Identifying the Gene Regulatory Network of the Starvation-Induced Transcriptional Activator Nla28.

Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene regulatory network associated with Nla28, a transcriptional activator/enhancer binding protein (EBP) and a key regulator of the early starvation response. Previous work showed that Nla28 directly regulates EBP genes that are important for fruiting body development. However, the Nla28 regulatory network is likely to be much larger because hundreds of starvation-induced genes are downregulated in a nla28 mutant strain. To identify candidates for direct Nla28-mediated transcription, we analyzed the downregulated genes using a bioinformatics approach. Nine potential Nla28 target promoters (29 genes) were discovered. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the nine promoters along with three previously identified EBP gene promoters were indeed in vivo targets of Nla28. These results also suggested that Nla28 used tandem, imperfect repeats of an 8-bp sequence for promoter binding. Interestingly, eight of the new Nla28 target promoters were predicted to be intragenic. Based on mutational analyses, the newly identified Nla28 target loci contained at least one gene that was important for starvation-induced development. Most of these loci contained genes predicted to be involved in metabolic or defense-related functions. Using the consensus Nla28 binding sequence, bioinformatics, and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions, such as regulatory, metabolic, and cell envelope biogenesis, were assigned to many genes. IMPORTANCE In bacteria, starvation leads to profound changes in behavior and physiology. Some of these changes have economic and health implications because the starvation response has been linked to the formation of biofilms, virulence, and antibiotic resistance. To better understand how starvation contributes to changes in bacterial physiology and resistance, we identified the putative starvation-induced gene regulatory network associated with Nla28, a transcriptional activator from the bacterium Myxoccocus xanthus. We determined the mechanism by which starvation-responsive genes were activated by Nla28 and showed that several of the genes were important for the formation of a highly resistant cell type.

Bacterial glycocalyx integrity drives multicellular swarm biofilm dynamism.

Exopolysaccharide (EPS) layers on the bacterial cell surface are key determinants of biofilm establishment and maintenance, leading to the formation of higher-order 3D structures that confer numerous survival benefits to a cell community. In addition to a specific cell-associated EPS glycocalyx, we recently revealed that the social δ-proteobacterium Myxococcus xanthus secretes a novel biosurfactant polysaccharide (BPS) to the extracellular milieu. Together, secretion of the two polymers (EPS and BPS) is required for type IV pilus (T4P)-dependent swarm expansion via spatio-specific biofilm expression profiles. Thus the synergy between EPS and BPS secretion somehow modulates the multicellular lifecycle of M. xanthus. Herein, we demonstrate that BPS secretion functionally alters the EPS glycocalyx via destabilization of the latter, fundamentally changing the characteristics of the cell surface. This impacts motility behaviors at the single-cell level and the aggregative capacity of cells in groups via cell-surface EPS fibril formation as well as T4P production, stability, and positioning. These changes modulate the structure of swarm biofilms via cell layering, likely contributing to the formation of internal swarm polysaccharide architecture. Together, these data reveal the manner by which the combined secretion of two distinct polymers induces single-cell changes that modulate swarm biofilm communities.

ICP-MS characterization of seven North American snake venoms.

Snake venoms are inherently complex. They are mixtures of multiple enzymes, peptides, lipids, carbohydrates, nucleosides, and metal ions. Metal ions make up a small portion of a snake's venom but play outsized roles in enzyme function and stability. Unlike enzyme primary structure, which is easily predicted from genomic sequences, a venom's metal ion content must be measured directly. We leveraged the high throughput and sensitivity of inductively coupled plasma mass spectrometry to analyze the metal ion content of seven North American snake venoms. All venoms were collected from snakes reared at one location, so we could discount variation from environmental or geographical factors. We profiled 71 metal isotopes. Selenium isotopes were consistently high across all venoms tested. When each venom's toxicity was graphed as a function of each different metal isotope, the only strong relationships between metal content and toxicity were for magnesium isotopes.

The Effect of Simulated Field Storage Conditions on Dental Restorative Materials for Military Field Use.

INTRODUCTION: Dental readiness, one critical component of medical readiness, is adversely impacted by dental emergencies. Many dental emergencies require restorative materials such as glass ionomers, resins, and zinc oxide eugenols to remedy them. The Authorized Dental Allowance List (ADAL) and Authorized Medical Allowance List (AMAL) contain the equipment and materials used by Navy dentists to treat Sailors and Marines. These supplies are subjected to harsh storage conditions on deployments. Much is known about how materials behave when stored at room temperature, but less is known about how their properties are affected after exposure to high temperatures and humidity. We subjected five dental restorative materials to storage in aggravated conditions, and then tested them to determine which products are more robust. MATERIALS AND METHODS: Unopened packages of Fuji Triage, Fuji IX GP (both GC America Inc., Alsip, Illinois), TPH Spectra ST Low Viscosity, Intermediate Restorative Material (both Dentsply Sirona, York, Pennsylvania), and Herculite XRV (Kerr Corporation, Orange, California) were exposed to 0, 5, or 10 days' storage at 30-60°C with 95% relative humidity. After storage in these aggravated conditions, we tested the compressive strength, hardness, elastic modulus, flexural strength, flexural modulus, sorption, and solubility of each material. RESULTS: The physical properties of all materials were affected by storage in aggravated conditions, though the properties of some materials degraded more than others. Both glass ionomers, Fuji Triage (P = 0.0012) and Fuji IX GP (P = 0.0031), and the composite Herculite XRV (P = 0.0253) lost compressive strength after 5 or 10 days in aggravated conditions. The hardness values for all materials were affected (P < 0.05) by the aggravated conditions, though the elastic modulus of TPH Spectra was not affected (P > 0.05). None of the materials lost flexural strength (P > 0.05) or had changes in their flexural modulus (P > 0.05). The water sorption behavior of Fuji Triage (P = 0.0426) and Fuji IX GP (P = 0.0201) changed after 10 days of aggravated storage, and the solubility of all materials was altered by the harsh conditions. CONCLUSION: Some materials degrade more than others in aggravated conditions. Both resin composite materials were more resistant to high temperatures and humidity levels than the glass ionomers tested. These changes in physical characteristics should be considered when reviewing or optimizing the ADAL/AMAL for different projected operational environments.

Construction of a genetically modified T7Select phage system to express the antimicrobial peptide 1018.

Bacteriophage therapy was an ascendant technology for combating bacterial infections before the golden age of antibiotics, but the therapeutic potential of phages was largely ignored after the discovery of penicillin. Recently, with antibiotic-resistant infections on the rise, these phages are receiving renewed attention to combat problematic bacterial infections. Our approach is to enhance bacteriophages with antimicrobial peptides, short peptides with broad-spectrum antibiotic or antibiofilm effects. We inserted coding sequences for 1018, an antimicrobial peptide previously shown to be an effective broad-spectrum antimicrobial and antibiofilm agent, or the fluorescent marker mCherry, into the T7Select phage genome. Transcription and production of 1018 or mCherry began rapidly alter E. coli cultures were infected with genetically modified phages. mCherry fluorescence, which requires a 90 min initial maturation period, was observed in infected cultures after 2 h of infection. Finally, we tested phages expressing 1018 (1018 T7) against bacterial planktonic cultures and biofilms, and found the 1018 T7 phage was more effective than the unmodified T7Select phage at both killing planktonic cells and eradicating established biofilms, validating our phage-driven antimicrobial peptide expression system. The combination of narrow-spectrum phages delivering relatively high local doses of broad-spectrum antimicrobials could be a powerful method to combat resistant infections. The experiments we describe prove this combination is feasible in vitro, but further testing and optimization are required before genetically modified phages are ready for use in vivo.

Bacterial Surface Spreading Is More Efficient on Nematically Aligned Polysaccharide Substrates.

Biofilm-forming bacteria typically deposit layers of polysaccharides on the surfaces they inhabit; hence, polysaccharides are their immediate environment on such surfaces. Previously, we showed that many biofilm-forming bacteria preferentially spread in the direction of aligned and densely packed polysaccharide fibers in compressed substrates, a behavior we referred to as polymertropism. This arrangement of polysaccharide fibers is likely to be similar to that found in the "slime" trails deposited by many biofilm-forming bacteria and would explain previous observations that bacteria tend to follow these trails of polysaccharides. Here, we show that groups of cells or flares spread more rapidly on substrates containing aligned and densely packed polysaccharide fibers. Flares also persist longer, tend to hold their trajectories parallel to the long axes of polysaccharide fibers longer, and ultimately show an increase in displacement away from their origin. On the basis of these findings and others, we propose a model for polymertropism. Namely, we suggest that the packing of the aligned polymers increases the efficiency of surface spreading in the direction of the polymer's long axes; therefore, bacteria tend to spread more rapidly in this direction. Additional work suggests that bacteria can leverage polymertropism, and presumably more efficient surface spreading, for a survival advantage. In particular, when two bacterial species were placed in close proximity and in competition with each other, the ability of one species to move rapidly and directly away from the other by utilizing the aligned polymers of compressed agar substrates led to a clear survival benefit.IMPORTANCE The directed movement of bacteria on compressed substrates was first described in the 1940s and referred to as elasticotaxis (R. Y. Stanier, J Bacteriol 44:405-412, 1942). More recently, this behavior was referred to as polymertropism, as it seems to be a response to the nematic alignment and tight packing of polymers in the substrate (D. J. Lemon, X. Yang, P. Srivastava, Y. Y. Luk, A. G. Garza, Sci Rep 7:7643, 2017, https://doi.org/10.1038/s41598-017-07486-0). The data presented here suggest that bacteria are more efficient at surface spreading when the polymers in the substrate are arranged in this manner. These data also suggest that bacteria can leverage polymertropism, and presumably more efficient surface spreading, for a survival advantage. Namely, one bacterial species was able to use its strong polymertropism response to escape from and survive competition with another species that normally outcompetes it.

Polymertropism of rod-shaped bacteria: movement along aligned polysaccharide fibers.

In nature, bacteria often live in surface-associated communities known as biofilms. Biofilm-forming bacteria typically deposit a layer of polysaccharide on the surfaces they inhabit; hence, polysaccharide is their immediate environment on many surfaces. In this study, we examined how the physical characteristics of polysaccharide substrates influence the behavior of the biofilm-forming bacterium Myxococcus xanthus. M. xanthus responds to the compression-induced deformation of polysaccharide substrates by preferentially spreading across the surface perpendicular to the axis of compression. Our results suggest that M. xanthus is not responding to the water that accumulates on the surface of the polysaccharide substrate after compression or to compression-induced changes in surface topography such as the formation of troughs. These directed surface movements do, however, consistently match the orientation of the long axes of aligned and tightly packed polysaccharide fibers in compressed substrates, as indicated by behavioral, birefringence and small angle X-ray scattering analyses. Therefore, we suggest that the directed movements are a response to the physical arrangement of the polymers in the substrate and refer to the directed movements as polymertropism. This behavior might be a common property of bacteria, as many biofilm-forming bacteria that are rod-shaped and motile on soft surfaces exhibit polymertropism.

Modeling Legionnaires' disease outbreaks: estimating the timing of an aerosolized release using symptom-onset dates.

BACKGROUND: Over the last 30 years, there have been a number of reported Legionnaires' disease outbreaks resulting from the release of causative organisms from aerosol-producing devices. METHODS: We model a Legionnaires' disease epidemic curve as the convolution of an infection-time distribution (representing the aerosolized release) and an incubation-period distribution. The model is fitted to symptom-onset data from specific outbreaks to estimate the start and end dates of the release. We also develop this retrospective "back-calculation" model into a prospective "real-time" model that can estimate the final size of an ongoing outbreak, in addition to the timing of its release. RESULTS: In the retrospective analysis, the estimated release end dates were generally earlier than reported end dates. This suggests that, in many outbreaks, the release might have already ended by the time the source was reportedly cleaned or closed. Prospective analysis showed that valid estimates of the release start date could be achieved early in the outbreak, the total number of cases could be reasonably determined shortly after the release had ended, and estimates of the release end date could be satisfactorily achieved in the latter stages of the outbreak. CONCLUSIONS: This model could be used in the course of a Legionnaires' disease outbreak to provide early estimates of the total number of cases, thus helping to inform public-health planning. Toward the end of the outbreak, estimates of the release end date could help corroborate standard epidemiologic, environmental, and microbiologic investigations that seek to identify the source.