Cross-Activation of Two Nitrogenase Gene Clusters by CnfR1 or CnfR2 in the Cyanobacterium Anabaena variabilis

ABSTRACT In Anabaena variabilis, the nif1 genes, which are activated by CnfR1, produce a Mo-nitrogenase that is expressed only in heterocysts. Similarly, the nif2 genes, which are activated by CnfR2, make a Mo-nitrogenase that is expressed only in anaerobic vegetative cells. However, CnfR1, when it was expressed in anaerobic vegetative cells under the control of the cnfR2 promoter or from the Co2+-inducible coaT promoter, activated the expression of both nifB1 and nifB2. Activation of nifB2, but not nifB1, by CnfR1 required NtcA. Thus, expression of the nif1 system requires no heterocyst-specific factor other than CnfR1. In contrast, CnfR2, when it was expressed in heterocysts under the control of the cnfR1 promoter or from the coaT promoter, did not activate the expression of nifB1 or nifB2. Thus, activation of the nif2 system in anaerobic vegetative cells by CnfR2 requires additional factors absent in heterocysts. CnfR2 made from the coaT promoter activated nifB2 expression in anaerobic vegetative cells grown with fixed nitrogen; however, oxygen inhibited CnfR2 activation of nifB2 expression. In contrast, activation of nifB1 and nifB2 by CnfR1 was unaffected by oxygen. CnfR1, which does not activate the nifB2 promoter in heterocysts, activated the expression of the entire nif2 gene cluster from a nifB2::nifB1::nifB2 hybrid promoter in heterocysts, producing functional Nif2 nitrogenase in heterocysts. However, activity was poor compared to the normal Nif1 nitrogenase. Expression of the nif2 cluster in anaerobic vegetative cells of Nostoc sp. PCC 7120, a strain lacking the nif2 nitrogenase, resulted in expression of the nif2 genes but weak nitrogenase activity. IMPORTANCE Cyanobacterial nitrogen fixation is important in the global nitrogen cycle, in oceanic productivity, and in many plant and fungal symbioses. While the proteins that mediate nitrogen fixation have been well characterized, the regulation of this complex and expensive process is poorly understood in cyanobacteria. Using a genetic approach, we have characterized unique and overlapping functions for two homologous transcriptional activators CnfR1 and CnfR2 that activate two distinct nitrogenases in a single organism. We found that CnfR1 is promiscuous in its ability to activate both nitrogenase systems, whereas CnfR2 depends on additional cellular factors; thus, it activates only one nitrogenase system.

. Expression of cnfR2 under the control of the cnfR1 promoter or cnfR1 under the control of the cnfR2 promoter. Expression from P cnfR1 :cnfR2 (BP870) and P cnfR2 :cnfR1 (BP871)was determined by RT-qPCR in aerobic cells grown 24 h -N +O 2 , leading to formation of heterocysts that activate the cnfR1 promoter, or anaerobic cells grown 6 h -N -O 2 , leading to vegetative cells that activate the cnfR2 promoter. Expression of cnfR1 and cnfR2 was normalized to rnpB and the values on the y-axis represents the relative log 2 fold differences in expression Statistical analysis: P < 0.001 (***). The horizontal bars below the P-values provide statistical comparisons of the means for the two values immediately below the ends of the bar and do not include values between these ends. Vector with a promoterless lacZ for promoter fusions and a Tc r cassette for selection of promoter fragments; allows for recombination into the frtBC region of the chromosome; Km r Tc r (4) pBP870 PcnfR1:cnfR2 fusion was inserted between the BglII and SacI sites of pBP639.
This study pBP890 2.1-kb PCR fragment, containing the oriT site and aadA (Sp r Sm r ) cassette, was generated from pBP716 using primers FosOriTSp-L2 + FosOriTSp-R2 and recombineered into pAAWZ1787 at the Cm r cassette.
This study pBP907 PcnfR2:cnfR1cnfR2HTH fusion was created using fusion PCR; PcnfR2 and cnfR1:cnfR2HTH were amplified from pBP873 using primer set patB2-L201 + P2patB1-R and from pBP872 using primer set P2patB1-L + patB2-R(Fus), respectively, fused into a single fragment, and then inserted between the BglII and SacI sites of pBP639.
This study pBP910 PcnfR1:cnfR2cnfR1HTH fusion was created using fusion PCR; PcnfR1 and cnfR2:cnfR1HTH were amplified from pBP872 using primer set patB1L102 + P1patB2-R and from pBP873 using primer set P1patB2-L + patB1-R(Fus), respectively, fused into a single fragment, and then inserted between the BglII and SacI sites of pBP639.
This study pBP920 PcnfR1:cnfR1 (wild-type) was amplified by PCR using primers patB1L102 and patB1-R(Fus) and then inserted between the BglII and SacI sites of pBP639.
This study pBP921 PcnfR2:cnfR2 (wild-type) was amplified by PCR using primers patB2L-201 and patB2-R(Fus) and then inserted between the BglII and SacI sites of pBP639.
This study pBP1142 PcoaT:cnfR1 fusion was created using fusion PCR; PcoaT and cnfR1 fragments were amplified from Synechocystis sp. PCC 6803 using primer set pcoaR-L(BglII) + pcoaT:cnfR1-R and from pBP920 using primer set pcoaT:cnfR1-L + patB1-R(Fus), respectively, fused into a single fragment, digested with BglII and SacI, and then inserted between the BglII and SacI sites of pBP744.