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.

The motile TnphoA mutant IC24 of Proteus mirabilis U6450 generates an aberrant swarming colony, and was shown to be impaired in swarm cell differentiation, i.e. cell elongation and hyperflagellation, causing delayed and slower population migration across a solid growth medium. Levels of transcript from the flagellin filament gene fliC, the flagellar master operon flhDC, and the leucine-responsive regulatory protein gene lrp, a regulator of swarming differentiation, were reduced in IC24 mutant swarm cells. The transposon had inserted into a gene encoding a putative P-type ATPase closely related to those transporting cations across bacterial membranes. This ppa gene (Proteus P-type ATPase) was maximally expressed in differentiated swarm cells. The data suggest an effect of ion homeostasis on swarm cell differentiation, possibly mediated via the lrp-flhDC pathway.

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.

A TnphoA mutant of Proteus mirabilis was isolated, which had lost the ability to swarm, yet was still motile. The transposon had inserted into flgN, a flagella gene encoding a 147‐amino‐acid protein of undefined function. Proteus flgN is arranged in an operon with the class III anti‐σ28 gene, flgM, flanked by the class II genes, flgA, flgBCD and flhBA, and a novel putative virulence‐related gene. The flgN mutation caused a substantial reduction in cell surface‐associated flagellin, particularly during differentiation to the normally hyperflagellated swarm cell. This was not due to an effect on flagella gene expression or a typical defect in the flagella export apparatus as there was no class III gene downregulation by FlgM feedback, or intracellular flagellin accumulation. Loss of FlgN nevertheless caused a severe reduction in the incorporation of pulse‐labelled flagellin into the membrane/flagellum fraction of differentiating cells. Substantial amounts of both non‐oligomeric flagellin and flagellin degradation products appeared in the extracellular medium, although the few mature filaments made by the mutant were no more sensitive to proteolysis than those of the wild type. FlgN appeared soluble and active in the cytosol. The data suggest that the function of FlgN is to facilitate the initiation of flagella filament assembly, a role that may be especially critical in attaining the much higher concentration of surface flagellin required for swarming. Proteus FlgN has leucine zipper‐like motifs arranged on potential amphipathic helices, a feature conserved in cytosolic chaperones for the exported substrates of flagella‐related type III virulence systems. While gel filtration of FlgN from the soluble cell fraction did not establish an interaction with flagellin, it indicated that FlgN may associate with an unknown component and/or form an oligomer.

Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical rods into long, aseptate swarm cells that overexpress flagella and virulence factors, particularly haemolysin, A non-swarming Tn5phoA mutant was unable to synthesize flagella, to fully elongate or to induce high levels of the toxin. The mutation lay within a 2091 bp gene encoding a homologue of the Escherichia coli FlhA belonging to a family of proteins that are required for assembly of flagella or virulence proteins and that are suggested to act either directly in membrane translocation and/or in regulating synthesis of the export apparatus. In trans expression of multicopy flhA restored cell elongation and migration and generated differentiation-specific hyperexpression of flagellin and toxin genes to levels above those seen in the wild-type strain. Transcription of flhA was strongly induced during differentiation, from its own putative sigma(28) promoter, The results suggest a mechanistic coupling of flagella assembly and swarm-cell differentiation.

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 TnphoA 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.

A transposon mutant of Escherichia coli 5K was isolated which reduced 10- to 50-fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within the rfaP gene (required for attachment of phosphate-containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene. The loss in cytolytic activity of the secreted HlyA protein was not related to the HlyC-directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate-zonal centrifugation the mutant-secreted HlyA migrated as a larger species than the wild type. The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide-independent secretion process.

The three different pore-forming RTX-toxins of Actinobacillus pleuropneumoniae are reviewed, and new and uniform designations for these toxins and their genes are proposed. The designation ApxI (for Actinobacillus pleuropneumoniae RTX-toxin I) is proposed for the RTX-toxin produced by the reference strains for serotypes 1, 5a, 5b, 9, 10 and 11, which was previously named haemolysin I (HlyI) or cytolysin I (ClyI). This protein is strongly haemolytic and shows strong cytotoxic activity towards pig alveolar macrophages and neutrophils; it has an apparent molecular mass in the range 105 to 110 kDa. The genes of the apxI operon will have the designations apxIC, apxIA, apxIB, and apxID for the activator, the structural gene and the two secretion genes respectively. The designation ApxII is proposed for the RTX-toxin which is produced by all serotype reference strains except serotype 10 and which was previously named App, HlyII, ClyII or Cyt. This protein is weakly haemolytic and moderately cytotoxic and has an apparent molecular mass between 103 and 105 kDa. The genes of the apxII operon will have the designations apxIIC for the activator gene and apxIIA for the structural toxin gene. In the apxII operon, no genes for secretion proteins have been found. Secretion of ApxII seems to occur via the products of the secretion genes apxIB and apxID of the apxI operon. The designation ApxIII is proposed for the nonhaemolytic RTX-toxin of the reference strains for serotypes 2, 3, 4, 6 and 8, which was previously named cytolysin III (ClyIII), pleurotoxin (Ptx), or macrophage toxin (Mat).(ABSTRACT TRUNCATED AT 250 WORDS)

SummarySwarming by Proteus mirabilis involves differentiation of typical short vegetative rods into filamentous hyper‐flagellated swarm cells which undergo cycles of rapid and co‐ordinated population migration across surfaces and exhibit high levels of virulence gene expression. By supplementing a minimal growth medium (MGM) unable to support swarming migration we identified a single amino acid, glutamine, as sufficient to signal initiation of cell differentiation and migration. Bacteria isolated from the migrating edge of colonies grown for 8h with glutamine as the only amino acid were filamentous and synthesized the characteristic high levels of flagellin and haemolysin. In contrast, addition of the other 19 common amino acids (excluding glutamine) individually or in combination did not initiate differentiation even after 24 h, cells remaining typical vegetative rods with basal levels of haemolysin and flagellin. The glutamine analogue γ‐glutamyl hydroxamate (GH) inhibited swarming but not growth of P. mirabilis on glutamine MGM and transposon mutants defective in glutamine uptake retained their response to glutamine signalling and its inhibition by GH, suggesting that differentiation signalling by glutamine may be transduced independently of the cellular glutamine transport system. Levels of mRNA transcribed from the haemolysin (hpmA) and flagellin (fliC) genes were low in vegetative cells grown on MGM without glutamine or with glutamine and GH, but were specifically increased c. 40‐fold during glutamine‐dependent differentiation. In liquid glutamine—MGM cultures, differentiation to filamentous hyper‐flagellated hyper‐haemorytic swarm cells occurred early in the exponential phase of growth, and increased concomitantly with the concentration of glutamine from a 0.1 mM threshold up to 10 mM. Differentiation in liquid culture was completely inhibited by GH but was further stimulated c. 30% in the absence of GH by the viscosity agent polyvinylpy‐rollidone (PVP). Chemotaxis assays of bacterial cells in which the viscosity of liquid media was varied by PVP to allow either swimming or swarming motility demonstrated that glutamine was chemoattractive specifically to differentiated swarming cells.

The genetic determinant encoding the synthesis and secretion of hemolysin I (HlyI; gene designation, hlyI) by Actinobacillus pleuropneumoniae serotype 1 4074T was cloned in the lambda vector EMBL4. A 10.2-kb fragment that encoded hemolytic activity in the phage lysate was aligned by Southern blot hybridization to genes hlyC, hlyA, hlyB, and hlyD of the Escherichia coli hemolysin operon, and expression of the A. pleuropneumoniae genes in E. coli revealed that they have the same functions as their E. coli analogs: hlyIC encodes a protein that activates inactive 105-kDa prohemolysin I (encoded by hlyIA) to active hemolysin I, while hlyIB and hlyID are necessary for HlyIA secretion. Northern (RNA) hybridization of A. pleuropneumoniae RNA revealed that the gene cluster is transcribed as two RNA species, a major one of 3.5 kb, corresponding to hlyICA, and a second, minor one of 7.5 kb, corresponding to the whole operon, hlyICABD. The level of hlyI mRNA was substantially higher in A. pleuropneumoniae 4074T cells grown in the presence of Ca2+, supporting the view that the expression of the hlyI determinant is Ca2+ regulated. Parallel RNA hybridization with random gene probes suggested that this Ca2+ regulation is specific for the hlyI determinant.

The secreted hemolytic activity produced by Actinobacillus pleuropneumoniae serotype 2 reference strain is thermolabile, inactivated by proteinase K and requires Ca2+ as cofactor for its hemolytic activity. Purification of the hemolytic activity resulted in a fraction containing two proteins, one of 105 kDa and one of 125 kDa. These two proteins could be further separated by preparative SDS polyacrylamide gel electrophoresis. This purification step, resulted in loss of the hemolytic activity. Polyclonal antibodies were made against each of these proteins in rabbits. Neutralization experiments showed that antibodies made against the 105 kDa protein could neutralize the hemolytic activity produced by A. pleuropneumoniae serotype 2, while antibodies made against the 125 kDa protein were unable to neutralize the hemolytic activity. The 105 kDa protein therefore, is the hemolysin of A. pleuropneumoniae serotype 2, known as HlyII. This protein is closely related immunologically to the hemolysin I (HlyI) from A. pleuropneumoniae serotype 1. DNA::DNA hybridization experiments performed by the Southern blot method using the cloned structural gene of HlyI from A. pleuropneumoniae serotype 1 demonstrate that the structural genes of the two hemolysins (hlyIA and hlyIIA) are different and show at least 30% heterology. This confirms that HlyI and HlyII are two different proteins, although they have a very similar molecular weight and show strong immunological cross reactions.

The DNA sequence of the gene encoding the structural protein of hemolysin I (HlyI) of Actinobacillus pleuropneumoniae serotype 1 strain 4074 was analyzed. The nucleotide sequence shows a 3,072-bp reading frame encoding a protein of 1,023 amino acids with a calculated molecular size of 110.1 kDa. This corresponds to the HlyI protein, which has an apparent molecular size on sodium dodecyl sulfate gels of 105 kDa. The structure of the protein derived from the DNA sequence shows three hydrophobic regions in the N-terminal part of the protein, 13 glycine-rich domains in the second half of the protein, and a hydrophilic C-terminal area, all of which are typical of the cytotoxins of the RTX (repeats in the structural toxin) toxin family. The derived amino acid sequence of HlyI shows 42% homology with the hemolysin of A. pleuropneumoniae serotype 5, 41% homology with the leukotoxin of Pasteurella haemolytica, and 56% homology with the Escherichia coli alpha-hemolysin. The 13 glycine-rich repeats and three hydrophobic areas of the HlyI sequence show more similarity to the E. coli alpha-hemolysin than to either the A. pleuropneumoniae serotype 5 hemolysin or the leukotoxin (while the last two are more similar to each other). Two types of RTX hemolysins therefore seem to be present in A. pleuropneumoniae, one (HlyI) resembling the alpha-hemolysin and a second more closely related to the leukotoxin. Ca(2+)-binding experiments using HlyI and recombinant A. pleuropneumoniae prohemolysin (HlyIA) that was produced in E. coli shows that HlyI binds 45Ca2+, probably because of the 13 glycine-rich repeated domains. Activation of the prohemolysin is not required for Ca2+ binding.

SummaryThe gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild‐type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemotytic activity were attained similarly by the E. coli post‐translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram‐negative human and animal pathogens.

High-resolution electrophoresis of larval and adult hemoglobins of Xenopus laevis reveals stage-specific differences in the number and mobility of the globin chains. To establish the relationship between the globin chains and the previously described globin genes, the corresponding mRNAs were hybrid-selected from total erythroblast RNA by representative cDNA clones, and translated in vitro. Electrophoretic separation of the translation products allowed identification of a major and a minor α-globin chain in the larval and adult stages. This also holds for the adult β-chains, however in the larval stage a difference in abundance is only detectable in the β-mRNAs, but not in the translation products, because they comigrate. The fact that major and minor globin chains can be assigned to genes, which are located in two clusters, suggests that the related genes are expressed coordinately, but at different levels. Analysis of the globin patterns during development reveals that transition from the larval to the adult globin chains coincides with metamorphosis. Moreover, there is evidence of two globin chains that are only expressed in early larval stages and hence might be related to additional larval β-globin genes of as yet unknown genomic location.

The switch from larval to adult globin synthesis during amphibian metamorphosis was analyzed by polyacrylamide gel electrophoresis in acid urea‐Triton X‐100. 4–6 slowly migrating components were detected in larvae and at least 4 faster components appear at metamorphosis and persist in the adult stage. To relate the globin subunits to the mRNA sequences, isolated as cDNA clones, mRNAs were hybrid‐selected from total RNA of larval and adult erythrocytes and translated in a wheat germ system. The translation products were identified by comparison with the globin pattern of hemolysates