Reaction of acylated homoserine lactone bacterial signaling molecules with oxidized halogen antimicrobials.

Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.

Degradation pathway of homoserine lactone bacterial signal molecules by halogen antimicrobials identified by liquid chromatography with photodiode array and mass spectrometric detection.

The degradation pathway of acylated homoserine lactone bacterial signaling molecules by oxidizing hypochlorite and stabilized hypobromite antimicrobials has been characterized. A reversed-phase HPLC separation using a cyano column was developed to detect the parent lactones, lactone-hydrolysis products, and halogenation products. Elucidation of the structures of the reaction products was done with the aid of online photodiode array UV spectroscopy and atmospheric pressure chemical ionization mass spectrometry. Quantitative output of the HPLC method was also used to estimate the kinetics of the degradation pathway. The results of this work found that only beta-keto-amide signal molecules are halogenated, where normal amide signals are not, and may represent one possible mechanism for control of industrial biofilms.

Sequence analysis of the region downstream from a peptidoglycan hydrolase-encoding gene from Staphylococcus aureus NCTC8325.

The nucleotide (nt) sequence of a 4.7-kb DNA fragment downstream from a peptidoglycan hydrolase-encoding gene (lytA) from Staphylococcus aureus NCTC8325 was determined. Sequencing revealed three open reading frames (ORFs) of 513, 447 and 879 bp with consensus ribosome-binding sites located upstream from the ATG start codons. Results from in vitro transcription-translation analysis and maxicell experiments suggested that the 447-bp ORF was the one being actively expressed. Comparison of the amino acid (aa) sequences of the ORFs with the aa sequences in the NCBI Entrez database (Release 4.0, April 1993) did not show any significant homology to any sequenced polypeptides. However, nt sequences downstream from lytA showed perfect homology to the bacteriophage phi 11 attachment site (attP) and integration site (attB), and significant homology to downstream regions of the staphylokinase (sak) and exfoliative toxin A (eta) genes of S. aureus.