Construction of p16S lux , a Novel Vector for Improved Bioluminescent Labeling of Gram-Negative Bacteria

Abstract

A novel vector has been constructed for the constitutive luminescent tagging of gram-negative bacteria by site-specific integration into the 16S locus of the bacterial chromosome. A number of gram-negative pathogens were successfully tagged using this vector, and the system was validated during murine infections of living animals.Bioluminescent imaging (BLI) has become a valuable tool for studying bacterial infections in real time in small animal models. Various species of gram-negative bacteria have been rendered luminescent using a variety of approaches. The species include Escherichia coli (3,4,11,12), Salmonella enterica serovar Typhimurium (1, 2), Yersinia enterocolitica (7), Brucella melitensis (9), Pseudomonas aeruginosa (5), and Citrobacter rodentium (13). In E. coli, both firefly luciferase (3, 4) and the bacterial lux system (11) have been used to provide the luminescent signal. However, the use of firefly luciferase is somewhat limited in that the substrate for the reaction (luciferin) must be added exogenously. For this reason, most researchers favor the use of the lux operon from Photorhabdus luminescens, in which the substrate for the luminescence reaction is synthesized endogenously, allowing accurate real-time in vivo tracking of bacterial localization. To date, the lux operon has been provided either on a plasmid (2-4, 11, 12) or via transposon mutagenesis (1,5,7,9,13). However, both of these methods have significant limitations. When animals are infected with strains carrying a bioluminescence plasmid and monitored over several days, there is the possibility of plasmid loss, resulting in underrepresentation of bacterial infection. When transposon mutagenesis is utilized, a bank of transposon integrants must be screened for strains with sufficient light emission, and the integrating transposon has the potential to influence local gene expression, bacterial fitness, and bacterial pathogenesis.We therefore sought to develop a method to reproducibly label gram-negative bacteria by site-directed chromosomal integration using a constitutively expressed luminescence reporter system.We recently reported the construction of pPL2luxP help , a chromosomal integration vector containing a synthetic lux operon derived from Photorhabdus luminescens (where P help indicates a highly expressed Listeria promoter) (8) for real-time monitoring of Listeria monocytogenes infections in mice (10). Our construct was based on combining the lux operon with the backbone of pGh9::ISS1, a thermosensitive E. coli/gram-positive shuttle vector which integrates randomly into the bacterial chromosome as a consequence of the presence of ISS1 (6). To construct p16Slux, a fragment containing the constitutive luxP help construct was cloned in pGh9::ISS1, yielding pGhlux. The ISS1 element was then excised and replaced with a fragment of the E. coli DH10B 16S rRNA genes, obtained by PCR using KOD Hot Start DNA polymerase (Merck, Nottingham, United Kingdom) primers 16S_fwd_Econew (5Ј-CTGATGAATTCCAGGTGTAGCGGT GAAATG-3Ј) and 16S_rev_XhoI ...

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STAT3 Activation in Th17 and Th22 Cells Controls IL-22–Mediated Epithelial Host Defense during Infectious Colitis

“…For our study, the bioluminescent strain ICC 169 of C. rodentium was used (30). Inoculation was performed via oral gavage of 8 3 10 9 CFU in a total volume of 200 ml PBS.…”

Bacterial luciferase reporters: The Swiss army knife of molecular biology

“…53,75 For the vast majority of organisms investigated by BLI, luminescence correlates well with bacterial numbers or colony forming units during active growth in vitro. However, in a number of organisms a significant drop in luminescence was observed during batch cultures upon entering stationary growth phase possibly due to a change in the availability of co-factors, substrate or both 49,94,96 (Riedel CU, unpublished observation). Since bacteria are actively replicating during infection, this problem has no consequences for in vivo BLI of bacterial infections.…”

“…93 Moreover, we were able to use the P help lux construct to tag various Gram-negative bacteria using a plasmid that integrates into a 16S ribosomal RNA gene. 94 This has allowed for the in vivo monitoring infections of Citrobacter rodentium, P. aeruginosa and S. typhimurium. 94,95 It is worth mentioning, that the use of a defined integration site has several advantages over transposon-based systems besides avoiding problems with the virulence traits of a bacterium.…”

“…94 This has allowed for the in vivo monitoring infections of Citrobacter rodentium, P. aeruginosa and S. typhimurium. 94,95 It is worth mentioning, that the use of a defined integration site has several advantages over transposon-based systems besides avoiding problems with the virulence traits of a bacterium. By using a defined promoter and integration at a defined site a direct comparison of different strains is possible as shown for three different strains of L. monocytogenes (10403S, EGDe, F2365) 46 or the a ΔagrD mutant and its isogenic wild type parent strain.…”