Identification and regulation of genes for cobalamin transport in the cyanobacterium Synechococcus sp. strain PCC 7002.
Adam A. Perez, Dmitry A. Rodionov, Donald A. Bryant
Journal of Bacteriology
The cyanobacterium Synechococcus sp. strain PCC 7002 is a cobalamin auxotroph and utilizes this coenzyme solely for the synthesis of L-methionine by methionine synthase (MetH). Synechococcus sp. strain PCC 7002 is unable to synthesize cobalamin de novo, and due to the large size of this tetrapyrrole, an active transport system must exist for cobalamin uptake. Surprisingly, no cobalamin transport system was identified in the initial annotation of the genome of this organism. Using more sophisticated in silico prediction tools, a btuB-cpdA-btuC-btuF operon, encoding components putatively required for B12 uptake (btu) system, was identified. The expression of these genes was predicted to be controlled by a cobalamin riboswitch. Global transcriptional profiling by RNA sequencing (RNA-seq) of a cobalamin-independent, Synechococcus sp. strain PCC 7002 strain grown in the absence and presence of cobalamin confirmed regulation of the btu operon by cobalamin. Pérez et al. (2016, J. Bacteriol., submitted), developed a, cobalamin-dependent, yellow fluorescent protein (YFP) reporter system in a Synechococcus sp. strain PCC 7002 strain that had been genetically modified to allow cobalamin-independent growth. This reporter system was exploited to validate components of the btu uptake system by assessing the ability of targeted mutants to transport cobalamin. The btuB promoter and a variant counterpart mutated in an essential element of the predicted cobalamin riboswitch were fused to a yfp reporter. The combined data indicate that the btuB-cpdA-btuF-btuC operon in this cyanobacterium is transcriptionally regulated by a cobalamin riboswitch. IMPORTANCE: Using a cobalamin-regulated reporter system for expression of yellow fluorescent protein, genes previously misidentified as encoding subunits of a siderophore transporter were shown to encode components of cobalamin uptake in the cyanobacterium Synechococcus sp. strain PCC 7002. This study demonstrates the importance of experimental validation of in silico predictions and provides a general scheme for in vivo verification of similar cobalamin transport systems. A putative cobalamin riboswitch was identified in Synechococcus sp. strain PCC 7002. This riboswitch acts as a potential transcriptional attenuator of the btu operon that encodes the components of the cobalamin active transport system.
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