Contribution of NADH oxidase to aerobic metabolism of Streptococcus pyogenes

Abstract

An understanding of how the heme-deficient gram-positive bacterium Streptococcus pyogenes establishes infections in O(2)-rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of S. pyogenes and other lactic acid bacteria which directly catalyzes the four-electron reduction of O(2) to H(2)O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O(2) conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O(2) conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O(2) tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H(2)O(2) in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H(2)O(2) and rescued the growth defect of the mutants under high-O(2) conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O(2) with carbohydrate limitation.

FIG. 1

S. pyogenes contains a single nox homologue. (A) The arrows represent the directions of transcription of open reading frames, which are contained within the same chromosomal region as nox. Information presented under the arrows describes the genes to which the open reading frames have the highest homology. A possible factor-independent terminator located 3′ of the ldh homologue was identified by the method of Brendel and Trifonov (5). (B) The S. pyogenes nox homologue (S.py.; GenBank accession no. AF101442) contains all of the signature residues characteristic of E. faecalis nox (E.f., GenBank accession no. X68847) (44) and S. pneumoniae nox (S.pn.; GenBank accession no. AF014458), including the cysteine sulfenic acid redox center, the NADH contact region (box 2), and FAD-binding regions (boxes 1 and 3).

FIG. 2

Construction of nox null mutants. Insertional inactivation of nox in two unrelated strains of S. pyogenes was accomplished by first amplifying a region internal to the nox coding region (noxtrunc) and then inserting the fragment into integrational vector pCIV2. The resulting element, pNOX1, contains a kanamycin resistance determinant (striped bar) and the noxtrunc region (empty bar) of JRS4. Recombination between homologous regions of pNOX1 and the S. pyogenes JRS4 and HSC12 chromosomes (indicated by the large X between the plasmid and chromosomal restriction maps) generated JNOX1 and HNOX1, respectively, which have a chromosomal structure that contains two truncated and inactive versions of nox (indicated by the zigzag line). E, EcoRI; H, HindIII; N, NcoI; Nd, NdeI; P, PstI; Sc, ScaI; Sm, SmaI; ORI, origin of replication.

FIG. 3

Characterization of the nox mutants on solid medium. Both wild-type SAM1 and the nox null mutant JNOX1 were grown under high (ambient atmosphere; see Materials and Methods)- and low-O₂ conditions on solid THY medium at 37°C. Wild-type SAM1 was grown for 14 h, and the nox mutant JNOX1 was grown for >20 h.

FIG. 4

Glucose affects the growth of the JNOX1 mutant and the NOXase activity of the SAM1 wild-type strain. (A) Glucose rescues the JNOX1 mutant growth defect in liquid C medium. Wild-type SAM1 and mutant JNOX1 were cultured for 20 h under low-O₂ conditions (static growth) in liquid C medium. Cultures were then diluted to an optical density at 600 nm (OD₆₀₀) of 0.015 in liquid C medium and grown again under low-O₂ conditions in the presence or absence of 1% glucose at 37°C. The OD₆₀₀ was measured every hour for 7 h. Data presented are representative of at least five independent experiments. (B) Glucose affects NOXase specific activity. At the 4-, 5-, and 6-h time points, aliquots were removed from the cultures described in the legend to panel A and assayed for NOXase specific activity. Data presented represent the mean and standard deviation of samples analyzed in triplicate and are representative of at least five independent experiments.