Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with ⌬PGM mutant cells reaching sizes ϳ3-fold larger than those of the wild type (WT). The ⌬PGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the ⌬PGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the ⌬PGM mutant. In analysis of the aborted infectious process, we found that ⌬PGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the ⌬PGM mutant and later challenged with a lethal dose of WT S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae ⌬PGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.With increased development of intensive operations, disease has become a significant hurdle to the profitable culture of fish and shellfish. Streptococcal infections in fish, in particular those produced by the pathogen Streptococcus iniae, have increased markedly with intensification of aquaculture practices (37). S. iniae causes a fatal meningoencephalitis and is associated with large-scale mortality in a wide variety of marine and freshwater cultured fish species, as well as in wild species (5,39,46). More than 30 species of fish have documented susceptibility to S. iniae disease, including trout (10), yellowtail (19), tilapia (39), barramundi (6), and hybrid striped bass (HSB) (11). S. iniae is distributed globally and is estimated to cause yearly economic losses of hundreds of millions of dollars. Occasionally, S. iniae can cause serious zoonotic infections in humans who injure themselves while handling infected fish (20,42).Relatively little is known of the genetics of S. iniae or of the pathogenic mechanisms underlying its virulence. Here, we describe a severely attenuated mutant of S. iniae, identified through random transposon mutagenesis and direct screening for virulence in HSB. Analysis of the transposon insertion site revealed a disruption of an open reading frame (ORF) with similarity to bacterial phosphoglucomutase (pgm) genes. The enzyme phosphoglucomutase (PGM) interconverts glucose-6-phosphate an...