The generation of multiple co‐existing mal‐regulatory mutations through polygenic evolution in glucose‐limited populations of Escherichia coli

Abstract

The multicomponent glucose transport system of Escherichia coli was used to study the polygenic basis of increased fitness in prolonged nutrient-limited, continuous cultures. After 280 generations of glucose-limited growth, nearly all bacteria in four independent chemostat populations exhibited increased glucose transport and contained multiple, stable mutations. Fitter bacteria increased outer membrane permeability for glucose through overexpression of the LamB glycoporin. Three classes of mutation influenced LamB levels as well as regulation of other mal genes. Low-level mal/lamB constitutivity resulted from mlc mutations acquired in all populations as well as changes at another uncharacterized locus. Larger increases in transporter content resulted from widespread acquisition of a regulatory malT-con mutation in fit isolates. The malT mutations sequenced from 67 adapted isolates were all single base substitutions resulting in amino acid replacements in the N-terminal third of the MalT activator protein. Analysis of malT-con sequences revealed a mutational spectrum distinct from that found in plate-selected malT mutants, suggesting that mutational pathways were affected by environmental factors. A second major finding was the remarkable allele diversity in malT within a population derived from a single clone, with at least 11 different alleles co-existing in a population. The multiplicity of alleles (as well as those found in adaptive mgl changes in the accompanying study) suggest that the periodic selection events observed previously in such populations are not a major factor in reducing genetic diversity. A simple model is presented for the generation of genetic heterogeneity in bacterial populations undergoing polygenic selection.

Metabolic Changes Associated With Adaptive Diversification in Escherichia coli

“…This group of genes has already been identified as involved in adaptation to a better glucose uptake rate in other evolution experiments. For example, in all replicate populations of a 280-generation experiment performed under continuous growth (chemostat culture) on glucose, the mal/lamB genes were found overexpressed, leading to a significant increase in glucose uptake (Notley-McRobb and Ferenci 1999;see Kurlandzka et al 1991 for other examples). This improvement is probably linked to the LamB protein, known as being the most important contributor to glucose uptake under nutrient limitation (Death et al 1993).…”

“…Classic genetic and physiological microbial models, such as yeast and bacteria, offer a powerful alternative. By coupling evolution experiments and genetic investigations in these microorganisms, several studies have identified cellular functions involved in evolutionary processes (e.g., Treves et al 1998;Notley-McRobb and Ferenci 1999;Cooper et al 2001;Spencer et al 2007). However, in most of the cases, these studies focus on specific candidate genes, and general investigations of metabolic changes are rare (but see Kurlandzka et al 1991;Cooper et al 2003;Pelosi et al 2006).…”

The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics

“…(v) Phenotypes 3, 4 and 5 are generated from phenotype 2, 3 and 4, respectively, in the same way. It should be noted that we use the term phenotype here not for a cell line carrying a defined mutation but for a group of different mutants that have the same improved kinetic properties with respect to µ max and K s on glucose [compare with Notley-McRobb & Ferenci (2000, 1999a]. This process is illustrated in Fig.…”

“…Notley-McRobb & Ferenci (2000) estimated that in their glucose-limited chemostats (Dl 0n3 h −" ), containing 2-3i10"! cells in total, 10% mutants with an improved glucose affinity were generated at each replication round due to loss-of-function mutations in mgl and mlc.…”

Genomic Sequencing Reveals Regulatory Mutations and Recombinational Events in the Widely Used MC4100 Lineage ofEscherichia coliK-12

“…According to citations, this transposable reporter construct has been used in more than 100 studies of gene regulation but has not been fully sequenced. placMu50 was introduced into MC4100 to generate MC4100(MuLac) strain BW2952, the ancestor strain in experimental evolution experiments, because mal expression is a useful marker for detecting an assortment of regulatory mutations in evolving cultures (9,23).…”