Summary spxB-encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signalling, and oxidative stress. We report here the first cis-and transacting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semitransparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency ª 3 ¥ 10

Acetyl phosphate (AcP) is a small-molecule metabolite that can act as a phosphoryl group donor for response regulators of two-component systems (TCSs). The serious human respiratory pathogen Streptococcus pneumoniae (pneumococcus) synthesizes AcP by the conventional pathway involving phosphotransacetylase and acetate kinase, encoded by pta and ackA, respectively. In addition, pneumococcus synthesizes copious amounts of AcP and hydrogen peroxide (H 2 O 2 ) by pyruvate oxidase, which is encoded by spxB. To assess possible roles of AcP in pneumococcal TCS regulation and metabolism, we constructed strains with combinations of spxB, pta, and ackA mutations and determined their effects on ATP, AcP, and H 2 O 2 production. Unexpectedly, ⌬ackA mutants were unstable and readily accumulated primary suppressor mutations in spxB or its positive regulator, spxR, thereby reducing H 2 O 2 and AcP levels, and secondary capsule mutations in cps2E or cps2C. ⌬ackA ⌬spxB mutants contained half the cellular amount of ATP as a ⌬spxB or spxB ؉ strain. Acetate addition and anaerobic growth experiments suggested decreased ATP, rather than increased AcP, as a reason that ⌬ackA mutants accumulated spxB or spxR suppressors, although experimental manipulation of the AcP amount was limited. This finding and other considerations suggest that coping with endogenously produced H 2 O 2 may require energy. Starting with a ⌬spxB mutant, we constructed ⌬pta, ⌬ackA, and ⌬pta ⌬ackA mutants. Epistasis and microarray experiment results were consistent with a role for the SpxB-Pta-AckA pathway in expression of the regulons controlled by the WalRK Spn , CiaRH Spn , and LiaSR Spn TCSs involved in sensing cell wall status. However, AcP likely does not play a physiological role in TCS sensing in S. pneumoniae.

Adaptation to endogenous oxidative stress is an integral aspect of Streptococcus pneumoniae colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H2O2 and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in S. pneumoniae.

Nitric oxide (NO·) is a ubiquitous molecular mediator in biology. Many signaling actions of NO· generated by mammalian NO· synthase (NOS) result from targeting of the haem moiety of soluble guanylate cyclase. Some pathogenic and environmental bacteria also produce a NOS that is evolutionary related to the mammalian enzymes, but a bacterial haem-containing receptor for endogenous enzymatically-generated NO· has not been previously identified. Here we show that NOS of the human pathogen Staphylococcus aureus, in concert with an NO·-metabolising flavohaemoprotein, regulates electron transfer by targeting haem-containing cytochrome oxidases under microaerobic conditions to maintain membrane bioenergetics. This process is essential for staphylococcal nasal colonisation and resistance to the membrane-targeting antibiotic daptomycin, and demonstrates the conservation of NOS-derived NO·-haem receptor signaling between bacteria and mammals.

The biosynthesis of histidine in Escherichia coli and Salmonella typhimurium has been an important model system for the study of relationships between the flow of intermediates through a biosynthetic pathway and the control of the genes encoding the enzymes that catalyze the steps in a pathway. This article provides a comprehensive review of the histidine biosynthetic pathway and enzymes, including regulation of the flow of intermediates through the pathway and mechanisms that regulate the amounts of the histidine biosynthetic enzymes. In addition, this article reviews the structure and regulation of the histidine ( his ) biosynthetic operon, including transcript processing, Rho-factor-dependent “classical” polarity, and the current model of his operon attenuation control. Emphasis is placed on areas of recent progress. Notably, most of the enzymes that catalyze histidine biosynthesis have recently been crystallized, and their structures have been determined. Many of the histidine biosynthetic intermediates are unstable, and the histidine biosynthetic enzymes catalyze some chemically unusual reactions. Therefore, these studies have led to considerable mechanistic insight into the pathway itself and have provided deep biochemical understanding of several fundamental processes, such as feedback control, allosteric interactions, and metabolite channeling. Considerable recent progress has also been made on aspects of his operon regulation, including the mechanism of pp(p)Gpp stimulation of his operon transcription, the molecular basis for transcriptional pausing by RNA polymerase, and pathway evolution. The progress in these areas will continue as sophisticated new genomic, metabolomic, proteomic, and structural approaches converge in studies of the histidine biosynthetic pathway and mechanisms of control of his biosynthetic genes in other bacterial species.

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