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

Sample collection and processing

Shime saba sashimi sample was collected from a local supermarket in Malaysia and processed within half an hour following collection. Five grams of sample was stomached (mixing of sample in a sterile plastic bag by applying forces to the outside of the bag) using Stomacher^® 400 circulator (Seward, West Sussex, UK) and homogenized in 50 ml of peptone water and then spread on MacConkey (MAC) agar. The culture plates were incubated overnight at 28 °C.

Bacterial strains, media and culture conditions

C. neteri SSMD04, Chromobacterium violaceum CV026, Erwinia carotovora GS101 and E. carotovora PNP22 were maintained in Luria Bertani (LB) medium at 28 °C. lux-based biosensor Escherichia coli (pSB401) was grown in LB supplemented with tetracycline (20 µg/mL) at 37 °C. All broth cultures were incubated with shaking (220 rpm).

Species identification of isolate SSMD04

Detection of AHL production in C. neteri SSMD04

AHL-type QS activity of C. neteri SSMD04 was screened using biosensor C. violaceum CV026. This is performed by cross streaking C. neteri SSMD04 against C. violaceum CV026 (McClean et al., 1997). E. carotovora GS101 and E. carotovora PNP22 were used as positive and negative controls, respestively (Jones et al., 1993).

AHL extraction

C. neteri SSMD04 was cultured overnight at 28 °C in LB broth (100 mL) supplemented with 50 mM of 3-(N-morpholino)propanesulfonic acid (MOPS) (pH5.5). Culture supernatant was collected by centrifugation and organic compounds were subsequently extracted twice with equal volume of acidified ethyl acetate (AEA) (0.1% v/v glacial acetic acid). The extracts were air dried and reconstituted in 1 mL of AEA, transferred into sterile microcentrifuge tubes and air dried again. The extracts were later used for detection of AHL by lux-based biosensor E. coli (pSB401) as well as triple quadrupole LC/MS.

AHL identification by triple quadrupole LC/MS

AHL extracts were reconstituted in acetonitrile (ACN) prior to LC/MS analysis as described before (Lau et al., 2013), with slight modifications. In brief, mobile phase A was water with 0.1% v/v formic acid and mobile phase B used was ACN with 0.1% formic acid. The flow rate used was 0.5 mL/min. The gradient profile was set to: A:B 80:20 at 0 min, 50:50 at 7 min, 50:50 at 7.10 min, 80:20 at 12 min, 80:20 at 12.10 min, 20:80 at 14 min, 20:80 at 14.10 min. Precursor ion scan mode was carried out in positive ion mode with Q1 set to monitor m/z 90 to m/z 400 and Q3 set to monitor for m/z 102 which is characteristics of lactone ring moiety. ACN was also used as a blank.

Measurement of bioluminescence

E. coli (pSB401) (Winson et al., 1998) was used as biosensor for the detection of exogenous short chain AHLs present in the extracts. The biosensor strain was cultured in LB broth supplemented with tetracycline (20 µg/mL). The overnight culture was then diluted to an OD₆₀₀ of 0.1 with fresh LB broth with tetracycline. The diluted E. coli culture was used to resuspend the extracts from Section ‘AHL extraction,’ prior to being dispensed into a 96-well optical bottom microtitre plate. Cell density bioluminescence measurements were carried out by Infinite M200 luminometer-spectrophotometer (Tecan, Männedorf, Switzerland) over a period of 24 h. Diluted E. coli culture without extracts was read for normalization and sterile broth was used as negative control. The results were displayed as relative light units (RLU)/OD₄₉₅ nm against incubation time.

Lipase activity

The lipase activity of C. neteri SSMD04 was tested in a medium consisted of (in w/v) 0.05% yeast extract, 0.1% tryptone, 1% NaCl, and 1.5% agar supplemented with 0.5% (v/v) corn oil. The oil forms an opaque suspension in the agar. Bacteria culture was streaked on the agar and incubated at 28 °C for 48 h. The enzymatic activity is visualized by a halo zone formed around the colonies caused by the breakdown of lipids.

luxI/R homologues search and analysis

Whole genome of C. neteri SSMD04 was sequenced and annotated (Chan et al., 2014). The luxI/R homologues were searched on RAST using the “Genome Browser” function. The “Annotation Overview” function on RAST was used to make locus comparison of cneI/R pair and the orphan luxR with other genomes. Multiple sequence alignment of LuxR-type proteins was done with ClustalW. ESPript (Robert & Gouet, 2014) was used for the presentation of the alignment.

Species identification of C. neteri SSMD04

Seven 16S rRNA gene sequences were found in C. neteri SSMD04 genome, of which 6 were identical and the other has 2 SNPs. Both variants were included in phylogenetic analysis with other sequences of Cedecea spp. available in GenBank. As can be seen, 16S rRNA gene sequences of C. neteri SSMD04 and other C. neteri strains appeared in a monophyletic clade (Fig. 1).

The Biology Gen III microbial identification system was also used to assess the identity of C. neteri SSMD04 biochemically. The system identified this strain to be C. neteri with a probability and similarity of 0.697. The positive reaction in sucrose well and D-sorbitol well agrees with the report of Farmer 3rd et al. (1982).

Detection of AHL-type QS activity in C. neteri SSMD04 using AHL biosensor

Since AHL-type QS has been known to regulate virulence and food spoilage traits in some bacteria, we hypothesized that C. neteri exhibits QS activity as well. In order to investigate the presence of AHL-type QS activity in C. neteri SSMD04, it was streaked on LBA against biosensor C. violaceum CV026, which would show purple pigmentation due to the production of violacein in the presence of short chain AHLs. The result shown in Fig. 2 indicated the presence of exogenous AHLs in C. neteri SSMD04 culture. E. carotovora GS101, a known AHL producing Erwinia strain, and E. carotovora PNP22, an AHL-synthase mutant were chosen as positive and negative controls, respectively.

C. neteri SSMD04 also activated lux-based biosensor E. coli (pSB401) which produces bioluminescence in the presence of short chain AHLs (Fig. 3).

AHL identification by triple quadrupole LC/MS

The use of both biosensors (C. violaceum CV026 and E. coli (pSB401)) does not give information on the specific type of AHL produced since each detects a variety of AHLs. Therefore, triple quadrupole LC/MS was employed in the identification of the AHL produced by C. neteri SSMD04. The extracted-ion chromatogram (EIC) generated from the triple quadrupole LC/MS system showed a peak with the same retention time as that of the synthetic N-butyryl-homoserine lactone (C4-HSL) standard, which was constantly present in all three replicates (Fig. 4). Analysis of the mass spectrum (MS) data revealed the presence of a peak with mass-to-charge ratio (m/z) of 172 (Fig. 5), which is consistent with the previously reported value (Ortori et al., 2011). This implication was strengthened by the presence of a product ion peak (m/z = 102), confirming the presence of a homoserine lactone ring, the invariant structure of AHLs. This result is in agreement with the results of the biosensor strains, C. violaceum CV026 and E. coli (pSB401), as both respond to C4-HSL.

Lipase activity

C. neteri SSMD04 was streaked on medium containing 0.5% (v/v) corn oil and incubated overnight to test for its lipase activity. The halo zone was visible around the colonies after 24 h of incubation, but it was more visible after 48 h of incubation (Fig. 6).

luxI/R homologues search and analysis

From the data generated by NCBI prokaryotic genome annotation pipeline, a 636 bp luxI homologue, hereafter named cneI, was found in the genome. This gene shares 70% base pair similarity with N-acyl homoserine lactone synthase croI of Citrobacter rodentium ICC168. Analysis of amino acid sequence of cneI using InterPro (Mitchell et al., 2015) identified the presence of an acyl-CoA-N-acyltransferase domain, the structural domain of N-acyl homoserine lactone synthetases (Gould, Schweizer & Churchill, 2004; Watson et al., 2002).

Eight bp away from cneI is a sequence encoding a hypothetical protein, potentially the cognate receptor, a luxR homologue (cneR). The coding region was found to be 705 bp long and share 70% similarity with croR of C. rodentium. Analysis of this protein reveals two signature domains, the autoinducer-binding domain and the C-terminal effector.

In order to investigate the relatedness of cneI/R and croI/R, the organization of cneI/R and their flanking region was examined and comparison to another C. neteri genome, M006, as well as C. rodentium ICC168 was made (Fig. 7). As can be seen, gene organization of cneI/R and their flanking region is highly similar in both C. neteri strains, but displays no similarity with croI/R and their flanking region. Interestingly, the genome of C. davisae DSM 4568 harbours no luxI/R homologous pair, suggesting that AHL-type quorum sensing is not present in all members of the genus Cedecea.

Apart from that, a sequence potentially encoding an orphan luxR type receptor (723 bp) was also found within the genome. This luxR homologue shares 69% sequence homology to luxR homologue of Enterobacter asburiae L1. Multiple sequence alignment of this orphan LuxR, CneR, and other canonical LuxR-type proteins (Fig. 8) reveals the presence of conserved sites, residues 57, 61, 70, 71, 85, 113, 178, 182, 188 (TraR sequence numbering used as a reference), which are present in at least 95% of LuxR-type proteins (Whitehead et al., 2001; Zhang et al., 2002).

Orphan LuxR is known to be present in E. coli and Salmonella which allows response to exogenous AHLs (Ahmer, 2004). The function of the orphan LuxR in C. neteri SSMD04 is not known. However, genome comparison of C. neteri SSMD04 and M006, C. davisae DSM4568, C. rodentium ICC168, S. enterica subsp. enterica serovar Choleraesuis str. SC-B67, and E. coli K12 shows a considerable degree of conservation (Fig. 9). This is especially true for the 3 genomes of the genus Cedecea, despite the absence of canonical luxI/R pair in C. davisae DSM4568 genome.