Abstract
Bacillus cereus and other Gram-positive bacteria elaborate pili via a sortase D-catalyzed transpeptidation mechanism from major and minor pilin precursor substrates. After cleavage of the LPXTG sorting signal of the major pilin, BcpA, sortase D forms an amide bond between the C-terminal threonine and the amino group of lysine within the YPKN motif of another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the BcpA sorting signal, resulting in a covalent bond between BcpA and the cell wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby positioning BcpB at the tip of pili. Thus, unique attributes of the sorting signals of minor pilins provide Gram-positive bacteria with a universal mechanism ordering assembly of pili.
Highlights
Pili of Gram-positive bacteria comprised either two or three different pilin subunits synthesized as cytoplasmic precursors with N-terminal signal peptides and C-terminal sorting signals (P1 precursors) [14, 18]
Plasmids—The following plasmids contain B. cereus pilus genes cloned under control of the spac promoter of pLM5 [12] and were described previously: pJB39, pJB12, pJB28, pJB32, and pJB48 [15]. pJB182 encodes for bcpA-srtD-bcpB⌬VIPNTGG719, which contains a seven-residue deletion in BcpB, encompassing the IPNTG sorting signal motif and one residue flanking each end (VIPNTGG719). pJB12 was used as a template in site-directed mutagenesis with Pfu polymerase, as described previously [15], with primer pairs P128 and P129 Table 1. pJB139 encodes for bcpA-srtD-bcpB with a MH6 tag inserted six nucleotides upstream from the codons encoding for the IPNTG motif in bcpB. pJB139 was created by quick change with primer pair P237/P238 and pJB12 template DNA
The Sorting Signal of the Minor Pilin Is Required for Its Assembly into Pili—When transformed with pJB12, a plasmid encoding the B. cereus pilin operon, B. anthracis Sterne forms pili comprising both the major pilin subunit, BcpA, and the minor pilin subunit, BcpB (Fig. 1A) [15]
Summary
Plasmids—The following plasmids contain B. cereus pilus genes cloned under control of the spac promoter of pLM5 [12] and were described previously: pJB39 (bcpA-srtD), pJB12 (bcpA-srtD-bcpB), pJB28 (bcpAK162A-srtD-bcpB), pJB32 (bcpAsrtDC207A-bcpB), and pJB48 (bcpALAVAA-srtD-bcpB) [15]. pJB182 encodes for bcpA-srtD-bcpB⌬VIPNTGG719, which contains a seven-residue deletion in BcpB, encompassing the IPNTG sorting signal motif and one residue flanking each end (VIPNTGG719). pJB12 was used as a template in site-directed mutagenesis with Pfu polymerase, as described previously [15], with primer pairs P128 and P129 Table 1. pJB139 encodes for bcpA-srtD-bcpB with a MH6 tag inserted six nucleotides upstream from the codons encoding for the IPNTG motif in bcpB. pJB139 was created by quick change with primer pair P237/P238 and pJB12 template DNA. Plasmids—The following plasmids contain B. cereus pilus genes cloned under control of the spac promoter of pLM5 [12] and were described previously: pJB39 (bcpA-srtD), pJB12 (bcpA-srtD-bcpB), pJB28 (bcpAK162A-srtD-bcpB), pJB32 (bcpAsrtDC207A-bcpB), and pJB48 (bcpALAVAA-srtD-bcpB) [15]. PJB182 encodes for bcpA-srtD-bcpB⌬VIPNTGG719, which contains a seven-residue deletion in BcpB, encompassing the IPNTG sorting signal motif and one residue flanking each end (VIPNTGG719). PJB12 was used as a template in site-directed mutagenesis with Pfu polymerase, as described previously [15], with primer pairs P128 and P129 Table 1. PJB139 encodes for bcpA-srtD-bcpB with a MH6 tag inserted six nucleotides upstream from the codons encoding for the IPNTG motif in bcpB. PJB139 was created by quick change with primer pair P237/P238 and pJB12 template DNA. Amplified product was digested with NheI and ligated, and non-methylated plasmid DNA was digested with DpnI before transformation into Escherichia coli DH5-␣. pJB139 was amplified with primer pairs
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