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.     

Inhibition of Swarming in Proteus spp. by Tannic Acid

Swarming in all 27 strains of Proteus spp. tested was inhibited by the presence of 0.02% (w/v) tannic acid in the nutrient medium. Cells from colonies on this medium were nearly all short forms but were motile and piliated. The swarm-inhibition effect was not reversed by the addition of calcium chloride. The growth of other bacterial species was inhibited to varying extents by tannic acid: Gram positive cocci ( Micrococcus, Sarcina , and Staphylococcus spp.) were particularly sensitive. The relative resistance of Gram negative bacteria and the swarm-inhibition of Proteus spp. could be due to binding of tannic acid to proteins in the outer membrane of the cell wall.     

Characterization of Proteus mirabilis Precocious Swarming Mutants: Identification of rsbA, Encoding a Regulator of Swarming Behavior

Proteus mirabilis swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull’s-eye colony often associated with cultures of P. mirabilis. How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the P. mirabilis genome encoding RsbA (regulator of swarming behavior) and the P. mirabilis homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a rsbA mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of rsbA. Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.     

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.     

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.     

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.     

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.     

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.     

pH Measurements of Agar Culture Media by Using a Flat-Bottom Electrode

A new design, flat-bottom combination pH electrode was evaluated for utility in determining the pH of prepared agar media by surface contact of the electrode with the agar media.     

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.     

Growth of Desulfovibrio on the Surface of Agar Media

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.     

Growth and Plating Efficiency of Methanococci on Agar Media

Plating techniques for cultivation of methanogenic bacteria have been optimized for two members of the genus Methanococcus. Methanococcus maripaludis and Methanococcus voltae were cultivated on aerobically and anaerobically prepared agar media. Maintenance of O₂ levels below 5 μl/liter within an anaerobic glove box was necessary during plating and incubation for 90% recovery of plated cells. Under an atmosphere of H₂, CO₂, and H₂S (79:20:1), 2 to 3 days of incubation at 37°C were sufficient for the formation of visible colonies. The viability of plated cells was significantly affected by the growth phase of the culture, H₂S concentration, and the volume of medium per plate. In addition, colony size of methanococci was affected by agar type, as well as by the concentrations of agar, H₂S, and bicarbonate.     

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.