Laboratory Strains of Bacillus subtilis Do Not Exhibit Swarming Motility

We redemonstrate that SwrA is essential for swarming motility in Bacillus subtilis, and we reassert that laboratory strains of B. subtilis do not swarm. Additionally, we find that a number of other genes, previously reported to be required for swarming in laboratory strains, are dispensable for robust swarming motility in an undomesticated strain. We attribute discrepancies in the literature to a lack of reproducible standard experimental conditions, selection for spontaneous swarming suppressors, inadvertent genetic linkage to swarming mutations, and auxotrophy.Many species of bacteria are capable of flagellum-mediated swimming motility in liquid broth. Of those species, a subset is also capable of a related, but genetically separable, form of flagellum-mediated surface movement called swarming motility (17). Examples of swarming-proficient species include Proteus mirabilis, Vibrio parahaemolyticus, Serratia marcescens, Escherichia coli, Salmonella enterica, and Bacillus subtilis (1, 15, 16, 20, 28). In general, swarming requires a surfactant or wetting agent to reduce surface tension, an increase in flagellar number per cell, and other genetic features that are distinct from swimming (7, 14).There is confusion in the literature concerning the genetic requirements of the swarming phenotype of B. subtilis. It is generally accepted that the ancestral undomesticated strain B. subtilis 3610 exhibits robust swarming motility (18, 20, 33). Swarming motility of strain 3610 requires the production of a secreted surfactant, called surfactin (6, 20), to reduce surface tension and permit surface spreading, and it also requires the protein SwrA to activate flagellar biosynthesis gene expression and increase the number of flagella on the cell surface (5, 20). Some reports claim that domesticated derivatives of 3610, such as the commonly used laboratory strain 168, are also swarming proficient (10, 18, 19, 24). Strain 168, however, is defective in both surfactin production (9, 25) and SwrA (5, 21, 31), and thus, swarming 168 strains challenge the genetic definition of swarming motility. Our lab has never observed swarming in laboratory strains, and here we investigated swarming motility in a reportedly swarming-proficient 168 strain.We obtained a reportedly swarming-proficient 168 strain (13) (generous gift of Simone Séror, Orsay University, Paris-Sud, France) (Table ​(Table1)1) and compared its swarming phenotype to that of 3610 under our standard conditions (20). Swarm plates were prepared one day prior to use with 25 ml of LB medium (10 g Bacto tryptone, 5 g Bacto yeast extract, 5 g NaCl per liter) fortified with 0.7% Bacto agar. To minimize water on the agar surface and thus minimize the potentially confounding influence of swimming motility, plates were dried 20 min prior to inoculation and 10 min postinoculation open-faced in a laminar flow hood. For qualitative swarm assays, plates were centrally inoculated with cells from a freshly grown overnight colony using a sterile stick. For quantitative swarm expansion assays, 1 ml of cells grown to mid-exponential phase (optical density at 600 nm [OD₆₀₀], 0.5) was resuspended in PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄ per liter, pH 7.0) containing 0.5% India ink (Higgins) to an OD₆₀₀ of 10 and centrally spotted (10 μl). Swarm expansion was measured at 0.5-h intervals along a transect on the plate. Plates were incubated at 37°C in 20 to 30% humidity. Whereas strain 3610 was swarming proficient, strain 168 (Orsay) was swarming deficient (Fig. ​(Fig.1A).1A). Thus, strain 168 (Orsay) appeared to behave similarly to all other laboratory strains we have tested previously (20, 21).Open in a separate windowFIG. 1.Swarming motility on LB and B media. In qualitative plate images, colonized agar appears white and uncolonized agar appears black on LB and B media, as indicated. Swarming cells colonize a larger surface area than nonswarming cells. All strains are derivatives of strain 3610 unless otherwise indicated. Bar, 2 cm. (A) Quantitative swarm expansion assays on solid medium and growth in liquid medium of the indicated strains on LB medium (closed symbols) and on B medium (open symbols). To indicate variability in a particular experiment, we have reproduced the quantitative swarm expansion assay of strain 3610 on LB and B media with error bars in Fig. S5 in the supplemental material. (B) Quantitative swarm expansion assays on LB (closed symbols) and B (open symbols) media. The following strains were used: DS3337 (sfp), DS2415 (swrA), DS5106 (168 swrA⁺), DS5758 (168 sfp⁺), and DS5759 (168 swrA⁺ sfp⁺). In all assays, B medium was made according to reference 2 except for strain DS5759, for which B medium was supplemented with 780 μM threonine to compensate for thrC auxotrophy. (C) Swarm plates of the indicated strains on LB medium made with equal parts peptone instead of tryptone. (D) Quantitative swarm expansion assays of the indicated 3610-derived mutant strains on LB medium (closed symbols) and on B medium (open symbols). The following strains were used: DS72 (yvzB), DS2268 (epr), DS3903 (phrC), DS4978 (rapC), DS4979 (oppD), DS2509 (swrB), and DS3649 (degU). All points are averages for three replicates.

TABLE 1.

Strains

A Regional Initiative to Reduce Skin Infections amongst Aboriginal Children Living in Remote Communities of the Northern Territory,Australia

Background

Linked to extreme rates of chronic heart and kidney disease, pyoderma is endemic amongst Aboriginal children in Australia''s Northern Territory (NT). Many of those with pyoderma will also have scabies. We report the results of a community-based collaboration within the East Arnhem Region, which aimed to reduce the prevalence of both skin infections in Aboriginal children.

Methodology/Principal Findings

Commencing September 2004, we conducted an ecological study that included active surveillance for skin infections amongst children aged <15 years in five remote East Arnhem communities over a three year period. Screening was undertaken by trained local community workers, usually accompanied by another project team member, using a standard data collection form. Skin infections were diagnosed clinically with the aid of a pictorial flip chart developed for the purpose. Topical 5% permethrin was provided for age-eligible children and all household contacts whenever scabies was diagnosed, whilst those with pyoderma were referred to the clinic for treatment in accordance with current guidelines. In addition, annual mass scabies treatment (5% permethrin cream) was offered to all community residents in accordance with current guidelines but was not directly observed. Pyoderma and scabies prevalence per month was determined from 6038 skin assessments conducted on 2329 children. Pyoderma prevalence dropped from 46.7% at baseline to a median of 32.4% (IQR 28.9%–41.0%) during the follow-up period – an absolute reduction of 14.7% (IQR 4.7%–16.8%). Compared to the first 18 months of observation, there was an absolute reduction in pyoderma prevalence of 18 cases per 100 children (95%CI −21.0, −16.1, p≤0.001) over the last 18 months. Treatment uptake increased over the same period (absolute difference 13.4%, 95%CI 3.3, 23.6). While scabies prevalence was unchanged, the prevalence of infected scabies (that is with superimposed pyoderma) decreased from 3.7% (95%CI 2.4, 4.9) to 1.5% (95%CI 0.7, 2.2), a relative reduction of 59%.

Conclusion

Although pyoderma prevalence remained unacceptably high, there was a substantial reduction overall with improvements in treatment uptake a critical factor. More acceptable alternatives, such as cotrimoxazole for pyoderma and ivermectin as a community-wide scabicide, warrant further investigation in these settings. We are encouraged by progress made through this work, where local action was led by local community members and primary health care providers with external training and support.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00884728","term_id":"NCT00884728"}}NCT00884728     

Multi-species integrative biclustering

We describe an algorithm, multi-species cMonkey, for the simultaneous biclustering of heterogeneous multiple-species data collections and apply the algorithm to a group of bacteria containing Bacillus subtilis, Bacillus anthracis, and Listeria monocytogenes. The algorithm reveals evolutionary insights into the surprisingly high degree of conservation of regulatory modules across these three species and allows data and insights from well-studied organisms to complement the analysis of related but less well studied organisms.     

Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.     

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.     

Chronic cigarette smoking causes hypertension, increased oxidative stress, impaired NO bioavailability, endothelial dysfunction, and cardiac remodeling in mice

Cigarette smoking is a major independent risk factor for cardiovascular disease. While the association between chronic smoking and cardiovascular disease is well established, the underlying mechanisms are incompletely understood, partly due to the lack of adequate in vivo animal models. Here, we report a mouse model of chronic smoking-induced cardiovascular pathology. Male C57BL/6J mice were exposed to whole body mainstream cigarette smoke (CS) using a SCIREQ "InExpose" smoking system (48 min/day, 5 days/wk) for 16 or 32 wk. Age-matched, air-exposed mice served as nonsmoking controls. Blood pressure was measured, and cardiac MRI was performed. In vitro vascular ring and isolated heart experiments were performed to measure vascular reactivity and cardiac function. Blood from control and smoking mice was studied for the nitric oxide (NO) decay rate and reactive oxygen species (ROS) generation. With 32 wk of CS exposure, mice had significantly less body weight gain and markedly higher blood pressure. At 32 wk of CS exposure, ACh-induced vasorelaxation was significantly shifted to the right and downward, left ventricular mass was significantly larger along with an increased heart-to-body weight ratio, in vitro cardiac function tended to be impaired with high afterload, white blood cells had significantly higher ROS generation, and the blood NO decay rate was significantly faster. Thus, smoking led to blunted weight gain, hypertension, endothelial dysfunction, leukocyte activation with ROS generation, decreased NO bioavailability, and mild cardiac hypertrophy in mice that were not otherwise predisposed to disease. This mouse model is a useful tool to enable further elucidation of the molecular and cellular mechanisms of smoking-induced cardiovascular diseases.     

Stress-induced changes in gene interactions in human cells

Cells respond to variable environments by changing gene expression and gene interactions. To study how human cells response to stress, we analyzed the expression of >5000 genes in cultured B cells from nearly 100 normal individuals following endoplasmic reticulum stress and exposure to ionizing radiation. We identified thousands of genes that are induced or repressed. Then, we constructed coexpression networks and inferred interactions among genes. We used coexpression and machine learning analyses to study how genes interact with each other in response to stress. The results showed that for most genes, their interactions with each other are the same at baseline and in response to different stresses; however, a small set of genes acquired new interacting partners to engage in stress-specific responses. These genes with altered interacting partners are associated with diseases in which endoplasmic reticulum stress response or sensitivity to radiation has been implicated. Thus, our findings showed that to understand disease-specific pathways, it is important to identify not only genes that change expression levels but also those that alter interactions with other genes.