Enhanced high copy number plasmid maintenance and heterologous protein production in an <i>Escherichia coli</i> biofilm

O’Connell, Heather A.; Niu, Chen; Gilbert, Eric S

doi:10.1002/bit.21240

Cited by 27 publications

(39 citation statements)

References 45 publications

(38 reference statements)

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“…1) (5). Few other references to increased plasmid copy number in biofilms were found, although enhanced maintenance of high-copy-number plasmids has been demonstrated (24). Davies and Geesey found that plasmid copy number was ϳ1.5 times higher in Pseudomonas aeruginosa biofilms than in planktonic cells (25) but, to our knowledge, this observation has not been further investigated.…”

Section: Discussionmentioning

confidence: 82%

Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Cook

Dunny

2013

Antimicrob Agents Chemother

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Biofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity in Enterococcus faecalis cells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four different E. faecalis plasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

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Section: Discussionmentioning

confidence: 82%

Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Cook

Dunny

2013

Antimicrob Agents Chemother

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“…Indeed, although many efforts are being directed toward fighting biofilm formation or engineering strains with reduced biofilm formation abilities for biotechnological utilization (76), applications which exploit the biofilm formation abilities of harmless bacteria in a medical environment are presently being considered (19). A recent study demonstrating that, compared to planktonic cells, E. coli biofilms display enhanced high-copy-number-plasmid maintenance and heterologous protein production is also encouraging the use of biofilms in industrial applications (54). We showed in an in vitro experiment that a commensal MG1655 strain that constitutively expresses the Ag43 autotransporter adhesin (MG1655kmPcLflu) was more efficient than the isogenic MG1655⌬flu strain at competing for biofilm formation with a pathogenic enteroaggregative E. coli strain (Fig.…”

Section: Discussionmentioning

confidence: 99%

Tight Modulation of Escherichia coli Bacterial Biofilm Formation through Controlled Expression of Adhesion Factors

Quéré

Ghigo

et al. 2007

Appl Environ Microbiol

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Despite the economic and sanitary problems caused by harmful biofilms, biofilms are nonetheless used empirically in industrial environmental and bioremediation processes and may be of potential use in medical settings for interfering with pathogen development. Escherichia coli is one of the bacteria with which biofilm formation has been studied in great detail, and it is especially appreciated for biotechnology applications because of its genetic amenability. Here we describe the development of two new genetic tools enabling the constitutive and inducible expression of any gene or operon of interest at its native locus. In addition to providing valuable tools for complementation and overexpression experiments, these two compact genetic cassettes were used to modulate the biofilm formation capacities of E. coli by taking control of two biofilmpromoting factors, autotransported antigen 43 adhesin and the bscABZC cellulose operon. The modulation of the biofilm formation capacities of E. coli or those of other bacteria capable of being genetically manipulated may be of use both for reducing and for improving the impact of biofilms in a number of industrial and medical applications.Most natural and artificial surfaces available in the environment are prone to bacterial colonization. Following the initial adhesion event, cell-to-cell adhesion and the secretion of an extracellular matrix rapidly lead to the formation of a surfaceattached multicellular structure known as a biofilm (3,43,75,84). Besides being resistant to environmental shear forces, biofilm communities are also phenotypically more resistant to antibiotic and biocide treatments, a trait that poses important sanitary and economic problems. Indeed, biofilms formed by bacterial pathogens on medically relevant surfaces are difficult to eradicate and are thus often involved in the development of infections (12,13,48). Moreover, industrial biofouling resulting from bacterial biofilm formation is a major cause of pipe biocorrosion and reduces the efficiency of pharmaceutical or food bioprocesses (2,10,70,71).While recent studies have focused mainly on the negative impact of biofilms, bacterial biofilms can also have valuable applications, including those involving bioremediation and wastewater treatment bioreactor processes and the improvement of biomineralization or plant-bacteria symbiosis (1,14,40,42,52,62,73,74,80). Beneficial biofilms may also have medical applications, and the use of protective innocuous bacterial biofilms that interfere with the development of bacterial pathogens is considered a promising approach (19).Escherichia coli is a gram-negative enterobacterium that has been used extensively as a model to study biofilm development due to its relevance to the human biotic environment and its genetic amenability. In E. coli, various cell surface appendages were shown to be necessary to achieve mature biofilm development (78). Flagella, type I fimbriae, and curli are implicated in early adhesion steps, while the production of a polysaccharide-rich m...

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“…11 The temporal single-cell measurements showed that the biofilm heterogeneity increases over time. O' Connell et al 10 studied the dynamics of fluorescence during biofilm development by flow cytometry and detected three populations of E. coli cells with differing levels of GFP expression in 24-h biofilms.…”

Section: Discussionmentioning

confidence: 99%

“…11 The concentration gradients of the chemicals dissolved in the interstitial fluid within the biofilm matrix promote differences in bacterial enzymatic activities in different areas of the biofilms 12,13 and can create variation at the gene and protein levels. 10,[14][15][16] In this work, a protocol using fluorescence imaging is proposed for quantifying the dynamics of protein expression within a biofilm population at both bulk and singlecell levels. It should be noted that the entire protocol required only an epifluorescence…”

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confidence: 99%

“…10 During recombinant protein production, it is important to not only monitor the total amount of proteins produced by the culture using bulk methods such as fluorometry or standard fluorescence microscopy but also evaluate the distribution of this protein in individual cells. For instance, knowing the fraction of protein-producing and nonproducing cells may help in optimizing the operational parameters for maximum strain performance.…”

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confidence: 99%

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Temporal variation of recombinant protein expression in Escherichia coli biofilms analysed at single-cell level

Gomes

Carvalho

Briandet

et al. 2016

Process Biochemistry

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Highlights Epifluorescence microscopy and image processing enable single-cell expression analysis. Escherichia coli biofilm heterogeneity increased during biofilm development. Fluorescence heterogeneity was correlated with spatial heterogeneity. 2 AbstractBioprocesses based on surface-associated microorganisms are emerging in environmental and industrial areas owing to the physiological specificities and heterogeneities of biofilm cells. This study describes a simple and accurate method for evaluating recombinant protein expression at a single-cell scale during Escherichia coli biofilm development. The model recombinant protein used was enhanced Green Fluorescent Protein (eGFP), as its intrinsic fluorescence allows the quantification of expression at both population and single-cell levels. Specific cell fluorescence intensity sharply increased during the first 4 days of biofilm cultivation and thereafter decreased abruptly to reach a low-level plateau until the end of the experiment. During biofilm development, the population became increasingly heterogeneous with regard to eGFP expression. Three distinct biofilm types were observed along the experimental time: one with a homogeneous population (days 3-5), the second with a moderately heterogeneous population (days 6-8) and the third with a strongly heterogeneous population (days 9-11). Observation of E. coli biofilms by confocal laser scanning microscopy demonstrated marked spatial heterogeneity, with the cells actively producing eGFP restricted to the top layer of the biofilm. The proposed methodology allows a fine analysis of the recombinant protein expression within E. coli biofilms, and it may be used for optimizing the processing conditions. Keywords: Biofilm; Escherichia coli; recombinant protein expression; fluorescence microscopy; single-cell scale; spatial heterogeneity Introduction 3The Gram-negative bacterium Escherichia coli is one of the most commonly used recombinant protein production hosts 1 due to its ability to grow rapidly and to high densities on inexpensive substrates, its well-known genetics and the availability of various systems for gene expression.2 Several bacteria such as E. coli naturally grow in a community attached to a substratum and not in liquid cultures. The biocatalytic potential of these bacterial communities, termed biofilms, can be attributed to their high cell density; the former feature is widely used for wastewater treatment 3 and also for the production of industrial chemicals such as ethanol, butanol and lactic acid. 4,5 Recombinant protein production in biofilms has been mostly studied in the context of waste biodegradation 6,7 ; however, this strategy could also be advantageous in other processes such as the biosynthesis of pharmaceutical intermediates 8 and catalysts for the food industry. In fact, using a recombinant Aspergillus niger strain, which contained a gene encoding the glucoamylase-GFP fusion protein, Talabardon and Yang 9 showed that higher amounts of GFP (Green Fluorescent Protein) and glucoamylase...

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Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Cited by 27 publications

References 45 publications

Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Tight Modulation of Escherichia coli Bacterial Biofilm Formation through Controlled Expression of Adhesion Factors

Temporal variation of recombinant protein expression in Escherichia coli biofilms analysed at single-cell level

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