Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 103 and 5 × 104 cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.

Interest in public engagement with science activities has grown in recent decades, especially engagement through social media and among graduate students. Research on scientists’ views of engagement, particularly two-way engagement and engagement through social media, is sparse, particularly research examining graduate students’ views. We compare graduate students and faculty in biological and physical sciences at a land-grant, research-intensive university in their views on engagement. We find that both groups overwhelmingly believe that public input in decision-making around science issues is important, and hold largely pro-engagement attitudes. Graduate students, however, have somewhat more optimistic views of engagement through social media and on the appropriateness of discussing science controversy on social media. We discuss implications for graduate education and future engagement.

Biofilm formation is a complex process that requires a number of transcriptional, proteomic, and physiological changes to enable bacterial survival. The lipid membrane presents a barrier to communication between the machinery within bacteria and the physical and chemical features of their extracellular environment, and yet little is known about how the membrane influences biofilm development. We found that depleting the anionic phospholipid cardiolipin reduces biofilm formation in Escherichia coli cells by as much as 50%. The absence of cardiolipin activates the regulation of colanic acid synthesis (Rcs) envelope stress response, which represses the production of flagella, disrupts initial biofilm attachment, and reduces biofilm growth. We demonstrate that a reduction in the concentration of cardiolipin impairs translocation of proteins across the inner membrane, which we hypothesize activates the Rcs pathway through the outer membrane lipoprotein RcsF. Our study demonstrates a molecular connection between the composition of membrane phospholipids and biofilm formation in E. coli and suggests that altering lipid biosynthesis may be a viable approach for altering biofilm formation and possibly other multicellular phenotypes related to bacterial adaptation and survival. IMPORTANCE There is a growing interest in the role of lipid membrane composition in the physiology and adaptation of bacteria. We demonstrate that a reduction in the anionic phospholipid cardiolipin impairs biofilm formation in Escherichia coli cells. Depleting cardiolipin reduced protein translocation across the inner membrane and activated the Rcs envelope stress response. Consequently, cardiolipin depletion produced cells lacking assembled flagella, which impacted their ability to attach to surfaces and seed the earliest stage in biofilm formation. This study provides empirical evidence for the role of anionic phospholipid homeostasis in protein translocation and its effect on biofilm development and highlights modulation of the membrane composition as a potential method of altering bacterial phenotypes related to adaptation and survival.

15Biofilm formation is a complex process that requires a number of transcriptional, 16 proteomic, and physiological changes to enable bacterial survival. The lipid membrane 17 presents a barrier to communication between the machinery within bacteria and the 18 physical and chemical features of their extracellular environment, and yet little is 19 known about how the membrane influences biofilm development. We found that 20 depleting the anionic phospholipid cardiolipin reduces biofilm formation in Escherichia 21 coli cells by as much as 50%. The absence of cardiolipin activates the Rcs envelope stress 22 Importance 35There is a growing interest in the role of lipid membrane composition in the physiology 36 and adaptation of bacteria. We demonstrate that a reduction in the anionic 37 phospholipid cardiolipin impairs biofilm formation in Escherichia coli cells. Depleting 38 cardiolipin reduced protein translocation across the inner membrane and activated the 39 Rcs envelope stress response. Consequently, cardiolipin depletion produced cells 40 lacking assembled flagella, which impacted their ability to attach to surfaces and seed 41 the earliest stage in biofilm formation. This study provides empirical evidence for the 42 role of anionic phospholipid homeostasis in protein translocation and its effect on 43 biofilm development, and highlights modulation of the membrane composition as a 44 potential method of altering bacterial phenotypes related to adaptation and survival. 45 46 47 the composition and properties of membranes and biofilm formation is largely 87 7 Initiation of the Rcs signaling system leads to the phosphorylation of RcsB, 107 enabling it to dimerize and function as a transcriptional regulator (28). Phosphorlyated 108 RcsB can also form a heterodimer with the auxiliary protein RcsA (29); the RcsB-RcsB 109 and RcsA-RcsB complexes control the expression of a number of genes involved in acid 110 resistance, as well as the colanic acid and curli synthesis operons, osmotically inducible 111peroxidase osmC, and the small regulatory RNA rprA (30-32). We found that activation 112 of the Rcs pathway has little or no effect on Δcls cells growing planktonically. 113 Disrupting the Rcs system is sufficient to restore surface attachment in cls mutants, 114suggesting that Rcs activation is responsible for the biofilm defects we observe. The 115 data we describe supports a model in which CL reduction impairs the translocation of 116 proteins across the inner membrane (IM), which initiates the Rcs stress response, 117 leading to a downstream reduction in biofilm formation. 118 119Results 120 Cardiolipin affects surface attachment 121Previous studies have established that in the exponential phase of growth, CL 122 represents ~5% of total lipid composition in E. coli cells. Under certain environmental 123 conditions, such as high osmolarity, low pH, or upon entry into stationary phase, CL 124 content increases by as much as 200% (33). As nothing was known at the onset of this 125 study regarding how lipid composition ...

We have utilized our recently developed method to characterize lipid areas of various phospholipids with varying numbers of carbons and double bonds. In the case of lipids with unsaturated fatty acid chains our results suggest that lipid areas change with increasing hydrocarbon chain length, but not linearly-lateral lipid area is the result of the fine balance between the hydrocarbon chain length and double bond position. Furthermore, we discovered that the most dramatic change in lipid area occurs after the introduction of the first double bond to the lipid's acyl chains. Besides their importance in biology, lipid areas play a central role in molecular dynamics (MD) simulations, where their inconsistencies have been highlighted by the disparate results arising from MD simulations using different force fields. Since MD force fields are considered to be "well tuned" if they are able to reproduce experimental data, more reliable experimental information is necessary for their future development. 3388-Pos Board B493 Cardiolipin, a Key Component to Mimic the E. coli Bacterial Membrane in Model System Membranes Sílvia Lopes, Cristina Neves, Peter Eaton, Paula Gameiro. The phase transition temperatures of several lipidic systems were determined using two different techniques: dynamic light scattering (DLS) and steadystate fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize different lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for E. coli membranes. Given the important role of cardiolipin (CL) in natural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared to those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture.