Escherichia coli K12 Group 2 capsular biosynthesis does not appear to be thermoregulated by an RpoS-dependent mechanism

09/01/2016

Kira Elsbernd, Jonathan Frew, Gunjan Mhapankar, Megan Wheatley​

Volume 20
Fall 2015 / Winter 2016

Capsule production in Escherichia coli strains K12 and K30 requires the transcription factor RfaH which is regulated by unknown mechanisms in E. coli. However, in Salmonella enterica serovar typhi, RfaH is regulated by the stress-response sigma factor RpoS. In E. coli low temperatures have been shown to increase both RpoS expression and capsular polysaccharide production. However, more recent studies of have reported conflicting results concerning the temperature dependence of capsule regulation with reports of both 2-fold and 10-fold increases in capsule synthesis at 21˚C compared to 37˚C. We hypothesized that RpoS would be up regulated at 21˚C compared to 37˚C, resulting in increased capsule production. To investigate this, we grew E. coli strains K12, K30, and ΔrpoS of K12 at 21˚C and 37˚C and compared capsule production quantitatively using an adapted phenol-sulphuric acid assay and qualitatively using Maneval’s staining protocol and light microscopy. We observed low carbohydrate concentrations and variable results with the capsule quantification assay, but were able to confirm previous results showing that Δ(wza)K30 mutant strain of E. coli K30 is capsule deficient. Microscopy showed thicker capsule associated with wild type K30 compared to Δ(wza)K30, but we did not observe any differences in capsule between 21°C and 37°C. To investigate whether RpoS activity is thermoregulated, we transformed K12 and mutant ΔrpoS E. coli with a dsrB-lacZ β-galactosidase reporter plasmid. dsrB is a downstream gene regulated by RpoS, and was thus used as a readout for RpoS activity. Our results failed to demonstrate the expected thermoregulation of RpoS, and an RpoS mutant exhibited significantly greater β-galactosidase activity than a K12dsrB at 37°C. β-galactosidase activity units were higher than expected, suggesting that using a multi-copy plasmid to introduce the dsrB promoter::LacZ translational fusion reporter is not a sensitive method for measuring rpoS transcription levels. Our results warrant additional experimentation to determine the relationship between RpoS, temperature, and capsule production in E. coli K12 and K30 strains. Furthermore, alternative methods of measuring capsule production and RpoS expression should be explored.