Mutation of the murC and murB impairs heterocyst differentiation in Anabaena sp.
strain PCC 7120
Patrick Videau1,4, Orion S. Rivers1, Blake Ushijima1, Reid T. Oshiro1,5, Min Joo Kim1,
Benjamin Philmus2 and Loralyn M. Cozy3,#
1
Department of Microbiology, University of Hawaii, Honolulu, HI 96822, USA
2
Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University,
Corvallis, OR 97331
3
Department of Biology, Illinois Wesleyan University, 1312 Park Street PO Box 2900,
Bloomington, IL 61702, USA
Current Address: 4Department of Pharmaceutical Sciences, College of Pharmacy, Oregon
State University, Corvallis, OR 97331, 5Department of Biology, Indiana University,
Bloomington, IN 47405
#
Correspondence: LM Cozy, Illinois Wesleyan University, Department of Biology, 1312
Park Street PO Box 2900, Bloomington, IL 61702, USA
Email: lcozy@iwu.edu
Supplementary Material
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 PpetE-murC on a plasmid (yellow triangles), the C6 mutant harboring PpetE-murB
on a plasmid (green triangles), and the C6 mutant harboring PpetE-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.
Figure S2. Heterocyst formation, but not pattern formation, is impaired in strains UHM350
and UHM351 in copper replete conditions. The wild type harboring pPJAV328 (PpetEmurC/B, PpatS-YFP; also representative of pPJAV329; A and B), UHM350 (conditional
ΔmurB mutant harboring PpatS-YFP; C and D), and UHM351 (conditional ΔmurC mutant
harboring PpatS-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 PpatS-YFP. Carets indicate heterocysts. Bar, 10 μm.
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.
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.
Figure S5. Conditional mutants UHM350 (ΔmurB mutant harboring PpatS-YFP) and
UHM351 (ΔmurC mutant harboring PpatS-YFP) produce a wild type pattern of patS
expression at 9 h after the removal of combined nitrogen. The wild type harboring
pPJAV328 (PpatS-YFP; this image is representative of PpatS-YFP on pPJAV329 and PpatSGFP on pAM1951 (3); A), UHM350 (PpatS-YFP; B), and UHM351 (PpatS-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.
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
ATATAGGATCCATGAAAATTTCCCAGGCAGTTGGAAACGCC
murB-BamHI-F
murB-OEX-F
murB-OEX-BglII-R
murB-SacI-R
CAGGTTAGGAGAACGCCATGAAAATTTCCCAGGCAGTTGGAAA
CGCC
ATATAAGATCTTCACGCCGCCTGAAATTCACCCAGCATTTTGAC
ATATAGAGCTCTCACGCCGCCTGAAATTCACCCAGCATTTTGAC
murC-BamHI-F
ATATGGATCCATGAATAATAGTGTAGATTTTGGCGGTAGACC
murC-OEX-F
CAGGTTAGGAGAACGCCATGAATAATAGTGTAGATTTTGGCGG
TAG
ATATAAGATCTTCACAATGTGGCTGTTGCAGGTGCGCAC
ATATAGAGCTCTCACAATGTGGCTGTTGCAGGTGCGCACAGTG
CTGCCTCAGCTATGAGCAAGTGTCC
TGATTACTCCTCTTATCACCACACCACAC
TATTGCTTTGACATGCAAAGAATTTCTTTCCCGATATGACG
CTTTGCATGTCAAAGCAATATTTTCCGATTCCTTACTGGTGTTGG
murC-OEX-BglII-R
murC-SacI-R
PmurC-F
PmurC-R
PmurC-NtcAmut-F
PmurC-NtcAmut-R
PpatS-OEX-R
PpatS-XKS-MunI-F
CGCCGCTGCTCATGTATATCTCCTTCTTAAATCTAGCGCTCATC
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.
1
2
3
4
5
6
7
8
9
10
11
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.
2
3
4
5
6
7
8
9
10
-23
-28
-48
-48
0.20 0.97 0.62 0.60 1.3 × 10
5.9 × 10
1.6 × 10
7.8 × 10
0.16
0.13 0.46 0.29 1.9 × 10-26 1.5 × 10-30 1.6 × 10-49 6.7 × 10-49
0.88
-38
-45
-75
-74
0.54 0.46 1.6 × 10
1.7 × 10
5.6 × 10
7.4 × 10
0.07
-23
-26
-45
-45
0.91 8.3 × 10
1.0 × 10
2.0 × 10
8.2 × 10
0.46
3.9 × 10-51 9.2 × 10-60 8.2 × 10-45 2.0 × 10-45
0.46
-59
-56
0.002
4.4 × 10
9.9 × 10
4.3 × 10-48
5.1 × 10-55 5.4 × 10-51 2.2 × 10-55
0.47
1.4 × 10-83
9.4 × 10-85
11
0.17
0.90
0.08
0.46
0.46
2.0 × 10-42
7.2 × 10-49
9.1 × 10-75
9.1 × 10-75
0.98
*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.
1997. 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 36:11556-11563.
2. Nishida, S., K. Kurokawa, M. Matsuo, K. Sakamoto, K. Ueno, K. Kita, and K.
Sekimizu. 2006. Identification and characterization of amino acid residues essential for
the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from
Staphylococcus aureus. J. Biol. Chem. 281:1714-1724.
3. Yoon, H.-S., and J. W. Golden. 1998. Heterocyst pattern formation controlled by a
diffusible peptide. Science 282:935-938.
12
0.77
0.17
0.63
0.71
0.66
1.2 × 10-75
1.9 × 10-89
1.2 × 10-99
1.2 × 10-99
0.06
0.09