Nitric oxide (NO) contributes to protection from tuberculosis (TB). It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis (Mtb) growth, which prevents subsequent pathological inflammation. In contrast, we report NO primarily protects mice by repressing an interleukin-1 and 12/15-lipoxygenase dependent neutrophil recruitment cascade that promotes bacterial replication. Using Mtb mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports Mtb growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes TB in patients. The human 12/15 lipoxygenase ortholog, ALOX12, is expressed in cavitary TB lesions, the abundance of its products correlate with the number of airway neutrophils and bacterial burden, and a genetic polymorphism that increases ALOX12 expression is associated with TB risk. These data suggest that Mtb exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and nonpathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.

Background: KATmt is the first identified cAMP-regulated protein lysine acetylase in mycobacteria. Results: KATmt acylates fatty acyl CoA ligases in vivo in a cAMP-dependent manner, thus regulating their activity. Conclusion: Mycobacteria utilize KATmt to regulate the metabolic pool of acetyl and propionyl CoA. Significance: We provide novel paradigms for linking cAMP signaling and fatty acid metabolism in mycobacteria.

SUMMARY M. tuberculosis (Mtb) survives a hostile environment within the host that is shaped in part by oxidative stress. The mechanisms used by Mtb to resist these stresses remain ill-defined because the complex combination of oxidants generated by host immunity is difficult to accurately recapitulate in vitro. We performed a genome-wide genetic interaction screen to comprehensively delineate oxidative stress resistance pathways necessary for Mtb to resist oxidation during infection. Our analysis predicted functional relationships between the superoxide-detoxifying enzyme (SodA), an integral membrane protein (DoxX), and a predicted thiol-oxidoreductase (SseA). Consistent with that, SodA, DoxX and SseA form a membrane-associated oxidoreductase complex (MRC) that physically links radical detoxification with cytosolic thiol homeostasis. Loss of any MRC component correlated with defective recycling of mycothiol and accumulation of cellular oxidative damage. This previously uncharacterized coordination between oxygen radical detoxification and thiol homeostasis is required to overcome the oxidative environment Mtb encounters in the host.

We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in M. smegmatis and M. tuberculosis. By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 attP site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an attB site and a drug selection marker, we show both formation of a chromosomal attP site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

California shows the way for biosecurity in commercial gene synthesis To the Editor-On 21 January, California took a major step to increase biosecurity in commercial gene synthesis, introducing legislation that requires all scientists purchasing gene synthesis products to use companies that perform screening on customers and the sequences they order. If enacted, this legislation would make it a competitive advantage for companies to take biosecurity seriously. Here, we argue that the US federal government and other governments should emulate California's actions. Assembly member Rudy Salas (assembly district 32) introduced the legislation, which requires not only that customers use companies that perform biosecurity screening but also that companies offering DNA synthesis services in California perform sequence screening 1. These restrictions would make it harder for a potential nefarious actor to access genetic material for making pathogenic viruses de novo, such as smallpox, Ebola or influenza. The de novo synthesis of known pathogens, particularly small viruses, is listed as one of the most pressing biodefense risks by a 2018 report from the National Academies of Sciences, Engineering and Medicine 2. Many commercial gene synthesis companies already voluntarily screen customer orders to make sure that they are both selling to scientists working in regulated research institutions and not

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Little is known about iron efflux transporters within bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P 1B4 -ATPase, in cytoplasmic Fe 2؉ efflux has been proposed. We report here the distinct roles of mycobacterial P 1B4 -ATPases in the homeostasis of Co 2؉ Iron is an essential micronutrient required for numerous biological processes as it is used as a prosthetic group by several different enzymes (1, 2). However, in excess, it can be toxic due to its participation in Fenton chemistry and potential mismetallation in non-iron-containing metalloproteins. In this context, damage of iron-sulfur centers and mononuclear iron enzymes produced by various redox stresses are particular contributors to iron dyshomeostasis and consequent toxicity (3-6). Characterization of bacterial Fe 2ϩ homeostasis has mainly been focused in mechanisms of uptake (by divalent metal, siderophore, and heme transporters), transcriptional regulation (by Fur and IdeR systems), and Fe 2ϩ sequestration (by bacterioferritin and Dps proteins) (2, 7-9). Nevertheless, studies have suggested that cation diffusion facilitators and iron-citrate transporters participate in Fe 2ϩ efflux (10 -12). We recently observed that Bacillus subtilis PfeT, a P 1B4 -ATPase, confers Fe 2ϩ tolerance (13). PfeT is expressed under the control of PerR in response to peroxide exposure (14). Initial biochemical characterization showed that Fe 2ϩ activates isolated PfeT ATPase, leading to a higher V max than generated by Co 2ϩ , which is the proposed substrate of P 1B4 -ATPases (13,(15)(16)(17). Interestingly, phenotypic analysis of Listeria monocytogenes lacking the P 1B4 -ATPase FrvA showed a role of this ATPase in resistance to heme toxicity (18). These observations suggest a significant role of this subfamily of P-type ATPases in Fe 2ϩ homeostasis (13,14). P 1B4 -ATPases present in prokaryotes and plant chloroplasts are part of the large family of P-type ATPases (15,19,20). P-type ATPases are polytopic membrane proteins that transport a variety of ions using the energy provided by ATP hydrolysis (21-23). The P 1B subgroup includes proteins responsible for the efflux of cytoplasmic transition metals including Cu ϩ , Zn 2ϩ , Co 2ϩ , and Ni 2ϩ (19,22,23). The specificity of their transmembrane metal binding sites (TM-MBSs) 2 is determined by invariant amino acid sequences in their last three transmembrane segments (TMs) (17, 19, 24 -26). However, activation by non-cognate substrates has been reported for most P 1B -ATPase subgroups (22,27