Abstract:
UNLABELLED:Our goal was to develop a robust tagging method that can be used to track bacterial strains in vivo To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonella and Escherichia coli and Tn7 transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ(70)-dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7 system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10(-8) to 10(-10) in Salmonella enterica serovar Typhimurium and E. coli APEC O1, whereas the existing Tn7 system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivo animal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics. IMPORTANCE:This article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system in Salmonella and E. coli, and we predict that it will be widely applicable to other bacterial strains within the Enterobacteriaceae This technology will allow for improved in vivo analysis of bacterial pathogens.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Shivak DJ,MacKenzie KD,Watson NL,Pasternak JA,Jones BD,Wang Y,DeVinney R,Wilson HL,Surette MG,White APdoi
10.1128/AEM.01346-16subject
Has Abstractpub_date
2016-07-29 00:00:00pages
4931-43issue
16eissn
0099-2240issn
1098-5336pii
AEM.01346-16journal_volume
82pub_type
杂志文章abstract::Symbiotic microorganisms are widespread in nature and can play a major role in the ecology and evolution of animals. The aphid-Serratia symbiotica bacterium interaction provides a valuable model to study the mechanisms behind these symbiotic associations. The recent discovery of cultivable S. symbiotica strains with a...
journal_title:Applied and environmental microbiology
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doi:10.1128/AEM.02445-18
更新日期:2019-05-02 00:00:00
abstract::Lyophilizatin of 13 cyanobacterial cultures belonging to seven genera was attempted in a variety of suspending substances. All organisms survived lyophilization when suspended in lamb serum. Some of the organisms could be successfully lyophilized in horse serum, beef serum, fetal bovine serum, or human ascites fluid. ...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.32.6.777-780.1976
更新日期:1976-12-01 00:00:00
abstract::The conversion of ochratoxin C to ochratoxin A was studied in rats after oral and intravenous administration. The concentration of ochratoxin A in the blood as a function of time was the same after oral administration of equivalent amounts of either ochratoxin C or ochratoxin A. The maximum ochratoxin A concentrations...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.48.1.41-42.1984
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abstract::Similar populations of hypochlorite-treated spores were enumerated from two crops of Clostridium botulinum 12885A produced by the same procedure; however, germination required different L-alanine concentrations. Lactate permitted the germination of spores from both crops with suboptimal L-alanine concentrations. The d...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.45.4.1415-1417.1983
更新日期:1983-04-01 00:00:00
abstract::In Europe the use of the growth promoter avoparcin is considered to have selected for vancomycin-resistant enterococci (VRE). Sweden ceased using avoparcin in 1986, and only occasional cases of VRE from hospitals have been reported since 1995. Within the framework of a European study, samples from urban raw sewage, tr...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/aem.68.6.2838-2842.2002
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abstract::Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized t...
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doi:10.1128/AEM.02322-07
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abstract::Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing ...
journal_title:Applied and environmental microbiology
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doi:10.1128/AEM.01486-12
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abstract::Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC,...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.03015-10
更新日期:2011-05-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.64.10.4089-4092.1998
更新日期:1998-10-01 00:00:00
abstract::A modified glucoamylase gene (glaA) with an extra Thr- and Ser-rich Gp-I domain (T. Semimaru, M. Goto, K. Furukawa, and S. Hayashida, Appl. Environ. Microbiol. 61:2885-2890, 1995) was introduced into a mutant parental host, Aspergillus awamori var. kawachi, in which the original glaA gene had been completely deleted a...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.63.7.2940-2943.1997
更新日期:1997-07-01 00:00:00
abstract::In this study, we used chemostat cultures to analyze the quantitative effects of the specific growth rate and respiration on the metabolism in Lactococcus lactis CHCC2862 and on the downstream robustness of cells after freezing or freeze-drying. Under anaerobic conditions, metabolism remained homofermentative, althoug...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.02785-19
更新日期:2020-03-02 00:00:00
abstract::The genus Sepiola (Cephalopoda: Sepiolidae) contains 10 known species that occur in the Mediterranean Sea today. All Sepiola species have a light organ that contains at least one of two species of luminous bacteria, Vibrio fischeri and Vibrio logei. The two Vibrio species coexist in at least four Sepiola species (S. a...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/aem.66.8.3550-3555.2000
更新日期:2000-08-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.57.12.3429-3432.1991
更新日期:1991-12-01 00:00:00
abstract::The objective of this study was to develop a molecular detection method that better estimates the potential risk associated with the presence of Vibrio vulnificus. For that purpose, we applied seminested reverse transcription-PCR (RT-PCR) to viable but nonculturable (VBNC) populations of V. vulnificus and targeted the...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/aem.68.11.5641-5646.2002
更新日期:2002-11-01 00:00:00
abstract::The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in vi...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.05140-11
更新日期:2011-07-01 00:00:00
abstract::Many people in Japan often detect an unpleasant odor generated from laundry that is hung to dry indoors or when using their already-dried laundry. Such an odor is often described as a "wet-and-dirty-dustcloth-like malodor" or an "acidic or sweaty odor." In this study, we isolated the major microorganisms associated wi...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.07816-11
更新日期:2012-05-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.53.5.969-973.1987
更新日期:1987-05-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.53.12.2808-2814.1987
更新日期:1987-12-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.44.4.864-870.1982
更新日期:1982-10-01 00:00:00
abstract::In earlier studies, the pathway of toluene and m- and p-xylene degradation (TOL pathway) in Pseudomonas putida (pWW0) was found to be subject to catabolite repression when the strain was grown at the maximal rate on glucose or succinate in the presence of an inducer. This report describes catabolite repression of the ...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.62.2.601-606.1996
更新日期:1996-02-01 00:00:00
abstract::Wood-degrading fungi use a sequence of oxidative and hydrolytic mechanisms to loosen lignocellulose and then release and metabolize embedded sugars. These temporal sequences have recently been mapped at high resolution using directional growth on wood wafers, revealing previously obscured dynamics as fungi progressive...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.00159-18
更新日期:2018-08-01 00:00:00
abstract::Protease-negative variants were shown to outcompete the wild-type strains of Streptococcus cremoris E(8), HP, and Wg(2) at pH values higher than 6.0 in milk. For S. cremoris E(8) this process was studied in more detail. At lower pH values the wild type had a selective advantage. This pH-dependent selection was not fou...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.53.2.309-314.1987
更新日期:1987-02-01 00:00:00
abstract::Bacterial degradation of biphenyl and polychlorinated biphenyls proceeds by a well-studied pathway which produces benzoate and 2-hydroxypent-2,4-dienoate (or, in the case of polychlorinated biphenyls, the chlorinated derivatives of these compounds). Pseudomonas cepacia P166 utilizes 4-chlorobiphenyl for growth and pro...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.61.2.443-447.1995
更新日期:1995-02-01 00:00:00
abstract::A mutant of Pseudomonas sp. strain HBP1, originally isolated on 2-hydroxybiphenyl, was selected for the ability to grow on 2-propylphenol as the sole carbon and energy source. In the mutant strain, which was designated as Pseudomonas sp. strain HBP1 Prp, the cellular induction mechanism involved in the synthesis of th...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.59.3.860-866.1993
更新日期:1993-03-01 00:00:00
abstract::The preparation of DNA by alcohol precipitation in the presence of Na1 was used to enable the direct detection of Listeria monocytogenes in the amount of 10(3) CFU/0.5 g of sample. This procedure produces PCR-quality DNA directly from foods, such as soft cheese and minced meat. ...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.61.10.3745-3747.1995
更新日期:1995-10-01 00:00:00
abstract::A "repair-detection" procedure consisting of pour plating of food samples with Trypticase soy agar, followed by 1-h repair incubation at room temperature and subsequent overlay with violet red bile agar, was found to be an effective method for the detection of injuried and uninjuried coliforms from dairy products. Thi...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.35.4.820-822.1978
更新日期:1978-04-01 00:00:00
abstract::The role of light as an energy source for slightly aereated cultures of halobacteria was studied, using continuous cultures with low nutrient concentrations and a low oxygen supply. A series of experiments were carried out with non-illuminated and differently illuminated cultures and with different oxygen transfer rat...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.45.3.868-871.1983
更新日期:1983-03-01 00:00:00
abstract::A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportion...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.70.8.4432-4439.2004
更新日期:2004-08-01 00:00:00