E Unibus Plurum: Genomic Analysis of an Experimentally Evolved Polymorphism in Escherichia coli

Microbial populations founded by a single clone and propagated under resource limitation can become polymorphic. We sought to elucidate genetic mechanisms whereby a polymorphism evolved in Escherichia coli under glucose limitation and persisted because of cross-feeding among multiple adaptive clones. Apart from a 29 kb deletion in the dominant clone, no large-scale genomic changes distinguished evolved clones from their common ancestor. Using transcriptional profiling on co-evolved clones cultured separately under glucose-limitation we identified 180 genes significantly altered in expression relative to the common ancestor grown under similar conditions. Ninety of these were similarly expressed in all clones, and many of the genes affected (e.g., mglBAC, mglD, and lamB) are in operons coordinately regulated by CRP and/or rpoS. While the remaining significant expression differences were clone-specific, 93% were exhibited by the majority clone, many of which are controlled by global regulators, CRP and CpxR. When transcriptional profiling was performed on adaptive clones cultured together, many expression differences that distinguished the majority clone cultured in isolation were absent, suggesting that CpxR may be activated by overflow metabolites removed by cross-feeding strains in co-culture. Relative to their common ancestor, shared expression differences among adaptive clones were partly attributable to early-arising shared mutations in the trans-acting global regulator, rpoS, and the cis-acting regulator, mglO. Gene expression differences that distinguished clones may in part be explained by mutations in trans-acting regulators malT and glpK, and in cis-acting sequences of acs. In the founder, a cis-regulatory mutation in acs (acetyl CoA synthetase) and a structural mutation in glpR (glycerol-3-phosphate repressor) likely favored evolution of specialists that thrive on overflow metabolites. Later-arising mutations that led to specialization emphasize the importance of compensatory rather than gain-of-function mutations in this system. Taken together, these findings underscore the importance of regulatory change, founder genotype, and the biotic environment in the adaptive evolution of microbes

Detection in Soil of a Deletion in an Engineered DNA Sequence by Using DNA Probes

Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes. After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes. In addition to the expected bands of hybridization, a new band corresponding to the loss of vector sequences from the chromosome while retaining the nptII gene was observed for one of the strains. The more stressful conditions encountered in soil appeared to increase the frequency of loss of the vector sequences from this strain

Use of Gene Probes to Aid in Recovery and Identification of Functionally Dominant 2,4-Dichlorophenoxyacetic Acid-Degrading Populations in Soil

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to soils in microcosms, and degradation was monitored after each of five repeated additions. Total DNAs were isolated from soil bacterial communities after each 2,4-D treatment. The DNA samples were analyzed on slot blots and Southern blots by using a tfdA gene probe subcloned from plasmid pJP4 and a Spa probe derived from a different 2,4-D-degrading isolate, a Sphingomonas paucimobilis strain. 2,4-D applied to soil was quickly degraded by indigenous microbial populations. As determined by slot blot analyses of DNA from a Michigan soil, the increase in hybridization signal in response to 2,4-D treatments was greater with the Spa probe than with the tfdA probe. In contrast, the DNA from a Saskatchewan soil exhibited an increase in hybridization signal with the tfdA probe. This indicated that a population with 2,4-D-degradative gene sequences different from the tfdA gene sequence was dominant in the Michigan site, but not in the Saskatchewan site. A Southern blot analysis of DNA from Michigan soil showed that the dominant 2,4-D-degrading population was S. paucimobilis 1443. A less dominant 2,4-D-degrading population was detected with the tfdA probe; further analysis revealed that this population was a Pseudomonas pickettii 712. These gene probe analyses revealed that an important population carrying out 2,4-D degradation was not detected when the canonical tfdA gene probe was used. After a series of new strains were isolated, we identified a probe to detect and identify the dominant members of this new group

Tri-Trophic Linkages in Disease: Pathogen Transmission to Rainbow Trout Through Stonefly Prey

ABSTRACT Relationships between macroinvertebrates and microorganisms in aquatic environments are only poorly understood despite the fact that many aquatic macroinvertebrates feed on microbial biofilms during some life stage. Better understanding of trophic interactions between microbial biofims, macroinvertebrates, and fish may also help control fish diseases and loss of natural resources. It has also been suggested that pollution, habitat fragmentation, and poor water quality may contribute to increased pathogenesis and mortality in fish. Increased disease incidence is difficult to assess, however, in part because of the complexity of pathogen transmission dynamics. Several environmental pathogens exist whose reservoir(s) and means of transmission remain poorly understood, highlighting the need to study pathogen ecology and interactions with organisms other than susceptible hosts. Aeromonas salmonicida is rarely isolated from freshwater sediments. However, stoneßynymphswere found to frequently harbor A. salmonicida and were shown to preferentially feed on the bacterium. Rainbow trout juveniles were presented with different feeding regimes to determine the transmission capacity of nymphs, and all fish fed stoneflies harboring A. salmonicida expressed symptoms of disease. Although current rates of furunculosis in freshwater ecosystems are unknown, trout primarily feed on stoneflies when water oxygen levels are high and temperatures are low (winter months), which is presumed to correspond to high resistance to the pathogen. Given that furunculosis is associated with physiological stress and higher water temperatures, its natural incidence may change in response to global or regional climatological effects

Analysis of Competition in Soil among 2,4-Dichlorophenoxyacetic Acid-Degrading Bacteria

Competition among indigenous and inoculated 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was studied in a native Kansas prairie soil following 2,4-D additions. The soil was inoculated with four different 2,4-D-degrading strains at densities of 10(3) cells per g of soil; the organisms used were Pseudomonas cepacia DBO1(pJP4) and three Michigan soil isolates, strain 745, Sphingomonas paucimobilis 1443, and Pseudomonas pickettii 712. Following 2,4-D additions, total soil DNA was extracted and analyzed on Southern blots by using a tfdA gene probe which detected three of the strains and another probe that detected the fourth strain, S. paucimobilis 1443, which belongs to a different class of 2,4-D degraders. P. cepacia DBO1(pJP4), a constructed strain, outcompeted the other added strains and the indigenous 2,4-D-degrading populations. The S. paucimobilis population was the secondary dominant population, and strain 745 and P. pickettii were not detected. Relative fitness coefficients determined in axenic broth cultures predicted the outcome of competition in soil for some but not all strains. Lag time was shown to be a principal determinant of competitiveness among the strains, but the lag times were significantly reduced in mixed broth cultures, which changed the competitive outcome. Plasmids containing the genes for the 2,4-D pathway were important determinants of competitiveness since plasmid pKA4 in P. cepacia DBO1 resulted in the slower growth characteristic of its original host, P. pickettii, rather than the rapid growth observed when this strain harbors pJP4

Genetic and Phenotypic Diversity of 2,4-Dichlorophenoxyacetic Acid (2,4-D)-Degrading Bacteria Isolated from 2,4-D-Treated Field Soils

Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis). A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species. Hybridization analysis revealed four groups. Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis. Group II, which exhibited homology only with the tfdA4 gene, was a small group and was probably a subset of group I. All group I and II strains had plasmids. Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains. One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area. The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe. The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group. The group III strains were identified as S. paucimobilis. The group IV strains, which hybridized to neither the tfd probe nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses. Most of group IV strains could not be identified by the FAME analysis. Strains belonging to groups I and III were more frequently recovered from soils that had greater field exposure to 2,4-D, suggesting that they were the best competitors for 2,4-D under field conditions. The selection regimen which we used led to two successful but dissimilar groups; the members of one group were similar at the plasmid level but not at the organism level, and the members of the other group were similar at the organism level. Since the members of the latter group are ecologically successful and degradative genes unlike tfd genes, they deserve more attention

Wildlife fecal microbiota exhibit community stability across a longitudinal semi-controlled non-invasive sampling experiment

Wildlife microbiome studies are being used to assess microbial links with animal health and habitat. The gold standard of sampling microbiomes directly from captured animals is ideal for limiting potential abiotic influences on microbiome composition, yet fails to leverage the many benefits of non-invasive sampling. Application of microbiome-based monitoring for rare, endangered, or elusive species creates a need to non-invasively collect scat samples shed into the environment. Since controlling sample age is not always possible, the potential influence of time-associated abiotic factors was assessed. To accomplish this, we analyzed partial 16S rRNA genes of fecal metagenomic DNA sampled non-invasively from Rocky Mountain elk (Cervus canadensis) near Yellowstone National Park. We sampled pellet piles from four different elk, then aged them in a natural forest plot for 1, 3, 7, and 14 days, with triplicate samples at each time point (i.e., a blocked, repeat measures (longitudinal) study design). We compared fecal microbiota of each elk through time with point estimates of diversity, bootstrapped hierarchical clustering of samples, and a version of ANOVA–simultaneous components analysis (ASCA) with PCA (LiMM-PCA) to assess the variance contributions of time, individual and sample replication. Our results showed community stability through days 0, 1, 3 and 7, with a modest but detectable change in abundance in only 2 genera (Bacteroides and Sporobacter) at day 14. The total variance explained by time in our LiMM-PCA model across the entire 2-week period was not statistically significant (p>0.195) and the overall effect size was small (<10% variance) compared to the variance explained by the individual animal (p<0.0005; 21% var.). We conclude that non-invasive sampling of elk scat collected within one week during winter/early spring provides a reliable approach to characterize fecal microbiota composition in a 16S rDNA survey and that sampled individuals can be directly compared across unknown time points with minimal bias. Further, point estimates of microbiota diversity were not mechanistically affected by sample age. Our assessment of samples using bootstrap hierarchical clustering produced clustering by animal (branches) but not by sample age (nodes). These results support greater use of non-invasive microbiome sampling to assess ecological patterns in animal systems