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.

Negative feedback from a Proteus class II flagellum export defect to the flhDC master operon controlling cell division and flagellum assembly.

The Proteus mirabilis flagellum class I flhDC operon was isolated, and its transcript was shown to originate from a sigma70 promoter 244 bp 5' of flhD and 29 bp 3' of a putative cyclic AMP receptor protein-binding site. Expression of this regulatory master operon increased strongly as cells differentiated into elongated hyperflagellated swarm filaments, and cell populations artificially overexpressing flhDC migrated sooner and faster. A class II flhA transposon mutant was reduced in flagellum class III gene expression, as would be expected from the FlgM anti-sigma28 accumulation demonstrated in Salmonella typhimurium, but was unexpectedly also reduced in cell elongation. Here, we show that levels of flhDC transcript were ca. 10-fold lower in this flagellum export mutant, indicating that in cells defective in flagellum assembly, there is additional negative feedback via flhDC. In support of this view, artificial overexpression of flhDC in the flhA mutant restored elongation but not class III flagellum gene transcription.

A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator.

Proteus swarming is the rapid cyclical population migration across surfaces by elongated cells that hyperexpress flagellar and virulence genes. The mini-Tn5 transposon mutant mns2 was isolated as a tight nonswarming mutant that did not elongate or upregulate flagellar and hemolysin genes. Individual cell motility was retained but was reduced. The transposon had inserted in the gene encoding the global transcriptional regulator Lrp (leucine-responsive regulatory protein), expression of which was upregulated in differentiating swarm cells. Swarming was restored to the lrp mutant by artificial overexpression of the flhDC flagellar regulatory master operon. Lrp may be a key component in generating or relaying signals that are required for flagellation and swarming, possibly acting through the flhDC operon.

ATP-dependent import of a lumenal protein by isolated thylakoid vesicles.

The 33 kd protein of the photosynthetic oxygen-evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non-chloroplast proteins and precursors of stromal proteins are not imported.