Isolation and Characterization of Biofilm Formation-Defective Mutants of Staphylococcus aureus

Abstract

Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY, srrA, mgrA, and fmtA, a putative DEAD-box helicase, two members of the zinc-metallo-␤ lactamase/␤-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis. Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non-ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non-ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.

DEAD-Box RNA Helicases in Gram-Positive RNA Decay

“…This was first noticed by Tu Quoc et al (2007) who observed a reduced capacity for producing biofilm in a cshA mutant of the S. aureus S30 strain, which is normally a strong biofilm producer. Moreover, cshA mutants exhibit stabilization of their agrBDCA mRNA, which is responsible for quorum sensing in S. aureus (Oun et al, in preparation).…”

“…In Bacillus cereus, RNA helicases from the DEAD-box family were identified by mutagenesis to be required for survival at low temperatures, pH, and oxidative stress (Pandiani et al, 2010). Similarly, Tn916 mutagenesis identified an RNA helicase from the Gram-positive Clostridium perfringens to be involved in the adaptive response to oxidative stress (Briolat and Reysset, 2002), and random transposon mutagenesis identified a RNA helicase involved in the regulation of phenolic acid metabolism in Lactobacillus plantarum (Gury et al, 2004) and a RNA helicase (CshA) in S. aureus involved in biofilm formation (Tu Quoc et al, 2007). So far, however, their molecular function remains mostly elusive.…”

Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

“…CodY is activated through direct interaction with the BCAAs as well as GTP (18 -20, 23) and, in its active state, represses transcription of upwards of 100 genes (17,24,25). Depletion of BCAAs and GTP results in derepression of CodY target genes, the products of which are involved in a range of cellular processes depending on the species, including sporulation (23), biofilm formation (26,27), protein degradation and utilization (22), and amino acid metabolism and transport (17,24). More recently, CodY has emerged as a regulator of virulence in S. aureus (28)(29)(30), implicating CodY as an important link between the nutrient status of the cell and virulence.…”

Staphylococcus epidermidis SrrAB Regulates Bacterial Growth and Biofilm Formation Differently under Oxic and Microaerobic Conditions

“…6). In the S. aureus srrA transposon mutant, PIA synthesis was increased, whereas biofilm formation was decreased (23). This suggests that the mechanisms of biofilm formation regulated by SrrAB in S. epidermidis differ from those of S. aureus.…”

“…), suggesting a role in the retarded growth of S. aureus under anaerobic conditions (19). In addition, a transposon mutation in srrA resulted in reduction of biofilm formation in S. aureus, although PIA production was increased, suggesting that in S. aureus srrAB affects biofilm formation via an ica-independent pathway (23).…”