Mutation of the murc and murb impairs heterocyst differentiation in Anabaena sp. strain PCC 7120

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1 Mutation of the murc and murb impairs heterocyst differentiation in Anabaena sp. strain PCC 7120 Patrick Videau 1,4, Orion S. Rivers 1, Blake Ushijima 1, Reid T. Oshiro 1,5, Min Joo Kim 1, Benjamin Philmus 2 and Loralyn M. Cozy 3,# 1 Department of Microbiology, University of Hawaii, Honolulu, HI 96822, USA 2 Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR Department of Biology, Illinois Wesleyan University, 1312 Park Street PO Box 2900, Bloomington, IL 61702, USA Current Address: 4 Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, 5 Department of Biology, Indiana University, Bloomington, IN # Correspondence: LM Cozy, Illinois Wesleyan University, Department of Biology, 1312 Park Street PO Box 2900, Bloomington, IL 61702, USA lcozy@iwu.edu Supplementary Material

2 Figure S1. The C6 transposon mutant with an insertion in the murc (alr5065) gene produces a decreased percentage of heterocysts. Heterocysts were enumerated from cultures of the wild type (red squares), C6 transposon mutant (blue circles), the C6 mutant harboring P pete -murc on a plasmid (yellow triangles), the C6 mutant harboring P pete -murb on a plasmid (green triangles), and the C6 mutant harboring P pete -murc/b on a plasmid (black diamonds) at 0 to 72 h after the removal of combined nitrogen in triplicate and averaged. Error bars represent the standard deviation.

3 Figure S2. Heterocyst formation, but not pattern formation, is impaired in strains UHM350 and UHM351 in copper replete conditions. The wild type harboring ppjav328 (P pete - murc/b, P pats -YFP; also representative of ppjav329; A and B), UHM350 (conditional ΔmurB mutant harboring P pats -YFP; C and D), and UHM351 (conditional ΔmurC mutant harboring P pats -YFP; E and F) imaged 9 h (A, C, and E) or 24 h (B, D, and F) after the removal of combined nitrogen from media containing 2 μm copper. Top: brightfield; bottom: yellow fluorescence from P pats -YFP. Carets indicate heterocysts. Bar, 10 μm.

4 Figure S3. Amino acid alignment of the Anabaena MurC with MurC from seven other bacterial genera as previously described (1). Identical amino acids are noted by an asterisk while those described as invariant in the previous study are denoted by a plus.

5 Figure S4. Amino acid alignment of the Anabaena MurB with MurB from 15 other bacterial genera as previously described (2). Identical amino acids are noted by an asterisk while those described as highly conserved in the previous study are denoted by a plus.

6 Figure S5. Conditional mutants UHM350 (ΔmurB mutant harboring P pats -YFP) and UHM351 (ΔmurC mutant harboring P pats -YFP) produce a wild type pattern of pats expression at 9 h after the removal of combined nitrogen. The wild type harboring ppjav328 (P pats -YFP; this image is representative of P pats -YFP on ppjav329 and P pats - GFP on pam1951 (3); A), UHM350 (P pats -YFP; B), and UHM351 (P pats -YFP; C) were cultured in media lacking copper and imaged 9 h after the removal of combined nitrogen. Top: brightfield; Bottom: green (A) or yellow (B and C) fluorescence from the pats transcriptional fusion. Bar, 10 μm.

7 Table S1. Oligonucleotide primers used in this study. Oligonucleotide* Sequence alr5066-up-fm475l GAGCTCGATACTGAAGCTGACTACACTGTTACTA alr5066-up-rm475l CAGCATTTTCCCGGGGGCGTTTCCAACTGCCTGGGAAATTTTC alr5067-int-bamhi-f ATATAGGATCCACAGGAAAAGGACAAGGATTTGGCTTCGGC alr5067-int-saci-r ATATAGAGCTCGTTACCAAGTCGGAAAGTAAATCTGC alr5067-up-out TTGTTATTCAACGCACACG alr5067-dn-out CATGCGTTCTCGCATTGTAG murb-insert-f ATATAGGATCCGCATTTACTTCTTATAGAGTCGGG murb-insert-r TATATGAGCTCCATCAGGAGAAAGTACTTGAGCGCTAAC murc-insert-f ATATAGGATCCGGTCGGCAAGTCAACCTTGCCTCAAGTAG murc-insert-r TATATGAGCTCGCCAGAAGCATTGCGTAGCAACTCACGCAC murb-bamhi-f ATATAGGATCCATGAAAATTTCCCAGGCAGTTGGAAACGCC murb-oex-f murb-oex-bglii-r murb-saci-r murc-bamhi-f murc-oex-f murc-oex-bglii-r murc-saci-r PmurC-F PmurC-R PmurC-NtcAmut-F PmurC-NtcAmut-R CAGGTTAGGAGAACGCCATGAAAATTTCCCAGGCAGTTGGAAA CGCC ATATAAGATCTTCACGCCGCCTGAAATTCACCCAGCATTTTGAC ATATAGAGCTCTCACGCCGCCTGAAATTCACCCAGCATTTTGAC ATATGGATCCATGAATAATAGTGTAGATTTTGGCGGTAGACC CAGGTTAGGAGAACGCCATGAATAATAGTGTAGATTTTGGCGG TAG ATATAAGATCTTCACAATGTGGCTGTTGCAGGTGCGCAC ATATAGAGCTCTCACAATGTGGCTGTTGCAGGTGCGCACAGTG CTGCCTCAGCTATGAGCAAGTGTCC TGATTACTCCTCTTATCACCACACCACAC TATTGCTTTGACATGCAAAGAATTTCTTTCCCGATATGACG CTTTGCATGTCAAAGCAATATTTTCCGATTCCTTACTGGTGTTGG PpatS-OEX-R CGCCGCTGCTCATGTATATCTCCTTCTTAAATCTAGCGCTCATC PpatS-XKS-MunI-F CAATTGCTCGAGATGGTACCATGTCGACTGAATTTGTTTTGGGA ACACTTAAG PpetE-OEX-R GGCGTTCTCCTAACCTGTAGTTTTATTTTTC PpetE-XhoI-F TATATCTCGAGGCTGAGGTACTGAGTACACAGC prl-out-saci-r AACGTTGTTGCCATTGC Turbo-OEX-F GAAGGAGATATACATGAGCAGCGGCGCCCTGCTGTTCCACGGC YFP-MunI-R ATATACAATTGTCAGCTGGTGTCTCCGGAAC * Oligonucleotides are shown in the 5 to 3 direction.

8 Table S2. Statistical comparison of filament lengths between various strains of Anabaena* grown in BG-11 using two-tailed t-tests and presented as p-values *Strains: Wild type with 2 μm copper (1), wild type + ppjav329 with 2 μm copper (2), wild type + ppjav328 with 2 μm copper (3), wild type + ppjav329 without copper (4), wild type + ppjav328 without copper (5), UHM350 with 2 μm copper (6), UHM351 with 2 μm copper (7), UHM350 without copper (8), UHM351 without copper (9), UHM350 cured of ppjav308 with 2 μm copper (10), UHM351 cured of ppjav309 with 2 μm copper (11), and BJP002 with 2 μm copper (Δalr5067; 12). References 1. Bouhss, A., D. Mengin-Lecreulx, D. Blanot, J. van Heijenoort, and C. Parquet Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:Lalanine ligase from Escherichia coli. Biochemistry 36: Nishida, S., K. Kurokawa, M. Matsuo, K. Sakamoto, K. Ueno, K. Kita, and K. Sekimizu Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus. J. Biol. Chem. 281: Yoon, H.-S., and J. W. Golden Heterocyst pattern formation controlled by a diffusible peptide. Science 282: