Experimental evolution of gallium resistance in Escherichia coli

Abstract

Background and Objectives Metallic antimicrobial materials are of growing interest due to their potential to control pathogenic and multidrug-resistant bacteria. Yet we do not know if utilizing these materials can lead to genetic adaptations that produce even more dangerous bacterial varieties. Methodology Here we utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance. Results By day 10 of evolution, increased gallium resistance was evident in populations cultured in medium containing a sublethal concentration of gallium. Furthermore, these populations showed increased resistance to ionic silver and iron (III), but not iron (II) and no increase in traditional antibiotic resistance compared with controls and the ancestral strain. In contrast, the control populations showed increased resistance to rifampicin relative to the gallium-resistant and ancestral population. Genomic analysis identified hard selective sweeps of mutations in several genes in the gallium (III)-resistant lines including: fecA (iron citrate outer membrane transporter), insl1 (IS30 tranposase) one intergenic mutations arsC →/→ yhiS; (arsenate reductase/pseudogene) and in one pseudogene yedN ←; (iapH/yopM family). Two additional significant intergenic polymorphisms were found at frequencies > 0.500 in fepD ←/→ entS (iron-enterobactin transporter subunit/enterobactin exporter, iron-regulated) and yfgF ←/→ yfgG (cyclic-di-GMP phosphodiesterase, anaerobic/uncharacterized protein). The control populations displayed mutations in the rpoB gene, a gene associated with rifampicin resistance. Conclusions This study corroborates recent results observed in experiments utilizing pathogenic Pseudomonas strains that also showed that Gram-negative bacteria can rapidly evolve resistance to an atom that mimics an essential micronutrient and shows the pleiotropic consequences associated with this adaptation. Lay summary We utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance.

Experimental Evolution of Magnetite Nanoparticle Resistance in Escherichia coli

“…However, in prior studies we have deployed more direct methods to test whether the genomic variants we discovered should and actually do confer resistance. For example, we have utilized computational modeling of 3-dimensional structures of altered proteins to test their affinity for ionic metals [ 12 , 15 , 42 ]. These studies showed the alteration in the protein caused by the variant resulting from the selective sweep did reduce or increase the affinity to the target metal compared to the controls/ancestor.…”

“…E. coli MG1655 (ATCC #47076) was used in this study because it does not have plasmids and its circular chromosome is composed of 4,641,652 nucleotides (GenBank: GenBank: 117 NC_000913.3; Riley et al 2006). All of our previous studies of ionic and nanoparticle resistance have used this strain [ 13 , 14 , 15 ]. This strain is the ancestor of all the selection treatments (FeNP and Controls) evaluated in this study.…”

Too much of a good thing

“…Both G210C and A526T were also detected in our 45-day iron (II) adapted populations [ 27 ]. In red are two mutations (N169K and G400S) which were detected in gallium (iron analog) resistant strains [ 38 ] and as shown, map to similar regions as the iron resistant mutations. ( B ) The iron-citrate complexed structure (PDB1PO3) was also used to map two of the three iron resistant residues (D87Y lies in an unstructured region and therefore could not be mapped) and the gallium mutations.…”

“…Second, gallium is an iron analog. In our previous work, this strain of E. coli also acquires mutations in fecA as a result of gallium resistance which could account for the increased optical densities in the adapted strain in the metal [ 38 ]. What is most concerning is the multi-drug resistance acquired in this strain, which indicates that these strains have the potential to be even more harmful than originally anticipated.…”

Transporter Engineering for Microbial Manufacturing

“…Adaptive evolution strategy can be used to obtain strains with an evident product tolerance, [ 81 ] substrate utilization, and cell survival phenotype after evolution. [ 40,82 ] The goal of adaptive evolution is to obtain transporter mutants with higher transport efficiency, substrate specificity, or conversely, mutants with better substrate compatibility. For instance, an adaptive evolution was conducted using the hexose transporter CiGXS1 FIM in yeasts, and glucose–xylose cotransport mutants were selected based on their survival on xylose and glucose plates.…”