Studies On The Expression Of The bgl Operon Of Escherichia Coli In Stationary Phase

Abstract

The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to induce its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. The experiments described in this thesis were carried out to test these possibilities. In cultures exposed to prolonged stationary phase, majority of the bacterial population dies and a few mutants that have the ability to scavenge the nutrients released by the dying cell mass survive. Bgl+ mutants were found to be enriched in twenty-eight-day-old Luria Broth grown cultures of E. coli that are wild type for bgl but carry the rpoS819 allele. Out of the five Bgl+ mutants that were isolated, four carried a mutation in the hns locus while one of them, ZK819-97, had an activating mutation linked to the bgl operon. Further analysis of ZK819-97 by DNA sequencing revealed the existence of a single C to T transition at the CAP binding site in the regulatory region. ZK819-97 was chosen for further analysis. Competition assays were carried out in which Bgl+ strain, ZK819-97 (Strr), and the parental Bgl- strain, ZK820 (Nalr), were grown independently for twenty-four hours in Luria Broth and then mixed in 1:1,000 (v/v) ratio reciprocally, without addition of fresh nutrients. ZK819-97, when present in minority, was found to increase in number and take over the parental strain, ZK820, i.e. ZK819-97 showed a Growth Advantage in Stationary Phase phenotype. To determine whether the GASP phenotype of ZK819-97 is associated with the bgl locus, the bgl allele from this strain was transferred by P1 transduction to its parental strain, ZK819. The resulting strain, ZK819-97T (Bgl+, Tetr), when competed with the parental strain, ZK819 Tn5 (Bgl-, Kanr), also showed a GASP phenotype when present in minority in the mixed cultures. To reconfirm this further, the bgl locus was deleted from ZK819-97T. The resulting strain, ZK819-97Δbgl, showed a loss of the GASP phenotype. When the bglB locus was disrupted in ZK819-97T, the resulting strain, ZK819-97ΔB, also failed to show a GASP phenotype, indicating that the phospho-β-glucosidase B activity is essential for this phenotype. The strain, ZK819-IS1, carrying an activating IS1 insertion within the bgl regulatory region also showed a GASP phenotype, confirming that this phenotype of the Bgl+ strain is independent of the nature of the activating mutation. All the above mentioned strains used in the competition assays carry a mutant allele of rpoS, rpoS819. Introduction of the wild type rpoS allele in these strains resulted in the loss of the GASP phenotype of the Bgl+ strain, suggesting that the two mutations work in a concerted manner. The Bgl+ strain was found to show the GASP phenotype only when present in minority of 1:1,000 or 1:10,000 in the mixed cultures and showed a slight disadvantage at higher ratios, indicating that the GASP phenotype of the Bgl+ strain is a frequency dependent phenomenon. In competition assays carried out between 24-hour-old cultures of Bgl+ and Bgl- strains resuspended in five-day-old spent medium prepared from a wild type E. coli strain, Bgl+ strain did not show any extra or early GASP phenotype. In addition, a reporter strain, which has a lacZ transcriptional fusion with the activated bgl promoter, was resuspended in spent medium prepared from a five-day-old culture of wild type strain of E. coli and bgl promoter activity was measured by β-galactosidase assay. The bgl promoter did not show any induction in this medium. These experiments suggest the absence of any β-glucoside like molecules in the spent medium within the sensitivity of these assays. A reporter strain that has a lacZ transcriptional fusion to the wild type bgl promoter was used to measure the expression level of this promoter during exponential and stationary phase of growth in LB. Expression of the wild type as well as various activated promoters of bgl was found to be enhanced in stationary phase. To investigate a possible role of the rpoS encoded stationary phase specific sigma factor, RpoS (σs), and another stationary phase factor, Crl, known to be important for the regulation of many genes of the σs regulon, the bgl promoter activity measurements were carried out in the presence or the absence of RpoS and/or Crl. RpoS along with Crl was found to negatively regulate the expression of wild type as well as activated promoters of bgl, both in exponential and stationary phase. In the absence of the negative regulation by RpoS and Crl, the increase in the bgl promoter activity was more pronounced as compared to that in its presence. rpoS and crl mutations are common in nature and it has been suggested that crl deletion gives a growth advantage to the strain in stationary phase. To test this possibility crl deletion was created in wild type as well as in attenuated rpoS allele background. The strain carrying the crl deletion was found to have a growth advantage in stationary phase over the wild type strain in the presence of wild type rpoS allele, while it shows a slight disadvantage in combination with mutant rpoS. Over expression of LeuO or BglJ is known to activate the bgl operon. To study a possible role of these factors in the regulation of the bgl expression in stationary phase, the bgl promoter activity was measured in strains that were deleted for leuO and/or bglJ, in the absence or presence of crl. These studies indicated that BglJ had a moderate effect on the bgl promoter activity in stationary phase in the absence of Crl but not in its presence. LeuO did not have a significant effect on the bgl promoter activity in either condition. Thus under the conditions tested, the physiological increase in the levels of LeuO and BglJ in stationary phase was insufficient to regulate the bgl expression. Preliminary results show that the bgl operon might be involved in the regulation of oppA, an oligopeptide transporter subunit, in stationary phase. Implications of these findings are discussed. The studies reported in this thesis highlight the involvement of the bgl operon of E. coli in stationary phase. This could be mediated by genetic as well as physiological mechanisms. This study also underscores the importance of observing organisms closer to their natural context and the need to reconsider the concept of ‘cryptic genes’.