[6] Genetic approaches to study of biofilms

O’Toole, George A.; Pratt, Leslie A.; Watnick, Paula I.; Newman, Dianne K.; Weaver, Valerie B.; Kolter, Roberto

doi:10.1016/s0076-6879(99)10008-9

Cited by 772 publications

(644 citation statements)

References 36 publications

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“…The wells were washed three times with water and air-dried. The dye was solubilized with 80 % ethanol and A 550 was measured (O'Toole et al, 1999). SDS sensitivity test.…”

Section: Methodsmentioning

confidence: 99%

Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis

et al. 2010

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Pathogenic strains of mycobacteria produce copious amounts of glutamine synthetase (GS) in the culture medium. The enzyme activity is linked to synthesis of poly-a-L-glutamine (PLG) in the cell walls. This study describes a glnA-1 mutant of Mycobacterium bovis that produces reduced levels of GS. The mutant was able to grow in enriched 7H9 medium without glutamine supplementation. The glnA-1 strain contained no detectable PLG in the cell walls and showed marked sensitivity to different chemical and physical stresses such as lysozyme, SDS and sonication. The sensitivity of the mutant to two antitubercular drugs, rifampicin and D-cycloserine, was also increased. The glnA-1 strain infected THP-1 cells with reduced efficiency and was also attenuated for growth in macrophages. A Mycobacterium smegmatis strain containing the M. bovis glnA-1 gene survived longer in THP-1 cells than the wild-type strain and also produced cell wall-associated PLG. The M. bovis mutant was not able to replicate in the organs of BALB/c mice and was cleared within 4-6 weeks of infection. Disruption of the glnA-1 gene adversely affected biofilm formation on polystyrene surfaces. The results of this study demonstrate that the absence of glnA-1 not only attenuates the pathogen but also affects cell surface properties by altering the cell wall chemistry of the organism via the synthesis of PLG; this may be a target for drug development.

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“…The wells were washed three times with water and air-dried. The dye was solubilized with 80 % ethanol and A 550 was measured (O'Toole et al, 1999). SDS sensitivity test.…”

Section: Methodsmentioning

confidence: 99%

Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis

et al. 2010

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“…To assess biofilm formation during static conditions, an adaptation of the 96-well microtitre dish biofilm assay developed by O'Toole and co-workers (O'Toole & Kolter, 1998;O'Toole et al, 1999) based on viable counts was performed. An overnight culture of P. mirabilis HI4320 and the Dpst mutants was diluted 1 : 40 into fresh LB and allowed to grow at 37 uC with shaking for 2 h. Pooled human urine was inoculated with 25 ml of each 2 h culture into polystyrene six-well plates (Becton Dickinson) in triplicate.…”

Section: Methodsmentioning

confidence: 99%

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

et al. 2009

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Proteus mirabilis causes urinary tract infections (UTIs) in individuals requiring long-term indwelling catheterization. The pathogenesis of this uropathogen is mediated by a number of virulence factors and the formation of crystalline biofilms. In addition, micro-organisms have evolved complex systems for the acquisition of nutrients, including the phosphate-specific transport system, which has been shown to be important in biofilm formation and pathogenesis. A functional Pst system is important during UTIs caused by P. mirabilis HI4320, since transposon mutants in the PstS periplasmic binding protein and the PstA permease protein were attenuated in the CBA mouse model of UTI. These mutants displayed a defect in biofilm formation when grown in human urine. This study focuses on a comparison of the proteomes during biofilm and planktonic growth in phosphate-rich medium and human urine, and microscopic investigations of biofilms formed by the pst mutants. Our data suggest that (i) the Dpst mutants, and particularly the DpstS mutant, are defective in biofilm formation, and (ii) the proteomes of these mutants differ significantly from that of the wild-type. Therefore, since the Pst system of P. mirabilis HI4320 negatively regulates biofilm formation, this system is important for the pathogenesis of these organisms during complicated UTIs. INTRODUCTIONProteus mirabilis -a member of the family Enterobacteriaceae -is the most common aetiological agent responsible for complicated urinary tract infections (UTIs) (Mobley, 1996). Subpopulations at higher risk for infection by this pathogen include those with long-term indwelling catheterization as well as those with structural and functional abnormalities within the urinary tract (Mobley, 1996). Clinical syndromes associated with P. mirabilis include cystitis and pyelonephritis, with possible complications from stone formation and bacteraemia (Mobley, 1996). These micro-organisms survive in the urinary tract by virtue of their production of a battery of virulence factors, including urease, flagella, fimbriae, haemolysin and IgA protease (Belas, 1996;Mobley et al., 1994;Musher et al., 1975;Peerbooms et al., 1984; Walker et al., 1999), and formation of crystalline biofilm on indwelling catheters (Stickler et al., 1993).Biofilm formation -one of the most important mechanisms of pathogenicity of this micro-organism in the urinary tract -may be defined as a community of surface-attached bacteria encased in an extracellular matrix consisting of secreted carbohydrates, proteins and DNA. Indeed, biofilms have been associated with the virulence of a number of pathogens (Castelli et al., 2006). Organisms in these communities display phenotypic differences from planktonic cells, including a slower rate of growth and increased resistance to antibiotics (Lewis, 2001;Stoodley et al., 2002). P. mirabilis biofilms in urine are unique in that the extracellular polysaccharide matrix is enmeshed with struvite and carbonate apatite crystals (Jones et al., 2006).Abbreviations: CLSM, confocal ...

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“…Motility plates to detect swimming, swarming, and twitching were prepared as described (12). Biofilms were grown statically in polypropylene tubes for 2 days at 32°C in Hepes-buffered succinate medium, after which growth was measured and the resulting biofilms were visualized by staining with crystal violet (13).…”

Section: Methodsmentioning

confidence: 99%

Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis

Ochsner

Snyder

Vasil

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

189

226

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A novel secretion pathway originally found in plants has recently been discovered in bacteria and termed TAT, for ''twin-arginine translocation,'' with respect to the presence of an Arg-Arg motif in the signal sequence of TAT-secreted products. However, it is unknown whether the TAT system contributes in any way to virulence through the secretion of factors associated with pathogenesis or stress response. We found that the opportunistic pathogen Pseudomonas aeruginosa produces several virulence factors that depend on the TAT system for proper export to the periplasm, outer membrane, or extracellular milieu. We identified at least 18 TAT substrates of P. aeruginosa and characterized the pleiotropic phenotypes of a tatC deletion mutant. The TAT system proved essential for the export of phospholipases, proteins involved in pyoverdine-mediated ironuptake, anaerobic respiration, osmotic stress defense, motility, and biofilm formation. Because all these traits have been associated with virulence, we studied the role of TAT in a rat lung model. A tatC mutant did not cause the typical multifocal pulmonary abscesses and did not evoke a heavy inflammatory host response compared with wild type, indicating that tatC mutant cells are attenuated for virulence. Because the TAT apparatus is well conserved among important bacterial pathogens yet absent in mammalian cells, it represents a potential target for novel antimicrobial compounds.

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[6] Genetic approaches to study of biofilms

Cited by 772 publications

References 36 publications

Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis

Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis

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