Streptomyces sungeiensis SD3 as a Microbial Chassis for the Heterologous Production of Secondary Metabolites.

We present the newly isolated Streptomyces sungeiensis SD3 strain as a promising microbial chassis for heterologous production of secondary metabolites. S. sungeiensis SD3 exhibits several advantageous traits as a microbial chassis, including genetic tractability, rapid growth, susceptibility to antibiotics, and metabolic capability supporting secondary metabolism. Genomic and transcriptomic sequencing unveiled the primary metabolic capabilities and secondary biosynthetic pathways of S. sungeiensis SD3, including a previously unknown pathway responsible for the biosynthesis of streptazone B1. The unique placement of S. sungeiensis SD3 in the phylogenetic tree designates it as a type strain, setting it apart from other frequently employed Streptomyces chassis. This distinction makes it the preferred chassis for expressing biosynthetic gene clusters (BGCs) derived from strains within the same phylogenetic or neighboring phylogenetic clade. The successful expression of secondary biosynthetic pathways from a closely related yet slow-growing strain underscores the utility of S. sungeiensis SD3 as a heterologous expression chassis. Validation of CRISPR/Cas9-assisted genetic tools for chromosomal deletion and insertion paved the way for further strain improvement and BGC refactoring through rational genome editing. The addition of S. sungeiensis SD3 to the heterologous chassis toolkit will facilitate the discovery and production of secondary metabolites.

A PilZ domain protein interacts with the transcriptional regulator HinK to regulate type VI secretion system in Pseudomonas aeruginosa.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.

Impact of the host microbiota on fungal infections: New possibilities for intervention?

Many human fungal pathogens are opportunistic. They are primarily benign residents of the human body and only become infectious when the host's immunity and microbiome are compromised. Bacteria dominate the human microbiome, playing an essential role in keeping fungi harmless and acting as the first line of defense against fungal infection. The Human Microbiome Project, launched by NIH in 2007, has stimulated extensive investigation and significantly advanced our understanding of the molecular mechanisms governing the interaction between bacteria and fungi, providing valuable insights for developing future antifungal strategies by exploiting the interaction. This review summarizes recent progress in this field and discusses new possibilities and challenges. We must seize the opportunities presented by researching bacterial-fungal interplay in the human microbiome to address the global spread of drug-resistant fungal pathogens and the drying pipelines of effective antifungal drugs.

Characterization of the Biosynthetic Gene Cluster and Shunt Products Yields Insights into the Biosynthesis of Balmoralmycin.

Angucyclines are a family of structurally diverse, aromatic polyketides with some members that exhibit potent bioactivity. Angucyclines have also attracted considerable attention due to the intriguing biosynthetic origins that underlie their structural complexity and diversity. Balmoralmycin (compound 1) represents a unique group of angucyclines that contain an angular benz[α]anthracene tetracyclic system, a characteristic C-glycosidic bond-linked deoxy-sugar (d-olivose), and an unsaturated fatty acid chain. In this study, we identified a Streptomyces strain that produces balmoralmycin and seven biosynthetically related coproducts (compounds 2-8). Four of the coproducts (compounds 5-8) are novel compounds that feature a highly oxygenated or fragmented lactone ring, and three of them (compounds 3-5) exhibited cytotoxicity against the human pancreatic cancer cell line MIA PaCa-2 with IC50 values ranging from 0.9 to 1.2 µg/mL. Genome sequencing and CRISPR/dCas9-assisted gene knockdown led to the identification of the ~43 kb balmoralmycin biosynthetic gene cluster (bal BGC). The bal BGC encodes a type II polyketide synthase (PKS) system for assembling the angucycline aglycone, six enzymes for generating the deoxysugar d-olivose, and a hybrid type II/III PKS system for synthesizing the 2,4-decadienoic acid chain. Based on the genetic and chemical information, we propose a mechanism for the biosynthesis of balmoralmycin and the shunt products. The chemical and genetic studies yielded insights into the biosynthetic origin of the structural diversity of angucyclines. IMPORTANCE Angucyclines are structurally diverse aromatic polyketides that have attracted considerable attention due to their potent bioactivity and intriguing biosynthetic origin. Balmoralmycin is a representative of a small family of angucyclines with unique structural features and an unknown biosynthetic origin. We report a newly isolated Streptomyces strain that produces balmoralmycin in a high fermentation titer as well as several structurally related shunt products. Based on the chemical and genetic information, a biosynthetic pathway that involves a type II polyketide synthase (PKS) system, cyclases/aromatases, oxidoreductases, and other ancillary enzymes was established. The elucidation of the balmoralmycin pathway enriches our understanding of how structural diversity is generated in angucyclines and opens the door for the production of balmoralmycin derivatives via pathway engineering.

Comprehensive Interactome Analysis for the Sole Adenylyl Cyclase Cyr1 of Candida albicans.

Cyr1, the sole adenylyl cyclase of the fungal pathogen Candida albicans, is a central component of the cAMP/protein kinase A signaling pathway that controls the yeast-to-hypha transition. Cyr1 is a multivalent sensor and integrator of various external and internal signals. To better understand how these signals are relayed to Cyr1 to regulate its activity, we sought to establish the interactome of Cyr1 by using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to identify the proteins that coimmunoprecipitated with Cyr1. The method identified 36 proteins as candidates for authentic Cyr1-interacting partners, together with two known Cyr1-binding proteins, Cap1 and Act1. Fourteen identified proteins belonged to three functional groups, including actin regulation, cell wall components, and mitochondrial activities, that are known to play important roles in cell morphogenesis. To validate the proteomics data, we used biochemical and genetic methods to characterize two cell wall-related proteins, Mp65 and Sln1. First, coimmunoprecipitation confirmed their physical association with Cyr1. Second, deleting either MP65 or SLN1 resulted in severe defects in filamentation on serum plates. This study establishes the first Cyr1 interactome and uncovers a potential role for cell wall proteins in directly regulating Cyr1 activity to determine growth forms in C. albicans. IMPORTANCE A critical virulence trait of the human fungal pathogen Candida albicans is its ability to undergo the yeast-to-hypha transition in response to diverse environmental and cellular stimuli. Previous studies suggested that the sole adenylyl cyclase of C. albicans, Cyr1, is a multivalent signal sensor and integrator synthesizing cAMP to activate the downstream hypha-promoting events through the cAMP/protein kinase A pathway. To fully understand how Cyr1 senses and processes multiple stimuli to generate appropriate signal outputs, it was necessary to identify and characterize Cyr1-interacting partners. This study employed SILAC-based quantitative proteomic approaches and identified 36 Cyr1-associated proteins, many having functions associated with hyphal morphogenesis. Coimmunoprecipitation verified two cell surface proteins, Mp65 and Sln1. Furthermore, genetic and phenotypic analyses demonstrated the cAMP-dependent roles of these two proteins in determining hyphal growth. Our study establishes the first Cyr1 interactome and uncovers new Cyr1 regulators that mediate cell surface signals to influence the growth mode of C. albicans.

Biosynthesis of Tasikamides via Pathway Coupling and Diazonium-Mediated Hydrazone Formation.

Naturally occurring hydrazones are rare despite the ubiquitous usage of synthetic hydrazones in the preparation of organic compounds and functional materials. In this study, we discovered a family of novel microbial metabolites (tasikamides) that share a unique cyclic pentapeptide scaffold. Surprisingly, tasikamides A-C (1-3) contain a hydrazone group (C═N─N) that joins the cyclic peptide scaffold to an alkyl 5-hydroxylanthranilate (AHA) moiety. We discovered that the biosynthesis of 1-3 requires two discrete gene clusters, with one encoding a nonribosomal peptide synthetase (NRPS) pathway for assembling the cyclic peptide scaffold and another encoding the AHA-synthesizing pathway. The AHA gene cluster encodes three ancillary enzymes that catalyze the diazotization of AHA to yield an aryl diazonium species (diazo-AHA). The electrophilic diazo-AHA undergoes nonenzymatic Japp-Klingemann coupling with a ß-keto aldehyde-containing cyclic peptide precursor to furnish the hydrazone group and yield 1-3. The studies together unraveled a novel mechanism whereby specialized metabolites are formed by the coupling of two biosynthetic pathways via an unprecedented in vivo Japp-Klingemann reaction. The findings raise the prospect of exploiting the arylamine-diazotizing enzymes (AAD) for the in vivo synthesis of aryl compounds and modification of biological macromolecules.

From Jekyll to Hyde: The Yeast-Hyphal Transition of Candida albicans.

Candida albicans is a major fungal pathogen of humans, accounting for 15% of nosocomial infections with an estimated attributable mortality of 47%. C. albicans is usually a benign member of the human microbiome in healthy people. Under constant exposure to highly dynamic environmental cues in diverse host niches, C. albicans has successfully evolved to adapt to both commensal and pathogenic lifestyles. The ability of C. albicans to undergo a reversible morphological transition from yeast to filamentous forms is a well-established virulent trait. Over the past few decades, a significant amount of research has been carried out to understand the underlying regulatory mechanisms, signaling pathways, and transcription factors that govern the C. albicans yeast-to-hyphal transition. This review will summarize our current understanding of well-elucidated signal transduction pathways that activate C. albicans hyphal morphogenesis in response to various environmental cues and the cell cycle machinery involved in the subsequent regulation and maintenance of hyphal morphogenesis.

Pathway Retrofitting Yields Insights into the Biosynthesis of Anthraquinone-Fused Enediynes.

Anthraquinone-fused enediynes (AQEs) are renowned for their distinctive molecular architecture, reactive enediyne warhead, and potent anticancer activity. Although the first members of AQEs, i.e., dynemicins, were discovered three decades ago, how their nitrogen-containing carbon skeleton is synthesized by microbial producers remains largely a mystery. In this study, we showed that the recently discovered sungeidine pathway is a "degenerative" AQE pathway that contains upstream enzymes for AQE biosynthesis. Retrofitting the sungeidine pathway with genes from the dynemicin pathway not only restored the biosynthesis of the AQE skeleton but also produced a series of novel compounds likely as the cycloaromatized derivatives of chemically unstable biosynthetic intermediates. The results suggest a cascade of highly surprising biosynthetic steps leading to the formation of the anthraquinone moiety, the hallmark C8-C9 linkage via alkyl-aryl cross-coupling, and the characteristic epoxide functionality. The findings provide unprecedented insights into the biosynthesis of AQEs and pave the way for examining these intriguing biosynthetic enzymes.

Role of the oral microbiome, metabolic pathways, and novel diagnostic tools in intra-oral halitosis: a comprehensive update.

Halitosis or oral malodor is one of the most common reasons for the patients' visit to the dental clinic, ranking behind only dental caries and periodontitis. In the present times, where social and professional communications are becoming unavoidable, halitosis has become a concern of growing importance. Oral malodor mostly develops due to the putrefaction of substrates by the indigenous bacterial populations. Although culture-based studies have provided adequate information on halitosis, the high throughput omics technologies have amplified the resolution at which oral microbial community can be examined and has led to the detection of a broader range of taxa associated with intra-oral halitosis (IOH). These microorganisms are regulated by the interactions of their ecological processes. Thus to develop effective treatment strategies, it is important to understand the microbial basis of halitosis. In the current review, we provide an update on IOH in context to the role of the oral microbiome, metabolic pathways involved, and novel diagnostic tools, including breathomics. Understanding oral microbiota associated with halitosis from a broader ecological perspective can provide novel insights into one's oral and systemic health. Such information can pave the way for the emergence of diagnostic tools that can revolutionize the early detection of halitosis and various associated medical conditions.

The Proteome of Community Living Candida albicans Is Differentially Modulated by the Morphologic and Structural Features of the Bacterial Cohabitants.

Candida albicans is a commensal polymorphic and opportunistic fungus, which usually resides as a small community in the oral cavities of a majority of humans. The latter eco-system presents this yeast varied opportunities for mutualistic interactions with other cohabitant oral bacteria, that synergizes its persistence and pathogenicity. Collectively, these communities live within complex plaque biofilms which may adversely affect the oral health and increase the proclivity for oral candidiasis. The proteome of such oral biofilms with myriad interkingdom interactions are largely underexplored. Herein, we employed limma differential expression analysis, and cluster analysis to explore the proteomic interactions of C. albicans biofilms with nine different common oral bacterial species, Aggregatibacter actinomycetemcomitans, Actinomyces naeslundii, Fusobacterium nucleatum, Enterococcus faecalis, Porphyromonas gingivalis, Streptococcus mutants, Streptococcus sanguinis, Streptococcus mitis, and Streptococcus sobrinus. Interestingly, upon exposure of C. albicans biofilms to the foregoing heat-killed bacteria, the proteomes of the fungus associated with cellular respiration, translation, oxidoreductase activity, and ligase activity were significantly altered. Subsequent differential expression and cluster analysis revealed the subtle, yet significant alterations in the C. albicans proteome, particularly on exposure to bacteria with dissimilar cell morphologies, and Gram staining characteristics.

Correction: Discovery, biosynthesis and antifungal mechanism of the polyene-polyol meijiemycin.

Correction for 'Discovery, biosynthesis and antifungal mechanism of the polyene-polyol meijiemycin' by Zhen Jie Low et al., Chem. Commun., 2020, DOI: .

Proteomics Analysis of Candida albicans dnm1 Haploid Mutant Unraveled the Association between Mitochondrial Fission and Antifungal Susceptibility.

Candida albicans is a major fungal pathogen, accounting for approximately 15% of healthcare infections with associated mortality as high as 40% in the case of systemic candidiasis. Antifungal agents for C. albicans infections are limited, and rising resistance is an inevitable problem. Therefore, understanding the mechanism behind antifungal responses is among the top research focuses in combating Candida infections. Herein, the recently developed C. albicans haploid model is employed to examine the association between mitochondrial fission, regulated by Dnm1, and the pathogen's response to antifungals. Proteomic analysis of dnm1Δ and its wild-type haploid parent, GZY803, reveal changes in proteins associated with mitochondrial structures and functions, cell wall, and plasma membrane. Antifungal susceptibility testing revealed that dnm1Δ is more susceptible to SM21, a novel antifungal, than GZY803. Analyses of reactive oxygen species release, antioxidant response, lipid peroxidation, and membrane damages uncover an association between dnm1Δ and the susceptibility to SM21. Dynasore-induced mitochondrial inhibition in SC5314 diploids corroborate the findings. Interestingly, Dynasore-primed SC5314 cultures exhibit increased susceptibility to all antifungals tested. These data suggest an important contribution of mitochondrial fission in antifungal susceptibility of C. albicans. Hence, mitochondrial fission can be a potential target for combined therapy in anti-C. albicans treatment.

Discovery, biosynthesis and antifungal mechanism of the polyene-polyol meijiemycin.

Produced by a newly isolated Streptomycetes strain, meijiemycin is a gigantic linear polyene-polyol that exhibits structural features not seen in other members of the polyene-polyol family. We propose a biosynthetic mechanism and demonstrate that meijiemycin inhibits hyphal growth by inducing the aggregation of ergosterol and restructuring of the fungal plasma membrane.

Integrated Genomic and Metabolomic Approach to the Discovery of Potential Anti-Quorum Sensing Natural Products from Microbes Associated with Marine Samples from Singapore.

With 70% of the Earth's surface covered in water, the marine ecosystem offers immense opportunities for drug discovery and development. Due to the decreasing rate of novel natural product discovery from terrestrial sources in recent years, many researchers are beginning to look seaward for breakthroughs in new therapeutic agents. As part of an ongoing marine drug discovery programme in Singapore, an integrated approach of combining metabolomic and genomic techniques were initiated for uncovering novel anti-quorum sensing molecules from bacteria associated with subtidal samples collected in the Singapore Strait. Based on the culture-dependent method, a total of 102 marine bacteria strains were isolated and the identities of selected strains were established based on their 16S rRNA gene sequences. About 5% of the marine bacterial organic extracts showed quorum sensing inhibitory (QSI) activity in a dose-dependent manner based on the Pseudomonas aeruginosa QS reporter system. In addition, the extracts were subjected to mass spectrometry-based molecular networking and the genome of selected strains were analysed for known as well as new biosynthetic gene clusters. This study revealed that using integrated techniques, coupled with biological assays, can provide an effective and rapid prioritization of marine bacterial strains for downstream large-scale culturing for the purpose of isolation and structural elucidation of novel bioactive compounds.

MS/MS-Based Molecular Networking Approach for the Detection of Aplysiatoxin-Related Compounds in Environmental Marine Cyanobacteria.

Certain strains of cyanobacteria produce a wide array of cyanotoxins, such as microcystins, lyngbyatoxins and aplysiatoxins, that are associated with public health issues. In this pilot study, an approach combining LC-MS/MS and molecular networking was employed as a rapid analytical method to detect aplysiatoxins present in four environmental marine cyanobacterial samples collected from intertidal areas in Singapore. Based on 16S-ITS rRNA gene sequences, these filamentous cyanobacterial samples collected from Pulau Hantu were determined as Trichodesmium erythraeum, Oscillatoria sp. PAB-2 and Okeania sp. PNG05-4. Organic extracts were prepared and analyzed on LC-HRMS/MS and Global Natural Product Social Molecular Networking (GNPS) for the presence of aplysiatoxin-related molecules. From the molecular networking, six known compounds, debromoaplysiatoxin (1), anhydrodebromoaplysiatoxin (2), 3-methoxydebromoaplysiatoxin (3), aplysiatoxin (4), oscillatoxin A (5) and 31-noroscillatoxin B (6), as well as potential new analogues, were detected in these samples. In addition, differences and similarities in molecular networking clusters related to the aplysiatoxin molecular family were observed in extracts of Trichodesmium erythraeum collected from two different locations and from different cyanobacterial species found at Pulau Hantu, respectively.

Identification of a biosynthetic gene cluster for the polyene macrolactam sceliphrolactam in a Streptomyces strain isolated from mangrove sediment.

Streptomyces are a genus of Actinobacteria capable of producing structurally diverse natural products. Here we report the isolation and characterization of a biosynthetically talented Streptomyces (Streptomyces sp. SD85) from tropical mangrove sediments. Whole-genome sequencing revealed that Streptomyces sp. SD85 harbors at least 52 biosynthetic gene clusters (BGCs), which constitute 21.2% of the 8.6-Mb genome. When cultivated under lab conditions, Streptomyces sp. SD85 produces sceliphrolactam, a 26-membered polyene macrolactam with unknown biosynthetic origin. Genome mining yielded a putative sceliphrolactam BGC (sce) that encodes a type I modular polyketide synthase (PKS) system, several ß-amino acid starter biosynthetic enzymes, transporters, and transcriptional regulators. Using the CRISPR/Cas9-based gene knockout method, we demonstrated that the sce BGC is essential for sceliphrolactam biosynthesis. Unexpectedly, the PKS system encoded by sce is short of one module required for assembling the 26-membered macrolactam skeleton according to the collinearity rule. With experimental data disfavoring the involvement of a trans-PKS module, the biosynthesis of sceliphrolactam seems to be best rationalized by invoking a mechanism whereby the PKS system employs an iterative module to catalyze two successive chain extensions with different outcomes. The potential violation of the collinearity rule makes the mechanism distinct from those of other polyene macrolactams.

N-Lipidated Peptide Dimers: Effective Antibacterial Agents against Gram-Negative Pathogens through Lipopolysaccharide Permeabilization.

Treating infections caused by multidrug-resistant Gram-negative pathogens is challenging, and there is concern regarding the toxicity of the most effective antimicrobials for Gram-negative pathogens. We hypothesized that conjugating a fatty acid moiety onto a peptide dimer could maximize the interaction with lipopolysaccharide (LPS) and facilitate the permeabilization of the LPS barrier, thereby improving potency against Gram-negative pathogens. We systematically designed a series of N-lipidated peptide dimers that are active against Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE). The optimized lipid length was 6-10 carbons. At these lipid lengths, the N-lipidated peptide dimers exhibited strong LPS permeabilization. Compound 23 exhibited synergy with select antibiotics in most of the combinations tested. 23 and 32 also displayed rapid bactericidal activity. Importantly, 23 and 32 were nonhemolytic at 10 mg/mL, with no cellular or in vivo toxicity. These characteristics suggest that these compounds can overcome the limitations of current Gram-negative-targeted antimicrobials such as polymyxin B.

Amino acid modified xanthone derivatives: novel, highly promising membrane-active antimicrobials for multidrug-resistant Gram-positive bacterial infections.

Antibiotic resistance is a critical global health care crisis requiring urgent action to develop more effective antibiotics. Utilizing the hydrophobic scaffold of xanthone, we identified three components that mimicked the action of an antimicrobial cationic peptide to produce membrane-targeting antimicrobials. Compounds 5c and 6, which contain a hydrophobic xanthone core, lipophilic chains, and cationic amino acids, displayed very promising antimicrobial activity against multidrug-resistant Gram-positive bacteria, including MRSA and VRE, rapid time-kill, avoidance of antibiotic resistance, and low toxicity. The bacterial membrane selectivity of these molecules was comparable to that of several membrane-targeting antibiotics in clinical trials. 5c and 6 were effective in a mouse model of corneal infection by S. aureus and MRSA. Evidence is presented indicating that 5c and 6 target the negatively charged bacterial membrane via a combination of electrostatic and hydrophobic interactions. These results suggest that 5c and 6 have significant promise for combating life-threatening infections.