Ivan Gusarov

Sensing Small Molecules by Nascent RNA: A Mechanism to Control Transcription in Bacteria

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.

Endogenous Nitric Oxide Protects Bacteria Against a Wide Spectrum of Antibiotics

Its 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. Bacteria deploy nitric oxide synthases to counter oxidative stress from natural toxins and antibiotic drugs. 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.

Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages

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.

Control of Intrinsic Transcription Termination by N and NusA: The Basic Mechanisms

Intrinsic transcription termination plays a crucial role in regulating gene expression in prokaryotes. After a short pause, the termination signal appears in RNA as a hairpin that destabilizes the elongation complex (EC). We demonstrate that negative and positive termination factors control the efficiency of termination primarily through a direct modulation of hairpin folding and, to a much lesser extent, by changing pausing at the point of termination. The mechanism controlling hairpin formation at the termination point relies on weak protein interactions with single-stranded RNA, which corresponds to the upstream portion of the hairpin. Escherichia coli NusA protein destabilizes these interactions and thus promotes hairpin folding and termination. Stabilization of these contacts by phage lambda N protein leads to antitermination.

Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner

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.

Bacterial Nitric Oxide Extends the Lifespan of C. elegans

Nitric oxide (NO) is an important signaling molecule in multicellular organisms. Most animals produce NO from L-arginine via a family of dedicated enzymes known as NO synthases (NOSes). A rare exception is the roundworm Caenorhabditis elegans, which lacks its own NOS. However, in its natural environment, C. elegans feeds on Bacilli that possess functional NOS. Here, we demonstrate that bacterially derived NO enhances C. elegans longevity and stress resistance via a defined group of genes that function under the dual control of HSF-1 and DAF-16 transcription factors. Our work provides an example of interspecies signaling by a small molecule and illustrates the lifelong value of commensal bacteria to their host.

Methicillin-resistant Staphylococcus aureus bacterial nitric oxide synthase affects antibiotic sensitivity and skin abscess development

Background: Methicillin-resistant Staphylococcus aureus (MRSA) generates NO through bacterial NO synthase (bNOS). Results: Loss of bNOS increases MRSA sensitivity to host neutrophils, cathelicidin antimicrobial peptides, and cell envelope-active antibiotics. Conclusion: bNOS influences MRSA disease pathology. Significance: Future development of bNOS-specific inhibitors could provide dual activities to reduce MRSA pathology and increase antibiotic effectiveness. Staphylococcus aureus infections present an enormous global health concern complicated by an alarming increase in antibiotic resistance. S. aureus is among the few bacterial species that express nitric-oxide synthase (bNOS) and thus can catalyze NO production from l-arginine. Here we generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. Loss of bNOS increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS also promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin. Surprisingly, bNOS-deficient strains gained resistance to aminoglycosides, suggesting that the role of bNOS in antibiotic susceptibility is more complex than previously observed in Bacillus species. Finally, the MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model. Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Future development of specific bNOS inhibitors could be an attractive option to simultaneously reduce MRSA pathology and enhance its susceptibility to commonly used antibiotics.

Methods of walking with the RNA polymerase.

Publisher Summary This chapter discusses the walking system with RNA polymerase. It provides an opportunity to study elongation complexes (ECs) stalled virtually at any desired position along the DNA. The walking procedure was initially based on a fully functional 6-histidine- tagged Escherichia coli (E. coli) RNAP immobilized on Ni++-chelating agarose beads. This technique has been used successfully in elucidating the mechanisms of pausing, arrests, intrinsic termination, and antitermination. The principle behind solid-phase walking is that the initial EC immobilized on a solid support undergoes rounds of washing (to remove the unincorporated nucleotide triphosphate [NTP] substrates), followed by addition of the incomplete set of NTPs (three or less) that allow transcription to proceed to the next DNA position corresponding to the first missing NTP. The chapter discusses preparation of RNAP, NTP substrates and DNA template. His-tagged-based walking and biotin-tagged-based walking technique is also discussed. The roadblock is a site-specific DNA binding protein that stops the EC completely without termination at any distance from the promoter. Two proteins have been described as removable roadblocks, the lac repressor and the mutant form of EcoRI restriction endonuclease, EcoRQ111. Many different NTP analogs can be utilized by E. coli RNAP and incorporated in the nascent RNA at specific positions during walking.

S-Nitrosylation Signaling in Escherichia coli

Bacteria respond to nitric oxide and oxygen in their environment using two different posttranslational modifications of the same transcription factor to promote expression of protective genes specific to each gas. Most bacteria generate nitric oxide (NO) either aerobically by NO synthases or anaerobically from nitrite. Far from being a mere by-product of nitrate respiration, bacterial NO has diverse physiological roles. Many proteins undergo NO-mediated posttranslational modification (S-nitrosylation) in anaerobically grown Escherichia coli. The regulation of one such protein, OxyR, represents a redox signaling paradigm in which the same transcription factor controls different protective genes depending on its S-nitrosylation versus S-oxidation status. We discuss a structural model that may explain the remarkable stability and specificity of OxyR S-nitrosylation.

Analysis of the Intrinsic Transcription Termination Mechanism and Its Control

Publisher Summary This chapter describes approaches that permit dissecting the termination process in several steps and identifying individual roles of the terminator elements, the hairpin and T-stretch, in each step. To study the basic mechanism of intrinsic termination and its control, an in vitro reconstituted system is used that contains Escherichia coli (E.coli) RNAP, a linear DNA template with a strong promoter and the intrinsic terminator, and pure elongation factors, NusA and N. E. coli NusA protein works as a general termination factor, which significantly increases the efficiency of many intrinsic terminators in vitro and in vivo. The trapped complex (TC) represents a unique configuration of the elongation complexes (EC) that occurs exclusively at the intrinsic termination points. Pausing at the tR2 termination point is determined by the downstream portion of its T-stretch. Pausing provides an additional time for the hairpin to form and is required for efficient termination under physiological conditions. To exclude the kinetic component (pausing) from the analysis of termination and to study the “mechanistic” component of the termination process in real time, the slow termination approach has been developed. This approach utilizes a modified A1-tR2 template carrying point substitutions within the T-stretch, rendering the RNA: DNA hybrid strong enough to delay the hairpin folding.