MICROBIOLOGY Correction for “BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm,” by Laura Hobley, Adam Ostrowski, Francesco V. Rao, Keith M. Bromley, Michael Porter, Alan R. Prescott, Cait E. MacPhee, Daan M. F. van Aalten, and Nicola R. Stanley-Wall, which appeared in issue 33, August 13, 2013, of Proc Natl Acad Sci USA (110:13600–13605; first published July 31, 2013; 10.1073/pnas.1306390110).
The authors wish to note the following: “Ongoing investigations into mechanism of BslA self-assembly led us to question the validity of the in vivo phenotype exhibited by the L77D BslA variant protein. We uncovered that the strain thought to carry a mutation of residue leucine 77 to aspartate in fact contained the wild-type bslA coding region. Investigations revealed that the original heterologous bslA coding region had not been sequenced upon introduction to the Bacillus subtilis chromosome and therefore it was not noticed that the desired mutation was lacking. Thus, we constructed a strain carrying the coding region with the L77D mutation and have established that the BslA L77D is unable to reinstate wild type biofilm formation to the bslA mutant; meaning that the colony formed remained wetting. (See corrected Fig. 6 and corrected Table S2.) Consistent with these findings, the BslA L77D protein was localized within pellicle biofilms to media fraction (see Fig. S5C). These findings are entirely consistent with our model of BslA function and do not impact the validity of our original conclusions.” The corrected Fig. 6, its legend, corrected Fig. S5, its legend, corrected Table S2, and corrected Table S3 appear below. The SI has been corrected online.
Fig. 6.
In vivo biofilm analysis of the hydrophobic cap of BslA. (A) Complex colony morphologies of strains containing leucine/isoleucine-to-lysine mutations in the β-sheets CAP2 and CAP3 alongside wild-type (NCIB3610), bslA− (NRS2097), and bslA+ (NRS2299) controls. (B) Pellicle morphology of the CAP2 and CAP3 mutants shown in A. (C) Sessile water-drop analysis of colony hydrophobicity of CAP2 and CAP3 mutants. Colonies were grown as for morphology analysis and 5-µL water drops placed on top. (D) Complex colony morphologies of strains containing mutations in the central CAP1 β-sheet. (E) Pellicle morphology of the CAP1 mutants shown in D. (F) Water-droplet analysis of colony hydrophobicity of CAP1 mutants. Table S3 gives strain details.
Table S2.
In vivo characterization of the hydrophobic cap
| Mutation | Colony morphology | Pellicle morphology | Water drop assay (mean contact angle ± SE) |
| Wild-type | Wild-type | Wild-type | Nonwetting 128.9° ± 1.3 |
| bslA | Null | Null | Wetting 24.7° ± 2.7 |
| bslA+ | Wild-type | Wild-type | Nonwetting 127.2° ± 3.4 |
| L76 | |||
| I | Wild-type | Wild-type | Nonwetting 124.8° ± 2.2 |
| K | Intermediate (almost null) | Null | Nonwetting 120.2° ± 13.1 |
| D | Intermediate | Intermediate | Nonwetting 127.6° ± 10.7 |
| L77 | |||
| I | Wild-type | Wild-type | Nonwetting 118.0° ± 8.3 |
| K | Null | Null | Wetting 49.6° ± 4.5 |
| D | Null | Null | Wetting 40° ± 0.4 |
| L79 | |||
| I | Wild-type | Wild-type | Nonwetting 122.1° ± 7.4 |
| K | Null | Null | Wetting 23.7° ± 5.0 |
| D | Null | Null | Wetting 18.5° ± 7.4 |
| L119K | Wild-type | Wild-type | Nonwetting 115.1° ± 8.0 |
| L121K | Intermediate | Null | Nonwetting 128.5° ± 1.0 |
| L123K | Intermediate (almost null) | Null | Nonwetting 130.5° ± 2.0 |
| L124K | Intermediate | Wild-type | Nonwetting 133.4° ± 0.8 |
| L153K | Intermediate | Null | Nonwetting 123.9° ± 1.7 |
| I155K | Intermediate | Null | Nonwetting 128.9° ± 1.9 |
Fig. S5.
Protein production and stability of in vivo BslA hydrophobic cap mutants. Western blot analysis using a BslA-specific primary antibody of protein extracted from whole complex colonies of (A) CAP2 and CAP3 mutants (as shown in Fig. 6A) and (B) CAP1 mutants (Fig. 6D). Colonies were grown for 48 h at 30 °C before extraction. (C) Pellicles of CAP1 mutant strains, grown for 18 h at 37 °C, were separated into pellicle (P, containing both cells and the biofilm matrix) and media (M) fractions. Western blots were probed with a BslA-specific primary antibody. Pellicles with wild-type morphology show that most BslA protein is found within the P fraction, whereas those with altered pellicle morphology have BslA protein in both the P and M fractions. When BslA is present in the media a second, lower-molecular-weight degradation product appears.
Table S3.
Full list of strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant genotype/description | Source/construction*,† |
| Strain | ||
| MC1061 | E. coli F'lacIQ lacZM15 Tn10 (tet) | E. coli Genetic Stock Centre |
| BL21 (DE3) | F– ompT hsdSB(rB−, mB−) gal dcm (DE3) | 1 |
| B834 (DE3) | F− ompT hsdSB(rB− mB−) gal dcm met (DE3) | Novagen |
| NCIB3610 | prototroph | BGSC |
| 168 | trpC2 | BGSC |
| JH642 | trpC2 pheA1 | 2 |
| NRS1471 | JH642 sacA::Phy-spank-gfpmut2 (kan) | 3 |
| NRS1473 | 3610 sacA::Phy-spank-gfpmut2 (kan) | SPP1 NRS1471 → 3610 |
| NRS2097 | 3610 bslA::cat | 4 |
| NRS2289 | 3610 sacA::PbslA-gfpmut2 (kan) | 5 |
| NRS2299 | 3610 bslA::cat amyE::Phy-spank-bslA-lacI (spc) | 4 |
| NRS2394 | 3610 sacA::PtapA-gfpmut2 (kan) | 6 |
| NRS3790 | 3610 bslA::cat amyE::Phy-spank-bslA-lacI (spc) sacA::Phy-spank-gfpmut2 (kan) | SPP1 NRS1471 → NRS2299 |
| NRS3812 | 3610 bslA::cat sacA::Phy-spank-gfpmut2 (kan) | SPP1 NRS1471 → NRS2097 |
| NRS3820 | 3610 bslA::cat amyE::Phy-spank-bslAL76I-lacI (spc) | SPP1 NRS3969 → NRS2097 |
| NRS3969 | 168 amyE::Phy-spank-bslAL76I-lacI (spc) | pNW1104 →168 |
| NRS4156 | 168 amyE::Phy-spank-bslAL76D-lacI (spc) | pNW1143 →168 |
| NRS4158 | 168 amyE::Phy-spank-bslAL76K-lacI (spc) | pNW1144 →168 |
| NRS4171 | 3610 bslA::cat amyE::Phy-spank-bslAL76D-lacI (spc) | SPP1 NRS4156 → NRS2097 |
| NRS4175 | 3610 bslA::cat amyE::Phy-spank-bslAL76K-lacI (spc) | SPP1 NRS4158 → NRS2097 |
| NRS4437 | 168 amyE::Phy-spank-bslAL79I-lacI (spc) | pNW1158 →168 |
| NRS4439 | 168 amyE::Phy-spank-bslAL79D-lacI (spc) | pNW1157 →168 |
| NRS4442 | 3610 bslA::cat amyE::Phy-spank-bslAL79I-lacI (spc) | SPP1 NRS4437 → NRS2097 |
| NRS4446 | 3610 bslA::cat amyE::Phy-spank-bslAL79D-lacI (spc) | SPP1 NRS4439 → NRS2097 |
| NRS4475 | 168 amyE::Phy-spank-bslAL79K-lacI (spc) | pNW1165 →168 |
| NRS4481 | 3610 bslA::cat amyE::Phy-spank-bslAL79K-lacI (spc) | SPP1 NRS4475 → NRS2097 |
| NRS4515 | 168 amyE::Phy-spank-bslAL98M-lacI (spc) | pNW1178 →168 |
| NRS4516 | 168 amyE::Phy-spank-bslAL77I-lacI (spc) | pNW1179 →168 |
| NRS4517 | 168 amyE::Phy-spank-bslAL77K-lacI (spc) | pNW1180 →168 |
| NRS5065 | 168 amyE::Phy-spank-bslAL77D-lacI (spc) | pNW1461 →168 |
| NRS4520 | 3610 bslA::cat amyE::Phy-spank-bslAL98M-lacI (spc) | SPP1 NRS4515 → NRS2097 |
| NRS4522 | 3610 bslA::cat amyE::Phy-spank-bslAL77I-lacI (spc) | SPP1 NRS4516 → NRS2097 |
| NRS4524 | 3610 bslA::cat amyE::Phy-spank-bslAL77K-lacI (spc) | SPP1 NRS4517 → NRS2097 |
| NRS5071 | 3610 bslA::cat amyE::Phy-spank-bslAL77D-lacI (spc) | SPP1 NRS5065 → NRS2097 |
| NRS4552 | 168 amyE::Phy-spank-bslAL119K-lacI (spc) | pNW1190 →168 |
| NRS4553 | 168 amyE::Phy-spank-bslAL121K-lacI (spc) | pNW1191 →168 |
| NRS4554 | 168 amyE::Phy-spank-bslAL123K-lacI (spc) | pNW1192 →168 |
| NRS4555 | 168 amyE::Phy-spank-bslAL124K-lacI (spc) | pNW1193 →168 |
| NRS4556 | 168 amyE::Phy-spank-bslAL153K-lacI (spc) | pNW1194 →168 |
| NRS4557 | 168 amyE::Phy-spank-bslAI155K-lacI (spc) | pNW1195 →168 |
| NRS4559 | 3610 bslA::cat amyE::Phy-spank-bslAL119K-lacI (spc) | SPP1 NRS4552 → NRS2097 |
| NRS4561 | 3610 bslA::cat amyE::Phy-spank-bslAL121k-lacI (spc) | SPP1 NRS4553 → NRS2097 |
| NRS4563 | 3610 bslA::cat amyE::Phy-spank-bslAL123K-lacI (spc) | SPP1 NRS4554 → NRS2097 |
| NRS4565 | 3610 bslA::cat amyE::Phy-spank-bslAL124K-lacI (spc) | SPP1 NRS4555 → NRS2097 |
| NRS4567 | 3610 bslA::cat amyE::Phy-spank–bslAL153K-lacI (spc) | SPP1 NRS4556 → NRS2097 |
| NRS4569 | 3610 bslA::cat amyE::Phy-spank-bslAI155K-lacI (spc) | SPP1 NRS4557 → NRS2097 |
| Plasmid | ||
| pUC19 | High-copy-number cloning vector | 7 |
| pDR111 | B. subtilis integration vector for IPTG-induced expression | 8 |
| pGEX-6P-1 | Vector for overexpression of GST-fused proteins | GE Healthcare |
| pNW518 | pDR111-bslA | 4 |
| pNW690 | pUC19-bslA | This work |
| pNW693 | pUC19-bslAL76I | This work |
| pNW1104 | pDR111-bslAL76I | This work |
| pNW1128 | pGEX-6P-1-TEV-bslA42–181 | This work |
| pNW1129 | pUC19-bslAL76D | This work |
| pNW1136 | pUC19-bslAL76K | This work |
| pNW1143 | pDR111-bslAL76D | This work |
| pNW1144 | pDR111-bslAL76K | This work |
| pNW1149 | pUC19-bslAL79K | This work |
| pNW1150 | pUC19-bslAL79D | This work |
| pNW1151 | pUC19-bslAL79I | This work |
| pNW1157 | pDR111-bslAL79D | This work |
| pNW1158 | pDR111-bslAL79I | This work |
| pNW1160 | pGEX-6P-1-bslA48–172, L98M | This work |
| pNW1162 | pGEX-6P-1-TEV-bslA42–181, L79K | This work |
| pNW1165 | pDR111-bslAL79K | This work |
| pNW1174 | pUC19-bslAL98M | This work |
| pNW1175 | pUC19-bslAL77I | This work |
| pNW1176 | pUC19-bslAL77K | This work |
| pNW1177 | pUC19-bslAL77D | This work |
| pNW1178 | pDR111-bslAL98M | This work |
| pNW1179 | pDR111-bslAL77I | This work |
| pNW1180 | pDR111-bslAL77K | This work |
| pNW1461 | pDR111-bslAL77D | This work |
| pNW1182 | pUC19-bslAL119K | This work |
| pNW1183 | pUC19-bslAL121K | This work |
| pNW1184 | pUC19-bslAL123K | This work |
| pNW1185 | pUC19-bslAL124K | This work |
| pNW1186 | pUC19-bslAL153K | This work |
| pNW1187 | pUC19-bslAI155K | This work |
| pNW1188 | pGEX-6-P-TEV-bslA42–181, L76K | This work |
| pNW1189 | pGEX-6-P-TEV-bslA42–181, L77K | This work |
| pNW1190 | pDR111-bslAL119K | This work |
| pNW1191 | pDR111-bslAL121K | This work |
| pNW1192 | pDR111-bslAL123K | This work |
| pNW1193 | pDR111-bslAL124K | This work |
| pNW1194 | pDR111-bslAL153K | This work |
| pNW1195 | pDR111-bslAI155K | This work |
| pNW1196 | pGEX-6P-1-bslA48–172 | This work |
| Primer | Sequence 5′ – 3′ | Use |
| NSW1337 | TACCGTCCAAACACGATTCTCAGCCTTGGCG | L76I mutagenesis |
| NSW1338 | CGCCAAGGCTGAGAATCGTGTTTGGACGGTA | L76I mutagenesis |
| NSW1517 | GCATCTCGAGTTATTAGTTGCAACCGCAAGGC | bslA42–181 cloning |
| NSW1537 | CAGGGGCCCCTGGGATCCGAAAATTTATATTTTCAAATGAGAACACAGTCTACA | bslA42–181 cloning |
| NSW1539 | TTACCGTCCAAACACGAAACTCAGCCTTGGCGTTA | L76K mutagenesis |
| NSW1540 | TAACGCCAAGGCTGAGTTTCGTGTTTGGACGGTAA | L76K mutagenesis |
| NSW1541 | TTACCGTCCAAACACGGATCTCAGCCTTGGCGTTA | L76D mutagenesis |
| NSW1542 | TAACGCCAAGGCTGAGATCCGTGTTTGGACGGTAA | L76D mutagenesis |
| NSW1571 | AAACACGCTTCTCAGCATTGGCGTTATGGAGTTTA | L79I mutagenesis |
| NSW1572 | TAAACTCCATAACGCCAATGCTGAGAAGCGTGTTT | L79I mutagenesis |
| NSW1573 | AAACACGCTTCTCAGCAAAGGCGTTATGGAGTTTA | L79K mutagenesis |
| NSW1574 | TAAACTCCATAACGCCTTTGCTGAGAAGCGTGTTT | L79K mutagenesis |
| NSW1575 | AAACACGCTTCTCAGCGATGGCGTTATGGAGTTTA | L79D mutagenesis |
| NSW1576 | TAAACTCCATAACGCCATCGCTGAGAAGCGTGTTT | L79D mutagenesis |
| NSW1585 | AAACACGAAAGACACAATGAACGGAAATGCCTTGC | L98M mutagenesis |
| NSW1586 | GCAAGGCATTTCCGTTCATTGTGTCTTTCGTGTTT | L98M mutagenesis |
| NSW1591 | CCGTCCAAACACGCTTATTAGCCTTGGCGTTATGG | L77I mutagenesis |
| NSW1592 | CCATAACGCCAAGGCTAATAAGCGTGTTTGGACGG | L77I mutagenesis |
| NSW1593 | CCGTCCAAACACGCTTAAAAGCCTTGGCGTTATGG | L77K mutagenesis |
| NSW1594 | CCATAACGCCAAGGCTTTTAAGCGTGTTTGGACGG | L77K mutagenesis |
| NSW1595 | CCGTCCAAACACGCTTGATAGCCTTGGCGTTATGG | L77D mutagenesis |
| NSW1596 | CCATAACGCCAAGGCTATCAAGCGTGTTTGGACGG | L77D mutagenesis |
| NSW1597 | AACAGTAAGAGTTCCTAAAGCACTTGATTTGTTAG | L119K mutagenesis |
| NSW1598 | CTAACAAATCAAGTGCTTTAGGAACTCTTACTGTT | L119K mutagenesis |
| NSW1599 | AAGAGTTCCTTTGGCAAAAGATTTGTTAGGAGCTG | L121K mutagenesis |
| NSW1704 | CAGCTCCTAACAAATCTTTTGCCAAAGGAACTCTT | L121K mutagenesis |
| NSW1705 | TCCTTTGGCACTTGATAAATTAGGAGCTGGCGAAT | L123K mutagenesis |
| NSW1706 | ATTCGCCAGCTCCTAATTTATCAAGTGCCAAAGGA | L123K mutagenesis |
| NSW1707 | TTTGGCACTTGATTTGAAAGGAGCTGGCGAATTCA | L124K mutagenesis |
| NSW1708 | TGAATTCGCCAGCTCCTTTCAAATCAAGTGCCAAA | L124K mutagenesis |
| NSW1709 | TGCGGAGAATAAATCAAAAAGCATCGGAAATAAAT | L153K mutagenesis |
| NSW1710 | ATTTATTTCCGATGCTTTTTGATTTATTCTCCGCA | L153K mutagenesis |
| NSW1711 | GAATAAATCATTAAGCAAAGGAAATAAATTTTACG | I155K mutagenesis |
| NSW1712 | CGTAAAATTTATTTCCTTTGCTTAATGATTTATTC | I155K mutagenesis |
| NSW1713 | CTGTTCCAGGGGCCCCTGGGATCCGCTTCATTGTTCGCAACAATCAC | bslA48–172 cloning |
| NSW1714 | CTCGAGTTAGTTGCAACCGCATCATTAAGTGCTGCGCTTAGCCACGTC | bslA48–172 cloning |
BSGC represents the Bacillus genetic stock center.
The direction of strain construction is indicated with DNA or phage (SPP1) (→) recipient strain.