LuxR solos in Photorhabdus species

Abstract

Bacteria communicate via small diffusible molecules to mediate group-coordinated behavior, a process designated as quorum sensing. The basic molecular quorum sensing system of Gram-negative bacteria consists of a LuxI-type autoinducer synthase producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor detecting the AHLs to control expression of specific genes. However, many proteobacteria possess one or more unpaired LuxR-type receptors that lack a cognate LuxI-like synthase, referred to as LuxR solos. The enteric and insect pathogenic bacteria of the genus Photorhabdus harbor an extraordinarily high number of LuxR solos, more than any other known bacteria, and all lack a LuxI-like synthase. Here, we focus on the presence and the different types of LuxR solos in the three known Photorhabdus species using bioinformatics analyses. Generally, the N-terminal signal-binding domain (SBD) of LuxR-type receptors sensing AHLs have a motif of six conserved amino acids that is important for binding and specificity of the signaling molecule. However, this motif is altered in the majority of the Photorhabdus-specific LuxR solos, suggesting the use of other signaling molecules than AHLs. Furthermore, all Photorhabdus species contain at least one LuxR solo with an intact AHL-binding motif, which might allow the ability to sense AHLs of other bacteria. Moreover, all three species have high AHL-degrading activity caused by the presence of different AHL-lactonases and AHL-acylases, revealing a high quorum quenching activity against other bacteria. However, the majority of the other LuxR solos in Photorhabdus have a N-terminal so-called PAS4-domain instead of an AHL-binding domain, containing different amino acid motifs than the AHL-sensors, which potentially allows the recognition of a highly variable range of signaling molecules that can be sensed apart from AHLs. These PAS4-LuxR solos are proposed to be involved in host sensing, and therefore in inter-kingdom signaling. Overall, Photorhabdus species are perfect model organisms to study bacterial communication via LuxR solos and their role for a symbiotic and pathogenic life style.

Keywords: LuxR solos; cell-cell communication; entomopathogenic bacteria; quorum quenching; quorum sensing.

Figure 1

Presence and domain structure of the LuxR solos in P. luminescens, P. asymbiotica, and P. temperata. LuxR-type receptors share a modular domain structure, with a N-terminal signal-binding domain (SBD) (gray box) and a C-terminal DNA-binding domain (DBD) with the conserved “HTH LUXR” motif (gray hexagon), which is illustrated in the upper part. The N-terminus is marked with an “N” and the C-terminus with a “C.” The LuxR solos of Photorhabdus species, P. luminescens TT01, P. temperata NC19, and P. asymbiotica ATCC43949, are grouped into three types based on the different N-terminal domain. The “HTH LUXR” motif (SMART00421) is indicated by a hexagon, the AHL-domain (PFAM03472: “Autoind_bind”-domain) and the PAS4-domain (PFAM08448: “PAS_4”-domain) by boxes. LuxR solos marked with an asterisk additionally have an N-terminal transmembrane domain (not illustrated). Homologous proteins or cluster are marked with the colors red, purple, orange and green. LuxR solos were identified using SMART 7 software (Letunic et al., 2012) and BLAST software (Altschul et al., 1990).

Figure 2

Conserved amino acid motifs of LuxR-type proteins with different domains and their corresponding signaling molecules. Upper part: Motif of the six conserved amino acid positions in typical AHL-sensors. Protein sequences of LuxR from Vibrio fischeri, TraR from Agrobacterium tumefaciens, SdiA from Escherichia coli, QscR and LasR from Pseudomonas aeruginosa were used to generate the alignment. Middle part: Motif of the six conserved amino acids positions of PluR (Plu4562) from P. luminescens, PluR (Pte1335) from P. temperata and PauR (Pau4062) from P. asymbiotica. PluR from P. luminescens and from P. temperata are sensing photopyrones as signaling molecule, however this is yet unidentified for PauR. Lower part: Motif of the six conserved amino acid positions of the overall 80 PAS4-LuxR solos in all three Photorhabdus species, whereas the corresponding signal molecules are yet unknown but possible are eukaryotic hormones. All alignments were generated with CLC Mainworkbench 7 software (CLC Bio Qiagen, Hilden, Germany). The sequence logo was made with WebLogo3 (Crooks et al., 2004).

Figure 3

Phylogenetic tree of the LuxR solos present in P. luminescens, P. temperata, and P. asymbiotica. Protein sequences of the overall LuxR solos of the three Photorhabdus species, of LuxR from Vibrio fischeri, of TraR from Agrobacterium tumefaciens, of SdiA from Escherichia coli as well as of QscR and LasR from Pseudomonas aeruginosa were aligned and a phylogenetic tree was generated. Based on this alignment the different amino acid motifs at the six conserved positions were identified by deviation from the WYDPWG-motif in the SBD of AHL-sensors. A special focus on the amino acids at positions of the WYDPWG-motif is shown from the inner to the outer circle. The amino acid W57, with respect to TraR, is marked in brown, Y61 in red, D70 in purple, P71 in dark red, W85 in pink and G113 in light pink, however substitutions within this positions are marked in different colors. LuxR-type proteins with an AHL-binding domain are marked with a red dash, LuxR-type proteins with a PAS4-domain are marked with a black dash and LuxR-type proteins with a N-terminal yet undefined domain are marked with a blue dash. Alignment and radial phylogenetic tree was generated with the CLC Mainworkbench 7 (CLC Bio Qiagen, Hilden, Germany). The scale bar indicates the length of the branches.

Figure 4

Quorum quenching by Photorhabdus species culture fluids. Bioluminescence of Vibrio harveyi wild-type BB120 was used as read-out to analyze degradative activity of Photorhabdus spec. supernatants. The supernatants of P. luminescens, P. temperata, and P. asymbiotica were harvested after 4 and 7 days of growth and added to the BB120 reporter strain in the mid-exponential growth phase, when bioluminescence occurs. As controls V. harveyi supernatant and LM medium, harvested after 8 h and 1 day of growth, was added. Luminescence (RLU) was measured before (black bars) and after (gray bars) the addition of the Photorhabdus spec. supernatant and optical density (OD) was measured before (black circles) and after (white circles) the addition of the Photorhabdus spec. supernatant. Error bars represent standard deviation of at least three independently performed experiments. RLU, relative light units.

Figure 5

Life and infection cycle of Photorhabdus spec. and involvement of the different LuxR solos. Life cycle of P. luminescens, P. temperata, and P. asymbiotica (left panel). The bacteria colonize the upper gut of heterorhabditid nematodes that invade insect larvae. After release into the insect's hemolymph the bacteria produce several toxins that rapidly kill the prey. After death of the insect host, the bacteria degrade the cadaver, and additionally support nematode development. When the cadaver is depleted from nutrients, bacteria and nematode re-associate and leave the carcass in search for a new victim. The pathogenic part of the cycle is drawn in red, the symbiotic part in blue. P. asymbiotica can additionally infect humans by inducing systemic and soft tissue infections (right panel). Putative involvement of the different LuxR solos at steps of the infection process or host sensing is indicated by the gray boxes (see text for details).