Fig 1.
Domain organization of LytS/LytTR histidine kinase/response regulator.
HKs of the LytS family contain a 5TM-5TMR_LYT domain (pfam07694) and a GAF domain, which together form the input domain. The cytosolic DHp [harboring a conserved histidine (His)] and CA modules in the HKs serve as the transmitter domain. The RR belongs to the LytTR-type family of transcriptional activators and comprises a CheY-like receiver domain [harboring a conserved aspartate (Asp)], and a LytTR-type effector domain made up of a 10-stranded β-fold DNA-binding domain.
Table 1.
Gene names as indicated are taken from the NCBI database and represent LytS-type histidine kinase/response regulator systems and their putative target genes. The abbreviations (Ab.) listed are used in the further figures.
Fig 2.
Phylogenetic tree of LytS-type histidine kinases in γ-proteobacteria.
(A) Based on a local alignment search using the NCBI RefSeq protein database, 1,521 individual LytS-type sequences were identified, and amino acid identity was calculated using the progressive alignment algorithm of the CLC Main Workbench 7.6 software. The phylogenetic tree was created using CLC’s high-accuracy neighbor-joining algorithm with 100 bootstrap replicates. Reassigned genera are given in black for the predominant members of the Enterobacteriaceae and in grey in the case of other γ-proteobacterial families. All genes encoding BtsS- or YpdA-type proteins in the set of species included in the tree are listed in S1 Table. The lengths of the branches of the tree represent the relative amount of evolutionary divergence between any two sequences in the tree. (B) Summary of the relative abundance of BtsS and YpdA sequences detected, and their distribution among Enterobacteriaceae and other γ-proteobacteria based on the phylogenetic tree.
Fig 3.
Comparison of BtsS/BtsR and YpdA/YpdB pairs in a subset of representative γ-proteobacterial species.
(A) Full-length TCS proteins as well as their individual domains were aligned and analyzed for percentage of sequence identity with respect to the E. coli homologs (100%, green). The absence of YpdA/YpdB is marked with ‘Ø’. (B) In addition, promoter sequences of the identified target genes (Table 1) were scanned and RR-binding motifs were deduced. No YpdB target promoter motifs were detected in species in which the YpdA/YpdB-type TCS was either absent or could not be unambiguously identified and are marked with ‘absent’. Species, in which the known DNA-binding motif of BtsR or YpdB was undetectable, are marked with ‘unidentified’.
Fig 4.
Activation of the yjiY promoter in various γ-proteobacterial species growing in LB medium.
Previous studies reported transient BtsS/BtsR-mediated yjiY promoter activation in E. coli grown in LB medium shortly before cells enter stationary phase [9]. Each species was transformed with an E. coli yjiY-lux reporter plasmid, and luminescence levels (blue) as well as growth (black dotted line) were measured over time. Escherichia coli (Ec), Citrobacter freundii (Cf), Salmonella enterica (Se), Enterobacter aerogenes (Ea), Xenorhabdus szentirmaii (Xs), Serratia marcescens (Sm), Yersinia enterocolitica (Ye), Vibrio harveyi (Vh) and Aeromonas hydrophila (Ah). Degrees of divergence of the BtsS-type proteins of the selected species (based on phylogenetic tree in Fig 2A) to the E. coli BtsS are indicated above the graphs (in black—members of the Enterobacteriaceae, in grey—members of other γ-proteobacterial families).
Fig 5.
Activation of the yhjX promoter in selected γ-proteobacterial species grown in minimal medium containing 0.4% (wt/vol) pyruvate as C-source.
Previous studies have reported constitutive YpdA/YpdB-mediated activation of the yhjX promoter in E. coli cultivated in minimal medium supplemented with pyruvate [7]. Each species was transformed with an E. coli yhjX-lux reporter plasmid, and luminescence levels (red) as well as growth (black dotted line) were measured over time. For further details, see the legend to Fig 4.
Table 2.
Maximal accumulation of external pyruvate in the supernatant during growth in LB medium.
Quantitative measurement of external pyruvate concentrations of all selected strains grown in LB medium (largest values out of one independent experiment). Plots of extracellular pyruvate concentrations over time are displayed in Fig 6.
Fig 6.
Extracellular concentrations of pyruvate during growth of the indicated γ-proteobacterial species in LB medium are plotted against time after inoculation.
At the times indicated, cells were harvested, and pyruvate levels in the cell-free supernatant were quantified by hydrophilic interaction liquid chromatography. All experiments were performed in triplicate, and the error bars indicate the standard deviations of the means. Abbreviations as in Fig 4.
Fig 7.
Schematic depiction of the functional links between the LytS/LytTR-type TCSs.
The high-affinity BtsS/BtsR and low-affinity YpdA/YpdB TCSs are interconnected, which results in either the production of YjiY, a CstA-like transport protein, or YhjX, a putative antiporter of the MF superfamily. Dashed lines indicate predicted components and effects.