Christopher Phippen

Influence of Iron on Production of the Antibacterial Compound Tropodithietic Acid and Its Noninhibitory Analog in Phaeobacter inhibens

ABSTRACT Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography–high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens, Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe3+ coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [FeIII(TDA)2] x complex. The pigment could also be produced by precipitation of pure TDA with FeCl3. Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound.

Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator NunF

Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun–nup regulon. Organization of the regulon is similar to clusters found in other CLP‐producing pseudomonads except for the border regions where putative LuxR‐type regulators are located. This study focuses on understanding the regulatory role of the LuxR‐type‐encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real‐time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun–nup regulon. Swarming and biofilm formation was reduced in a nunF knockout mutant suggesting that these CLPs may also play a role in these phenomena as observed in other pseudomonads. Fusion of the nunF promoter region to mCherry showed that nunF is strongly upregulated in response to carbon sources indicating the presence of a fungus suggesting that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens.

Influence of Niche-Specific Nutrients on Secondary Metabolism in Vibrionaceae

UNLABELLED Many factors, such as the substrate and the growth phase, influence biosynthesis of secondary metabolites in microorganisms. Therefore, it is crucial to consider these factors when establishing a bioprospecting strategy. Mimicking the conditions of the natural environment has been suggested as a means of inducing or influencing microbial secondary metabolite production. The purpose of the present study was to determine how the bioactivity of Vibrionaceae was influenced by carbon sources typical of their natural environment. We determined how mannose and chitin, compared to glucose, influenced the antibacterial activity of a collection of Vibrionaceae strains isolated because of their ability to produce antibacterial compounds but that in subsequent screenings seemed to have lost this ability. The numbers of bioactive isolates were 2- and 3.5-fold higher when strains were grown on mannose and chitin, respectively, than on glucose. As secondary metabolites are typically produced during late growth, potential producers were also allowed 1 to 2 days of growth before exposure to the pathogen. This strategy led to a 3-fold increase in the number of bioactive strains on glucose and an 8-fold increase on both chitin and mannose. We selected two bioactive strains belonging to species for which antibacterial activity had not previously been identified. Using ultrahigh-performance liquid chromatography-high-resolution mass spectrometry and bioassay-guided fractionation, we found that the siderophore fluvibactin was responsible for the antibacterial activity of Vibrio furnissii and Vibrio fluvialis These results suggest a role of chitin in the regulation of secondary metabolism in vibrios and demonstrate that considering bacterial ecophysiology during development of screening strategies will facilitate bioprospecting. IMPORTANCE A challenge in microbial natural product discovery is the elicitation of the biosynthetic gene clusters that are silent when microorganisms are grown under standard laboratory conditions. We hypothesized that, since the clusters are not lost during proliferation in the natural niche of the microorganisms, they must, under such conditions, be functional. Here, we demonstrate that an ecology-based approach in which the producer organism is allowed a temporal advantage and where growth conditions are mimicking the natural niche remarkably increases the number of Vibrionaceae strains producing antibacterial compounds.

Phylogenetic distribution of roseobacticides in the Roseobacter group and their effect on microalgae: Distribution of roseobacticides and their effect

The Roseobacter-group species Phaeobacter inhibens produces the antibacterial tropodithietic acid (TDA) and the algaecidal roseobacticides with both compound classes sharing part of the same biosynthetic pathway. The purpose of this study was to investigate the production of roseobacticides more broadly in TDA-producing roseobacters and to compare the effect of producers and non-producers on microalgae. Of 33 roseobacters analyzed, roseobacticide production was a unique feature of TDA-producing P. inhibens, P. gallaeciensis and P. piscinae strains. One TDA-producing Phaeobacter, 27-4, did not produce roseobacticides, possibly due to a transposable element. TDA-producing Ruegeria and Pseudovibrio did not produce roseobacticides. Addition of roseobacticide-containing bacterial extracts affected the growth of the microalgae Rhodomonas salina, Thalassiosira pseudonana and Emiliania huxleyi, while growth of Tetraselmis suecica was unaffected. During co-cultivation, growth of E. huxleyi was initially stimulated by the roseobacticide producer DSM 17395, while the subsequent decline in algal cell numbers during senescence was enhanced. Strain 27-4 that does not produce roseobacticides had no effect on algal growth. Both bacterial strains, DSM 17395 and 27-4, grew during co-cultivation presumably utilizing algal exudates. Furthermore, TDA-producing roseobacters have potential as probiotics in marine larviculture and it is promising that the live feed Tetraselmis was unaffected by roseobacticides-containing extracts.

The FlbA-regulated predicted transcription factor Fum21 of Aspergillus niger is involved in fumonisin production

Aspergillus niger secretes proteins throughout the colony except for the zone that forms asexual spores called conidia. Inactivation of flbA that encodes a regulator of G-protein signaling results in colonies that are unable to reproduce asexually and that secrete proteins throughout the mycelium. In addition, the ΔflbA strain shows cell lysis and has thinner cell walls. Expression analysis showed that 38 predicted transcription factor genes are differentially expressed in strain ΔflbA. Here, the most down-regulated predicted transcription factor gene, called fum21, was inactivated. Growth, conidiation, and protein secretion were not affected in strain Δfum21. Whole genome expression analysis revealed that 63 and 11 genes were down- and up-regulated in Δfum21, respectively, when compared to the wild-type strain. Notably, 24 genes predicted to be involved in secondary metabolism were down-regulated in Δfum21, including 10 out of 12 genes of the fumonisin cluster. This was accompanied by absence of fumonisin production in the deletion strain and a 25% reduction in production of pyranonigrin A. Together, these results link FlbA-mediated sporulation-inhibited secretion with mycotoxin production.

Growth on Chitin Impacts the Transcriptome and Metabolite Profiles of Antibiotic-Producing Vibrio coralliilyticus S2052 and Photobacterium galatheae S2753

The bacterial family Vibrionaceae (vibrios) is considered a major player in the degradation of chitin, the most abundant polymer in the marine environment; however, the majority of studies on the topic have focused on a small number of Vibrio species. In this study, we analyzed the genomes of two vibrios to assess their genetic potential for the degradation of chitin. We then used transcriptomics and metabolomics to demonstrate that chitin strongly affects these vibrios at both the transcriptional and metabolic levels. We observed a strong increase in production of secondary metabolites, suggesting an ecological role for these molecules during chitin colonization in the marine environment. ABSTRACT Members of the Vibrionaceae family are often associated with chitin-containing organisms, and they are thought to play a major role in chitin degradation. The purpose of the present study was to determine how chitin affects the transcriptome and metabolome of two bioactive Vibrionaceae strains, Vibrio coralliilyticus and Photobacterium galatheae. We focused on chitin degradation genes and secondary metabolites based on the assumption that these molecules in nature confer an advantage to the producer. Growth on chitin caused upregulation of genes related to chitin metabolism and of genes potentially involved in host colonization and/or infection. The expression of genes involved in secondary metabolism was also significantly affected by growth on chitin, in one case being 34-fold upregulated. This was reflected in the metabolome, where the antibiotics andrimid and holomycin were produced in larger amounts on chitin. Other polyketide synthase/ nonribosomal peptide synthetase (PKS-NRPS) clusters in P. galatheae were also strongly upregulated on chitin. Collectively, this suggests that both V. coralliilyticus and P. galatheae have a specific lifestyle for growth on chitin and that their secondary metabolites likely play a crucial role during chitin colonization. IMPORTANCE The bacterial family Vibrionaceae (vibrios) is considered a major player in the degradation of chitin, the most abundant polymer in the marine environment; however, the majority of studies on the topic have focused on a small number of Vibrio species. In this study, we analyzed the genomes of two vibrios to assess their genetic potential for the degradation of chitin. We then used transcriptomics and metabolomics to demonstrate that chitin strongly affects these vibrios at both the transcriptional and metabolic levels. We observed a strong increase in production of secondary metabolites, suggesting an ecological role for these molecules during chitin colonization in the marine environment.

Phaeobacter piscinae sp. nov., a species of the Roseobacter group and potential aquaculture probiont

Four heterotrophic, antimicrobial, motile, marine bacterial strains, 27-4T, 8-1, M6-4.2 and S26, were isolated from aquaculture units in Spain, Denmark and Greece. All four strains produced the antibiotic compound tropodithietic acid, which is a key molecule in their antagonism against fish pathogenic bacteria. Cells of the strains were Gram-reaction-negative, rod-shaped and formed star-shaped aggregates in liquid culture and brown-coloured colonies on marine agar. The predominant cellular fatty acids were C18 : 1ω7c, C16 : 0, C11 methyl C18 : 1ω7c and C16 : 0 2-OH, and the polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an aminolipid, a phospholipid and an unidentified lipid. The strains grew optimally at 31-33 °C. Growth was observed at a salt concentration between 0.5 and 5-6 % NaCl with an optimum at 2-3 %. The pH range for growth of the strains was from pH 6 to 8-8.5 with an optimum at pH 7. Based on 16S rRNA gene sequence analysis, the strains are affiliated with the genus Phaeobacter. The genome sequences of the strains have a DNA G+C content of 60.1 % and share an average nucleotide identity (ANI) of more than 95 %. The four strains are distinct from the type strains of the closely related species Phaeobactergallaeciensis and Phaeobacterinhibens based on an ANI of 90.5-91.7 and 89.6-90.4 %, respectively, and an in silico DNA-DNA hybridization relatedness of 43.9-46.9 and 39.8-41.9 %, respectively. On the basis of phylogenetic analyses as well as phenotypic and chemotaxonomic properties, the isolates are considered to represent a novel species, for which the name Phaeobacter piscinae sp. nov. is proposed. The type strain is 27-4T (=DSM 103509T=LMG 29708T).

Phylogenetic distribution of roseobacticides in the Roseobacter group and their effect on microalgae

The Roseobacter-group species Phaeobacter inhibens produces the antibacterial tropodithietic acid (TDA) and the algaecidal roseobacticides with both compound classes sharing part of the same biosynthetic pathway. The purpose of this study was to investigate the production of roseobacticides more broadly in TDA-producing roseobacters and to compare the effect of producers and non-producers on microalgae. Of 33 roseobacters analyzed, roseobacticide production was a unique feature of TDA-producing P. inhibens, P. gallaeciensis and P. piscinae strains. One TDA-producing Phaeobacter strain, 27-4, was unable to produce roseobacticides, possibly due to a transposable element. TDA-producing Ruegeria mobilis and Pseudovibrio did not produce roseobacticides. Addition of roseobacticide-containing bacterial extracts affected the growth of the microalgae Rhodomonas salina, Thalassiosira pseudonana and Emiliania huxleyi, while growth of Tetraselmis suecica was unaffected. During co-cultivation, growth of E. huxleyi was initially stimulated by the roseobacticide producer DSM 17395, while the subsequent decline in algal cell numbers during senescence was enhanced. Strain 27-4 that does not produce roseobacticides had no effect on algal growth. Both bacterial strains, DSM 17395 and 27-4, grew during co-cultivation presumably utilizing algal exudates. Furthermore, TDA-producing roseobacters have potential as probiotics in marine larviculture and it is promising that the live feed Tetraselmis was unaffected by roseobacticides-containing extracts. Originality-significance statement Some Roseobacter-group bacteria produce the antibacterial compound tropodithetic acid (TDA) and have potential as probiotics in marine aquaculture. However, a few of these strains additionally produce algaecidal compounds, the roseobacticides, which would restrict their use in marine larviculture where algae are used as live feed for fish larvae. We herein found that roseobacticides are limited to TDA-producing Phaeobacter strains and were not biosynthesized by TDA-producers outside this genus. Roseobacticides affected several strains of microalgae, but not the chlorophyte that is used as live feed in the aquaculture industry. Thus, the application of Roseobacter strains as probiotics is not hampered. Furthermore, these results demonstrate how Roseobacter-group strains act as gardeners of microalgae and thereby would be involved in environmental processes on a larger scale.

Novofumigatonin biosynthesis involves a non-heme iron-dependent endoperoxide isomerase for orthoester formation

Novofumigatonin (1), isolated from the fungus Aspergillus novofumigatus, is a heavily oxygenated meroterpenoid containing a unique orthoester moiety. Despite the wide distribution of orthoesters in nature and their biological importance, little is known about the biogenesis of orthoesters. Here we show the elucidation of the biosynthetic pathway of 1 and the identification of key enzymes for the orthoester formation by a series of CRISPR-Cas9-based gene-deletion experiments and in vivo and in vitro reconstitutions of the biosynthesis. The novofumigatonin pathway involves endoperoxy compounds as key precursors for the orthoester synthesis, in which the Fe(II)/α-ketoglutarate-dependent enzyme NvfI performs the endoperoxidation. NvfE, the enzyme catalyzing the orthoester synthesis, is an Fe(II)-dependent, but cosubstrate-free, endoperoxide isomerase, despite the fact that NvfE shares sequence homology with the known Fe(II)/α-ketoglutarate-dependent dioxygenases. NvfE thus belongs to a class of enzymes that gained an isomerase activity by losing the α-ketoglutarate-binding ability.Novofumigatonin is a meroterpenoid found in the fungus Aspergillus novofumigatus. Here, the authors elucidate the biosynthetic pathway of novofumigatonin and show that the endoperoxidase NvfI and the endoperoxide isomerase NvfE are involved in it.