Quorum sensing activity of Citrobacter amalonaticus L8A, a bacterium isolated from dental plaque

Abstract

Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium.

Figure 1. Score-orientated dendrogram of strain L8A.

Bacterial strain L8A was clustered hierarchically based on the protein mass spectra patterns with distance value normalized to maximum value of 1,000.

Figure 2. Preliminary AHL screening with cross streak bioassay.

(A) Short chain AHL screening of strain L8A with CV026. E. carotovora PNP22 (negative control “−”) and E. carotovora GS101 (positive control “+”) were included for comparison. (B) AHL screening of strain L8A with E. coli [pSB401]. E. carotovora PNP22 (negative control “−”) and E. carotovora GS101 (positive control “+”) were included for comparison.

Figure 3. Mass spectrometry analysis of spent supernatants extract C. amalonaticus strain L8A.

(A) Mass spectra of C4-HSL (m/z 172.0000) (marked by arrow). (B) Mass spectra of C6-HSL (m/z 200.3000) (marked by arrow). (C) Mass spectra of C8-HSL (m/z 228.2000) (marked by arrow). (D) Mass spectra of C16-HSL (m/z 340.0000) (marked by arrow).

Figure 4. Maximum likelihood phylogenetic tree of C. amalonaticus L8A 16S rRNA sequence and other closely related sequences.

The phylogenetic analysis of 16S rRNA of the strain L8A was hierarchical clustered under C. amalonaticus using MEGA 5.1.

Figure 5. Phylogenetic tree based on QS synthase gene in L8A.

In silico study of QS synthase gene of genome strain L8A was determined and represented in maximum likelihood phylogenetic tree.

Figure 6. QS synthase gene and regulatory gene in L8A.

C. amalonaticus L8A QS genes were synteny analyzed using Easyfig (red arrow indicated the QS synthase gene and green arrow indicated the QS transcriptional regulator gene).

Figure 7. Comparison chart of eight QS-associated Gene Ontology terms using QuickGO (showing in highlight).

Different relationships between the terms are indicated by unique colour of arrows as depicted in the right panel.

Figure 8. BRIG visualization of multiple C. amalonaticus genomes available from NCBI database.

The innermost rings show GC skew (purple/green) and GC content (black). The third innermost ring shows L8A genome and followed by strains 3e8A (CDQV01) and 3e8B (CDQX01) isolated from undernourished Malawian children’s gut. The remaining rings represent the ground beef isolate GTA-817-RBA-P2 (LAMY01) and environmental strains YG6 (LIGA01) and YG8 (LIGB01) prior the outermost ring with complete genome strain Y19.