Biology subjects

Mote, K. B.

Publications and source records attributed to Mote, K. B..

3 recordsLinked to original sources

bioRxiv2025

Genetic evidence for a periplasmic protein as a component for a subset of NtrYX two-component systems

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, Bordetella pertussis, to persist in the lower respiratory tract. The plrSR genes are in the middle of a six-gene cluster whose regulation and roles during infection were unknown. rsmB and plrP are often found 5 to plrSR homologs in {beta}- and {gamma}- proteobacteria, while trkAH are often found 3 to plrSR homologs in -proteobacteria. We investigated these genes to determine if they have a functional link to plrSR. 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 rsmB and a stronger promoter in the 3 end of plrS. Additionally, our results indicate that PlrP functions as a third component of the PlrSR TCS. Genetic manipulations of plrP, plrS, and plrR indicate that PlrP is essential in vitro and inhibits PlrS phosphatase activity, likely through PlrSs PDC domain. Since our results indicate that PlrR can be phosphorylated by another unknown phosphodonor in vitro, 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 in vivo survival, and PlrP affects PlrS activity in vivo. Given that plrP homologs always colocalize with ntrYX homologs, we propose that PlrP may fulfill similar functions in other {beta}- and {gamma}-proteobacteria that encode NtrYX- family TCSs, including nonpathogens. ImportanceBordetella species, including B. pertussis, 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 plrS and plrR, identifying one promoter within the first gene that drives expression of the second gene, which we named plrP, as well as plrS, and another promoter near the 3 end of plrS that drives expression of plrR and the downstream trkAH genes. Our data indicate that the plrP gene product is an essential third component of the PlrSR TCS, functioning to prevent PlrS from acting as a strong phosphatase in vitro. Comparative analyses suggest that PlrP homologs are present, and may function similarly, in NtrYX-family TCSs in other {beta}- and {gamma}-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.

microbiology↗

bioRxiv2024

DNA duplication-mediated activation of a two-component regulatory system serves as a bet-hedging strategy for Burkholderia thailandensis

Burkholderia thailandensis strain E264 (BtE264) and close relatives stochastically duplicate a 208.6 kb region of chromosome I via RecA-dependent recombination between two nearly identical insertion sequence elements. Because homologous recombination occurs at a constant, low level, populations of BtE264 are always heterogeneous, but cells containing two or more copies of the region (Dup+) have an advantage, and hence predominate, during biofilm growth, while those with a single copy (Dup-) are favored during planktonic growth. Moreover, only Dup+ bacteria form efficient biofilms within 24 hours in liquid medium. We determined that duplicate copies of a subregion containing genes encoding an archaic chaperone-usher pilus (aplFABCDE) and a two-component regulatory system (bubSR) are necessary and sufficient for generating efficient biofilms and for conferring a selective advantage during biofilm growth. BubSR functionality is required, as deletion of either bubS or bubR, or a mutation predicted to abrogate phosphorylation of BubR, abrogates biofilm formation. However, duplicate copies of the aplFABCDE genes are not required. Instead, we found that BubSR controls expression of aplFABCDE and bubSR by activating a promoter upstream of aplF during biofilm growth or when the 208.6 kb region, or just bubSR, are duplicated. Single cell analyses showed that duplication of the 208.6 kb region is sufficient to activate BubSR in 75% of bacteria during planktonic (BubSR OFF) growth conditions. Together, our data indicate that the combination of deterministic two-component signal transduction and stochastic, duplication-mediated activation of that TCS form a bet-hedging strategy that allows BtE264 to survive when conditions shift rapidly from those favoring planktonic growth to those requiring biofilm formation, such as may be encountered in the soils of Southeast Asia and Northern Australia. Our data highlight the positive impact that transposable elements can have on the evolution of bacterial populations. Author summaryTransposable elements naturally accumulate within genomes in all kingdoms of life. When present in the same orientation, a pair of homologous elements can act as substrates for DNA recombination reactions that can duplicate and delete intervening sequences - giving rise to genetically heterogenous populations. We showed here that Burkholderia thailandensis strain E264 uses this mechanism to amplify genes encoding a two-component regulatory system and an archaic chaperone usher pilus, priming the cells for rapid biofilm formation. The formation of a small subpopulation of biofilm-ready bacteria serves as a bet- hedging strategy, ensuring overall population survival should conditions change rapidly from those in which planktonic growth is optimal to those in which adherence and biofilm formation is required.

microbiology↗

bioRxiv2024

Cytochrome oxidase requirements in Bordetella reveal insights into evolution towards life in the mammalian 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. AUTHOR SUMMARYCytochrome oxidases, critical components for aerobic respiration, have been shown to be vital for pathogenesis and tissue tropism in several bacterial species. However, the majority of the research has focused on facultative anaerobes and infections of microoxic to anaerobic host environments, like the gut. We sought to understand the role of cytochrome oxidases during respiratory infection by Bordetella bronchiseptica, an obligate aerobe, performing the first analysis of cytochrome oxidases in an extracellular respiratory pathogen that we know of. By comparing B. bronchiseptica to the closely related B. pertussis, a strictly human-specific pathogen and the causative agent of whooping cough, we found three cytochrome oxidases that are important for growth and survival within the mammalian respiratory tract. We also found that a bo3-type cytochrome oxidase, predicted to have a low affinity for oxygen and therefore best suited to ambient air levels of oxygen, was sufficient for both the establishment of infection and persistence in the respiratory tract in mice. Our findings reveal the importance of low affinity cytochrome oxidases in respiratory pathogens, and emphasize the need to study the physiology of diverse pathogens.

microbiology↗