Swarming was assessed on 1.5% Luria-Bertani (LB) agar plates, while vegetative cell motility was assessed on 0.3% LB agar. Mutants were selected on chloramphenicol (80 μg ml −1). mirabilis U6450 was mutagenized with mini-Tn5Cm ( 19). Mutagenesis and characterization of the mutant.
We show that the disrupted gene, so far unique to Proteus, influences the shape of swarm cells and so enhances the multicellular alignment essential for population migration. Here we describe a second swarming-defective transposon mutant of this class, MNS185, which is similarly unimpaired in differentiation and cell motility. This mutant, FC18, mutated in the putative sugar transferase gene cmfA, was unable to make an extracellular polysaccharide required for mass cell movement and so was impaired in the mechanics of translocation ( 17). In contrast, only one swarming-defective transposon mutant has been shown to be unimpaired in differentiation and cell motility. Characterization of Proteus transposon mutants and multicopy suppressors of these mutants has identified many genes involved in swarming, and these have been shown or are presumed to influence differentiation and flagellar gene expression ( 3, 6, 7, 11, 16, 18, 19). These conditions result primarily from increased transcription of the flagellar master operon flhDC, which is the principal regulator of flagellar assembly and which also modulates cell division, integrating a variety of signals ( 11– 13). Regular cycles of migration and consolidation generate a characteristic pattern of concentric terraces ( 30, 35).įlagellar gene expression is strongly upregulated and septation is repressed during differentiation into swarm cells ( 2, 4, 16). In Proteus, migration ceases periodically, and continued growth is accompanied by increased septation and decreased flagellar density (consolidation). These align closely along their long axes, forming two-dimensional rafts that migrate by coordinated flagellar action within a film of hydrated polysaccharide secreted by the bacteria, causing rapid extension of the colony boundary ( 2, 35). In Proteus, it is characterized by the differentiation of short motile vegetative cells at the edge of a growing colony into extremely elongated hyperflagellated swarm cells. Swarming, the coordinated migration of multicellular colonies, is best known in Proteus mirabilis ( 2, 20, 30, 32, 35) but is also evident to various degrees in other motile flagellated species ( 2, 12, 20, 29). While complex multicellular behavior in bacteria is obvious in a relatively small number of species, e.g., fruiting body formation in myxobacteria and sporulation in streptomycetes ( 21, 23), the ability to form organized colonial communities is common ( 10, 32). mirabilis to become enlarged and ellipsoidal. Consistent with this view, overexpression of the ccmA gene caused cells of both Escherichia coli and P. 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. 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. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. 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. Two forms of CcmA were identified for wild-type P. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm.
Multiple copies of the wild-type gene, called ccmA, for curved cell morphology, restored swarming to the mutant. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. 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. 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. Swarming in Proteus mirabilis is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells.
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