Phage-encoded colanic acid-degrading enzyme permits lytic phage infection of a capsule-forming resistant mutant Escherichia coli strain

Abstract

In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria.

FIG 1

Isolation of a T7-resistant mutant (S3 mt) overproducing colanic acid. (A) Total RNA from wild-type or mutant E. coli was isolated, and mRNA levels of rcsA, lon, and wza were measured using real-time reverse transcription-PCR. Glyceraldehyde-3-phosphate dehydrogenase was used as the internal control. Three independent experiments were performed. (B) l-Fucose from wild-type and mutant bacteria was quantified. Three independent experiments were performed. (C) Bacteria were stained with phosphotungstic acid and were observed under a transmission electron microscope. Bars, 0.5 μm.

FIG 2

Point mutation in the lon gene promoter in S3 mutant bacteria. (A) The lon promoter regions from wild-type (wt) and mutant (S3 mt) bacteria were PCR amplified, and sequences were aligned (ClustalW). (B) Reporter plasmids containing the luciferase gene under the control of either the wild-type or the mutant lon promoter were constructed. Each plasmid was transformed into wild-type bacteria, and a luciferase assay was performed. Three independent experiments were performed. (C) The wild-type lon promoter and lon gene were cloned into the pBluescript vector and were used for the transformation of S3 mutant bacteria. Colanic acid from the wild type, the S3 mutant, and the transformant was quantified. Three independent experiments were performed.

FIG 3

Increase in biofilm formation by an E. coli phage-resistant mutant. (A) Wild-type or S3 mutant bacteria were cultured in 96-well polystyrene plates for 24 h. The culture medium was discarded, and the surface was stained with 0.4% crystal violet. (B) Wild-type bacteria were grown in 96-well polystyrene plates either in the presence or in the absence of phage T7. Biofilm was measured by staining with 0.4% crystal violet. (C) Wild-type bacteria were grown in the presence of phage T7 for 48 h. Biofilm was scraped, streaked onto an LB plate, and allowed to form colonies. Ten colonies were picked, and colanic acid from each colony was quantitated.

FIG 4

S3 mutant bacteria inhibited adsorption of phage T7. (A) Wild-type and S3 mutant bacteria were infected with phage T7 at an MOI of 0.1, and free phages in the culture were quantitated at the indicated time points. Three independent experiments were performed. (B) Genomic DNA of T7 was isolated and was used for electroporation into wild-type or S3 mutant bacteria. Cell growth was observed at 0 and 2 h postinfection in a spectrophotometer (600 nm). Plaques were observed on plates from lysates obtained after a 2-h incubation.

FIG 5

Bacteriophage PBECO4 infects E. coli mutant S3. (A) Growth of phage PBECO4 in the E. coli mutant S3 was observed. (B) The indicated amounts of purified recombinant CAE (product of PBECO4 ORF547) were added to cultures of S3 mutant bacteria. Colanic acid production from each culture was quantitated. (C) Purified recombinant CAE (product of PBECO4 ORF547) was added to cultures of S3 mutant bacteria for the indicated times. Colanic acid production from each culture was quantitated.

FIG 6

Cocktail effect of phages T7 and PBECO4. (A) S3 mutant bacteria were infected with either T7, PBECO4, or a cocktail (T7 plus PBECO4), and the resulting plaques were observed. Black arrows indicate typical (small) plaques of PBECO4; white arrows indicate larger plaques, not typical of PBECO4. (B) Genomic DNA was isolated from plaques of T7, PBECO4, or the larger-plaque phage in the cocktail treatment. PCR was performed with T7-specific primers. (C) Growth of the E. coli S3 mutant in the presence of either T7, PBECO4, or the phage cocktail. (D) Growth of the T7-resistant mutant of an E. coli clinical isolate (F-939) in the presence of either T7, PBECO4, or the phage cocktail. (E) Cocktail effect of purified recombinant colanic acid-degrading enzyme (CAE) and phage T7. The growth of the E. coli S3 mutant with the indicated amounts of CAE was observed. Three independent experiments were performed. (F) S3 mutant bacteria were incubated in a polystyrene 96-well plate in the presence of either SM buffer alone, T7, PBECO4, or a cocktail (T7 plus PBECO4) for 24 h. Biofilm was measured after staining with 0.4% crystal violet. (G) Wild-type bacteria were incubated in a polystyrene 96-well plate in the presence of either SM buffer alone, T7, PBECO4, or a cocktail (T7 plus PBECO4) for 48 h. Biofilm was measured after staining with 0.4% crystal violet.