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Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA-smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere–nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B . pertussis , the causal agent of human whooping cough, and B . bronchiseptica , which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B . bronchiseptica , which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B . pertussis , which evolved from a B . bronchiseptica -like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1 , ctaCDFGE1 , and cyoABCD1 . To test the hypothesis that the three cytochrome oxidases encoded within the B . pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B . bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo . No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B . pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B . bronchiseptica producing only the three B . pertussis -conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens.

Bordetella pertussis causes the acute disease whooping cough and survives only in the human respiratory tract, while Bordetella bronchiseptica causes long-term, chronic infections in a broad range of mammals and can also survive in extra-host environments. These bacteria produce a nearly identical set of virulence factors, including adenylate cyclase toxin (ACT), a protein that is modified by the addition of acyl chains. Acylation is required for ACT to cause hemolysis and for efficient intoxication of host cells in vitro . We found that ACT acylation is also important, but not absolutely required, during infection. We also discovered differences in ACT production, acylation, and secretion between B. bronchiseptica and B. pertussis that may contribute to the different virulence strategies of these species. This study highlights the advantage of conducting comparative analyses between closely related species to better understand the evolution of virulence.

To be effective pathogens, bacteria must adapt to the microenvironments they infect within a host. The respiratory tract can be broadly divided into two environments: the upper and lower respiratory tracts. The classical bordetellae, including Bordetella bronchiseptica, infect both the upper and lower respiratory tracts of their hosts and must therefore adjust their physiologies as they transition between these environments. This dissertation investigates three aspects of B. bronchiseptica adaptation to the respiratory tract. First, we investigated which genes are expressed specifically in the lower respiratory tract, focusing on one putative gene: BB1259. Our data indicate that BB1259 is not required for infection despite its putative expression pattern. Second, we biochemically characterized a two-component regulatory system, PlrSR, required for persistent infection in the lower respiratory tract. We found that PlrSR is a canonical two component system, with PlrS functioning as both a kinase and phosphatase to PlrR. We also showed that PlrS phosphatase activity, in addition to PlrS kinase activity, is required during infection. Third, we interrogated the role of cytochrome oxidases during infection, focusing on the evolutionary adaptation within the classical bordetellae towards life within the mammalian respiratory tract. No individual cytochrome oxidase was necessary during infection. However, the combination of the three cytochrome oxidases conserved between B. bronchiseptica and the closely related B. pertussis were sufficient for infection, with one cytochrome oxidase, CyoABCD1, being sufficient for infection in both mouse models tested. Understanding adaptation to the respiratory tract environment could lead to new therapeutic targets and thus address the public health risk still caused by the classical bordetellae despite widespread vaccination. In addition, researching how bacteria survive within the different environments of the respiratory tract also increases our knowledge of host physiology in areas that are difficult to assess directly like the surface of epithelial cells. Overall, this work contributes broadly to our understanding of the physiology of respiratory pathogens within the respiratory tract.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis, the causal agent of human whooping cough, and B. bronchiseptica, which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica, which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis, which evolved from a B. bronchiseptica-like ancestor but now only survives only in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1, and cyoABCD1. To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo. No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis-conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also showed that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on respiration requirements for bacteria that colonize the respiratory tract, the environment at the surface of the ciliated epithelium, and the evolution of virulence in bacterial pathogens.Competing Interest StatementThe authors have declared no competing interest.

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against the pertussis toxin but does not prevent transmission or colonization. Cases of B. pertussis infections are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past three days in the lung, suggesting PlrSR works in a phosphorylation dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 and Glu522 were essential for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS can both phosphotransfer to and exert phosphatase activity towards PlrR. Unexpectedly, PlrR forms a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics. Competing Interest Statement The authors have declared no competing interest.

Centromeres play a fundamental role in chromosome segregation. Although originally thought to be silent chromosomal regions, centromeres are actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA smFISH foci fluctuate in their levels over the cell cycle and do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Our results demonstrate that alpha-satellite expression occurs through RNA Polymerase II-dependent transcription, but does not require centromere proteins and other cell division components. Instead, our work implicates centromere-nucleolar associations as the major factor regulating alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels, explaining variations in alpha-satellite RNA between cell lines. In addition, alpha-satellite transcript levels increase substantially when the nucleolus is disrupted. Together, our results are inconsistent with a direct, physical role for alpha-satellite transcripts in cell division processes, and instead support a role for ongoing transcription in promoting centromere chromatin dynamics. The control of alpha-satellite transcription by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions. Competing Interest Statement

Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant CENP-A. Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres, but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery, but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.

PlrSR, a member of the NtrYX family of two-component regulatory systems (TCSs), is required for the classical bordetellae, including the causative agent of whooping cough, , to persist in the lower respiratory tract. The genes are in the middle of a six-gene cluster whose regulation and roles during infection were unknown. and are often found 5' to homologs in β- and γ- proteobacteria, while are often found 3' to homologs in ⍺-proteobacteria. We investigated these genes to determine if they have a functional link to . We found that this gene cluster does not function as an operon. Rather, it contains two internal promoters: a weaker promoter in the 3' end of and a stronger promoter in the 3' end of Additionally, our results indicate that PlrP functions as a third component of the PlrSR TCS. Genetic manipulations of and indicate that PlrP is essential and inhibits PlrS phosphatase activity, likely through PlrS's PDC domain. Since our results indicate that PlrR can be phosphorylated by another unknown phosphodonor , limiting PlrS phosphatase activity ensures PlrR∼P is not dephosphorylated to lethally low levels. Using natural-host models, we determined that high levels of PlrR∼P are required for survival, and PlrP affects PlrS activity . Given that homologs always colocalize with homologs, we propose that PlrP may fulfill similar functions in other β- and γ-proteobacteria that encode NtrYX- family TCSs, including nonpathogens. species, including , the causal agent of whooping cough, cause respiratory infections in humans and other animals. Their PlrSR two component regulatory systems, members of the NtrYX family, are required for survival in the lower respiratory tract. We characterized the six-gene cluster that includes and , identifying one promoter within the first gene that drives expression of the second gene, which we named , as well as plrS, and another promoter near the 3' end of that drives expression of and the downstream genes. Our data indicate that the gene product is an essential third component of the PlrSR TCS, functioning to prevent PlrS from acting as a strong phosphatase . Comparative analyses suggest that PlrP homologs are present, and may function similarly, in NtrYX-family TCSs in other β- and γ-proteobacteria. Our results are important because they provide insight into how bacteria sense and respond to their environment, including those they experience while causing human infection, and this understanding could inform therapeutic and vaccine development.

In in vivo tissue engineering, many implanted cells die because of hypoxic conditions immediately postimplantation. The aim of this study was to determine whether delayed myoblast implantation, at day 4 or 7, improves myoblast survival compared with implantation at day 0 in an in vivo arterio-venous loop (AB loop) chamber model. In adult inbred Sprague-Dawley rats, an AB loop was inserted into a plastic chamber (day 0). In Group I, day 0, two million DiI-labeled (neonatal inbred) myoblasts were implanted around the AB loop. In Groups II and III, day 0, the AB loop was created and inserted into a novel delayed cell seeding chamber, and 4 (Group II) or 7 days (Group III) later the delay chamber was seeded with 2 million DiI-labeled myoblasts. Constructs were harvested 7-day postmyoblast implantation, for morphometric determination of DiI/DAPI-positive myoblasts/mm 2 , and percent vascular volume on Griffonia simplicifolia lectin (endothelial cell marker)–labeled tissue sections. Control (nonmyoblast seeded) and experimental (myoblast seeded) constructs demonstrated similar capillary and tissue growth patterns. DiI/DAPI-labeled myoblasts/mm 2 appeared in similar numbers in constructs implanted at days 0 and 4, but increased markedly in day-7 implanted constructs. The percent vascular volume increased significantly ( p  = 0.03) over time. A positive correlation existed between myoblast survival and construct vascularity ( p  = 0.017). In conclusion, delaying myoblast implantation to 7-day postconstruct assembly, when new capillary growth is well established, significantly correlates with increased myoblast survival and indicates that cell seeding in regenerative procedures should always occur into an established vascular bed.