Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria

Taton, Arnaud; Unglaub, Federico; Wright, Nicole E.; Zeng, Wei Yue; Paz‐Yepes, Javier; Brahamsha, Bianca; Palenik, Brian; Peterson, Todd C.; Haerizadeh, Farzad; Golden, Susan S.; Golden, James W.

doi:10.1093/nar/gku673

Cited by 146 publications

(177 citation statements)

References 73 publications

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“…Another attractive production strain, Synechocystis sp. strain WHSyn, was isolated from an estuary in Woods Hole, MA, and can grow in a wide range of salinities (22).…”

Section: Resultsmentioning

confidence: 99%

“…For S. elongatus, integrative plasmids pAM5044 to pAM5049 and pAM5051 containing regions homologous to neutral site 1 were constructed and then introduced by natural transformation (20). RSF1010-based replicative plasmids pAM5052 to pAM5057 and pAM5059 harboring a mobA Y25F mutation (21,22) were introduced into the remaining cyanobacterial strains by conjugation from E. coli strain AM1359 (23,24) carrying the cargo plasmid. Strain AM1359 contains plasmids pRL443 for conjugation and pRL623 for methylation of donor DNA (25).…”

Section: Methodsmentioning

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

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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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“…Another attractive production strain, Synechocystis sp. strain WHSyn, was isolated from an estuary in Woods Hole, MA, and can grow in a wide range of salinities (22).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Schmidt

Golden

2014

Appl Environ Microbiol

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114

115

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“…A complementation plasmid was constructed in the pBAD24 vector. The Pseudomonas isolate LPH1 cobalamin synthase (cobV) deletion mutant was constructed through gene replacement with an aacC1 cassette (57). A complementation plasmid was constructed in an RSF1010 vector bearing an aadA cassette (conferring resistance to streptomycin and spectinomycin) and a P conII promoter with translational regulation via theophylline riboswitch variant E (58).…”

Section: Methodsmentioning

confidence: 99%

An Amoebal Grazer of Cyanobacteria Requires Cobalamin Produced by Heterotrophic Bacteria

Beld

Brahamsha

2017

Appl Environ Microbiol

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Amoebae are unicellular eukaryotes that consume microbial prey through phagocytosis, playing a role in shaping microbial food webs. Many amoebal species can be cultivated axenically in rich media or monoxenically with a single bacterial prey species. Here, we characterize heterolobosean amoeba LPG3, a recent natural isolate, which is unable to grow on unicellular cyanobacteria, its primary food source, in the absence of a heterotrophic bacterium, a Pseudomonas species coisolate. To investigate the molecular basis of this requirement for heterotrophic bacteria, we performed a screen using the defined nonredundant transposon library of Vibrio cholerae, which implicated genes in corrinoid uptake and biosynthesis. Furthermore, cobalamin synthase deletion mutations in V. cholerae and the Pseudomonas species coisolate do not support the growth of amoeba LPG3 on cyanobacteria. While cyanobacteria are robust producers of a corrinoid variant called pseudocobalamin, this variant does not support the growth of amoeba LPG3. Instead, we show that it requires cobalamin that is produced by the Pseudomonas species coisolate. The diversity of eukaryotes utilizing corrinoids is poorly understood, and this amoebal corrinoid auxotroph serves as a model for examining predator-prey interactions and micronutrient transfer in bacterivores underpinning microbial food webs.IMPORTANCE Cyanobacteria are important primary producers in aquatic environments, where they are grazed upon by a variety of phagotrophic protists and, hence, have an impact on nutrient flux at the base of microbial food webs. Here, we characterize amoebal isolate LPG3, which consumes cyanobacteria as its primary food source but also requires heterotrophic bacteria as a source of corrinoid vitamins. Amoeba LPG3 specifically requires the corrinoid variant produced by heterotrophic bacteria and cannot grow on cyanobacteria alone, as they produce a different corrinoid variant. This same corrinoid specificity is also exhibited by other eukaryotes, including humans and algae. This amoebal model system allows us to dissect predator-prey interactions to uncover factors that may shape microbial food webs while also providing insight into corrinoid specificity in eukaryotes.KEYWORDS amoeba, corrinoids, microbial interactions, vitamin B 12 U nicellular organisms dominate the eukaryotic phylogenetic tree of life. Although protists make up a significant part of natural microbial communities, they have been largely overlooked and are understudied as simple model eukaryotes (1, 2). While culture-independent metagenomic studies provide insight into the abundance and natural diversity of protists in environmental settings (3), in-depth characterization requires the isolation and cultivation of individual species. A large range of eukaryotic microalgal species have been cultivated and studied, particularly as a source of renewable biofuel compounds in recent years (4, 5); however, besides microalgae, there are only a few protist microbes developed as model systems. Amoebae are...

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“…To generate an RSF1010 derivative that is functional in A. oris, the ampicillin resistance gene of pCVD047, a cyanobacterial broad-host-range vector (16), was replaced with the Kan r cassette from pJRD215 (17). Briefly, with primers pCVD047-noAmp-F and pCVD047-noAmp-R for an inverse PCR, an amplicon containing the pCVD047 sequence without the ampicillin resistance gene was generated.…”

Section: Methodsmentioning

confidence: 99%

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris

Siegel

Ton‐That

2016

J Bacteriol

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The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motifcontaining proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity. A ctinomyces oris is a key colonizer in the development of dental plaque or oral biofilms because of its ability to adhere to many oral colonizers and host surfaces. This multiadhesive property is attributed to the presence of two types of fimbriae or pili called type 1 and 2 fimbriae (1, 2). Type 1 fimbriae-made of the shaft fimbrillin FimP and the tip fimbrillin FimQ-mediate bacterial adherence to proline-rich proteins that coat tooth enamel (2-4). Type 2 fimbriae-composed of the shaft fimbrillin FimA and the tip fimbrillin FimB-promote polymicrobial interactions or bacterial coaggregation, biofilm formation, and adhesion to host cells (2,5,6). A. oris also encodes 14 cell wall-anchored proteins (AcaA to AcaN), one of which, CafA (previously named AcaF), has been shown to be the major coaggregation factor that forms a distinct fimbrial tip of type 2 fimbriae (7). It was recently shown that the cell wall-anchored protein AcaC (named GspA) is glycosylated, and its glycosylation appears to be coupled to protein secretion and sortase SrtA-catalyzed cell wall anchoring (8). Shared features found in these cell wall-anchored proteins and pilins (or fimbrillins) are an N-terminal signal peptide and a C-terminal cell wall sorting signal (CWSS). The N-terminal signal...

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Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria

Cited by 146 publications

References 73 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

An Amoebal Grazer of Cyanobacteria Requires Cobalamin Produced by Heterotrophic Bacteria

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris

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