Metabolic commensalism and competition in a two-species microbial consortium

We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source. The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats. The numbers of CFU of P. putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers. When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P. putida R1 (500:1), whereas under similar growth conditions in biofilms, P. putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1). In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms. Insertion into P. putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P. putida R1 cells at different positions in the biofilms. Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm. In the initial phase of biofilm development, the growth activity of P. putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6. High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P. putida R1. After a few days, Acinetobacter strain C6 colonies were overgrown by P. putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm. In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P. putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6. We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1. In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.     

Development and dynamics of Pseudomonas sp. biofilms

Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.     

Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities

Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of forming biofilms on surfaces as a survival strategy. It exhibits a large variety of competition/virulence factors, such as three types of motilities: flagellum-mediated swimming, flagellum-mediated swarming, and type IV pilus-mediated twitching. A strategy frequently used by bacteria to survive changing environmental conditions is to create a phenotypically heterogeneous population by a mechanism called phase variation. In this report, we describe the characterization of phenotypic variants forming small, rough colonies that spontaneously emerged when P. aeruginosa 57RP was cultivated as a biofilm or in static liquid cultures. These small-colony (S) variants produced abundant type IV fimbriae, displayed defective swimming, swarming, and twitching motilities, and were impaired in chemotaxis. They also autoaggregated in liquid cultures and rapidly initiated the formation of strongly adherent biofilms. In contrast, the large-colony variant (parent form) was poorly adherent, homogeneously dispersed in liquid cultures, and produced scant polar fimbriae. Further analysis of the S variants demonstrated differences in a variety of other phenotypic traits, including increased production of pyocyanin and pyoverdine and reduced elastase activity. Under appropriate growth conditions, cells of each phenotype switched to the other phenotype at a fairly high frequency. We conclude that these S variants resulted from phase variation and were selectively enriched when P. aeruginosa 57RP was grown as a biofilm or in static liquid cultures. We propose that phase variation ensures the prior presence of phenotypic forms well adapted to initiate the formation of a biofilm as soon as environmental conditions are favorable.     

Phenotypic variation of Pseudomonas putida and P. tolaasii affects the chemotactic response to Agaricus bisporus mycelial exudate.

The chemotactic response of wild-type Pseudomonas putida and P. tolaasii, and a phenotypic variant of each species, to Agaricus bisporus mycelial exudate was examined. Both P. putida, the bacterium responsible for initiating basidiome development of A. bisporus, and P. tolaasii, the causal organism of bacterial blotch disease of the mushroom, displayed a positive chemotactic response to Casamino acids and to A. bisporus mycelial exudate. The response was both dose- and time-dependent and marked differences were observed between the response time of the wild-type strains and their phenotypic variants. Phenotypic variants responded rapidly to both attractants and reached a maximum response after 10-20 min, whereas the wild-types took 45-60 min. The differences are partly explained by the more rapid swimming speed of the phenotypic variants. Both variants responded maximally to similar concentrations of Casamino acids and mycelial exudates. Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (Mr less than 2000) thermostable compounds. Sugars present in the exudate did not elicit a chemotactic response in any isolate, but a mixture of 14 amino acids detected in the exudate accounted for between 50 and 75% of the chemotactic response of the fungal exudate.     

Characterization of colony morphology variants isolated from Streptococcus pneumoniae biofilms

In this study, we report the isolation of colony morphology variants from Streptococcus pneumoniae serotype 3 biofilms. The colony variants differed in colony size (large, medium, and small) and their mucoid appearance on blood agar. The small nonmucoid variant (SCV) emerged during the initial attachment stage of S. pneumoniae biofilm formation and dominated over the course of biofilm growth. Mucoid variants appeared at later biofilm developmental stages. The reduction in colony size/mucoidy correlated with a decrease in capsule production and an increase in initial attachment. The large mucoid variant formed flat unstructured biofilms, failed to aggregate in liquid culture, and adhered poorly to solid surfaces. In contrast, SCVs autoaggregated in liquid culture, hyperadhered to solid surfaces, and formed biofilms with significant three-dimensional structure, mainly in the form of microcolonies. The variants showed similar antibiotic resistance/susceptibility based on a modified Kirby-Bauer test and when grown as biofilms. However, antimicrobial treatment of S. pneumoniae biofilms altered the colony variant's distribution and mainly affected the most interior areas of biofilm microcolonies. To further explore the nature of the variants, the capsule biosynthetic operon (cps3DSUM) was explored in greater detail. The genetic analysis indicated that the emergence of nonmucoid variants was due to a deletion comprising cps3DSU as well as additional genes upstream of the cps3 operon. Overall, our findings suggest that in vitro biofilm formation of S. pneumoniae serotype 3 coincides with the emergence of colony variants with distinct genotypic and phenotypic characteristics.     

Construction of artificially structured microbial consortia (ASMC) using dielectrophoresis: examining bacterial interactions via metabolic intermediates within environmental biofilms

The construction of artificial biofilms with defined internal architectures is described. Bacterial cells are suspended in a low conductivity medium, guided to specific areas in a microelectrode array by dielectrophoresis (DEP), and then immobilised using the flocculating agent poly(ethylenimine). Multispecies biofilms can be constructed by introducing different species at different times. The rapid construction of such biofilms with defined internal architectures provides, when combined with visual reporters of gene activity, a powerful new method for the investigation of the effects of the spatial organisation on interactions between bacterial species in biofilms. To demonstrate the utility of the technique as a method for investigating metabolic interactions in biofilms, aggregates were constructed from Acinetobacter sp. C6 and Pseudomonas putida::gfp. The Acinetobacter degrades benzyl alcohol, overproducing benzoate, which in turn is consumed by the Pseudomonas strain. The P. putida has a chromosomally expressed cassette encoding a gfp downstream of the promoter which controls degradation of benzoate, making the interaction between the two strains in the metabolism of benzyl alcohol visible by the production of green fluorescent protein (GFP). Microscopic observation of the biofilms, including the use of confocal laser scanning microscopy (CLSM), confirmed that metabolic exchange occurred. In addition, it was observed that the bacteria appear to have a preferred biofilm architecture, with P. putida in the bottom layer, and Acinetobacter at the top.     

A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion

A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 microM parathion (146 mg l(-1)) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis-Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 microM p-nitrophenol (83 mg l(-1)) and P. putida KT2440 pSB337 inhibited by 1,000 microM p-nitrophenol (139 mg l(-1)). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.     

Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P. putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable exopolysaccharide genes was found to form biofilm with a structure similar to wild-type biofilm, but with a stability lower than that of wild-type biofilm. Based on our data, we suggest that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however, exopolysaccharides play a role as structural stabilizers.     

Characterization of starvation-induced dispersion in Pseudomonas putida biofilms

The biofilm lifestyle, where microbial cells are aggregated because of expression of cell-to-cell interconnecting compounds, is believed to be of paramount importance to microbes in the environment. Because microbes must be able to alternate between sessile and planktonic states, it is anticipated that they must be able to regulate their ability to form biofilm and to dissolve biofilm. We present an investigation of a biofilm dissolution process occurring in flow-chamber-grown Pseudomonas putida biofilms. Local starvation-induced biofilm dissolution appears to be an integrated part of P. putida biofilm development that causes characteristic structural rearrangements. Rapid global dissolution of entire P. putida biofilms was shown to occur in response to carbon starvation. Genetic analysis suggested that the adjacent P. putida genes PP0164 and PP0165 play a role in P. putida biofilm formation and dissolution. PP0164 encodes a putative periplasmic protein of previously unknown function, and PP0164 mutant bacteria are sticky, and unable to reduce their adhesiveness and dissolve their biofilm in response to carbon starvation. PP0165 encodes a putative transmembrane protein containing GGDEF and EAL domains, and PP0165 mutant bacteria are unable to increase their adhesiveness and form biofilm. We suggest that the PP0164 and PP0165 proteins are involved in the regulation of the adhesiveness of the bacteria; the PP0165 protein through c-di-GMP signalling, and the PP0164 protein as a transducer of the signal.     

Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy.

As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe. The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy, demonstrating that P. putida was present throughout the biofilm. Acridine orange staining revealed a very heterogeneous structure of the fully hydrated biofilm, with cell-free channels extending from the surface into the biofilm. This indicated that toluene may penetrate to deeper layers of the biofilm, and consequently P. putida may be actively degrading toluene in all regions of the biofilm. Furthermore, measurements of growth rate-related parameters for P. putida showed reduced rRNA content and cell size (relative to that in a batch culture), indicating that the P. putida population was not degrading toluene at a maximal rate in the biofilm environment. Assuming that the rRNA content reflected the cellular activity, a lower toluene degradation rate for P. putida present in the biofilm could be estimated. This calculation indicated that P. putida was responsible for a significant part (65%) of the toluene degraded by the entire community.     

Resistance to oxidative stress caused by ceftazidime and piperacillin in a biofilm of Pseudomonas.

The capacity to form a biofilm was evaluated in Pseudomonas aeruginosa isolated from patients with lung and urinary infections. Adherence, development of microcolonies and slime formation varied in the studied strains. P. aeruginosa P63 isolated from cystic fibrosis (CF) exhibited important microcolony formation with the densest biofilm, and was selected to study the oxidative stress produced with ceftazidime and piperacillin by means of chemiluminescence (CL) in cell suspensions and biofilm. P. aeruginosa strain P63 was compared with P69; both were sensitive to ceftazidime and showed increase of reactive species of oxygen (ROS) in the presence of this antibiotic. P. aeruginosas P69 exhibited resistance to piperacillin and low ROS production, while piperacillin-sensitive strain P63 showed high oxidative stress with this antibiotic. Piperacillin stimulated oxidative stress, increasing ROS production only in the sensitive strain. Higher antibiotic concentrations were necessary to augment ROS in bacteria biofilm than in suspension. Incubation of P63 strain with ceftazidime or piperacillin in the presence of its own extracellular matrix (EM) or sodium alginate stimulated lesser oxidative stress and slower decrease of ROS than in the absence of these polysaccharides. A variant, V(10), obtained from strain P63 showed more sensitivity to the antibiotics than the wild-type, and concomitantly exhibited higher production of ROS in the presence of both the antibiotics studied.     

Metabolic Commensalism and Competition in a Two-Species Microbial Consortium

We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source. The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats. The numbers of CFU of P. putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers. When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P. putida R1 (500:1), whereas under similar growth conditions in biofilms, P. putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1). In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms. Insertion into P. putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P. putida R1 cells at different positions in the biofilms. Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm. In the initial phase of biofilm development, the growth activity of P. putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6. High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P. putida R1. After a few days, Acinetobacter strain C6 colonies were overgrown by P. putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm. In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P. putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6. We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1. In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.     

Distribution of bacterial growth activity in flow-chamber biofilms.

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.     

Interspecific diversity reduces and functionally substitutes for intraspecific variation in biofilm communities

Diversity has a key role in the dynamics and resilience of communities and both interspecific (species) and intraspecific (genotypic) diversity can have important effects on community structure and function. However, a critical and unresolved question for understanding the ecology of a community is to what extent these two levels of diversity are functionally substitutable? Here we show, for a mixed-species biofilm community composed of Pseudomonas aeruginosa, P. protegens and Klebsiella pneumoniae, that increased interspecific diversity reduces and functionally substitutes for intraspecific diversity in mediating tolerance to stress. Biofilm populations generated high percentages of genotypic variants, which were largely absent in biofilm communities. Biofilms with either high intra- or interspecific diversity were more tolerant to SDS stress than biofilms with no or low diversity. Unexpectedly, genotypic variants decreased the tolerance of biofilm communities when experimentally introduced into the communities. For example, substituting P. protegens wild type with its genotypic variant within biofilm communities decreased SDS tolerance by twofold, apparently due to perturbation of interspecific interactions. A decrease in variant frequency was also observed when biofilm populations were exposed to cell-free effluents from another species, suggesting that extracellular factors have a role in selection against the appearance of intraspecific variants. This work demonstrates the functional substitution of inter- and intraspecific diversity for an emergent property of biofilms. It also provides a potential explanation for a long-standing paradox in microbiology, in which morphotypic variants are common in laboratory grown biofilm populations, but are rare in diverse, environmental biofilm communities.     

Development of Spatial Distribution Patterns by Biofilm Cells

Confined spatial patterns of microbial distribution are prevalent in nature, such as in microbial mats, soil communities, and water stream biofilms. The symbiotic two-species consortium of Pseudomonas putida and Acinetobacter sp. strain C6, originally isolated from a creosote-polluted aquifer, has evolved a distinct spatial organization in the laboratory that is characterized by an increased fitness and productivity. In this consortium, P. putida is reliant on microcolonies formed by Acinetobacter sp. C6, to which it attaches. Here we describe the processes that lead to the microcolony pattern by Acinetobacter sp. C6. Ecological spatial pattern analyses revealed that the microcolonies were not entirely randomly distributed and instead were arranged in a uniform pattern. Detailed time-lapse confocal microscopy at the single-cell level demonstrated that the spatial pattern was the result of an intriguing self-organization: small multicellular clusters moved along the surface to fuse with one another to form microcolonies. This active distribution capability was dependent on environmental factors (carbon source and oxygen) and historical contingency (formation of phenotypic variants). The findings of this study are discussed in the context of species distribution patterns observed in macroecology, and we summarize observations about the processes involved in coadaptation between P. putida and Acinetobacter sp. C6. Our results contribute to an understanding of spatial species distribution patterns as they are observed in nature, as well as the ecology of engineered communities that have the potential for enhanced and sustainable bioprocessing capacity.     

Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilm development

A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa, phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using anti-Pf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa, including a putative postsegregational killing operon.     

The effect of a competitor on a model adaptive radiation

The ecological forces shaping adaptive radiations are of great interest to evolutionary ecologists. Here, we experimentally test the hypothesis that the diversification of a lineage should be limited in the presence of competition from another taxon. We do this by studying a model microbial adaptive radiation (the generation of phenotypic diversity in asexual lineages of the bacterium Pseudomonas fluorescens) in the presence or absence of a competitor (Pseudomonas putida). In a spatially heterogeneous environment, the competitor P. putida reduced P. fluorescens population size only slightly and had no effect on diversification. In a spatially homogeneous environment, the competitor reduced P. fuoresecens population size to a much greater extent. Again the final extent of diversification in P. fluorescens was not affected by the competitor, but early diversification was accelerated. In this environment, P. putida suppressed the growth of a common variant of P. fluorescens and directly or indirectly facilitated the growth of a rare morph. Our results suggest that competition experienced by diversifying lineages may have complex effects on adaptive radiations not fully captured by current theory.     

An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress

The novel insertion sequence ISS12 plays a key role in the tolerance of Pseudomonas putida S12 to sudden toluene stress. Under normal culturing conditions the P. putida S12 genome contained seven copies of ISS12. However, a P. putida S12 population growing to high cell density after sudden addition of a separate phase of toluene carried eight copies. The survival frequency of cells in this variant P. putida S12 population was 1000 times higher than in "normal" P. putida S12 populations. Analysis of the nucleotide sequence flanking the extra ISS12 insertion revealed integration into the srpS gene. srpS forms a gene cluster with srpR and both are putative regulators of the solvent resistance pump SrpABC. SrpABC makes a major contribution to solvent tolerance in P. putida S12 and is induced by toluene. The basal level of srp promoter activity in the P. putida S12 variant was seven times higher than in wild-type P. putida S12. Introduction of the intact srpRS gene cluster in the variant resulted in a dramatic decrease of survival frequency after a toluene shock. These findings strongly suggest that interruption of srpS by ISS12 up-regulates expression of the solvent pump, enabling the bacterium to tolerate sudden exposure to lethal concentrations of toxic solvents. We propose that P. putida S12 employs ISS12 as a mutator element to generate diverse mutations to swiftly adapt when confronted with severe adverse conditions.