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. 2012;12(4):3814-30.
doi: 10.3390/s120403814. Epub 2012 Mar 26.

A variant quorum sensing system in Aeromonas veronii MTCC 3249

Kamlesh Jangid  1 , Perunninakulath S Parameswaran, Yogesh S Shouche
Affiliations

A variant quorum sensing system in Aeromonas veronii MTCC 3249

Kamlesh Jangid et al. Sensors (Basel). 2012.

Abstract

We have investigated the quorum sensing control in Aeromonas veronii MTCC 3249, originally isolated as A. culicicola from the midgut of Culex quinquefasciatus. Based on biosensor assays, the bacterium showed constant production of multiple acyl-homoserine lactones (AHLs) with increasing cell-density. The luxRI gene homologs, acuR (A. culicicola transcriptional Regulator) and acuI (A. culicicola autoInducer) were successfully amplified by inverse-PCR. Sequence analysis indicated acuRI were divergent from all known quorum sensing gene homologs in Aeromonas. Two localized regions in the C-terminal autoinducer binding domain of acuR showed indels suggesting variations in autoinducer specificity. Further, only a single copy of the quorum sensing genes was detected, suggesting a tight regulation of mechanisms under its control. Chromatography and further chemical analysis identified two AHLs in the culture supernatant: 6-carboxy-HHL (homoadipyl homoserine lactone), a novel AHL, and N-tetradecanoylhomoserine lactone. The existence of a potentially variant quorum sensing system might therefore, reflect in some way the ecological strategies adopted by this bacterium in the mosquito midgut.

Keywords: 6-carboxy-HHL; AHL; Aeromonas; acuI; acuR; luxRI homolog; mosquito midgut; quorum sensing.

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Figures

Figure 1.
Figure 1.
Strategy for Inverse PCR amplification of acuRI from A. veronii.
Figure 2.
Figure 2.
Expression of gfp by E. coli JM109 pJBA89 in response to AHL production by A. veronii MTCC 3249.
Figure 3.
Figure 3.
Growth dependent AHL production by A. veronii MTCC 3249. (a) Fold induction of bioluminescence in E. coli JM109 with pSB401 as a function of increasing AHL concentration in the culture supernatant; (b) Constant production of AHLs by A. veronii in the culture supernatant proportional to its CFU/mL.
Figure 4.
Figure 4.
luxRI homolog sequences of A. hydrophila (ahyRI, X89469), A. salmonicida (asaRI, U65741), and A. veronii (acuRI, AY989817). luxI homologs (red), luxR homologs (green), and ygg homolog (purple). Region of dyad symmetry, ><, not homologous to the lux box consensus sequence (blue region). PstI site (underlined).
Figure 4.
Figure 4.
luxRI homolog sequences of A. hydrophila (ahyRI, X89469), A. salmonicida (asaRI, U65741), and A. veronii (acuRI, AY989817). luxI homologs (red), luxR homologs (green), and ygg homolog (purple). Region of dyad symmetry, ><, not homologous to the lux box consensus sequence (blue region). PstI site (underlined).
Figure 4.
Figure 4.
luxRI homolog sequences of A. hydrophila (ahyRI, X89469), A. salmonicida (asaRI, U65741), and A. veronii (acuRI, AY989817). luxI homologs (red), luxR homologs (green), and ygg homolog (purple). Region of dyad symmetry, ><, not homologous to the lux box consensus sequence (blue region). PstI site (underlined).
Figure 5.
Figure 5.
acuRI copy number in A. veronii MTCC 3249 using southern hybridization. Lane 1: 1 kb Plus DNA marker (Invitrogen); lane 2: HindIII digest; and lane 3: PstI digest.
Figure 6.
Figure 6.
TLC analysis of AHLs produced by A. veronii MTCC 3249. Lane 1: mixture of synthetic AHL standards, HHL and OHL; and lane 2: acetonitrile reconstituted extract of AHLs from the culture supernatant.
Figure 7.
Figure 7.
IR spectra of C. violaceum CV026 positive HPLC fraction from culture supernatant of A. veronii MTCC 3249.
Figure 8.
Figure 8.
MS/MS scan of C. violaceum CV026 positive HPLC fraction from culture supernatant of A. veronii MTCC 3249.
Figure 9.
Figure 9.
Chemical structures of the AHLs secreted by A. veronii MTCC 3249. (a) Compound-1: N-tetradecanoylhomoserine lactone; (b) Compound-2: 6-carboxy-HHL.
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