Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion.

IMPORTANCE: To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

Transmembrane Transcription Regulators Are Widespread in Bacteria and Archaea.

To adapt and proliferate, bacteria must sense and respond to the ever-changing environment. Transmembrane transcription regulators (TTRs) are a family of one-component transcription regulators that respond to extracellular information and influence gene expression from the cytoplasmic membrane. How TTRs function to modulate expression of their target genes while localized to the cytoplasmic membrane remains poorly understood. In part, this is due to a lack of knowledge regarding the prevalence of TTRs among prokaryotes. Here, we show that TTRs are highly diverse and prevalent throughout bacteria and archaea. Our work demonstrates that TTRs are more common than previously appreciated and are enriched within specific bacterial and archaeal phyla and that many TTRs have unique transmembrane region properties that can facilitate association with detergent-resistant membranes. IMPORTANCE One-component signal transduction systems are the major class of signal transduction systems among bacteria and are commonly cytoplasmic. TTRs are a group of unique one-component signal transduction systems that influence transcription from the cytoplasmic membrane. TTRs have been implicated in a wide array of biological pathways critical for both pathogens and human commensal organisms but were considered to be rare. Here, we demonstrate that TTRs are in fact highly diverse and broadly distributed in bacteria and archaea. Our findings suggest that transcription factors can access the chromosome and influence transcription from the membrane in both archaea and bacteria. This study challenges thus the commonly held notion that signal transduction systems require a cytoplasmic transcription factor and highlights the importance of the cytoplasmic membrane in directly influencing signal transduction.

Achieving Single-Molecule Tracking of Subcellular Regulation in Bacteria during Real-Time Environmental Perturbations.

Bacteria rely on protein systems for regulation in response to external environmental signals. Single-molecule fluorescence imaging and tracking has elucidated the complex mechanism of these protein systems in a variety of bacteria. We recently investigated Vibrio cholerae, the Gram-negative bacterium responsible for the human cholera disease, and its regulation of the production of toxins and virulence factors through the membrane-localized transcription factors TcpP and ToxR. These experiments determined that TcpP and ToxR work cooperatively under steady-state conditions, but measurements of how these dynamical interactions change over the course of environmental perturbations were precluded by the traditional preparation of bacterial cells confined on agarose pads. Here, we address this gap in technology and access single-molecule dynamics during real-time changes by implementing two alternative sample preparations: microfluidic devices and chitosan-coated coverslips. We report the first demonstration of single-molecule tracking within live bacterial cells in a microfluidic device. Additionally, using the chitosan-coated coverslips, we show that real-time environmental changes impact TcpP-PAmCherry dynamics, activating a virulence condition in the bacteria about 45 min after dropping to pH 6 and about 20 min after inducing ToxR expression. These new technology advances open our ability for new experiments studying a variety of bacteria with single-molecule imaging and tracking during real-time environmental perturbations.

Vibrio cholerae requires oxidative respiration through the bd-I and cbb3 oxidases for intestinal proliferation.

Vibrio cholerae respires both aerobically and anaerobically and, while oxygen may be available to it during infection, other terminal electron acceptors are proposed for population expansion during infection. Unlike gastrointestinal pathogens that stimulate significant inflammation leading to elevated levels of oxygen or alternative terminal electron acceptors, V. cholerae infections are not understood to induce a notable inflammatory response. To ascertain the respiration requirements of V. cholerae during infection, we used Multiplex Genome Editing by Natural Transformation (MuGENT) to create V. cholerae strains lacking aerobic or anaerobic respiration. V. cholerae strains lacking aerobic respiration were attenuated in infant mice 105-fold relative to wild type, while strains lacking anaerobic respiration had no colonization defect, contrary to earlier work suggesting a role for anaerobic respiration during infection. Using several approaches, including one we developed for this work termed Comparative Multiplex PCR Amplicon Sequencing (CoMPAS), we determined that the bd-I and cbb3 oxidases are essential for small intestinal colonization of V. cholerae in the infant mouse. The bd-I oxidase was also determined as the primary oxidase during growth outside the host, making V. cholerae the only example of a Gram-negative bacterial pathogen in which a bd-type oxidase is the primary oxidase for energy acquisition inside and outside of a host.

SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics.

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we develop a method for the case of multiple interconverting species undergoing free diffusion and introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of single-particle trajectory datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.

The draft genome of the hyperthermophilic archaeon Pyrodictium delaneyi strain hulk, an iron and nitrate reducer, reveals the capacity for sulfate reduction.

Pyrodictium delaneyi strain Hulk is a newly sequenced strain isolated from chimney samples collected from the Hulk sulfide mound on the main Endeavour Segment of the Juan de Fuca Ridge (47.9501 latitude, -129.0970 longitude, depth 2200 m) in the Northeast Pacific Ocean. The draft genome of strain Hulk shared 99.77% similarity with the complete genome of the type strain Su06T, which shares with strain Hulk the ability to reduce iron and nitrate for respiration. The annotation of the genome of strain Hulk identified genes for the reduction of several sulfur-containing electron acceptors, an unsuspected respiratory capability in this species that was experimentally confirmed for strain Hulk. This makes P. delaneyi strain Hulk the first hyperthermophilic archaeon known to gain energy for growth by reduction of iron, nitrate, and sulfur-containing electron acceptors. Here we present the most notable features of the genome of P. delaneyi strain Hulk and identify genes encoding proteins critical to its respiratory versatility at high temperatures. The description presented here corresponds to a draft genome sequence containing 2,042,801 bp in 9 contigs, 2019 protein-coding genes, 53 RNA genes, and 1365 hypothetical genes.