Table S1Plasmids and primers

Plasmids / Derivation and relevant characteristicsa / Reference or source
pHB1071 / AprSmr/Spr(with a promoterless npt), pUC19 ligated to pHB912, to provide multiple cloning sites upstream of the gfp reporter gene in the E. coli-Anabaena shuttle vector / Wang and Xu, 2005
pHB4786 / Apr,PCRfragment (Anabaenachromosomal bp 613750-614579)upstream of patAamplifiedusingprimersPpatA-L-f andPpatA-L-r,clonedinto pMD18-T / This study
pHB4795 / Apr,PCRfragment (Anabaenachromosomal bp 613750-614579bearing a 30-bp deletion frombp 614087 to 614116) upstream of patA generated by overlap PCR (Horton et al, 1989)using PpatA-L-f/PpatA-t-r and PpatA-t-f/PpatA-L-r as the primer pairs, Anabaena chromosomalDNA as the template, cloned into pMD18-T
pHB4797 / SmrSpr,the fragment upstream of patA (-1~-829)excisedfrompHB4786withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pHB4805 / SmrSpr,the fragment upstream of patA (-1~-829)minus the HetR binding site excisedfrompHB4795withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pHB4942 / Apr,PCRfragment (Anabaenachromosomal bp 613657-614046)upstream of patAamplifiedusingprimers PpatA-297 and PpatA+92,clonedinto pMD18-T
pHB4943 / Apr,PCRfragment (Anabaenachromosomal bp 614363-614579)upstream of patAamplifiedusingprimers PpatA-L-f and PpatA-614,clonedinto pMD18-T
pHB4948 / Apr,PCRfragment (Anabaenachromosomal bp218081-218616)upstream of alr0202amplifiedusingprimersPalr0202-f and Palr0202-r,clonedinto pMD18-T
pHB4949 / Apr,thePCRfragment (Anabaenachromosomal bp 612288-614579)carryingpatAamplifiedusingprimers PpatA-L-f and patA-End,clonedinto pMD18-T
pHB4950 / Apr,PCRfragment (Anabaenachromosomal bp3906284-3907350)upstream of alr3234amplifiedusingprimersUalr3234-f and Ualr3234-r,clonedinto pMD18-T
pHB4951 / Apr,PCRfragment (Anabaenachromosomal bp 612288-614579bearing a 30-bp deletion from bp 614087 to 614116) containing the full-length promoter and the encoding region of patA generated by overlap PCR using PpatA-L-f/PpatA-t-r and PpatA-t-f/patA-End as the primer pairs, AnabaenachromosomalDNA as the template, cloned into pMD18-T
pHB4954 / Apr,PCRfragment (Anabaenachromosomal bp3906284-3907350bearing a 29-bp deletion frombp 3906496 to 3906524) upstream of alr3234 generated by overlap PCR using Ualr3234-f/Ualr3234-t-r and Ualr3234-t-f/Ualr3234-r as the primer pairs, Anabaena chromosomalDNA as the template, cloned into pMD18-T
pHB4955 / Apr,PCRfragment (Anabaenachromosomal bp218081-218616bearing a 24-bp deletion frombp 218356 to 218379
) upstream of alr0202 generated by overlap PCR using Palr0202-f/Palr0202-t-r and Palr0202-t-f/ Palr0202-r as the primer pairs, Anabaena chromosomalDNA as the template, cloned into pMD18-T
pHB5049 / SmrSpr,the fragment containing the full-length promoter and the encoding region of patA excisedfrompHB4949withSmaIandPstI,ligated withSmaI andSse8387I-digestedpHB1071
pHB5050 / SmrSpr,the fragment containing the full-length promoter minus the HetRbinding site and the encoding region of patA excisedfrompHB4951withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pHB5054 / SmrSpr,the fragment overlapping the 5′-end of patA (+92~-297) excisedfrompHB4942withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pHB5055 / SmrSpr,the fragment in the upstream region of PpatA (-615~-830) excisedfrompHB4943withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pHB5062 / SmrSpr,thefragment overlapping the 5′-end of alr3234 (+133~-933)excisedfrompHB4950withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pHB5063 / SmrSpr,thefragment overlapping the 5′-end of alr3234 (+133~-933) minus the conserved HetRbinding site excisedfrompHB4954withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pHB5064 / SmrSpr,the fragment upstream of alr0202 (-81~-616)excisedfrompHB4948withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pHB5065 / SmrSpr,the fragment upstream of alr0202 (-81~-616) minus the conserved HetRbinding site excisedfrompHB4955withSmaIandPstI,ligated withSmaI/Sse8387I-digestedpHB1071
pMD18-T / Apr, T-vector / Takara
Primers / Sequences (5′→3′)
M1-f / Biotin-gcacgttggctcaaacccatcagtagcaaaagtattatct
M1-r / agataatacttttgctactgatgggtttgagccaacgtgc
M2-f / Biotin-agttagatattcaaacccatcagtagcaaaagtattatct
M2-r / agataatacttttgctactgatgggtttgaatatctaact
M3-f / Biotin-agttattggcatgctcccatcagtagcaaaagtattatct
M3-r / agataatacttttgctactgatgggagcatgccaataact
M4-f / Biotin-agttattggctcaaatatgacagtagcaaaagtattatct
M4-r / agataatacttttgctactgtcatatttgagccaataact
M5-f / Biotin-agttattggctcaaacccatagactgcaaaagtattatct
M5-r / agataatacttttgcagtctatgggtttgagccaataact
M6-f / Biotin-agttattggctcaaacccatcagtatagcgagtattatct
M6-r / agataatactcgctatactgatgggtttgagccaataact
M7-f / Biotin-agttattggctcaaacccatcagtagcaaacacgctatct
M7-r / agatagcgtgtttgctactgatgggtttgagccaataact
M8-f / Biotin-agttattggctcaaacccatcagtagcaaaagtatgcatg
M8-r / catgcatacttttgctactgatgggtttgagccaataact
Palr0202-40-f / Biotin-tcagttgcggaatagcccagcaaaatgcctccagtttcgt
Palr0202-30-f / Biotin-tcagttgcggaatagcccagcaaaatgcct
Palr0202-40-r / acgaaactggaggcattttgctgggctattccgcaactga
Palr0202-30-r / aggcattttgctgggctattccgcaactga
Palr3234-40-f / Biotin-tcgccctggggatatcgcccacaataagcgtatcctcctc
Palr3234-30-f / Biotin-cgccctggggatatcgcccacaataagcgt
Palr3234-40-r / gaggaggatacgcttattgtgggcgatatccccagggcga
Palr3234-30-r / acgcttattgtgggcgatatccccagggcg
Palr0202-f / gattatttccacttcagcacaca
Palr0202-r / gaccattcatccatcaagacct
Palr0202-t-f / tcaccgcctttcttgccagtttcgtgagcgtcataactt
Palr0202-t-r / cgctcacgaaactggcaagaaaggcggtgaagttctttg
pDU1-1 / acttccgccgtgctgctgac
pDU1-2 / gttatgggtgacgcggttgtg
patA-End / ccacggttcagaaacgattg
PhepA-40-f / Biotin-ttatggtgggcaatgcccaccataatacttatatttcaga
PhepA-30-f / Biotin-ttatggtgggcaatgcccaccataatactt
PhepA-40-r / tctgaaatataagtattatggtgggcattgcccaccataa
PhepA-30-r / aagtattatggtgggcattgcccaccataa
PhetP-40-f / Biotin-taggcgaggggtctaacccctcattacctagtagaaaatg
PhetP-30-f / Biotin-aggcgaggggtctaacccctcattacctag
PhetP-40-r / cattttctactaggtaatgaggggttagacccctcgccta
PhetP-30-r / ctaggtaatgaggggttagacccctcgcct
PhetR-40-f / Biotin-taaatccgggtgcagcccaaaagtcttaaaaatagcaaat
PhetR-30-f / Biotin-taaatccgggtgcagcccaaaagtcttaaa
PhetR-40-r / atttgctatttttaagacttttgggctgcacccggattta
PhetR-30-r / tttaagacttttgggctgcacccggattta
PhetZ-40-f / Biotin-aatgtgagggtctagcccagcaggtgggattagagaaaca
PhetZ-30-f / Biotin-aatgtgagggtctagcccagcaggtgggat
PhetZ-40-r / tgtttctctaatcccacctgctgggctagaccctcacatt
PhetZ-30-r / atcccacctgctgggctagaccctcacatt
PpatA-40-f / Biotin-agttattggctcaaacccatcagtagcaaaagtattatct
PpatA-30-f / Biotin-agttattggctcaaacccatcagtagcaaa
PpatA-40-r / agataatacttttgctactgatgggtttgagccaataact
PpatA-30-r / tttgctactgatgggtttgagccaataact
PpatA+92 / accagtgattgtcttcccagtta
PpatA-297 / aatgaaagatacagaaaagcaacc
PpatA-614 / actaaaagctaggttactccaa
PpatA-L-f / tcaggattggggacaagtg
PpatA-L-r / ggcgatcgctctgttattaa
PpatA-t-f / cacagataatactatacgtgtagtacttaaggtactgt
PpatA-t-r / aagtactacacgtatagtattatctgtgtttttaccgc
rnpB-1 / gccgaaggaactatggttgg
rnpB-2 / aagccgggttctgttctctg
Ualr3234-f / atttaggctttaatggtaacttga
Ualr3234-r / tcctgatacataacttcccctg
Ualr3234-t-f / aggaaacgatagtcatatcctcctcatcgactacgaag
Ualr3234-t-r / tcgatgaggaggatatgactatcgtttccttgagtttta

aAp, ampicillin; Sm, streptomycin; Sp, spectinomycin; unless stated otherwise, the template for PCR reactions was Anabaenasp. genomic DNA.

References

Horton RM, Hunt HD, Ho SN, et al.Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene,1989,77: 61-68

Wang Y, Xu X. Regulation by hetC of genes required for heterocyst differentiation and cell division in Anabaena sp. strain PCC 7120. JBacteriol,2005,187:8489-8493