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Turning down tolerance Persister cells, which are found in abundance in biofilms, adopt a quiescent state and survive antimicrobial treatments, seeding disease recurrence and incubating new resistance mutations. Building on work implicating the reactive small-molecule hydrogen sulfide in bacterial defense against antibiotics, Shatalin et al. conducted a structure-based screen for inhibitors of a bacterial hydrogen sulfide–producing enzyme and found a group of inhibitors that act through an allosteric mechanism (see the Perspective by Mah). These inhibitors potentiated bactericidal antibiotics in vitro and in mouse infection models. They also suppressed persister bacteria and disrupted biofilm formation. This strategy of taking out persister cells may be promising for treating recalcitrant infections and holding the line against drug-resistant bacteria. Science , abd8377, this issue p. 1169 ; see also abj3062, p. 1153

The biological mediator hydrogen sulfide (H(2)S) is produced by bacteria and has been shown to be cytoprotective against oxidative stress and to increase the sensitivity of various bacteria to a range of antibiotic drugs. Here we evaluated whether bacterial H(2)S provides resistance against the immune response, using two bacterial species that are common sources of nosocomial infections, Escherichia coli and Staphylococcus aureus. Elevations in H(2)S levels increased the resistance of both species to immune-mediated killing. Clearances of infections with wild-type and genetically H(2)S-deficient E. coli and S. aureus were compared in vitro and in mouse models of abdominal sepsis and burn wound infection. Also, inhibitors of H(2)S-producing enzymes were used to assess bacterial killing by leukocytes. We found that inhibition of bacterial H(2)S production can increase the susceptibility of both bacterial species to rapid killing by immune cells and can improve bacterial clearance after severe burn, an injury that increases susceptibility to opportunistic infections. These findings support the role of H(2)S as a bacterial defense mechanism against the host response and implicate bacterial H(2)S inhibition as a potential therapeutic intervention in the prevention or treatment of infections.

Many prokaryotic species generate hydrogen sulfide (H(2)S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine β-synthase, cystathionine γ-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H(2)S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H(2)S suppresses this effect. Moreover, in bacteria that normally produce H(2)S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics.

It's a Gas Many antibiotics, including beta-lactams, aminoglycosides, and quinolones, kill bacteria (at least in part) by oxidative stress. Gusarov et al. (p. 1380 ) show that nitric oxide (NO) produced by bacterial NO synthases (bNOS) protects bacteria, including Staphylococcus aureus and Bacillus anthracis , against toxic agents they may encounter in the soil or in host organisms. Thus, bNOS activity is specifically induced in response to antibiotics and, in turn, activates the expression of another key antioxidant enzyme: superoxide dismutase. Hence, NO-mediated antibiotic resistance not only operates by direct chemical modification of toxic molecules, but also alleviates oxidative stress caused by naturally occurring antibiotics.

Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen— Bacillus anthracis —critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages. The mechanism underlying bNOS-dependent resistance to macrophage killing relies on NO-mediated activation of bacterial catalase and suppression of the damaging Fenton reaction. Our results demonstrate that pathogenic bacteria use their own NO as a key defense against the immune oxidative burst, thereby establishing bNOS as an essential virulence factor. Thus, bNOS represents an attractive antimicrobial target for treatment of anthrax and other infectious diseases.

Monitoring of gene knockouts is a new microarray-based genetic technique used for genome-wide identification of conditionally essential genes in bacteria, We have developed a new microarray-based genetic technique, named MGK (Monitoring of Gene Knockouts), for genome-wide identification of conditionally essential genes. MGK identified bacterial genes that are critical for fitness in the absence of aromatic amino acids, and was further applied to identify genes whose inactivation causes bacterial cell death upon exposure to the bacteriostatic antibiotic chloramphenicol. Our findings suggest that MGK can serve as a robust tool in functional genomics studies.

Thiamin and riboflavin are precursors of essential coenzymes-thiamin pyrophosphate (TPP) and flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD), respectively. In Bacillus spp, genes responsible for thiamin and riboflavin biosynthesis are organized in tightly controllable operons. Here, we demonstrate that the feedback regulation of riboflavin and thiamin genes relies on a novel transcription attenuation mechanism. A unique feature of this mechanism is the formation of specific complexes between a conserved leader region of the cognate RNA and FMN or TPP. In each case, the complex allows the termination hairpin to form and interrupt transcription prematurely. Thus, sensing small molecules by nascent RNA controls transcription elongation of riboflavin and thiamin operons and possibly other bacterial operons as well.

Many prokaryotic species generate hydrogen sulfide (H2S) and nitric oxide (NO) enzymatically, from cysteine and arginine, respectively, in their natural environments. Both gases are small freely diffusible signaling molecules that are known to be involved in numerous physiological and pathological processes in mammals. However the biochemistry and physiological role of these gases in bacteria remains largely unknown. We have shown that inactivation of H2S producing enzymes (cystathionine beta-synthase, cystathionine gamma lyase, or 3-mercaptopyruvate sulfurtransferase) and NO-synthase in several Gram (+) and Gram (−) bacteria render them highly sensitive to different classes of antibiotics (Gusarov et al., Science 325 (2009) 1380–1384; Shatalin et al. Science 334 (2011) 986–990). We also presented evidence that Bacillus anthracis-derived NO is critical at the early stage of infection (Shatalin et al. PNAS 105 (2008) 1009–1013). Here we show that: (1) cbs/cse and nos mutations change Bacilli global gene transcription profile; (2) apore formation process in cbs/cse and nos mutants ofB. anthracis is affected; (3) virulence of cbs/cse and nos mutants of B. anthracis is diminished. These results demonstrate that bacterial H2S and NO are an important virulence factors, and that enzymes generated these gases may serve as an attractive target for antimicrobial therapy.