During infection, Streptococcus pneumoniae exists mainly in sessile biofilms rather than in planktonic form, except during sepsis. The capacity to form biofilms is believed to be important for nasopharyngeal colonization as well as disease pathogenesis, but relatively little is known about the regulation of this process. Here, we investigated the effect of exogenous iron [Fe(III)] as well as the role of luxS (encoding S-ribosylhomocysteine lyase) on biofilm formation by S. pneumoniae D39. Fe(III) strongly enhanced biofilm formation at concentrations of >50 M, while Fe(III) chelation with deferoxamine was inhibitory. Importantly, Fe(III) also upregulated the expression of luxS in wild-type D39. A luxS-deficient mutant (D39luxS) failed to form a biofilm, even with Fe(III) supplementation, whereas a derivative overexpressing luxS (D39luxS؉) exhibited enhanced biofilm formation capacity and could form a biofilm without added Fe(III). D39luxS exhibited reduced expression of the major Fe(III) transporter PiuA, and the cellular [Fe(III)] was significantly lower than that in D39; in contrast, D39luxS؉ had a significantly higher cellular [Fe(III)] than the wild type. The release of extracellular DNA, which is an important component of the biofilm matrix, also was directly related to luxS expression. Similarly, genetic competence, as measured by transformation frequency as well as the expression of competence genes comD, comX, comW, cglA, and dltA and the murein hydrolase cbpD, which is associated with fratricide-dependent DNA release, all were directly related to luxS expression levels and were further upregulated by Fe(III). Moreover, mutagenesis of cbpD blocked biofilm formation. We propose that competence, fratricide, and biofilm formation are closely linked in pneumococci, and that luxS is a central regulator of these processes. We also propose that the stimulatory effects of Fe(III) on all of these parameters are due to the upregulation of luxS expression, and that LuxS provides for a positive Fe(III)-dependent amplification loop by increasing iron uptake.

The thiol-containing tripeptide glutathione is an important cellular constituent of many eukaryotic and prokaryotic cells. In addition to its disulfide reductase activity, glutathione is known to protect cells from many forms of physiological stress. This report represents the first investigation into the role of glutathione in the Gram-positive pathogen Streptococcus pneumoniae. We demonstrate that pneumococci import extracellular glutathione using the ABC transporter substrate binding protein GshT. Mutation of gshT and the gene encoding glutathione reductase (gor) increases pneumococcal sensitivity to the superoxide generating compound paraquat, illustrating the importance of glutathione utilization in pneumococcal oxidative stress resistance. In addition, the gshT and gor mutant strains are hypersensitive to challenge with the divalent metal ions copper, cadmium, and zinc. The importance of glutathione utilization in pneumococcal colonization and invasion of the host is demonstrated by the attenuated phenotype of the gshT mutant strain in a mouse model of infection.

b -galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans . Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P AdhC (or P CopA ) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Sitedirected mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

In Neisseria gonorrhoeae, the MerR family transcription factor NmlR activates 3 operons in response to disulfide stress. In the present study, we show that trxB, a monocistronic operon under the control of NmlR, encodes a functional thioredoxin reductase. It is shown that neisserial TrxB has biochemical properties similar to those of its homologue from Escherichia coli. Analysis of a trxB mutant of N. gonorrhoeae showed that it was more sensitive to disulfide stress and to stress induced by organic hydroperoxides, superoxide, and nitric oxide than wild-type gonococcus. TrxB was found to be essential for the microaerobic induction of aniA and norB, the genes encoding nitrite reductase and nitric oxide reductase, respectively. The importance of TrxB during natural infection was demonstrated by the fact that the survival of gonococci within human cervical epithelial cells, as well as biofilm formation on these cells, was greatly reduced for a trxB mutant compared with a wild-type strain.

c Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that is carried asymptomatically in the nasopharynx by up to 70% of the human population. Translocation of the bacteria into internal sites can cause a range of diseases, such as pneumonia, otitis media, meningitis, and bacteremia. This transition from nasopharynx to growth at systemic sites means that the pneumococcus needs to adjust to a variety of environmental conditions, including transition metal ion availability. Although it is an important nutrient, iron potentiates oxidative stress, and it is established that in S. pneumoniae, expression of iron transport systems and proteins that protect against oxidative stress are regulated by an orphan response regulator, RitR. In this study, we investigated the effect of iron and manganese ion availability on the growth of a ritR mutant. Deletion of ritR led to impaired growth of bacteria in high-iron medium, but this phenotype could be suppressed with the addition of manganese. Measurement of metal ion accumulation indicated that manganese prevents iron accumulation. Furthermore, the addition of manganese also led to a reduction in the amount of hydrogen peroxide produced by bacterial cells. Studies of virulence in a murine model of infection indicated that RitR was not essential for pneumococcal survival and suggested that derepression of iron uptake systems may enhance the survival of pneumococci in some niches.

NGO0579 is annotated copA in the Neisseria gonorrhoeae chromosome, suggesting that it encodes a cation-transporting ATPase specific for copper ions. Compared to wild-type cells, a copA mutant was more sensitive to killing by copper ions but not to other transition metals. The mutant also accumulated a greater amount of copper, consistent with the predicted role of CopA as a copper efflux pump. The copA mutant showed a reduced ability to invade and survive within human cervical epithelial cells, although its ability to form a biofilm on the surface of these cells was not significantly different from that of the wild type. In the presence of copper, the copA mutant exhibited increased sensitivity to killing by nitrite or nitric oxide. Therefore, we concluded that copper ion efflux catalyzed by CopA is linked to the nitrosative stress defense system of Neisseria gonorrhoeae. These observations suggest that copper may exert its effects as an antibacterial agent in the innate immune system via an interaction with reactive nitrogen species.

estD encodes a carboxylic ester hydrolase and is part of the NmlR regulon in Neisseria gonorrhoeae. An estD mutant was found to be susceptible to nitrite and to S-nitrosoglutathione. This mutant was also unable to infect and survive within human cervical epithelial cells, and it showed reduced ability to form a biofilm on these cells. We conclude that esterase D is an integral part of the nitrosative stress defense system of N. gonorrhoeae and that it has potential importance in pathogenesis.

The NmlR sp transcription factor of Streptococcus pneumoniae is shown to induce adhC (alcohol dehydrogenase) expression in the presence of both formaldehyde and methylglyoxal. nmlR sp and adhC mutant strains display altered and opposite aerobic growth phenotypes. The nmlR sp strain exhibits increased resistance to high oxygen tension, attributable to decreased H 2 O 2 production, which correlated with downregulation of carbamoyl phosphate synthase (carB). This indicates a possible role for AdhC in aldehyde metabolism and a broader role for NmlR sp in the regulation of carbon metabolism.