Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, PR China
Correspondence
Xudong Xu
xux{at}ihb.ac.cn
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ABSTRACT |
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INTRODUCTION |
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In the timetable of heterocyst development, genes required for initiation and morphogenesis are basically divided into two groups: (1) transcription of hetR, a gene essential to initiation of heterocyst development, and of hetC, a gene required for very early heterocyst development, is upregulated within 3·5 h after deprivation of fixed nitrogen (Black & Wolk, 1993; Xu & Wolk, 2001
); (2) from 5 to 10 h, hepA, hetM and devA, the genes involved in formation of the heterocyst envelope, are induced (Holland & Wolk, 1990
; Cai & Wolk, 1997
; Maldener et al., 1994
). hetN is induced between 6 and 12 h after transfer into nitrogen-free medium (Bauer et al., 1997
). hetR, hetC, hepA, devA and hetN are expressed specifically or primarily in heterocysts or proheterocysts.
patB is a gene required for nitrogen fixation but not morphogenesis of heterocysts. Its deduced product resembles a transcriptional regulator with a Fe4S4 ferredoxin region near its N-terminus and a helixturnhelix motif near its C-terminus. A patB deletion mutant is unable to fix nitrogen, while a patB mutant defective in its N-terminal ferredoxin domain or without the C-terminal helixturnhelix domain grows very slowly and produces multiple contiguous heterocysts in stationary-phase culture in nitrogen-free medium. Genes regulated by patB and those regulating patB are unknown. patB is specifically expressed in heterocysts starting from 12 to 18 h after nitrogen step-down (Liang et al., 1992; Jones et al., 2003
).
Two-component signal transduction systems are important machineries for bacteria to regulate cell differentiation and other physiological processes in response to environmental or intracellular changes (Albright et al., 1989). The simplest two-component regulatory systems consist of a sensor histidine kinase, often located in the cytoplasmic membrane, and a cytoplasmic response regulator. Upon sensing a certain signal, an input domain of the sensor histidine kinase modulates the activity of its transmitter domain, which then auto-phosphorylates an internal histidine residue and transfers the phosphoryl group to a response regulator. Consequently, transcription of particular genes, or various other functions, are regulated. However, many prokaryotic signalling systems have multiple components, interconnections with other regulatory circuits or feedback loops (Stock et al., 2000
).
In the genome of Anabaena sp. PCC7120, there are 203 two-component signal transduction genes, 73 encoding sensory kinases, 77 encoding response regulators and 53 encoding hybrid sensory kinases and response regulators (Kaneko et al., 2001). patA, a gene involved in patterning of heterocysts, is predicted to be a response regulator without a DNA-binding domain (Liang et al., 1992
). hepK, a gene involved in the formation of the polysaccharide layer, encodes a sensor histidine kinase, which controls the induction of hepA by interacting with DevRA (Zhu et al., 1998
; Zhou & Wolk, 2003
). In the work described here, by transposon mutagenesis of Anabaena sp. PCC 7120, we obtained a heterocyst development mutant, 1017, whose heterocyst envelope could not be stained by alcian blue, indicating that the polysaccharide layer was lacking or that heterocyst development was blocked at an early stage. Inverse PCR and DNA sequencing localized the transposon to the N-terminal portion of a two-component histidine kinase gene.
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METHODS |
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Transposon mutagenesis and selection of mutants.
Anabaena sp. PCC 7120 was mutagenized with Tn5-1087b as described by Ernst et al. (1992). The resulting transconjugants were inoculated on erythromycin-containing BG11 plates for further growth, then tested on BG110 plates for their capacity for diazotrophic growth under aerobic conditions. The mutants that turned yellow were tested in nitrogen-free liquid medium. To observe the polysaccharide layer of heterocysts, filaments grown in BG110 were treated with 0·05 % (w/v) alcian blue for 5 min (Hebbar & Curtis, 2000
).
Construction of a gene library for Anabaena sp. PCC 7120.
Total DNA of Anabaena sp. PCC 7120 was partially digested with Sau3AI at 4 °C until most DNA fragments were between 0·5 and 9 kb, as revealed by gel electrophoresis. DNA fragments of 29 kb were then retrieved from the agarose gel and ligated with BamHI-cut and dephosphorylated pRL25C (Wolk et al., 1988). The ligated products were electroporated into E. coli DH10B, resulting in more than 25 000 kanamycin-resistant colonies, of which about 60 % contained inserts averaging 3·6 kb in size.
Identification of transposon-interrupted genes and DNA fragments.
One microgram of genomic DNA was completely digested with AluI and self-ligated in 200 µl reaction mixture at 16 °C for 20 h. PCR amplifications were conducted using 2 µl of the ligation product as the template and 1087b-2 and 1087b-3 (Table 1) as the primers. The PCR products were sequenced with primer 1087b-1 (Table 1
). Plasmid DNA extracted from complemented mutants was electroporated into E. coli DH10B and sequenced with primers pRL25V-1 and pRL25Cseq from both ends. Locations of the sequences were determined by searching the Kazusa genome database (www.kazusa.or.jp/cyano/cyano.html). The alr0117 region was sequenced from PCR products generated using primers alr0117-4 and alr0117-5.
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Extraction of RNA.
Total RNA was extracted according to a method described for Frankia (Xu et al., 2002) with modifications. About 200 ml of Anabaena sp. PCC 7120 grown in BG11, or 24 h after transfer into BG110, was collected by centrifugation, washed once with 10 ml TE (10 mM Tris/HCl and 1 mM EDTA, pH 8·0), and resuspended in 2 ml LETS (100 mM LiCl, 10 mM EDTA, 10 mM Tris/HCl, 1 % SDS, pH 8·0), which was then mixed with 2 ml glass beads (kept in double-distilled H2O) and 2 ml phenol/chloroform (1 : 1, v/v). The cells were broken by four rounds of vortexing (1 min) intermitted by cooling on ice (1 min) and the mixture was centrifuged at 6000 r.p.m. for 15 min. The supernatant was transferred into Eppendorf tubes. After addition of 0·2 M LiCl and 2·5 vols ethanol to each tube, the nucleic acids were precipitated at -70 °C, spun down and dissolved in 300 µl diethyl pyrocarbonate (DEPC)-treated double-distilled H2O. The nucleic acid solution was treated with 6 units RNase-free DNase I (Takara) in the presence of 100 units RNasin (Takara) at 37 °C for 40 min and repeated for six to eight rounds until no PCR product could be detected with the primers for RT-PCR. After each round, total RNA was precipitated and dissolved in RNase-free double-distilled H2O.
PCR and RT-PCR.
PCRs were conducted in 50 µl volumes containing 10 mM Tris/HCl (pH 8·3), 50 mM KCl, 2·5 mM MgCl2, 50 µM dNTPs, 100 pmol of each of the primers, 2 units Taq DNA polymerase (MBI) and appropriate template DNA. The reactions were initiated by 94 °C for 5 min, followed by 30 cycles of: 94 °C 1 min, 60 °C 1 min and 72 °C 1 min, and a final extension at 72 °C for 5 min. For RT-PCR, first-strand cDNA was synthesized in a 25 µl reaction system containing 50 mM Tris/HCl (pH 8·3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 50 µM dNTPs, 25 units RNasin, 200 units M-MLV reverse transcriptase (Promega), 2 µg total RNA and 0·5 µg random primers at 37 °C for 60 min. The relative concentration of cDNA was evaluated after serial dilutions by PCR using primers glnA-1 and glnA-2 and adjusted to the same level according to the brightness of PCR bands. Two microlitres of the adjusted cDNA was used for PCR to detect the induced expression of genes. The primers are listed in Table 1.
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RESULTS |
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DISCUSSION |
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alr0117 encodes a two-component histidine kinase with a transmembrane segment at its 5'-terminus. Using RT-PCR, we tested the whole-filament transcription of this gene before and after nitrogen deprivation and found no upregulation of the gene. However, the result of such a whole-filament test does not exclude the possibility that the gene was upregulated in proheterocysts and downregulated in other vegetative cells. It should also be noted that the RT-PCR results provide qualitative rather than quantitative evaluation of gene expression. Using the same method, we found that the induction of hepA and patB was blocked in an alr0117 null mutant. The regulatory effect on hepA, the first gene known to be essential for the formation of the polysaccharide layer, explains the lack of that layer from the heterocyst envelope in mutant 1017. hetM and hglE, the two genes involved in formation of the glycolipid layer, and hetN, which is required for maintenance of heterocyst pattern, remained inducible in alr0117 : : Tn5-1087b. Because the upregulation of hetM and hetN is known to occur between the onset of induced expression of hepA and patB, alr0117 may control multiple genes that are apparently unrelated to each other during heterocyst development.
It is clear that both alr0117 and hepK control the expression of hepA and the formation of the polysaccharide layer. However, it was not reported whether hepK also affects the expression of patB or other genes. One possibility is that hepK only controls the expression of hepA; therefore it acts downstream of alr0117. The second possibility is that hepK controls other gene(s) in addition to hepA; therefore hepK and alr0117 may act in the same or different regulatory systems with overlapping effects.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 August 2003;
revised 5 November 2003;
accepted 7 November 2003.
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