Pyrogallol impairs staphylococcal biofilm formation via induction of bacterial oxidative stress.

AIMS: To examine the effect of the phenolic compound pyrogallol on staphylococcal biofilm formation. METHODS AND RESULTS: In crystal violet biofilm assays, pyrogallol-reduced biofilm formation in Staphylococcus epidermidis ATCC 35984, Staph. epidermidis NRRL-B41021, Staphylococcus aureus USA300, and Staph. aureus Newman, without significantly impairing bacterial viability. Pyrogallol-mediated impairment of biofilm formation was likely due to induction of bacterial oxidative stress, as its effect was greater in catalase-deficient versus WT Staph. aureus, and biofilm production was rescued by exogenous catalase. The effect of pyrogallol on staphylococcal biofilm formation mirrored that of the known oxidant hydrogen peroxide, which also reduced biofilm formation in a dose-dependent manner. CONCLUSIONS: Pyrogallol reduces biofilm formation in S. aureus and Staph. epidermidis in a mechanism involving induction of bacterial oxidative stress.

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System.

In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing.IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. In contrast, we show that targeted degradation and acquisition of new immunity elements require highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets.

Involvement of mismatch repair in the reciprocal control of motility and adherence of uropathogenic Escherichia coli.

Type 1 fimbriae and flagella, two surface organelles critical for colonization of the urinary tract by uropathogenic Escherichia coli (UPEC), mediate opposing virulence objectives. Type 1 fimbriae facilitate adhesion to mucosal cells and promote bacterial persistence in the urinary tract, while flagella propel bacteria through urine and along mucous layers during ascension to the upper urinary tract. Using a transposon screen of the E. coli CFT073 fim locked-ON (L-ON) mutant, a construct that constitutively expresses type 1 fimbriae and represses motility, we identified six mutants that exhibited a partial restoration of motility. Among these six mutated genes was mutS, which encodes a component of the methyl-directed mismatch repair (MMR) system. When complemented with mutS in trans, motility was again repressed. To determine whether the MMR system, in general, is involved in this reciprocal control, we characterized the effects of gene deletions of other MMR components on UPEC motility. Isogenic deletions of mutS, mutH, and mutL were constructed in both wild-type CFT073 and fim L-ON backgrounds. All MMR mutants showed an increase in motility in the wild-type background, and ΔmutH and ΔmutS mutations increased motility in the fim L-ON background. Cochallenge of the wild-type strain with an MMR-defective strain showed a subtle but significant competitive advantage in the bladder and spleen for the MMR mutant using the murine model of ascending urinary tract infection after 48 h. Our findings demonstrate that the MMR system generally affects the reciprocal regulation of motility and adherence and thus could contribute to UPEC pathogenesis during urinary tract infections.