Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions.

The ability to perform controlled experiments with bioaerosols is a fundamental enabler of many bioaerosol research disciplines. A practical alternative to using hazardous biothreat agents, e.g., for detection equipment development and testing, involves using appropriate model organisms (simulants). Several species of Gram-negative bacteria have been used or proposed as biothreat simulants. However, the appropriateness of different bacterial genera, species, and strains as simulants is still debated. Here, we report aerobiological stability characteristics of four species of Gram-negative bacteria (Pantoea agglomerans, Serratia marcescens, Escherichia coli, and Xanthomonas arboricola) in single-cell particles and cell clusters produced using four spray liquids (H2O, phosphate-buffered saline[PBS], spent culture medium[SCM], and a SCM-PBS mixture). E. coli showed higher stability in cell clusters from all spray liquids than the other species, but it showed similar or lower stability in single-cell particles. The overall stability was higher in cell clusters than in single-cell particles. The highest overall stability was observed for bioaerosols produced using SCM-containing spray liquids. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research.IMPORTANCE The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns.

Mechanisms of Bacterial (Serratia marcescens) Attachment to, Migration along, and Killing of Fungal Hyphae.

We have found a remarkable capacity for the ubiquitous Gram-negative rod bacterium Serratia marcescens to migrate along and kill the mycelia of zygomycete molds. This migration was restricted to zygomycete molds and several basidiomycete species. No migration was seen on any molds of the phylum Ascomycota. S. marcescens migration did not require fungal viability or surrounding growth medium, as bacteria migrated along aerial hyphae as well.S. marcescens did not exhibit growth tropism toward zygomycete mycelium. Bacterial migration along hyphae proceeded only when the hyphae grew into the bacterial colony. S. marcescens cells initially migrated along the hyphae, forming attached microcolonies that grew and coalesced to generate a biofilm that covered and killed the mycelium. Flagellum-defective strains of S. marcescens were able to migrate along zygomycete hyphae, although they were significantly slower than the wild-type strain and were delayed in fungal killing. Bacterial attachment to the mycelium does not necessitate type 1 fimbrial adhesion, since mutants defective in this adhesin migrated equally well as or faster than the wild-type strain. Killing does not depend on the secretion of S. marcescens chitinases, as mutants in which all three chitinase genes were deleted retained wild-type killing abilities. A better understanding of the mechanisms by which S. marcescens binds to, spreads on, and kills fungal hyphae might serve as an excellent model system for such interactions in general; fungal killing could be employed in agricultural fungal biocontrol.

EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model.

Serratia marcescens is a soil- and water-derived bacterium that secretes several host-directed factors and causes hospital infections and community-acquired ocular infections. The putative two-component regulatory system composed of EepR and EepS regulates hemolysis and swarming motility through transcriptional control of the swrW gene and pigment production through control of the pigA-pigN operon. Here, we identify and characterize a role for EepR in regulation of exoenzyme production, stress survival, cytotoxicity to human epithelial cells, and virulence. Genetic analysis supports the model that EepR is in a common pathway with the widely conserved cyclic-AMP receptor protein that regulates protease production. Together, these data introduce a novel regulator of host-pathogen interactions and secreted-protein production.

The Rcs signal transduction pathway is triggered by enterobacterial common antigen structure alterations in Serratia marcescens.

The enterobacterial common antigen (ECA) is a highly conserved exopolysaccharide in Gram-negative bacteria whose role remains largely uncharacterized. In a previous work, we have demonstrated that disrupting the integrity of the ECA biosynthetic pathway imposed severe deficiencies to the Serratia marcescens motile (swimming and swarming) capacity. In this work, we show that alterations in the ECA structure activate the Rcs phosphorelay, which results in the repression of the flagellar biogenesis regulatory cascade. In addition, a detailed analysis of wec cluster mutant strains, which provoke the disruption of the ECA biosynthesis at different levels of the pathway, suggests that the absence of the periplasmic ECA cyclic structure could constitute a potential signal detected by the RcsF-RcsCDB phosphorelay. We also identify SMA1167 as a member of the S. marcescens Rcs regulon and show that high osmolarity induces Rcs activity in this bacterium. These results provide a new perspective from which to understand the phylogenetic conservation of ECA among enterobacteria and the basis for the virulence attenuation detected in wec mutant strains in other pathogenic bacteria.

Motility enhancement of bacteria actuated microstructures using selective bacteria adhesion.

Microrobots developed by the technological advances are useful for application in various fields. Nevertheless, they have limitations with respect to their actuator and motility. Our experiments aim to determine whether a bioactuator using the flagellated bacteria Serratia marcescens would enhance the motility of microrobots. In this study, we investigate that the flagellated bacteria Serratia marcescens could be utilized as actuators for SU-8 microstructures by bovine serum albumin-selective patterning. Firstly, we analyze the adherence of the bacteria to the SU-8 micro cube by selective patterning using 5% BSA. The results show that number of attached-bacteria in the uncoated side of the selectively- coated micro cube with BSA increased by 200% compared with that in all sides of the non treated micro cube. Secondly, the selectively BSA coated micro cube had 210% higher motility than the uncoated micro cube. The results revealed that the bacteria patterned to a specific site using 5% BSA significantly increase the motility of the bacteria actuated microstructure.

The use of chromogenic bacteria as coloured substitutes for pathogens: a simple strategy during design and development of a new method for sample pretreatment.

AIMS: In the present study, chromogenic (red) bacteria were used to simulate actual target bacteria during set-up and optimization of an isolation process of bacteria, designed for food samples. Isolation of bacteria from food in the context of molecular biological detection of food pathogens is a multistep process. Development of such a separation method requires continuous monitoring of the location of the presumable targets in the sample tubes. Therefore, red-coloured pigmented bacteria were used as substitutes for the actual target bacteria, during the establishment of a new sample preparation technique. METHODS AND RESULTS: The chromogenic bacteria Micrococcus roseus and Serratia marcescens were confirmed to withstand the physical (e.g. centrifugal forces) and chemical (e.g. lysis buffer composition) conditions required during establishment of the new technique. Furthermore, the suitability of these model bacteria to substitute for the actual target pathogens (Salmonella enterica subsp. enterica serovar Typhimurium and Listeria monocytogenes) was assured by testing the physical properties of the model bacteria with respect to the proposed separation methods. CONCLUSION: Visibility of the pigmented bacteria within the complex sample matrices served to allocate bacterial content during the various steps necessary for finalization of the method protocol. The presumptive bacterial targets can be allocated simply by visualization of their bright red colour silhouetted against the background sample matrix. SIGNIFICANCE AND IMPACT OF STUDY: The use of pigmented bacteria as substitutes for actual colourless target bacteria during design and development of a bacterial isolation method is a simple and inexpensive application. It saves a huge amount of time and resources, as the proof of principle of new methods is possible in rapid succession.

Hexavalent molybdenum reduction to molybdenum blue by S. marcescens strain Dr. Y6.

A molybdate-reducing bacterium has been locally isolated. The bacterium reduces molybdate or Mo(6+) to molybdenum blue (molybdate oxidation states of between 5+ and 6+). Different carbon sources such as acetate, formate, glycerol, citric acid, lactose, fructose, glucose, mannitol, tartarate, maltose, sucrose, and starch were used at an initial concentration of 0.2% (w/v) in low phosphate media to study their effect on the molybdate reduction efficiency of bacterium. All of the carbon sources supported cellular growth, but only sucrose, maltose, glucose, and glycerol (in decreasing order) supported molybdate reduction after 24 h of incubation. Optimum concentration of sucrose for molybdate reduction is 1.0% (w/v) after 24 h of static incubation. Ammonium sulfate, ammonium chloride, valine, OH-proline, glutamic acid, and alanine (in the order of decreasing efficiency) supported molybdate reduction with ammonium sulfate giving the highest amount of molybdenum blue after 24 h of incubation at 0.3% (w/v). The optimum molybdate concentration that supports molybdate reduction is between 15 and 25 mM. Molybdate reduction is optimum at 35 degrees C. Phosphate at concentrations higher than 5 mM strongly inhibits molybdate reduction. The molybdenum blue produced from cellular reduction exhibits a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as Serratia marcescens Strain Dr.Y6 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny.

The function of SpnR and the inhibitory effects by halogenated furanone on quorum sensing in Serratia marcescens AS-1.

By secretion and detection of a series of signaling molecules, bacteria are able to coordinate gene expression as a community, to regulate a variety of important phenotypes, from virulence factor production to biofilm formation to symbiosis related behaviours such as bioluminescence. This widespread signaling mechanism is called quorum sensing. There are several quorum sensing systems described in Serratia. Serratia marcescens AS-1, isolated from soil, had the LuxI/LuxR homologues called SpnI/SpnR. S. marcescens AS-1 produced two kinds of N-acyl-L-homoserine lactones, N-hexanoyl-L-homoserine lactone and N-(3-oxohexanoyl)-L-homoserine lactone as signal molecules, which involved in quorum sensing to control the gene expression in response to increased cell density. By gene replacement method, the spnR mutant was constructed, named S. marcescens AS-1R. SpnR acted as a negative regulator for the production of prodigiosin, swarming motility and biofilm formation, which were regulated by quorum sensing. Halogenated furanone, known as a natural inhibitor of quorum sensing, could effectively inhibit the quorum sensing of S. marcescens AS-1 but without interrupting AHL-SpnR interaction. All results will be helpful to understand the mechanisms of halogenated furanone inhibition on quorum sensing and the potential application of halogenated furanone in effectively preventing infection disease caused by Serratia strains.

Ultrasound-assisted (R)-phenylephrine whole-cell bioconversion by S. marcescens N10612.

The strain Serratia marcescens N10612 is used to perform the bioconversion of 1-(3-hydroxyphenyl)-2-(methyamino)-ethanone (HPMAE) to (R)-phenylephrine ((R)-PE), which is an ephedrine drug substitute. The use of an ultrasound approach is found to improve the efficiency of the (R)-PE bioconversion. The optimization of the (R)-PE bioconversion is carried out by means of statistical experiment design. The optimal conditions obtained are 1.0mM HPMAE, 18.68 g/L glucose and ultrasound power of 120 W, where the predicted specific rate of the (R)-PE bioconversion is 31.46 ± 2.22 (ìmol/h/g-cells) and the experimental specific rate is 33.27 ± 1.46 (ìmol/h/g-cells), which is 3-fold higher than for the operation under ultrasound power of 200 W (11.11 ìmol/h/g-cells) and 4.3-fold higher than for the shaking operation (7.69 ìmol/h/g-cells). The kinetics study of the bioconversion also shows that under the ultrasound operation, the optimal rate (Vmax) of the (R)-PE bioconversion increases from 7.69 to 11.11 (µmol/h/g-cells) and the substrate inhibition constant (KSi) increases from 1.063 mM for the shaking operation to 1.490 mM for ultrasound operation.

Analysis and description of the evolution of alginate immobilised cells systems.

Different immobilised cells models, including very simple ones, can be useful in the fitting of experimental results. However, goodness or the ability to extrapolate results needs to be in accordance with basic observations and these will also suggest models to be proposed. In this paper, observations of calcium alginate/bacteria systems are used to show the ability of basic models to fit classic observations, as well as how new observations, in this case from electronic microscopy, oblige us to think about more complex mechanisms and mathematical treatments. Nevertheless it is not only important to discuss the model type, but also the type of kinetics assumed in the interior of the beads, as well as the internal structure, the boundary conditions related to bead shredding and cell escape and finally, geometrical effects.

[Chitinase from Serratia marcescens BKM B-851]. / Khitinaza Serratia marcescens BKM B-851

The chitinase biosynthesis was studied during the cultivation of the strain of Serratia marcescens BKM B-851 with a high chitinolytic activity. Under submerged cultivation of bacterial cells on the medium containing demineralized crab shell extracellular chitinase showed maximum activity on the 3rd day. Cells of S. marcescens BKM B-851 synthesized chitinase as an adaptive enzyme. Chitinase obtained from the culture liquid by ammonium sulphate precipitation was then dialyzed and liophylized. It displayed optimum hydrolysis of colloid chitin at pH 7-8 and 50 degrees C and of native chitin at 30 degrees C.

[Activity of intracellular and extracellular nuclease according to the phases of growth of pigment and pigment-free strains of Serratia marcescens]. / Aktivnost' vnutri- i vnekletochnoi nukleazy po fazam rosta pigmentnogo i bespigmentnogo shtammov Serratia marcescens

Changes in the activity of intracellular and extracellular nuclease of pigment and pigment-free strains of Serratia marcescens were studied. The activity of intra- and extracellular nuclease of the pigment-free strain was higher than that of the pigment strain at all growth stages of the microorganism. The activity of intracellular nuclease in the lag-phase was higher than in the phase of exponential growth of both strains. Prior to cell division the enzyme activity declined in both strains. At the beginning of the stationary phase the activity of intracellular nuclease was relatively stable in both strains. By the end of the stationary phase the activity of intracellular nuclease of the pigment-free strain increased 4--6 fold and that of the pigment strain remained unchanged. Simultaneously the activity of extracellular nuclease of the pigment-free strain increased and that of the pigment strain grew but slightly.

[Effect of mitomycin on the biosynthesis of extracellular endonuclease by Serratia marcescens]. / Vliianie mitomitsina na biosintez vnekletochnoi éndonukleazy Serratia marcescens.

The effect of mitomycin C on extracellular endonuclease activity of Serratia marcescens was studied. It was shown that in a concentration of 0.02-1.0 micrograms/ml, mitomycin C markedly increased biosynthesis of the endonuclease by growing and washed cells, the cell productivity being increased 80-100 times. The highest increase in the cell productivity was observed when mitomycin C was added to the cells at the end of the growth exponential phase. The increase in the activity of the extracellular endonuclease was due to the de novo synthesis of the enzyme since it was inhibited by chloramphenicol.

[Automated biochemical analysis based on the use of electrosorption systems]. / Avtomatizirovannyi biokhimicheskii analiz s ispol'zovaniam elektrosorbtsionnykh sistem.

An engineering method to separate biocomponents that is suitable for discrete and flow-type autoanalyzers is proposed. The principle is based on using electrosorption systems; it provides, to a high reproducibility, the separation of cells and biomacromolecules from another components of samples to be analysed and makes it possible to solve analytical problems which require the application of several currently available separation systems.

Collective motion of spherical bacteria.

A large variety of motile bacterial species exhibit collective motions while inhabiting liquids or colonizing surfaces. These collective motions are often characterized by coherent dynamic clusters, where hundreds of cells move in correlated whirls and jets. Previously, all species that were known to form such motion had a rod-shaped structure, which enhances the order through steric and hydrodynamic interactions. Here we show that the spherical motile bacteria Serratia marcescens exhibit robust collective dynamics and correlated coherent motion while grown in suspensions. As cells migrate to the upper surface of a drop, they form a monolayer, and move collectively in whirls and jets. At all concentrations, the distribution of the bacterial speed was approximately Rayleigh with an average that depends on concentration in a non-monotonic way. Other dynamical parameters such as vorticity and correlation functions are also analyzed and compared to rod-shaped bacteria from the same strain. Our results demonstrate that self-propelled spherical objects do form complex ordered collective motion. This opens a door for a new perspective on the role of cell aspect ratio and alignment of cells with regards to collective motion in nature.

(1)H, (13)C and (15)N resonance assignments of the periplasmic signalling domain of HasR, a TonB-dependent outer membrane heme transporter.

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that internalize nutrients such as vitamin B12, metal complexes, heme, some carbohydrates, etc. In addition to their transport activity, several TBDTs are also involved in a signalling cascade from the cell surface into the cytoplasm, via their periplasmic signalling domain. Here we report the backbone and side chain resonance assignments of the signalling domain of HasR, a TonB-dependent outer membrane heme transporter from Serratia marcescens as a first step towards its structural study.

RssAB signaling coordinates early development of surface multicellularity in Serratia marcescens.

Bacteria can coordinate several multicellular behaviors in response to environmental changes. Among these, swarming and biofilm formation have attracted significant attention for their correlation with bacterial pathogenicity. However, little is known about when and where the signaling occurs to trigger either swarming or biofilm formation. We have previously identified an RssAB two-component system involved in the regulation of swarming motility and biofilm formation in Serratia marcescens. Here we monitored the RssAB signaling status within single cells by tracing the location of the translational fusion protein EGFP-RssB following development of swarming or biofilm formation. RssAB signaling is specifically activated before surface migration in swarming development and during the early stage of biofilm formation. The activation results in the release of RssB from its cognate inner membrane sensor kinase, RssA, to the cytoplasm where the downstream gene promoters are located. Such dynamic localization of RssB requires phosphorylation of this regulator. By revealing the temporal activation of RssAB signaling following development of surface multicellular behavior, our findings contribute to an improved understanding of how bacteria coordinate their lifestyle on a surface.

Computational and experimental study of chemotaxis of an ensemble of bacteria attached to a microbead.

Micro-objects propelled by whole cell actuators, such as flagellated bacteria, are being increasingly studied and considered for a wide variety of applications. In this work we present theoretical and experimental investigations of chemotactic motility of a 10 µm diameter microbead propelled by an ensemble of attached flagellated bacteria. The stochastic model presented here encompasses the behavior of each individual bacterium attached to the microbead in a spatiotemporally varying chemoattractant field. The computational model shows that in a chemotactic environment, the ensemble of bacteria, although constrained, propel the bead in a chemotactic manner with a 67% enhancement in displacement to distance ratio (defined as directionality) compared to nonchemotactic propulsion. The simulation results are validated experimentally. Close agreement between theory and experiments demonstrates the possibility of using the presented model as a predictive tool for other similar biohybrid systems.