Article Figures & Data
Figures
- FIG. 1.
Luminescence of luxS mutants in the symbiotic light organ. Animal luminescence was monitored during the initial stages of E. scolopes colonization by V. fischeri wild-type (solid diamonds), ainS mutant (solid squares), luxS mutant (solid triangles), and ainS luxS mutant (solid circles) strains. For each time point, mean values of 24 animals were calculated and standard errors of the mean are indicated. The experiment was repeated with the same outcome.
- FIG. 2.
Colonization competence of quorum-sensing mutants. Colonization levels of the ainS mutant (A), luxS mutant (S), ainS luxS mutant (A S), and ainS-luxS-luxO (A S O) mutant relative to V. fischeri wild type (wt) were measured at 24 and 48 h postinoculation. Each bar represents the mean value of 15 animals with the associated standard errors. The experiment was conducted twice with the same outcome.
- FIG. 3.
Relationship between luminescence and quorum-sensing signal activity during growth of V. fischeri in culture. The relative level of LuxS signal activity (solid diamonds) in the culture supernatant is given as a percentage of the level produced by the positive control, an overnight V. harveyi BB152 culture (see Materials and Methods). The concentration of the AinS signal, C8-HSL (solid circles), in the V. fischeri culture was determined with synthetically produced C8-HSL as standard. Luminescence of the culture (crosses) is presented for comparison. Shown is a representative experiment; standard deviation bars are indicated.
- FIG. 4.
Transcriptional activity of luxS and ainS in culture. β-Galactosidase activity of V. fischeri wild-type cells carrying either a luxS::lacZ fusion (solid diamonds) or an ainS::lacZ fusion (open diamonds) in trans on low-copy-number plasmids pCL152 and pCL154, respectively, was measured during growth. Shown are the cumulative data of three cultures; standard deviation bars were smaller than the symbols.
- FIG. 5.
Conceptual model for the regulation of luminescence and colonization genes by V. fischeri LuxS and AinS. Arrows indicate positive, inducing effects, and bars indicate negative, inhibitory ones. (See Discussion for explanation.)
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Description Source or reference Strains V. fischeri ES114 Wild-type isolate from E. scolopes light organ 5 ainS mutant CL21 ainS gene partially deleted and replaced by chloramphenicol resistance (cat) marker 24 litR mutant PMF8 litR gene inactivated by insertion of kanamycin resistance marker (kanR) 10 luxI mutant VCW2G7 luxI gene inactivated by a frameshift mutation 24 luxO mutant CL42 luxO gene inactivated by insertion of kanamycin resistance marker (kanR) 24 luxR mutant CL53 luxR gene inactivated by insertion of erythromycin resistance marker (ermR) This study luxS mutant CL39 luxS gene inactivated by insertion of kanamycin resistance marker (kanR) This study ainS luxS mutant CL41 Double mutant carrying mutations as described above for CL21 and CL39 This study luxS luxO mutant CL90 Double mutant carrying mutations as described above for CL39 and CL42 This study ainS luxS luxO mutant CL91 Triple mutant carrying mutations as described above for CL21, CL39 and CL42 This study V. harveyi BB120 Wild type 1 BB152 AI-2 producer 1 BB170 AI-2 sensor 1 BB886 AI-2 sensor mutant 1 Plasmids pCL112 2.1-kb HaeIII fragment carrying ainS gene in pVO8 24 pCL115 2.7-kb V. fischeri ES114 DNA with luxS gene cloned into pEVS79 This study pCL122 pCL115 with random TnKan insertion 70 bp downstream of the luxS transcriptional start site This study pCL125 1.1-kb V. fischeri ES114 DNA with luxS gene cloned into pVO8 This study pCL145 1.8-kb V. fischeri ES114 DNA with luxO gene cloned into pEVS79 24 pCL149 Allelic exchange vector pEVS79 carrying partially deleted luxR gene replaced by erythromycin resistance (erm) marker This study pCL152 pCL112 with lacZ gene from pKV124 cloned into EcoRV site approximately 200 bp downstream of translational start site This study pCL154 pCL125 with the lacZ gene from pKV124 cloned into the EcoRV site approximately 200 bp downstream of translational start site This study pCL155 pCL145 with luxO gene inactivated by insertion of ermR-marker into NsiI site This study pEVS79 Allelic exchange vector, camR 41 pEVS94 Source of the ermR cassette 41 pKV30 Cloning vector carrying partially deleted luxR gene replaced by erythromycin resistance (erm) marker 44 pKV124 Source of lacZ gene 47 pMU106 Allelic exchange vector carrying partially deleted ainS gene replaced by chloramphenicol resistance (cat) marker 24 pVO8 V. fischeri cloning vector, ermR 45 - TABLE 2.
LuxS activity of V. fischeri a
Strainb % LuxS activity in V. harveyi reporter strain: BB170 (AI-2 sensor) BB886 (AI-2 sensor mutant) V. harveyi Wild-type BB120 147 (14) 100 (37) AI-1 mutant BB152 100 (18) 1 (12) V. fischeri Wild type (vector) 49 (3) 0 (1) luxS (vector) 0 (3) 1 (2) Wild type (pCL125) 44 (8) 2 (2) luxS (pCL125) 41 (16) 2 (2) ↵ a The V. harveyi reporter strains BB170 (AI-2 sensor) and BB886 (AI-2 sensor mutant) were incubated in medium containing 10% (vol/vol) culture supernatants of either the V. fischeri wild type or the luxS mutant carrying in trans either a vector control (pVO8) or a functional copy of the luxS gene on pCL125 (Table 1). Shown is the normalized response of a representative experiment. Standard errors are indicated in parentheses.
↵ b The addition of culture supernatants from either wild-type V. harveyi BB120 (which produces both V. harveyi AI-1, an acyl-HSL, and AI-2) or V. harveyi BB152 (which produces only AI-2) served as positive controls.
- TABLE 3.
Luminescence of V. fischeri luxS mutantsa
Strain type Luminescence values (10−2 quanta s−1 cell−1) with addition ofb: None C8-HSL 3-Oxo-C6-HSL Wild type 5.7 (0.1) 8.2 (1.1) 17,000 (3,900) luxS 4.2 (0.2) 5.1 (0.4) 22,000 (3,600) ainS BDc 4.3 (0.2) 5 (1) ainS luxS BD 2.6 (0.1) 4 (1) luxO 6.8 (0.8) 12.2 (2.9) 24,000 (4,000) luxS luxO 8.3 (1.1) 17.2 (4.0) 37,000 (1,500) ↵ a Specific luminescence values are the means of cultures with an OD600 of between 1.0 and 5.0; standard errors of the means are indicated in parentheses. The results shown are from a representative experiment.
↵ b Cultures were grown in SWT either without additions or with the addition of either 120 nM C8-HSL or 120 nM 3-oxo-C6-HSL.
↵ c BD, below detection (<0.02 quanta s−1 cell−1).
- TABLE 4.
Transcriptional activity of V. fischeri luxS and ainS
Strain type β-Galactosidase activity (Miller units) from fusion toa: luxS ainS Wild type 205 (46) 17.8 (4.4) ainS 189 (15) 2.0 (0.1) ainS (+ C8-HSL)b NDc 17.2 (4.1) luxS 204 (23) 16.0 (4.1) ainS luxS 216 (35) 1.9 (0.1) luxO 225 (46) 18.9 (3.9) ainS luxO ND 15.1 (6.0) litR 181 (30) 2.7 (0.4) luxR 167 (24) 17.4 (4.3) luxI 206 (36) 17.4 (3.8) ↵ a Shown is transcriptional activity of luxS and ainS in different genetic backgrounds. β-Galactosidase activity was measured during growth and averaged for several measurements taken at ODs of between 1 and 5. Shown is a representative experiment, with standard errors in parentheses.
↵ b The ainS mutant was grown in the presence of 120 nM C8-HSL.
↵ c ND, not determined.
