Parallel changes in global protein profiles during long-term experimental evolution in Escherichia coli - PubMed (original) (raw)

Parallel changes in global protein profiles during long-term experimental evolution in Escherichia coli

Ludovic Pelosi et al. Genetics. 2006 Aug.

Abstract

Twelve populations of Escherichia coli evolved in and adapted to a glucose-limited environment from a common ancestor. We used two-dimensional protein electrophoresis to compare two evolved clones, isolated from independently derived populations after 20,000 generations. Exceptional parallelism was detected. We compared the observed changes in protein expression profiles with previously characterized global transcription profiles of the same clones; this is the first time such a comparison has been made in an evolutionary context where these changes are often quite subtle. The two methodologies exhibited some remarkable similarities that highlighted two different levels of parallel regulatory changes that were beneficial during the evolution experiment. First, at the higher level, both methods revealed extensive parallel changes in the same global regulatory network, reflecting the involvement of beneficial mutations in genes that control the ppGpp regulon. Second, both methods detected expression changes of identical gene sets that reflected parallel changes at a lower level of gene regulation. The protein profiles led to the discovery of beneficial mutations affecting the malT gene, with strong genetic parallelism across independently evolved populations. Functional and evolutionary analyses of these mutations revealed parallel phenotypic decreases in the maltose regulon expression and a high level of polymorphism at this locus in the evolved populations.

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Figures

F<sc>igure</sc> 1.—

Figure 1.—

Two-dimensional protein gel electrophoresis of the ancestor and an evolved clone. Protein extracts were prepared from cultures grown in DM250 medium for 24 hr at 37°. Laboratory-made dry strips (18 cm, pH range from 4 to 8) were hydrated in 500-μg protein samples from the ancestral strain (A) and from an evolved clone isolated at generation 20,000 from population Ara + 1 (B). The steady-state isoelectrofocalization patterns were analyzed by 12% acrylamide-bis SDS–PAGE and Coomassie blue staining. Protein spots numbered M1 and M2 did not show any variation among the three genotypes analyzed but are shown as internal standards (□) (see Table 1). (○) Protein spots modified in both evolved clones. Spots decreasing or disappearing in both evolved clones are marked on the ancestral protein pattern (A); spots increasing or appearing in both evolved clones are marked on the evolved protein pattern (B). (▵) Protein spots modified only in the Ara + 1 evolved clone. (▿) Protein spots showing decreased intensity only in the Ara − 1 evolved clone. The insets show enlarged parts of each gel with the maltoporin LamB and the maltose-binding protein MalE (open arrowheads). Mw, molecular weight; pI, isoelectric point.

F<sc>igure</sc> 2.—

Figure 2.—

Schematic structure of the MalT protein and location of evolved mutant alleles. The protein is composed of four domains (D

anot

2001; R

ichet

et al. 2005): DT1 (residues 1–241), DT2 (residues 250–436), DT3 (residues 437–806), and DT4 (residues 807–901). DT1 binds ATP and contains surface determinants involved in the binding of repressor proteins, including Aes, MalK, and MalY. DT2 and DT3 are involved in maltotriose binding and represent putative multimerization domains. DT4 corresponds to the DNA-binding domain and presumably recruits RNA polymerase. The malT gene was sequenced in one clone isolated at 20,000 generations from each of the 12 populations designated Ara − 1–Ara − 6 and Ara + 1–Ara + 6. Amino-acid substitutions deduced from the mutations identified in the evolved clones are indicated using the one-letter code, and the residue number refers to its position in the MalT polypeptide. In both the Ara + 1 and Ara + 2 populations, small internal deletions were found in malT (their size and position relative to the malT translational start codon are shown).

F<sc>igure</sc> 3.—

Figure 3.—

Phenotypic tests of MalT activity. The ancestral strain is labeled 606. One evolved clone was isolated at 20,000 generations from each of the 12 populations designated Ara − 1–Ara-6 and Ara + 1–Ara + 6. A malT_-deleted strain (Δ_malT) served as a control. (A) Growth ability in DM250 maltose at 37°. +, Growth after 8 hr; −, no growth even after 24 hr. (B) Relative level of LamB protein. Cellular extracts were prepared from all strains after overnight culture in DM250 medium. Each sample contained 20 μg of total protein and was subjected to 12% SDS–PAGE and immunoblot analysis with polyclonal antibodies against LamB (anti-LamB) or RpoA (anti-RpoA) as a control. (C) The number of mutations identified in malT in each population is indicated. Δ, deletion allele; Anc, ancestral state.

F<sc>igure</sc> 4.—

Figure 4.—

Fitness effects of two evolved malT mutations and a mutation with upregulated expression of malT, all moved into the ancestral genetic background. Competitions were performed in the same medium used in the long-term evolution experiment. Error bars are 95% confidence intervals based on six replicate competition assays for each genotype. From left to right, the four genotypes are as follows: 606 is the ancestral strain with the ancestral malT allele; malTAra+1 and malTAra−1 are alleles that evolved in the focal populations Ara + 1 and Ara − 1, respectively, which were then moved into the ancestral chromosome; and malTup is an upregulated allele also moved into the ancestral chromosome.

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