Processing of DNA Clamp Loader Subunit DnaX Is Important in the Absence of Caulobacter Cell Division Inhibitors

Abstract

The DNA clamp loader is critical to the processivity of DNA replication and to the coordination of synthesis on the leading and lagging strands. In bacteria, the major subunit of the clamp loader, DnaX, has two forms: the essential full‐length DnaX tau and a shorter DnaX gamma. These forms are conserved across bacterial species and three distinct mechanisms have been found to create them such as ribosomal frameshift or transcriptional slippage. In Caulobacter crescentus, the full‐length DnaX tau is partially proteolyzed by ClpXP into two shorter DnaX gamma forms. Despite being discovered over 30 years ago, the purpose of maintaining multiple DnaX forms is unknown.To examine the purpose of encoding two forms, we created a nonproteolyzable allele of DnaX (tau‐only) which had no observable growth or morphological defects but showed a mild sensitivity to DNA damage. Adding an additional plasmid encoded copy of dnaX(tau‐only) to the dnaX(tau‐only) strain causes growth defects, filamentation, and increased RecA induction. These defects are not observed when adding a plasmid copy of dnaX(tau‐only) to the wild type strain, suggesting that an excess of DnaX tau is toxic, and likely causes replication stress.To better define the pathways that are affected in the absence of DnaX gamma, we performed transposon sequencing analysis to identifying genes that cause synthetic interactions with the dnaX(tau‐only) allele. We found that the insertions in two cell division inhibitors, sidA and didA, were underrepresented in the dnaX(tau‐only) strain compared to wild type. Consistent with this finding, we observed that adding a plasmid copy of dnaX‐tau‐only in a ΔsidA or ΔdidA strain causes an extreme growth defect and filamentation. Interestingly, although sidA is expressed as part of the RecA/LexA‐dependent DNA damage response in Caulobacter, we do not see a similar filamentation when we add an exogenous copy of the dnaX‐tau‐only allele to a ΔrecA strain. This indicates that the uninduced level of sidA expressing is sufficient to suppress the growth and filamentation phenotypes. The observation that cell division inhibitors are critical in the absence of proper DnaX processing suggests that the coordination of DNA replication and cell division is not intact in the dnaX‐tau‐only allele.