Journal of Experimental Microbiology and Immunology (JEMI) Vol. 15: 52 – 58 Copyright © April 2011, M&I UBC Assessing the Role of SpoT and RelA in Capsular Polysaccharide Synthesis after Treatment with Sub-lethal Concentrations of Kanamycin to Confer Decreased Antibiotic Sensitivity in Escherichia coli Wesley Chenne, Louisa Ng, and Mary Rose Pambid Department of Microbiology & Immunology, University of British Columbia Resistance to various antibiotics has been associated with increased capsular polysaccharide production through activation of RelA and SpoT, regulators of the stringent response, through their respective synthesis and hydrolysis of guanosine tetraphosphate in Escherichia coli. In order to further characterize the role of SpoT and RelA in capsular polysaccharide production and in conferring antibiotic resistance, growth patterns and induced capsular polysaccharide levels of various strains were determined following sub- lethal treatment with kanamycin and further treatment at inhibitory levels of kanamycin. Contrary to previous reports that capsular polysaccharide confers antibiotic resistance, it was found that spoT mutants produced the least capsular polysaccharide but had higher levels of resistance in comparison to relA mutants, which produced the most polysaccharide but exhibited lower resistance. As expected, both these mutants exhibited lower resistance to kanamycin as a result of pre-treatment than the wild-type strain. Thus, we propose that bacterial survival and resistance development upon antibiotic administration is dependent upon a myriad of factors that stem from the regulation of guanosine tetraphosphate which not only include capsular polysaccharide levels, but also other pathways leading to and resulting from the induction of the stringent response. For Escherichia coli, a general response to the production of ppGpp, but primarily serves as the environmental stressors, such as nutrient starvation and hydrolase for the degradation of ppGpp to ensure that the administration of antibiotics, is the induction of the cell homeostasis can be achieved upon return to normal stringent response (16), which promotes cell survival at environmental conditions, as the decrease in ppGpp the expense of exponential cellular growth. The results in downregulation of rpoS and a reduction in the stringent response is regulated by the expression of the expression of σS (14). rpoS gene and its end product, sigma factor s (σS) (6, Upon exposure to sub-lethal levels of KAN or other 11). This expression is upregulated by the activity of aminoglycosides, a downstream effect that has been the alarmone guanosine tetraphosphate (ppGpp) in observed is the induction and up-regulation of capsular conjunction with dskA (15, 21). In turn, the two major polysaccharides (CPS) (1). CPS has been shown to cellular factors that have been implicated in the confer protective effects for E. coli by putatively production of ppGpp are SpoT and RelA (21). When limiting the entry and activity of KAN and other cells are treated with aminoglycosides such as aminoglycosides (20). Recent studies with relA mutants kanamycin (KAN), its activity leads to the simultaneous have been performed with regards to the effects of upregulation of both RelA and SpoT activity (1). KAN RelA on conferring lessened antibiotic sensitivity after binds to the 30S subunit of the ribosome, leading to pre-treatment with sublethal levels of KAN, but they blockage of the anti-codon/codon binding site and have shown inconclusive evidence for its exact role (1). limitation of protein synthesis (3, 10). RelA binds to Moreover, the role of SpoT in the relationship between these blocked ribosomes and mediates the conversion conferring lessened antibiotic sensitivity and the of ATP and GTP to AMP and ppGpp (19). Moreover, production of CPS upon sub-lethal treatments of KAN KAN also causes membrane damage and a decrease in requires more characterization. intracellular ferrous iron via the induction of the We hypothesized that upon pre-treatment with sub- bacteriocidal Fenton hydroxyl radical reactions (10), lethal levels of KAN, the presence of both SpoT and which induces SpoT activity and the production of RelA would confer the greatest decrease in antibiotic ppGpp (18) However, SpoT is not only implicated in sensitivity in a parental wildtype strain, followed by 52 Journal of Experimental Microbiology and Immunology (JEMI) Vol. 15: 52 – 58 Copyright © April 2011, M&I UBC relA, spoT, and relA/spoT mutant strains. To study the TABLE 1. Bacterial strains acquired from E. coli Genetic Stock effects of these gene products, isogenic mutant strains Center. were assayed for initial sensitivity to KAN, then pre- Strain Genotype treated with sub-lethal levels of the antibiotic prior to WG1 (WT) conducting measures of growth at different inhibitory F+, ΔrfbB51, F1-1 concentrations. To further elaborate the role of CPS in AT-2 (relA) Hfr(PO22), relA1, metB1 conferring a decrease in antibiotic sensitivity, the induced amounts of CPS were also qualitatively and 58-161 (spoT) F+, bio-1(Unst), spoT1, metB1, creC510 quantitatively assayed following this initial sub-lethal AB301 (relA/spoT) Hfr(PO21), relA1, spoT1, metB1 pre-treatment with KAN. In comparison to relA mutants, spoT mutants were shown to have higher resistance despite exhibiting lower levels of CPS production. In Antibiotic sensitivity test. The sensitivity of each strain of E. coli fact, the relA mutant was shown to produce the highest to KAN was tested by growing each strain at four different levels of levels of CPS, yet its resistance fell short of both the antibiotic based on the MIC of the strain. For each strain, four different flasks containing 90 ml of M9 minimal salts media were spoT and the wild type strain. Finally, the spoT/relA inoculated with 10 ml of each pretreated culture. KAN was added at double mutant displayed slightly lower CPS production concentrations of 1 MIC, 2 MIC, and 3 MIC to three of the flasks; the than the wild-type, but had the lowest levels of last flask was not subjected to KAN treatment. After measuring and recording the initial turbidity of the cultures, the bacteria were resistance; this suggests that other than the induction of incubated at 37°C in a shaking water bath, and further turbidity CPS due to spoT and relA, there are also other cellular measurements were taken and recorded at 15, 30, 60, 100, and 160 processes that result from these two regulators involved min. following the first measurement. The cultures were grown overnight and the final turbidity was read and recorded. All turbidity in the induction of antibiotic resistance. readings were performed using a Spectronic 20D spectrophotometer with wavelength set at 460 nm, using M9 minimal salts media as a MATERIALS AND METHODS blank. CPS staining. E. coli capsules were stained with Congo Red and Bacterial strain. E. coli strains WG1 (WT), AT-2(relA), 58- Maneval’s solution (2) by preparing air dried and heat fixed slides of 161(spoT), AB301 (relA, spoT) as described in Table 1 were obtained pretreated and untreated cells on glass slides. Slides were visualized from the Coli Genetic Stock Centre, Yale University. by light microscopy at 1000x magnification. Images were acquired by Preparation of M9 minimal salts media supplemented with aligning a Samsung ST50 digital camera with the ocular lens at ISO vitamins and amino acids. M9 minimal media (1) was prepared with 100, f/56, and exposure time of 1/28 seconds. 0.4% glycerol as the carbon source. A amino acid supplement was Capsule isolation. CPS was isolated by following a modified prepared by adding 0.6 g of Casamino Acids (Bacto #223030) in 200 version of the methods outlined by Lu et a (10). 60 ml of each ml of distilled water, 0.6 g of L-tryptophan (Sigma #T0254), 0.6 g of pretreated and untreated bacterial cultures were centrifuged at 17,000 L-glutamine (Sigma #G5763), and 0.6 g of L-asparagine (Difco 0583- x g for 20 min. The supernatants were discarded and the pellets were 12) in 200 ml of distilled water. Biotin solution was prepared by resuspended in 40 ml of PBS (137 mM NaCl, 2.7 mM KCl 1.4mM adding 20 mg of D-biotin (Sigma #B4501) to 120 ml of distilled H2O Na2HPO4, 1.8 mM KH2PO4). Following addition of ice cold acetone, and filter sterilized using a 45 um filter. Biotin was added to a final samples were centrifuged at 6,000 x g for 10 min. After discarding concentration of 10 µg/ml. the supernatants and resuspending the pellets, the samples were Overnight (ON) cultures. Overnight (ON) cultures of each E. coli transferred into 1 ml/cm Spectra/Por® molecularporous membrane strain were prepared by inoculating 10 ml of supplemented M9 dialysis tubes with molecular weight cut-offs at 6,000 – 8,000 kDa. minimal salts media with a loopful of bacteria and incubating in an air The samples were dialyzed against 2 litres of distilled water at 4°C shaker with mild aeration (50-100 rpm) at 37°C. for 48 hours, after which a lyophilizer was used to dry the isolated Minimum Inhibitory Concentration (MIC) assay. ON cultures expolymers overnight. Each dried sample of expolymers was were diluted by a factor of 1000, and 100 ul of each diluted strain was dissolved in 3 ml of 10 mM MgCl2. Ribonuclease A (Fermentas added to the wells of two rows of a 96 well plate. Stock KAN #EN0531) and Deoxyribonuclease I (Sigma #2326670) were added to solution at 20 mg/ml was diluted to 1 mg/ml, and 100 µl was added to the dissolved samples, which were incubated for 3 hours at 37°C in a each well of the first column. Serial half dilutions were performed shaking water bath. Pronase (Boehringer Mannheim #165921) was across the rows by transferring 100 µl from the wells in the first added to the samples, which were incubated at 37°C in a shaking column to the wells of the next column, leaving the last column water bath overnight.