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SadB Is Required for the Transition from Reversible to Irreversible Attachment during Biofilm Formation by Pseudomonas aeruginosa PA14. Nicky C. Caiazza, 2004.Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state . Here we report a new class of P . aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation . The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P . aeruginosa PA14 and encodes a protein of unknown function . Complementation analysis and phage-mediated transduction demonstrated that the transposon insertion in PA5346 was the cause of the biofilm-defective phenotype . Examination of flow cell-grown biofilms showed that the sadB199 mutant could initiate surface attachment but failed to form microcolonies despite being proficient in both twitching and swimming motility . Closer examination of early attachment revealed an increased number of the sadB199 mutant cells arrested at reversible attachment, functionally defined as adherence via the cell pole . A positive correlation among biofilm formation, irreversible attachment, and SadB level was demonstrated, and furthermore, RpoN and FleR appear to negatively affect SadB levels . Fractionation studies showed that the SadB protein is localized to the cytoplasm, and with the use of GPS-linker scanning mutagenesis, the C-terminal portion of SadB was shown to be dispensable for function, whereas the two putative domains of unknown function and the linker region spanning these domains were required for function . We discuss the results presented here in the context of microbial development as it applies to biofilm formation . Two-Component Signal Transduction in Enterococcus faecalis. Lynn Hancock, 2002. Firm but Slippery Attachment of Deinococcus geothermalis. M. Kolari, 2002.Bacterial biofilms impair the operation of many industrial processes . Deinococcus geothermalis is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria . It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known . High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of D . geothermalis E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium . Extensive slime matrix was absent from the D . geothermalis E50051 biofilms . AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed . We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible . In spite of this, AFM imaging of living D . geothermalis E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells . The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface . Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water . Biofilms were evaluated for their strength of attachment . D . geothermalis E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of Burkholderia cepacia F28L1 or the well-characterized biofilm former Staphylococcus epidermidis O-47 . Deinococcus radiodurans strain DSM 20539T also formed tenacious biofilms . This paper shows that D . geothermalis has firm but laterally slippery attachment not reported before for a nonmotile species . Putative Interhelical Interactions within the PheP Protein Revealed by Second-Site Suppressor Analysis. C. Dogovski, 2003.Highly conserved glycine residues within span I and span II of the phenylalanine and tyrosine transporter PheP were shown to be important for the function of the wild-type protein . Replacement by amino acids with increasing side chain volume led to progressive loss of transport activity . Second-site suppression studies performed with a number of the primary mutants revealed a tight packing arrangement between spans I and II that is important for function and an additional interaction between spans I and III . We also postulate that a third motif, GXXIG, present in span I and highly conserved within different members of the amino acid-polyamine-organocation family, may function as a dimerization motif . Surprisingly, other highly conserved residues, such as Y60 and L41, could be replaced by various residues with no apparent loss of activity .
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