Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography.

Proteus mirabilis is a Gram-negative Gammaproteobacterium and a major causative agent of urinary tract infections in humans. It is characterized by its ability to switch between swimming motility in liquid media and swarming on solid surfaces. Here, we used cryo-electron tomography and subtomogram averaging to reveal the structure of the flagellar motor of P. mirabilis at nanometer resolution in intact cells. We found that P. mirabilis has a motor that is structurally similar to those of Escherichia coli and Salmonella enterica, lacking the periplasmic elaborations that characterize other more specialized gammaproteobacterial motors. In addition, no density corresponding to stators was present in the subtomogram average suggesting that the stators are dynamic. Finally, several assembly intermediates of the motor were seen that support the inside-out assembly pathway.

Antibiofilm effect of Nocardiopsis sp. GRG 1 (KT235640) compound against biofilm forming Gram negative bacteria on UTIs.

Urinary tract infections (UTIs) are diverse public health complication and caused by range of pathogens, however mostly Gram negative bacteria cause significant life threatening risks to different populations. The prevalence rate and antimicrobial resistance among the Gram negative uropathogens alarmed significantly heighten the economic burden of these infections. In this study, we investigated the antibiofilm efficiency of Pyrrolo [1,2-a] pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) extracted from endophytic actinomycetes Nocardiopsis sp. GRG 1 (KT235640) against P. mirabilis and E. coli. The extracted compound was characterized through TLC, HPLC, GC-MS, LC-MS and confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM). The compound, Pyrrolo [1,2-a] pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) inhibits both bacterial biofilm formation as well as reduces the viability of preformed biofilms. Furthermore, CLSM image shows cell shrinkage, disorganized cell membrane and loss of viability. The SEM result also confirms the cell wall degradation in treated cells of the bacteria. Hence, the Pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) is active against P. mirabilis and E. coli.

Hemolytic Escherichia coli Inhibits Swarming and Differentiation of Proteus mirabilis.

Swarming is a hallmark of Proteus mirabilis, whether common gram-negative bacilli affect the swarming of P. mirabilis is still unclear. In this study, we found that P. mirabilis swarming was inhibited by Escherichia coli ATCC25922, but was not affected by Klebsiella pneumoniae, Acinetobacter baumannii, or Pseudomonas aeruginosa strains. The migration distance of P. mirabilis when mixed with E. coli ATCC25922 was strongly reduced, and the inhibition of the swarming of P. mirabilis by E. coli ATCC25922 was dependent on cell density. In addition, initiation of P. mirabilis swarming was delayed by E. coli ATCC25922. Among clinical isolates, including gram-negative bacilli and gram-positive cocci, only hemolytic E. coli inhibited the swarming of P. mirabilis. In summary, hemolytic E. coli inhibited the swarming and differentiation of P. mirabilis.

Mechanism of Kin-Discriminatory Demarcation Line Formation between Colonies of Swarming Bacteria.

Swarming bacteria use kin discrimination to preferentially associate with their clonemates for certain cooperative behaviors. Kin discrimination can manifest as an apparent demarcation line (a region lacking cells or with much lower cell density) between antagonist strains swarming toward each other. In contrast, two identical strains merge with no demarcation. Experimental studies suggest contact-dependent killing between different strains as a mechanism of kin discrimination, but it is not clear whether this killing is sufficient to explain the observed patterns. Here, we investigate the formation of demarcation line with a mathematical model. First, using data from competition experiments between kin discriminating strains of Myxococcus xanthus and Proteus mirabilis, we found the rates of killing between the strains to be highly asymmetric, i.e., one strain kills another at a much higher rate. Then, to investigate how such asymmetric interactions can lead to a stable demarcation line, we construct reaction-diffusion models for colony expansion of kin-discriminatory strains. Our results demonstrate that a stable demarcation line can form when both cell movement and cell growth cease at low nutrient levels. Further, our study suggests that, depending on the initial separation between the inoculated colonies, the demarcation line may move transiently before stabilizing. We validated these model predictions by observing dynamics of merger between two M. xanthus strains, where one strain expresses a toxin protein that kills a second strain lacking the corresponding antitoxin. Our study therefore provides a theoretical understanding of demarcation line formation between kin-discriminatory populations, and can be used for analyzing and designing future experiments.

Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis.

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram-negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ-like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ-driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.

A random graph model of density thresholds in swarming cells.

Swarming behaviour is a type of bacterial motility that has been found to be dependent on reaching a local density threshold of cells. With this in mind, the process through which cell-to-cell interactions develop and how an assembly of cells reaches collective motility becomes increasingly important to understand. Additionally, populations of cells and organisms have been modelled through graphs to draw insightful conclusions about population dynamics on a spatial level. In the present study, we make use of analogous random graph structures to model the formation of large chain subgraphs, representing interactions between multiple cells, as a random graph Markov process. Using numerical simulations and analytical results on how quickly paths of certain lengths are reached in a random graph process, metrics for intercellular interaction dynamics at the swarm layer that may be experimentally evaluated are proposed.

Shelter in a Swarm.

Flagella propel bacteria during both swimming and swarming, dispersing them widely. However, while swimming bacteria use chemotaxis to find nutrients and avoid toxic environments, swarming bacteria appear to suppress chemotaxis and to use the dynamics of their collective motion to continuously expand and acquire new territory, barrel through lethal chemicals in their path, carry along bacterial and fungal cargo that assists in exploration of new niches, and engage in group warfare for niche dominance. Here, we focus on two aspects of swarming, which, if understood, hold the promise of revealing new insights into microbial signaling and behavior, with ramifications beyond bacterial swarming. These are as follows: how bacteria sense they are on a surface and turn on programs that promote movement and how they override scarcity and adversity as dense packs.

Effects of confinement, surface-induced orientations and strain on dynamical behaviors of bacteria in thin liquid crystalline films.

We report on the organization and dynamics of bacteria (Proteus mirabilis) dispersed within lyotropic liquid crystal (LC) films confined by pairs of surfaces that induce homeotropic (perpendicular) or hybrid (homeotropic and parallel orientations at each surface) anchoring of the LC. By using motile vegetative bacteria (3 µm in length) and homeotropically aligned LC films with thicknesses that exceed the length of the rod-shaped cells, a key finding reported in this paper is that elastic torques generated by the LC are sufficiently large to overcome wall-induced hydrodynamic torques acting on the cells, thus leading to LC-guided bacterial motion near surfaces that orient LCs. This result extends to bacteria within LC films with hybrid anchoring, and leads to the observation that asymmetric strain within a hybrid aligned LC rectifies motions of motile cells. In contrast, when the LC film thickness is sufficiently small that confinement prevents alignment of the bacteria cells along a homeotropically aligned LC director (achieved using swarm cells of length 10-60 µm), the bacterial cells propel in directions orthogonal to the director, generating transient distortions in the LC that have striking "comet-like" optical signatures. In this limit, for hybrid LC films, we find LC elastic stresses deform the bodies of swarm cells into bent configurations that follow the LC director, thus unmasking a coupling between bacterial shape and LC strain. Overall, these results provide new insight into the influence of surface-oriented LCs on dynamical bacterial behaviors and hint at novel ways to manipulate bacteria using confined LC phases that are not possible in isotropic solutions.

Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis.

UNLABELLED: A key regulator of swarming in Proteus mirabilis is the Rcs phosphorelay, which represses flhDC, encoding the master flagellar regulator FlhD4C2. Mutants in rcsB, the response regulator in the Rcs phosphorelay, hyperswarm on solid agar and differentiate into swarmer cells in liquid, demonstrating that this system also influences the expression of genes central to differentiation. To gain a further understanding of RcsB-regulated genes involved in swarmer cell differentiation, transcriptome sequencing (RNA-Seq) was used to examine the RcsB regulon. Among the 133 genes identified, minC and minD, encoding cell division inhibitors, were identified as RcsB-activated genes. A third gene, minE, was shown to be part of an operon with minCD. To examine minCDE regulation, the min promoter was identified by 5' rapid amplification of cDNA ends (5'-RACE), and both transcriptional lacZ fusions and quantitative real-time reverse transcriptase (qRT) PCR were used to confirm that the minCDE operon was RcsB activated. Purified RcsB was capable of directly binding the minC promoter region. To determine the role of RcsB-mediated activation of minCDE in swarmer cell differentiation, a polar minC mutation was constructed. This mutant formed minicells during growth in liquid, produced shortened swarmer cells during differentiation, and exhibited decreased swarming motility. IMPORTANCE: This work describes the regulation and role of the MinCDE cell division system in P. mirabilis swarming and swarmer cell elongation. Prior to this study, the mechanisms that inhibit cell division and allow swarmer cell elongation were unknown. In addition, this work outlines for the first time the RcsB regulon in P. mirabilis. Taken together, the data presented in this study begin to address how P. mirabilis elongates upon contact with a solid surface.

Arginine promotes Proteus mirabilis motility and fitness by contributing to conservation of the proton gradient and proton motive force.

Swarming contributes to Proteus mirabilis pathogenicity by facilitating access to the catheterized urinary tract. We previously demonstrated that 0.1-20 mmol/L arginine promotes swarming on normally nonpermissive media and that putrescine biosynthesis is required for arginine-induced swarming. We also previously determined that arginine-induced swarming is pH dependent, indicating that the external proton concentration is critical for arginine-dependent effects on swarming. In this study, we utilized survival at pH 5 and motility as surrogates for measuring changes in the proton gradient (ΔpH) and proton motive force (µH(+) ) in response to arginine. We determined that arginine primarily contributes to ΔpH (and therefore µH(+) ) through the action of arginine decarboxylase (speA), independent of the role of this enzyme in putrescine biosynthesis. In addition to being required for motility, speA also contributed to fitness during infection. In conclusion, consumption of intracellular protons via arginine decarboxylase is one mechanism used by P. mirabilis to conserve ΔpH and µH(+) for motility.

Dynamic self-assembly of motile bacteria in liquid crystals.

This paper reports an investigation of dynamical behaviors of motile rod-shaped bacteria within anisotropic viscoelastic environments defined by lyotropic liquid crystals (LCs). In contrast to passive microparticles (including non-motile bacteria) that associate irreversibly in LCs via elasticity-mediated forces, we report that motile Proteus mirabilis bacteria form dynamic and reversible multi-cellular assemblies when dispersed in a lyotropic LC. By measuring the velocity of the bacteria through the LC (8.8 ± 0.2 µm s(-1)) and by characterizing the ordering of the LC about the rod-shaped bacteria (tangential anchoring), we conclude that the reversibility of the inter-bacterial interaction emerges from the interplay of forces generated by the flagella of the bacteria and the elasticity of the LC, both of which are comparable in magnitude (tens of pN) for motile Proteus mirabilis cells. We also measured the dissociation process, which occurs in a direction determined by the LC, to bias the size distribution of multi-cellular bacterial complexes in a population of motile Proteus mirabilis relative to a population of non-motile cells. Overall, these observations and others reported in this paper provide insight into the fundamental dynamic behaviors of bacteria in complex anisotropic environments and suggest that motile bacteria in LCs are an exciting model system for exploration of principles for the design of active materials.

Activity of Proteus mirabilis FliL is viscosity dependent and requires extragenic DNA.

Proteus mirabilis is a urinary tract pathogen and well known for its ability to move over agar surfaces by flagellum-dependent swarming motility. When P. mirabilis encounters a highly viscous environment, e.g., an agar surface, it differentiates from short rods with few flagella to elongated, highly flagellated cells that lack septa and contain multiple nucleoids. The bacteria detect a surface by monitoring the rotation of their flagellar motors. This process involves an enigmatic flagellar protein called FliL, the first gene in an operon (fliLMNOPQR) that encodes proteins of the flagellar rotor switch complex and flagellar export apparatus. We used a fliL knockout mutant to gain further insight into the function of FliL. Loss of FliL results in cells that cannot swarm (Swr(-)) but do swim (Swm(+)) and produces cells that look like wild-type swarmer cells, termed "pseudoswarmer cells," that are elongated, contain multiple nucleoids, and lack septa. Unlike swarmer cells, pseudoswarmer cells are not hyperflagellated due to reduced expression of flaA (the gene encoding flagellin), despite an increased transcription of both flhD and fliA, two positive regulators of flagellar gene expression. We found that defects in fliL prevent viscosity-dependent sensing of a surface and viscosity-dependent induction of flaA transcription. Studies with fliL cells unexpectedly revealed that the fliL promoter, fliL coding region, and a portion of fliM DNA are needed to complement the Swr(-) phenotype. The data support a dual role for FliL as a critical link in sensing a surface and in the maintenance of flagellar rod integrity.

Flagellum density regulates Proteus mirabilis swarmer cell motility in viscous environments.

Proteus mirabilis is an opportunistic pathogen that is frequently associated with urinary tract infections. In the lab, P. mirabilis cells become long and multinucleate and increase their number of flagella as they colonize agar surfaces during swarming. Swarming has been implicated in pathogenesis; however, it is unclear how energetically costly changes in P. mirabilis cell morphology translate into an advantage for adapting to environmental changes. We investigated two morphological changes that occur during swarming--increases in cell length and flagellum density--and discovered that an increase in the surface density of flagella enabled cells to translate rapidly through fluids of increasing viscosity; in contrast, cell length had a small effect on motility. We found that swarm cells had a surface density of flagella that was ∼5 times larger than that of vegetative cells and were motile in fluids with a viscosity that inhibits vegetative cell motility. To test the relationship between flagellum density and velocity, we overexpressed FlhD(4)C(2), the master regulator of the flagellar operon, in vegetative cells of P. mirabilis and found that increased flagellum density produced an increase in cell velocity. Our results establish a relationship between P. mirabilis flagellum density and cell motility in viscous environments that may be relevant to its adaptation during the infection of mammalian urinary tracts and movement in contact with indwelling catheters.

Changes in peptidoglycan structure and metabolism during differentiation of Proteus mirabilis into swarmer cells.

The O-acetylation of peptidoglycan in Gram-negative bacteria occurs specifically at the C-6 hydroxyl group of muramoyl residues. The level of peptidoglycan O-acetylation was found to decrease from 51% to 29% upon differentiation of Proteus mirabilis vegetative cells to swarmers. This decrease was accompanied by a change in the muropeptide composition of the peptidoglycan. In particular, the content of anhydromuropeptides increased, while the amount of Lys-Lys-muropeptides arising from bound lipoprotein decreased. These changes together with a shift in proportion of larger muropeptides suggested a decrease in average chain length of the muropeptides from swarmer cells. Zymography using SDS-PAGE gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the activity of specific autolysins during the differentiation of vegetative to swarming cells of P. mirabilis. A 43 kDa autolysin with increased specificity for O-acetylated peptidoglycan was detected in vegetative cells, but its activity appeared to decrease as the cells began to differentiate, while the levels of 3 other autolysins with apparent specificity for non-O-acetylated peptidoglycan increased. These changes are discussed in relation to the autolysin profile of the bacteria and the changes in peptidoglycan composition with cell differentiation.

Perturbation of FliL interferes with Proteus mirabilis swarmer cell gene expression and differentiation.

Proteus mirabilis is a dimorphic, motile bacterium often associated with urinary tract infections. Colonization of urinary tract surfaces is aided by swarmer cell differentiation, which is initiated by inhibition of flagellar rotation when the bacteria first contact a surface. Mutations in fliL, encoding a flagellar structural protein with an enigmatic function, result in the inappropriate production of differentiated swarmer cells, called pseudoswarmer cells, under noninducing conditions, indicating involvement of FliL in the surface sensing pathway. In the present study, we compared the fliL transcriptome with that of wild-type swarmer cells and showed that nearly all genes associated with motility (flagellar class II and III genes) and chemotaxis are repressed. In contrast, spontaneous motile revertants of fliL cells that regained motility yet produced differentiated swarmer cells under noninducing conditions transcribed flagellar class II promoters at consistent levels. Expression of umoA (a known regulator of swarmer cells), flgF, and flgI increased significantly in both swarmer and pseudoswarmer cells, as did genes in a degenerate prophage region situated immediately adjacent to the Rcs phosphorelay system. Unlike swarmer cells, pseudoswarmers displayed increased activity, rather than transcription, of the flagellar master regulatory protein, FlhD(4)C(2), and analyses of the fliL parent strain and its motile revertants showed that they result from mutations altering the C-terminal 14 amino acids of FliL. Collectively, the data suggest a functional role for the C terminus of FliL in surface sensing and implicate UmoA as part of the signal relay leading to the master flagellar regulator FlhD(4)C(2), which ultimately controls swarmer cell differentiation.

Radial and spiral stream formation in Proteus mirabilis colonies.

The enteric bacterium Proteus mirabilis, which is a pathogen that forms biofilms in vivo, can swarm over hard surfaces and form a variety of spatial patterns in colonies. Colony formation involves two distinct cell types: swarmer cells that dominate near the surface and the leading edge, and swimmer cells that prefer a less viscous medium, but the mechanisms underlying pattern formation are not understood. New experimental investigations reported here show that swimmer cells in the center of the colony stream inward toward the inoculation site and in the process form many complex patterns, including radial and spiral streams, in addition to previously-reported concentric rings. These new observations suggest that swimmers are motile and that indirect interactions between them are essential in the pattern formation. To explain these observations we develop a hybrid model comprising cell-based and continuum components that incorporates a chemotactic response of swimmers to a chemical they produce. The model predicts that formation of radial streams can be explained as the modulation of the local attractant concentration by the cells, and that the chirality of the spiral streams results from a swimming bias of the cells near the surface of the substrate. The spatial patterns generated from the model are in qualitative agreement with the experimental observations.

Regulation of gene expression during swarmer cell differentiation in Proteus mirabilis.

The gram-negative bacterium Proteus mirabilis can exist in either of two cell types, a vegetative cell characterized as a short rod and a highly elongated and hyperflagellated swarmer cell. This differentiation is triggered by growth on solid surfaces and multiple inputs are sensed by the cell to initiate the differentiation process. These include the inhibition of flagellar rotation, the accumulation of extracellular putrescine and O-antigen interactions with a surface. A key event in the differentiation process is the upregulation of FlhD(2)C(2), which activates the flagellar regulon and additional genes required for differentiation. There are a number of genes that influence FlhD(2)C(2) expression and the function of these genes, if known, will be discussed in this review. Additional genes that have been shown to regulate gene expression during swarming will also be reviewed. Although P. mirabilis represents an excellent system to study microbial differentiation, it is largely understudied relative to other systems. Therefore, this review will also discuss some of the unanswered questions that are central to understanding this process in P. mirabilis.

Interaction of intracorporeal lithotripters with Proteus mirabilis inoculated inside artificial calcium and struvite stones.

BACKGROUND AND PURPOSE: Renal calculi may contain bacteria that can remain active inside the stone and produce bacteremia and/or endotoxemia after lithotripsy. Urinary tract infection associated with urinary stones represents high morbidity. The purpose of this research was to use novel artificial struvite stones inoculated with living bacteria and to study the effect of four different intracorporeal lithotripters on bacterial inactivation after in vitro lithotripsy. MATERIALS AND METHODS: Two types of artificial kidney stone models (calcium sulphate and mixed struvite-calcium sulphate) were manufactured and infected with Proteus mirabilis. Stones were fractured using either electrohydraulic, laser, ultrasonic, or pneumatic lithotripters. Bacterial viability was determined before and after the lithotripsy. RESULTS: Bacterial inactivation was not affected by the stone matrix; ie, calcium or struvite. The four tested lithotripters were almost equally efficient at reducing the viability of P mirabilis in both the low and the high energy setting. CONCLUSIONS: We were able to obtain novel artificial struvite stones infected with bacteria. Intracorporeal lithotripters are efficient at reducing the viability of P mirabilis in vitro. Tested stone materials play a minor role regarding inactivation. Whether the bactericidal effect reported is desirable or not is still to be answered, because the presence of endotoxin from cell lysis may increase the risk of urosepsis.

An explanatory model to validate the way water activity rules periodic terrace generation in Proteus mirabilis swarm.

This paper explains the biophysical principles which, according to us, govern the Proteus mirabilis swarm phenomenon. Then, this explanation is translated into a mathematical model, essentially based on partial differential equations. This model is then implemented using numerical methods of the finite volume type in order to make simulations. The simulations show most of the characteristics which are observed in situ and in particular the terrace generation.

The effect of monoterpenes on swarming differentiation and haemolysin activity in Proteus mirabilis.

Urinary tract infection by Proteus mirabilis depends on several virulence properties that are coordinately regulated with swarming differentiation. Here we report the antibacterial and anti-swarming effect of seventeen terpenoids, and the effect of subinhibitory concentrations of five selected terpenoids on swarming, biofilm formation and haemolysin activity. The results showed that all the terpenes evaluated, particularly oxygenated terpenoids, inhibited P. mirabilis with MIC values ranging between 3 and 10 mg/L. Moreover, citral, citronellol and geraniol effectively inhibit P. mirabilis swarming in a dose dependent manner, reducing swimming/swarming cell differentiation and haemolysin activity at 1/10 MIC concentration. The inhibition of P. mirabilis swarming and virulence factor expression by selected oxygenated terpenoids suggest that essential oils with high concentration of these compounds have the potential to be developed as products for preventing P. mirabilis infections.