Identification of the oriC region and its influence on heterocyst development in the filamentous cyanobacterium Anabaena sp. strain PCC 7120.

Anabaena sp. strain PCC 7120 (Anabaena PCC 7120) is a filamentous, nitrogen-fixing cyanobacterium. Upon deprivation of combined nitrogen, about 5-10 % of the cells become heterocysts, i.e. cells devoted to N(2) fixation. Heterocysts are intercalated among vegetative cells and distributed in a semi-regular pattern, and adjacent heterocysts are rarely observed. Previously, we showed that the cell cycle could play a regulatory function during heterocyst development, although the mechanism involved remains unknown. As a further step to understand this phenomenon, we identified the oriC region for chromosomal DNA replication, located between dnaA and dnaN. The oriC region of Anabaena PCC 7120 was able to support the self-replication of a plasmid in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Surprisingly, integration of the oriC region into the chromosome of Anabaena PCC 7120 through homologous recombination led to much slower cell growth in the absence of a combined-nitrogen source and to multiple contiguous proheterocysts after prolonged incubation. Real-time RT-PCR showed that expression of two heterocyst-related genes, hetR and hetN, was altered in these strains: hetR expression remained high 48 h after induction, and hetN increased to high levels after induction for 12 h. These results suggest that the balance between oriC and DnaA could be important for heterocyst development.

[1]  C. Wolk,et al.  Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena , 1993, Molecular microbiology.

[2]  R. Haselkorn,et al.  Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120. , 1991, Genes & development.

[3]  W. Messer,et al.  Interaction of the Initiator Protein DnaA of Escherichia coli with Its DNA Target (*) , 1995, The Journal of Biological Chemistry.

[4]  J. Meeks,et al.  Regulation of Cellular Differentiation in Filamentous Cyanobacteria in Free-Living and Plant-Associated Symbiotic Growth States , 2002, Microbiology and Molecular Biology Reviews.

[5]  C. Zhang.  A gene encoding a protein related to eukaryotic protein kinases from the filamentous heterocystous cyanobacterium Anabaena PCC 7120. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Chater,et al.  Cluster of DnaA Boxes Involved in Regulation of Streptomyces Chromosome Replication: from In Silico to In Vivo Studies , 2006, Journal of bacteriology.

[7]  C. Wolk,et al.  [83] Conjugal transfer of DNA to cyanobacteria , 1988 .

[8]  M. Thyssen,et al.  Relationship among Several Key Cell Cycle Events in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120 , 2006, Journal of bacteriology.

[9]  Feng Gao,et al.  Origins of replication in Cyanothece 51142 , 2008, Proceedings of the National Academy of Sciences.

[10]  W. Buikema,et al.  The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120 , 2001, Molecular microbiology.

[11]  C. Wolk,et al.  Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences , 1990, Journal of bacteriology.

[12]  C. Helmstetter,et al.  Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli , 1993, Molecular microbiology.

[13]  Robert Haselkorn,et al.  Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena , 1985, Nature.

[14]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[15]  Cheng-Cai Zhang,et al.  Developmental Regulation of the Cell Division Protein FtsZ in Anabaena sp. Strain PCC 7120, a Cyanobacterium Capable of Terminal Differentiation , 2000, Journal of bacteriology.

[16]  A. Danchin,et al.  Universal replication biases in bacteria , 1999, Molecular microbiology.

[17]  Jennifer R. Moran,et al.  Transcriptional Regulation of the Heterocyst Patterning Gene patA from Anabaena sp. Strain PCC 7120 , 2010, Journal of bacteriology.

[18]  T. Arcondéguy,et al.  Inhibition of Cell Division Suppresses Heterocyst Development in Anabaena sp. Strain PCC 7120 , 2006, Journal of bacteriology.

[19]  S. Shestakov,et al.  Transformation in the cyanobacterium Synechocystis sp. 6803 , 1982 .

[20]  A. Ernst,et al.  Heterocyst Metabolism and Development , 1994 .

[21]  R. Haselkorn,et al.  Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. Vioque.  The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria. , 1997, Nucleic acids research.

[23]  K. Sekimizu,et al.  Loss of flagellation in dnaA mutants of Escherichia coli , 1994, Journal of bacteriology.

[24]  P. Marraccini,et al.  A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis , 1993, Plant Molecular Biology.

[25]  Jean-Michel Claverie,et al.  Phylogeny.fr: robust phylogenetic analysis for the non-specialist , 2008, Nucleic Acids Res..

[26]  W. Messer.  The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication. , 2002, FEMS microbiology reviews.

[27]  T. A. Brown,et al.  A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. , 1990, Nucleic acids research.

[28]  T. Black,et al.  Analysis of a Het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing , 1994, Journal of bacteriology.

[29]  C. Wolk,et al.  Conjugal transfer of DNA to cyanobacteria. , 1988, Methods in enzymology.

[30]  R. Haselkorn,et al.  Suppression of heterocyst differentiation in Anabaena PCC 7120 by a cosmid carrying wild-type genes encoding enzymes for fatty acid synthesis. , 1997, FEMS microbiology letters.

[31]  K. Skarstad,et al.  Limiting DNA replication to once and only once , 2000, EMBO reports.

[32]  J. Errington,et al.  A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis. , 2009, Genes & development.

[33]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[34]  W. Messer,et al.  DnaA initiator—also a transcription factor , 1997, Molecular microbiology.

[35]  S. Cebrat,et al.  Where does bacterial replication start? Rules for predicting the oriC region. , 2004, Nucleic acids research.

[36]  H. Krisch,et al.  In vitro insertional mutagenesis with a selectable DNA fragment. , 1984, Gene.

[37]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.