Detection of NO-synthase activity of lactobacilli by fluorescent staining

Abstract

-argin-ine, and functions as one of the universal regulators ofcell and tissue metabolism. It also functions as the maincytotoxic/cytostatic effector of the cellular immunitysystem. In mammalian cells, NO production is cata-lyzed by the family of NO synthase (NOS) isoenzymes[1]. The process of bacterial NO synthesis, in contrastto the eukaryotic one, is still far from being fully under-stood. An NO synthase mechanism of nitric-oxide pro-duction alternative to denitrification was initiallyrevealed in the microorganisms of the genus Nocardia[2]. Later, genes that encode proteins homologous tothe mammalian NOS oxygenase domain were identi-fied in the genomes of a number of gram-positive bac-teria [3]. As yet, there are few works on the isolation,purification, and study of the properties of bacterial NOsynthases [4–6].NO synthase is supposed to be present in Lactoba-cillus plantarum [7]. Previously, we have applied EPR(electron paramagnetic resonance) to establish thecapability of L. plantarum 8P-A3 to synthesize nitricoxide. This strain was shown not to produce NO viadenitrification but to have a NO-synthase activity, likemammalian cells [8].This work presents the first evidence of NO-syn-thase nitric-oxide production in L. plantarum obtainedby the method of fluorescent staining.The culture of L. plantarum 8P-A3 [8] isolated fromthe Lactobacterin Dried preparation (Biomed, Russia)was a test subject. MRS medium of known composition[8] was used for bacterial growth. One ml of 18-h cul-ture was added into 50-ml test tubes containing 30 mlof the medium with or without L-arginine (100 µM)and incubated without mixing at 37°e. The growthkinetics was determined at 590 nm on a Lambda 35double-beam spectrophotometer, Perkin Elmer Instru-ments (USA). For fluorescent detection of nitric oxide,30 ml of the culture from the stationary growth phase(48 h) was precipitated by centrifugation at 4000 g for20 min in an Avanti