Chemical Biology of Chromosome Segregation in Pseudomonas aeruginosa

Abstract

Chromosome duplication and cell division are two critical events of a cell’s life cycle that occur with high precision across diverse environmental conditions. Inheriting a single copy of a chromosome by each daughter cell is essential for survival. An important aspect ensuring this is chromosome organization by condensins and other nucleoid-associated proteins, which, through varied levels of compaction, enables numerous DNA-dependent processes.

Passing down a fully duplicated and condensed chromosome during cell division is not a trivial job for bacteria since they efficiently adapt to the constantly changing environments and conditions that may cause DNA damage or interrupt normal progression and coordination between cell cycle events. Unlike in eukaryotes, chromosome duplication and cell division occur concurrently. To ensure the coordinated progression of the cell cycle and viability of offspring, bacteria employ a variety of pathways, some of which are known, while others are still unknown or yet to be discovered, for coordinating DNA duplication with cell division. The role of global chromosome compaction in the process of DNA duplication and cell division also remains unknown. Therefore, a better understanding of these complex processes and, more importantly, the mechanism of proteins involved in these processes could significantly enhance our existing knowledge. This, in turn, can enable us to manipulate chromosomes for various applications, find new targets for drug discovery, and even develop complementary approaches to genetic manipulations to explore cellular mechanisms of these processes in bacteria.

The current study investigated the role of two redundant systems, condensins and ParB, in the organization and assembly of replisome, segrosome, and divisome at the midcell and cell quarters in P. aeruginosa. P. aeruginosa is a Gram-negative, opportunistic human pathogen infecting immunocompromised patients and is the major cause of mortality and morbidity in patients suffering from cystic fibrosis. It has a reputation for being notoriously resistant to multiple antibiotics, making infections caused by P. aeruginosa life-threatening. Till now, the vast majority of studies have focused on multi-drug resistant aspects of P. aeruginosa, but lately, it has been gaining attention in the scientific world as an interesting model for cell cycle studies due to the discovery of new proteins and pathways associated with the cell cycle. Previously, our lab discovered a new class of condensins, MksB, a relative of MukB, that might have comparable functions in DNA compaction and segregation in P. aeruginosa and prompted us to further explore other life cycle facets of this fascinating yet deadly pathogen. Our lab also found that the condensins (SMC and MksB) and the ParABS system, mediating dynamics, and positioning of origin of replication (oriC) are synthetically lethal. Deletion of either affected oriC dynamics, global chromosome layout, and, most importantly, disrupted chromosome segregation process as evidenced by the production of anucleate cells. These results piqued our curiosity, and further investigations revealed that the condensins and the ParABS system have additional biological roles in the biogenesis of bacterial cells.

Condensins and the ParABS system are presently thought to be involved in chromosome compaction and segregation. In the first project (Chapter 3), we investigated which proteins assembled and occupied the midcell and cell quarters. Next, we explored the sequence of events at the midcell and cell quarters during the cell cycle and proposed a mechanism for the biogenesis of new bacterial cells governed by condensins and ParB. Using fluorescence microscopy, we tracked the dynamics of key proteins involved in chromosome replication, segregation, and cell division relative to oriC. Our findings revealed that these processes are closely linked, with all three occurring at the midcell in newborn cells and later on at cell quarters.

After characterizing and establishing the role of condensins and the ParABS system in chromosome duplication and cell division process, we next characterized and employed chemical probes to study the mechanism and role of condensins and ParB in these processes. P. aeruginosa, like many other Gram-negative bacteria, poses a significant in healthcare settings due to its ability to develop antibiotic resistance. The rise in multi-drug-resistant bacteria is swiftly diminishing the effectiveness of current antibiotics, resulting in high mortality and morbidity rates, and increased public health challenges due to bacterial infections. Since most of the drugs that are currently in late-stage clinical trials are analogs of the antibiotics towards which bacteria are rapidly becoming resistant, there is a dire need to develop or screen new antibacterials that are unique and mechanistically different from the existing ones. Based on the synthetic lethality of condensins (SMC and MksB) and ParB, we developed an assay to screen a library of fungal extracts for potential inhibitors of chromosome segregation. Specifically, we screened for compounds that inhibited the ParABS system (Chapter 4) and condensins (Chapter 5) based on their increased activity in condensin and ParB mutants, respectively.

In the second project (Chapter 4), we report the discovery of a family of compounds extracted from Humicola sp. that had enhanced activity condensin mutants and were later characterized as the first known inhibitors of ParB protein. Specifically, we found new analogs of sterigmatocystin, one of which, 4-hydroxy-sterigmatocystin, 4HS, displayed an antibacterial activity and induced the phenotype typical for parAB mutants, including defects in chromosome segregation and cell division. The data describe an inhibitor of the ParAB pathway and expand the known spectrum of activities of sterigmatocystin to include bacterial chromosome segregation. Furthermore, 4HS was also found to disrupt oriC dynamics in a manner similar to ParB.

In the third project (Chapter 5), we discovered compounds derived from Aspergillus sp. that were synergistic with the deletion of parB. These compounds inhibited cell growth and induced cell lysis in P. aeruginosa. The hit compounds were found to be analogs of previously reported asteltoxins that inhibited respiration. Our subsequent experiments confirmed that our hit compound, 176LJ22M, also inhibited membrane bioenergetics by suppressing ATP levels and dissipating proton motive force (PMF), leading to growth inhibition and cell lysis. Comparison with known inhibitors of respiration further revealed that 176LJ22M not only inhibits respiration but also targets chromosome segregation, as evidenced by the increased frequency of anucleate cells.

Overall, in the current thesis, the fundamental concepts about the cell cycle and the associated proteins involved in its progression were first established. This was followed by the development of complementary approaches to genetic manipulations to explore the cellular mechanism of chromosome segregation in P. aeruginosa. Additionally, chemical probes were discovered that targeted key proteins involved in chromosome segregation, including condensins and the ParABS system. These probes enabled deeper insights into the roles of these proteins in the segregation of chromosomes during the cell cycle.

Keywords: chromosome segregation, condensins, ParABS system, cell cycle, chemical probes, sterigmatocystin, asteltoxin, Pseudomonas aeruginosa