The Rare Codon AGA Is Involved in Regulation of Pyoluteorin Biosynthesis in Pseudomonas protegens Pf-5

The soil bacterium Pseudomonas protegens Pf-5 can colonize root and seed surfaces of many plants, protecting them from infection by plant pathogenic fungi and oomycetes. The capacity to suppress disease is attributed to Pf-5's production of a large spectrum of antibiotics, which is controlled by complex regulatory circuits operating at the transcriptional and post-transcriptional levels. In this study, we analyzed the genomic sequence of Pf-5 for codon usage patterns and observed that the six rarest codons in the genome are present in all seven known antibiotic biosynthesis gene clusters. In particular, there is an abundance of rare codons in pltR, which encodes a member of the LysR transcriptional regulator family that controls the expression of pyoluteorin biosynthetic genes. To test the hypothesis that rare codons in pltR influence pyoluteorin production, we generated a derivative of Pf-5 in which 23 types of rare codons in pltR were substituted with synonymous preferred codons. The resultant mutant produced pyoluteorin at levels 15 times higher than that of the wild-type Pf-5. Accordingly, the promoter activity of the pyoluteorin biosynthetic gene pltL was 20 times higher in the codon-modified stain than in the wild-type. pltR has six AGA codons, which is the rarest codon in the Pf-5 genome. Substitution of all six AGA codons with preferred Arg codons resulted in a variant of pltR that conferred increased pyoluteorin production and pltL promoter activity. Furthermore, overexpression of tRNAUCUArg, the cognate tRNA for the AGA codon, significantly increased pyoluteorin production by Pf-5. A bias in codon usage has been linked to the regulation of many phenotypes in eukaryotes and prokaryotes but, to our knowledge, this is the first example of the role of a rare codon in the regulation of antibiotic production by a Gram-negative bacterium.Keywords: Pseudomonas protegens, pyoluteorin, rare codon, AGA codon, regulatio

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:L-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism. IMPORTANCE: Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program

Nontoxic Strains of Cyanobacteria Are the Result of Major Gene Deletion Events Induced by a Transposable Element

Blooms that are formed by cyanobacteria consist of toxic and nontoxic strains. The mechanisms that result in the occurrence of nontoxic strains are enigmatic. All the nontoxic strains of the filamentous cyanobacterium Planktothrix that were isolated from 9 European countries were found to have lost 90% of a large microcystin synthetase (mcy) gene cluster that encoded the synthesis of the toxic peptide microcystin (MC). Those strains still contain the flanking regions of the mcy gene cluster along with remnants of the transposable elements that are found in between. The majority of the strains still contain a gene coding for a distinct thioesterase type II (mcyT), which is putatively involved in MC synthesis. The insertional inactivation of mcyT in an MC-producing strain resulted in the reduction of MC synthesis by 94 ± 2% (1 standard deviation). Nontoxic strains that occur in shallow lakes throughout Europe form a monophyletic lineage. A second lineage consists of strains that contain the mcy gene cluster but differ in their photosynthetic pigment composition, which is due to the occurrence of strains that contain phycocyanin or large amounts of phycoerythrin in addition to phycocyanin. Strains containing phycoerythrin typically occur in deep-stratified lakes. The rare occurrence of gene cluster deletion, paired with the evolutionary diversification of the lineages of strains that lost or still contain the mcy gene cluster, needs to be invoked in order to explain the absence or dominance of toxic cyanobacteria in various habitats

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens

Secondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. In Pseudomonas spp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations in gacS or gacA (Gac(-) mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac(-) mutants in Pseudomonas protegens strain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac(-) mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac(-) mutants: a reduced proportion of Gac(-) mutants accumulated when P. protegens Pf-5 was cocultured with Bacillus subtilis than in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution of P. protegens, assessed by the accumulation of Gac(-) mutants. IMPORTANCE Many microorganisms produce antibiotics, which contribute to ecologic fitness in natural environments where microbes constantly compete for resources with other organisms. However, biosynthesis of antibiotics is costly due to the metabolic burdens of the antibiotic-producing microorganism. Our results provide an example of the fitness trade-off associated with antibiotic production. Under noncompetitive conditions, antibiotic biosynthesis led to accumulation of spontaneous mutants lacking a master regulator of antibiotic production. However, relatively few of these spontaneous mutants accumulated when a competitor was present. Results from this work provide information on the evolution of antibiotic biosynthesis and provide a framework for their discovery and regulation

RNA-seq Analysis Reveals That an ECF σ Factor, AcsS, Regulates Achromobactin Biosynthesis in Pseudomonas syringae pv. syringae B728a

Iron is an essential micronutrient for Pseudomonas syringae pv. syringae strain B728a and many other microorganisms; therefore, B728a has evolved methods of iron acquirement including the use of iron-chelating siderophores. In this study an extracytoplasmic function (ECF) sigma factor, AcsS, encoded within the achromobactin gene cluster is shown to be a major regulator of genes involved in the biosynthesis and secretion of this siderophore. However, production of achromobactin was not completely abrogated in the deletion mutant, implying that other regulators may be involved such as PvdS, the sigma factor that regulates pyoverdine biosynthesis. RNA-seq analysis identified 287 genes that are differentially expressed between the AcsS deletion mutant and the wild type strain. These genes are involved in iron response, secretion, extracellular polysaccharide production, and cell motility. Thus, the transcriptome analysis supports a role for AcsS in the regulation of achromobactin production and the potential activity of both AcsS and achromobactin in the plant-associated lifestyle of strain B728a

MIBiG 3.0 : a community-driven effort to annotate experimentally validated biosynthetic gene clusters

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/

Investigations into the microcystin gene cluster from Hapalosiphon hibernicus BZ-3-1 and Planktothrix agardhii CYA 126/8

Thesis (M.S.)--University of Hawaii at Manoa, 2006.Includes bibliographical references (leaves 72-75).xiv, 75 leaves, bound ill. 29 cmHapalosiphon hibernicus BZ-3-1 is a terrestrial cyanobacterium isolated from a soil sample from Maui, Hawaii. It was shown to produce microcystin-LA during screening for protein phosphatase inhibitors. Using a PCR approach involving degenerate primer mixes, which were derived from comparison of the three known gene clusters previously sequenced from Microcystis aeruginosa, Planktothrix agardhii and Anabaeno circinalis. Using the primer mixes the genes mcyA. -B. -C. -G. -N, and -J were amplified in almost their entirety. The genes mcyD and -E did not give amplicons under similar conditions and therefore needed to be amplified in a slightly different manner. Fragments from the 5' and 3' end of each gene (I kb) were amplified with the degenerate primer mixes and then the known fragments were connected together using the gathered sequence. After sequencing the genes, the organization of the gene cluster was established by connecting PCR. The resulting gene cluster yielded two operons, mcyABC and mcyGDJEFIH, which are transcribed in opposite directions from a putative bi-directional promoter. The H. hibernicus cluster shows the greatest similarity to the Anabaena cluster (mcyA. -B. -D. -G. -J) and the nodularin gene cluster from Nodularia (mcy-E. F. -f). It was noticed that the Planktothrix cluster was the only one to contain mcyT and be deficient in mcyI and -F. Genetic studies were then undertaken to disrupt mcyT using homologous recombination. The DNA was introduced into the cyanobacterial cells via electroporation and resistance to chloramphenicol was used to select for the mutant. The mutant was analyzed for peptide production and it was discovered that microcystin biosynthesis had been reduced to 5-10% of that found in the wild type. During characterization of the mutant peptide production a new anabeanopeptin derivative was found Structure determination was accomplished using NMR and HR-MS techniques. Stereochemistry of the amino acid residues was determined by the advanced Marfey analysis using LC-MS

Formation of the Ureido Bond in Anabaenopeptins by ApnA from Planktothrix agardhii CYA 126/8.

The process leading to the formation of the ureido bond linking lysine and tyrosine/arginine, respectively, in the anabaenopeptins has been reconstituted in vitro. Trapping experiments suggest that an enzyme bound lysine -carbamic acid is an intermediate. Labeling experiments with 18O-bicarbonate suggest that a carbamic acid mixed anhydride is formed from the tyrosine/arginine thioester and subsequently rearranged to yield the ureido-dipeptide

Metabolomics Analysis Reveals Both Plant Variety and Choice of Hormone Treatment Modulate Vinca Alkaloid Production in Catharanthus Roseus.

The medicinal plant Catharanthus roseusproduces numerous secondary metabolites of interest for the treatment of many diseases—most notably for the terpene indole alkaloid (TIA) vinblastine, which is used in the treatment of leukemia and Hodgkin’s lymphoma. Historically, methyl jasmonate (MeJA) has been used to induce TIA production, but in the past, this has only been investigated in either whole seedlings, cell culture, or hairy root culture. In this work, we investigate the induction capabilities of MeJA and ethylene, a different phytohormone, in both the shoots and roots of two varieties of C. roseus. Using LCMS and RT-qPCR, we demonstrate the importance of variety selection, as we observe markedly different induction patterns of important TIA precursor compounds. Additionally, both phytohormone choice and concentration have significant effects on TIA biosynthesis. Finally, our study suggests that several early-induction pathway steps as well as pathway-specific genes are likely to be transcriptionally regulated. Our findings highlight the need for a complete set of ’omics resources in commonly used C. roseus varieties.</p