Genome-Wide Identification of Genes Necessary for Biofilm Formation by Nosocomial Pathogen Stenotrophomonas maltophilia Reveals that Orphan Response Regulator FsnR Is a Critical Modulator

Abstract

Stenotrophomonas maltophilia is a Gram-negative bacterial pathogen of increasing concern to human health. Most clinical isolates of S. maltophilia efficiently form biofilms on biotic and abiotic surfaces, making this bacterium resistant to a number of antibiotic treatments and therefore difficult to eliminate. To date, very few studies have investigated the molecular and regulatory mechanisms responsible for S. maltophilia biofilm formation. Here we constructed a random transposon insertion mutant library of S. maltophilia ATCC 13637 and screened 14,028 clones. A total of 46 nonredundant genes were identified. Mutants of these genes exhibited marked changes in biofilm formation, suggesting that multiple physiological pathways, including extracellular polysaccharide production, purine synthesis, transportation, and peptide and lipid synthesis, are involved in bacterial cell aggregation. Of these genes, 20 putatively contributed to flagellar biosynthesis, indicating a critical role for cell motility in S. maltophilia biofilm formation. Genetic and biochemical evidence demonstrated that an orphan response regulator, FsnR, activated transcription of at least two flagellum-associated operons by directly binding to their promoters. This regulatory protein plays a fundamental role in controlling flagellar assembly, cell motility, and biofilm formation. These results provide a genetic basis to systematically study biofilm formation of S. maltophilia.

Systematic Mutational Analysis of Histidine Kinase Genes in the Nosocomial Pathogen Stenotrophomonas maltophilia Identifies BfmAK System Control of Biofilm Development

“…The biofilm-to-growth ratios of nine mutants (the ⌬Smlt0158, ⌬Smlt0595, ⌬Smlt0882, ⌬Smlt1421, ⌬Smlt1636, ⌬Smlt1785, ⌬Smlt2380, ⌬Smlt2710, and ⌬Smlt3944 mutants) were significantly increased, whereas the other 38 mutants, including the ⌬Smlt0278 (phoQ), ⌬Smlt2324 (ravS), and ⌬Smlt4106 (hpaS) mutants, exhibited decreased biofilm-togrowth ratios. Of these, the Smlt2324 (ravS) mutation led to a decrease in biofilm that was also observed in our previous study using random, large-scale transposon mutagenesis (17). It is interesting to note that, although insertional inactivation of the rpfC ortholog (Smlt2234) resulted in a significant decrease in the biofilm-to-growth ratio (Fig.…”

“…The insertional inactivation mutants (by homologous, single-crossover methodology) and in-frame deletion (by homologous, double-crossover methodology) S. maltophilia mutants were constructed using the suicide vectors pK18mob and pK18mobsacB, respectively, as previously reported (17,18). The primers used to amplify the relevant DNA sequences are listed in Table 2.…”

“…For the bacterial swimming motility assays, the strains were inoculated into semisolid 0.1% NYG agar with a toothpick and cultured for 12 h at 28°C, and the diameters of the swimming zones were measured. For biofilm quantification, the crystal violet staining method used was reported previously (17). S. maltophilia cultures were inoculated quantitatively into NYG medium within 96-well polystyrene plates, and the plates were incubated at 28°C for 5 h without shaking.…”

“…Indeed, only HK RpfC, which is responsible for regulating cell-cell communication and virulence factor expression in the close-relative bacteria Xanthomonas spp., has been shown to control diffusible signaling factor (DSF)-triggered modulation in S. maltophilia (14)(15)(16). Our previous study identified FsnR, an orphan RR that controls transcription of flagellar genes and biofilm formation (17), but the cognate HK for FsnR remains unclear.…”

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

“…Biofilm formation may be an important adaptation enabling the dominance of Halanaerobium across hydraulically fractured shales. Although biofilm-related genes are not detected in all surface Halanaerobium genomes 19 , these shale genomes encode genes for flagellar motility and cellular aggregation (for example, polysaccharide production and diguanylate cyclase) 20 (Supplementary Table 7). Biofilm, organic acid and H 2 production, together with the capacity to reduce thiosulfate to sulfide (using three copies of the rhodanese-like thiosulfate:cyanide sulfur-transferase gene 3 ), implicate a role for shale Halanaerobium in steel corrosion and reservoir souring 21 .…”