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.     

Putrescine Importer PlaP Contributes to Swarming Motility and Urothelial Cell Invasion in Proteus mirabilis

Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437–446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.     

The ITS region as a target for characterization of fungal communities using emerging sequencing technologies

In a previous work, it was observed that the swarming of polyamine-deficient Proteus mirabilis ( speB :: sm ) was severely inhibited on Luria–Bertani (LB) swarming plates (LBSw) (LB, 0.5% glucose, 0.5% agar), and it was clarified that extracellular putrescine was important as a signaling molecule for the induction of swarming in P. mirabilis . However, a polyamine-deficient strain (delta- speAB delta- speC ) of Escherichia coli swarmed as well as the parental strain on LBSw plates. We report that the swarming phenotype of a polyamine-deficient E. coli strain is dependent on spermidine and PotABCD, a spermidine importer.     

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.     

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.     

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.     

Isolation of lacZ fusions to Proteus mirabilis genes regulated by intercellular signaling: potential role for the sugar phosphotransferase (Pts) system in regulation

Using a mini-Tn5lacZ1 reporter transposon, lacZ fusions have been identified in Proteus mirabilis that are activated by the accumulation of self-produced extracellular signals. Genes identified by this approach include putative homologs of pgm, nlpA and two genes of unknown function. The extracellular signal(s) involved in activation were resistant to the effects of acid and alkali. The signal required for activation of (nlpA) cma482::lacZ was sensitive to protease, suggesting the signal is a peptide or small protein. The signals behaved as polar molecules and were not extractable with ethyl acetate. A mini-Tn5Cm insertion was identified in a probable ptsI homolog that blocked activation of the cma134::lacZ fusion by an extracellular signal. The ptsI mutation did not alter extracellular signal production and may have a role in signal response.     

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.     

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.     

mini-Tn7 insertion in bacteria with secondary, non-glmS-linked attTn7 sites: example Proteus mirabilis HI4320

We previously constructed a series of mini-Tn7 chromosome integration vectors that, when provided only with the site-specific transposition machinery, generally transpose to a naturally evolved, neutral attTn7 site that is located 25-bp downstream of the glmS gene. Here we provide a protocol for application of the mini-Tn7 system in Proteus mirabilis as an example of a bacterium with a secondary attTn7 site that is not linked to glmS but, in this case, located in the carAB operon. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. mirabilis by transformation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. When transposon-containing cells are selected on rich medium, insertions occur at both attTn7 sites with equal efficiency and frequency. Because carA mutants are arginine and pyrimidine auxotrophs, single-site insertions at the glmS attTn7 sites can be obtained by selection on minimal medium. From start to verification of the insertion events, the whole procedure takes 5 d. This chromosome integration system in P. mirabilis provides an important tool for animal and biofilm studies based on this bacterium. Vectors are available for gene complementation and expression, gene fusion analyses and tagging with a green fluorescent protein (GFP)-encoding reporter gene.     

Association of pyrogenic exotoxin genes with pharyngitis and rheumatic fever/rheumatic heart disease among Indian isolates of Streptococcus pyogenes

AIM: To monitor the presence of various pyrogenic exotoxin genes in strains of Streptococcus pyogenes isolated in India. METHODS & RESULTS: Isolates recovered from pharyngitis (52) and rheumatic fever (RF)/ rheumatic heart disease (RHD) (8) patients were analysed for the presence of toxin genes, speA, speB and speF, by PCR. The specificity of the products was confirmed by restriction enzyme digestion and Southern hybridization. Among the 60 isolates studied, the incidence of speA, speB and speF were 5(8.3%), 56(93.3%) and 53(88.3%), respectively. The expression of these genes was established in representative isolates by RT-PCR. CONCLUSIONS: Comparative analysis of frequency of the speA, speB and speF genes, among pharyngitis and RF/RHD associated isolates, showed higher incidence in RF/RHD (25%, 100%,100%) as compared to pharyngitis patients (5.8%, 92.3%, 86.5%), respectively. SIGNIFICANCE OF STUDY: The presence of the speA gene, which is usually associated with scarlet fever or toxic shock-like syndrome, within few Indian isolates may be indicative of new virulent strains circulating within the Indian community. High distribution of toxin genes among RF/RHD compared to pharyngitis isolates indicate their possible role in increased virulence.     

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.     

Changes in the organization of the outer membrane of Proteus mirabilis during swarming: freeze-fracture structure and membrane fluidity analysis.

Freeze-fracture studies of short, nonswarming Proteus mirabilis revealed the characteristic gram-negative profile of fractured inner membrane with densely packed particles and sectioned outer membrane with little or no fracture plane. Long swarming cells, however, fractured easily along both the inner membrane and a second membrane, probably the outer membrane. The inner membrane had a typical profile, whereas the outer membrane had fewer but more prominent particles. Isolation and purification of the inner and outer membranes of the short and long bacteria and examination of them with electron paramagnetic resonance measurements after spinlabeling supported the above observations. The outer membrane of swarmer cells allowed higher mobility of the spin label than did the outer membrane of the nonswarming short cells, which showed a typical rigid profile. These results suggest that regions of lipid bilayer appear in the outer membrane during swarmer formation. Previous observation of the behavior and biochemistry of P. mirabilis during swarming are discussed in light of these results.     

Frequency of genes speA,speB, and speC in Streptococcus pyogenes strains and the identification of the infective agent by polymerase chain reaction

In cultures of S. pyogenes isolated from patients and carriers in different territories of the Russian Federation the genes of erythorogenic toxins A, B and C (speA, speB and specC) were detected. The possibility of the identification of S. pyogenes by means of PCR on the basis of primers to erythrogenic toxin B was determined. Gene speB was detected in all S. pyogenes cultures under study and proved to be species specific. Genes speA and speC were detected, respectively, in 29.4% and 9.35% of the S. pyogenes cultures under study. A test system for the identification of S. pyogenes on the basis of primers to gene speB was developed. The prospects for the detection of genes speA and speC for intraspecific typing of this infective agent were evaluated.     

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.