K-105. Functional Study of Carboxysome Operon Promoter(s) from Halothiobacillus neapolitanus

S. Neidler1, F. Cai1, J. M. Shively2,1, G. C. Cannon1, S. Heinhorst1;
1The Univ. of Southern Mississippi, Hattiesburg, MS, 2Clemson Univ., Clemson, SC.

The carboxysome is a microcompartment found in many autotrophic bacteria. This bacterial “organelle” functions to enhance CO2 fixation by sequestering D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) within a proteinaceous shell and providing a catalytic advantage for the enzyme. The genes encoding the two RubisCO subunits and the carboxysome shell proteins are organized as an operon (cso) in chemoautotrophs and in many marine photoautotrophs, notably the Prochlorococci. A previous study in our lab established a transcriptional profile of the cso operon from the sulfur oxidizer, Halothiobacillus neapolitanus, and identified the putative promoter upstream from the first gene in the operon. However, phenotypes of insertion mutants and transcript end analyses suggest the possibility that additional, internal promoter(s) might also exist. We have generated constructs in broad-host-range promoter probe vectors that allow us to assess the promoter activity of selected target regions from the H. neapolitanus cso operon. The main cso promoter activated transcription of the reporter gene in Escherichia coli, while the results with internal promoter candidates were less clear. Movement of the constructs into H. neapolitanus and analysis of promoter deletion mutants in that bacterium are underway to quantify promoter activity and clarify some of the ambiguities observed in E. coli.