Simultaneous gene inactivation and promoter reporting in cyanobacteria

详细信息    查看全文

• 作者：Kangming Chen ; Xinyi Xu ; Liping Gu…
• 关键词：Cyanobacteria ; GFP reporter ; Single ; crossover recombination ; Nitrogen fixation ; GTP ; binding elongation factor
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2015
• 出版时间：February 2015
• 年：2015
• 卷：99
• 期：4
• 页码：1779-1793
• 全文大小：4,155 KB
• 参考文献：1. Allen MB, Arnon DI (1955) Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by / Anabaena cylindrica Lemm. Plant Physiol 30(4):366-72. doi:10.2307/4258938 CrossRef
  2. Andersson CR, Tsinoremas NF, Shelton J, Lebedeva NV, Yarrow J, Min H, Golden SS (2000) Application of bioluminescence to the study of circadian rhythms in cyanobacteria. Method Enzymol 305:527-42. doi:10.1016/S0076-6879(00)05511-7 CrossRef
  3. Argueta C, Yuksek K, Summers M (2004) Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in / Nostoc punctiforme. J Microbiol Methods 59(2):181-88. doi:10.1016/j.mimet.2004.06.009 CrossRef
  4. Bauer CC, Haselkorn R (1995) Vectors for determining the differential expression of genes in heterocysts and vegetative cells of / Anabaena sp. strain PCC 7120. J Bacteriol 177(11):3332-336
  5. Berendt S, Lehner J, Zhang YV, Rasse TM, Forchhammer K, Maldener I (2012) Cell wall amidase AmiC1 is required for cellular communication and heterocyst development in the cyanobacterium / Anabaena PCC 7120 but not for filament integrity. J Bacteriol 194(19):5218-227. doi:10.1128/JB.00912-12 CrossRef
  6. Bijlsma JJE, Lie-A-Ling M, Nootenboom IC, Vandenbroucke-Grauls CMJE, Kusters JG (2000) Identification of loci essential for the growth of / Helicobacter pylori under acidic conditions. J Infect Dis 182(5):1566-569. doi:10.1086/315855 CrossRef
  7. Biswas I, Gruss A, Ehrlich SD, Maguin E (1993) High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175(11):3628-635
  8. Borthakur D, Haselkorn R (1989) Tn5 mutagenesis of / Anabaena sp. strain PCC 7120: isolation of a new mutant unable to grow without combined nitrogen. J Bacteriol 171(10):5759-761
  9. Bryant DA (1994) The molecular biology of cyanobacteria. Kluwer, Dordrecht CrossRef
  10. Buikema WJ, Haselkorn R (1991) Isolation and complementation of nitrogen fixation mutants of the cyanobacterium / Anabaena sp. Strain PCC 7120. J Bacteriol 173(6):1879-885
  11. Buikema WJ, Haselkorn R (2001) Expression of the / Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions. Proc Natl Acad Sci U S A 98(5):2729-734. doi:10.1073/pnas.051624898 CrossRef
  12. Cai YP, Wolk CP (1990) Use of a conditionally lethal gene in / Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol 172(6):3138-145
  13. Caldon CE, Yoong P, March PE (2001) Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function. Mol Microbiol 41(2):289-97. doi:10.1046/j.1365-2958.2001.02536.x CrossRef
  14. Carrasco CD, Ramaswamy KS, Ramasubramanian TS, Golden JW (1994) / Anabaena / xisF gene encodes a developmentally regulated site-specific recombinase. Genes Dev 8(1):74-3. doi:10.1101/gad.8.1.74
  15. Casey ES, Grossman A (1994) In vivo and in vitro characterization of the light-regulated / cpcB2A2 promoter of / Fremyella diplosiphon. J Bacteriol 176(20):6362-374
  16. Chapman JS, Meeks JC (1987) Conditions for mutagenesis of the nitrogen-fixing cyanobacterium / Anabaena variabilis. J Gen Microbiol 133(1):111-18. doi:10.1099/00221287-133-1-111
  17. Chen K, Gu L, Xiang X, Lynch M, Zhou R (2012) Identification and characterization of five intramembrane metalloproteases in / Anabaena variabilis. J Bacteriol 194(22):6105-115. doi:10.1128/jb.01366-12 CrossRef
  18. C
• 作者单位：Kangming Chen (1)
  Xinyi Xu (1)
  Liping Gu (1)
  Michael Hildreth (1)
  Ruanbao Zhou (1)
  
  1. Department of Biology and Microbiology, South Dakota State University, Brookings, SD, 57007, USA
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0?% of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72?h, decreased 49.6?% compared to wild-type. Thus, we discovered that All2508?may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms.