Genomic characterization of non-O1, non-O139 Vibrio cholerae reveals genes for a type III secretion system

Dziejman et al. 10.1073/pnas.0409918102.

Supporting Information

Files in this Data Supplement:

Supporting Text

Construction of AM-19226 Genomic Library. A total of 150 m g of AM-19226 genomic DNA was prepared by standard methods and sheared by nebulization into fragments ranging from 1.5 to 4 kb in size. The DNA fragment ends were blunted by incubation with T4 Polymerase at 25°C for 10 min, followed by addition of Klenow polymerase and incubation first for 10 min at 25°C, and then for 2 h at 16°C. Repaired DNA fragments of 2-4 kb in size were recovered by agarose gel electropphoresis and extraction by using a QIAquick gel extraction kit (Qiagen, Valencia, CA). The DNA was then ligated into the pSMART by vector using the Lucigen CloneSmart system according to manufacturer’s specifications, followed by electroporation into cells and plating on LB agar containing ampicillin. A Genetix Q-Pix robot was then used to pick individual colonies into 96-well round-bottom plates containing 200 m l of LB containing 120 m g/ml ampicillin. Plates were shaken for 18 h at 37°C and transferred to a BIOMEK FX for plasmid purification using the SPRINTPREP system (Agencourt) according to manufacturer’s protocols. The resulting plasmid DNA was resuspended in 50 m l of deionized water and transferred to a 384-well plate for sequencing.

Primer Pairs. The following primer pairs (U = upstream primer, D = downstream primer) were used to produce DNA for use as probes: U-vcsN2, ATGCGTCGACAGTTGAGCCAATTCCATT; D-vcsN2, ATGCTCTAGACGACCAAACGAGATAATG; U-vcsC2, ATGCGTCGACGTTACCGATGCTATGGGT; D-vcsC2, ATGCTCTAGAAGTCGGTTGTTTCGGTAA; U-vcsV2, ATGCAGATCTTTTGGCTCACTTGATGGG; D-vcsV2, ATGCGTCGACGCCACATCATTGCTTGCT; U-vspD, ATCGTCTAGAACTCGAAGAGCAGAAAAAAGC; D-vspD, ATCGGTCGACCTTCCCGCTTTTGATGAAATG.

Fig. 4. Southern Analysis of V. cholerae non-O1, non-O139 strains. Genomic DNA from each strain was digested with EcoRI and transferred to a nylon membrane, which was probed by using PCR products generated from strain AM-19226 ORFs vcsV2 and vspD. Lanes 1, 22, and 37 do not contain genomic DNA, and lane 38 contains genomic DNA from the TTSS⁺ V. parahaemolyticus strain VP47. A complete description of each lane can be found in Table 5.

Table 6. This spreadsheet represents the absent (yellow) and present (blue) assignments for individual genes for each strain based on array analysis, and is included to provide a higher resolution of Fig. 1. Unless indicated in the text, assignments have not been validated by other methods. Therefore, absent gene assignments may in some cases reflect sequence divergence. The results are derived from the fluorescence ratios for at least three independent arrays for each strain.

• Early Edition
• Archives
• Online Submission

• Feature Articles

  (Expanded research articles of exceptional breadth)

• Commentaries

  (Expert commentary on leading research)

• Letters

  (Online-only letters to the editor)

• Inaugural Articles

  (Articles by recently elected NAS members)

• PNAS Profiles

  (Biographical profiles of Academy members)

• Sustainability Science

  (Interactions of human and environmental systems)

• Collected Papers

  (Editorials, Perspectives, Reviews, Colloquia, etc.)

• Special Features

  (Solicited articles on exceptional research topics)

• Sackler Colloquia

  (Scientific reports held under NAS auspices)

• Podcasts

  Brief podcasts with leading scientists