Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

Treatment of Multidrug-Resistant Vancomycin-Resistant Enterococcus faecium Hardware-Associated Vertebral Osteomyelitis with Oritavancin plus Ampicillin.

Weekly oritavancin plus ampicillin continuous infusion combination therapy was used to successfully treat a deep spine vancomycin-resistant Enterococcus faecium infection associated with hardware. Checkerboard and time-kill assays confirmed synergy between these two antibiotics. Further synergies of oritavancin and ampicillin with rifampin or the endogenous human antimicrobial peptide cathelicidin LL-37 were demonstrated.

Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

Mutual exclusivity of hyaluronan and hyaluronidase in invasive group A Streptococcus.

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.

An efficient in vivo-inducible CRISPR interference system for group A Streptococcus genetic analysis and pathogenesis studies.

While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacterium's poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically, it has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based single guide RNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just 2 days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets. IMPORTANCE: While group A Streptococcus (GAS) remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference (CRISPRi) for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS and, together with SpyBrowse, represents a valuable resource for researchers in the GAS field.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from Marine Cyanobacterium Moorena producens JHB Discovered via Coculture with Candida albicans.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies on this strain led to the discovery of several novel compounds such as hectochlorins and jamaicamides. However, bioinformatic analyses of its genome indicate the presence of numerous cryptic biosynthetic gene clusters that have yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was cocultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that coculture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria.

Severe soft tissue infection caused by a non-beta-hemolytic Streptococcus pyogenes strain harboring a premature stop mutation in the sagC gene.

We recovered a non-beta-hemolytic Streptococcus pyogenes strain from a severe soft tissue infection. In this isolate, we detected a premature stop codon within the sagC gene of the streptolysin S (SLS) biosynthetic operon. Reintroduction of full-length sagC gene on a plasmid vector restored the beta-hemolytic phenotype to our clinical isolate, indicating that the point mutation in sagC accounted for loss of hemolytic activity. To the best of our knowledge, this is the first report to demonstrate that a severe soft tissue infection can be caused by a non-beta-hemolytic S. pyogenes strain lacking a functional SagC.

Unrecognized Potent Activities of Colistin Against Clinically Important mcr+ Enterobacteriaceae Revealed in Synergy with Host Immunity.

Colistin (COL) is a cationic cyclic peptide that disrupts negatively-charged bacterial cell membranes and frequently serves as an antibiotic of last resort to combat multidrug-resistant Gram-negative bacterial infections. Emergence of the horizontally transferable plasmid-borne mobilized colistin resistance (mcr) determinant and its spread to Gram-negative strains harboring extended-spectrum ß-lactamase and carbapenemase resistance genes threatens futility of our chemotherapeutic arsenal. COL is widely regarded to have zero activity against mcr+ patients based on standard antimicrobial susceptibility testing (AST) performed in enriched bacteriological growth media; consequently, the drug is withheld from patients with mcr+ infections. However, these standard testing media poorly mimic in vivo physiology and omit host immune factors. Here we report previously unrecognized bactericidal activities of COL against mcr-1+ isolates of Escherichia coli (EC), Klebsiella pneumoniae (KP), and Salmonella enterica (SE) in standard tissue culture media containing the physiological buffer bicarbonate. Moreover, COL promoted serum complement deposition on the mcr-1+ Gram-negative bacterial surface and synergized potently with active human serum in pathogen killing. At COL concentrations readily achievable with standard dosing, the peptide antibiotic killed mcr-1+ EC, KP, and SE in freshly isolated human blood proved effective as monotherapy in a murine model of mcr-1+ EC bacteremia. Our results suggest that COL, currently ignored as a treatment option based on traditional AST, may in fact benefit patients with mcr-1+ Gram negative infections based on evaluations performed in a more physiologic context. These concepts warrant careful consideration in the clinical microbiology laboratory and for future clinical investigation of their merits in high risk patients with limited therapeutic options.

A residue in helical conformation in the native state adopts a ß-strand conformation in the folding transition state despite its high and canonical Φ-value.

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved ß-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state.

Bicarbonate Effects on Antibacterial Immunity and Mucus Glycobiology in the Cystic Fibrosis Lung: A Review With Selected Experimental Observations.

The primary defect in cystic fibrosis (CF) is abnormal chloride and bicarbonate transport in the cystic fibrosis transmembrane conductance regulator (CFTR) epithelial ion channel. The apical surface of the respiratory tract is lined by an airway surface liquid layer (ASL) composed of mucin comprising mainly MUC5A and MUC5B glycoproteins. ASL homeostasis depends on sodium bicarbonate secretion into the airways and secretion deficits alter mucus properties leading to airway obstruction, inflammation, and infections. Downstream effects of abnormal ion transport in the lungs include altered intrinsic immune defenses. We observed that neutrophils killed Pseudomonas aeruginosa more efficiently when it had been exposed to sodium bicarbonate, and formation of neutrophil extracellular traps (NETs) by neutrophils was augmented in the presence of increasing bicarbonate concentrations. Physiological levels of bicarbonate sensitized P. aeruginosa to the antimicrobial peptide cathelicidin LL-37, which is present in both lung ASL and in NETs. Sodium bicarbonate has various uses in clinical medicine and in the care of CF patients, and could be further explored as a therapeutic adjunct against Pseudomonas infections.

Inhibition of histamine accumulation by novel histamine-degrading species of Staphylococcus sp. isolated from goats and sheep milk.

Histamine is an active amine compound that occurs in various fermented foods that may cause adverse effects on the human health. Certain microorganisms are able to degrade histamine by an oxidative deamination reaction. Therefore, the present study aimed to quantify histamine-forming and/or -degrading activity of the isolates derived from milk of goat and sheep herds, in Iran, by the capillary zone electrophoresis (CZE) method; and we evaluated the molecular characteristics of staphylococcal isolates. Among 243 staphylococcal isolates, 29 histamine-degrading bacteria were identified. One of these isolates, identified as Staph. epidermidis, No. 605, exhibited the highest activity compared to others, degrading available histamine to 58.33% within 24 h. By polymerase chain reaction (PCR) analysis, the isolate, No. 605 that exhibited remarkable histamine-degrading activity lacked the genes encoding coagulase and DNase, nor did it harbor any of the five classical enterotoxin genes. This is the first report to show that seven Staphylococcus species, including Staph. chromogenes, Staph. aureus, Staph. haemolyticus, Staph. epidermidis, Staph. pseudintermedius, Staph. agnetis, and Staph. hyicus, were able to degrade histamine, which were hitherto not known to have this capacity. Therefore, histamine-degrading activity is a definite criterion to introduce fermenting organisms able to decrease histamine content in different food products.

Effect of eliminating hdcA gene of Staphylococcus epidermidis TYH1 on Histamine production.

Background and Objectives: The possible adverse effect of histamine on human health has made it a detrimental aspect to the quality and safety of many fermented food products especially fish sauce. Materials and Methods: In the present study, hdcA gene in Staphylococcus epidermidis TYH1 was knocked out and its effect on histamine production was evaluated. HdcA encodes histidine decarboxylase, an enzyme that produces histamine from histidine. Both strains of TYH1, the wild type (WT) and mutant (ΔhdcA) were then incubated in tryptic soy broth (TSB) supplemented with histidine (0.5 mM). The histamine content determined by capillary zone electrophoretic (CZE) analysis. Safety assessment of this mutant of food origin was conferred by virulence genes. Results: It was found that S. epidermidis TYH1 exhibited production of histamine (50.09 ± 0.06 µg/mL), while ΔhdcA strain of TYH1 exhibited no histamine forming activity. Safety assessment of ΔhdcA revealed the presence of nuc gene, while superantigenic toxins and coa genes were not observed. Therefore, it has the ability to be used as a starter culture to decrease the histamine content in any fermented food products. Conclusion: Our study findings may contribute to provide a novel approach of promoting the food safety of fish sauce and other fermented food products regarding the regulation of histamine content.

Clostridiolysin S, a post-translationally modified biotoxin from Clostridium botulinum.

Through elaboration of its botulinum toxins, Clostridium botulinum produces clinical syndromes of infant botulism, wound botulism, and other invasive infections. Using comparative genomic analysis, an orphan nine-gene cluster was identified in C. botulinum and the related foodborne pathogen Clostridium sporogenes that resembled the biosynthetic machinery for streptolysin S, a key virulence factor from group A Streptococcus responsible for its hallmark beta-hemolytic phenotype. Genetic complementation, in vitro reconstitution, mass spectral analysis, and plasmid intergrational mutagenesis demonstrate that the streptolysin S-like gene cluster from Clostridium sp. is responsible for the biogenesis of a novel post-translationally modified hemolytic toxin, clostridiolysin S.

Identifying the effect of vancomycin on health care-associated methicillin-resistant Staphylococcus aureus strains using bacteriological and physiological media.

BACKGROUND: The evolving antibiotic-resistant behavior of health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern. They are resistant to a broad class of antibiotics such as macrolides, aminoglycosides, fluoroquinolones, and many more. FINDINGS: The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (cation-adjusted Mueller-Hinton broth) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behavior of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments. CONCLUSIONS: The study reveals the vancomycin resistance behavior of HA-MRSA USA100 strains in dual media conditions using wide-ranging experiments.

The novel polysaccharide deacetylase homologue Pdi contributes to virulence of the aquatic pathogen Streptococcus iniae.

The aquatic zoonotic pathogen Streptococcus iniae represents a threat to the worldwide aquaculture industry and poses a risk to humans who handle raw fish. Because little is known about the mechanisms of S. iniae pathogenesis or virulence factors, we established a high-throughput system combining whole-genome pyrosequencing and transposon mutagenesis that allowed us to identify virulence proteins, including Pdi, the polysaccharide deacetylase of S. iniae, that we describe here. Using bioinformatics tools, we identified a highly conserved signature motif in Pdi that is also conserved in the peptidoglycan deacetylase PgdA protein family. A Deltapdi mutant was attenuated for virulence in the hybrid striped bass model and for survival in whole fish blood. Moreover, Pdi was found to promote bacterial resistance to lysozyme killing and the ability to adhere to and invade epithelial cells. On the other hand, there was no difference in the autolytic potential, resistance to oxidative killing or resistance to cationic antimicrobial peptides between S. iniae wild-type and Deltapdi. In conclusion, we have demonstrated that pdi is involved in S. iniae adherence and invasion, lysozyme resistance and survival in fish blood, and have shown that pdi plays a role in the pathogenesis of S. iniae. Identification of Pdi and other S. iniae virulence proteins is a necessary initial step towards the development of appropriate preventive and therapeutic measures against diseases and economic losses caused by this pathogen.

Genetic Determinants Enabling Medium-Dependent Adaptation to Nafcillin in Methicillin-Resistant Staphylococcus aureus.

Antimicrobial susceptibility testing standards driving clinical decision-making have centered around the use of cation-adjusted Mueller-Hinton broth (CA-MHB) as the medium with the notion of supporting bacterial growth, without consideration of recapitulating the in vivo environment. However, it is increasingly recognized that various medium conditions have tremendous influence on antimicrobial activity, which in turn may have major implications on the ability of in vitro susceptibility assays to predict antibiotic activity in vivo. To elucidate differential growth optimization and antibiotic resistance mechanisms, adaptive laboratory evolution was performed in the presence or absence of the antibiotic nafcillin with methicillin-resistant Staphylococcus aureus (MRSA) TCH1516 in either (i) CA-MHB, a traditional bacteriological nutritionally rich medium, or (ii) Roswell Park Memorial Institute (RPMI), a medium more reflective of the in vivo host environment. Medium adaptation analysis showed an increase in growth rate in RPMI, but not CA-MHB, with mutations in apt, adenine phosphoribosyltransferase, and the manganese transporter subunit, mntA, occurring reproducibly in parallel replicate evolutions. The medium-adapted strains showed no virulence attenuation. Continuous exposure of medium-adapted strains to increasing concentrations of nafcillin led to medium-specific evolutionary strategies. Key reproducibly occurring mutations were specific for nafcillin adaptation in each medium type and did not confer resistance in the other medium environment. Only the vraRST operon, a regulator of membrane- and cell wall-related genes, showed mutations in both CA-MHB- and RPMI-evolved strains. Collectively, these results demonstrate the medium-specific genetic adaptive responses of MRSA and establish adaptive laboratory evolution as a platform to study clinically relevant resistance mechanisms.IMPORTANCE The ability of pathogens such as Staphylococcus aureus to evolve resistance to antibiotics used in the treatment of infections has been an important concern in the last decades. Resistant acquisition usually translates into treatment failure and puts patients at risk of unfavorable outcomes. Furthermore, the laboratory testing of antibiotic resistance does not account for the different environment the bacteria experiences within the human body, leading to results that do not translate into the clinic. In this study, we forced methicillin-resistant S. aureus to develop nafcillin resistance in two different environments, a laboratory environment and a physiologically more relevant environment. This allowed us to identify genetic changes that led to nafcillin resistance under both conditions. We concluded that not only does the environment dictate the evolutionary strategy of S. aureus to nafcillin but also that the evolutionary strategy is specific to that given environment.

Genetic and biochemical modulation of sialic acid O-acetylation on group B Streptococcus: phenotypic and functional impact.

Group B Streptococcus (GBS) is an important human pathogen and a model system for studying the roles of bacterial glycosylation in host-microbe interactions. Sialic acid (Sia), expressed prominently in the GBS capsular polysaccharide (CPS), mimics mammalian cell surface Sia and can interact with host Sia-binding proteins to subvert immune clearance mechanisms. Our earlier work has shown that GBS partially O-acetylates CPS Sia residues and employs an intracellular O-acetylation/de-O-acetylation cycle to control the final level of this surface Sia modification. Here, we examine the effects of point mutations in the NeuD O-acetyltransferase and NeuA O-acetylesterase on specific glycosylation phenotypes of GBS, pinpointing an isogenic strain pair that differs dramatically in the degree of the O-acetyl modification (80% versus 5%) while still expressing comparable levels of overall sialylation. Using these strains, higher levels of O-acetylation were found to protect GBS CPS Sia against enzymatic removal by microbial sialidases and to impede engagement of human Siglec-9, but not to significantly alter the ability of GBS to restrict complement C3b deposition on its surface. Additional experiments demonstrated that pH-induced migration of the O-acetyl modification from the 7- to 9-carbon position had a substantial impact on GBS-Siglec-9 interactions, with 7-O-acetylation exhibiting the strongest interference. These studies show that both the degree and position of the GBS O-acetyl modification influence Sia-specific interactions relevant to the host-pathogen relationship. We conclude that native GBS likely expresses a phenotype of intermediate Sia O-acetylation to strike a balance between competing selective pressures present in the host environment.

Bacterial phenotype variants in group B streptococcal toxic shock syndrome.

We conducted genetic and functional analyses of isolates from a patient with group B streptococcal (GBS) necrotizing fasciitis and toxic shock syndrome. Tissue cultures simultaneously showed colonies with high hemolysis (HH) and low hemolysis (LH). Conversely, the HH and LH variants exhibited low capsule (LC) and high capsule (HC) expression, respectively. Molecular analysis demonstrated that the 2 GBS variants were of the same clonal origin. Genetic analysis found a 3-bp deletion in the covR gene of the HH/LC variant. Functionally, this isolate was associated with an increased growth rate in vitro and with higher interleukin-8 induction. However, in whole blood, opsonophagocytic and intracellular killing assays, the LH/HC phenotype demonstrated higher resistance to host phagocytic killing. In a murine model, LH/HC resulted in higher levels of bacteremia and increased host mortality rate. These findings demonstrate differences in GBS isolates of the same clonal origin but varying phenotypes.

Enhanced topical delivery of non-complexed molecular iodine for Methicillin-resistant Staphylococcus aureus decolonization.

Staphylococcus aureus, a leading cause of serious human infections in both healthcare and community settings, is increasingly difficult to control due to expanding resistance to multiple antibiotic classes. Methicillin-resistant S. aureus (MRSA) strains have disseminated on a global scale and are associated with adverse patient outcomes, increased hospital stays, and significant economic costs to the healthcare system. A proximal step in S. aureus infection is colonization of the nasal mucosa, and effective strategies to decolonize high risk patients to reduce the risk of invasive infection and nosocomial spread represent an important clinical priority. With rising resistance to mupirocin, the most common antibiotic utilized for nasal MRSA decontamination, we are examining the use of pure molecular iodine (I2)-based formulations for this indication. Recently, an iodophor formulation of povidone-iodine (PVP-I) has shown significant promise for nasal MRSA decontamination by swabbing the anterior nares of patients in hospital settings, but the I2 concentration in this treatment is less than 0.01% of total iodine species present and like all providone-iodine formulations causes skin staining. Here we determine that a novel non-staining formulation of I2 combined with the safe organic emollient glycerin delivers high local concentrations of the active antimicrobial entity (I2) with minimal evaporative loss, exhibits activity at ∼1 part per million against MRSA and other important Gram-positive and -negative human pathogens. This formulation for I2 topical delivery produced similar reductions in mean bacterial burden and was associated with fewer treatment failures (<2-logfold reduction) than PVP-I in a murine model of MRSA nasal decontamination. Formulations of I2 in glycerin emollient merit further exploration as topical disinfectants for human medical indications.

Refactoring the Cryptic Streptophenazine Biosynthetic Gene Cluster Unites Phenazine, Polyketide, and Nonribosomal Peptide Biochemistry.

The disconnect between the genomic prediction of secondary metabolite biosynthetic potential and the observed laboratory production profile of microorganisms is well documented. While heterologous expression of biosynthetic gene clusters (BGCs) is often seen as a potential solution to bridge this gap, it is not immune to many challenges including impaired regulation, the inability to recruit essential building blocks, and transcriptional and/or translational silence of the biosynthetic genes. Here we report the discovery, cloning, refactoring, and heterologous expression of a cryptic hybrid phenazine-type BGC (spz) from the marine actinomycete Streptomyces sp. CNB-091. Overexpression of the engineered spz pathway resulted in increased production and chemical diversity of phenazine natural products belonging to the streptophenazine family, including bioactive members containing an unprecedented N-formylglycine attachment. An atypical discrete adenylation enzyme in the spz cluster is required to introduce the formylglycine moiety and represents a phylogenetically distinct class of adenylation proteins.