The Antimicrobial Activity of Marinocine, Synthesized by Marinomonas mediterranea , Is Due to Hydrogen Peroxide Generated by Its Lysine Oxidase Activity

Abstract

Marinocine is a broad-spectrum antibacterial protein synthesized by the melanogenic marine bacterium Marinomonas mediterranea. This work describes the basis for the antibacterial activity of marinocine and the identification of the gene coding for this protein. The antibacterial activity is inhibited under anaerobic conditions and by the presence of catalase under aerobic conditions. Marinocine is active only in culture media containing L-lysine. In the presence of this amino acid, marinocine generates hydrogen peroxide, which causes cell death as confirmed by the increased sensitivity to marinocine of Escherichia coli strains mutated in catalase activity. The gene coding for this novel enzyme was cloned using degenerate PCR with primers designed based on conserved regions in the antimicrobial protein AlpP, synthesized by Pseudoalteromonas tunicata, and some hypothetical proteins. The gene coding for marinocine has been named lodA, standing for lysine oxidase, and it seems to form part of an operon with a second gene, lodB, that codes for a putative dehydrogenase flavoprotein. The identity of marinocine as LodA has been demonstrated by N-terminal sequencing of purified marinocine and generation of lodA mutants that lose their antimicrobial activity. This is the first report on a bacterial lysine oxidase activity and the first time that a gene encoding this activity has been cloned.

Hydrogen Peroxide Linked to Lysine Oxidase Activity Facilitates Biofilm Differentiation and Dispersal in Several Gram-Negative Bacteria

“…1). Such a mode of action was first discovered for the AlpP homologue LodA produced by M. mediterranea (31). Interestingly, our results show that the substrate lysine is produced endogenously by the biofilm bacteria and consequently allows for AlpP/LodA activity.…”

“…Sucrose-and streptomycin-resistant colonies were screened for kanamycin sensitivity, and gene insertion confirmed by PCR with the primers CAUDIR4-CAUREV5 (which gives a 670-bp fragment in wild-type and first recombination mutants maintaining locus NP_419374) and CAUDIR3-CAU-REV5 (which gives a 2.3-kb fragment in WT and a 2.1-kb fragment in CAU-MUR1 as a result of the substitution of locus NP_419374 with the smaller streptomycin resistance marker). Deletion of the C. violaceum alpP/lodA homologue was achieved by using a previously described method (31). Briefly, a 500-bp region of the gene containing the fourth and fifth conserved domains, out of a total of eight in the homologous proteins, was amplified by PCR using the primers CVDIR2nco and CVREV1 (Table 2).…”

SO-LAAO, a Novell-Amino Acid Oxidase That EnablesStreptococcus oligofermentansTo OutcompeteStreptococcus mutansby Generating H2O2from Peptone

“…So far, all the described LAAOs, except the lysine oxidase of Marinomonas mediterranea (21), are flavoproteins possessing a FAD binding domain (37). It is reported that LAAOs possess another characteristic sequence motif, RxGGR, and this motif is also present in several families of flavoproteins, including achacin and aplysianin A, monoamine oxidase, corticosteroidbinding proteins, and tryptophan 2-monooxygenases (37).…”

“…LAAOs, which catalyze the oxidative deamination of amino acids to yield ammonia, hydrogen peroxide, and ketoacids with oxygen consumed (9), have been widely detected in snake and insect venoms (1,30) and in some fungi, algae, and bacteria (17,25,38). According to the substrate spectra, LAAOs can be divided into two categories, one with a broad spectrum of substrates, like the LAAO of Rhodococcus opacus DSM 43250, which catalyzes not only almost all the 20 L-amino acids but also some derivatives (9), and another with a restricted substrate spectrum, like lysine oxidase of Marinomonas mediterranea, which uses lysine exclusively (21). However, SO-LAAO of S. oligofermentans in this study can represent a novel amino acid oxidase by showing detectable activity against only seven L-amino acids (Table 1).…”

Regulation of the Marinomonas mediterranea Antimicrobial Protein Lysine Oxidase by l -Lysine and the Sensor Histidine Kinase PpoS

“…It synthesizes an antimicrobial protein that was initially named marinocine and was later named LodA (24,25). LodA is a novel enzyme (EC 1.4.3.20) very specific for L-Lys that catalyzes the reaction L-lysine ϩ O 2 ϩ H 2 O32-aminoadipate 6-semialdehyde ϩ NH 3 ϩ H 2 O 2 .…”