Thursday, November 15, 2012

Role of rpoS in E. coli 0157:H7 strain H32 biofilm development and survival

Bacterial strain development can be aided by EZ-Tn5™ Transposomes for generating mutations in certain genes for study of important characteristics. It is well-known that the E. coli strains that harbor the serotype O157:H7 can cause several diseases in humans; the strain studied by Sheldon et al. carries the rpoS gene that controls cell survival in pathogenic E. coli by providing resistance factors to various stressors, and is also responsible for biofilm development. In this study, a custom transposome was constructed using the EZ-Tn5™ Custom Transposome™ Construction Kits in the pMOD-2 vector and contained a Kanamycin resistance gene and a GFP gene. The transposon was complexed with the EZ-Tn5 Transposase to form the Transposome, which was electroporated into the H32 strain of E. coli O157:H7 using standard procedures. Kanamycin-resistant mutants were isolated and the locations of the transposon insertion points in the chromosome determined by sequencing. The GFP functionality allowed easy quantification of microbes in the biofilms by confocal microscopy. A subset of the GFP-containing cells was further modified to be RpoS-deficient using lambda-Red recombinase to inactivate the rpoS gene to provide a suitable control to compare with the GFP-tagged, RpoS wild type E. coli. Both strains of the E. coli cells were further used to study biofilm formation and compared for survivability in samples of water from various sources. Biofilm mass formation and cell survivability were assayed.

The researchers found that the protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. Confocal scanning laser microscopy revealed a nutrient-dependent role of rpoS in biofilm formation. The enhanced biofilm formation of the rpoS mutant did not translate to increased survival in sterile double-distilled water, filter-sterilized lake water, or unfiltered lake water. While the rpoS recombinant mutant showed an overall cell viability reduction the wild type cells showed significantly less loss of viability, depending on the water source. However, the survival rates of the detached biofilm-derived rpoS+ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.
ResearchBlogging.orgSheldon JR, et al. (2012). Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival. Applied and environmental microbiology, 78 (23), 8331-9 PMID: 23001657

Friday, November 9, 2012

Tn5 as an Insect Gene Vector

Few non-microbial successes for the use of EZ-Tn5™ transposons have been reported, and it is worth going back into the archives to highlight novel uses of the EZ-Tn5™ system (previously known as EZ::Tn™). David O’Brochta and his research team at the University of Maryland report success on using a custom EZ-Tn5 transposon to transfect and insert a custom transposon into the nucleus of the mosquito Aedes aegypti (strain Orlando). The transposon was constructed using the EZ-Tn5 pMOD-2 vector and was developed to include the following markers: (1) The DsRed gene, a marker that conveys autofluorescence when expressed, and (2) a brain and ganglion-specific promoter to tag the Aedes aegypti germ line. A second round of experiments used the EZ-Tn5 KAN-1 Transposome  (since replaced with the EZ-Tn5 KAN-2 Transposome product) to tag the plasmid pUCSacRB, previously documented as a useful tool in determining resistance to sucrose at concentrations >5%. Insertion of the Transposome into the SacRB gene of the plasmid inactivates it and results in resistance to sucrose-based lysis. The plasmid was transfected into the embryonic cells by microinjection. The embryos were permitted to develop after which the gDNA was isolated and transformed into E. coli DH10B and Kanamycin-resistant clones isolated. Evidence for the physical integration of the Tn5::3xP3DsRed element was obtained from Southern hybridization and transposon display experiments.

The authors found that further optimization of the system for the control of self-integration and transposition efficiency would be useful. The results demonstrate the successful use of the EZ-Tn5 Transposon system for developing transposon-based mutagenesis assays for non-bacterial gene expression studies.

ResearchBlogging.orgRowan KH et al. (2004). Tn5 as an insect gene vector. Insect biochemistry and molecular biology, 34 (7), 695-705 PMID: 15242711

Thursday, November 1, 2012

Transposon mutagenesis identifies uropathogenic Escherichia coli biofilm factors

Transposon mutagenesis using EZ-Tn5™ Transposomes has been a common method for determining gene activity and location of pathogenesis features in medically important bacteria. Hadjifrangiskou et al. demonstrate the use of the <EZ-Tn5 <R6Kγori/KAN-2> Tnp Transposome Kit to identify and rescue a specific pathogenesis marker in uropathogenic E. coli that appears to be responsible for biofilm formation in catheters and biotic surfaces. The biofilms in these environments are responsible for rapid formation of intracellular bacterial communities within bladder epithelial cells and protects the communities from the host immune system; what remains unknown is the nature of the interaction and relationship between the intracellular bacterial communities and what factors are responsible for biofilm buildup inside the human host. It is also well known that the presence of flagella and pili are essential for biofilm formation, and using transposon mutagenesis with the concept of knocking out flagellar “motor” genes may have an impact on biofilm formation.

To search for markers responsible for this phenomenon, three different strains were transformed with the EZ-Tn5 <R6Kλori/KAN-2> Transposome. Mutant E. coli that showed biofilm defects after mutagenesis were selected and the insertion points of the transposon identified through multiple rounds of sequencing using a modified Random Amplification of Transposon Ends (RATE procedure). Other assays performed to determine loss of biofilm formation included motility assays, Hemagglutination assays, immunoblot analysis, and mouse infections followed by microscopy to characterize any molecular changes that arose from the mutagenesis procedure.  The results showed numerous changes in biofilm formation and bacterial consortia assembly characteristics due to the transposon insertional mutagenesis, resulting in reduced to total loss of the ability to form bacterial communities, and a subgroup of these mutants generated with the EZ-Tn5 Transposome were unable to form  biofilms when tested in a mouse model system.

The experiments reveal that a complex variety of regulatory and effector genes that express under multiple conditions, again demonstrating the utility of Transposon mutagenesis using EZ-Tn5 Transposomes for elucidating bacterial pathogenesis factors.

ResearchBlogging.orgHadjifrangiskou, M. et al. (2012). Transposon Mutagenesis Identifies Uropathogenic Escherichia coli Biofilm Factors. Journal of bacteriology, 194 (22), 6195-205 PMID: 22984258