Isolation and molecular characterization of a stationary phase promoter useful for gene expression in Gordonia

Singh, Pooja; Chachan, Sahil; Singhi, Divya; Srivastava, Preeti

doi:10.1016/j.gene.2016.07.018

Cited by 12 publications

(6 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, in another Gram-positive bacteria, Gordonia sp. IITR100, a SPP was identified and the β-galactosidase activities were up to 600 miller units ( Singh et al, 2016 ). However, the β-galactosidase activities vary with respect to strain, copy number of plasmid, growth medium, temperature, etc., so it is difficult to assess the strength of promoter based on Miller units alone.…”

Section: Development Of Gene Expression Systems Using Stationary Phasmentioning

confidence: 99%

Molecular Basis of Stationary Phase Survival and Applications

2017

Self Cite

View full text Add to dashboard Cite

Stationary phase is the stage when growth ceases but cells remain metabolically active. Several physical and molecular changes take place during this stage that makes them interesting to explore. The characteristic proteins synthesized in the stationary phase are indispensable as they confer viability to the bacteria. Detailed knowledge of these proteins and the genes synthesizing them is required to understand the survival in such nutrient deprived conditions. The promoters, which drive the expression of these genes, are called stationary phase promoters. These promoters exhibit increased activity in the stationary phase and less or no activity in the exponential phase. The vectors constructed based on these promoters are ideal for large-scale protein production due to the absence of any external inducers. A number of recombinant protein production systems have been developed using these promoters. This review describes the stationary phase survival of bacteria, the promoters involved, their importance, regulation, and applications.

show abstract

Section: Development Of Gene Expression Systems Using Stationary Phasmentioning

confidence: 99%

Molecular Basis of Stationary Phase Survival and Applications

2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…They are of interest because of their potential in industrial and environmental microbiology, with capacities for synthesis and biodegradation of particular chemical compounds, including rubber (17, 25–27). However, there are few genetic tools available, and those that are available are mostly restricted to pRC4-derived plasmids and some expression systems (28–30). A large number of phages infecting Gordonia hosts have been reported, and these span a considerable range of genetic diversity (31–36).…”

Section: Introductionmentioning

confidence: 99%

Yet More Evidence of Collusion: a New Viral Defense System Encoded by Gordonia Phage CarolAnn

Montgomery

Bustamante

Dedrick

et al. 2019

mBio

View full text Add to dashboard Cite

Temperate phages play important roles in the physiology of their bacterial hosts and establish a lysogenic relationship with the host through which prophage-expressed genes confer new phenotypes. A key phenotype is prophage-mediated defense against heterotypic viral attack, in which temperate phages collude with their bacterial host to prevent other phages from attacking, sometimes with exquisite specificity. Such defense systems have been described in Pseudomonas and Mycobacterium phages but are likely widespread throughout the microbial community. Here, we describe a novel prophage-mediated defense system encoded by Gordonia phage CarolAnn, which defends against infection by unrelated phages grouped in cluster CZ. CarolAnn genes 43 and 44 are coexpressed with the repressor and are necessary and sufficient to confer defense against phage Kita and its close relatives. Kita and these relatives are targeted through Kita gene 53, a gene that is of unknown function but which is the location of defense escape mutations that overcome CarolAnn defense. Expression of Kita gene 53 is toxic to Gordonia terrae in the presence of CarolAnn genes 43 and 44, suggesting that defense may be mediated by an abortive infection type of mechanism. CarolAnn genes 43 and 44 are distant relatives of mycobacteriophage Sbash genes 31 and 30, respectively, which also confer viral defense but use a different targeting system. IMPORTANCE Prophage-mediated viral defense systems play a key role in microbial dynamics, as lysogeny is established relatively efficiently, and prophage-expressed genes can strongly inhibit lytic infection of other, unrelated phages. Demonstrating such defense systems in Gordonia terrae suggests that these systems are widespread and that there are a multitude of different systems with different specificities for the attacking phages.

show abstract

“…Minimization of putative promoters. Although any novel promoter characterized for a nonmodel organism can be an enabling technology in and of itself, optimization through minimization can reduce promoter size for easier cloning (i.e., by Gibson assembly) and has resulted in the identification of important regulatory elements such as UTR elements, enhancer regions, or sigma factors (15,29,30). We used the two strongest promoter candidates, PDR_1261 and PrpmB, a relatively small promoter with moderate activity, PrplL, and the smallest novel promoter, PgroEL, for minimization.…”

Section: Resultsmentioning

confidence: 99%

Discovery and Characterization of NativeDeinococcus radioduransPromoters for Tunable Gene Expression

Chen

Sherman

Chu

et al. 2019

Appl Environ Microbiol

View full text Add to dashboard Cite

The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of Deinococcus radiodurans for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in D. radiodurans using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in D. radiodurans. Of these top eight, two promoter regions, PDR_1261 and PrpmB, were stronger and more consistent than the most widely used promoter sequence in D. radiodurans, PgroES. Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in Escherichia coli or were D. radiodurans specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in D. radiodurans. IMPORTANCE The tolerance of the extremophile, Deinococcus radiodurans, to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in D. radiodurans, very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for D. radiodurans via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, PgroES. This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in D. radiodurans. The promoters identified in this study will facilitate metabolic engineering of D. radiodurans and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.R ecent work regarding the biological engineering of extremophiles has increased interest in their use as a robust chassis for metabolic engineering. The appeal of using extremophiles in various applications is largely due to their ability to survive conditions toxic to traditional engineering strains. One extremophile considered attractive is Deinococcus radiodurans, a Gram-positive bacteria known for its tolerance to ionizing radiation, heavy metal exposure, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens (1-3). In the context of its applications in bioremediation,

show abstract

Isolation and molecular characterization of a stationary phase promoter useful for gene expression in Gordonia

Cited by 12 publications

References 34 publications

Molecular Basis of Stationary Phase Survival and Applications

Molecular Basis of Stationary Phase Survival and Applications

Yet More Evidence of Collusion: a New Viral Defense System Encoded by Gordonia Phage CarolAnn

Discovery and Characterization of NativeDeinococcus radioduransPromoters for Tunable Gene Expression

Contact Info

Product

Resources

About