Mutations of interest

Amongst the sequenced lineages, there were instances of the same mutations being detected in independent lineages. Such instances of parallelism suggest that either these mutations confer a selective advantage or serve as a mutation hotspot. Examples of such mutations include BPS, indels and GCRs, as described below.

5.2.2.4.2.1REL4536 ΔsbcC::cat-1K-1 and REL4536 ΔsbcC::cat-1K-8

REL4536 ΔsbcC::cat-1K-1 and REL4536 ΔsbcC::cat-1K-8, for instance, exhibited two instances of parallelism; a roughly 33 kb deletion and a 5 bp indel was detected in clones from both lineages. As these lineages had always been maintained on separate plates during the course of evolution, we are confident that these mutations arose independently in the lineages (Table A.7). It seems likely that the 33 kb deletion was

mediated via homologous recombination of IS3 elements flanking the region. A total of

30 genes were deleted and are listed in Table A.8. Many of the deleted genes encode

hypothetical proteins of unknown function and are likely to be associated with cryptic prophage P22 activity. Genes involved in transcription regulation were also deleted. For example, appY, which encodes the transcriptional regulator AppY, is induced by the

global regulator ArcAB. In turn, ArcAB induces the expression of genes involved in anaerobic energy metabolism, survival at stationary phase and phosphate starvation (294). Another deleted gene of interest is envY, which encodes the DNA-binding

transcriptional regulator EnvY. EnvY is involved in the regulation of genes encoding cellular envelope proteins that are required for survival at low temperatures and during stationary phase (295). Loss of this region of the genome has previously been reported (296) and was also observed in E. coli REL4536 clones evolved under anaerobic

conditions for 2,000 generations (205) by Finn (2015). However, the selective advantage conferred by this deletion is not immediately apparent.

The 5 bp indel, found in both the REL4536 ΔsbcC::cat-1K-1 and REL4536 ΔsbcC::cat- 1K-8 sequenced clones, restored the original coding frame of the pseudogene dcuS.

161 DcuS, along with DcuR, is part of a two component regulatory system; DcuS is a dicarboxylate-sensing histidine kinase that reports the external dicarboxylate concentration to DcuR, while DcuR regulates genes involved in the anaerobic fumarate respiratory system (297). Hence, it is possible that this mutation may have reactivated an anaerobic respiratory pathway (297) and subsequently, been selected for. The same mutation was also observed in all sequenced E. coli REL4536 clones evolved under

anaerobic conditions for 2,000 generations by Finn (2015) (205).

5.2.2.4.2.2The adhE gene

Amongst the 10 sequenced clones, four different non-synonymous mutations in adhE

were detected (Table 5.6). The adhE gene encodes AdhE, which can function as an

alcohol dehydrogenase and a coenzyme A-dependent acetaldehyde dehydrogenase. Under conditions where fermentation is possible, AdhE can catalyse the reduction of acetyl-CoA to ethanol. Alternatively, during aerobic growth, AdhE can catalyse the oxidation of acetaldehyde to acetyl-CoA (298). In summary, AdhE is involved in the maintenance of the redox balance under anaerobic conditions and previous studies have reported that E. coli cells were not able to grow under anaerobic growth conditions

without the gene. The same study also observed that additional mutations in the pta

gene rescued the cells and allowed them to grow (298).

Table 5.6. Details of adhE mutations detected in this study.

Lineage Reference

Position* Change Mutation type Amino Acid Change

REL4536 ΔsbcC::cat-1K-12 1,439,509 A Æ G Transition Glu (568) Gly REL4536 ΔsbcC::cat-1K-14 1,439,821 A Æ C Transversion Asp (672) Ala REL4536 AN-1K-3 1,438,030 A Æ G Transition Tyr (75) Cys REL4536 AN-1K-4 1,439,673 G Æ A Transition Ala (623) Thr REL4536 AN-1K-6 1,438,030 A Æ G Transition Tyr (75) Cys

*_{Position of mutation on reference}

E. coli REL4536 genome.

As different mutations in adhE were found in independent lineages, with or without an

active sbcC gene, it is likely that mutations in adhE generally confer a selective

advantage to growth under anaerobic conditions. Lending support to this theory are findings by Finn (2015), where BPSs in adhE were also observed in all sequenced

162

E. coli REL4536 clones evolved under anaerobic conditions for 2,000 generations

(205). All of the BPSs listed in Table 5.6 resulted in the substitution of an amino acid

residue for another, and so, were likely to confer a conformational change of the protein. Thus, it is possible that AdhE protein function was disrupted, which may result in the diversion of the fermentation pathway to increased succinate or acetate production to serve as additional energy sources for growth (299, 300).

5.2.2.4.2.3IS insertions

Other instances of the same mutations being detected in independent lineages involved IS elements. For example, IS150 insertion was detected in cycA at the same position

(4,381,583 in the REL4536 reference genome) in both REL4536 ΔsbcC::cat-1K-8 and REL4536 ΔsbcC::cat-1K-14 clones (Table A.7). Encoded by cycA¸ the CycA

transporter is involved in the uptake of glycine, serine and alanine. It has previously been demonstrated that cells with inactivated cycA exhibited decreased transport of the

three amino acids and were resistant to d-cycloserine inhibition (301). While it is possible that these mutations could have derived from cross contamination between lineages, as both REL4536 ΔsbcC::cat-1K-8 and REL4536 ΔsbcC::cat-1K-14 lineages had been maintained on the same plates, IS150 insertion was found at exactly the same

position in sequenced REL4536 AN clones (for lineages 1, 3, 4 and 6). Therefore, it is probable that this position is a hotspot for IS150 insertion, which may aid in adaptation

to either the anaerobic environment or the 24-well growth vessels.

IS150 insertion was also detected in ydiQ at the same position (974,185 in the REL4536

reference genome) in the REL4536 ΔsbcC::cat-1K-6, REL4536 ΔsbcC::cat-1K-7 and REL4536 ΔsbcC::cat-1K-14 clones (Table A.7), suggesting the possibility of this

position being another hotspot for IS150 insertion. YdiQ, encoded for by ydiQ, is a

putative electron transport flavoprotein subunit that may be involved in the electron transport chain between the fatty acid oxidation and respiration pathways under anaerobic conditions (302). Additionally, IS150 insertion was also detected in ydiU at

the same position (963,716 in the REL4536 reference genome) in the REL4536

ΔsbcC::cat-1K-1, REL4536 ΔsbcC::cat-1K-6, REL4536 ΔsbcC::cat-1K-7 and

REL4536 ΔsbcC::cat-1K-14 clones, suggesting this position may be another hotspot for IS150 insertion. YdiU, encoded for by ydiU, is a conserved protein with unknown

163 function. However, the advantages of inactivating both ydiQ and ydiU via insertion of

IS elements remain unclear.

Another instance of parallelism was detected in clones from REL4536 ΔsbcC::cat-1K-7 and REL4536 ΔsbcC::cat-1K-14, where an IS150 element was deleted from the pflB

gene, resulting in the reactivation of the gene (Table A.7). The pflB gene encodes

pyruvate formate lyase I, an enzyme involved in the conversion of pyruvate to formate during anaerobic fermentation. Therefore, the presence of a functional pflB gene under

anaerobic conditions should be beneficial and advantageous under anaerobic growth conditions. Moreover, reactivation of the pflB gene was also reported by Finn (2015) for

some sequenced REL4536 clones evolved under anaerobic and fluctuating conditions for 2,000 generations (205). Additionally, a C Æ T transition in the pflB gene at the

same position (1,766,332 in the REL4536 reference genome) was detected in the REL4536 ΔsbcC::cat-1K-14 and REL4536 AN-1K-4 clones. As this mutation was synonymous, and thus did not result in the substitution of amino acids, the beneficial effect of this mutation is not immediately apparent.