Transcript Analysis of the CSO Operon and Characterization of Two Sets of Conserved Bacterial Microcompartment Genes in the Model Organism Halothiobacillus neapolitanus
This study was designed to achieve better understanding of (1) how carboxysome genes are regulated and expressed to yield with precise relative ratios and (2) the in vivo roles of two sets of conserved bacterial microcompartment genes, namely the three csoS1 and two csoS4 genes of H. neapolitanus , in the biogenesis and function of the carboxysome. For the first goal, a detailed transcriptional profile of carboxysomal genes in H. neapolitanus was established using absolute quantification real-time RT-PCR and transcript ends analysis. This transcriptional profile revealed that a single promoter, denoted cso promoter, was located upstream from the clustered carboxysomal genes. Transcripts of all nine carboxysomal genes were detectable but were present at different levels. In vivo activities of the cso promoter and selected internal non-coding regions within the carboxysome operon were further examined by using a promoter reporter vector and by generating a cso promoter deletion mutant. Both experiments established the cso promoter as the only promoter in the carboxysome operon. This result, together with the transcriptional profile of carboxysomal genes, strongly implied that the primary transcript of the H. neapolitanus cso operon undergoes differential processing and/or decay and results in different expression levels of individual carboxysomal proteins. To address the functions of three CsoS1 paralogs, in vivo and in vitro experiments were performed. Fusing a tetracysteine motif to the C-terminus of CsoS1A confirmed that all three CsoS1 proteins are carboxysome components. A truncated csoS1B mutant and a site-directed mutant of csoS1A were generated to address the functions of the C-terminal extension of the CsoS1B polypeptide and of the central pore in the CsoS1A hexamer, respectively. An attempt was made to gain better insights into the spatial orientations of CsoS1 proteins and other carboxysome shell components via chemical modification on the carboxysome surface. Finally, single and double knockout mutants were generated for functional study of csoS4A and csoS4B genes. Phenotypic and kinetic characterization of these mutants revealed that lack of CsoS4 proteins resulted in a more permeable shell which cannot provide the catalytic advantage to r ibu lose 1,5- bis phosphate c arboxylase/o xygenase (RubisCO).