Phenotypic variation caused by variation in the relative copy number of pDU1-based plasmids expressing the GAF domain of Pkn41 or Pkn42 in Anabaena sp. PCC 7120

Yang, Yang; Huang, Xiaozhen; Wang, Li; Risoul, Véronique; Zhang, Cheng‐Cai; Chen, Wenli

doi:10.1016/j.resmic.2012.10.010

Cited by 20 publications

(20 citation statements)

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“…The use of RSF1010-based plasmids allows the same constructs to be characterized in several different strains and mimics a common way in which the riboswitch devices are likely to be used. Both genome and plasmid copy numbers can vary substantially between cyanobacterial strains and under different growth and genetic conditions (32)(33)(34). The genome copy number can range from monoploid to polyploid for different strains and can vary between different growth phases (33).…”

Section: Resultsmentioning

confidence: 99%

“…strain PCC 6803 (32). However, although the copy number of empty shuttle plasmids may be relatively low and stable under different growth conditions, shuttle vectors containing genes that are a metabolic burden on cells can have variable copy numbers (34). Therefore, absolute gene expression levels cannot be reliably compared between different strains or growth phases but rather only between different constructs and induction conditions in the same strain under the same growth conditions.…”

Section: Resultsmentioning

confidence: 99%

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Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

Schmidt

Golden

2014

Appl Environ Microbiol

117

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Cyanobacteria are photosynthetic bacteria that are currently being developed as biological production platforms. They derive energy from light and carbon from atmospheric carbon dioxide, and some species can fix atmospheric nitrogen. One advantage of developing cyanobacteria for renewable production of biofuels and other biological products is that they are amenable to genetic manipulation, facilitating bioengineering and synthetic biology. To expand the currently available genetic toolkit, we have demonstrated the utility of synthetic theophylline-responsive riboswitches for effective regulation of gene expression in four diverse species of cyanobacteria, including two recent isolates. We evaluated a set of six riboswitches driving the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942, Leptolyngbya sp. strain BL0902, Anabaena sp. strain PCC 7120, and Synechocystis sp. strain WHSyn. We demonstrated that riboswitches can offer regulation of gene expression superior to that of the commonly used isopropyl-␤-D-thiogalactopyranoside induction of a lacI q -P trc promoter system. We also showed that expression of the toxic protein SacB can be effectively regulated, demonstrating utility for riboswitch regulation of proteins that are detrimental to biomass accumulation. Taken together, the results of this work demonstrate the utility and ease of use of riboswitches in the context of genetic engineering and synthetic biology in diverse cyanobacteria, which will facilitate the development of algal biotechnology.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

Schmidt

Golden

2014

Appl Environ Microbiol

117

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“…To check if the phenotype of the all1549⍀sp/sm mutant was due to the disruption of the rel ana gene, a complementation test was carried out. Plasmid pRL25T-C1549, constructed using a pDU1-based shuttle vector (32,45) harboring the entire open reading frame (ORF) of rel ana as well as its promoter region, was transferred into mutant strain all1549⍀sp/ sm. The complemented strain, called C1549, grows at a rate slightly slower than that of the wild type when nitrate is present in the growth medium.…”

Section: Resultsmentioning

confidence: 99%

ppGpp Metabolism Is Involved in Heterocyst Development in the Cyanobacterium Anabaena sp. Strain PCC 7120

et al. 2013

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Here we show that all1549 (here designated rel ana ) in Anabaena, homologous to relA/spoT, is upregulated in response to nitrogen deprivation and predominantly localized in vegetative cells. The disruption of rel ana strongly affects the synthesis of ppGpp, and the resulting mutant, all1549⍀sp/sm, fails to form heterocysts and to grow in the absence of a combined-nitrogen source. This phenotype can be complemented by a wild-type copy of rel ana . Although the upregulation of hetR is affected in the mutant, ectopic overexpression of hetR cannot rescue the phenotype. However, we found that the mutant rapidly loses its viability, within a time window of 3 to 6 h, following the deprivation of combined nitrogen. We propose that ppGpp plays a major role in rebalancing the metabolic activities of the cells in the absence of the nitrogen source supply and that this regulation is necessary for filament survival and consequently for the success of heterocyst differentiation.

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“…Colonies arose upon transformation with the LfiA/B-expressing construct but did not grow upon re-streaking on fresh plates. Lack of fluorescence signal in some of the depicted cells is likely due to the phenotypic variation of copy numbers of the pRL25C plasmid in different cells within an Anabaena filament 68 . Scale bars: 5 µm.…”

Section: Supplementary Informationmentioning

confidence: 99%

Two novel heteropolymer-forming proteins maintain multicellular shape of the cyanobacteriumAnabaenasp. PCC 7120

Springstein

Nürnberg²,

Kieninger

et al. 2019

Preprint

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23The determinants of bacterial cell shape are extensively studied in unicellular forms. 24Nonetheless, the mechanisms that shape bacterial multicellular forms remain understudied. 25Here we study coiled-coil rich proteins (CCRPs) in the multicellular cyanobacterium Anabaena 26 sp. PCC 7120 (hereafter Anabaena). Our results reveal two CCPRs, termed LfiA and LfiB (for 27 linear filament), which assemble into a heteropolymer that traverses the longitudinal cell axis. 28Two additional CCRPs, CypS (for cyanobacterial polar scaffold) and CeaR (for cyanobacterial 29 elongasome activity regulator), form a polar proteinaceous scaffold and regulate MreB activity, 30 respectively. Deletion mutants of these CCRPs are characterized by impaired filament shape 31 and decreased viability. Our results indicate that the four CCRPs form a proteinaceous network 32 that stabilizes the Anabaena multicellular filament. We propose that this cytoskeletal network 33 is essential for the manifestation of the linear filament phenotype in Anabaena. 34 (a multi-enzyme complex), is a regulator of longitudinal PG biogenesis, and thus it plays a 49 crucial role in the adaption to different environments and prokaryotic multicellularity. 50The key hallmarks of permanent bacterial multicellularity are morphological 51 differentiation and a well-defined and reproducible shape, termed patterned multicellularity 3 . 52Unlike biofilms, patterned multicellular structures are the result of either coordinated swarming 53 or developmental aggregation behavior as in myxobacteria 9 . Additional factors include cell 54 division, proliferation and cell differentiation as in sporulating actinomycetes 10 and 55 cyanobacterial filaments 11,12 . In myxobacteria as well as in actinomycetes, it has been shown 56 that patterned multicellular traits are dependent on the coordinated function of different coiled-57 coil-rich proteins (CCRPs). Reminiscent of eukaryotic intermediate filaments (IFs) 13,14 , many 58 bacterial CCRPs were shown to perform analogous cytoskeletal functions through their ability 59 to self-assemble into distinct filaments in vitro and in vivo [15][16][17][18][19] . Unlike FtsZ or MreB 20,21 , 60 bacterial IF-like CCRPs do not require any additional co-factors for polymerization in vitro 22 . 61For example, in Myxococcus xanthus, the coordinated swarming and aggregation into fruiting 62 bodies is mediated by its gliding motility 23 , which strictly depends on the filament-forming 63 CCRP AglZ 24 . AglZ is organized in a large multi-protein complex that governs gliding motility 64 in synergy with MreB, which still retained its PG synthesis function but was also co-opted for 65 gliding motility in M. xanthus [25][26][27][28] . Actinobacteria, such as Streptomyces species, grow by 66 building new cell wall (i.e. PG) only at the cell poles, independent of MreB 29,30 , which is 67 strikingly different from how most other bacteria grow 31 . This characteristic polar growth mode 68 is organized by a cytoskeletal network of at least three CCRPs -DivIVA...

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Phenotypic variation caused by variation in the relative copy number of pDU1-based plasmids expressing the GAF domain of Pkn41 or Pkn42 in Anabaena sp. PCC 7120

Cited by 20 publications

References 43 publications

Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

ppGpp Metabolism Is Involved in Heterocyst Development in the Cyanobacterium Anabaena sp. Strain PCC 7120

Two novel heteropolymer-forming proteins maintain multicellular shape of the cyanobacteriumAnabaenasp. PCC 7120

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