INCREASED FREE DNAA DECREASES THE FREQUENCY OF HETEROCYSTS IN ANABAENA SP. PCC 7120
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摘要: 研究在模式生物鱼腥蓝细菌Anabaena sp. PCC 7120中, 以DnaA为研究对象, 探究蓝细菌细胞周期中复制起始和异形胞分化之间的关系。结果显示: 在有氮环境中, DnaA蛋白缺失或过表达并不影响细胞增殖和异形胞的分化。在缺氮环境下, DnaA缺失突变株Malr2009的异形胞分化频率(8.57%)与野生型(8.64%)间无显著差别, 且该菌株增殖速率与野生型相比也无显著差异, DnaA蛋白缺失没有影响蓝细菌突变株(Malr2009)的异形胞分化频率和增殖速率。但DnaA蛋白过表达菌株Oalr2009的异形胞分化频率降低了20%, 其在第12天A750约为1.2, 细胞增殖速率快于野生型(第12天时A750约为0.9), 增殖速率提高了30%。综上结果表明在鱼腥蓝细菌PCC 7120中, 虽然DnaA不是细胞生长过程所必需的, 但在缺氮条件下, 游离DnaA增加会抑制异形胞分化频率。
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关键词:
- 鱼腥蓝细菌PCC 7120 /
- DnaA /
- DnaA box /
- oriC /
- 异形胞
Abstract: This study investigated the role of DnaA in the relationship between cell cycle and heterocyst differentiation using Anabaena PCC 7120 as the model organism. When providing combined nitrogen, deletion or overexpression of DnaA did not affect cyanobacterial proliferation and heterocyst differentiation. Upon deprivation of combined nitrogen, DnaA deletion (Malr2009) showed no difference in heterocyst frequency (8.57%) compared with the wild type(8.64%) at 24h, and the proliferation rate of Malr2009 was similar to the wild type, DnaA deletion did not affect cyanobacterial frequency of heterocysts or proliferation rate, but the overexpression of DnaA protein (Oalr2009) reduced the frequency of heterocysts by 20% compared with wild type, and increased the cell proliferation rate by 30% with about 1.2 at the 12th day, higher than that of the wild type (about 0.9 at the 12th day). In conclusion, although DnaA is not necessary for the growth of Anabaena sp. PCC 7120 cells, but under deprivation of combined nitrogen, the increased free DnaA inhibited the frequency of heterocysts.-
Key words:
- Anabaena sp. PCC 7120 /
- DnaA /
- DnaA box /
- oriC /
- Heterocyst
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图 1 pCT-alr2009(A)和pCint2M-alr2009(B)载体示意图
Figure 1. The vector map of pCT-alr2009 (A) and pCint2M-alr2009 (B)
图 4 五种菌株异形胞间营养细胞间隔数频数的统计
Figure 4. Frequency of the number of the vegetative cells between heterocysts in the five strains
图 5 五种菌株分别在供氮(A)和缺氮(B)条件下的生长状况
Figure 5. Growth curves of the five strains under nitrogen replete (A) and nitrogen starvation (B) conditions
表 1 实验中所用主要引物
Table 1. Primers used in this study
引物名称
Primer name序列Sequence (5′—3′) P1 GGTAACAACAAGATGATGATGGAAATGCCCATTGAC P2 CTCCAGATCCACCTCATGGTTTTCGAGAACGGCTAT P3 CATCTTGTTGTTACCTCCTTAGCA P4 GGTGGATCTGGAGGTAGTGGTTCTGGTGGTGGTAGCAC P5 GCAGAAATTCGATATCTAGATCCGTGAGGGTTGCCATCTG P6 AGCGCTACCGACGCTAGTGCTTTACCACCTTTGCTGCTAATC P7 ACCGGATCATCAGTACTCCCTGATCATAAAAATTCGACGTTTTC P8 CGCAACGTTGTTGCCATTGTCGGCGTTCAATGGTCAC P9 AGCACTAGCGTCGGTAGCGCT P10 GGGAGTACTGATGATCCGGT 1-up ACGGTTTACAAGCATAAAGCGATCCTAAAGCCTGTGAAATTAAC 1-dw GTGGTGGTGGTGCTCTTAGGTATGTTGGCGCATGAGAT 2-dw GTTAGCAGCCGGATCTCAG 3-up GCAGAAATTCGATATCTAGATCTGCACAGTGTTGATAGGAATA 3-dw GATGTTTAACTTTGTTTTAGGGCGACT 4-up CCGCGCGGCAGCCATTCATCACAGCTATTCGTAACG 4-dw GTGGTGGTGGTGCTCTAATTGAAGCGAGGCTGTGG 
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表 2 缺氮24h后异形胞频率
Table 2. Heterocysts frequency after 24h of nitrogen starvation
指标Index WT MoriC MdnaAN Malr2009 Oalr2009 异形胞Heterocyst 280 290 290 385 257 营养细胞Vegetative cells 2962 2176 2183 4106 3430 异形胞频率Heterocysts frequency (%) 8.64 11.76 11.73 8.57 6.97
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Adams D G, Carr N G. Heterocyst differentiation and cell division in the cyanobacterium Anabaena cylindrica: effect of high light intensity [J]. Journal of Cell Science, 1981, 49(1): 341-352. doi: 10.1242/jcs.49.1.341
Zhang C C, Laurent S, Sakr S, et al. Heterocyst differentiation and pattern formation in cyanobacteria: a chorus of signals [J]. Molecular Microbiology, 2006, 59(2): 367-375. doi: 10.1111/j.1365-2958.2005.04979.x
Golden J W, Yoon H S. Heterocyst development in Anabaena [J]. Current Opinion in Microbiology, 2003, 6(6): 557-563. doi: 10.1016/j.mib.2003.10.004
Kumar K, Mella-Herrera R A, Golden J W. Cyanobacterial heterocysts [J]. Cold Spring Harbor Perspectives in Biology, 2010, 2(4): a000315.
Muro-Pastor A M, Hess W R. Heterocyst differentiation: from single mutants to global approaches [J]. Trends in Microbiology, 2012, 20(11): 548-557. doi: 10.1016/j.tim.2012.07.005
Meeks J C, Elhai J. Regulation of cellular differentiation in filamentous cyanobacteria in free-living and plant-associated symbiotic growth states [J]. Microbiology and Molecular Biology Reviews, 2002, 66(1): 94-121. doi: 10.1128/MMBR.66.1.94-121.2002
Zhou Y, Chen W L, Wang L, et al. Identification of the oriC region and its influence on heterocyst development in the filamentous cyanobacterium Anabaena sp. strain PCC 7120 [J]. Microbiology, 2011, 157(7): 1910-1919. doi: 10.1099/mic.0.047241-0
Ozaki S, Fujimitsu K, Kurumizaka H, et al. The DnaA homolog of the hyperthermophilic eubacterium Thermotoga maritima forms an open complex with a minimal 149-bp origin region in an ATP-dependent manner [J]. Genes to Cells, 2006, 11(4): 425-438. doi: 10.1111/j.1365-2443.2006.00950.x
Marczynski G T, Shapiro L. Control of chromosome replication in Caulobacter crescentus [J]. Annual Review of Microbiology, 2002, 56(1): 625-656. doi: 10.1146/annurev.micro.56.012302.161103
Mackiewicz P, Zakrzewska-Czerwinska J, Zawilak A, et al. Where does bacterial replication start? Rules for predicting the oriC region [J]. Nucleic Acids Research, 2004, 32(13): 3781-3791. doi: 10.1093/nar/gkh699
Hu S. Research on coordination mechanism between cell cycle and heterocyst differentiation in Anabaena sp. strain PCC 7120 [D]. Wuhan: Huazhong Agricultural University, 2015: 24-26.
Ohbayashi R, Watanabe1 S, Ehira S, et al. Diversification of DnaA dependency for DNA replication in cyanobacterial evolution [J]. The ISME Journal, 2015, 10(5): 1113-1121.
Ohbayashi R, Nakamachi A, Hatakeyama T S, et al. Coordination of polyploid chromosome replication with cell size and growth in a Cyanobacterium [J]. Microbiology, 2019, 10(2): e00510-e00519.
Watanabe S, Ohbayashi R, Kanesaki Y, et al. Intensive DNA replication and metabolism during the lag phase in Cyanobacteria [J]. PLoS One, 2015, 10(9): e0136800. doi: 10.1371/journal.pone.0136800
Watanabe S, Ohbayashi R, Shiwa Y, et al. Light-dependent and asynchronous replication of cyanobacterial multi-copy chromosomes [J]. Molecular Microbiology, 2012, 83(4): 856-865. doi: 10.1111/j.1365-2958.2012.07971.x
Fukushima S, Itaya M, Kato H, et al. Reassessment of the in vivo functions of DNA polymerase I and RNase H in bacterial cell growth [J]. Journal of Bacteriology, 2007, 189(23): 8575-8583. doi: 10.1128/JB.00653-07
William D D, Garry W B. Coupling the initiation of chromosome replication to cell size in Escherichia coli [J]. Current Opinion of Microbiology, 2003, 6(2): 146-150. doi: 10.1016/S1369-5274(03)00026-2
Yoshikawa H, Haas M. On the regulation of the initiation of DNA replication in bacteria [J]. Cold Spring Harbor Symposia on Quantitative Biology, 1968(33): 843-855.
Shereda R D, Kozlov A G, Lohman T M, et al. SSB as an organizer/mobilizer of genome maintenance complexes [J]. Critical Reviews in Biochemistry and Molecular Biology, 2008, 43(5): 289-318. doi: 10.1080/10409230802341296
Fekete R A, Venkova-Canova T, Park K, et al. IHF-dependent activation of P1 plasmid origin by dnaA [J]. Molecular Microbiology, 2006, 62(6): 1739-1751. doi: 10.1111/j.1365-2958.2006.05479.x
Felczak M M, Simmons L A, Kaguni J M. An essential tryptophan of Escherichia coli DnaA protein functions in oligomerization at the E. coli replication origin [J]. Journal of Biological Chemistry, 2005, 280(26): 24627-24633. doi: 10.1074/jbc.M503684200
Kurokawa K, Nishida S, Emoto A, et al. Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli [J]. The EMBO Journal, 1999, 18(23): 6642-6652. doi: 10.1093/emboj/18.23.6642
Schaper S, Messer W. Interaction of the initiator protein DnaA of Escherichia coli with its DNA target [J]. The Journal of Biological Chemistry, 1995, 270(29): 17622-17626. doi: 10.1074/jbc.270.29.17622
Fuller R S, Funnell B E, Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites [J]. Cell, 1984, 38(3): 889-900. doi: 10.1016/0092-8674(84)90284-8
Chen A H, Afonso B, Silver P A, et al. Spatial and temporal organization of chromosome duplication and segregation in the cyanobacterium Synechococcus elongatus PCC 7942 [J]. PLoS One, 2012, 7(10): e0047837.
Richter S, Hagemann M, Messer W. Transcriptional analysis and mutation of a dnaA-like gene in Synechocystis sp. strain PCC 6803 [J]. Journal of Bacteriology, 1998, 180(18): 4946-4949. doi: 10.1128/JB.180.18.4946-4949.1998
Sakr S, Jeanjean R, Zhang C C, et al. Inhibition of cell division suppresses heterocyst development in Anabaena sp. strain PCC 7120 [J]. Journal of Bacteriology, 2006, 188(4): 1396-1404. doi: 10.1128/JB.188.4.1396-1404.2006
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