Characterization of Proteus mirabilis precocious swarming mutants: identification of rsbA, encoding a regulator of swarming behavior

Abstract

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.

FIG. 1

Swarming behavior of the precocious phenotype. The swarming behaviors of the wild-type parent (BB2000) (A) and a Tn5-generated precocious mutant (BB2235) (B) were compared. Cells were grown overnight in L broth and washed in 1 × PBS, and a 5-μl spot of inoculum was added to the center of the agar. The plates were incubated at 37°C and observed after 4 h. Precocious swarming behavior is characterized by a decreased initial lag period prior to swarming onset, resulting in a larger swarming colony compared to that of the parent.

FIG. 2

Swarming velocity of precocious mutants during cyclic phases of swarmer cell differentiation and behavior. The swarming behaviors of a precocious swarming mutant (BB2235) and wild-type cells were characterized through two cycles of migration and consolidation by measuring the translocation distance at the swarming-colony edge at 30-min intervals according to the procedure of Gygi et al. (33). Comparison of velocity between wild-type and precocious swarming cells indicates that there are no defects in speed but that the precocious mutants start swarming much sooner (at least 30 min) than the wild type. Error bars, standard errors of the means (n = 5).

FIG. 3

Phenotype of precocious mutants. In addition to an early onset of swarming behavior, precocious mutants show other phenotypic differences from the wild type, including retention of the differentiated swarmer cell morphology when grown under noninducing conditions, swarming movement on minimal agar medium, and non-wild-type chemotaxis patterns. The upper panels compare the cell morphology of the wild-type cells (A) and the precocious swarming mutant BB2235 (B) after 8 h of growth in L broth at 37°C. Precocious mutants possess the characteristics of swarmer cells (elongated, polyploid, nonseptate cells with numerous flagella) when they are in liquid medium (a noninducing environment), while the wild-type cells remain undifferentiated swimmer cells. The wild type does not swarm on minimal glycerol medium (C), but after 48 h of incubation at 37°C, the precocious mutants show signs of swarming behavior (D). The lower panels compare the chemotactic behavior (a swimmer cell phenotype) in relation to ribose. The wild-type cells (E) form a tighter and more discrete series of chemotactic rings than the precocious mutants (F). This difference may be due to a higher percentage of swarmer cells in the precocious mutant population compared to the wild type when P. mirabilis is grown in the semisolid Mot agar.

FIG. 4

Location of the Tn5 insertion on the P. mirabilis chromosome. The insertion point of the mini-Tn5–Cm transposon in each of the six precocious mutants (B) was determined by using either IPCR cloning and subsequent nucleotide sequence analysis (BB2235), cloning of Cm^r fragments from precocious mutant chromosomal DNA (BB2231 and BB2233), Southern blot analysis (A) with probes to sites within the region, or PCR amplification of segments of the region adjacent to the Tn5 insertions (16). The oligonucleotides used to locate each transposon insertion point are listed in Table 1. The transposon insertions clustered in two loci. Precocious mutants BB2231, BB2232, and BB2235 were found to have Tn5 insertions in rsbA, while BB2233, BB2234, and BB2236 were located in the 3′ end of rcsC. The nucleotide sequence of 5,458 bp encoding rsbA, rcsB, and the 3′ portion of rcsC has been submitted to the DDBJ/EMBL/GenBank databases under accession no. AF071215.

FIG. 5

Alignment of RsbA domains with those of related bacterial sensor proteins. Underlined spaces indicate gaps introduced to optimize alignments. Amino acids that are shared among all five proteins are shown in white on a solid background, and those shared by 3 or 4 of the proteins are shaded. Boldface letters indicate alignment of the conserved histidine and aspartate residues. BLASTP analysis P values for the overall alignments of these four proteins with RsbA were 1 × 10⁻¹²⁵ for E. coli YojN (29% identity, 50% similarity), 1 × 10⁻¹⁸ for E. coli ResC (19% identity, 39% similarity), 6 × 10⁻¹⁷ for V. harveyi LuxQ (22% identity, 41% similarity), and 3 × 10⁻¹² for E. coli EvgS (22% identity, 41% similarity).

FIG. 6

Molecular analysis of the rsbA mutation producing the precocious phenotype. Based on analysis of Tn5::2235, a truncated rsbA gene (nt 1117 to 3076) containing a Cm^r gene cassette (′rsbA::cam::rsbA) inserted at the HpaI site (nt 2121) was used to construct a precocious swarming mutant via allelic-exchange mutagenesis. Selection for chloramphenicol-resistant colonies was followed by a screen for plasmid carriage (Ap^r or Ap^s) and the precocious swarming phenotype. Two types of mutations were observed. The first (A) involved a single crossover (Campbell integration) that resulted in a merodiploid containing a cam gene inserted in the whole rsbA gene, the plasmid, and a truncated copy of rsbA. These mutants had the precocious swarming phenotype. The second type of single crossover (B) resulted in a wild-type copy of rsbA, the plasmid, and the truncated rsbA plus cam gene insert. These colonies were wild type for swarming.

FIG. 7

Initiation of P. mirabilis swarming behavior is density dependent. (A) Swarming onset compared to the density of the inoculum. (B) Swarming onset when a constant density of cells is maintained but the ratio of living to UV-killed cells varies. (C) Swarming onset when the ratio of P. mirabilis to E. coli cells changes but the total cell density remains constant. The horizontal axis shows the number of P. mirabilis cells in the inoculum as CFU. Error bars, standard errors of the means (n = 4).

FIG. 8

Precocious swarming mutants retain density dependence. The density-dependent initiation of swarming behavior in wild-type and precocious swarming cells was compared. The initial inoculum concentration was varied in 10-fold increments from 2 × 10⁶ to 2 × 10² cells (CFU) delivered in 5-μl-aliquot droplets to the L-agar surface. The onset time of swarming behavior was then recorded. Error bars, standard errors of the mean (n = 4).

FIG. 9

Overexpression of RsbA and complementation of precocious mutants. pMM309, a multicopy plasmid (pCR2.1) with an intact rsbA gene, was introduced into wild-type cells and precocious mutant MM100. Swarming behavior was then observed by measuring the time of initiation of swarming migration on L agar. A 5-μl aliquot containing 2 × 10⁶ cells was used as an inoculum. Error bars, standard errors of the means (n = 4).

FIG. 10

Membrane topology of native RsbA and of the mutant RsbA producing precocious swarming. RsbA has amino acid motifs indicative of membrane spanning sensory proteins. Computer-assisted motif searches (37) suggest a possible topology for RsbA that separates an N-terminal regulatory domain from a C-terminal transmitter domain. This model also predicts that the mutation in MM100 removes the N-terminal portion of the protein, thus eliminating the regulatory functions while retaining the transmitter domain.