Deciphering the evolutionary significance of maintaining cryptic genetic systems by bacteria

Abstract

Cryptic genes are phenotypically silent DNA sequences with the potential to code for a function, but remain inactive during the normal life span of the organism. However they can be activated by a single mutational event such as recombination, deletion, insertions or point mutation, resulting in a discernible phenotype. The bgl operon of E. coli, involved in the uptake and breakdown of plant derived aromatic -glucosides salicin and arbutin, is one of the well studied examples of cryptic genetic systems. The present study was aimed at understanding the causes for the conservation of the silent bgl operon without being lost over evolutionary time. Antimicrobial resistance is one of the most serious problems in the clinical context since microorganisms are equipped with distinct defense mechanisms to meet the challenges posed by the widespread presence of antibiotics in the environment. The non-heritable transient phenomenon of resisting low doses of antibiotics is broadly termed as phenotypic antibiotic resistance. It has been observed earlier that wild type E. coli shows phenotypic antibiotic resistance when exposed to salicylate and related compounds by inducing the expression of the marRAB (multiple antibiotic resistance) operon. Salicylate being structurally similar to the breakdown products of the β-glucosides salicin and arbutin, viz. saligenin and hydroquinone respectively, suggested the possibility that bacteria having the functional form of the bgl operon and are actively metabolizing salicin, can exhibit phenotypic antibiotic resistance. Results presented in Chapter 2 show that E. coli and Klebsiella strains carrying inducible or constitutive form of the bgl operon show marA dependent antibiotic resistance to low doses of antibiotics through enhanced expression of different multidrug efflux pumps. The aglycone product released during salicin metabolism viz. saligenin is responsible for conferring the phenotype. The prolonged state of low level of resistance can give rise to a genetically resistant population of mutants as the frequency of mutants that show heritable resistance increases when Bgl+ bacteria are exposed to salicin and sub-lethal doses of nalidixic acid. Most of the identified mutations were clinically relevant and primarily localized to the QRD (quinolone resistance determining region) locus of the gyrA subunit of DNA gyrase. There was no fitness cost associated with genetic resistance as the mutants showed no growth disadvantage in competition experiments performed between the sensitive parent and the mutants. These findings indicate that presence of the activated form of the bgl operon in the genome facilitates the survival of bacteria in environments in which both aromatic β-glucosides as well as antibiotics are present. In addition to facilitating β-glucosides metabolism, the activated bgl operon has been shown to confer growth advantage in stationary phase by enhancing the metabolic capability of the cell. BglG, the positive regulator of the operon, has been implicated in the regulation of several downstream target genes in stationary phase. BglG is a sequence specific RNA binding protein that binds as a homodimer at a 32 nucleotide long sequence termed as ribonucleic antiterminator (RAT). A genome wide comparative transcriptome analysis of ΔbglG and bglG (WT) strains performed earlier revealed that bglG deletion led to down-regulation of several metabolic pathways including lipopolysaccharide (LPS) biosynthesis. The downregulation of LPS biosynthesis pathway resulted in a permeability defect in the bglG deletion strain. Studies described in Chapter 3 addresses the morphological changes associated with the ΔbglG strain and the mechanistic details of the regulation of the genes involved in LPS synthesis and transport by BglG in stationary phase. The DNA binding transcription factor GadE, involved in the regulation of genes required for pH maintenance, was significantly downregulated in the bglG deletion strain as seen in the transcriptome analysis described above. Earlier studies have also shown that gadE is one of the key positive regulators of genes involved in LPS core biosynthesis. Using electrophoretic mobility shift assays it could be demonstrated that BglG binds gadE mRNA that carries a putative RAT sequence and the binding increases the stability of gadE mRNA by an unknown mechanism. Partial loss in permeability barrier of the strain was also observed upon deletion of gadE. These observations suggest that GadE is one of the downstream targets of BglG and brings about the regulation of the composition of cell wall in stationary phase. One possible reason for the retention of the silent form of the bgl operon in wild isolates is a fitness cost associated with the activated form under nonselective conditions. Work presented in Appendix was an attempt to investigate the possibility of the activated form of the operon conferring a growth disadvantage under nonselective conditions such as exponential growth phase in nutrient rich media. Exponential growth phase competition experiments using strains of different bgl genotypes could not identify any distinct growth disadvantage associated with the Bgl+ strain at least under the conditions tested. In light of these observations the epilogue section highlights how the bgl operon appears to be a dynamic component of the genome and its involvement in multiple survival strategies is expected to provide a strong selective force for its maintenance in the genome