New insights into genetic inheritance in bacteria

A multidisciplinary study reveals how a molecular switch controls the distribution of bacterial DNA

When a cell divides, each daughter cell must receive a complete set of genetic information. In 2019, scientists led by Max Planck Fellow Martin Thanbichler discovered a CTP-dependent molecular switch that controls this process. Now the authors were able to elucidate in an international team how the hydrolysis of CTP determines the dynamics of bacterial DNA segregation. Their findings could lead to the development of specific enzyme inhibitors with potential antimicrobial activity.

Nucleotides serve as essential cofactors in a multitude of biochemical reactions, based on their ability to store energy in the form of energy-rich chemical bonds. Their hydrolysis powers the majority of energy-requiring processes in the cell. Importantly, it also helps to trigger conformational changes in proteins that act as molecular switches in diverse cellular pathways. Therefore, the nucleo­tide-dependent regula­tion of protein function is a central topic in biology. A new multidisciplinary study led by Martin Thanbichler from the Max Planck Institute for Terrestrial Microbiology and Philipps-Universität Marburg has now elucidated a new role of nucleotide hydrolysis in bacterial DNA segregation.

In most bacteria, the distribution of newly copied DNA molecules depends on a three-part system known as the ParABS system. Recently, the scientists had discovered that its key protein ParB represents a novel class of nucleotide-dependent molecular switches that uses the nucleotide cytidine triphosphate (CTP). However, the precise role of CTP hydrolysis remained unclear. In their new publi­cation in the journal Molecular Cell, the authors now clarify the nucleotide-dependent conformational dynamics of ParB and show that the ParB CTPase activity determines the size and dynamics of the bacterial DNA partition complexes.

During bacterial cell division, hundreds of ParB dimers assemble into a large DNA-associated complex, the so-called partition complex, which serves as a docking site for the DNA segregation machinery. In doing so, they first bind to specific sequen­ce motifs (parS) in the target DNA molecules. CTP binding then allows their transition to a ring-like conforma­tion in which they embrace the DNA and slide away laterally from the initial binding site, in a process known as “spreading”. As a result, the parS sequence becomes avail­able again for the loading of new ParB dimers. In this way, even a single parS motif is sufficient to support the loading of hundreds of ParB rings on the DNA.

The new study demonstrates that the CTPase activity of ParB determines the size of the partition complex by limiting the sliding time of ParB rings and, thus, the degree of their spreading from parS motifs. CTP hydrolysis triggers ring opening, thereby promoting the unloading of ParB and its subsequent reasso­ci­ation with a parS motif. Using CTPase-deficient ParB variants, the scientists showed that this process is essential for ParABS-mediated DNA segregation. In the absence of CTPase activity, ParB forms hyperstable rings that remain associated longer with the DNA, leading to excessive spreading and severe DNA segre­gation defects.

ParB represents the first class of CTP-dependent molecular switches known to date, whereas all previously characterized molecular switches use the purine nucleotides ATP and GTP instead. Interestingly, the catalytic domain of ParB is conserved in other proteins. “It is likely that CTP binding and hydrolysis control the activity of various other protein families in a range of different cellular processes. Our findings thus pave the way to comprehensively understand the principles underlying the function of CTP-dependent molecular switches in biology,” says Manuel Osorio-Valeriano, the first author of the publication.

Additionally, this study provides the first detailed insights into the catalytic mechanism of CTP hydrolysis by ParB proteins. Professor Martin Thanbichler adds: “Our results could lead to the development of specific enzyme inhibitors with potential antimicrobial activity. Moreover, the new findings advance our understanding of the mechanistic aspects of bacterial DNA segregation and open new perspectives for the design of self-replicative minimal synthe­tic cells.”

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