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. 2015 Sep 1:3:e1216.
doi: 10.7717/peerj.1216. eCollection 2015.

Genome analysis of quorum sensing Cedecea neteri SSMD04 leads to identification of its novel signaling synthase (cneI), cognate receptor (cneR) and an orphan receptor

Kian-Hin Tan  1 , Jia-Yi Tan  1 , Wai-Fong Yin  1 , Kok-Gan Chan  1
Affiliations

Genome analysis of quorum sensing Cedecea neteri SSMD04 leads to identification of its novel signaling synthase (cneI), cognate receptor (cneR) and an orphan receptor

Kian-Hin Tan et al. PeerJ. .

Abstract

Cedecea neteri is a very rare human pathogen. We have isolated a strain of C. neteri SSMD04 from pickled mackerel sashimi identified using molecular and phenotypics approaches. Using the biosensor Chromobacterium violaceum CV026, we have demonstrated the presence of short chain N-acyl-homoserine lactone (AHL) type quorum sensing (QS) activity in C. neteri SSMD04. Triple quadrupole LC/MS analysis revealed that C. neteri SSMD04 produced short chain N-butyryl-homoserine lactone (C4-HSL). With the available genome information of C. neteri SSMD04, we went on to analyse and identified a pair of luxI/R homologues in this genome that share the highest similarity with croI/R homologues from Citrobacter rodentium. The AHL synthase, which we named cneI(636 bp), was found in the genome sequences of C. neteri SSMD04. At a distance of 8bp from cneI is a sequence encoding a hypothetical protein, potentially the cognate receptor, a luxR homologue which we named it as cneR. Analysis of this protein amino acid sequence reveals two signature domains, the autoinducer-binding domain and the C-terminal effector which is typical characteristic of luxR. In addition, we found that this genome harboured an orphan luxR that is most closely related to easR in Enterobacter asburiae. To our knowledge, this is the first report on the AHL production activity in C. neteri, and the discovery of its luxI/R homologues, the orphan receptor and its whole genome sequence.

Keywords: Autoinducer; Food microbiology; Mass spectrometry; N-acyl-homoserine lactone; N-butyryl-homoserine lactone; Quorum sensing.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Phylogenetic tree showing the position of C. neteri SSMD04 (green squares) relative to other Cedecea spp.
The maximum likelihood tree was inferred from 1,297 aligned positions of the 16S rDNA sequences using Hasegawa-Kishino-Yano substitution model. Boostrap values are represented at the nodes. The scale denotes the number of substitutions per nucleotide position. Serratia marcescens strain HokM was used as outgroup.
Figure 2
Figure 2. Screening for AHL-type QS activity of C. neteri SSMD04 using the biosensor C. violaceum CV026.
The positions of C. neteri SSMD04, E. carotovora PNP22, E. carotovora GS101 are indicated by arrows, whereas C. violaceum CV026 was streaked perpendicularly against the tested strain at the periphery of the plate.
Figure 3
Figure 3. Detection of AHL by E. coli (pSB401).
Relative light unit (RLU)/OD495 against incubation time of cultures of E. coli (pSB401) in the presence of extracted AHLs (square plots) and negative control (circle plots).
Figure 4
Figure 4. EIC of C. neteri SSMD04 extract.
The data represented three replicates of the extract against synthetic C4-HSL, labelled as “std.” ACN was used as blank.
Figure 5
Figure 5. Mass spectrometry analysis of C. neteri SSMD04 spent supernatant extract.
Product ions of the peak seen which shows that the extract of C. neteri SSMD04 contains C4-HSL.
Figure 6
Figure 6. C. neteri SSMD04 grown on medium containing 0.5% corn oil.
The halo zone around the colonies indicate lipase activity.
Figure 7
Figure 7. Schematic representation of cneI/R locus of C. neteri SSMD04 in comparison with C. neteri M006 and C. rodentium ICC168 (GenBank ID: CP0009458.1 and FN543502.1, respectively).
Species and strain are shown at the left. Each gene is numbered and labelled accordingly. Homologous genes are represented in the same colour. The arrows show the orientation of the gene. Overlapping genes are arranged below the line.
Figure 8
Figure 8. Multiple sequence alignment of CneR, C. neteri SSMD04 orphan LuxR (Orphan) with five other canonical QS LuxR-type proteins.
TraR, CroR, LuxR, LasR, and RhlR (GenBank ID: 282950058, 59482356, 299361, 541657, 1117981, 740696391, 689266442, respectively) are LuxR-type proteins involved in quorum sensing from A. tumefaciens, C. rodentium, Vibrio fischeri, Pseudomonas aeruginosa (LasR and RhlR), respectively. Identical residues are denoted by a vertical filled bar, and conserved residues are denoted by unfilled box. TraR residue numbering is shown above the alignment as reference.
Figure 9
Figure 9. Organization of C. neteri SSMD04 orphan luxR and its flanking genes in comparison with other selected species C. davisae DSM4568, C. neteri M006, C. rodentium ICC168, Salmonella enterica subsp. enterica serovar Ch.
Organization of C. neteri SSMD04 orphan luxR and its flanking genes in comparison with other selected species C. davisae DSM4568, C. neteri M006, C. rodentium ICC168, Salmonella enterica subsp. enterica serovar Choleraesuis str. SC-B67 and E. coli K12 (GenBank ID: 513473511, 690276415, 282947233, 62178570, 556503834, respectively).
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