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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 Fe(3+) 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 [Fe(III)(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.

SUBMITTER: D'Alvise PW

PROVIDER: S-EPMC4711134 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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

D'Alvise Paul W PW Phippen Christopher B W CB Nielsen Kristian F KF Gram Lone L

Applied and environmental microbiology 20151030 2

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, wh ...[more]

PMID: 26519388

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Project description:Bacteriophages have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. In this study, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and determined its mechanism of infection. Phaeobacter virus MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. The genomic sequence of MD18 displayed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8, but genomic and phylogenetic analyses, assessing host range and a search of available metagenomes are all consistent with the conclusion that Phaeobacter phage MD18 is a novel lytic phage. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, Gene Ontology (GO) term enrichment, and protein associations revealed that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens.IMPORTANCE Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

Project description:Roseobacterclade bacteria are abundant in surface waters and are among the most metabolically diverse and ecologically significant species. This group includes opportunistic symbionts that associate with micro- and macroalgae. We have proposed that one representative member,Phaeobacter inhibens, engages in a dynamic symbiosis with the microalgaEmiliania huxleyi In one phase, mutualistically beneficial molecules are exchanged, including theRoseobacter-produced antibiotic tropodithietic acid (TDA), which is thought to protect the symbiotic interaction. In an alternative parasitic phase, triggered by algal senescence, the bacteria produce potent algaecides, the roseobacticides, which kill the algal host. Here, we employed genetic and biochemical screens to identify the roseobacticide biosynthetic gene cluster. By using a transposon mutagenesis approach, we found that genes required for TDA synthesis-thetdaoperon andpaacatabolon-are also necessary for roseobacticide production. Thus, in contrast to the one-cluster-one-compound paradigm, thetdagene cluster can generate two sets of molecules with distinct structures and bioactivities. We further show that roseobacticide production is quorum sensing regulated via anN-acyl homoserine lactone signal (3-OH-C10-HSL). To ensure tight regulation of algaecide production, and thus of a lifestyle switch from mutualism to parasitism, roseobacticide biosynthesis necessitates the presence of both an algal senescence molecule and a quorum sensing signal.IMPORTANCEMarineRoseobacterspecies are abundant in the oceans and engage in symbiotic interactions with microscopic algae. One member,P. inhibens, produces the antibiotic TDA and a growth hormone thought to protect and promote algal growth. However, in the presence of molecules released by senescing algae, the bacteria produce potent algaecides, the roseobacticides, which kill the host. We examined the regulatory networks and biosynthetic genes required for roseobacticide production. We found thatP. inhibensuses largely the same set of genes for production of both TDA and roseobacticides, thus providing a rare case in which one gene cluster synthesizes two structurally and functionally distinct molecules. Moreover, we found roseobacticide production to be regulated by quorum sensing. Thus, two small molecules, the algal metabolite and the quorum-sensing signal, ensure tight control in the production of roseobacticides. These results highlight the role of small molecules in regulating microbial symbioses.

Dataset Information

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

Publications

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

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