Methods for the genetic manipulation of marine bacteria

Zeaiter, Zahraa; Mapelli, Francesca; Crotti, Elena; Borin, Sara

doi:10.1016/j.ejbt.2018.03.003

Cited by 25 publications

(30 citation statements)

References 133 publications

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“…Fortunately, both electroporation and conjugation strategies applicable in marine bacteria are being developed. [21][22][23] Single gene products from marine Proteobacteria have since the early eighties been cloned and expressed in heterologous hosts. 24 However, transferring and heterologous expression of larger, more complex biosynthetic pathways is challenging in part because of the size (10-100 kbp) in part because of nuances in regulatory elements or metabolic background between the native producers and the engineering expression hosts.…”

Section: Introductionmentioning

confidence: 99%

Marine Proteobacteria as a source of natural products: advances in molecular tools and strategies

et al. 2019

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Section: Introductionmentioning

confidence: 99%

Marine Proteobacteria as a source of natural products: advances in molecular tools and strategies

et al. 2019

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“…Basic Protocol 2 describes the successful electroporation of competent cells with exogenous DNA. Across a number of bacterial species, the presence of salts is known to influence the uptake of foreign DNA (Altermark et al, 2007;Wang et al, 2015;Zeaiter et al, 2018). Upon electroporation, standard Amp R , ampicillin resistance (100 μg/ml); Kan R , kanamycin resistance (100 μg/ml).…”

Section: Understanding Resultsmentioning

confidence: 99%

“…Salts are known to influence the uptake of foreign DNA by bacterial species (Altermark, Niiranen, Willassen, Smalås, & Moe, 2007;Wang et al, 2015;Zeaiter, Mapelli, Crotti, & Borin, 2018). In V. cholerae, for example, the presence of sodium increases the activity of endonuclease Dns (Altermark et al, 2007), thus hindering DNA uptake.…”

Section: Presence Of Saltmentioning

confidence: 99%

Improved Method for Transformation of Vibrio vulnificus by Electroporation

2020

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Vibrio vulnificus, an emergent human pathogen, causes fulminant septicemia with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, the factors to conclusively indicate the virulence potential of V. vulnificus strains remain largely unknown. Understanding the pathogenesis of this bacterium at a molecular level is severely hindered by inefficiencies in transformation, for instance, due to the presence of a periplasmic nuclease, Vvn. Currently, successful transformation of V. vulnificus is nearly impossible due to lack of mobilizable plasmids for the bacterium, requiring (i) very high DNA concentrations, (ii) plasmid linearization, (iii) development of novel V. vulnificus‐derived plasmids, or (iv) time‐consuming conjugation‐based methods. To overcome these limitations, we describe a rapid, efficient, and reproducible electroporation protocol to effectively transform widely available plasmids, with different copy numbers and antibiotic resistances, into phylogenetically distant strains of V. vulnificus. Cells are made competent in high concentrations of sucrose devoid of cations and recovered from electroporation using a high‐salinity recovery medium. Compared to existing methods for transformation of V. vulnificus, significantly higher efficiencies are obtained using this improved protocol. Rapid and effective transformations can markedly improve molecular analyses of V. vulnificus leading to a greater understanding of its virulence potential. This is crucial to develop rapid detection methods which have the potential to prevent future outbreaks. The electroporation protocol described here may be particularly useful for optimizing transformation of other nuclease‐producing bacteria. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent cells Basic Protocol 2: Transformation of cells by electroporation

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“…The inducers of IPTG and L-arabinose at the concentrations as indicated were added. The 2,6-diaminophenedioic acid (DAP) at a final concentration of 50 μg/mL was dosed for cultivating E. coli WM3064, in which RP4 Tra function is integrated into the genome and is auxotrophic for DAP (41).…”

Section: Experimental Methodsmentioning

confidence: 99%

Developing a population-state decision system for intelligently reprogramming extracellular electron transfer in Shewanella oneidensis

Tang

Fan

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The unique extracellular electron transfer (EET) ability has positioned electroactive bacteria (EAB) as a major class of cellular chassis for genetic engineering aimed at favorable environmental, energy, and geoscience applications. However, previous efforts to genetically enhance EET ability have often impaired the basal metabolism and cellular growth due to the competition for the limited cellular resource. Here, we design a quorum sensing-based population-state decision (PSD) system for intelligently reprogramming the EET regulation system, which allows the rebalanced allocation of the cellular resource upon the bacterial growth state. We demonstrate that the electron output from Shewanella oneidensis MR-1 could be greatly enhanced by the PSD system via shifting the dominant metabolic flux from initial bacterial growth to subsequent EET enhancement (i.e., after reaching a certain population-state threshold). The strain engineered with this system achieved up to 4.8-fold EET enhancement and exhibited a substantially improved pollutant reduction ability, increasing the reduction efficiencies of methyl orange and hexavalent chromium by 18.8- and 5.5-fold, respectively. Moreover, the PSD system outcompeted the constant expression system in managing EET enhancement, resulting in considerably enhanced electron output and pollutant bioreduction capability. The PSD system provides a powerful tool for intelligently managing extracellular electron transfer and may inspire the development of new-generation smart bioelectrical devices for various applications.

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Methods for the genetic manipulation of marine bacteria

Cited by 25 publications

References 133 publications

Marine Proteobacteria as a source of natural products: advances in molecular tools and strategies

Marine Proteobacteria as a source of natural products: advances in molecular tools and strategies

Improved Method for Transformation of Vibrio vulnificus by Electroporation

Developing a population-state decision system for intelligently reprogramming extracellular electron transfer in Shewanella oneidensis

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