Jyl S. Matson

Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane.

Single‐molecule tracking in live Vibrio cholerae reveals that ToxR recruits the membrane‐bound virulence regulator TcpP to the toxT promoter

Vibrio cholerae causes the human disease cholera by producing a potent toxin. The V. cholerae virulence pathway involves an unusual transcription step: the bitopic inner‐membrane proteins TcpP and ToxR activate toxT transcription. As ToxT is the primary direct transcription activator in V. cholerae pathogenicity, its regulation by membrane‐localized activators is key in the disease process. However, the molecular mechanisms by which membrane‐localized activators engage the transcription process have yet to be uncovered in live cells. Here we report the use of super‐resolution microscopy, single‐molecule tracking, and gene knockouts to examine the dynamics of individual TcpP proteins in live V. cholerae cells with < 40 nm spatial resolution on a 50 ms timescale. Single‐molecule trajectory analysis reveals that TcpP diffusion is heterogeneous and can be described by three populations of TcpP motion: one fast, one slow, and one immobile. By comparing TcpP diffusion in wild‐type V. cholerae to that in mutant strains lacking either toxR or the toxT promoter, we determine that TcpP mobility is greater in the presence of its interaction partners than in their absence. Our findings support a mechanism in which ToxR recruits TcpP to the toxT promoter for transcription activation.

Regulated intramembrane proteolysis of the virulence activator TcpP in Vibrio cholerae is initiated by the tail-specific protease (Tsp)

Vibrio cholerae uses a multiprotein transcriptional regulatory cascade to control expression of virulence factors cholera toxin and toxin‐co‐regulated pilus. Two proteins in this cascade are ToxR and TcpP – unusual membrane‐localized transcription factors with relatively undefined periplasmic domains and transcription activator cytoplasmic domains. TcpP and ToxR function with each other and two other membrane‐localized proteins, TcpH and ToxS, to activate transcription of toxT, encoding the direct activator of toxin and pilus genes. Under some conditions, TcpP is degraded in a two‐step proteolytic pathway known as regulated intramembrane proteolysis (RIP), thereby inactivating the cascade. The second step in this proteolytic pathway involves the zinc metalloprotease YaeL; V. cholerae cells lacking YaeL accumulate a truncated yet active form of TcpP termed TcpP*. We hypothesized that a protease acting prior to YaeL degrades TcpP to TcpP*, which is the substrate of YaeL. In this study, we demonstrate that a C‐terminal protease called Tsp degrades TcpP to form TcpP*, which is then acted upon by YaeL. We present evidence that TcpH and Tsp serve to protect full‐length TcpP from spurious proteolysis by YaeL. Cleavage by Tsp occurs in the periplasmic domain of TcpP and requires residues TcpPA172 and TcpPI174 for wild‐type activity.

Sterilization of Biofilm on a Titanium Surface Using a Combination of Nonthermal Plasma and Chlorhexidine Digluconate

Nosocomial infections caused by opportunistic bacteria pose major healthcare problem worldwide. Out of the many microorganisms responsible for such infections, Pseudomonas aeruginosa is a ubiquitous bacterium that accounts for 10–20% of hospital-acquired infections. These infections have mortality rates ranging from 18 to 60% and the cost of treatment ranges from

Evolution of a global regulator: Lrp in four orders of γ-Proteobacteria

20,000 to

A putative Vibrio cholerae two-component system controls a conserved periplasmic protein in response to the antimicrobial peptide polymyxin B

80,000 per infection. The formation of biofilms on medical devices and implants is responsible for the majority of those infections. Only limited progress has been made to prevent this issue in a safe and cost-effective manner. To address this, we propose employing jet plasma to break down and inactivate biofilms in vitro. Moreover, to improve the antimicrobial effect on the biofilm, a treatment method using a combination of jet plasma and a biocide known as chlorhexidine (CHX) digluconate was investigated. We found that complete sterilization of P. aeruginosa biofilms can be achieved after combinatorial treatment using plasma and CHX. A decrease in biofilm viability was also observed using confocal laser scanning electron microscopy (CLSM). This treatment method sterilized biofilm-contaminated surfaces in a short treatment time, indicating it to be a potential tool for the removal of biofilms present on medical devices and implants.

Antimicrobial Effectiveness of Regular Dielectric-Barrier Discharge (DBD) and Jet DBD on the Viability of Pseudomonas aeruginosa

BackgroundBacterial global regulators each regulate the expression of several hundred genes. In Escherichia coli, the top seven global regulators together control over half of all genes. Leucine-responsive regulatory protein (Lrp) is one of these top seven global regulators. Lrp orthologs are very widely distributed, among both Bacteria and Archaea. Surprisingly, even within the phylum γ-Proteobacteria (which includes E. coli), Lrp is a global regulator in some orders and a local regulator in others. This raises questions about the evolution of Lrp and, more broadly, of global regulators.ResultsWe examined Lrp sequences from four bacterial orders of the γ-Proteobacteria using phylogenetic and Logo analyses. The orders studied were Enterobacteriales and Vibrionales, in which Lrp plays a global role in tested species; Pasteurellales, in which Lrp is a local regulator in the tested species; and Alteromonadales, an order closely related to the other three but in which Lrp has not yet been studied. For comparison, we analyzed the Lrp paralog AsnC, which in all tested cases is a local regulator. The Lrp and AsnC phylogenetic clusters each divided, as expected, into subclusters representing the Enterobacteriales, Vibrionales, and Pasteuralles. However the Alteromonadales did not yield coherent clusters for either Lrp or AsnC. Logo analysis revealed signatures associated with globally- vs. locally- acting Lrp orthologs, providing testable hypotheses for which portions of Lrp are responsible for a global vs. local role. These candidate regions include both ends of the Lrp polypeptide but not, interestingly, the highly-conserved helix-turn-helix motif responsible for DNA sequence specificity.ConclusionsLrp and AsnC have conserved sequence signatures that allow their unambiguous annotation, at least in γ-Proteobacteria. Among Lrp orthologs, specific residues correlated with global vs. local regulatory roles, and can now be tested to determine which are functionally relevant and which simply reflect divergence. In the Alteromonadales, it appears that there are different subgroups of Lrp orthologs, one of which may act globally while the other may act locally. These results suggest experiments to improve our understanding of the evolution of bacterial global regulators.

Genome Sequence of Klebsiella pneumoniae Urinary Tract Isolate Top52

The epidemic pathogen Vibrio cholerae senses and responds to different external stresses it encounters in the aquatic environment and in the human host. One stress that V. cholerae encounters in the host is exposure to antimicrobial peptides on mucosal surfaces. We used massively parallel cDNA sequencing (RNA-Seq) to quantitatively identify the transcriptome of V. cholerae grown in the presence and absence of sub-lethal concentrations of the antimicrobial peptide polymyxin B. We evaluated the transcriptome of both wild type V. cholerae and a mutant carrying a deletion of vc1639, a putative sensor kinase of an uncharacterized two-component system, under these conditions. In addition to many previously uncharacterized pathways responding with elevated transcript levels to polymyxin B exposure, we confirmed the predicted elevated transcript levels of a previously described LPS modification system in response to polymyxin B exposure. Additionally, we identified the V. cholerae homologue of visP (ygiW) as a regulatory target of VC1639. VisP is a conserved periplasmic protein implicated in lipid A modification in Salmonellae. This study provides the first systematic analysis of the transcriptional response of Vibrio cholerae to polymyxin B, raising important questions for further study regarding mechanisms used by V. cholerae to sense and respond to envelope stress.

Random Transposon Mutagenesis of Vibrio cholerae

Bacterial biofilm formation on medical implants is a major cause of illness in patients and is, therefore, increasing healthcare costs due to extended hospital stays and the failure/disposal of contaminated implants. Only limited progress has been made to prevent or eradicate this growing problem. Effective approaches include inhibiting the initiation of biofilm growth by killing planktonic bacteria and breaking down existing biofilms. We propose to employ atmospheric pressure nonthermal regular dielectric-barrier discharge (DBD) and jet plasma to kill bacteria that have been grown planktonically or in biofilms. In this paper, Pseudomonas aeruginosa was grown either planktonically or on titanium coupons in a bioreactor under dynamic conditions to form mature biofilms. The planktonic bacteria and biofilms were exposed to regular DBD and jet plasma and bacterial survival was evaluated after treatment for various times and at different distances. Within 5 min of plasma treatment, we observed complete decontamination of planktonically grown bacteria, and within 15 min, we observed more than a 3 log reduction (99.9%) of bacteria grown as biofilms. The efficacy of plasma treatment was also visualized using scanning electron microscopy, where disruption of biofilm was found with an increasing treatment time. This paper also determined that jet plasma is more effective in treating biofilms than regular DBD plasma.

Infant Mouse Model of Vibrio cholerae Infection and Colonization

ABSTRACT Klebsiella pneumoniae is a significant cause of nosocomial infections, including ventilator-associated pneumonias and catheter-associated urinary tract infections. K. pneumoniae strain TOP52 #1721 (Top52) was isolated from a woman presenting with acute cystitis and subsequently characterized using various murine models of infection. Here we present the genome sequence of K. pneumoniae Top52.