Perturbation of FliL interferes with Proteus mirabilis swarmer cell gene expression and differentiation - PubMed (original) (raw)
Perturbation of FliL interferes with Proteus mirabilis swarmer cell gene expression and differentiation
Kathleen Cusick et al. J Bacteriol. 2012 Jan.
Abstract
Proteus mirabilis is a dimorphic, motile bacterium often associated with urinary tract infections. Colonization of urinary tract surfaces is aided by swarmer cell differentiation, which is initiated by inhibition of flagellar rotation when the bacteria first contact a surface. Mutations in fliL, encoding a flagellar structural protein with an enigmatic function, result in the inappropriate production of differentiated swarmer cells, called pseudoswarmer cells, under noninducing conditions, indicating involvement of FliL in the surface sensing pathway. In the present study, we compared the fliL transcriptome with that of wild-type swarmer cells and showed that nearly all genes associated with motility (flagellar class II and III genes) and chemotaxis are repressed. In contrast, spontaneous motile revertants of fliL cells that regained motility yet produced differentiated swarmer cells under noninducing conditions transcribed flagellar class II promoters at consistent levels. Expression of umoA (a known regulator of swarmer cells), flgF, and flgI increased significantly in both swarmer and pseudoswarmer cells, as did genes in a degenerate prophage region situated immediately adjacent to the Rcs phosphorelay system. Unlike swarmer cells, pseudoswarmers displayed increased activity, rather than transcription, of the flagellar master regulatory protein, FlhD(4)C(2), and analyses of the fliL parent strain and its motile revertants showed that they result from mutations altering the C-terminal 14 amino acids of FliL. Collectively, the data suggest a functional role for the C terminus of FliL in surface sensing and implicate UmoA as part of the signal relay leading to the master flagellar regulator FlhD(4)C(2), which ultimately controls swarmer cell differentiation.
Figures
Fig 1
Functional categories of genes whose expression significantly increased (A) or decreased (B) in swarmer cells (black bars) or pseudoswarmer cells (hatched bars). Genes were grouped according to their Gene Ontology (GO) categories assigned by GoMiner. Putatively annotated genes for which no GO terms have yet been assigned were categorized based on their COG or Kyoto Encyclopedia of Genes and Genomes (KEGG) ontology pathway (6) designations.
Fig 2
Verification of RNA-Seq gene expression analysis by qRT-PCR. Individual gene expression ratios were calculated using RPKM values generated via RNA-Seq and plotted against calculations done for the same gene using qRT-PCR on both total RNA and mRNA as templates. Each qRT-PCR assay was performed on the sample subject to RNA-Seq plus two independent samples. Pearson correlations of 0.964 (P = 5.5 × 10−7) and 0.898 (P = 7.5 × 10−5) were obtained when mRNA and total RNA served as the template, respectively, in the qRT-PCRs.
Fig 3
Swimming, swarming, and cellular elongation of wild-type and pseudoswarmer cells. Swimming (A) was measured using semisolid Mot agar, and swarming (B) was measured on LB agar. (C to E) Phase-contrast micrographs of BB2000 (C), BB2204 (D), and YL1001 (E) taken after growth in LB broth. (F and G) Determination of the percent elongated cells in the populations of BB2000 (●), BB2204 (■), and YL1001 (▴). (F) Percentage of cells with a length of >7 μm; (G) percentage of cells with a length >3-fold greater than the floating standard (see Materials and Methods). Error bars represent standard deviations (n > 400).
Fig 4
Expression of fliL operon genes and flaA in pseudoswarmer cells. (A) Organization of the fliL operon in BB2000, BB2204, and YL1001. Mini-Tn_5_-Cmr (∼2 kb) disrupts _fliL_2204 at nucleotide 440, and fliL_YL1001 is mutated by a 68-bp scar following excision of the transposon. The fliL promoter is indicated by a bent arrow, and open circles represent transcriptional terminators in the mini-Tn_5 transposon. (B) RT-PCR analysis of transcription of class II (fliL and fliM) and class III (flaA) flagellar genes, relative to rpoA (as a positive referential control). Lanes: 1, BB2000; 2, BB2204; 3, YL1001. RT(+) indicates complete reverse transcriptase PCR, while RT(−) lanes lack reverse transcriptase and serve as a control for DNA contamination.
Fig 5
Characterization of the fliL mutations resulting in the pseudoswarmer phenotype. Nucleotide (top) and deduced amino acid (bottom) sequences of the C terminus of FliL of BB2000 (A), BB2204 (B), and YL1001 (C) are shown. Brackets indicate the mini-Tn_5_-Cmr sequence, and underlining indicates IS elements of the transposon.
Fig 6
Transcription of hallmark genes in fliL pseudoswarmer strains. (A) Expression of the flhD gene (class I), the fliL, fliM, and fliA genes (all class II), and the flgM and flaA genes (both class III). (B) Expression of umoA to umo D genes. Filled bars, BB2204; open bars, YL1001. Expression in each pseudoswarmer strain was compared with the expression of the same gene in wild-type swimmer cells. A >1 change indicates that expression of the gene is greater in the pseudoswarmer cells than in swimmer cells, while a value of <1 means that expression decreases in the pseudoswarmer strain. Error bars indicate standard errors for three independent measurements from three biological samples.
References
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