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.