported that leuA2 genes in Pseudomonas spp. do not contain the upstream attenuator elements that are present in E. coli (43).

Assessment of the ability of the iiv19 leuA2 double mutant to colonize soil.
Understanding the role of iiv19 in survival in soil represented a rather complicated challenge, given the overlapping orientation of the two genes and the unknown precise structure of the iiv19 gene. Since the function of the leuA2 gene is well established, we reasoned that the role of iiv19 could potentially be deduced by complementing the leuA2 mutation biochemically in a double mutant. We therefore constructed the strain Pf0-119 from P. fluorescens Pf0-1; this mutant has a deletion in the genomic region spanning both leuA2 and the predicted iiv19 gene. As anticipated, Pf0-119 behaved as an auxotroph, requiring exogenous supplementation of leucine for growth in PMM.

The impact of the mutation on soil colonization was assessed by comparing Pf0-119 to the parent strain Pf0-1 in our sterile soil system (33). Growth of the two strains was monitored when they were inoculated alone in soil (i.e., each strain assessed individually) and in competitive soil (i.e., the two strains mixed together). To differentiate the strains, neutral kanamycin and streptomycin resistance markers were introduced into each chromosome (see Materials and Methods) and verified not to influence colonization of individual and competitive soil (data not shown). Pf0-1 introduced alone rapidly colonized soil, reaching the maximum population density afforded by the soil system at ca. 10⁸ CFU/g by the second day (Fig. 2A). Pf0-1 remained at this density even after three months following inoculation, provided that moisture loss was kept to a minimum (data not shown). In contrast, Pf0-119 exhibited a markedly reduced rate of colonization, gradually reaching a maximum of ca. 10⁷ CFU/g, about 10-fold less than Pf0-1 (Fig. 2A). Thus, the soil system appeared to contain sufficient amounts of leucine and/or leucine-containing peptides (LCP) to support the growth of the auxotrophic Pf0-119 but became limiting eventually. An alternative explanation is that leucine was not the limiting factor, but the absence of the iiv19 gene resulted in reduced fitness. However, supplementation of leucine to the soil system fully restored the colonization ability of Pf0-119 (Fig. 2A), indicating that the absence of the leuA2 gene was likely the main reason behind the observed defect. A concentration of 0.4 µmol/g was the minimum amount of leucine required for Pf0-119 to consistently reach the same population density as Pf0-1. This concentration is far below the level of hydrolysable leucine detected in a variety of natural soils (12, 21), and supplementation of soil with leucine neither increased nor decreased the population density of Pf0-1 (Fig. 2A).

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FIG. 2. Comparisons of growth of the Pf0-119 mutant and the Pf0-1 parent strains in individual and competitive soil assays with (filled symbols) or without (open symbols) leucine. (A) Pf0-1 (triangles) and Pf0-119 (circles) were introduced into individual soil systems and the data are presented as log10 CFU/g soil. (B) Equal amounts of Pf0-1 and Pf0-119 were mixed prior to being introduced into the same soil systems, and the data are presented as competitive index values. The error bars indicate the standard deviations of the means from at least three independent experiments.

Given the reduced colonization efficiency exhibited by the mutant in soil, we predicted that Pf0-1 would be dominant over Pf0-119 in competitive soil. Indeed, Pf0-119 was outcompeted by Pf0-1 by at least an order of magnitude (Fig. 2B). However, we observed an unexpected reversal in trend when leucine was added to the soil: Pf0-119 outcompeted Pf0-1 by two orders of magnitude (Fig. 2B). One explanation for this result may be that leucine had a toxic effect on Pf0-1, but this is unlikely since leucine supplementation did not adversely affect its ability to colonize soil alone (Fig. 2A). Alternatively, since iiv19 is known to be preferentially expressed in soil (33), the consequence of its activity under an elevated level of exogenous leucine may have somehow been manifested as a competitive defect. In order to address this hypothesis, it was necessary to study the effect of the mutation in each of these genes independently.

The absence of the iiv19 gene does not affect soil colonization.
There are two distinct classes of homologous genes, leuA and leuA2, that encode the 2-isopropylmalate synthase (43). Unlike most members of the class Gammaproteobacteria, Pseudomonas spp. possess the leuA2 homolog. Given that the two classes share only 30% sequence identity (43), it is unlikely that the iiv19 gene is also present antisense to the leuA homologs. We designed a hybrid construct by fusing the leuA2 promoter from Pf0-1 to the leuA gene from E. coli MG1655. We then introduced the hybrid construct into a noncoding region in the genome of Pf0-119 by using the Tn7 system. The resulting strain, Pf0-119PEleuA, was capable of growing in PMM without leucine, indicating that the E. coli leuA gene was correctly being transcribed by the native promoter from Pf0-1. Since Pf0-119PEleuA is capable of expressing a functional 2-isopropylmalate synthase while lacking the iiv19 gene, we had the opportunity to assess specifically the impact of the iiv19 mutation in the soil environment. In competition experiments, Pf0-119PEleuA was capable of colonizing the soil equally as well as Pf0-1, both in the presence and absence of leucine supplementation (Fig. 3A). These results indicate that iiv19 does not play a significant role in soil colonization and its presence did not contribute to the apparent reduced fitness of Pf0-1 in competition with Pf0-119 (Fig. 2B).

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FIG. 3. Growth of Pf0-119PEleuA and Pf0-1leuB compared to growth of Pf0-1 in soil with (filled symbols) or without (open symbols) leucine. (A) Competitive index values for soil inoculated with a mixture of the partially complemented mutant strain Pf0-119PEleuA and the parent strain Pf0-1. (B) Growth of the Pf0-1leuB mutant in soil inoculated with the individual strain. (C) Competitive index values for soil inoculated with a mixture of the Pf0-1leuB mutant and the parental Pf0-1 strain. The error bars indicate the standard deviations of the means from at least three independent experiments.

Although the iiv19 gene may not be present antisense to the leuA gene in E. coli, there exists the possibility that an unrelated iiv19-like element is present antisense to the E. coli leuA gene in Pf0-119PEleuA. If such an element indeed exists and is expressed in Pf0-119PEleuA, the lack of competition observed against Pf0-1 (Fig. 3A) does not necessarily refute a role of iiv19 in soil colonization. To address this issue, a leuB deletion mutant was constructed. The leuB gene encodes the 3-isopropylmalate dehydrogenase complex, which functions two steps after the leuA gene product in the leucine biosynthesis pathway (15). Pf0-1leuB is incapable of producing leucine but harbors the wild-type leuA2-iiv19 locus. Pf0-1leuB exhibited characteristics similar to those of Pf0-119: it had a reduced rate of colonization without leucine (Fig. 3B), but it outcompeted Pf0-1 when leucine was supplied exogenously (Fig. 3C). These results confirm that iiv19 is not involved in soil colonization, nor does its presence affect the decreased fitness of Pf0-1 in competition with Pf0-119.

Effect of leucine supplementation on isoleucine-valine pseudoauxotrophy in PMM.
Leucine, like other amino acids, has a transient existence in soil, be it free or as a component in peptides and proteins. Radiolabeled leucine is often utilized in assays to estimate bacterial growth rates in environmental samples, such as soil samples (1). It is not readily apparent why the addition of leucine, which is present at a limiting concentration in our soil system (Fig. 2A), negatively affects Pf0-1 only in competition against strains that cannot synthesize leucine (Fig. 2B and 3C).

Given the chemical and physical complexities of the soil environment, we explored the underlying mechanism behind this phenomenon under a more-defined setting. For growth in PMM, Pf0-119 requires 100 to 200 µM leucine, which is considered to be the general range needed to support the growth of most P. fluorescens auxotrophs (36). However, despite leucine supplementation, Pf0-119 consistently grew to a density less than that of Pf0-1 (Fig. 4A), and providing more leucine did not increase its density. In contrast, when cocultured with Pf0-1 in leucine-supplemented PMM, Pf0-119 reached a density much higher than that obtained in pure culture (Fig. 4A). A similar finding was observed when cells were grown on the surface of PMM solidified with agar (data not shown). Taken together, these results suggest that some factor other than leucine affected the relative fitness of Pf0-119, and it is somehow related to cohabitation in medium with Pf0-1. Cell-free supernatant of Pf0-1 grown in leucine-supplemented PMM did not have any noticeable effect on Pf0-119 and vice versa (data not shown), indicating that a secreted factor was presumably not involved. However, we cannot exclude the possibility that such a factor may have been unstable and/or below the effective concentration in our assays.

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FIG. 4. Effect of various branched-chain amino acids on the growth of Pf0-119 and Pf0-1 in PMM. Pf0-119 (black bars) and Pf0-1 (white bars) were grown overnight in PMM supplemented with one or more branched-chain amino acids. The data are presented as relative increases in cell density (log10 CFU/ml) compared to the initial density of ca. 104 CFU/ml. (A) Strains were inoculated in PMM supplemented with leucine individually or together (competitive) in the same tubes. In separate experiments, strains were inoculated in individual tubes (B) or the same tubes (C) containing PMM supplemented with leucine (L) or a combination of leucine, isoleucine, and valine (LIV). Each amino acid was added at 200 µM. The error bars indicate the standard deviations of the means from at least three independent experiments.

Amino acids can inhibit growth as a consequence of antagonism between related amino acids (13). In the case of leucine, an increased intracellular concentration inhibits the activity of acetohydroxy acid synthase (14). Since this enzyme performs the first common step in the biosynthesis of all branched-chain amino acids, inhibition of its activity halts growth, as the cells are no longer able to synthesize leucine, isoleucine, and valine. Growth resumes when these amino acids are exogenously supplied (30) or the acetohydroxy acid synthase is actively derepressed (19). To test the antagonism theory, Pf0-119 and Pf0-1 were individually grown in PMM supplemented with leucine alone or a cocktail of leucine, isoleucine, and valine. The addition of isoleucine and valine increased the density of Pf0-119 by almost an order of magnitude and also increased the density of Pf0-1 by a smaller margin (Fig. 4B). The addition of isoleucine and valine also closed the remaining gap between cocultured Pf0-119 and Pf0-1 values (Fig. 4C). These results collectively support the notion that leucine supplementation was indeed exerting an antagonistic influence on isoleucine and valine biosynthesis.

Extension of the amino acid antagonism theory into the soil environment.
Although the antagonism theory can account for most of the experimental data derived from the use of PMM, it is not readily applicable for an understanding of the outcome of the soil experiments. Unlike growth in PMM, leucine supplementation supported Pf0-119 colonization of soil as effectively as it supported Pf0-1 soil colonization, and it also supported dominance over Pf0-1 when the strains were placed in competition together. The antagonism theory was directly tested in soil by setting up groups of competitions between Pf0-119 and Pf0-1 in leucine-supplemented soil. To a given group, either histidine or a cocktail of isoleucine and valine was further added. The relative changes in the dominance of Pf0-119 over Pf0-1 following exposure to either isoleucine and valine or histidine are shown in Fig. 5. Relative fitness of 100% indicates that a given treatment did not influence Pf0-119's advantage over Pf0-1. The addition of isoleucine and valine significantly reduced the relative fitness, indicating that their presence made Pf0-1 more competitive. An alternative interpretation is that leucine supplementation in a competitive soil assay has a negative impact on Pf0-1 greater than its negative impact on Pf0-119. Histidine, which was included as a non-branched-chain amino acid control, did not influence the relative fitness, strengthening the relevance of the antagonism theory in the soil competitions.

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FIG. 5. Effect of adding branched-chain amino acids or histidine to leucine-supplemented soil. Equal amounts of Pf0-119 and Pf0-1 cells were mixed and introduced into leucine-supplemented soil. Each soil system was further supplemented with a mixture of isoleucine and valine (black bars) or histidine (white bars). The competitive index values derived from each treatment were compared to the competitive index values derived from soil supplemented with leucine alone, and the data are presented as percent relative fitness. One hundred percent relative fitness indicates that a given treatment generated the same competitive index values as leucine alone. The error bars indicate the standard deviations of the means from at least three independent experiments.

So what is the underlying basis for Pf0-1's elevated sensitivity to antagonism by leucine?
Leucine is taken up by the cell through two major routes: (i) as a free amino acid via the LIV transport systems, which are repressed by leucine or other amino acid substrates (17, 27), and (ii) as a component of peptides via peptide transport systems that are constitutively active (2). The rate of leucine metabolism is considered to be lower than the combined rates of transporting in LCP and cleaving them into free amino acids (39). Thus, in the presence of increasing levels of LCP, the intracellular accumulation rate of leucine would increase, eventually leading to isoleucine-valine pseudoauxotrophy. One of the obvious differences between Pf0-119 and Pf0-1 is that the former does not synthesize leucine de novo and the only source for intracellular accumulation would be through uptake. It is estimated that more than 99% of amino acids in soil exist in a polymeric state, such as in peptides and proteins (37). With these data taken together, the initial uptake rates of exogenous leucine and LCP would be the same for Pf0-119 and Pf0-1 in leucine-supplemented soil. However, the intracellular accumulation rate would be higher for Pf0-1, since it is capable of synthesizing leucine de novo and especially since none of the leucine-specific biosynthesis genes are predicted to contain the transcription attenuation motif (43). The net effect would be that inhibition of acetohydroxy acid synthase activity would occur more rapidly among the Pf0-1 cells and quicken the onset of pseudoauxotrophy. Such a relative effect would not be predicted to manifest as a colonization defect in soils inoculated with the strain alone (Fig. 2A).

Among the obvious physiological conditions that are difficult to represent in constantly shaken broth are spatial chemical gradations (e.g., branched-chain amino acids). One of the main physical differences between soil and PMM is that leucine was present only in the free form in the latter. Leucine uptake via the LIV transport systems is quickly repressed when the intracellular level reaches a certain point (27). Thus, Pf0-1 is not expected to be dominated by Pf0-119 in PMM as it was in soil. It is, however, less apparent why leucine-supplemented Pf0-119 grows to a higher density in the presence of Pf0-1 than on its own in PMM, especially when Pf0-1 does not appear to secrete any factors that benefit Pf0-119.

Relevance of findings.
Leucine has long been considered a major regulatory signal in E. coli, exerting both positive and negative effects, largely through the global transcriptional regulator Lrp (6). Fluctuating intracellular levels of leucine may have stimulated significant physiological changes that were not captured in our assays, or perhaps they were somehow reflected in Pf0-119's ability to reach a higher density in the presence of Pf0-1 in PMM. Although iiv19 was demonstrated to be a nonfactor in this study, the relevance of its existence antisense to leuA2 remains to be characterized. Because of technical difficulties associated with extracting mRNA from soil (32), we could not establish whether a transcript of iiv19 can be detected directly from soil. This finding is necessary to confirm that iiv19 is indeed expressed in soil, since IVET can detect only promoters or promoter-like elements. Moreover, the precise nature of each of the cryptic genes remains to be elucidated. There is at least one convincing piece of evidence that each member of an overlapping pair of genes, Pf1-0939 and iiv14, encodes a protein and that the iiv14 gene product plays a role in soil colonization (33a). Perhaps when a larger set is characterized in greater detail, a common pattern that may reveal the path toward function will emerge.

We have demonstrated that an auxotroph can outcompete its prototrophic parent in the soil environment, provided that the lacking amino acid is exogenously provided. Since soil bacteria are generally described to lead a lifestyle of feast and famine, selecting against genes that ensure survival for fleeting moments of advantage over your neighbors would be considered a poor evolutionary strategy. However, as shown via the unlikely conditions presented in this study, the potential to dominate as an auxotroph is clearly present in the genome of P. fluorescens Pf0-1. In addition, it appears that amino acid antagonism may represent a mechanism for competition in nature, as it was recently demonstrated that valine is actively secreted by various bacterial biofilms, including E. coli and P. aeruginosa (42). The practical significance of this study, however, may lie across the species barrier. There have been several reports of the isolation of significant numbers of P. aeruginosa auxotrophs, including leucine auxotrophs, from the lungs of cystic fibrosis patients and healthy individuals (3-5, 40). Although there has not yet been a strong claim regarding the mechanism driving auxotrophy selection, this mechanism has largely been explained as one of increased fitness over the prototrophs as a consequence of not expending energy for de novo biosynthesis. Unlike bulk soil, sputum represents an ideal environment where auxotrophy may be selected as a competitive trait, beyond the classic explanation described here. A recent microarray analysis indicated that, in sputum, amino acid biosynthesis genes are down-regulated and transport genes are up-regulated (25). From the outside looking in, this surely looks like an ideal place to invoke the amino acid antagonism theory.

 
We thank Julie Nicoll for her technical assistance with the soil assays.

This work was supported by USDA grant 2006-35604-16673 to S.B.L. W.K. was supported in part by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.

 
* Corresponding author. Mailing address: Center for Adaptation Genetics and Drug Resistance, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6764. Fax: (617) 636-0458. E-mail: Stuart.Levy{at}tufts.edu

Published ahead of print on 25 April 2008.

Present address: FAS Center for Systems Biology, Harvard University, Cambridge, MA.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, June 2008, p. 3644-3651, Vol. 74, No. 12
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