Regulation of the Swarming Inhibitor disA in Proteus mirabilis

The disA gene encodes a putative amino acid decarboxylase that inhibits swarming in Proteus mirabilis. 5′ rapid amplification of cDNA ends (RACE) and deletion analysis were used to identify the disA promoter. The use of a disA-lacZ fusion indicated that FlhD₄C₂, the class I flagellar master regulator, did not have a role in disA regulation. The putative product of DisA, phenethylamine, was able to inhibit disA expression, indicating that a negative regulatory feedback loop was present. Transposon mutagenesis was used to identify regulators of disA and revealed that umoB (igaA) was a negative regulator of disA. Our data demonstrate that the regulation of disA by UmoB is mediated through the Rcs phosphorelay.     

The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis

When two different strains of swarming Proteus mirabilis encounter one another on an agar plate, swarming ceases and a visible line of demarcation forms. This boundary region is known as the Dienes line and is associated with the formation of rounded cells. While the Dienes line appears to be the product of distinction between self and nonself, many aspects of its formation and function are unclear. In this work, we studied Dienes line formation using clinical isolates labeled with fluorescent proteins. We show that round cells in the Dienes line originate exclusively from one of the swarms involved and that these round cells have decreased viability. In this sense one of the swarms involved is dominant over the other. Close cell proximity is required for Dienes line formation, and when strains initiate swarming in close proximity, the dominant Dienes type has a significant competitive advantage. When one strain is killed by UV irradiation, a Dienes line does not form. Killing of the dominant strain limits the induction of round cells. We suggest that both strains are actively involved in boundary formation and that round cell formation is the result of a short-range killing mechanism that mediates a competitive advantage, an advantage highly specific to the swarming state. Dienes line formation has implications for the physiology of swarming and social recognition in bacteria.The gram-negative bacterium Proteus mirabilis is well known for its ability to differentiate into hyperflagellated, motile, and elongated swarmer cells that rapidly spread over a surface. When cultured on a nutrient agar plate, a strain of P. mirabilis typically is able to colonize the whole plate within 24 h. This phenomenon is both of interest in terms of the differentiation and survival strategy of the organism and of practical importance, as contamination of agar plates by swarming P. mirabilis is a common problem in diagnostic microbiology. When two different strains of P. mirabilis swarm on the same agar plate, a visible demarcation line with lower cell density forms at the intersection, and this line is known as a Dienes line (5, 6). A Dienes line is seen when both strains are swarming; it is not a property of the smaller vegetative cells (5). When two identical isolates meet, the swarming edges merge without formation of a Dienes line. This phenomenon has been used in epidemiological typing of clinical isolates (4, 20, 23, 28) and raises interesting questions concerning its mechanism and biological importance. Dienes typing in the clinical environment suggests that the number of Dienes types is large; 81 types were found in one study alone (25). Incompatibility between swarming strains may not be unique to P. mirabilis; a comparable process also appears to occur in Pseudomonas aeruginosa (19). Like many other bacteria, some P. mirabilis strains produce bacteriocins, termed proticines (3). It has been shown that Dienes line formation is not directly caused by a proticine, nor has any other secreted substance or cellular lysate been found to contribute to this phenomenon (27). There does, however, seem to be a circumstantial link between proticine production and Dienes line formation. In the 1970s Senior typed strains of P. mirabilis based on their proticine production and sensitivity (23, 24). He found that proticine production is not related to proticine sensitivity (apart from strains being resistant to their own proticine) but that there is a good correlation between a combined proticine production-sensitivity (P/S) type and Dienes line formation. Strains with the same P/S type do not form a Dienes line, whereas strains with different P/S types do. The more closely related the P/S types of two strains, the less clearly defined the Dienes line is, suggesting that relatedness of strains plays a central role. In contrast, a strain of Proteus vulgaris has been shown to be Dienes compatible with a strain of P. mirabilis with the same P/S type (24). Furthermore, Dienes incompatibility between otherwise identical strains can be triggered by phage lysogeny (2). In contrast to P/S type, the polysaccharide (O) or flagellar (H) serotype has been shown to have no relationship to Dienes line formation (25, 27).Research into the mechanism governing Dienes line formation in Proteus has revealed some important features. The Dienes line region contains many large and often rounded cells (5). The nature of these cells remains controversial. Dienes suggested that round cells originated from both strains but that viable round cells always originated from one of the two intersecting strains. He concluded from this that nonviable round cells should therefore be cells of the other strain (6). In contrast, Wolstenhome suggested that the round cells originated mostly from one of the two swarms involved and that only 50% of these cells were viable (32). It has also been noted that round cells occasionally seem to develop into stable L forms lacking a full cell wall and that they can grow to form tiny L-type colonies (5). Additionally, extracellular DNase has been found at the site of the Dienes line (1). The presence of this enzyme has been interpreted to be a result of cell lysis, as P. mirabilis is known to contain large amounts of DNase (22, 26). Recently, a cluster of six genes, termed ids (identification of self), has been linked to the incompatibility in interpenetration between two strains (and therefore formation of the visible demarcation line), although the function and expression of these genes are not understood yet (8).Despite the fact that Dienes line formation has been known for over 50 years, many questions concerning this phenomenon remain unanswered. Recent advances in imaging, molecular biology, and genomics offer new ways of investigating it. In this work, clinical isolates expressing fluorescent proteins were used to observe the cells involved in Dienes line formation in real time, to evaluate the fate of the round cells, and to test the role of extracellular material and direct cell-cell contact in this phenomenon. Furthermore, the biological role of the Dienes phenomenon in the competition between strains in different situations was investigated.     

Swarming characteristics of Proteus mirabilis under anaerobic and aerobic conditions

Nutrients have a pronounced effect on the growth and swarming behaviour of Proteus mirabilis 7002. Iron, zinc, amino acids, and dioxygen are important for rapid growth and normal swarming. Anaerobically grown cultures of P. mirabilis 7002 were unable to swarm on anaerobically maintained rich nutrient agar. Upon exposure to aerobic conditions, P. mirabilis 7002 resumed swarming behaviour. Scanning electron microscopy was used to demonstrate the presence of community organization and mature rafts during normal swarming. These results support the importance of dioxygen and redox status in cell differentiation.     

Characterization of Indole-Positive Proteus mirabilis

Thirteen indole-producing, swarming strains of Proteus were identified by additional biochemical testing as being Proteus mirabilis. These strains were characterized by 40 biochemical tests and by susceptibility testing to 11 antibiotics. All produced ornithine decarboxylase and were susceptible to members of the penicillin-cephalosporin groups of antibiotics. These indole-positive strains are similar to indole-negative P. mirabilis and are distinctly different from P. vulgaris. For greatest accuracy and to insure greatest clinical relevancy, P. mirabilis and P. vulgaris should be distinguished from one another in the laboratory by performing both the indole and ornithine decarboxylase tests.     

Loss of FliL Alters Proteus mirabilis Surface Sensing and Temperature-Dependent Swarming

Proteus mirabilis is a dimorphic motile bacterium well known for its flagellum-dependent swarming motility over surfaces. In liquid, P. mirabilis cells are 1.5- to 2.0-μm swimmer cells with 4 to 6 flagella. When P. mirabilis encounters a solid surface, where flagellar rotation is limited, swimmer cells differentiate into elongated (10- to 80-μm), highly flagellated swarmer cells. In order for P. mirabilis to swarm, it first needs to detect a surface. The ubiquitous but functionally enigmatic flagellar basal body protein FliL is involved in P. mirabilis surface sensing. Previous studies have suggested that FliL is essential for swarming through its involvement in viscosity-dependent monitoring of flagellar rotation. In this study, we constructed and characterized ΔfliL mutants of P. mirabilis and Escherichia coli. Unexpectedly and unlike other fliL mutants, both P. mirabilis and E. coli ΔfliL cells swarm (Swr⁺). Further analysis revealed that P. mirabilis ΔfliL cells also exhibit an alteration in their ability to sense a surface: e.g., ΔfliLP. mirabilis cells swarm precociously over surfaces with low viscosity that normally impede wild-type swarming. Precocious swarming is due to an increase in the number of elongated swarmer cells in the population. Loss of fliL also results in an inhibition of swarming at <30°C. E. coli ΔfliL cells also exhibit temperature-sensitive swarming. These results suggest an involvement of FliL in the energetics and function of the flagellar motor.     

Bacteriophage Typing of Proteus mirabilis, Proteus vulgaris, and Proteus morganii

A bacteriphage typing scheme for differentiating Proteus isolated from clinical specimens was developed. Twenty-one distinct patterns of lysis were seen when 15 bacteriophages isolated on 8 Proteus mirabilis, 1 P. vulgaris, and 1 P. morganii were used to type 162 of 189 (85.7%) P. mirabilis and P. vulgaris isolates. Seven phages isolated on 3 P. morganii were used to type 13 of 19 (68.4%) P. morganii isolates. Overall, 84.1% of the 208 isolates were lysed by at least 1 phage at routine test dilution (RTD) or 1,000 x RTD. Fifty isolates, retyped several weeks after the initial testing, showed no changes in lytic patterns. The phages retained their titers after storage at 4 C for several months. A computer analysis of the data showed that there was no relationship between the source of the isolate and bacteriophage type. This bacteriophage typing system may provide epidemiological information on strains involved in human infections.     

Differential Expression of Nonagglutinating Fimbriae and MR/P Pili in Swarming Colonies of Proteus mirabilis

The expression of nonagglutinating fimbriae (NAF) and mannose-resistant/Proteus-like (MR/P) pili in swarming colonies of Proteus mirabilis was investigated. Elongated swarmer cells do not express pili, and the relative number of bacteria expressing NAF during swarming and early consolidation phases was very low (<5%). Relative expression of NAF in a terrace increased to approximately 30% at 48 h. We also determined the expression of NAF and MR/P pili in two phenotypically distinguishable regions of each terrace. The expression of both NAF and MR/P pili was always higher in the region closer (proximal) to the middle of the colony than in the distal region of the terrace. The relative numbers of bacteria expressing NAF or MR/P pili in the proximal region were between 39.1 and 63% and between 5.9 and 7.7%, respectively. In the distal region, expression levels were between 20.8 and 27.3% and between 3.7 and 5. 6%, respectively. A time course experiment testing NAF expression in both the proximal and distal regions of a terrace indicated that NAF expression in the proximal regions was always higher than in the distal regions and increased to a plateau 40 to 50 h after the start of the swarming phase for any given terrace. These results indicate that expression of NAF or MR/P pili in swarming colonies of P. mirabilis is highly organized, spatially and temporally. The significance of this controlled differentiation remains to be uncovered.     

Isolation and Characterization of Antitumor Lipopolysaccharide from Proteus mirabilis

Systematic isolation of the cell constituents of Proteus mirabilis RMS–203 was performed to find out localization of antitumor principle only in the lipopolysaccharide (LPS) layer of the cell wall fraction.

LPS with strong antitumor activity was extracted from P. mirabilis RMS–203 by phenol-water method followed by purification on DEAE-Sephadex A–50 column chromatography.

The main components of purified LPS were galactose, hexosamine, 2-keto-deoxy-octonic acid (KDO), myristic acid, β-hydroxymyristic acid and α,ε-diaminopimelic acid.

The minimal effective dose of LPS against Ehrlich solid carcinoma in mice was 0.1~1.0 μg/mouse. LD₅₀ in mice and pyrogenicity in rabbits were 28 mg/kg and 10^?3–10^?5 μg/rabbit, respectively.     

Inhibitory Effects of Secondary Metabolites from the Red Alga Delisea pulchra on Swarming Motility of Proteus mirabilis

Abnormal, uncoordinated swarming motility of the opportunistic human pathogen Proteus mirabilis was seen when a crude extract of the Australian red alga Delisea pulchra was added to the medium. This occurred at concentrations at which growth rate, swimming motility, cell elongation, polynucleation, and hyperflagellation were not affected. One halogenated furanone from D. pulchra inhibited swarming motility at concentrations that did not affect growth rate and swimming motility. Other structurally similar D. pulchra furanones had no effect on swarming, suggesting considerable specificity in the effects of furanones on swarming motility by P. mirabilis.     

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.Abbreviation SDS sodium dodecylsulfate     

Functional Identification of the Proteus mirabilis Core Lipopolysaccharide Biosynthesis Genes

In this study, we report the identification of genes required for the biosynthesis of the core lipopolysaccharides (LPSs) of two strains of Proteus mirabilis. Since P. mirabilis and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up to the second outer-core residue, the functions of the common P. mirabilis genes was elucidated by genetic complementation studies using well-defined mutants of K. pneumoniae. The functions of strain-specific outer-core genes were identified by using as surrogate acceptors LPSs from two well-defined K. pneumoniae core LPS mutants. This approach allowed the identification of two new heptosyltransferases (WamA and WamC), a galactosyltransferase (WamB), and an N-acetylglucosaminyltransferase (WamD). In both strains, most of these genes were found in the so-called waa gene cluster, although one common core biosynthetic gene (wabO) was found outside this cluster.Gram-negative motile and frequently swarming bacteria of the genus Proteus and the family Enterobacteriaceae are opportunistic human pathogens (33). Currently, the genus consists of five species (Proteus mirabilis, P. penneri, P. vulgaris, P. myxofaciens, and P. hauseri) and three genomospecies (4, 5, and 6) (33, 35). P. mirabilis is a common uropathogen that causes urinary tract infections especially in individuals with functional or anatomical abnormalities of the urinary tract (52) and elderly persons undergoing long-term catheterization (53) but less frequently in normal hosts (43). Potentially serious complications arising from P. mirabilis infections include bladder and kidney stone formation, catheter obstruction due to the formation of encrusting biofilms, and bacteremia (reviewed in reference 2). This bacterium is found more frequently than Escherichia coli in kidney infections (14) and may be associated with rheumatoid arthritis (38). Studies aimed at the identification of P. mirabilis virulence factors showed that flagella and fimbriae (MR/P and PMF) are required for entry into and colonization of the bladder, respectively (reviewed in reference 12). Other important virulence factors are urease, hemolysin, and iron acquisition (12). More recently, an extracellular metalloprotease (37) and several putative DNA binding regulatory, cell-envelope related, and plasmid-encoded proteins have been identified by signature-tagged mutagenesis (8, 21).The lipopolysaccharide (LPS), as in other members of the family Enterobacteriaceae, consists of three domains, an endotoxic glycolipid (lipid A), an O-polysaccharide (O-PS) chain or O-antigen, and an intervening core oligosaccharide (OS) region. The O-antigen is the major surface antigen, and its serological O specificity, in contrast to that of other Gram-negative bacteria (31), is defined by the structure of the O-PS chain and that of the core OS (51). On the basis of immunospecificity, 60 O serogroups (28, 36) have been recognized in P. mirabilis and P. vulgaris, and several new Proteus O serogroups have been proposed for P. penneri (27, 55). The LPS is a potential Proteus virulence factor (42), and recently two mutants deficient in a glycosyltransferase and with attenuated virulence have been isolated and it has been speculated that this glycosyltransferase could be involved in LPS biosynthesis (21). LPS plays a significant role in the resistance of P. mirabilis to antimicrobial peptides (32), and LPS charge alterations may influence the swarming motility of the bacterium (3, 32). In addition, the core LPS is a charged OS which plays an important role in the biological activities of the LPS and the function of the bacterial outer membrane (10). In Proteus, the core OS structures of up to 34 strains of different O serogroups have been determined (51). These structures revealed that Proteus core OSs share a heptasaccharide fragment that includes a 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo) disaccharide, an l-glycero-α-d-manno-heptose (l,d-Hep) trisaccharide, and one residue each of d-glucose (d-Glc), d-galacturonic acid (d-GalA), and either d-glucosamine (d-GlcN) or d-galactosamine (d-GalN) (51). This common fragment is also found in the core LPSs of Klebsiella pneumoniae and Serratia marcescens (11, 41, 50). The rest of the Proteus core OS is quite variable, and it is possible to recognize up to 37 and 11 different structures in the genus and P. mirabilis, respectively (51). Some P. mirabilis core OS structures are characterized by the presence of unusual residues, such as, for instance, quinovosamine; an open-chain form of N-acetylgalactosamine (GalNAc); or unusual amino acids (51). In contrast, little is known about the genes encoding enzymes involved in core LPS biosynthesis in P. mirabilis, which makes detailed genetic analysis of the role of LPS in P. mirabilis pathogenesis difficult. Thus, we decided to identify these genes by using P. mirabilis strains R110 and 51/57, the whole structures of whose core LPSs are known (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Chemical structures of the core LPSs of P. mirabilis strains R110 and 51/57 (51), K. pneumoniae types 1 (50) and 2 (41), and S. marcescens N28b (11).     

Initiation of Swarming Motility by Proteus mirabilis Occurs in Response to Specific Cues Present in Urine and Requires Excess l-Glutamine

Proteus mirabilis, a leading cause of catheter-associated urinary tract infection (CaUTI), differentiates into swarm cells that migrate across catheter surfaces and medium solidified with 1.5% agar. While many genes and nutrient requirements involved in the swarming process have been identified, few studies have addressed the signals that promote initiation of swarming following initial contact with a surface. In this study, we show that P. mirabilis CaUTI isolates initiate swarming in response to specific nutrients and environmental cues. Thirty-three compounds, including amino acids, polyamines, fatty acids, and tricarboxylic acid (TCA) cycle intermediates, were tested for the ability to promote swarming when added to normally nonpermissive media. l-Arginine, l-glutamine, dl-histidine, malate, and dl-ornithine promoted swarming on several types of media without enhancing swimming motility or growth rate. Testing of isogenic mutants revealed that swarming in response to the cues required putrescine biosynthesis and pathways involved in amino acid metabolism. Furthermore, excess glutamine was found to be a strict requirement for swarming on normal swarm agar in addition to being a swarming cue under normally nonpermissive conditions. We thus conclude that initiation of swarming occurs in response to specific cues and that manipulating concentrations of key nutrient cues can signal whether or not a particular environment is permissive for swarming.     

可溶性组织因子突变体的基因构建、表达和性质

血浆中组织因子 (TF)的过量表达与许多病理过程密切相关。TF抑制物有可能防治这些疾病。设计了两种可溶性组织因子 (sTF)的突变体 (MCsTF和MFsTF) ,突变了协同催化的功能域 ,保留与因子VII/VIIa结合的功能域 ,使突变体竞争性抑制野生型TF的功能。用PCR的方法 ,对可溶性组织因子cDNA基因进行了点突变 ,并实现了在大肠杆菌中高效表达。rsTF、rMCsTF和rMFsTF的促凝活性研究表明 ,rMFsTF的激活X因子活性和促凝血作用相当于rsTF的 10 % ,而rMCsTF几乎完全失去了激活X因子活性和促凝血作用。rMCsTF和rMFsTF与VII/VIIa因子形成复合物对激活X因子的催化特异常数 (kcat/Km)分别是FVII/VIIa·rsTF的 2 .0 %和 3.7% ,也说明突变体与VII/VIIa形成的复合物对激活X因子催化活性显著降低。rMCsTF和rMFsTF对rsTF活性抑制动力学研究及体外活性研究表明 ,两种突变体均有抑制rsTF活性和抑制兔脑粉的促凝活性作用 ,抑制作用呈量效关系     

Abolition of Swarming of Proteus by p-Nitrophenyl Glycerin: General Properties

Para-nitrophenyl glycerin (PNPG) was shown to be an effective agent to abolish the swarming of Proteus mirabilis and Proteus vulgaris on predried solid culture media. The level required to abolish swarming varied with the strain of Proteus, the components of the medium, and also with the conditions of incubation. Generally 0.1 to 0.2 mM PNPG effectively abolished swarming for at least 24 h with aerobic incubation. Levels of PNPG that abolished swarming showed no effect upon the growth of the cells, little or no effect upon the motility characteristics of the organisms, and no effect upon the cellular morphology. PNPG was found to be freely water soluble, stable to autoclaving, and to retain biological activity for at least one month in prepared culture media stored under refrigeration.     

Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis

Nitrate reductase A has been solubilized from purified cytoplasmic membranes by extraction with terl-amyl alcohol. The resulting aqueous solution contained monomeric reductase which polymerized slowly to dimers and tetramers with sedimentation coefficients of respectively 10.5, 16 and 23 Svedbergunits. The polymerization could be stopped to some extent by addition of a small amount of Triton X-100. These distinct entities of nitrate reductase A were separable on electro-focusing, DEAE-column chromatography and polyacrylamide gel electrophoresis, and have been proved to consist of similar subunits with molecular weights of 104000, 63000, and 56000 daltons. The molecular weights of monomeric nitrate reductase A was found to be about 240000 daltons.Chlorate reductase C has been solubilized by a similar procedure, resulting in only monomeric enzyme. Chlorate reductase C exhibited a sedimentation coefficient of 7.7 Svedbergunits, an isoelectric point of pH=4.55 and a molecular weight of approx. 180000 daltons. It was found to consist of three subunits with molecular weights of 75000, 63000 and 56000 daltons. The latter two subunits are most probably common in nitrate reductase A and chlorate reductase C.     

Transduction of R Factors by a Proteus mirabilis Bacteriophage

A transducing phage, designated phim, was isolated from a lysogenic strain of Proteus mirabilis and was characterized with respect to its physical and genetic properties. The phage contains double-stranded deoxyribonucleic acid (DNA) with an S(20,w) degrees of 29 which corresponds to a molecular weight of 24 x 10(6) daltons. The base composition of phim DNA was estimated to be 40% guanine plus cytosine on the basis of the buoyant density of the DNA. phim carries out generalized transduction of chromosomal genes in P. mirabilis at a frequency of 5 x 10(-8) to 2 x 10(-6) per adsorbed phage. To obtain R-factor transduction, it was necessary to have a resident R factor in the recipient cells. In these experiments, different combinations of genetically distinguishable R factors were used in the donor and recipient cells. The frequencies of R-factor transduction were 10(-9) to 2 x 10(-8). The transduction of R factors using an R(-) recipient could not be detected. Transductant R factors were usually recombinant between donor and resident R factors. All of the transduced R factors were transferable by conjugation. A plausible explanation for the requirement for a resident R factor in the recipient cells is that phim transduces only a portion of the R-factor genome and therefore requires a resident R factor for genetic recombination. The reason for the low frequencies of R-factor transduction is not known, but some possible interpretations have been discussed.