A novel caulobacter nucleoid-associated protein and its effects on global chromosome accessibility [electronic resource]
Keywords: Chemistry.

The bacterium Caulobacter crescentus divides asymmetrically. This lends to its suitability as a model organism for studying the bacterial cell cycle as well as the asymmetry inherent in all life. Central to Caulobacter's cell cycle is its single circular chromosome, which encodes genetic elements whose expression patterns are coordinated in a temporally oscillating transcriptional network. But there is more to the bacterial chromosome than genetic elements like genes and their promoters; in reality, the chromosome is a DNA polymer with physical constraints, capabilities, and complexities outside of its role as a template for transcription. For instance, DNA in bacteria is supercoiled and compacted by three orders of magnitude. At the ~1Mb level, the E. coli nucleoid is characterized by insulated macrodomains and flexible unstructured regions. At the ~10 kb level the bacterial nucleoid is arranged into so-called microdomains by nucleoid-associated proteins (NAPs). This structuring of DNA functions in segregation, replication, decatenation, and double- and single-strand break repair. In the work presented in this thesis I leverage four Next Generation Sequencing (NGS)-based technologies to examine the regulation and function of four physical and non-genic features of the Caulobacter chromosome: its covalent modifications, essential intergenic regions, and global accessibility, as well as a novel, essential nucleoid-associated protein. First, in Chapter 2, I explore the covalent modification systems in Caulobacter using SMRT sequencing technologies. By measuring the methylation state of every base pair in the chromosome at five times in the cell cycle, I demonstrate that DNA methylation by the cell cycle-regulated methyltransferase CcrM is in fact dynamic, changing from fully methylated to hemimethylated at the time of replication fork passage. Importantly, the master transcriptional regulators ctrA and dnaA have promoters that become activated or repressed respectively once hemimethylated. From this perspective, the methylation data supports a model in which the replicating chromosome acts like a clock, with the cell cycle timing of ctrA activation and dnaA repression precisely synchronized with replication fork passage. I then use the SMRT sequencing data to predict which additional promoters besides PctrA and PdnaA are possibly controlled by methylation state. In addition, I also report that some of the CcrM target sites were found to be constitutively unmethylated. Work by our colleagues has shown that at one such unmethylated region of the chromosome called Gap 7, the DNA-binding proteins MucR1/2 block CcrM from its cognate site. In Chapter 3, I investigate Caulobacter's essential intergenic regions, called Gap regions. These non-genic elements of unknown function appeared as essential regions of the chromosome during Transposon-sequencing (Tn-seq) experiments. By performing molecular genetics experiments I demonstrated that at least some Gaps are nonessential. Furthermore, I deduce that at least two of those nonessential Gaps appeared in the Tn-seq screen because they are specifically protected from transposase, the enzyme that carries out transposition, in vivo. I end this chapter by exploring the effects of placing the intergenic regions encompassing Gaps onto high copy plasmids. These experiments demonstrate that such perturbations actually cause elongation and division defects in Caulobacter in a growth rate-dependent manner. I predict that some of these transposase-protected regions may be blocked by the binding of specific proteins that function in the Caulobacter cell cycle. In Chapter 4, I build further support for these conclusions by developing and performing ATAC-seq in Caulobacter. By measuring transposase accessibility of the entire chromosome in vivo, ATAC-seq more definitively and globally divides Gaps into non-disruptable regions, which are relatively protected from transposase enzyme, and true candidate essential regions. Thus, uncharacterized non-genic elements of the chromosome may perform essential functions in Caulobacter. I also performed ATAC-seq on a strain lacking the novel nucleoid-associated protein GapR, which binds over many Gaps in vivo, to test whether this protein is responsible for forming Gap regions where it binds the chromosome. While GapR does not affect local DNA accessibility, global analysis of ATAC-seq data provides evidence that GapR plays key roles in shaping megabase-scale properties of the Caulobacter nucleoid. In WT (wild type) cells, this global accessibility pattern is reminiscent of the macrodomain-level structure of the E. coli chromosome, which features insulated origin and terminus-proximal regions as well as two highly flexible origin-flanking regions. These domains compose the largest scale of chromosome organization known in prokaryotes and are thought to help control the fidelity of chromosome segregation. Such macrodomains have not been observed explicitly before in Caulobacter. Overall, ATAC-seq shows that the chromosome is varied in accessibility along its length, reflecting how transcription, local DNA structure, NAPs like GapR, and possibly other protein-DNA interactions, alongside other unknown factors, may together yield globally varying enzyme access to the nucleoid. In Chapter 5, I focus on GapR, a novel, conserved and essential DNA binding protein introduced above. ChIP-seq experiments revealed that GapR binds AT-rich DNA globally throughout the chromosome, including over a majority of Gap regions. Although GapR shares many similarities with the E. coli NAP H-NS, there are two important differences between these proteins. First, whereas H-NS represses transcription of many genes in E. coli, depletion-RNAseq experiments in which gene expression changes were measured after proteolytic removal of GapR from Caulobacter demonstrated that GapR does not control transcription. In addition, although GapR is essential in Caulobacter, hns is dispensable in E. coli. In the final Chapter I will explore efforts to elucidate the essential function of GapR. Perspectives on additional functions of GapR proposed by our colleagues, including the regulation of initiation of chromosome segregation, are also discussed. Altogether, the work in this chapter provides evidence that NAPs can perform multiple diverse functions, including those that help fine-tune cell cycle progression. These experiments highlight an integrated view of the bacterial cell cycle in which both genic as well as non-genic and physical elements of the chromosome play key roles. Further, they herald novel applications of NGS techniques as promising tools for microbiologists.