Recombinant production of the lantibiotic nisin using Corynebacterium glutamicum in a two-step process

Abstract Background The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products. Results We established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin. Conclusions We demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.

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Towards improved resistance of Corynebacterium glutamicum against nisin

10.1101/2021.08.09.454123 ◽

2021 ◽

Author(s):

Dominik Weixler ◽

Oliver Goldbeck ◽

Gerd Michael Seibold ◽

Bernhard Johannes Eikmanns ◽

Christian Ullrich Riedel

Keyword(s):

Antimicrobial Peptides ◽

Corynebacterium Glutamicum ◽

Cell Envelope ◽

Resistance Mechanisms ◽

Heterologous Production ◽

Human Pathogens ◽

Gram Positive Bacteria ◽

Lipid Ii ◽

Immunity Genes ◽

Abc Transporter Genes

The bacteriocin nisin is one of the best studied antimicrobial peptides. It is widely used as a food preservative due to its antimicrobial activity against various Gram-positive bacteria including human pathogens such as Listeria monocytogenes and others. The receptor of nisin is the universal cell wall precursor lipid II, which is present in all bacteria. Thus, nisin has a broad spectrum of target organisms. Consequently, heterologous production of nisin with biotechnological relevant organisms including Corynebacterium glutamicum is difficult. Nevertheless, bacteria have evolved several mechanisms of resistance against nisin and other cationic antimicrobial peptides (CAMPs). Here, we transferred resistance mechanisms described in other organisms to C. glutamicum with the aim to improve nisin resistance. The presented approaches included: expression of (i) nisin immunity genes nisI and/or nisFEG or (ii) nisin ABC-transporter genes of Staphylococcus aureus and its homologues of C. glutamicum, (iii) genes coding for enzymes for alanylation or lysinylation of the cell envelope to introduce positive charges, and/or (iv) deletion of genes for porins of the outer membrane. None of the attempts alone increased resistance of C. glutamicum more than two-fold. To increase resistance of C. glutamicum to levels that will allow heterologous production of active nisin at relevant titers, further studies are needed.

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Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4915-4926.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4915-4926 ◽

Cited By ~ 205

Author(s):

Michael B. Cooley ◽

William G. Miller ◽

Robert E. Mandrell

Keyword(s):

Escherichia Coli ◽

Arabidopsis Thaliana ◽

Salmonella Enterica ◽

Fluorescent Protein ◽

Enterohemorrhagic Escherichia Coli ◽

Plant Responses ◽

Human Pathogens ◽

E Coli ◽

Green Fluorescent ◽

Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

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Active Supervision: An Effective, Efficient, Low-Intensity Strategy to Support Student Success

Beyond Behavior ◽

10.1177/1074295618799343 ◽

2018 ◽

Vol 27 (3) ◽

pp. 153-159 ◽

Cited By ~ 1

Author(s):

Holly M. Menzies ◽

Kathleen Lynne Lane ◽

Wendy Peia Oakes ◽

Karen Ruth ◽

Emily D. Cantwell ◽

...

Keyword(s):

Student Engagement ◽

Student Success ◽

Disruptive Behavior ◽

Challenging Behavior ◽

Successful Implementation ◽

Step Process ◽

Low Intensity ◽

Practical Strategy

Active supervision is a practical strategy for increasing student engagement and decreasing student disruptive behavior. In this article, we describe a step-by-step process for using active supervision, with teaching tips to assist with successful implementation. Throughout the article we offer lessons from the field featuring the perspectives of practitioners who have used active supervision in classrooms that include students with challenging behavior.

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Construction of a Vitreoscilla Hemoglobin Promoter-Based Tunable Expression System for Corynebacterium glutamicum

Catalysts ◽

10.3390/catal8110561 ◽

2018 ◽

Vol 8 (11) ◽

pp. 561 ◽

Cited By ~ 4

Author(s):

Kei-Anne Baritugo ◽

Hee Taek Kim ◽

Mi Na Rhie ◽

Seo Young Jo ◽

Tae Uk Khang ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Fluorescent Protein ◽

Expression System ◽

Vitreoscilla Hemoglobin ◽

Aminobutyric Acid ◽

Vector System ◽

Efficient Production ◽

Gamma Aminobutyric Acid ◽

Recombinant Strains ◽

Gaba Production

Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, in this study, we developed a novel tunable promoter system based on repeats of the Vitreoscilla hemoglobin promoter (Pvgb). Tunable expression of green fluorescent protein (GFP) was investigated under one, four, and eight repeats of Pvgb (Pvgb, Pvgb4, and Pvgb8). The intensity of fluorescence in recombinant C. glutamicum strains increased as the number of Pvgb increased from single to eight (Pvgb8) repeats. Furthermore, we demonstrated the application of the new Pvgb promoter-based vector system as a platform for metabolic engineering of C. glutamicum by investigating 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) production in several C. glutamicum strains. The profile of 5-AVA and GABA production by the recombinant strains were evaluated to investigate the tunable expression of key enzymes such as DavBA and GadBmut. We observed that 5-AVA and GABA production by the recombinant strains increased as the number of Pvgb used for the expression of key proteins increased. The recombinant C. glutamicum strain expressing DavBA could produce higher amounts of 5-AVA under the control of Pvgb8 (3.69 ± 0.07 g/L) than the one under the control of Pvgb (3.43 ± 0.10 g/L). The average gamma-aminobutyric acid production also increased in all the tested strains as the number of Pvgb used for GadBmut expression increased from single (4.81–5.31 g/L) to eight repeats (4.94–5.58 g/L).

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A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes ◽

10.3390/pr7050291 ◽

2019 ◽

Vol 7 (5) ◽

pp. 291 ◽

Cited By ~ 1

Author(s):

Chih-Yu Wu ◽

Chao-Wei Huang ◽

Yu-Shin Nai ◽

Pei-Yu Chu ◽

Chung-Hsiung Wang ◽

...

Keyword(s):

Recombinant Proteins ◽

Posttranslational Modifications ◽

Mammalian Cells ◽

Insect Cells ◽

Fluorescent Protein ◽

Expression System ◽

Recombinant Baculovirus ◽

New Combination ◽

Vector System ◽

Baculovirus Expression Vector

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

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Dynamics in the expression of epigenetic modifiers and histone modifications in perinatal rat germ cells during de novo DNA methylation†

Biology of Reproduction ◽

10.1093/biolre/ioaa206 ◽

2020 ◽

Author(s):

Arlette Rwigemera ◽

Rhizlane El omri-Charai ◽

Laetitia L Lecante ◽

Geraldine Delbes

Keyword(s):

Dna Methylation ◽

Germ Cell ◽

Histone Modifications ◽

Germ Cells ◽

Posttranslational Modifications ◽

Fluorescent Protein ◽

De Novo ◽

Expression Patterns ◽

Dna Methyltransferases ◽

Epigenetic Modifiers

Abstract Epigenetic reprogramming during perinatal germ cell development is essential for genomic imprinting and cell differentiation; however, the actors of this key event and their dynamics are poorly understood in rats. Our study aimed to characterize the expression patterns of epigenetic modifiers and the changes in histone modifications in rat gonocytes at the time of de novo DNA methylation. Using transgenic rats expressing Green Fluorescent Protein (GFP) specifically in germ cells, we purified male gonocytes by fluorescent activated cell sorting at various stages of perinatal development and established the transcriptomic profile of 165 epigenetic regulators. Using immunofluorescence on gonad sections, we tracked six histone modifications in rat male and female perinatal germ cells over time, including methylation of histone H3 on lysines 27, 9, and 4; ubiquitination of histone H2A on lysine119; and acetylation of histone H2B on lysine 20. The results revealed the dynamics in the expression of ten-eleven translocation enzymes and DNA methyltransferases in male gonocytes at the time of de novo DNA methylation. Moreover, our transcriptomic data indicate a decrease in histone ubiquitination and methylation coinciding with the beginning of de novo DNA methylation. Decreases in H2AK119Ub and H3K27me3 were further confirmed by immunofluorescence in the male germ cells but were not consistent for all H3 methylation sites examined. Together, our data highlighted transient chromatin remodeling involving histone modifications during de novo DNA methylation. Further studies addressing how these dynamic changes in histone posttranslational modifications could guide de novo DNA methylation will help explain the complex establishment of the male germ cell epigenome.

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Effect of Route of Introduction and Host Cultivar on the Colonization, Internalization, and Movement of the Human Pathogen Escherichia coli O157:H7 in Spinach

Journal of Food Protection ◽

10.4315/0362-028x-72.7.1521 ◽

2009 ◽

Vol 72 (7) ◽

pp. 1521-1530 ◽

Cited By ~ 78

Author(s):

R. MITRA ◽

E. CUESTA-ALONSO ◽

A. WAYADANDE ◽

J. TALLEY ◽

S. GILLILAND ◽

...

Keyword(s):

Escherichia Coli ◽

Fluorescent Protein ◽

Sprinkler Irrigation ◽

Conclusive Evidence ◽

Escherichia Coli O157 ◽

Human Pathogens ◽

Soil Infiltration ◽

Fresh Tissue ◽

E Coli ◽

Over Time

Human pathogens can contaminate leafy produce in the field by various routes. We hypothesized that interactions between Escherichia coli O157:H7 and spinach are influenced by the route of introduction and the leaf microenvironment. E. coli O157:H7 labeled with green fluorescent protein was dropped onto spinach leaf surfaces, simulating bacteria-laden raindrops or sprinkler irrigation, and survived on the phylloplane for at least 14 days, with increasing titers and areas of colonization over time. The same strains placed into the rhizosphere by soil infiltration remained detectable on very few plants and in low numbers (102 to 106 CFU/g fresh tissue) that decreased over time. Stem puncture inoculations, simulating natural wounding, rarely resulted in colonization or multiplication. Bacteria forced into the leaf interior survived for at least 14 days in intercellular spaces but did not translocate or multiply. Three spinach cultivars with different leaf surface morphologies were compared for colonization by E. coli O157:H7 introduced by leaf drop or soil drench. After 2 weeks, cv. Bordeaux hosted very few bacteria. More bacteria were seen on cv. Space and were dispersed over an area of up to 0.3 mm2. The highest bacterial numbers were observed on cv. Tyee but were dispersed only up to 0.15 mm2, suggesting that cv. Tyee may provide protected niches or more nutrients or may promote stronger bacterial adherence. These findings suggest that the spinach phylloplane is a supportive niche for E. coli O157:H7, but no conclusive evidence was found for natural entry into the plant interior. The results are relevant for interventions aimed at minimizing produce contamination by human pathogens.

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A Novel Assay for Protein Phosphatase 2A (PP2A) Complexes In Vivo Reveals Differential Effects of Covalent Modifications on Different Saccharomyces cerevisiae PP2A Heterotrimers

Eukaryotic Cell ◽

10.1128/ec.4.6.1029-1040.2005 ◽

2005 ◽

Vol 4 (6) ◽

pp. 1029-1040 ◽

Cited By ~ 36

Author(s):

Matthew S. Gentry ◽

Yikun Li ◽

Huijun Wei ◽

Farhana F. Syed ◽

Sameer H. Patel ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Posttranslational Modifications ◽

Protein Phosphatase ◽

Cellular Localization ◽

Fluorescent Protein ◽

Protein Phosphatase 2A ◽

Catalytic Subunit ◽

Phosphatase 2A ◽

Covalent Modifications

ABSTRACT Protein phosphatase 2A (PP2A) catalytic subunit can be covalently modified at its carboxy terminus by phosphorylation or carboxymethylation. Determining the effects of these covalent modifications on the relative amounts and functions of different PP2A heterotrimers is essential to understanding how these modifications regulate PP2A-controlled cellular processes. In this study we have validated and used a novel in vivo assay for assessing PP2A heterotrimer formation in Saccharomyces cerevisiae: the measurement of heterotrimer-dependent localization of green fluorescent protein-PP2A subunits. This assay relies on the fact that the correct cellular localization of PP2A requires that it be fully assembled. Thus, reduced localization would occur as the result of the inability to assemble a stable heterotrimer. Using this assay, we determined the effects of PP2A C-subunit phosphorylation mimic mutations and reduction or loss of PP2A methylation on the formation and localization of PP2AB/Cdc55p and PP2AB ′ /Rts1p heterotrimers. Collectively, our findings demonstrate that phosphorylation and methylation of the PP2A catalytic subunit can influence its function both by regulating the total amount of specific PP2A heterotrimers within a cell and by altering the relative proportions of PP2AB/Cdc55p and PP2AB ′ /Rts1p heterotrimers up to 10-fold. Thus, these posttranslational modifications allow flexible, yet highly coordinated, regulation of PP2A-dependent signaling pathways that in turn modulate cell growth and function.

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Differences in Growth of Salmonella enterica and Escherichia coli O157:H7 on Alfalfa Sprouts

Applied and Environmental Microbiology ◽

10.1128/aem.68.6.3114-3120.2002 ◽

2002 ◽

Vol 68 (6) ◽

pp. 3114-3120 ◽

Cited By ~ 85

Author(s):

A. O. Charkowski ◽

J. D. Barak ◽

C. Z. Sarreal ◽

R. E. Mandrell

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

Salmonella Enterica ◽

Fluorescent Protein ◽

Human Pathogens ◽

E Coli ◽

Inoculum Dose ◽

Alfalfa Sprouts ◽

Green Fluorescent ◽

Alfalfa Seeds

ABSTRACT Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log10 on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log10. The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.

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Integrating Option Grid Patient Decision Aids in the Epic Electronic Health Record: Case Study at 5 Health Systems

Journal of Medical Internet Research ◽

10.2196/22766 ◽

2021 ◽

Vol 23 (5) ◽

pp. e22766

Author(s):

Peter Scalia ◽

Farhan Ahmad ◽

Danielle Schubbe ◽

Rachel Forcino ◽

Marie-Anne Durand ◽

...

Keyword(s):

Uterine Fibroid ◽

Decision Aids ◽

The United States ◽

Successful Implementation ◽

Patient Decision Aids ◽

Step Process ◽

Patient Decision ◽

Facilitators And Barriers ◽

Electronic Health

Background Some researchers argue that the successful implementation of patient decision aids (PDAs) into clinical workflows depends on their integration into electronic health records (EHRs). Anecdotally, we know that EHR integration is a complex and time-consuming task; yet, the process has not been examined in detail. As part of an implementation project, we examined the work involved in integrating an encounter PDA for symptomatic uterine fibroids into Epic EHR systems. Objective This study aims to identify the steps and time required to integrate a PDA into the Epic EHR system and examine facilitators and barriers to the integration effort. Methods We conducted a case study at 5 academic medical centers in the United States. A clinical champion at each institution liaised with their Epic EHR team to initiate the integration of the uterine fibroid Option Grid PDAs into clinician-facing menus. We scheduled regular meetings with the Epic software analysts and an expert Epic technologist to discuss how best to integrate the tools into Epic for use by clinicians with patients. The meetings were then recorded and transcribed. Two researchers independently coded the transcripts and field notes before categorizing the codes and conducting a thematic analysis to identify the facilitators and barriers to EHR integration. The steps were reviewed and edited by an Epic technologist to ensure their accuracy. Results Integrating the uterine fibroid Option Grid PDA into clinician-facing menus required an 18-month timeline and a 6-step process, as follows: task priority negotiation with Epic software teams, security risk assessment, technical review, Epic configuration; troubleshooting, and launch. The key facilitators of the process were the clinical champions who advocated for integration at the institutional level and the presence of an experienced technologist who guided Epic software analysts during the build. Another facilitator was the use of an emerging industry standard app platform (Health Level 7 Substitutable Medical Applications and Reusable Technologies on Fast Healthcare Interoperability Resources) as a means of integrating the Option Grid into existing systems. This standard platform enabled clinicians to access the tools by using single sign-on credentials and prevented protected health information from leaving the EHR. Key barriers were the lack of control over the Option Grid product developed by EBSCO (Elton B Stephens Company) Health; the periodic Epic upgrades that can result in a pause on new software configurations; and the unforeseen software problems with Option Grid (ie, inability to print the PDA), which delayed the launch of the PDA. Conclusions The integration of PDAs into the Epic EHR system requires a 6-step process and an 18-month timeline. The process required support and prioritization from a clinical champion, guidance from an experienced technologist, and a willing EHR software developer team.

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