The β-sliding clamp directs the localization of HdaA to the replisome in Caulobacter crescentus

Abstract

The initiation of chromosome replication is tightly regulated in bacteria to ensure that it takes place only once per cell cycle. In many proteobacteria, this process requires the ATP-bound form of the DnaA protein. The regulatory inactivation of DnaA (RIDA) facilitates the conversion of DnaA-ATP into replication-inactive DnaA-ADP, thereby preventing overinitiation. Homologues of the HdaA protein, together with the β-clamp of the DNA polymerase (DnaN), are required for this process. Here, we used fluorescence resonance energy transfer experiments to demonstrate that HdaA interacts with DnaN in live Caulobacter crescentus cells. We show that a QFKLPL motif in the N-terminal region of HdaA is required for this interaction and that this motif is also needed to recruit HdaA to the subcellular location occupied by the replisome during DNA replication. An HdaA mutant protein that cannot colocalize or interact with DnaN can also not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during RIDA in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated. In addition, we show that a conserved R145 residue located in the AAA+ domain of HdaA is also needed for the function of HdaA, although it does not affect the interaction of HdaA with DnaN in vivo. The AAA+ domain of HdaA may therefore be required during RIDA after the initial recruitment of HdaA to the replisome by DnaN.

Fig. 1.

The subcellular localization of HdaA during the C. crescentus cell cycle. Schematic showing the C. crescentus cell cycle. The shading indicates the distribution of replisome components and of HdaA in cells. SW, swarmer cell; ST, stalked cell; EPD, early predivisional cell; LPD, late predivisional cell.

Fig. 2.

The organization of the HdaA protein. Diagram of the HdaA domain structure. Mutant and truncated HdaA proteins created for this study are also shown below the diagram of the wild-type protein.

Fig. 3.

The N-terminal region of HdaA, but not its R145 finger, is necessary for the colocalization of HdaA with the β-sliding clamp of the replisome. (a) Cells from strains JC593, JC888 and JC708, expressing DnaN–CFP together with YFP–HdaA or YFP–HdaAR145A or YFP–HdaAΔN, respectively, were visualized by phase-contrast (Ph3) and fluorescence microscopy. Right panels correspond to overlays of the two fluorescence microscopy images (green colour indicates co-localization). Bars, 2 µm. (b) Statistical analysis of the results shown in (a). More than 160 cells of each strain were analysed. This table shows the percentage of cells that do not contain a fluorescent focus (no focus), that contain only DnaN–CFP foci (only CFP foci), that contain only YFP–HdaA foci (only YFP foci), that contain colocalized DnaN–CFP and YFP–HdaA foci (CFP and YFP foci colocalized) and that contain spatially dissociated DnaN–CFP and YFP–HdaA foci (CFP and YFP foci not colocalized). (c) The intracellular levels of YFP-tagged HdaA proteins in cell extracts from strains JC593, JC888 and JC708 were evaluated by immunoblot using antibodies raised against HdaA. Cells were cultivated to exponential phase in M2G medium, and 0.3 % xylose was added to the medium 1 h before sample collection for immunoblotting and fluorescence microscopy.

Fig. 4.

YFP–HdaA interacts with DnaN–CFP in live C. crescentus cells and this interaction requires the N-terminal region of HdaA but not the R145 finger of HdaA. Strains JC593, JC888 and JC708 (expressing DnaN–CFP together with YFP–HdaA or YFP–HdaAR145A or HdaAΔN, respectively), strain JC654 (expressing a YFP–CFP fusion protein) and strain JC809 (expressing YFP and CFP) were cultivated to exponential phase in M2G medium prior to the addition of 0.3 % xylose for 1 h. Live cells were then collected for acceptor photobleaching FRET analysis as previously described (Kentner & Sourjik, 2009) (also see Methods). The graph shows the mean increase in CFP emission measured after YFP photobleaching for each population of cells, corresponding to the apparent FRET efficiency. Values above 0.5 % indicate an interaction between the CFP- and the YFP-tagged proteins, while values above 1 % indicate a strong interaction (Kentner & Sourjik, 2009). As expected, the control strain JC654 gave a value above 1 %, while the control strain JC809 gave a value below 0.5 %. For each strain, at least three measurements were performed. Error bars indicate sd.

Fig. 5.

The N-terminal region of HdaA is not sufficient for co-localization with the replisome. (a) Cells of strains JC1094 (expressing DnaN–CFP and YFP–HdaA′_1–10), JC673 (expressing YFP–HdaA′_1–35) and JC674 (expressing YFP–HdaA′_1–50) were cultivated to exponential phase in M2G medium, and 0.3 % xylose was added to the medium 1 h before sample collection. Cells were visualized by phase-contrast (Ph3) and fluorescence microscopy. For strain JC1094, the right panel shows an overlay of the two fluorescence microscopy images. Bars, 2 µm. (b) The intracellular levels of YFP-tagged HdaA′ proteins in cell extracts from strains JC1094, JC673, JC674, JC780 (expressing YFP) and CB15N (wild-type, WT) were analysed by immunoblot using antibodies raised against GFP (also detecting YFP). Cells were cultivated to exponential phase in M2G medium and 0.3 % xylose (Xyl) or 0.2 % glucose (Glu) was added to media 6 h before sample collection.

Fig. 6.

Mutant YFP–HdaAR145A proteins form brighter fluorescent foci than YFP–HdaA in replicating predivisional cells. Swarmer cells from cultures of strains JC578 (expressing YFP–HdaA) and JC912 (expressing YFP–HdaAR145A) were isolated by centrifugation in a Percoll density gradient (Evinger & Agabian, 1977), resuspended in M2G medium containing 0.15 % xylose and allowed to progress synchronously through their cell cycle. Samples of each culture were collected after 120 min. The resulting predivisional cells were then visualized by fluorescence microscopy (same greyscale). Representative images acquired and visualized using identical settings are shown. Bars, 2 µm.