Genetic changes during a laboratory adaptive evolution process that allowed fast growth in glucose to an Escherichia coli strain lacking the major glucose transport system

Abstract

Background: Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), which is the major bacterial component involved in glucose transport and its phosphorylation, accumulate high amounts of phosphoenolpyruvate that can be diverted to the synthesis of commercially relevant products. However, these strains grow slowly in glucose as sole carbon source due to its inefficient transport and metabolism. Strain PB12, with 400% increased growth rate, was isolated after a 120 hours adaptive laboratory evolution process for the selection of faster growing derivatives in glucose. Analysis of the genetic changes that occurred in the PB12 strain that lacks PTS will allow a better understanding of the basis of its growth adaptation and, therefore, in the design of improved metabolic engineering strategies for enhancing carbon diversion into the aromatic pathways.

Results: Whole genome analyses using two different sequencing methodologies: the Roche NimbleGen Inc. comparative genome sequencing technique, and high throughput sequencing with Illumina Inc. GAIIx, allowed the identification of the genetic changes that occurred in the PB12 strain. Both methods detected 23 non-synonymous and 22 synonymous point mutations. Several non-synonymous mutations mapped in regulatory genes (arcB, barA, rpoD, rna) and in other putative regulatory loci (yjjU, rssA and ypdA). In addition, a chromosomal deletion of 10,328 bp was detected that removed 12 genes, among them, the rppH, mutH and galR genes. Characterization of some of these mutated and deleted genes with their functions and possible functions, are presented.

Conclusions: The deletion of the contiguous rppH, mutH and galR genes that occurred simultaneously, is apparently the main reason for the faster growth of the evolved PB12 strain. In support of this interpretation is the fact that inactivation of the rppH gene in the parental PB11 strain substantially increased its growth rate, very likely by increasing glycolytic mRNA genes stability. Furthermore, galR inactivation allowed glucose transport by GalP into the cell. The deletion of mutH in an already stressed strain that lacks PTS is apparently responsible for the very high mutation rate observed.

Figure 1

Isolation of the evolved PB12 strain. The isolation of PB12 has previously been reported and is included to provide orientation to the reader and for discussion purposes [10]. The evolutionary process that generated the PB12 strain initiated with the parental PB11 strain that lacks the PTS system. Deletion of this system generates a carbon stress response when PB11 is grown in glucose as the sole carbon source [9,13]. This strain that grows very slowly in glucose and generates white colonies (WC) in glucose-McConkey agar plates, was grown in a batch culture fermentor containing minimal medium with 2 g/l of glucose as the sole carbon source and 30 μg/ml of kanamycin. Under these conditions, a selection pressure is generated, favoring faster growing mutants. The culture was maintained until the stationary phase and then a continuous culture was initiated by feeding a glucose solution at progressively higher dilution rates in the same medium. Dotted line indicates the end of the batch culture and the start of the continuous culture. This procedure allowed the isolation of mutants according to their growth rates. Samples were monitored on glucose-McConkey agar plates to identify red colonies as an indicative of glucose utilization [Glc⁺ phenotype. Red colonies (RC) were detected after a period of 70 hr. The arrows indicate the isolation time for several Glc⁺ variants including PB12. Numbers indicate different dilution rates (D = h^-1). All the isolated colonies from this culture carry the same large deletion present in strain PB12 (data not shown). This figure was derived and modified from figure 1 from Flores et al. 2007 [10]

Figure 2

Comparative genomic maps of the JM101 and PB12 strains. Deletions detected in the PB12 strain. The small deletion is the result of the elimination of the PTS genes (ptsH, ptsI and crr) that was previously generated in the parental PB11 strain [7,9]. The largest of these deletions appeared during the laboratory adaptive evolution process (Figures 3 and 4).

Figure 3

Chromosomal deletion markers in the PB12 strain. The absence of a chromosomal fragment in the PB12 strain was confirmed by PCR and by Sanger resequencing. Ten genes were deleted and the galR and ptsP genes were fused. Section A shows the ptsP and galR genes that were amplified in the JM101, PB11 and PB12 strains: line 1, (M) molecular weight markers; lines 2, 3 and 4, ptsP amplification in the JM101, PB11 and PB12 strains, respectively; lines 5, 6 and 7, galR amplification in the JM101, PB11 and PB12 strains, respectively; line 8, amplification of the chromosomal region in the PB12 strain; line 9, (M) molecular weight markers; lines 10, 11 and 12, amplification of the chromosomal region using DNA from strains JM101, PB11 and PB12, respectively. Section B presents the oligonucleotides utilized for DNA amplifications. The left section (L) includes the oligonucleotides employed for the amplification of the ptsP and galR genes of the three strains (lines 2–7), and the right section (R) presents the entire chromosomal regions of the same three strains amplified using ptsP-fwd and galR-rv oligonucleotides (lines 8, 10–12). The nucleotide sequences of the oligos utilized are included in table S3 presented in additional file 4.

Figure 4

Chromosomal gene arrangement in the parental JM101 strain and in the evolved PB12 derivative. A) Gene organization in the chromosome of the parental strain. B) Gene deletion and chromosome rearrangement in the PB12 strain genome. Figure S1 presented in Additional file 3, includes the nucleotide sequence of the genomic region where the deletion occurred.