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. 2007 Sep;189(17):6447-56.
doi: 10.1128/JB.00657-07. Epub 2007 Jun 29.

Escherichia Coli K1-specific Bacteriophage CUS-3 Distribution and Function in Phase-Variable Capsular Polysialic Acid O Acetylation

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Escherichia Coli K1-specific Bacteriophage CUS-3 Distribution and Function in Phase-Variable Capsular Polysialic Acid O Acetylation

Michael R King et al. J Bacteriol. .
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Abstract

Escherichia coli K1 is the leading cause of human neonatal sepsis and meningitis and is important in other clinical syndromes of both humans and domestic animals; in this strain the polysialic acid capsule (K1 antigen) functions by inhibiting innate immunity. Recent discovery of the phase-variable capsular O acetylation mechanism indicated that the O-acetyltransferase gene, neuO, is carried on a putative K1-specific prophage designated CUS-3 (E. L. Deszo, S. M. Steenbergen, D. I. Freedberg, and E. R. Vimr, Proc. Natl. Acad. Sci. USA 102:5564-5569, 2005). Here we describe the isolation and characterization of a CUS-3 derivative (CUS-3a), demonstrating its morphology, lysogenization of a sensitive host, and the distribution of CUS-3 among a collection of 111 different K1 strains. The 40,207-bp CUS-3 genome was annotated from the strain RS218 genomic DNA sequence, indicating that most of the 63 phage open reading frames have their closest homologues in one of seven different lambdoid phages. Translational fusion of a reporter lacZ fragment to the hypervariable poly-Psi domain facilitated measurement of phase variation frequencies, indicating no significant differences between switch rates or effects on rates of the methyl-directed mismatch repair system. PCR analysis of poly-Psi domain length indicated preferential loss or gain of single 5'-AAGACTC-3' nucleotide repeats. Analysis of a K1 strain previously reported as "locked on" indicated a poly-Psi region with the least number of heptad repeats compatible with in-frame neuO expression. The combined results establish CUS-3 as an active mobile contingency locus in E. coli K1, indicating its capacity to mediate population-wide capsule variation.

Figures

FIG. 1.
FIG. 1.
TEM of negatively stained CUS-3 phage particles. Magnification: ×150,000 (A) and ×200,000 (B). The white arrow points to the tip of the portal with emanating polysialic acid depolymerase protein endo-N. Particle morphology is similar to that of the lytic K1-specific phage K1F (32), as well as podoviruses in general.
FIG. 2.
FIG. 2.
Virtual and real endonuclease digestion of CUS-3 DNA confirms phage isolation. CUS-3 particles from an EV36 lysate were collected by differential centrifugation, and the DNA was subjected to digestion with EcoR1 (lane 2). The virtual digestion profile of DNA from nucleotides 2621662 through 2661871 of the strain RS218 chromosomal contig is identical (lane 4) to the actual digestion product (lane 2). Lambda HindIII (lane 1) and Promega 1-kb ladder (lane 3) are shown for comparison. The sizes (in bp) of the virtual digestion products are given on the right.
FIG. 3.
FIG. 3.
Genetic organization and homologies of CUS-3. A. ORFs are shown as numbered, color-coded arrows indicating the predicted directions of transcription. Each color refers to the indicated phage with the closest homologue shown below the line, which indicates the CUS-3 region of the RS218 chromosome from attL to attR. ORFs with uncertain identity are shown in black, as are nucleotide boundary sequences (bsq). The white box in ORF3 indicates continuation of the bsq1 sequence defining the N terminus of the encoded endo-N structural gene product. B. Pustell matrix analysis (MacVector 9.0.2) of CUS-3 versus HK620. The identity diagonal from the upper left to lower right corner indicates nucleotide similarity between the respective phage genomes shown with the same orientation as the CUS-3 genome in panel A. Analysis parameters for a window size of 30 nucleotides were as follows: minimum score of 65%, hash value of 6, jump of 3.
FIG. 4.
FIG. 4.
Poly-Ψ domain length dictates the neuO reading frame. Strains harboring plasmids overexpressing neuO with the indicated numbers of AAGACTC repeats (poly-Ψ) were induced with IPTG (even-numbered lanes). Whole-cell extracts of uninduced (odd-numbered lanes) and induced strains were fractionated by polyacrylamide gel electrophoresis, and polypeptides were visualized by staining with Coomassie dye as previously described (34). Sizes of molecular mass markers (lane M) are given by on the left. Relative NeuO activity was measured in induced extracts as described previously, where a minus sign indicates no detectable transacetylation of a model substrate (11). Asterisks indicate overproduced, truncated gene products with molecular masses expected from the neuO sequence (accession number AY779018). The predicted mass of active NeuO (not visible in lane 6) is ca. 32 kDa. Each pair of extracts, starting from lane 1, represents the results obtained with plasmids pSX788, pSX789-1, pSX790, and pSX789-2, respectively.
FIG. 5.
FIG. 5.
Poly-Ψ variation in successive B→W transitions. The schematic diagram at the top indicates repeated passage of a founder B colony, chosen at random and found to have a domain length of 18 repeats (third lane from left). W colonies were chosen randomly and analyzed after successive switches. Poly-Ψ domain lengths were estimated as previously described (11), with ΦX174 HinFI and HaeIII size markers (first two left lanes, respectively) shown in bp on the left. Note that with the exception of the W colony at step 1, there was one predominant domain amplified after each successive switch.
FIG. 6.
FIG. 6.
Poly-Ψ domain length in strain C375. Primers amplifying the poly-Ψ domain (lane 1) or complete neuO gene (lane 2) were used to amplify DNA recovered from nonviable, freeze-dried samples of strain C375. The bracket at left indicates the region of the gel that would detect poly-Ψ domains with 1 to 40 5′-AAGACTC-3′ repeats. Lanes 3 and 4 represent ΦX174 HinFI and HaeIII size markers, respectively, with lengths given in bp on the right.

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