Identification of Novel Factors Involved in Modulating Motility of Salmonella enterica Serotype Typhimurium

Salmonella enterica serotype Typhimurium can move through liquid using swimming motility, and across a surface by swarming motility. We generated a library of targeted deletion mutants in Salmonella Typhimurium strain ATCC14028, primarily in genes specific to Salmonella, that we have previously described. In the work presented here, we screened each individual mutant from this library for the ability to move away from the site of inoculation on swimming and swarming motility agar. Mutants in genes previously described as important for motility, such as flgF, motA, cheY are do not move away from the site of inoculation on plates in our screens, validating our approach. Mutants in 130 genes, not previously known to be involved in motility, had altered movement of at least one type, 9 mutants were severely impaired for both types of motility, while 33 mutants appeared defective on swimming motility plates but not swarming motility plates, and 49 mutants had reduced ability to move on swarming agar but not swimming agar. Finally, 39 mutants were determined to be hypermotile in at least one of the types of motility tested. Both mutants that appeared non-motile and hypermotile on plates were assayed for expression levels of FliC and FljB on the bacterial surface and many of them had altered levels of these proteins. The phenotypes we report are the first phenotypes ever assigned to 74 of these open reading frames, as they are annotated as ‘hypothetical genes’ in the Typhimurium genome.     

RecA Protein Plays a Role in the Chemotactic Response and Chemoreceptor Clustering of Salmonella enterica

The RecA protein is the main bacterial recombinase and the activator of the SOS system. In Escherichia coli and Salmonella enterica sv. Typhimurium, RecA is also essential for swarming, a flagellar-driven surface translocation mechanism widespread among bacteria. In this work, the direct interaction between RecA and the CheW coupling protein was confirmed, and the motility and chemotactic phenotype of a S. Typhimurium ΔrecA mutant was characterized through microfluidics, optical trapping, and quantitative capillary assays. The results demonstrate the tight association of RecA with the chemotaxis pathway and also its involvement in polar chemoreceptor cluster formation. RecA is therefore necessary for standard flagellar rotation switching, implying its essential role not only in swarming motility but also in the normal chemotactic response of S. Typhimurium.     

Genetic transplantation: Salmonella enterica serovar Typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems

In Salmonella enterica serovar Typhimurium, sigma(28) and anti-sigma factor FlgM are regulatory proteins crucial for flagellar biogenesis and motility. In this study, we used S. enterica serovar Typhimurium as an in vivo heterologous system to study sigma(28) and anti-sigma(28) interactions in organisms where genetic manipulation poses a significant challenge due to special growth requirements. The chromosomal copy of the S. enterica serovar Typhimurium sigma(28) structural gene fliA was exchanged with homologs of Aquifex aeolicus (an extreme thermophile) and Chlamydia trachomatis (an obligate intracellular pathogen) by targeted replacement of a tetRA element in the fliA gene location using lambda-Red-mediated recombination. The S. enterica serovar Typhimurium hybrid strains showed sigma(28)-dependent gene expression, suggesting that sigma(28) activities from diverse species are preserved in the heterologous host system. A. aeolicus mutants defective for sigma(28)/FlgM interactions were also isolated in S. enterica serovar Typhimurium. These studies highlight a general strategy for analysis of protein function in species that are otherwise genetically intractable and a straightforward method of chromosome restructuring using lambda-Red-mediated recombination.     

Characterization of Photoactivated Singlet Oxygen Damage in Single-Molecule Optical Trap Experiments

Optical traps or “tweezers” use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments—the most common biological application of optical tweezers—and may guide the development of more robust experimental protocols.     

A new muscle contractile system composed of a thick filament lattice and a single actin filament

To bridge the gap between the contractile system in muscle and in vitro motility assay, we have devised an A-band motility assay system. A glycerinated skeletal myofibril was treated with gelsolin to selectively remove the thin filaments and expose a single A-band. A single bead-tailed actin filament trapped by optical tweezers was made to interact with the inside or the outer surface of the A-band, and the displacement of the bead-tailed filament was measured in a physiological ionic condition by phase-contrast and fluorescence microscopy. We observed large back-and-forth displacement of the filament accompanied by a large change in developed force. Despite this large tension fluctuation, we found that the average force was proportional to the overlap inside and outside the A-band up to approximately 150 nm and 300 nm from the end of the A-band, respectively. Consistent with the difference in the density of myosin molecules, the average force per unit length of the overlap inside the A-band (the time-averaged force/myosin head was approximately 1 pN) was approximately twice as large as that outside. Thus, we conclude that the A-band motility assay system described here is suitable for studying force generation on a single actin filament, and its sliding movement within a regular three-dimensional thick filament lattice.     

The structure of chromatophores from purple photosynthetic bacteria fused with lipid-impregnated collodion films determined by near-field scanning optical microscopy.

Lipid-impregnated collodion (nitrocellulose) films have been frequently used as a fusion substrate in the measurement and analysis of electrogenic activity in biological membranes and proteoliposomes. While the method of fusion of biological membranes or proteoliposomes with such films has found a wide application, little is known about the structures formed after the fusion. Yet, knowledge of this structure is important for the interpretation of the measured electric potential. To characterize structures formed after fusion of membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides with lipid-impregnated collodion films, we used near-field scanning optical microscopy. It is shown here that structures formed from chromatophores on the collodion film can be distinguished from the lipid-impregnated background by measuring the fluorescence originating either from endogenous fluorophores of the chromatophores or from fluorescent dyes trapped inside the chromatophores. The structures formed after fusion of chromatophores to the collodion film look like isolated (or sometimes aggregated, depending on the conditions) blisters, with diameters ranging from 0.3 to 10 microm (average approximately 1 microm) and heights from 0.01 to 1 microm (average approximately 0.03 microm). These large sizes indicate that the blisters are formed by the fusion of many chromatophores. Results with dyes trapped inside chromatophores reveal that chromatophores fused with lipid-impregnated films retain a distinct internal water phase.     

Uncovering a large set of genes that affect surface motility in Salmonella enterica serovar Typhimurium

We describe a large set of genes affecting motility in Salmonella enterica serovar Typhimurium. Identified in microarray experiments as displaying flagellar gene expression patterns or controlled by known flagellar regulators, we show that null mutations in these genes primarily affect swarming motility. Three genes function in chemotaxis.     

FimZ Is a Molecular Link between Sticking and Swimming in Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium produces two types of filamentous appendages on its surface. Fimbriae mediate adherence to tissues and cells via receptor-specific interactions, and flagella are the organelles of motility. These appendages play a role in colonization and dissemination, respectively, from infected surfaces and may be important components of bacterial survival. Increased expression of FimZ in serovar Typhimurium resulted in bacteria which were hyperfimbriated but were nonmotile in soft agar. This lack of motility was associated with down regulation of the flhDC master flagellar operon. Therefore, FimZ represents a molecular connection between flagella and fimbrial formation in serovar Typhimurium, indicating that the synthesis of flagella and fimbriae are oppositely controlled.     

Construction and calibration of an optical trap on a fluorescence optical microscope

The application of optical traps has come to the fore in the last three decades. They provide a powerful, sterile and noninvasive tool for the manipulation of cells, single biological macromolecules, colloidal microparticles and nanoparticles. An optically trapped microsphere may act as a force transducer that is used to measure forces in the piconewton regime. By setting up a well-calibrated single-beam optical trap within a fluorescence microscope system, one can measure forces and collect fluorescence signals upon biological systems simultaneously. In this protocol, we aim to provide a clear exposition of the methodology of assembling and operating a single-beam gradient force trap (optical tweezers) on an inverted fluorescence microscope. A step-by-step guide is given for alignment and operation, with discussion of common pitfalls.     

Optical trap as a tool for studying motor proteins

The review briefs the optical trap method, a modern experimental tool based on the recently discovered ability of light to trap and hold micron and submicron particles in a focused beam. The physical principle underlying the optical trap and the opportunities that it provides for studying the molecular nature of biological motility are considered. Several studies into the physical characteristics and functions of single motor protein molecules performed using the optical trap and recording nanometer displacements and piconewton forces are analyzed.     

不同微生物的单光束激光陷阱操纵

本文报导了分别采用Ｈｅ－Ｎｅ和Ａｒ＾＋激光器与光不显微镜构成的单光束激光陷阱操纵酵母菌、青霉等不同微生物的实验观察结果,讨论了操纵条件。研究表明：用单光束高会聚激光产生的梯度力操纵微生物体,其有效作用力的大小不仅与激光功率、波长、束腰半径和光束会聚角有关,还与微生物的大小、吸收系数、菌龄及培养方法等因素有关。     

Antimicrobial resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential, landfill, and natural environments in southern Ontario, Canada

To assess the impacts of different types of human activity on the development of resistant bacteria in the feces of wild small mammals, we compared the prevalences and patterns of antimicrobial resistance and resistance genes in generic Escherichia coli and Salmonella enterica isolates from fecal samples collected from wild small mammals living in four environments: swine farms, residential areas, landfills, and natural habitats. Resistance to antimicrobials was observed in E. coli isolates from animals in all environments: 25/52 (48%) animals trapped at swine farms, 6/69 (9%) animals trapped in residential areas, 3/20 (15%) animals trapped at landfills, and 1/22 (5%) animals trapped in natural habitats. Animals trapped on farms were significantly more likely to carry E. coli isolates with resistance to tetracycline, ampicillin, sulfisoxazole, and streptomycin than animals trapped in residential areas. The resistance genes sul2, aadA, and tet(A) were significantly more likely to be detected in E. coli isolates from animals trapped on farms than from those trapped in residential areas. Three S. enterica serotypes (Give, Typhimurium, and Newport) were recovered from the feces of 4/302 (1%) wild small mammals. All Salmonella isolates were pansusceptible. Our results show that swine farm origin is significantly associated with the presence of resistant bacteria and resistance genes in wild small mammals in southern Ontario, Canada. However, resistant fecal bacteria were found in small mammals living in all environments studied, indicating that environmental exposure to antimicrobials, antimicrobial residues, resistant bacteria, or resistance genes is widespread.     

FlgN Is Required for Flagellum-Based Motility by Bacillus subtilis

The assembly of the bacterial flagellum is exquisitely controlled. Flagellar biosynthesis is underpinned by a specialized type III secretion system that allows export of proteins from the cytoplasm to the nascent structure. Bacillus subtilis regulates flagellar assembly using both conserved and species-specific mechanisms. Here, we show that YvyG is essential for flagellar filament assembly. We define YvyG as an orthologue of the Salmonella enterica serovar Typhimurium type III secretion system chaperone, FlgN, which is required for the export of the hook-filament junction proteins, FlgK and FlgL. Deletion of flgN (yvyG) results in a nonmotile phenotype that is attributable to a decrease in hag translation and a complete lack of filament polymerization. Analyses indicate that a flgK-flgL double mutant strain phenocopies deletion of flgN and that overexpression of flgK-flgL cannot complement the motility defect of a ΔflgN strain. Furthermore, in contrast to previous work suggesting that phosphorylation of FlgN alters its subcellular localization, we show that mutation of the identified tyrosine and arginine FlgN phosphorylation sites has no effect on motility. These data emphasize that flagellar biosynthesis is differentially regulated in B. subtilis from classically studied Gram-negative flagellar systems and questions the biological relevance of some posttranslational modifications identified by global proteomic approaches.     

Alignment of biological microparticles by a polarized laser beam

The optical alignment of biological samples is of great relevance to microspectrometry and to the micromanipulation of single particles. Recently, Bayoudh et al. (J. Mod. Opt. 50:1581–1590, 2003) have shown that isolated, disk-shaped chloroplasts can be aligned in a controlled manner using an in-plane-polarized Gaussian beam trap, and suggested that this is due to their nonspherical shape. Here we demonstrate that the orientation of various micrometer-sized isolated biological particles, trapped by optical tweezers, can be altered in a controlled way by changing the plane of linear polarization of the tweezers. In addition to chloroplasts, we show that subchloroplast particles of small size and irregular overall shape, aggregated photosynthetic light-harvesting protein complexes as well as chromosomes can be oriented with the linearly polarized beam of the tweezers. By using a laser scanning confocal microscope equipped with a differential polarization attachment, we also measured the birefringence of magnetically oriented granal chloroplasts, and found that they exhibit strong birefringence with large local variations, which appears to originate from stacked membranes. The size and sign of the birefringence are such that the resulting anisotropic interaction with the linearly polarized laser beam significantly contributes to the torque orienting the chloroplasts.     

Salmonella enterica serotype Typhimurium ShdA is an outer membrane fibronectin-binding protein that is expressed in the intestine

The shdA gene is the only determinant known to be required for persistence of Salmonella enterica serotype Typhimurium (S. Typhimurium) in the murine caecum and for efficient and prolonged shedding of the organism with the faeces. To study the biological activity of the ShdA protein, we examined its expression and binding activity. ShdA was not detected with anti-ShdA antiserum in S. Typhimurium strain ATCC14028 grown in vitro, suggesting that this protein is not expressed under standard conditions of bacterial cultivation in the laboratory. However, in mice infected with S. Typhimurium, an immunofluorescence signal detected with anti-ShdA antiserum co-localized with that generated by anti-O4 antiserum in thin sections from the caecum. Expression of the cloned shdA gene from the T7 promoter in vitro resulted in detection of ShdA in the outer membrane of S. Typhimurium and in binding of fibronectin to the bacterial surface. Binding of purified glutathione-S-transferase (GST)-ShdA fusion protein to fibronectin was dose dependent and could be partially inhibited by preincubation with antifibronectin antibodies. GST-ShdA bound to connective tissue and the basement membrane in thin sections from the murine caecum in situ. A similar labelling pattern was produced when thin sections of the murine caecum were stained with antifibronectin antiserum. Collectively, these data demonstrate that ShdA is a surface-localized, fibronectin-binding protein whose expression is induced in vivo in the murine caecum, a tissue in which a cognate receptor of this outer membrane protein is expressed.     

The role of c-di-GMP signaling in an Aeromonas veronii biovar sobria strain

Aeromonas is a ubiquitous gram-negative bacterium that persists in the environment. It is shown that all isolates of persistent Aeromonas clones show strong biofilm formation ability. C-di-GMP regulates biofilm formation in many bacteria. To investigate the impact of c-di-GMP signaling, we introduced heterologous GGDEF and EAL domain proteins from Salmonella Typhimurium to an Aeromonas veronii biovar sobria strain. Overexpression of the GGDEF domain protein AdrA increased c-di-GMP concentration and biofilm formation and reduced motility. Production of the quorum-sensing signaling molecule C4-homoserine lactone and adhesion to aquatic plant duckweed and amoeba surfaces were enhanced. On the other hand, overexpression of the EAL domain protein YhjH decreased biofilm formation and increased motility.     

Why iron–dithiocarbamates ensure detection of nitric oxide in cells and tissues

The in vivo mechanism of NO trapping by iron-dithiocarbamate complexes is considered. Contrary to common belief, we find that in biological systems the NO radicals are predominantly trapped by ferric iron-dithiocarbamates. Therefore, the trapping leads to ferric mononitrosyl complexes which are diamagnetic and cannot be directly detected with Electron Paramagnetic Resonance spectroscopy. The ferric mononitrosyl complexes are far easily reduced to ferrous state with L-cysteine, glutathione, ascorbate or dithiocarbamate ligands than their non-nitrosyl counterpart. When trapping NO in oxygenated biological systems, the majority of trapped nitric oxide is found in diamagnetic ferric mononitrosyl iron complexes. Only a minority fraction of NO is trapped in the form of paramagnetic ferrous mononitrosyl iron complexes with dithiocarbamate ligands. Subsequent ex vivo reduction of biological samples sharply increases the total yield of the paramagnetic mononitrosyl iron complexes. Reduction also eliminates the overlapping EPR spectrum from Cu(2+)-dithiocarbamate complexes. This facilitates the quantification of yields from NO trapping.