# Biology and Regulation of Staphylococcusococcal Biofilm
## 4 Activity of the _ica_ Operon Encoded Enzymes
### 6 Slipped-Strand Mispairing or \"Streisinger Slippage\"  
The _icaADBC_ genes and IGR are rich in repetitive -TATTT- motives. Such tandem motives can lead to frameshift mutations through a mechanism called \"Streisinger slippage\",  
Figure 2: Organization and function of the _ica_ gene cluster in staphylococci. The cluster is composed of the _ica_ operon _icaADBC_ and the repressor gene _icaR_, which is inversely oriented to _icaA_. The approximately 160–170 nt long intergenic region (IGR) carries the promoters for _icaR_ and _icaA_ and operator sites. The _icaADBC_ operon encodes all enzymes necessary for PIA/dPNAG biosynthesis. IcaA is a cytoplasmic enzyme which has _N_-acetylglucosaminyltransferase activity using UDP-N-acetylglucosamine as a substrate; its activity is enhanced by IcaD, which acts as a co-enzyme. IcaB is a surface-attached poly-_N_-acetylglucosamine deacetyllase responsible for deacetylation of approximately every fourth _N_-acetylglucosamine molecule; its activity is essential for biofilm function. IcaC is membrane-localized and demonstrates O-succinyltransferase activity in approximately 6% of the dPNAG, rendering PIA/dPNAG anionic. IcaC plays also a role in the elongation of oligo _N_-acetylglucosamines. which can result in insertions, deletions, and duplications [95]. One such mutation was first described within the intergenic region, IGR, of the _ica_ gene cluster of _S. aureus_ SA113 by [PERSON] et al. in 2003 [93]. They identified a 5 bp TATTT deletion at the proposed IcaR operator site of IGR. This deletion affected the binding of IcaR and probably other secondary repressors as well, with the effect that _icaADBC_ was derepressed. This led to the hyperproduction of PIA/dPNAG and consequently the hyper-mucoid phenotype, as illustrated for MN8m in Figure 3. The 5 bp TATTT deletions with the hyper-mucoid phenotype were also observed in _S. aureus_ strains isolated from cystic fibrosis (CF) patients [96]. Mucoid _S. aureus_ strains are present in 8.6% of _S. aureus_-positive CF patients, and quite a high proportion of the strains carried a 5 bp-deletion (\(\Delta\)-TATTT), suggesting that highly mucoid strains might contribute to the severity of the CF disease [97; 98]. The TATT tetranucleotide repeats also play a role in the inactivation of _icaB_ and _icaC_. Since PIA/dPNAG-negative _S. aureus_ strains are frequently isolated from patients in clinics, a question ensued as to how such variants can arise. Beginning with the hyper-mucoid MN8 strain, PIA/dPNAG-negative variants were isolated and analyzed in more detail. All the mutants JB17, JB15, and JB12 were frameshift mutants in _icaB_ and _icaC_, respectively [99]. The JB-mutants suffered either a deletion, such as in JB17 and JB15, or an insertion, such as in JB12, leading each time to a frameshift mutation and the inactivation of the corresponding gene (Figure 3). The slipped-strand mispairing appeared to be reversible and was independent of RecA. The slippage normally occurs during DNA replication and is caused by a DNA polymerase error. This type of deletion event of nucleotides is common in many organisms and can be advantageous when it activates beneficial genes that enhance microbial survival in adverse environments; however, it can also be deleterious when it alters or suppress function of genes relevant to survival [100]. Another _ica_ regulatory mechanism was described in _S. epidermidis_. One of the first findings was an alternating insertion and excision of the insertion sequence element IS256 [101]. Figure 3: Phase variation of staphylococcal PIA/dPNAG expression. _icaADBC_ expression is controlled at different levels. (1) Repressor binding: IcaR binds to the operator site (op) of the intergenic region (IGR) and thus prevents _icaADBC_ transcription by blocking RNA polymerase binding. TcaR and Rob also appear to bind to IGR, but the exact binding needs to be verified. (2) Repetitive -TATT-motives cause deletions and insertions by slipped-strand mispairing during DNA replication.