Fig 1.
Transcriptional and translational levels of hexA expression.
The transcriptional level, by fusing the promoter of hexA to mCherry (A), as well as the translational level, by generating protein hybrids of HexA with mCherry under the control of the hexA promoter (B), were investigated. The reporter constructs PhexA-mCherry and PhexA-hexA-mCherry, respectively, were integrated into the chromosome and fluorescence intensities were measured after 5 h (early exponential phase), 24 h (mid-exponential phase), 30 h (stationary phase), and 48 h (late stationary phase) in the P. luminescens strains TT01-1°, TT01-2°, TT01-1°ΔhexA. The asterisk (*) indicates statistically significant differences with a p-value smaller than 0.05. The hash (#) indicates no statistically significant differences with a p-value bigger than 0.05. Error bars represent standard deviation of three independently performed experiments.
Fig 2.
Effect of HexA on the bioluminescence of P. luminescens.
Bioluminescence in P. luminescens TT01-1°, TT01-2° and TT01-1°ΔhexA under native conditions and with enhanced levels of HexA. Error bars represent standard deviation of three independently performed experiments (A). Promoter activity of luxCDABE at the population level. The respective reporter construct PluxC-mCherry was integrated into the chromosome and fluorescence intensities were measured after 24 h (mid-exponential growth phase) and 30 h (stationary growth phase) in the strains TT01-1°, TT01-2°, TT01-1°-ΔhexA. The hash (#) indicates no statistically significant differences with a p-value bigger than 0.05. (B). PluxC activity in strains TT01-1°, TT01-2° and TT01-1°ΔhexA at the single cell level. The scale depicts 10 μm. Representative images from one of three independently performed experiments are shown (C).
Fig 3.
Transcriptional levels of hfq in P. luminescens TT01-1°, TT01-2° and TT01-1°ΔhexA.
Promoter activity of hfq at the population level. The respective reporter construct Phfq-mCherry was integrated into the chromosome and fluorescence intensities were measured after 24 h (mid-exponential phase) and 30 h (stationary phase) in the strains TT01-1°, TT01-2°, TT01-1°ΔhexA. The asterisk (*) indicates statistically significant differences with a p-value smaller than 0.05. The hash (#) indicates no statistically significant differences with a p-value bigger than 0.05. Error bars represent standard deviation of three independently performed experiments (A). Phfq activity in TT01-1°, TT01-2° and TT01-1°ΔhexA at the single cell level. The scale depicts 10 μm. Representative images from one of three independently performed experiments at time point 48 h are shown (B).
Fig 4.
Cell clumping in P. luminescens TT01-1°, TT01-2° and TT01-1°ΔhexA.
PpcfA-mCherry activity and cell clumping in TT01-1°, TT01-2° and TT01-1°ΔhexA. The scale depicts 10 μM. Representative images from one of three independently performed experiments are shown (A). Promoter activity of pcfABCDEF at the population level in TT01-1°, TT01-2° and TT01-1°ΔhexA. The asterisk (*) indicates statistically significant differences with a p-value smaller than 0.05. Error bars represent standard deviation of three independently performed experiments (B).
Fig 5.
Effect of HexA on the PpcfA activity in the heterologous systems of E. coli ΔlrhA and Sh. oneidensis.
E. coli ΔlrhA and Sh. oneidensis were transformed with plasmids pBAD24-Para-pluR_Plac-hexA and pACYC-Plac-hexA_Para-pluR, respectively, in combination with plasmid pBBR-PpcfA-lux. In E. coli ΔlrhA the pluR expression was achieved via the addition of 0.1% (w/v) arabinose and in Sh. oneidensis 0.02% (w/v) arabinose was added for pluR expression. The values were measured as relative light units [RLU] divided by OD600nm (A). In the upper panel the promoter region of pcfA with the PluR binding site (PluR BS) is depicted. In E. coli ΔlrhA two different truncations s1 and s2 of the pcfA promoter were tested. Thereby, pluR induction was achieved via the addition of 1 mM IPTG and hexA expression was induced via 0.02% (w/v) or 0.2% arabinose (w/v) on plasmid pBAD24-Plac-pluR_Para-hexA (B). The figures represent three biological replicates; n.i.: non-induced, ind: induced; All values are given in percentage, relative to the respective maximum pluR induction.
Fig 6.
Direct binding of HexA to the pcfA promoter region.
The biotinylated PpcfA DNA-fragment was captured onto a streptavidin-coated (SA) sensor-chip. Different concentrations of His-tagged HexA (125 nM: purple line; 250 nM: dark blue line; 500 nM: light blue line; 1000 nM: green line; 2000 nM: yellow line) were passed over the chip. An overall affinity of KD 1.3 μM was determined, the association and dissociation rates were determined as ka = 1300 M*s and kd = 0.002 1/s, respectively. As a negative control for unspecific binding, the SA chip was coated with a sacB DNA fragment.
Fig 7.
Model of the versatile role of HexA controlling 1° and 2° specific phenotypes in P. luminescens.
HexA directly represses the promoter of the pcfABCDEF operon, which is responsible for the formation of cell clumps, and indirectly represses the translation of luxCDABE, presumably via sRNAs, and thereby diminishes light production in 2° cells.