scholarly journals A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

2015 ◽  
Vol 81 (9) ◽  
pp. 3176-3181 ◽  
Author(s):  
Christopher J. Pappas ◽  
Nadia Benaroudj ◽  
Mathieu Picardeau
Keyword(s):  
Genetic Manipulation ◽  
Shuttle Vector ◽  
Plasmid Vector ◽  
Wild Type ◽  
Pathogenic Leptospira ◽  
Content Type ◽  
Large Panel ◽  
Stable Plasmid ◽  
The Stability ◽  
Functional Copy

ABSTRACTLeptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including theparA,parB, andrepgenes into pMAT, a plasmid that cannot replicate inLeptospiraspp. and contains a backbone consisting of anaadAcassette,oriR6K, andoriTRK2/RP4. The inserted DNA segment was isolated from a 52-kb region withinLeptospiramayottensisstrain 200901116 that is not found in the closely related strainL. mayottensis200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use oftranscomplementation in the pathogenLeptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of theperRgene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel ofLeptospirastrains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation ofLeptospiraspp.

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

scholarly journals Oligopeptide Permease A5 Modulates Vertebrate Host-Specific Adaptation of Borrelia burgdorferi

2011 ◽  
Vol 79 (8) ◽  
pp. 3407-3420 ◽  
Author(s):  
B. V. Subba Raju ◽  
Maria D. Esteve-Gassent ◽  
S. L. Rajasekhar Karna ◽  
Christine L. Miller ◽  
Tricia A. Van Laar ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Immunoblot Analysis ◽  
Vertebrate Host ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Specific Adaptation ◽  
Protein Levels ◽  
Oligopeptide Permease

ABSTRACTBorrelia burgdorferi, the agent of Lyme disease, undergoes rapid adaptive gene expression in response to signals unique to its arthropod vector or vertebrate hosts. Among the upregulated genes under vertebrate host conditions is one of the five annotated homologs of oligopeptide permease A (OppA5, BBA34). A mutant lackingoppA5was constructed in an lp25-deficient isolate ofB. burgdorferistrain B31, and the minimal regions of infectivity were restored via a shuttle vector pBBE22 with or without an intact copy ofbba34. Immunoblot analysis of thebba34mutant revealed a reduction in the levels of RpoS, BosR, and CsrABbwith a concomitant reduction in the levels of OspC, DbpA, BBK32, and BBA64. There were no changes in the levels of OspA, NapA, P66, and three other OppA orthologs. Quantitative transcriptional analysis correlated with the changes in the protein levels. However, thebba34mutant displayed comparable infectivities in the C3H/HeN mice and the wild-type strain, despite the reduction in several pathogenesis-related proteins. Supplementation of the growth medium with increased levels of select components, notably sodium acetate and sodium bicarbonate, restored the levels of several proteins in thebba34mutant to wild-type levels. We speculate that the transport of acetate appears to contribute to the accumulation of key metabolites, like acetyl phosphate, that facilitate the adaptation ofB. burgdorferito the vertebrate host by the activation of the Rrp2-RpoN-RpoS pathway. These studies underscore the importance of solute transport to host-specific adaptation ofB. burgdorferi.

scholarly journals Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

2015 ◽  
Vol 81 (21) ◽  
pp. 7394-7402 ◽  
Author(s):  
Nan Li ◽  
Ting Qin ◽  
Xiao Lin Zhang ◽  
Bei Huang ◽  
Zhi Xin Liu ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Bacterial Pathogen ◽  
Infected Fish ◽  
Economic Losses ◽  
Flavobacterium Columnare ◽  
Wild Type ◽  
Single Strain ◽  
Content Type

ABSTRACTFlavobacterium columnareis an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. TheF. johnsoniaeompApromoter (PompA) was fused tosacBto construct a counterselectable marker forF. columnare.F. columnarecarrying PompA-sacBfailed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacBwas constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes,cslAandcslB, were deleted. The ΔcslAand ΔcslBmutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslAΔcslB) was completely deficient in chondroitin lyase activity. Cells ofF. columnarewild-type strain G4and of the chondroitin lyase-deficient ΔcslAΔcslBmutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors ofF. columnarebut may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylumBacteroidetes.

scholarly journals Improved Production of Propionic Acid in Propionibacterium jensenii via Combinational Overexpression of Glycerol Dehydrogenase and Malate Dehydrogenase from Klebsiella pneumoniae

2015 ◽  
Vol 81 (7) ◽  
pp. 2256-2264 ◽  
Author(s):  
Long Liu ◽  
Xin Zhuge ◽  
Hyun-dong Shin ◽  
Rachel R. Chen ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Malate Dehydrogenase ◽  
Propionic Acid ◽  
Shuttle Vector ◽  
Fumarate Hydratase ◽  
Glycerol Dehydrogenase ◽  
Potential Candidate ◽  
Wild Type ◽  
Metabolic Intermediate ◽  
Environmental Friendliness ◽  
Content Type

ABSTRACTMicrobial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineeringPropionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis inP. jenseniiby examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-typeP. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-typeP. jenseniito 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction inP. jenseniiand further improved PA titers via the coexpression of GDH and MDH, making the engineeredP. jenseniistrain a potential industrial producer of PA.

scholarly journals GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8+ T Cell Immunology in R. typhi Infection

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Matthias Hauptmann ◽  
Nicole Burkhardt ◽  
Ulrike Munderloh ◽  
Svenja Kuehl ◽  
Ulricke Richardt ◽  
...  
Keyword(s):  
T Cell ◽  
Genetic Manipulation ◽  
Interleukin 2 ◽  
Scid Mice ◽  
Wild Type ◽  
Rickettsia Typhi ◽  
Content Type ◽  
First Time

ABSTRACT Rickettsia typhi is the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae. We describe here for the first time the transformation of R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed R. typhi (R. typhi GFPuv) bacteria are viable, replicate with kinetics similar to those of wild-type R. typhi in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment in vitro and in vivo during infection of mice. CB17 SCID mice infected with R. typhi GFPuv succumb to the infection with kinetics similar to those for animals infected with wild-type R. typhi and develop comparable pathology and bacterial loads in the organs, demonstrating that the plasmid does not influence pathogenicity. In the spleen and liver of infected CB17 SCID mice, the bacteria are detectable by immunofluorescence microscopy in neutrophils and macrophages by histological staining. Finally, we show for the first time that transformed rickettsiae can be used for the detection of CD8+ T cell responses. GFP-specific restimulation of spleen cells from R. typhi GFPuv-infected BALB/c mice elicits gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion by CD8+ T cells. Thus, R. typhi GFPuv bacteria are a novel, potent tool to study infection with the pathogen in vitro and in vivo and the immune response to these bacteria.

scholarly journals Properties and Construction of Plasmid pFW213, a Shuttle Vector with the Oral Streptococcus Origin of Replication

2011 ◽  
Vol 77 (12) ◽  
pp. 3967-3974 ◽  
Author(s):  
Yi-Ywan M. Chen ◽  
Hui-Ru Shieh ◽  
Cheng-Tzer Lin ◽  
Shin-Yi Liang
Keyword(s):  
Shuttle Vector ◽  
Content Type ◽  
Reading Frame ◽  
E Coli ◽  
Chromosome Sequence ◽  
Theta Replication ◽  
The Stability ◽  
Functional Analyses ◽  
Replication Intermediates ◽  
Derivatives Of

ABSTRACTStreptococcus parasanguinisis among the most successful colonizers of the human body. Strain FW213 harbors a 7.0-kb cryptic plasmid, pFW213, with a copy number at 5 to 10 per chromosome. Sequence and functional analyses of pFW213 revealed that the open reading frame (ORF) encoding the replication protein (Rep) is essential for the replication of pFW213, and the putative plasmid addiction system (RelB and RelE) and an ORF (ORF6) with no known function are required for its stability. The minimal replicon of pFW213 contains therepgene and its 5′-flanking 390-bp region. Within the minimal replicon, an A/T-rich region followed by 5 contiguous 22-bp repeats was located 5′ of the ATG ofrep.No single-stranded replication intermediates were detected in the derivatives of pFW213, suggesting that pFW213 replicates via the theta replication mechanism. The minimal replicon was unstable in streptococcal hosts without selection, but the stability was greatly enhanced in derivatives containing the intactrelBEgenes. AStreptococcus-Escherichia colishuttle vector, pCG1, was constructed with the pFW213 replicon. Plasmid pCG1 features a multiple cloning region and a spectinomycin resistance determinant that is expressed in bothStreptococcusspp. andE. coli. Various streptococcal DNA fragments were cloned in pCG1, and the recombinant constructs were stably maintained in the streptococcal hosts. Since pCG1 is compatible with the most widely used streptococcal replicon, pVA380-1, pCG1 will provide a much needed tool allowing the cloning of two genes that work in concert in the same host.

scholarly journals A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

2016 ◽  
Vol 82 (14) ◽  
pp. 4421-4428 ◽  
Author(s):  
Gina L. Lipscomb ◽  
Jonathan M. Conway ◽  
Sara E. Blumer-Schuette ◽  
Robert M. Kelly ◽  
Michael W. W. Adams
Keyword(s):  
Plant Biomass ◽  
Genetic Manipulation ◽  
Optimal Growth ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Content Type ◽  
Background Strain ◽  
Caldicellulosiruptor Bescii ◽  
Kanamycin Selection ◽  
Integrating Vector

ABSTRACTCaldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in thepyrFgenome region. ThepyrFgene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand theC. besciigenetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs inC. bescii. These strains showed resistance to kanamycin at concentrations >50 μg · ml−1, whereas wild-typeC. besciiwas sensitive to kanamycin at 10 μg · ml−1. In addition, placement of the Cbhtkmarker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of thepyrEgene in wild-typeC. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. ThepyrEgene functioned as a counterselectable marker, likepyrF, and was used together with Cbhtkin the ΔpyrEbackground strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme.IMPORTANCECaldicellulosiruptor besciiis a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used withC. besciiis based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system forC. besciiusing the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism.

scholarly journals Enhanced Butanol Production Obtained by Reinforcing the Direct Butanol-Forming Route in Clostridium acetobutylicum

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Yu-Sin Jang ◽  
Jin Young Lee ◽  
Joungmin Lee ◽  
Jin Hwan Park ◽  
Jung Ae Im ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Clostridium Acetobutylicum ◽  
Metabolic Flux ◽  
Genetic Manipulation ◽  
High Titer ◽  
Strain Improvement ◽  
High Yield ◽  
Butanol Production ◽  
Wild Type ◽  
Content Type

ABSTRACTButanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation byClostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of theptaandbukgenes, encoding phosphotransacetylase and butyrate kinase, while theadhE1D485Ggene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD**) strain. By reinforcing the direct butanol-forming flux inC. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain within siturecovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production inC. acetobutylicumdescribed here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer.IMPORTANCERenewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered development of engineered clostridia having highly efficient and selective butanol production capability. Here we report for the first time the results of systems metabolic engineering studies of two butanol-forming routes and their relative importances in butanol production. Based on these findings, a metabolically engineeredClostridium acetobutylicumstrain capable of producing butanol to a high titer with high yield and selectivity could be developed by reinforcing the direct butanol-forming flux.

scholarly journals Use of a Novel Escherichia coli-Leuconostoc Shuttle Vector for Metabolic Engineering of Leuconostoc citreum To Overproduce d-Lactate

2012 ◽  
Vol 79 (5) ◽  
pp. 1428-1435 ◽  
Author(s):  
Han Seung Chae ◽  
Seung Hwan Lee ◽  
Ju-Hoon Lee ◽  
Si Jae Park ◽  
Pyung Cheon Lee
Keyword(s):  
Escherichia Coli ◽  
Lactococcus Lactis ◽  
Shuttle Vector ◽  
Plasmid Vector ◽  
Open Reading Frames ◽  
Erythromycin Resistance ◽  
Content Type ◽  
Enhanced Production ◽  
Leuconostoc Citreum ◽  
Theta Replication

ABSTRACTDetermination of the complete nucleotide sequence of a cryptic plasmid, pMBLT00, fromLeuconostoc mesenteroidessubsp.mesenteroidesKCTC13302 revealed that it contains 20,721 bp, a G+C content of 38.7%, and 18 open reading frames. Comparative sequence and mung been nuclease analyses of pMBLT00 showed that pMBLT00 replicates via the theta replication mechanism. A new, stableEscherichia coli-Leuconostocshuttle vector, pMBLT02, which was constructed from a theta-replicating pMBLT00 replicon and an erythromycin resistance gene of pE194, was successfully introduced intoLeuconostoc,Lactococcus lactis, andPediococcus. This shuttle vector was used to engineerLeuconostoc citreum95 to overproduced-lactate. TheL. citreum95 strain engineered using plasmid pMBLT02, which overexpressesd-lactate dehydrogenase, exhibited enhanced production of optically pured-lactate (61 g/liter, which is 6 times greater than the amount produced by the control strain) when cultured in a reactor supplemented with 140 g/liter glucose. Therefore, the shuttle vector pMBLT02 can serve as a useful and stable plasmid vector for further development of ad-lactate overproduction system in otherLeuconostocstrains andLactococcus lactis.

scholarly journals Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Kensuke Shima ◽  
Mary M. Weber ◽  
Christiane Schnee ◽  
Konrad Sachse ◽  
Nadja Käding ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Genetic Manipulation ◽  
Shuttle Vector ◽  
Chlamydia Psittaci ◽  
Zoonotic Transmission ◽  
Vector System ◽  
Multiple Cloning Site ◽  
Host Pathogen Interactions ◽  
Content Type ◽  
Host Pathogen

ABSTRACT The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in C. psittaci infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of C. psittaci using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian C. psittaci strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry includes genes encoding the green fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Target genes can be inserted at a multiple cloning site (MCS). Importantly, these genes can be regulated by a tetracycline-inducible (tet) promoter. Using the pCps-Tet-mCherry plasmid shuttle vector, we show the expression of GFP, as well as the induction of mCherry expression, in C. psittaci strain 02DC15, which belongs to the avian C. psittaci 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions. IMPORTANCE Psittacosis, caused by avian C. psittaci, has a major economic impact in the poultry industry worldwide and represents a significant risk for zoonotic transmission to humans. In the past decade, the tools of genetic manipulation have been improved for chlamydial molecular studies. While several genetic tools have been mainly developed in Chlamydia trachomatis, a stable gene-inducible shuttle vector system has not to date been available for C. psittaci. In this study, we adapted a C. trachomatis plasmid shuttle vector system to C. psittaci. We constructed a C. psittaci plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in C. psittaci. Importantly, exogeneous genes can be inserted at an MCS and are regulated by a tet promoter. The application of the pCps-Tet-mCherry shuttle vector system enables a promising new approach to investigate unknown gene functions of this pathogen.

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