Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869

• Published in: Applied and Environmental Microbiology 2006-11, Vol.72 (11), p.7183-7192
• Main Authors: Kikuchi, Yoshimi, Date, Masayo, Itaya, Hiroshi, Matsui, Kazuhiko, Wu, Long-Fei
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arthrobacter - enzymology; Arthrobacter - metabolism; Arthrobacter globiformis; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Cloning; Corynebacterium glutamicum; Corynebacterium glutamicum - genetics; Corynebacterium glutamicum - metabolism; Dextranase - metabolism; Escherichia coli; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Gene Deletion; Genomics; Gram-positive bacteria; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Industrial Microbiology - methods; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Microbiology; Molecular Sequence Data; Molecular structure; Physiology and Biotechnology; Polysaccharides - metabolism; Protein Folding; Sequence Analysis, DNA
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.01528-06

Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.