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.

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.

Structural analyses unravel the molecular mechanism of cyclic di-GMP regulation of bacterial chemotaxis via a PilZ adaptor protein.

The bacterial second messenger cyclic di-GMP (c-di-GMP) has emerged as a prominent mediator of bacterial physiology, motility, and pathogenicity. c-di-GMP often regulates the function of its protein targets through a unique mechanism that involves a discrete PilZ adaptor protein. However, the molecular mechanism for PilZ protein-mediated protein regulation is unclear. Here, we present the structure of the PilZ adaptor protein MapZ cocrystallized in complex with c-di-GMP and its protein target CheR1, a chemotaxis-regulating methyltransferase in Pseudomonas aeruginosa This cocrystal structure, together with the structure of free CheR1, revealed that the binding of c-di-GMP induces dramatic structural changes in MapZ that are crucial for CheR1 binding. Importantly, we found that restructuring and repositioning of two C-terminal helices enable MapZ to disrupt the CheR1 active site by dislodging a structural domain. The crystallographic observations are reinforced by protein-protein binding and single cell-based flagellar motor switching analyses. Our studies further suggest that the regulation of chemotaxis by c-di-GMP through MapZ orthologs/homologs is widespread in proteobacteria and that the use of allosterically regulated C-terminal motifs could be a common mechanism for PilZ adaptor proteins. Together, the findings provide detailed structural insights into how c-di-GMP controls the activity of an enzyme target indirectly through a PilZ adaptor protein.

Emerging paradigms for PilZ domain-mediated C-di-GMP signaling.

PilZ domain-containing proteins constitute a large family of bacterial signaling proteins. As a widely distributed protein domain for the binding of the second messenger c-di-GMP, the canonical PilZ domain contains a set of motifs that define the binding site for c-di-GMP and an allosteric switch for propagating local conformational changes. Here, we summarize some new insights gathered from recent studies on the commonly occurring single-domain PilZ proteins, YcgR-like proteins and PilZ domain-containing cellulose synthases. The studies collectively illuminate how PilZ domains function as cis- or trans-regulatory domains that enable c-di-GMP to control the activity of its cellular targets. Overall, the review highlights the diverse protein structure, biological function and regulatory mechanism of PilZ domain-containing proteins, as well as the challenge of deciphering the function and mechanism of orphan PilZ proteins.

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.

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.

A cyclic di-GMP-binding adaptor protein interacts with a chemotaxis methyltransferase to control flagellar motor switching.

The bacterial messenger cyclic diguanylate monophosphate (c-di-GMP) binds to various effectors, the most common of which are single-domain PilZ proteins. These c-di-GMP effectors control various cellular functions and multicellular behaviors at the transcriptional or posttranslational level. We found that MapZ (methyltransferase-associated PilZ; formerly known as PA4608), a single-domain PilZ protein from the opportunistic pathogen Pseudomonas aeruginosa, directly interacted with the methyltransferase CheR1 and that this interaction was enhanced by c-di-GMP. In vitro assays indicated that, in the presence of c-di-GMP, MapZ inhibited CheR1 from methylating the chemoreceptor PctA, which would be expected to increase its affinity for chemoattractants and promote chemotaxis. MapZ localized to the poles of P. aeruginosa cells, where the flagellar motor and other chemotactic proteins, including PctA and CheR1, are also located. P. aeruginosa cells exhibit a random walk behavior by frequently switching the direction of flagellar rotation in a uniform solution. We showed that binding of c-di-GMP to MapZ decreased the frequency of flagellar motor switching and that MapZ was essential for generating the heterogeneous motility typical of P. aeruginosa cell populations and for efficient surface attachment during biofilm formation. Collectively, the studies revealed that c-di-GMP affects flagellar motor output by regulating the methylation of chemoreceptors through a single-domain PilZ adaptor protein.