MorA defines a new class of regulators affecting flagellar development and biofilm formation in diverse Pseudomonas species

Abstract

Assembly of bacterial flagella is developmentally important during both planktonic cell growth and biofilm formation. Flagellar biogenesis is complex, requiring coordinated expression of over 40 genes, and normally commences during the log-to-stationary transition phase. We describe here a novel membrane-localized regulator, MorA, that controls the timing of flagellar development and affects motility, chemotaxis, and biofilm formation in Pseudomonas putida. MorA is conserved among diverse Pseudomonas species, and homologues are present in all Pseudomonas genomes sequenced thus far. In P. putida, the absence of MorA derepresses flagellar development, which leads to constitutive formation of flagella in the mutant cells in all growth phases. In Pseudomonas aeruginosa, the absence of MorA led to a reduction in biofilm formation. However, unlike the motility of P. putida, the motility of the P. aeruginosa mutants was unaffected. Our data illustrate a novel developmentally regulated sensory and signaling pathway for several properties required for virulence and ecological fitness of Pseudomonas species.

FIG. 1.

morA_Pp mutant exhibits enhanced swimming motility. The swimming motility of the wild type (WT) and the morA_Pp mutant in semisolid agar (0.4% [wt/vol] agar) was examined. The morA_Pp gene driven by its native promoter was expressed in the wild-type and morA_Pp mutant strains in trans on a broad-host-range plasmid (pGB1morA). The wild-type strain was P. putida parental strain PNL-MK25. The results are based on three independent experiments, each with five replicates.

FIG. 2.

morA_Pp in P. putida PNL-MK25 enhances swimming motility by regulating the timing of flagellar development. (A) Proportion of flagellated cells expressed as a percentage of the total number of wild-type (WT) and morA_Pp mutant cells counted by TEM. Counts were based on an average of 400 cells. The wild-type strain was P. putida parental strain PNL-MK25. (B) TEM of wild-type and morA_Pp mutant cells at the early log and log-to-stationary transition growth stages. Magnification, ×7,200.

FIG. 3.

morA affects biofilm formation in P. putida PNL-MK25 and P. aeruginosa PAO1. (A) Plasmids pGB1 and pGB1morA were introduced into both the wild-type strain (WT) and the morA mutant (morA_Pp::aacCI mutant strain). Biofilms that formed at 3 and 10 h after inoculation in polystyrene tubes were stained with 0.1% crystal violet. The wild-type strain was P. putida parental strain PNL-MK25. (B) Plasmids pUCP19 and pUPMR were introduced into both the wild-type strain and the morA mutant (morA_Pa::aacCI mutant strain), and biofilms that formed at 3 and 10 h after inoculation were detected as described above. The wild-type strain (WT) was P. aeruginosa parental strain PAO1. (C) Adherent cells in the biofilms formed on polystyrene surfaces at 3 and 10 h after inoculation were stained with crystal violet and examined by transmitted light microscopy (magnification, ×630). WT, wild type.

FIG. 4.

Expression of flagellin (fliC) is deregulated in the morA mutant strains. (A) Northern analysis of P. putida and P. aeruginosa wild-type and morA mutant cells harvested at the early log phase (OD₆₀₀, 0.3) (lanes E), the mid-log phase (OD₆₀₀, 1.0) (lanes M), and the log-to-stationary transition phase (OD₆₀₀, 1.7) (lanes T) with digoxigenin-labeled P. putida and P. aeruginosa fliC probes, respectively. (B) SDS-PAGE analysis of P. putida wild-type and morA_Pp mutant total protein extracted from cells harvested at the early log, mid-log, and log-to-stationary transition phases.

FIG. 5.

Conservation of MorA in Pseudomonas species. (A) Organization of the P. putida PNL-MK25 morA locus. The solid arrows show the directions of the genes. Mutant strains B12H and C3H carried single mTn5-gfp transposon insertions at positions 1453 and 1480 of morA, respectively. The targeted morA_Pp knockout mutant was generated by inserting a gentamicin cassette at the ClaI site located at position 1121. morA_Pp is flanked by a probable ABC transporter and a serine hydroxymethyltransferase gene, glyA. The 1.1-kb region used to probe PNL-MK25 genomic DNA is indicated. (B) Domain architecture of MorA family members in Pseudomonas species. The three conserved regions of the predicted MorA proteins are (i) a transmembrane domain(s) (vertical bars), (ii) sensory PAS and PAC domains, and (iii) catalytic GGDEF (DUF1) and EAL (DUF2) domains. The domains were predicted by using the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de). (C) GGDEF and EAL domains are highly conserved. Alignment of the GGDEF and EAL domains of MorA, Pflu3486, PP0672, and PA4601 was performed by using ClustalW (http://www.ebi.ac.uk/clustalw). The GGDEF and EAL domains are highlighted with black, identical amino acids are highlighted with dark gray, and similar amino acids are highlighted with light gray.

FIG. 6.

Membrane localization of P. putida MorA. Coomassie blue staining (left panel) and Western blot analysis (right panel) were performed with duplicate gels. Membranes were probed with anti-MorA antibody (A) or anti-GFP antibody (B) in cytoplasmic and membrane fractions of the P. putida wild type and morA_Pp mutant. Both strains expressed GFP from plasmid pGB3. Lane M, Bio-Rad Precision Plus protein standards; lane 1, P. putida wild-type cytoplasmic fraction; lane 2, P. putida wild-type membrane fraction; lane 3, P. putida morA_Pp mutant cytoplasmic fraction; lane 4, P. putida morA_Pp mutant membrane fraction. The predicted molecular mass of MorA is 145 kDa. The wild-type strain was P. putida parental strain PNL-MK25.