Article Figures & Data
Figures
- FIG. 1.
The dns-negative mutant is hypertransformable. The frequencies of chitin-induced transformation of a dns mutant and complemented strains were determined. Lane 1, A1552 (WT); lane 2, A1552/pBR322 (vector control); lane 3, A1552Δdns; lane 4, A1552Δdns/pBR322 (vector control); lane 5, A1552Δdns/pBR-Tet-VC0470-His (constitutive dns expression); lane 6, A1552Δdns/pBR-Promo-VC0470-His (indigenous promoter of dns). Results are from three or more independent experiments.
- FIG. 2.
Cell density-dependent natural transformation. Transformation frequencies (y axis) were determined on chitin surfaces with donor gDNA (2 μg) added at 0, 2, 4, 6, and 8 h after inoculation, as indicated on the x axis. Results are from three independent experiments.
- FIG. 3.
The nuclease Dns degrades extracellular DNA. Crab shell fragments immersed in DASW medium were inoculated without bacteria (control; lane 1) or with V. cholerae A1552 (WT; lane 2), A1552Δdns (lane 3), A1552ΔluxO (lane 4), and A1552ΔhapR (lane 5), respectively. Donor gDNA (2 μg) was added immediately at the time of inoculation; DNA was recovered from the supernatant after a 2-hour incubation period and visualized on an agarose gel. Undegraded donor gDNA (arrow) and degradation products (bar) are shown. Lane L, 1-kb DNA ladder (Invitrogen).
- FIG. 4.
The extracellular nuclease is active in vitro. Supernatants from 2-hour-old bacterial cultures on chitin surfaces were collected and tested for nuclease activity by adding 4 μg of gDNA. After incubation at 30°C for 5 to 60 min (as indicated at the top of the lanes), DNA was reisolated, and its quality was visualized on agarose gels. The bacterial cultures used were A1552ΔhapR (A), A1552 (WT) (B), A1552ΔluxO (C), and A1552Δdns (D). Undegraded (arrow) and degraded (bar) gDNA is indicated.
- FIG. 5.
Quorum-sensing-dependent expression of dns. A transcriptional fusion between the promoter of dns (VC0470) and lacZ was constructed (plasmid pBR-Promo[dns]-lacZ) and transferred into strains A1552ΔhapRΔlacZ (lane 1), A1552ΔlacZ (FY_Vc_0003; lane 2), and A1552ΔluxOΔlacZ (lane 3). The bacterial strains were grown in LB medium until they reached an OD600 of ∼0.8. dns expression is reflected by β-galactosidase activity (given in Miller units).
- FIG. 6.
Cell density-dependent degradation of extracellular DNA is a result of the nuclease Dns. Bacterial strains A1552/pBAD-tfoX (parental strain; open bars) and A1552Δdns/pBAD-tfoX (nuclease deletion mutant; filled bars) were rendered competent by artificially inducing tfoX. Samples were taken at the time points indicated on the x axis and checked for their OD600 (secondary y axis) and their frequency of transformation (primary y axis). The results are from three independent experiments. Student's t test results: *, statistically significant difference relative to latest time point at 4:15 (P < 0.05); **, statistically significant difference between early time points (2:15, 2:45, 3:15) and late time points (3:45, 4:15) (P < 0.001).
- FIG. 7.
Model depicting the regulation of dns by quorum sensing (based on reference 34). At low cell density (left), few autoinducer molecules are present in the environment. This is sensed by the quorum-sensing systems (CqsS and LuxPQ; not shown), and phosphate is transferred via LuxU to LuxO. LuxO∼P is active in conjunction with RpoN (σ54) and inhibits the synthesis of the HapR regulator by inducing the expression of small regulatory RNAs (sRNAs) (posttranscriptional control). Under these conditions, the gene for the extracellular nuclease Dns is expressed. At high cell density (right), the phosphorylation cascade is reversed and LuxO is dephosphorylated. As a consequence, HapR accumulates, shutting down dns expression. HapR-mediated induction of comEA expression also occurs, provided that chitin is present (23).
Tables
- TABLE 1.
Bacterial strains and plasmids
Strain or plasmid Genotype or descriptiona Source or reference Strains A1552 WT, O1 El Tor Inaba, Rifr 37 A1552Δdns A1552ΔVC0470 This study A1552Δxds A1552ΔVC2621 This study A1552ΔdnsΔxds A1552ΔVC0470ΔVC2621 This study A1552ΔhapR (ATN140) A1552ΔVC0583 23 A1552ΔhapRΔdns A1552ΔVC0583ΔVC0470 This study N16961 N16961 (WT sequenced strain) 15 N16961Δdns N16961ΔVC0470 This study FY_Vc_0003 (A1552ΔlacZ) A1552ΔVC2338 (smooth) 5 A1552ΔluxO (ATN120) A1552ΔVC1021 23 A1552ΔluxOΔlacZ A1552ΔVC1021ΔVC2338 This study A1552ΔhapRΔlacZ A1552ΔVC0583ΔVC2338 This study Plasmids pBR322 Apr Tcr 4 pBR-Promo[dns]-lacZ pBR322, −610 bp to ATG of VC0470 and lacZ This study pBR-Tet-VC0470-His pBR322, Tet promoter, VC0470, and six-His tag sequence This study pBR-Promo-VC0470-His pBR322, −610 bp and all of VC0470 and six-His tag sequence This study ↵ a Locus tags are given in accordance with reference 15.
- TABLE 2.
Oligonucleotide primers used in this study
Primer name Sequence VC0470 up for MCM67 5′-TTTTCTAGATGCCCAAGATGCAGATCGAGC-3′ VC0470 up rev MCM79 5′-TGCCCGAACTGATGGGCAGA-3′ VC0470 down for MCM92 5′-TCTGCCCATCAGTTCGGGCAAATCATCATAAAAGACGTAGAT-3′ VC0470 down rev MCM105 5′-TTTCCATGGGAGCAGAAAATCAAACAATG-3′ VC2621 up for MCM126 5′-TTTTCTAGAAAAGAAGCACAACTCGATCG-3′ VC2621 up rev MCM138 5′-CGCCGTCGCTAGAAAGTG-3′ VC2621 down for MCM150 5′-CACTTTCTAGCGACGGCGTGTTCTCATTTCCATGATGTAC-3′ VC2621 down rev MCM162 5′-TTTCCATGGGTTAGATAGACCGATTCAGCA-3′ VC0470-up-RBS 5′-GGCAATTTATGAGTGTTAAACTGATAAAAAACC-3′ VC0470-up-Promo#1 5′-GTTCGTTTTGATCTGCACTGCCAAGC-3′ VC0470-His-down 5′-TGCCCATCAATGATGATGATGATGATGGTTCGGGCATTGCTCACG-3′ VC0470-Promo-lacZ#2 5′-CTCTACGGCGTACATAAAAGACGTAGATAAGTAGGTTTTTTATCAG-3′ VC0470-Promo-lacZ#3 5′-TACGTCTTTTATGTACGCCGTAGAGCAAAGGCGTTATTGGCTTG-3′ VC0470-Promo-lacZ#4 5′-TTATTGTGGGGATGACGCTTTCACACG-3′ Dns-His-chrom-#2 5′-AGATTCTGCCCATCAATGATGATGATGATGATGGTTCGGGCATTGC-3′ Dns-His-chrom-#3 5′-CATCATCATTGATGGGCAGAATCTCACCCTGCCCCCTTGTATTTTGC-3′ - TABLE 4.
Transformation efficiencies of V. cholerae dns deletion strains
Strain Genotype or description Relative transformation frequency (±SD)a Crab shell associated A1552 WT 1b A1552Δdns ΔVC0470 339.4 (±179.4) A1552ΔhapR ΔVC0583 BDc A1552ΔhapRΔdns ΔVC0583 ΔVC0470 2.5 (±1.5) N16961 First sequenced WT strain (15) BDc N16961Δdns ΔVC0470 1.5 (±1.2) tfoX overexpressing A1552/pBAD-tfoX Parental strain; tfoX overexpression 1d A1552Δdns/pBAD-tfoX ΔVC0470; tfoX overexpression 166 (±99) A1552ΔhapR/pBAD-tfoX ΔVC0583; tfoX overexpression BDe A1552ΔhapRΔdns/pBAD-tfoX ΔVC0583 ΔVC0470; tfoX overexpression 1.9 (±2) ↵ a Relative difference to WT transformation frequency; averages are of three independent experiments. BD, below detection limit.
↵ b WT strain value set to 1; absolute transformation frequency equals 4.6 × 10−6.
↵ c Transformation is below the detection limit of 6.7 × 10−8.
↵ d Parental strain A1552/pBAD-tfoX value set to 1; absolute transformation frequency equals 1.5 × 10−7.
↵ e Transformation is below the detection limit of 4.0 × 10−9.
