Development of 225Ac-doped biocompatible nanoparticles for targeted alpha therapy.

Targeted alpha therapy (TAT) relies on chemical affinity or active targeting using radioimmunoconjugates as strategies to deliver α-emitting radionuclides to cancerous tissue. These strategies can be affected by transmetalation of the parent radionuclide by competing ions in vivo and the bond-breaking recoil energy of decay daughters. The retention of α-emitting radionuclides and the dose delivered to cancer cells are influenced by these processes. Encapsulating α-emitting radionuclides within nanoparticles can help overcome many of these challenges. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are a biodegradable and biocompatible delivery platform that has been used for drug delivery. In this study, PLGA nanoparticles are utilized for encapsulation and retention of actinium-225 ([225Ac]Ac3+). Encapsulation of [225Ac]Ac3+ within PLGA nanoparticles (Zave = 155.3 nm) was achieved by adapting a double-emulsion solvent evaporation method. The encapsulation efficiency was affected by both the solvent conditions and the chelation of [225Ac]Ac3+. Chelation of [225Ac]Ac3+ to a lipophilic 2,9-bis-lactam-1,10-phenanthroline ligand ([225Ac]AcBLPhen) significantly decreased its release (< 2%) and that of its decay daughters (< 50%) from PLGA nanoparticles. PLGA nanoparticles encapsulating [225Ac]AcBLPhen significantly increased the delivery of [225Ac]Ac3+ to murine (E0771) and human (MCF-7 and MDA-MB-231) breast cancer cells with a concomitant increase in cell death over free [225Ac]Ac3+ in solution. These results demonstrate that PLGA nanoparticles have potential as radionuclide delivery platforms for TAT to advance precision radiotherapy for cancer. In addition, this technology offers an alternative use for ligands with poor aqueous solubility, low stability, or low affinity, allowing them to be repurposed for TAT by encapsulation within PLGA nanoparticles.

Development of an Experimental Approach to Achieve Spatially Resolved Plant Root-Associated Metaproteomics Using an Agar-Plate System.

Plant-microbe interactions in the rhizosphere play a vital role in plant health and productivity. The composition and function of root-associated microbiomes is strongly influenced by their surrounding environment, which is often customized by their host. How microbiomes change with respect to space and time across plant roots remains poorly understood, and methodologies that facilitate spatiotemporal metaproteomic studies of root-associated microbiomes are yet to be realized. Here, we developed a method that provides spatially resolved metaproteome measurements along plant roots embedded in agar-plate culture systems, which have long been used to study plants. Spatially defined agar "plugs" of interest were excised and subsequently processed using a novel peptide extraction method prior to metaproteomics, which was used to infer both microbial community composition and function. As a proof-of-principle, a previously studied 10-member community constructed from a Populus root system was grown in an agar plate with a 3-week-old Populus trichocarpa plant. Metaproteomics was performed across two time points (24 and 48 h) for three distinct locations (root base, root tip, and a region distant from the root). The spatial resolution of these measurements provides evidence that microbiome composition and expression changes across the plant root interface. Interrogation of the individual microbial proteomes revealed functional profiles related to their behavioral associations with the plant root, in which chemotaxis and augmented metabolism likely supported predominance of the most abundant member. This study demonstrated a novel peptide extraction method for studying plant agar-plate culture systems, which was previously unsuitable for (meta)proteomic measurements.

Spatially co-registered wide-field nonlinear optical imaging of living and complex biosystems in a total internal reflection geometry.

Nonlinear optical microscopy that leverages an objective based total internal reflection (TIR) excitation scheme is an attractive means for rapid, wide-field imaging with enhanced surface sensitivity. Through select combinations of distinct modalities, one can, in principle, access complementary chemical and structural information for various chemical species near interfaces. Here, we report a successful implementation of such a wide-field nonlinear optical microscope system, which combines coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), second harmonic generation (SHG), and sum frequency generation (SFG) modalities on the same platform. The intense optical fields needed to drive these high order nonlinear optical processes are achieved through the use of femtosecond pulsed light in combination with the intrinsic field confinement induced by TIR over a large field of view. The performance of our multimodal microscope was first assessed through the experimental determination of its chemical fidelity, intensity and polarization dependences, and spatial resolution using a set of well-defined model systems. Subsequently, its unique capabilities were validated through imaging complex biological systems, including Hydrangea quercifolia pollen grains and Pantoea sp. YR343 bacterial cells. Specifically, the spatial distribution of different molecular groups in the former was visualized via vibrational contrast mechanisms of CARS, whereas co-registered TPF imaging allowed the identification of spatially localized intrinsic fluorophores. We further demonstrate the feasibility of our microscope for wide-field CARS imaging on live cells through independent characterization of cell viability using spatially co-registered TPF imaging. This approach to TIR enabled wide-field imaging is expected to provide new insights into bacterial strains and their interactions with other species in the rhizosphere in a time-resolved and chemically selective manner.

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe Pantoea sp. YR343.

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that Pantoea sp. YR343, a microbe isolated from Populus deltoides, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of Pantoea sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ ipdC mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in Pantoea sp. YR343. The Δ ipdC mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association.

Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7.

Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense, Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms.IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions.

Raman chemical imaging of the rhizosphere bacterium Pantoea sp. YR343 and its co-culture with Arabidopsis thaliana.

Chemical imaging of plant-bacteria co-cultures makes it possible to characterize bacterial populations and behaviors and their interactions with proximal organisms, under conditions closest to the environment in the rhizosphere. Here Raman micro-spectroscopy and confocal Raman imaging are used as minimally invasive probes to study the rhizosphere bacterial isolate, Pantoea sp. YR343, and its co-culture with model plant Arabidopsis thaliana by combining enhanced Raman spectroscopies with electron microscopy and principal component analysis (PCA). The presence of carotenoid pigments in the wild type Pantoea sp. YR343 was characterized using resonance Raman scattering, which was also used to confirm successful disruption of the crtB gene in an engineered carotenoid mutant strain. Other components of the Pantoea sp. YR343 cells were imaged in the presence of resonantly enhanced pigments using a combination of surface enhanced Raman imaging and PCA. Pantoea sp. YR343 cells decorated with Ag colloid synthesized ex situ gave spectra dominated by carotenoid scattering, whereas colloids synthesized in situ produced spectral signatures characteristic of flavins in the cell membrane. Scanning electron microscopy (SEM) of whole cells and transmission electron microscopy (TEM) images of thinly sliced cross-sections were used to assess structural integrity of the coated cells and to establish the origin of spectral signatures based on the position of Ag nanoparticles in the cells. Raman imaging was also used to characterize senescent green Arabidopsis thaliana plant roots inoculated with Pantoea sp. YR343, and PCA was used to distinguish spectral contributions from plant and bacterial cells, thereby establishing the potential of Raman imaging to visualize the distribution of rhizobacteria on plant roots.

Correction to Thermodynamic Characterization of a Thermostable Antibiotic Resistance Enzyme, the Aminoglycoside Nucleotidyltransferase (4').

Biochemistry 2012, 51 (45), 9147−9155. DOI: 10.1021/bi301126g. Page 9148. A corrected version of the Figure 2 legend appears here: Figure 2. Backbone of the ANT D80Y variant in ribbon representation. Two monomer subunits are colored red and green. Bound kanamycin A molecules are colored blue, and Mg-AMPCPP molecules are colored yellow (Protein Data Bank entry 1KNY).14 Page 9148 (last line). The sentence should read, "A thermostable variant of ANT, T130K, was obtained from thermophilic cyanobacterium T. elongatus."

Metabolic adaptations of Azospirillum brasilense to oxygen stress by cell-to-cell clumping and flocculation.

The ability of bacteria to monitor their metabolism and adjust their behavior accordingly is critical to maintain competitiveness in the environment. The motile microaerophilic bacterium Azospirillum brasilense navigates oxygen gradients by aerotaxis in order to locate low oxygen concentrations that can support metabolism. When cells are exposed to elevated levels of oxygen in their surroundings, motile A. brasilense cells implement an alternative response to aerotaxis and form transient clumps by cell-to-cell interactions. Clumping was suggested to represent a behavior protecting motile cells from transiently elevated levels of aeration. Using the proteomics of wild-type and mutant strains affected in the extent of their clumping abilities, we show that cell-to-cell clumping represents a metabolic scavenging strategy that likely prepares the cells for further metabolic stresses. Analysis of mutants affected in carbon or nitrogen metabolism confirmed this assumption. The metabolic changes experienced as clumping progresses prime cells for flocculation, a morphological and metabolic shift of cells triggered under elevated-aeration conditions and nitrogen limitation. The analysis of various mutants during clumping and flocculation characterized an ordered set of changes in cell envelope properties accompanying the metabolic changes. These data also identify clumping and early flocculation to be behaviors compatible with the expression of nitrogen fixation genes, despite the elevated-aeration conditions. Cell-to-cell clumping may thus license diazotrophy to microaerophilic A. brasilense cells under elevated oxygen conditions and prime them for long-term survival via flocculation if metabolic stress persists.

Quantifying platinum binding on protein-functionalized magnetic microparticles using single particle-ICP-TOF-MS.

This work describes an analytical procedure, single particle-inductively coupled plasma-time-of-flight-mass spectrometry (SP-ICP-TOF-MS), that was developed to determine the platinum binding efficiency of protein-coated magnetic microparticles. SP-ICP-TOF-MS is advantageous due to its ability to quasi-simultaneously detect all nuclides (7Li-242Pu), allowing for both platinum and iron (composition of magnetic microparticles) to be measured concurrently. This method subsequently allows for the differentiation between bound and unbound platinum. The 1 µm magnetic microparticles were fully characterized for their iron concentration, particle concentration, and trace element composition by bulk digestion-ICP-MS and SP-ICP-TOF-MS. The results of both approaches agreed with the certificate values. Using the single particle methodology the platinum loading was quantified to be to 0.18 ± 0.02 fg per particle and 0.32 ± 0.02 fg per particle, for the streptavidin-coated and azurin-coated microparticles, respectively. Both streptavidin-coated and the azurin-coated microparticles had a particle-platinum association of >65%. Platinum bound samples were also analyzed via bulk digestion-based ICP-MS. The bulk ICP-MS results overestimated platinum loading due to free platinum in the samples. This highlights the importance of single particle analysis for a closer inspection of platinum binding performance. The SP-ICP-TOF-MS approach offers advantages over typical bulk digestion methods by eliminating laborious sample preparation, enabling differentiation between bound/unbound platinum in a solution, and quantification of platinum on a particle-by-particle basis. The procedure presented here enables quantification of metal content per particle, which could be broadly implemented for other single particle applications.

Swimming motility plays a key role in the stochastic dynamics of cell clumping.

Dynamic cell-to-cell interactions are a prerequisite to many biological processes, including development and biofilm formation. Flagellum induced motility has been shown to modulate the initial cell-cell or cell-surface interaction and to contribute to the emergence of macroscopic patterns. While the role of swimming motility in surface colonization has been analyzed in some detail, a quantitative physical analysis of transient interactions between motile cells is lacking. We examined the Brownian dynamics of swimming cells in a crowded environment using a model of motorized adhesive tandem particles. Focusing on the motility and geometry of an exemplary motile bacterium Azospirillum brasilense, which is capable of transient cell-cell association (clumping), we constructed a physical model with proper parameters for the computer simulation of the clumping dynamics. By modulating mechanical interaction ('stickiness') between cells and swimming speed, we investigated how equilibrium and active features affect the clumping dynamics. We found that the modulation of active motion is required for the initial aggregation of cells to occur at a realistic time scale. Slowing down the rotation of flagellar motors (and thus swimming speeds) is correlated to the degree of clumping, which is consistent with the experimental results obtained for A. brasilense.

Microstructural and Rheological Transitions in Bacterial Biofilms.

Biofilms are aggregated bacterial communities structured within an extracellular matrix (ECM). ECM controls biofilm architecture and confers mechanical resistance against shear forces. From a physical perspective, biofilms can be described as colloidal gels, where bacterial cells are analogous to colloidal particles distributed in the polymeric ECM. However, the influence of the ECM in altering the cellular packing fraction (ϕ) and the resulting viscoelastic behavior of biofilm remains unexplored. Using biofilms of Pantoea sp. (WT) and its mutant (ΔUDP), the correlation between biofilm structure and its viscoelastic response is investigated. Experiments show that the reduction of exopolysaccharide production in ΔUDP biofilms corresponds with a seven-fold increase in ϕ, resulting in a colloidal glass-like structure. Consequently, the rheological signatures become altered, with the WT behaving like a weak gel, whilst the ΔUDP displayed a glass-like rheological signature. By co-culturing the two strains, biofilm ϕ is modulated which allows us to explore the structural changes and capture a change in viscoelastic response from a weak to a strong gel, and to a colloidal glass-like state. The results reveal the role of exopolysaccharide in mediating a structural transition in biofilms and demonstrate a correlation between biofilm structure and viscoelastic response.

The Azospirillum brasilense Che1 chemotaxis pathway controls swimming velocity, which affects transient cell-to-cell clumping.

The Che1 chemotaxis-like pathway of Azospirillum brasilense contributes to chemotaxis and aerotaxis, and it has also been found to contribute to regulating changes in cell surface adhesive properties that affect the propensity of cells to clump and to flocculate. The exact contribution of Che1 to the control of chemotaxis and flocculation in A. brasilense remains poorly understood. Here, we show that Che1 affects reversible cell-to-cell clumping, a cellular behavior in which motile cells transiently interact by adhering to one another at their nonflagellated poles before swimming apart. Clumping precedes and is required for flocculation, and both processes appear to be independently regulated. The phenotypes of a ΔaerC receptor mutant and of mutant strains lacking cheA1, cheY1, cheB1, or cheR1 (alone or in combination) or with che1 deleted show that Che1 directly mediates changes in the flagellar swimming velocity and that this behavior directly modulates the transient nature of clumping. Our results also suggest that an additional receptor(s) and signaling pathway(s) are implicated in mediating other Che1-independent changes in clumping identified in the present study. Transient clumping precedes the transition to stable clump formation, which involves the production of specific extracellular polysaccharides (EPS); however, production of these clumping-specific EPS is not directly controlled by Che1 activity. Che1-dependent clumping may antagonize motility and prevent chemotaxis, thereby maintaining cells in a metabolically favorable niche.

Thermodynamic characterization of a thermostable antibiotic resistance enzyme, the aminoglycoside nucleotidyltransferase (4').

The aminoglycoside nucleotidyltransferase (4') (ANT) is an aminoglycoside-modifying enzyme that detoxifies antibiotics by nucleotidylating at the C4'-OH site. Previous crystallographic studies show that the enzyme is a homodimer and each subunit binds one kanamycin and one Mg-AMPCPP, where the transfer of the nucleotidyl group occurs between the substrates bound to different subunits. In this work, sedimentation velocity analysis of ANT by analytical ultracentrifugation showed the enzyme exists as a mixture of a monomer and a dimer in solution and that dimer formation is driven by hydrophobic interactions between the subunits. The binding of aminoglycosides shifts the equilibrium toward dimer formation, while the binding of the cosubstrate, Mg-ATP, has no effect on the monomer-dimer equilibrium. Surprisingly, binding of several divalent cations, including Mg(2+), Mn(2+), and Ca(2+), to the enzyme also shifted the equilibrium in favor of dimer formation. Binding studies, performed by electron paramagnetic resonance spectroscopy, showed that divalent cations bind to the aminoglycoside binding site in the absence of substrates with a stoichiometry of 2:1. Energetic aspects of binding of all aminoglycosides to ANT were determined by isothermal titration calorimetry to be enthalpically favored and entropically disfavored with an overall favorable Gibbs energy. Aminoglycosides in the neomycin class each bind to the enzyme with significantly different enthalpic and entropic contributions, while those of the kanamycin class bind with similar thermodynamic parameters.

Quantifying biofilm propagation on chemically modified surfaces.

Conditions affecting biofilm formation differ among bacterial species and this presents a challenge to studying biofilms in the lab. This work leverages functionalized silanes to control surface chemistry in the study of early biofilm propagation, quantified with a semi-automated image processing algorithm. These methods support the study of Pantoea sp. YR343, a gram-negative bacterium isolated from the poplar rhizosphere. We found that Pantoea sp. YR343 does not readily attach to hydrophilic surfaces but will form biofilms with a "honeycomb" morphology on hydrophobic surfaces. Our image processing algorithm described here quantified the evolution of the honeycomb morphology over time, and found the propagation to display a logarithmic behavior. This methodology was repeated with a flagella-deficient fliR mutant of Pantoea sp. YR343 which resulted in reduced surface attachment. Quantifiable differences between Pantoea WT and ΔfliR biofilm morphologies were captured by the image processing algorithm, further demonstrating the insight gained from these methods.

In Situ Detection of Amino Acids from Bacterial Biofilms and Plant Root Exudates by Liquid Microjunction Surface-Sampling Probe Mass Spectrometry.

The plant rhizosphere is a complex and dynamic chemical environment where the exchange of molecular signals between plants, microbes, and fungi drives the development of the entire biological system. Exogenous compounds in the rhizosphere are known to affect plant-microbe organization, interactions between organisms, and ultimately, growth and survivability. The function of exogenous compounds in the rhizosphere is still under much investigation, specifically with respect to their roles in plant growth and development, the assembly of the associated microbial community, and the spatiotemporal distribution of molecular components. A major challenge for spatiotemporal measurements is developing a nondisruptive and nondestructive technique capable of analyzing the exogenous compounds contained within the environment. A methodology using liquid microjunction-surface sampling probe-mass spectrometry (LMJ-SSP-MS) and microfluidic devices with attached microporous membranes was developed for in situ, spatiotemporal measurement of amino acids (AAs) from bacterial biofilms and plant roots. Exuded arginine was measured from a living Pantoea YR343 biofilm, which resulted in a chemical image indicative of biofilm growth within the device. Spot sampling along the roots of Populus trichocarpa with the LMJ-SSP-MS resulted in the detection of 15 AAs. Variation in AA concentrations across the root system was observed, indicating that exudation is not homogeneous and may be linked to local rhizosphere architecture and different biological processes along the root.

Identification of a diguanylate cyclase expressed in the presence of plants and its application for discovering candidate gene products involved in plant colonization by Pantoea sp. YR343.

Microbial colonization of plant roots is a highly complex process that requires the coordination and regulation of many gene networks, yet the identities and functions of many of these gene products have yet to be discovered. Pantoea sp. YR343, a gamma-proteobacterium isolated from the rhizosphere of Populus deltoides, forms robust biofilms along the root surfaces of Populus and possesses plant growth-promoting characteristics. In this work, we identified three diguanylate cyclases in the plant-associated microbe Pantoea sp. YR343 that are expressed in the presence of plant roots. One of these diguanylate cyclases, DGC2884, localizes to discrete sites in the cells and its overexpression results in reduced motility and increased EPS production and biofilm formation. We performed a genetic screen by expressing this diguanylate cyclase from an inducible promoter in order to identify candidate gene products that may be involved in root colonization by Pantoea sp. YR343. Further, we demonstrate the importance of other domains in DGC2884 to its activity, which in combination with the genes identified by transposon mutagenesis, may yield insights into the mechanisms of plant association as well as the activity and regulation of homologous enzymes in medically and agriculturally relevant microbes.

The FtcR-Like Protein ActR in Azorhizobium caulinodans ORS571 Is Involved in Bacterial Motility and Symbiosis With the Host Plant.

Bacterial signal transduction pathways are important for a variety of adaptive responses to environment, such as two-component systems (TCSs). In this paper, we reported the characterization of a transcriptional regulator in Azorhizobium caulinodans ORS571, ActR, with an N-terminal receiver domain and one C-terminal OmpR/PhoB-type DNA binding domain. Sequence analysis showed that ActR shared a high similarity with FtcR regulator of Brucella melitensis 16M known to be involved in flagellar regulation. The structural gene of this regulator was largely distributed in Alphaproteobacteria, in particular in Rhizobiales and Rhodobacterales, and was located within clusters of genes related to motility functions. Furthermore, we studied the biological function of ActR in A. caulinodans grown at the free-living state or in association with Sesbania rostrata by constructing actR gene deletion mutant. In the free-living state, the bacterial flagellum and motility ability were entirely deleted, the expression of flagellar genes was downregulated; and the exopolysaccharide production, biofilm formation, and cell flocculation decreased significantly compared with those of the wild-type strain. In the symbiotic state, ΔactR mutant strain showed weakly competitive colonization and nodulation on the host plant. These results illustrated that FtcR-like regulator in A. caulinodans is involved in flagellar biosynthesis and provide bacteria with an effective competitive nodulation for symbiosis. These findings improved our knowledge of FtcR-like transcriptional regulator in A. caulinodans.

Application of Machine Learning Techniques to an Agent-Based Model of Pantoea.

Agent-based modeling (ABM) is a powerful simulation technique which describes a complex dynamic system based on its interacting constituent entities. While the flexibility of ABM enables broad application, the complexity of real-world models demands intensive computing resources and computational time; however, a metamodel may be constructed to gain insight at less computational expense. Here, we developed a model in NetLogo to describe the growth of a microbial population consisting of Pantoea. We applied 13 parameters that defined the model and actively changed seven of the parameters to modulate the evolution of the population curve in response to these changes. We efficiently performed more than 3,000 simulations using a Python wrapper, NL4Py. Upon evaluation of the correlation between the active parameters and outputs by random forest regression, we found that the parameters which define the depth of medium and glucose concentration affect the population curves significantly. Subsequently, we constructed a metamodel, a dense neural network, to predict the simulation outputs from the active parameters and found that it achieves high prediction accuracy, reaching an R 2 coefficient of determination value up to 0.92. Our approach of using a combination of ABM with random forest regression and neural network reduces the number of required ABM simulations. The simplified and refined metamodels may provide insights into the complex dynamic system before their transition to more sophisticated models that run on high-performance computing systems. The ultimate goal is to build a bridge between simulation and experiment, allowing model validation by comparing the simulated data to experimental data in microbiology.

A carotenoid-deficient mutant of the plant-associated microbe Pantoea sp. YR343 displays an altered membrane proteome.

Membrane organization plays an important role in signaling, transport, and defense. In eukaryotes, the stability, organization, and function of membrane proteins are influenced by certain lipids and sterols, such as cholesterol. Bacteria lack cholesterol, but carotenoids and hopanoids are predicted to play a similar role in modulating membrane properties. We have previously shown that the loss of carotenoids in the plant-associated bacteria Pantoea sp. YR343 results in changes to membrane biophysical properties and leads to physiological changes, including increased sensitivity to reactive oxygen species, reduced indole-3-acetic acid secretion, reduced biofilm and pellicle formation, and reduced plant colonization. Here, using whole cell and membrane proteomics, we show that the deletion of carotenoid production in Pantoea sp. YR343 results in altered membrane protein distribution and abundance. Moreover, we observe significant differences in the protein composition of detergent-resistant membrane fractions from wildtype and mutant cells, consistent with the prediction that carotenoids play a role in organizing membrane microdomains. These data provide new insights into the function of carotenoids in bacterial membrane organization and identify cellular functions that are affected by the loss of carotenoids.