New yeast recombineering tools for bacteria

Shanks, Robert M. Q.; Kadouri, Daniel E.; MacEachran, Daniel P.; O’Toole, George A.

doi:10.1016/j.plasmid.2009.05.002

Cited by 95 publications

(117 citation statements)

References 56 publications

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“…Plasmids were constructed with standard methods (41), including yeast gap repair (42) and Gibson assembly (43) using the Gibson assembly cloning kit (New England BioLabs, Ipswich, MA, USA), resulting in plasmids pAMG488 for deletion of Clo1313_0396, pCS2 for deletion of Clo1313_0089, and pCS6 for deletion of Clo1313_2023. Plasmids were isolated from Escherichia coli strain BL21, such that the DNA was not methylated by Dcm methylase in E. coli prior to transformation into C. thermocellum (44).…”

Section: Methodsmentioning

confidence: 99%

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

Appl Environ Microbiol

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Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ␤-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5-to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and ␣-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters. IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

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Section: Methodsmentioning

confidence: 99%

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

Appl Environ Microbiol

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“…Molecular biology. The N-terminal polyhistidine-tagged slpB, slpC, and slpD genes under the control of the P BAD promoter on p15a-based plasmid pMQ125 (39) were generated by replacing prtS on pMQ356 (23) but maintaining the N-terminal His 7 tag. This was done by digesting pMQ356 with SalI and SmaI that cut in the prtS gene and then replacing the entire prtS gene using primers that amplify the slp genes and have regions of homology to the His 7 tag and vector backbone.…”

Section: Methodsmentioning

confidence: 99%

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

et al. 2015

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The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Microbial keratitis (MK) is a blinding disease with poor visual outcomes even with effective antibiotics and antifungal agents (1-3). In addition to being a major cause of hospital-acquired infections, such as ventilator-associated pneumonia (4, 5), Serratia marcescens is a common cause of MK (1, 2, 6-8), yet the virulence factors involved in this process are poorly understood. In general, bacterial secreted factors, including hemolysins and proteases, contribute to the pathogenesis of bacterial corneal infections (9-12). There are several studies characterizing the importance of proteases from S. marcescens isolates in ocular infections, most recently reviewed by Matsumoto (13). Serralysin is a cytotoxic factor capable of killing mammalian cells in vitro (14,15). Purified serralysin is sufficient to cause keratitis when injected into rabbit eyes and promotes the spread of bacteria through the corneal stroma (16-18). Additionally, serralysin can degrade components of the human immune system, such as PAR-2, in vitro, and this may impact corneal pathogenesis (13,(19)(20)(21). Serralysin, and serralysin family metalloproteases, such as alkaline protease from Pseudomonas aeruginosa, can proteolyze mammalian cell surface proteins, thereby modulating cell physiology. A recent example is the protease-mediated activation of the epithelial sodium channel, lea...

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“…The advantages of yeast recombination are that multiple pieces of DNA can be sewn together seamlessly without multiple rounds of amplification, and without the need for ligation in vitro or the use of restriction sites. This method is efficient, robust and simple to perform (Gibson 2009, Shanks et al 2009.…”

mentioning

confidence: 99%

“…The observation that linear DNA fragments can efficiently stimulate recombination in Saccharomyces cerevisiae has led to the rapid development of powerful methods for DNA manipulation in yeast (Oldenburg et al 1997). One of these powerful methodologies is a yeastbased homologous recombination cloning technique (Shanks et al 2009). This technique is based on the ability of yeast cells to recombine sequences similar during the natural event of homologous recombination that occurs to repair DNA damage during replication (Kuzminov 2001, Orr-Weaver et al 1981.…”

mentioning

confidence: 99%

Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

Queiroz

Silva

Santos

et al. 2013

An. Acad. Bras. Ciênc.

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RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSCrepYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.

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New yeast recombineering tools for bacteria

Cited by 95 publications

References 56 publications

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

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