Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior

Proteus mirabilis is a dimorphic bacterium which exists in liquid cultures as a 1.5- to 2.0-microns motile swimmer cell possessing 6 to 10 peritrichous flagella. When swimmer cells are placed on a surface, they differentiate by a combination of events that ultimately produce a swarmer cell. Unlike the swimmer cell, the polyploid swarmer cell is 60 to 80 microns long and possesses hundreds to thousands of surface-induced flagella. These features, combined with multicellular behavior, allow the swarmer cells to move over a surface in a process called swarming. Transposon Tn5 was used to produce P. mirabilis mutants defective in wild-type swarming motility. Two general classes of mutants were found to be defective in swarming. The first class was composed of null mutants that were completely devoid of swarming motility. The majority of nonswarming mutations were the result of defects in the synthesis of flagella or in the ability to rotate the flagella. The remaining nonswarming mutants produced flagella but were defective in surface-induced elongation. Strains in the second general class of mutants, which made up more than 65% of all defects in swarming were motile but were defective in the control and coordination of multicellular swarming. Analysis of consolidation zones produced by such crippled mutants suggested that this pleiotropic phenotype was caused by a defect in the regulation of multicellular behavior. A possible mechanism controlling the cyclic process of differentiation and dediferentiation involved in the swarming behavior of P. mirabilis is discussed.     

Alterations in the cell envelope composition of Proteus mirabilis during the development of swarmer cells.

Long, swarming cells of Proteus mirabilis had different proportions of some lipopolysaccharide components when compared to short cells, either agar grown or broth grown. Fluorescence spectrophotometry of antibody binding, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the change was in the proportion of lipopolysaccharide with long O-antigenic sidechains, swarmer lipopolysaccharide relative to short sidechain lipopolysaccharide than the non-swarming cells. The proteins and phospholipids of the envelop remained the same during swarmer development. The results are discussed in relation to the increase in flagella synthesis and permeability to some antibacterial agents during swarmer development.     

Structural similarity of the membrane envelopes of rhizobial bacteroids and the host plasma membrane as revealed by freeze-fracturing.

The freeze-fracture technique was used to study the host plasma membrane and the membrane envelope of bacteroids in rhizobial root nodules of three host-rhizobium combinations. In all three combinations studied, the membrane envelopes of bacteroids are structurally similar to their host plasma membrane. However, the membrane appears to be reversed, because the number and arrangement of particles in the outer fractured face (face A, concave) and in the inner fractured face (face B, convex) of the host plasma membrane are seen, respectively, in the inner fractured face (face B, convex) and in the outer fractured face (face A, concave) of the membrane envelope of the bacteroids at an early stage. This reversion of the membrane surface is consistent with the hypothesis that the membrane envelopes of bacteroids are derived from the host plasma membrane during endocytotic engulfment.     

Urease activity related to the growth and differentiation of swarmer cells of Proteus mirabilis

Urease activity was measured using whole cells of both long (swarming) and short (nonswarming) populations of Proteus mirabilis from casein hydrolysate agar (CHA) and broth (CHB) cultures, and from brain heart infusion broth (BHIB) cultures. Urease is a constitutive enzyme for both long and short cells, but its activity was tremendously increased when urea was incorporated into the media. Urease production was also affected by culture age and media used. Before exponential phase, urease activity was very low, and it increased to its highest point after about 4 h in BHIB and 8 h in both CHA and CHB cultures at 37 degrees C. Long cells had higher urease activity than did short cells when grown on CHA, and was also expressed by two different strains cultured in BHIB. Strain PM23, in BHIB, was able to form long cells (swarming cells) to a maximum proportion after about 4 h, but strain IM47 could not differentiate in any of the liquid media. The former had more urease when swarming differentiation was initiated. PM23 grew relatively faster than IM47 when the former began to differentiate, but this fast growth could not be observed when nutrient broth or minimal medium was used. These observations suggest that long or swarming cells are "faster growing" rather than "nongrowing bacteria".     

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.     

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.     

Differential membrane phospholipid synthesis during the cell cycle of Caulobacter crescentus.

The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.     

Motility of three strains of Proteus mirabilis (G9, P11 and PM5C) in liquid suspension under different environmental conditions

The effect of compounds on the motility of Proteus mirabilis swarmer cells varies from one strain to another. The effect of compounds on the motility of swarmer cells is mainly at higher concentrations than the concentration used to inhibit swarming. Boric acid only affects the motility of strain G9 swarmer cells, whereas sodium deoxycholate prevented the motility of swarmer cells for three strains. Some antibiotics show their effect on the motility of swarmer cells in anaerobic areas, by slowing the movement of swarmer cells, followed by stopping the movement after a period of time or disappearance of the cells. The differentiation between the strains of Proteus species seems to be better in liquid suspension than on the solid medium.     

Swarmer cell differentiation in Proteus mirabilis

Under the appropriate environmental conditions, the gram-negative bacterium Proteus mirabilis undergoes a remarkable differentiation to form a distinct cell type called a swarmer cell. The swarmer cell is characterized by a 20- to 40-fold increase in both cell length and the number of flagella per cell. Environmental conditions required for swarmer cell differentiation include: surface contact, inhibition of flagellar rotation, a sufficient cell density and cell-to-cell signalling. The differentiated swarmer cell is then able to carry out a highly ordered population migration termed swarming. Genetic analysis of the swarming process has revealed that a large variety of distinct loci are required for this differentiation including: genes involved in regulation, lipopolysaccharide and peptidoglycan synthesis, cell division, ATP production, putrescine biosynthesis, proteolysis and cell shape determination. The process of swarming is important medically because the expression of virulence genes and the ability to invade cells are coupled to the differentiated swarmer cell. In this review, the genetic and environmental requirements for swarmer cell differentiation will be outlined. In addition, the role of the differentiated swarmer cell in virulence and its possible role in biofilm formation will be discussed.     

A novel gene involved in regulating the flagellar gene cascade in Proteus mirabilis

In this study, we identified a transposon insertion in a novel gene, designated disA, that restored swarming motility to a putrescine-deficient speA mutant of Proteus mirabilis. A null allele in disA also increased swarming in a wild-type background. The DisA gene product was homologous to amino acid decarboxylases, and its role in regulating swarming was investigated by examining the expression of genes in the flagellar cascade. In a disA mutant background, we observed a 1.4-fold increase in the expression of flhDC, which encodes FlhD(2)C(2), the master regulator of the flagellar gene cascade. However, the expressions of class 2 (fliA, flgM) and class 3 (flaA) genes were at least 16-fold higher in the disA background during swarmer cell differentiation. Overexpression of DisA on a high-copy-number plasmid did not significantly decrease flhDC mRNA accumulation but resulted in a complete block in mRNA accumulation for both fliA and flaA. DisA overexpression also blocked swarmer cell differentiation. The disA gene was regulated during the swarming cycle, and a single-copy disA::lacZ fusion exhibited a threefold increase in expression in swarmer cells. Given that DisA was similar to amino acid decarboxylases, a panel of decarboxylated amino acids was tested for effects similar to DisA overexpression, and phenethylamine, the product of phenylalanine decarboxylation, was capable of inhibiting both swarming and the expression of class 2 and class 3 genes in the flagellar regulon. A DisA-dependent decarboxylated amino acid may inhibit the formation of active FlhD(2)C(2) heterotetramers or inhibit FlhD(2)C(2) binding to DNA.     

Pattern formation and the fine structure of the developing cell wall in colonies of Pediastrum boryanum

A single-layered disc of peripheral pronged cells and central prongless cells impart the typical gear shape to colonies of Pediastrum, while the walls of each cell have a characteristic reticulate triangular pattern. The two-layered wall forms in the cells during colony formation following zoospore aggregation and adhesion. The uniformly thin outer layer reflects contours resulting from differential thickening in the reticulate pattern of the inner, thicker, more fibrillar and granular wall layer. The reticulate pattern thus imparted to the outer wall layer persists in empty zoosporangia following the release of zoospores. Columns of electron-dense material extend through the outer wall layer except at the ridges and centers of the reticulum. Following mitosis and cleavage, the resulting zoospores are extruded within a vesicle membrane consisting of the inner wall layer. Separation of this membrane from the parent cell occurs in material of the inner layer adjacent to the outer wall. Vesicles containing swarming zoospores also contain a granular material which appears to become associated with the aggregating and adhering cells of new colonies. Microtubules occur in zoospores prior to adherence but are absent during wall deposition.     

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.     

Major outer membrane proteins unique to reproductive cells of Hyphomonas jannaschiana.

Separation on the basis of molecular weight resolved three proteins specific to the swarmer cell of Hyphomonas jannaschiana. In the reproductive cell, 4 major proteins were identified as cytoplasmic and 10 were identified as envelope. Of these envelope proteins, one was common to both the inner and outer membranes, four were common to the inner membrane, and five were common to the outer membrane. Four of these outer membrane proteins were specific to the reproductive cell, and two of these proteins, with apparent molecular weights of 116,000 and 29,000, constituted 19% of the total cell protein and 54% of the outer membrane protein.     

Freeze-fracture studies of Cryptosporidium muris.

The attachment site of Cryptosporidium muris to host cells was investigated using the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with the host plasma membrane at the dense band, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was dramatically altered at the above two junctures. The outer parasitophorous membrane showed low IMP-density as compared to the host plasma membrane, although both membranes were continuous. The inner parasitophorous membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP. When the attachment sites of parasites and host cells were fractured, circular-shaped fractured faces were observed on both sites of the parasite and host cell. These exposed faces corresponded to the dense bands and were very similar in size in each parasite.     

Evidence that putrescine acts as an extracellular signal required for swarming in Proteus mirabilis

In a search for Proteus mirabilis genes that were regulated by cell-to-cell signalling, a lacZ fusion (cmr437::mini-Tn5lacZ) was identified that was repressed 10-fold by a self-produced extracellular signal from wild-type cells. However, the cmr437::mini-Tn5lacZ insertion itself led to a marked reduction in this extracellular repressing signal. The cmr437::mini-Tn5lacZ insertion was mapped to a speA homologue in P. mirabilis. Sequence analysis indicated that a speB homologue was encoded downstream of speA. Products of the SpeA and SpeB enzymes (agmatine and putrescine) were tested for repression of cmr437::lacZ. Agmatine did not have repressing activity. However, putrescine was an effective repressing molecule at concentrations down to 30 microM. A second prominent phenotype of the cmr437 (speA)::mini-Tn5lacZ insertion was a severe defect in swarming motility. This swarming defect was also observed in a strain containing a disruption of the downstream speB gene. Differentiation of the speB mutant to swarmer cells was delayed by two hours relative to wild-type cells. Furthermore, the speB mutant was unable to migrate effectively across agar surfaces and formed very closely spaced swarming rings. Exogenous putrescine restored both the normal timing of swarmer cell differentiation and the ability to migrate to speB mutants.     

Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants.

The outer membrane layer of the cell wall was isolated from wild-type Salmonella typhimurium LT2 as well as from its mutants producing lipopolysaccharides with shorter saccharide chains. Chemical analysis of these preparations indicated the following. (i) The number of lipopolysaccharide molecules per unit area was constant, regardless of the length of the saccharide side chain in lipopolysaccharide. (ii) In contrast, in "deep rough" (Rd or Re) mutants producing the lipopolysaccharides with very short saccharide chains, the amount of outer membrane protein per unit surface area decreased to about 60% of the value in the wild type. (iii) In the wild type, the amount of phospholipids is slightly less than what is needed to cover one side of the membrane as a monolayer. In comparison with the wild type, the outer membrane of Rd and Re mutants contains about 70% more phospholipids, which therefore must be distributed in both the outer and inner leaflets of the membrane. Freeze-fracture studies showed that the outer membrane of Re mutants were easily fractured, but fracture became increasingly difficult in strains producing lipopolysaccharides with longer side chains. The convex fracture face was always nearly smooth, but the concave fracture face or the outer half of the membrane was densely covered with particles 8 to 10 nm in diameter. The density of particles was decreased in Re mutants to the same extent as the reduction in proteins, suggesting the largely proteinaceous nature of particles. A model for the supramolecular structure of the outer membrane is presented on the basis of these and other results.     

The ability of Proteus mirabilis to sense surfaces and regulate virulence gene expression involves FliL, a flagellar basal body protein

Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL, fliF, and fliG, encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL- mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL+ in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL+ was expressed in Escherichia coli. These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB. These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression.     

Role of the Umo proteins and the Rcs phosphorelay in the swarming motility of the wild type and an O-antigen (waaL) mutant of Proteus mirabilis

Proteus mirabilis is a Gram-negative bacterium that exists as a short rod when grown in liquid medium, but during growth on surfaces it undergoes a distinct physical and biochemical change that culminates in the formation of a swarmer cell. How P. mirabilis senses a surface is not fully understood; however, the inhibition of flagellar rotation and accumulation of putrescine have been proposed to be sensory mechanisms. Our lab recently isolated a transposon insertion in waaL, encoding O-antigen ligase, that resulted in a loss of swarming but not swimming motility. The waaL mutant failed to activate flhDC, the class 1 activator of the flagellar gene cascade, when grown on solid surfaces. Swarming in the waaL mutant was restored by overexpression of flhDC in trans or by a mutation in the response regulator rcsB. To further investigate the role of the Rcs signal transduction pathway and its possible relationship with O-antigen surface sensing, mutations were made in the rcsC, rcsB, rcsF, umoB (igaA), and umoD genes in wild-type and waaL backgrounds. Comparison of the swarming phenotypes of the single and double mutants and of strains overexpressing combinations of the UmoB, UmoD, and RcsF proteins demonstrated the following: (i) there is a differential effect of RcsF and UmoB on swarming in wild-type and waaL backgrounds, (ii) RcsF inhibits UmoB activity but not UmoD activity in a wild-type background, and (iii) UmoD is able to modulate activity of the Rcs system.     

Regionalization of transmembrane glycoproteins in the plasma membrane of boar sperm head is revealed by fracture-label

We used fracture-label and surface labeling techniques to characterize the distribution and topology of wheat germ agglutinin (WGA) receptors in the plasma membrane of boar sperm heads. We show that freeze- fracture results in preferential, but not exclusive, partition of WGA- binding sites with the outer (exoplasmic) half of the plasma membrane. Labeling of the inner (protoplasmic) half of the membrane is significant, and is denser over the areas that overlie the acrosome. Exoplasmic membrane halves are uniformly labeled. Analysis of freeze- fracture replicas revealed that the distribution of intramembrane particles over protoplasmic faces parallels that of WGA-binding sites as observed by fracture-label. Coating of intact spermatozoa with cationized ferritin results in drastic reduction of the labeling of both protoplasmic and exoplasmic membrane halves. Labeling of sperm cells lysed by short hypotonic shock fails to reveal the presence of WGA-binding sites at the inner surface of the plasma membrane. We conclude that: (a) all WGA-binding glycoconjugates are exposed at the outer surface of the membrane; (b) some of these glycoconjugates correspond to transmembrane glycoproteins that, on fracture, partition with the inner half of the membrane; (c) these transmembrane proteins are accumulated in the region of the plasma membrane that overlies the acrosome; and (d) parallel distribution of intramembrane particles and WGA-binding glycoproteins provides renewed support for the view of particles as the morphological counterpart of integral membrane proteins.