Bacterial nitric oxide synthases (bNOS) are present in many Gram-positive species and have been demonstrated to synthesize NO from arginine in vitro and in vivo. However, the physiological role of bNOS remains largely unknown. We show that NO generated by bNOS increases the resistance of bacteria to a broad spectrum of antibiotics, enabling the bacteria to survive and share habitats with antibiotic-producing microorganisms. NO-mediated resistance is achieved through both the chemical modification of toxic compounds and the alleviation of the oxidative stress imposed by many antibiotics. Our results suggest that the inhibition of NOS activity may increase the effectiveness of antimicrobial therapy.

Bacterial nitric-oxide (NO) synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These "promiscuous" bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO donors for research and clinical needs.

Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N‐terminal coiled‐coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C‐terminal domain (CTD). We show that depending on pH, Gfh1‐CTD exists in two alternative orientations. At pH above 7, it assumes an inactive ‘flipped’ orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1‐CTD switches to an active ‘Gre‐like’ orientation, which enables Gfh1 to bind to and inhibit RNAP.

No abstract