Closely linked genetic loci required for swarm cell differentiation and multicellular migration by Proteus mirabilis

The pathogenic bacterium Proteus mirabilis exhibits a form of multicellular behaviour called swarming migration. This involves the differentiation of vegetative cells at the colony margin into swarm cells which are long, aseptate, multinucleate, hyper-flagellated filaments able to undergo repeated cycles of co-ordinated population migration and consolidation (reversion to vegetative cells). Transposon mutagenesis of uropathogenic P. mirabilis strain U6450 with Tn5 generated 4860 chromosomal insertions and, of these, 75 (1.6%) caused visibly abnormal swarming behaviour, indicating that at least 45 genes are involved in directing motility, cell differentiation and multicellular behaviour. While about one fifth of the swarm-defective mutants lacked flagella and were non-motile non-swarming (NMNS) the majority were normally flagellated and motile but were unable to form swarm cells (motile non-swarming, MNS), or were motile and able to form swarm cells but displayed aberrant patterns of multicellular migration (dendritic swarming, DS) or consolidation (frequent and infrequent consolidation, FC and IC). Restriction enzyme mapping of representative mutant DNAs by Southern hybridization with transposon DNA probes identified eight different mutated genetic loci within the five phenotypic classes. Subsequent Southern analysis of large restriction fragments separated by pulsed-field electrophoresis showed that these eight mutated loci required for motility, cell differentiation and multicellular migration were clustered on a region of DNA spanning approximately 8% of the 4.2 mbp P. mirabilis chromosome. Further linkage analysis showed that the DS locus involved in the ordered migration of the swarm cell population mapped separately from two main clusters of swarm loci, one cluster containing, within 112 kbp, genetic determinants of motility (NMNS) and also differentiation into swarm cells (MNS1, MNS2), and a second within a neighbouring 95 kbp DNA sequence containing three loci involved in the control of consolidation (FC, IC1, IC2).     

Extracellular slime associated with Proteus mirabilis during swarming.

Light microscopy, transmission electron microscopy, and scanning electron microscopy were used to visualize the extracellular slime of Proteus mirabilis swarm cells. Slime was observed with phase-contrast microscopy after fixation in hot sulfuric acid-sodium borate. Ruthenium red was used to stain slime for transmission electron microscopy. Copious quantities of extracellular slime were observed surrounding swarm cells; the slime appeared to provide a matrix through which the cells could migrate. Swarm cells were always found embedded in slime. These observations support the argument that swarming of P. mirabilis is associated with the production of large quantities of extracellular slime. Examination of nonswarming mutants of P. mirabilis revealed that a number of morphological changes, including cell elongation and increased flagellum synthesis, were required for swarm cell migration. It is still unclear whether extracellular slime production also is required for migration.     

Co-ordinate expression of virulence genes during swarm-cell differentiation and population migration of Proteus mirabilis

The uropathogenic Gram-negative bacterium Proteus mirabilis exhibits a form of multicellular behaviour termed swarming, which involves cyclical differentiation of typical vegetative cells into filamentous, multinucleate, hyperflagellate swarm cells capable of rapid and co-ordinated population migration across surfaces. We observed that differentiation into swarm cells was accompanied by substantial increases in the activities of intracellular urease and extracellular haemolysin and metalloprotease, which are believed to be central to the pathogenicity of P. mirabilis. In addition, the ability of P. mirabilis to invade human urothelial cells in vitro was primarily a characteristic of differentiated swarm cells, not vegetative cells. These virulence factor activities fell back as the cells underwent cyclical reversion to the vegetative form (consolidation), in parallel with the diagnostic modulation of flagellin levels on the cell surface. Control cellular alkaline phosphatase activities did not increase during differentiation or consolidation. Non-flagellated, nonmotile transposon insertion mutants were unable to invade urothelial cells and they generated only low-level activities of haemolysin, urease and protease (0-10% of wild type). Motile mutants unable to differentiate into swarm cells were comparably reduced in their haemolytic, ureolytic and invasive phenotypes and generated threefold less protease activity. Mutants that were able to form swarm cells but exhibited various aberrant patterns of swarming migration produced wild-type activities of haemolysin, urease and protease, but their ability to enter urothelial cells was three- to 10-fold lower.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic aspects of the structured cell population in a swarming colony of Proteus mirabilis

Proteus mirabilis forms a concentric-ring colony by undergoing periodic swarming. A colony in the process of such synchronized expansion was examined for its internal population structure. In alternating phases, i.e., swarming (active migration) and consolidation (growth without colony perimeter expansion), phase-specific distribution of cells differing in length, in situ mobility, and migration ability on an agar medium were recognized. In the consolidation phase, the distribution of mobile cells was restricted to the inner part of a new ring and a previous terrace. Cells composing the outer part of the ring were immobile in spite of their ordinary swimming ability in a viscous solution. A sectorial cell population having such an internal structure was replica printed on fresh agar medium. After printing, a transplant which was in the swarming phase continued its ongoing swarming while a transplanted consolidation front continued its scheduled consolidation. This shows that cessation of migration during the consolidation phase was not due to substances present in the underlying agar medium. The ongoing swarming schedule was modifiable by separative cutting of the swarming front or disruption of the ring pattern by random mixing of the pattern-forming cell population. The structured cell population seemed to play a role in characteristic colony growth. However, separation of a narrow consolidation front from a backward area did not induce disturbance in the ongoing swarming schedule. Thus, cells at the frontal part of consolidation area were independent of the internal cell population and destined to exert consolidation and swarming with the ongoing ordinary schedule.     

A novel membrane protein influencing cell shape and multicellular swarming of Proteus mirabilis

Swarming in Proteus mirabilis is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells. We describe a transposon mutant, MNS185, that was unable to swarm even though vegetative cells retained normal motility and the ability to differentiate into swarm cells. However, these elongated cells were irregularly curved and had variable diameters, suggesting that the migration defect results from the inability of these deformed swarm cells to align into multicellular rafts. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. Multiple copies of the wild-type gene, called ccmA, for curved cell morphology, restored swarming to the mutant. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. Two forms of CcmA were identified for wild-type P. mirabilis; they were full-length integral membrane CcmA1 and N-terminally truncated peripheral membrane CcmA2, both present at approximately 20-fold higher concentrations in swarm cells. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. Elongated cells of a ccmA null mutant were less misshapen than those of MNS185 and were able to swarm, albeit more slowly than wild-type cells. The truncated CcmA proteins may therefore interfere with normal morphogenesis, while the wild-type proteins, which are not essential for swarming, may enhance migration by maintaining the linearity of highly elongated cells. Consistent with this view, overexpression of the ccmA gene caused cells of both Escherichia coli and P. mirabilis to become enlarged and ellipsoidal.     

Periodic phenomena in Proteus mirabilis swarm colony development.

Proteus mirabilis colonies exhibit striking geometric regularity. Basic microbiological methods and imaging techniques were used to measure periodic macroscopic events in swarm colony morphogenesis. We distinguished three initial phases (lag phase, first swarming phase, and first consolidation phase) followed by repeating cycles of subsequent swarming plus consolidation phases. Each Proteus swarm colony terrace corresponds to one swarming-plus-consolidation cycle. The duration of the lag phase was dependent upon inoculation density in a way that indicated the operation of both cooperative and inhibitory multicellular effects. On our standard medium, the second and subsequent swarm phases displayed structure in the form of internal waves visible with reflected and dark-field illumination. These internal waves resulted from organization of the migrating bacteria into successively thicker cohorts of swarmer cells. Bacterial growth and motility were independently modified by altering the composition of the growth medium. By varying the glucose concentration in the substrate, it was possible to alter biomass production without greatly affecting the kinetics of colony surface area expansion. By varying the agar concentration in the substrate, initial bacterial biomass production was unaffected but colony expansion dynamics were significantly altered. Higher agar concentrations led to slower, shorter swarm phases and longer consolidation phases. Thus, colony growth was restricted by higher agar concentrations but the overall timing of the swarming-plus-consolidation cycles remained constant. None of a variety of factors which had significant effects on colony expansion altered terracing frequencies at 32 degrees C, but the length of the swarming-plus-consolidation cycle was affected by temperature and medium enrichment. Some clinical isolates displayed significant differences in terracing frequencies at 32 degrees C. Our results defined a number of readily quantifiable parameters in swarm colony development. The data showed no connection between nutrient (glucose) depletion and the onset of different phases in swarm colony morphogenesis. Several observations point to the operation of density-dependent thresholds in controlling the transitions between distinct phases.     

A cell-surface polysaccharide that facilitates rapid population migration by differentiated swarm cells of Proteus mirabilis

Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical vegetative rods into long, hyperflagellated, virulent swarm cells. A swarm-defective Tn phoA insertion mutant was isolated that was not defective in cell motility, differentiation or control of the migration cycle, but was specifically impaired in the ability to undergo surface translocation as a multicellular mass. The mutation, previously shown to compromise urinary tract virulence, was located within a 1112 bp gene that restored normal swarming of the mutant when expressed in trans . The gene encoded a 40.6 kDa protein that is related to putative sugar transferases required for lipopolysaccharide (LPS) core modification in Shigella and Salmonella . The immediately distal open reading frame encoded a protein that is related to dehydrogenases involved in the synthesis of LPS O-side-chains, enterobacterial common antigen and extracellular polysaccharide (PS). Gel electrophoresis and electron microscopy showed that the mutant still made LPS but it had lost the ability to assemble a surface (capsular) PS, which gas-liquid chromatography and mass spectrometry indicated to be an acidic type II molecule rich in galacturonic acid and galactosamine. We suggest that this surface PS facilitates translocation of differentiated cell populations by reducing surface friction.     

Evidence against the involvement of chemotaxis in swarming of Proteus mirabilis.

Nonswarming and nonchemotactic mutants of Proteus mirabilis were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light. These mutants were used in experiments to determine if chemotaxis is involved in the swarming of P. mirabilis. Nonchemotactic mutants failed to form chemotactic bands in a semisolid casein hydrolysate medium, yet they swarmed on the same medium containing 1.5% agar. Nonswarming mutants were attracted towards individual amino acids and components of tryptose. In cross-feeding experiments, no evidence was obtained to indicate the production of a diffusable chemical repellent. In studies with the wild-type P. mirabilis, no clear-cut negative chemotaxis was seen even though three different assays were used and numerous chemicals were tested. Additional evidence against the involvement of chemotaxis in swarming comes from finding that dialysis does not interfere with swarming; swarm cells will swarm immediately when transferred to fresh media, and swarm cells will swarm on an agar-water medium supplemented with a surfactant. These data indicate that chemotaxis is not involved in the swarming of P. mirabilis.     

ZapA, the IgA-degrading metalloprotease of Proteus mirabilis, is a virulence factor expressed specifically in swarmer cells

The IgA-degrading metalloprotease, ZapA, of the urinary tract pathogen Proteus mirabilis is co-ordinately expressed along with other proteins and virulence factors during swarmer cell differentiation. In this communication, we have used zapA to monitor IgA protease expression during the differentiation of vegetative swimmer cells to fully differentiated swarmer cells. Northern blot analysis of wild-type cells and beta-galactosidase measurements using a zapA:lacZ fusion strain indicate that zapA is fully expressed only in differentiated swarmer cells. Moreover, the expression of zapA on nutrient agar medium is co-ordinately regulated in concert with the cycles of cellular differentiation, swarm migration and consolidation that produce the bull's-eye colonies typically associated with P. mirabilis. ZapA activity is not required for swarmer cell differentiation or swarming behaviour, as ZapA- strains produce wild-type colony patterns. ZapA- strains fail to degrade IgA and show decreased survival compared with the wild-type cells during infection in a mouse model of ascending urinary tract infection (UTI). These data underscore the importance of the P. mirabilis IgA-degrading metalloprotease in UTI. Analysis of the nucleotide sequences adjacent to zapA reveals four additional genes, zapE, zapB, zapC and zapD, which appear to possess functions required for ZapA activity and IgA proteolysis. Based on homology to other known proteins, these genes encode a second metalloprotease, ZapE, as well as a ZapA-specific ABC transporter system (ZapB, ZapC and ZapD). A model describing the function and interaction of each of these five proteins in the degradation of host IgA during UTI is presented.     

Bacterial swarming: a biochemical time-resolved FTIR-ATR study of Proteus mirabilis swarm-cell differentiation

Fourier transform infrared spectroscopy was applied to the study of the differentiation process undergone by Proteus mirabilis. This bacterium exhibits a remarkable dimorphism, allowing the cells to migrate on a solid substratum in a concerted manner yielding characteristic ring patterns. We performed an in situ noninvasive analysis of biochemical events occurring as vegetative cells differentiate into elongated, multinucleate, nonseptate, and hyperflagellated swarm cells. The major findings arising from this study are (i) the real-time monitoring of flagellar filament assembly, (ii) the evidence for de novo synthesis of qualitatively different lipopolysaccharides (LPS) and/or exopolysaccharides (EPS) constituting the slime into which bacteria swarm, and (iii) the alteration in the membrane fatty acid composition with a concomitant 10 degrees C decrease in the gel/liquid crystal phase transition resulting in an elevated membrane fluidity in swarm cells at the growth temperature. The time course of events shows that the EPS-LPS syntheses are synchronous with membrane fatty acid alterations and occur about 1 h before massive flagellar filament assembly is detected. This study not only provided a time sketch of biochemical events involved in the differentiation process but also led to the identification of the major spectral markers of both vegetative and swarm cells. This identification will allow to resolve the time-space structure of P. mirabilis colonies by using infrared microscopy.     

Does water activity rule P. mirabilis periodic swarming? II. Viscoelasticity and water balance during swarming

Following the analysis of the biochemical and functional properties of the P. mirabilis extra cellular matrix performed in the first part of this study, the viscoelasticity of an actively growing colony was investigated in relation to water activity. The results demonstrate that the P. mirabilis colony exhibits a marked viscoelastic character likely due to both cell rafts and exoproduct H-bond networks. Besides, the water loss by evaporation during migration has been measured, whereas the experimental determination of the water diffusion coefficient in agar has allowed us to estimate the net water influx at the agar/colony interface. These data drive us to propose that a periodic increase of the water activity at the colony's periphery, mainly due to the drastic surface to volume ratio increase associated with swarming, causes the periodic and synchronous cessation of migration through the dissociation of exoproduct networks, which in turn strongly alters the matrix viscoelasticity.     

Differentiation of Serratia liquefaciens into swarm cells is controlled by the expression of the flhD master operon.

The velocity with which a swarming colony of Serratia liquefaciens colonizes the surface of a suitable solid substratum was controlled by modulating the expression of the flhD master operon. In liquid medium, the stimulation of flhD expression resulted in filamentous, multinucleate, and hyperflagellated cells that were indistinguishable from swarm cells isolated from the edge of a swarm colony. Thus, expression of the flhD master operon appears to play a central role in the process of swarm cell differentiation.     

Structural and serological studies on the O-antigen of Proteus mirabilis O14, a new polysaccharide containing 2-[(R)-1-carboxyethylamino]ethyl phosphate.

An O-specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain D-galactose, 2-acetamido-2-deoxy-D-glalactose, phosphate, N-(2-hydroxyethyl)-D-alanine (D-AlaEtn), and O-acetyl groups. Studies of the initial and O-deacetylated polysaccharides using one- and two-dimensional 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, NOESY, H-detected 1H,13C heteronuclear multiple-quantum coherence, and heteronuclear multiple-bond correlation experiments, demonstrated the following structure of the repeating unit: [equation: see text] This is the second bacterial polysaccharide reported to contain alpha-D-Galp6PAlaEtn, whereas the first one was the O-antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska-Pietrasik, D., Rozalski, A., Knirel, Y.A. & Kochetkov, N.K. (1990) Eur. J. Biochem., 188, 645-651]. Anti-(P. mirabilis O14) serum cross-reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O-antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O-antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup.     

低湿度培养诱生变形杆菌复杂群集运动增殖生长模式

探讨低湿度培养环境对奇异变形杆菌集群运动生长模式的影响。用含水量为90％的低湿度平板诱生群集运动增殖能力不同的内环与外环变形杆菌,通过菌落直径比值反映2种菌群集运动增殖能力差异。内环菌菌落直径较小,外环菌菌落直径较大,1、2,3代菌落直径比值分别为（1．83±0．17）、（3．71±0．12）、（4．51±0。12）。外环菌集群运动增殖速率大于内环菌,且有随着培养代数增多差异增大的趋势,直观反映了一种环境变化即可对系统产生巨大的影响。     

Induction of in vitro human lymphocyte migration by interleukin 3, interleukin 4, and interleukin 6.

The effects of interleukin 3 (IL 3), IL 4, IL 6, and interferon-gamma (IFN-gamma) on lymphocyte migration have been investigated and compared with those of transforming growth factor-beta 1 (TGF-beta 1), granulocyte colony stimulating factor (GCSF), and macrophage colony stimulating factor (MCSF). Potent, temperature-dependent stimulation of lymphocyte migration was obtained in response to IL 3 and IL 4 (ED50 less than 10(-11) M and less than 10(-13) M, respectively) and this migration was abolished in the presence of 3 micrograms ml-1 cytochalasin B. IL 6 and IFN-gamma were less active (ED50 greater than or equal to 10(-9) M and greater than or equal to 10(-8) M, respectively), maximal migration in response to IFN-gamma being only 30% above background as compared with approximately 250% for IL 3 and IL 4. TGF-beta 1, GCSF, and MCSF failed to stimulate lymphocyte migration in doses similar to those used for IL 3, IL 4, and IL 6. The presence of antisera to IL 3, IL 4, and IL 6 specifically inhibited lymphocyte migration induced by the corresponding cytokines (IC50 values being 1/10,000, greater than 1/30,000, and greater than 1/30,000 dilution of antibody, respectively). Cross-desensitization experiments using IL 3 and IL 4 demonstrated that neither IL 3 nor IL 4 were able to stimulate dose-related lymphocyte migration in cells preincubated with IL 3. Cells preincubated with IL 4 were only stimulated by a supraoptimal concentration of IL 4 (10(-11) M). The induction of lymphocyte migration by IL 3, IL 4, and IL 6 therefore appears to be a specific and potentially important effect of these cytokines. Cross-desensitization of lymphocytes by IL 3 and IL 4 raises the possibility that the induction of lymphocyte migration by these cytokines may occur through a common postreceptor signal transduction mechanism.     

Immunofluorescent evidence of Proteus mirabilis swarm cell formation on sterilized rat feces.

Swarming Proteus spp. were detected with the use of proteometry (a most-probable-number technique) in the fecal material of selected animal species and in raw sewage from a local sewage treatment plant. Proteus spp. were not detected in any of several soil and freshwater samples examined. Since rat feces harbored high numbers of Proteus mirabilis compared with other habitats examined, we chose to examine it for the possibility of supporting swarming. Immunofluorescent studies with a strain-specific conjugate revealed the morphogenesis of short forms into elongated swarm cells upon the surface of sterilized rat feces that had been inoculated with short forms of P. mirabilis. the same phenomenon was not observed consistently when nonsterile rat feces were inoculated and examined with immunofluorescence.     

Colony fissioning in honey bees: size and significance of the swarm fraction

During colony founding in honey bees, a portion of a colony’s workforce (the “swarm fraction”) departs with the old mother queen in a swarm while the remaining workforce stays with a new daughter queen in the parental nest. There is little quantitative information about swarm fraction size and about how swarm fraction size affects the growth and survival of mother-queen and daughter-queen colonies. We measured (a) the swarm fraction in naturally fissioning honey bee colonies, (b) the growth and survival of mother-queen colonies as a function of swarm size, and (c) the growth and survival of mother-queen and daughter-queen colonies as a function of the swarm fraction. We found an average swarm fraction of 0.75. We also found a significant positive effect of swarm size and swarm fraction on the growth (i.e., comb built, brood produced, food stored, and weight gained) and the survival of mother-queen colonies. We found no effect of swarm fraction on the survival of daughter-queen colonies. Evidently, a honey bee colony must devote a large majority of its workforce to a swarm so that the mother-queen colony can grow sufficiently rapidly to survive its first winter.