Efficient Electrotransformation of Bacteroides fragilis

Abstract

This study describes refined electroporation parameters for efficient transformation of Bacteroides fragilis by plasmids prepared from laboratory strains of Escherichia coli. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four E. coli-Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable for B. fragilis transformation, and it generated 2-to 900-fold more transformants (about 10 4 transformants per g pLYL05 DNA) than the other plasmids. Numerous microbes inhabit the human intestine, and the number of microbial cells can be nearly 10 11 cells per g of feces. The colon is the most densely populated environment in the human body, and the genus Bacteroides, which contains Gramnegative, obligate anaerobes, is one of the most abundant genera in the intestinal microbiota. In the clinical setting, Bacteroides strains are considered opportunistic pathogens that can occasionally cause infections, such as peritoneal abscesses, appendicitis, and septicemia (3,4,23). In the genus Bacteroides, B. fragilis is considered the most virulent species, and its capsular polysaccharides are especially linked to its pathogenesis (8, 13, 22). However, recent reports have highlighted the symbiotic properties of B. fragilis since the capsular polysaccharide displayed by this species can modify the human immune system (21). Another example of a host-Bacteroides interaction is a B. thetaiotaomicron interaction; this species can repress host inflammatory responses by promoting the binding of peroxisome proliferator-activated receptor gamma (PPAR-␥) to the NF-b subunit RelA (11). B. thetaiotaomicron is also known to induce production of antimicrobial peptides from Paneth cells in host intestinal epithelia (10).The recent accumulation of genomic information about several Bacteroides species (2) has enabled us to search for the genes responsible for the symbiotic properties of intestinal Bacteroides described above. However, screening for genes involved in host-Bacteroides symbioses requires development of a suitable, simple, and efficient genetic manipulation system. Although transconjugation by filter mating can be performed with Bacteroides species (19, 24 ), this technique is labor-intensive and time-consuming. Electroporation is a simple and reproducible procedure that is widely used for introduction of foreign nucleic acids into various bacteria. While Smith et al. previously described an electroporation protocol for Bacteroides species that could transform Bacteroides with plasmids from homologous hosts, plasmids derived from Escherichia coli laboratory strains could be transformed into Bacteroides only with difficulty (20). Recently, Patrick et al. described an efficient electrotransformation method for B. fragilis using purified T7 antirestriction protein Ocr...

PhoB Regulates the Survival of Bacteroides fragilis in Peritoneal Abscesses

“…Briefly, DNA fragments upstream and downstream of the region being deleted were separately PCR-amplified and fused by a second PCR amplification via an overlapping regions incorporated into the primer sequences. The resultant PCR products were ligated into pKK100 [18], [19]. The targeting plasmids were electroporated into B. fragilis strain YCH46 as described previously [18], [19].…”

“…The resultant PCR products were ligated into pKK100 [18], [19]. The targeting plasmids were electroporated into B. fragilis strain YCH46 as described previously [18], [19]. The diploids, in which targeting plasmid integrated into the chromosome via a single genetic crossover, were selected on GAM agar plates containing Em.…”

Characterization of a gene cluster for sialoglycoconjugate utilization in <I>Bacteroides fragilis</I>

“…The resultant PCR products were ligated into pKK100 (23). The targeting plasmids were electroporated into B. fragilis YCH46 as described previously (23,24). Diploids, in which the targeting plasmid integrated into the chromosome via a single genetic crossover, were selected on GAM agar plates containing Em.…”

Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation

“…Markerless gene deletion using the double-crossover method has been achieved in various bacteria (19,28,31,35,38). Generally, a low frequency of the second crossover event is an obstacle to successful gene deletion, and strategies for efficiently selecting second-crossover recombinants are required.…”