Phaeobacter Inhibens Research Articles

Causes and Consequences of a Variant Strain of Phaeobacter inhibens With Reduced Competition.

Phaeobacter inhibens 2.10 is an effective biofilm former and colonizer of marine surfaces and has the ability to outcompete other microbiota. During biofilm dispersal P. inhibens 2.10 produces heritable phenotypic variants, including those that have a reduced ability to inhibit the co-occurring bacterium Pseudoalteromonas tunicata. However, the genetic changes that underpin the phenotypic variation and what the ecological consequences are for variants within the population are unclear. To answer these questions we sequenced the genomes of strain NCV12a1, a biofilm variant of P. inhibens 2.10 with reduced inhibitory activity and the P. inhibens 2.10 WT parental strain. Genome wide analysis revealed point mutations in genes involved in synthesis of the antibacterial compound tropodithietic acid (TDA) and indirectly in extracellular polymeric substances (EPS) production. However, confocal laser scanning microscopy analyses found little differences in biofilm growth between P. inhibens 2.10 WT (parental) and NCV12a1. P. inhibens NCV12a1 was also not outcompeted in co-cultured biofilms with P. tunicata, despite its reduced inhibitory activity, rather these biofilms were thicker than those produced when the WT strain was co-cultured with P. tunicata. Notably, dispersal populations from biofilms of P. inhibens NCV12a1 had a higher proportion of WT-like morphotypes when co-cultured with P. tunicata. These observations may explain why the otherwise non-inhibiting variant persists in the presence of a natural competitor, adding to our understanding of the relative importance of genetic diversification in microbial biofilms.

The marine bacterium Phaeobacter inhibens secures external ammonium by rapid buildup of intracellular nitrogen stocks.

ABSTRACTReduced nitrogen species are key nutrients for biological productivity in the oceans. Ammonium is often present in low and growth-limiting concentrations, albeit peaks occur during collapse of algal blooms or via input from deep sea upwelling and riverine inflow. Autotrophic phytoplankton exploit ammonium peaks by storing nitrogen intracellularly. In contrast, the strategy of heterotrophic bacterioplankton to acquire ammonium is less well understood. This study revealed the marine bacterium Phaeobacter inhibens DSM 17395, a Roseobacter group member, to have already depleted the external ammonium when only ∼⅓ of the ultimately attained biomass is formed. This was paralleled by a three-fold increase in cellular nitrogen levels and rapid buildup of various nitrogen-containing intracellular metabolites (and enzymes for their biosynthesis) and biopolymers (DNA, RNA and proteins). Moreover, nitrogen-rich cells secreted potential RTX proteins and the antibiotic tropodithietic acid, perhaps to competitively secure pulses of external ammonium and to protect themselves from predation. This complex response may ensure growing cells and their descendants exclusive provision with internal nitrogen stocks. This nutritional strategy appears prevalent also in other roseobacters from distant geographical provenances and could provide a new perspective on the distribution of reduced nitrogen in marine environments, i.e. temporary accumulation in bacterioplankton cells.

A Novel Microbial Culture Chamber Co-cultivation System to Study Algal-Bacteria Interactions Using Emiliania huxleyi and Phaeobacter inhibens as Model Organisms.

Our understanding of microbial natural environments combines in situ experimentation with studies of specific interactions in laboratory-based setups. The purpose of this work was to develop, build and demonstrate the use of a microbial culture chamber enabling both in situ and laboratory-based studies. The design uses an enclosed chamber surrounded by two porous membranes that enables the comparison of growth of two separate microbial populations but allowing free exchange of small molecules. Initially, we tested if the presence of the macroalga Fucus vesiculosus inside the chamber affected colonization of the outer membranes by marine bacteria. The alga did indeed enrich the total population of colonizing bacteria by more than a factor of four. These findings lead us to investigate the effect of the presence of the coccolithophoric alga Emiliania huxleyi on attachment and biofilm formation of the marine bacterium Phaeobacter inhibens DSM17395. These organisms co-exist in the marine environment and have a well-characterized interdependence on secondary metabolites. P. inhibens attached in significantly higher numbers when having access to E. huxleyi as compared to when exposed to sterile media. The experiment was carried out using a wild type (wt) strain as well as a TDA-deficient strain of P. inhibens. The ability of the bacterium to produce the antibacterial compound, tropodithietic acid (TDA) influenced its attachment since the P. inhibens DSM17395 wt strain attached in higher numbers to a surface within the first 48 h of incubation with E. huxleyi as compared to a TDA-negative mutant. Whilst the attachment of the bacterium to a surface was facilitated by presence of the alga, however, we cannot conclude if this was directly affected by the algae or whether biofilm formation was dependent on the production of TDA by P. inhibens, which has been implied by previous studies. In the light of these results, other applications of immersed culture chambers are suggested.

Acyl-group specificity of AHL synthases involved in quorum-sensing in Roseobacter group bacteria.

N-Acylhomoserine lactones (AHLs) are important bacterial messengers, mediating different bacterial traits by quorum sensing in a cell-density dependent manner. AHLs are also produced by many bacteria of the marine Roseobacter group, which constitutes a large group within the marine microbiome. Often, specific mixtures of AHLs differing in chain length and oxidation status are produced by bacteria, but how the biosynthetic enzymes, LuxI homologs, are selecting the correct acyl precursors is largely unknown. We have analyzed the AHL production in Dinoroseobacter shibae and three Phaeobacter inhibens strains, revealing strain-specific mixtures. Although large differences were present between the species, the fatty acid profiles, the pool for the acyl precursors for AHL biosynthesis, were very similar. To test the acyl-chain selectivity, the three enzymes LuxI1 and LuxI2 from D. shibae DFL-12 as well as PgaI2 from P. inhibens DSM 17395 were heterologously expressed in E. coli and the enzymes isolated for in vitro incubation experiments. The enzymes readily accepted shortened acyl coenzyme A analogs, N-pantothenoylcysteamine thioesters of fatty acids (PCEs). Fifteen PCEs were synthesized, varying in chain length from C4 to C20, the degree of unsaturation and also including unusual acid esters, e.g., 2E,11Z-C18:2-PCE. The latter served as a precursor of the major AHL of D. shibae DFL-12 LuxI1, 2E,11Z-C18:2-homoserine lactone (HSL). Incubation experiments revealed that PgaI2 accepts all substrates except C4 and C20-PCE. Competition experiments demonstrated a preference of this enzyme for C10 and C12 PCEs. In contrast, the LuxI enzymes of D. shibae are more selective. While 2E,11Z-C18:2-PCE is preferentially accepted by LuxI1, all other PCEs were not, except for the shorter, saturated C10–C14-PCEs. The AHL synthase LuxI2 accepted only C14 PCE and 3-hydroxydecanoyl-PCE. In summary, chain-length selectivity in AHLs can vary between different AHL enzymes. Both, a broad substrate acceptance and tuned specificity occur in the investigated enzymes.

The Bacterial Symbiont Phaeobacter inhibens Shapes the Life History of Its Algal Host Emiliania huxleyi

Marine microbes form host-associated biofilm communities that are shaped by complex interactions between bacteria and their host. The roseobacter Phaeobacter inhibens exploits both symbiotic and pathogenic niches while interacting with its microalgal host Emiliania huxleyi. During co-cultivation over extended periods with E. huxleyi, we show that P. inhibens selectively kills two host cell types, the diploid calcifying strain and the haploid flagellated strain. Meanwhile, various non-calcifying diploid strains are resistant to this pathogen or the pathogen is avirulent to this cell type. This differential pathogenesis has the potential of dramatically altering the composition of E. huxleyi blooms, which are typically dominated by the roseobacter-susceptible calcifying strain. This cell type makes calcite plates, which are an important sink in the marine carbon cycle and forms part of the marine paleobotanic record. P. inhibens kills the haploid cells, which have been proposed as critical to the survival of the algae, as they readily escape both eukaryotic predation and viral infection. Consequently, bacteria such as P. inhibens could influence E. huxleyi’s life history by selective pathogenesis, thereby altering the composition of cell types within its populations and its bloom-bust lifestyle.

Effect of TDA-producing Phaeobacter inhibens on the fish pathogen Vibrio anguillarum in non-axenic algae and copepod systems.

SummaryThe expanding aquaculture industry plays an important role in feeding the growing human population and with the expansion, sustainable bacterial disease control, such as probiotics, becomes increasingly important. Tropodithietic acid (TDA)‐producing Phaeobacter spp. can protect live feed, for example rotifers and Artemia as well as larvae of turbot and cod against pathogenic vibrios. Here, we show that the emerging live feed, copepods, is unaffected by colonization of the fish pathogen Vibrio anguillarum, making them potential infection vectors. However, TDA‐producing Phaeobacter inhibens was able to significantly inhibit V. anguillarum in non‐axenic cultures of copepod Acartia tonsa and the copepod feed Rhodomonas salina. Vibrio grew to 106 CFU ml−1 and 107 CFU ml−1 in copepod and R. salina cultures, respectively. However, vibrio counts remained at the inoculum level (104 CFU ml−1) when P. inhibens was also added. We further developed a semi‐strain‐specific qPCR for V. anguillarum to detect and quantify the pathogen in non‐axenic systems. In conclusion, P. inhibens efficiently inhibits the fish larval pathogen V. anguillarum in the emerging live feed, copepods, supporting its use as a probiotic in aquaculture. Furthermore, qPCR provides an effective method for detecting vibrio pathogens in complex non‐axenic live feed systems.

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, 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.

Tritonibacter horizontis gen. nov., sp. nov., a member of the Rhodobacteraceae, isolated from the Deepwater Horizon oil spill.

A heterotrophic, Gram-stain-negative, aerobic, sodium-requiring and motile bacterium was isolated from oil-contaminated surface water of the Gulf of Mexico during the Deepwater Horizon oil spill. Strain O3.65T showed highest 16S rRNA gene sequence similarity to Phaeobacter gallaeciensis BS107T and Phaeobacter inhibens T5T, both with 98.3 %, respectively. Based on complete genome analysis, highest similarity was observed to species of the genus Ruegeria. Strain O3.65T exhibited a broad salinity, temperature and pH range of 0.5-10 % NaCl, 4-45 °C and 5.5-9.0, respectively. The DNA G+C content of strain O3.65T was 61.5 mol%. The major respiratory lipoquinone was ubiquinone-10 (Q-10), the most dominant fatty acids (>1 %) comprised 18 : 1ω7c and 18 : 1ω7c 11-methyl, 10 : 0 3OH, 12 : 1 3OH, 14 : 1 3OH/3-oxo-14 : 0, 16 : 0, 16 : 0 2OH, 18 : 1 2OH and 12 : 1. The polar lipid pattern indicated presence of phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid, two unidentified phospholipids and seven unidentified lipids. On Difco marine broth agar, strain O3.65T formed smooth, shiny white to beige and convex colonies with regular edges. Phylogenetic, phylogenomic and phenotypic differences revealed that strain O3.65T represents a new species of a novel genus within the family Rhodobacteraceae, for which we propose the name Tritonibacter horizontis gen. nov., sp. nov. The type strain of the type species is O3.65T (=DSM 101689T=LMG 29740T).

The fate of lysine: Non-targeted stable isotope analysis reveals parallel ways for lysine catabolization in Phaeobacter inhibens.

For a detailed investigation of the degradation of lysine in Phaeobacter inhibens DSM 17395, stable isotope experiments with uniformly 13C labeled L-lysine were carried out with lysine adapted cells and the metabolites were analyzed using GC/MS and HPLC/MS. A non-targeted stable isotope analysis was used which compares labeled and not labeled samples to determine the Mass Isotopomer Distribution not only for known metabolites but for all labeled compounds in our GC/MS analysis. We show that P. inhibens uses at least two parallel pathways for the first degradation steps of lysine. Further investigations identified L-pipecolate as an L-lysine degradation intermediate in P. inhibens. The analysis of HPLC/MS data as well as the labeling data of tricarboxylic acid (TCA) cycle intermediates show that L-lysine is not only catabolized directly to acetyl-CoA but also via the ethylmalonyl-CoA-pathway, leading to entry points into the TCA cycle via acetyl-CoA, succinyl-CoA, and malate. Altogether the presented data give a detailed insight into the catabolization of L-lysine following the fate of 13C labeled carbon via several ways into the TCA cycle.

Characterisation of the l-Cystine β-Lyase PatB from Phaeobacter inhibens: An Enzyme Involved in the Biosynthesis of the Marine Antibiotic Tropodithietic Acid.

The l-cystine β-lyase from Phaeobacter inhibens is involved in the biosynthesis of the sulfur-containing antibiotic tropodithietic acid. The recombinant enzyme was obtained by heterologous expression in Escherichia coli and biochemically characterised by unambiguous chemical identification of the products formed from the substrate l-cystine, investigation of the substrate spectrum, determination of the enzyme kinetics, sequence alignment with closely related homologues and site-directed mutagenesis to identify a highly conserved lysine residue that is critical for functionality. PatB from P. inhibens is a new member of the small group of characterised l-cystine β-lyases and the first example of an enzyme with such an activity that is required for the biosynthesis of an antibiotic. A comparison of PatB to previously reported enzymes with l-cystine β-lyase activity from bacteria and plants is given.

The Exometabolome of Two Model Strains of the Roseobacter Group: A Marketplace of Microbial Metabolites.

Recent studies applying Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) showed that the exometabolome of marine bacteria is composed of a surprisingly high molecular diversity. To shed more light on how this diversity is generated we examined the exometabolome of two model strains of the Roseobacter group, Phaeobacter inhibens and Dinoroseobacter shibae, grown on glutamate, glucose, acetate or succinate by FT-ICR-MS. We detected 2,767 and 3,354 molecular formulas in the exometabolome of each strain and 67 and 84 matched genome-predicted metabolites of P. inhibens and D. shibae, respectively. The annotated compounds include late precursors of biosynthetic pathways of vitamins B1, B2, B5, B6, B7, B12, amino acids, quorum sensing-related compounds, indole acetic acid and methyl-(indole-3-yl) acetic acid. Several formulas were also found in phytoplankton blooms. To shed more light on the effects of some of the precursors we supplemented two B1 prototrophic diatoms with the detected precursor of vitamin B1 HET (4-methyl-5-(β-hydroxyethyl)thiazole) and HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine) and found that their growth was stimulated. Our findings indicate that both strains and other bacteria excreting a similar wealth of metabolites may function as important helpers to auxotrophic and prototrophic marine microbes by supplying growth factors and biosynthetic precursors.

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).

The Composite 259-kb Plasmid of Martelella mediterranea DSM 17316T–A Natural Replicon with Functional RepABC Modules from Rhodobacteraceae and Rhizobiaceae

A multipartite genome organization with a chromosome and many extrachromosomal replicons (ECRs) is characteristic for Alphaproteobacteria. The best investigated ECRs of terrestrial rhizobia are the symbiotic plasmids for legume root nodulation and the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. RepABC plasmids represent the most abundant alphaproteobacterial replicon type. The currently known homologous replication modules of rhizobia and Rhodobacteraceae are phylogenetically distinct. In this study, we surveyed type-strain genomes from the One Thousand Microbial Genomes (KMG-I) project and identified a roseobacter-specific RepABC-type operon in the draft genome of the marine rhizobium Martelella mediterranea DSM 17316T. PacBio genome sequencing demonstrated the presence of three circular ECRs with sizes of 593, 259, and 170-kb. The rhodobacteral RepABC module is located together with a rhizobial equivalent on the intermediate sized plasmid pMM259, which likely originated in the fusion of a pre-existing rhizobial ECR with a conjugated roseobacter plasmid. Further evidence for horizontal gene transfer (HGT) is given by the presence of a roseobacter-specific type IV secretion system on the 259-kb plasmid and the rhodobacteracean origin of 62% of the genes on this plasmid. Functionality tests documented that the genuine rhizobial RepABC module from the Martelella 259-kb plasmid is only maintained in A. tumefaciens C58 (Rhizobiaceae) but not in Phaeobacter inhibens DSM 17395 (Rhodobacteraceae). Unexpectedly, the roseobacter-like replication system is functional and stably maintained in both host strains, thus providing evidence for a broader host range than previously proposed. In conclusion, pMM259 is the first example of a natural plasmid that likely mediates genetic exchange between roseobacters and rhizobia.

Roseochelin B, an Algaecidal Natural Product Synthesized by the Roseobacter Phaeobacter inhibens in Response to Algal Sinapic Acid.

The secondary metabolome of the representative Roseobacter, Phaeobacter inhibens, was examined in response to algal sinapic acid. In addition to roseobacticides, sinapic acid induced the production of two new natural products, roseochelin A and B, which were characterized by NMR and X-ray crystallography. Functional assays showed that roseochelin B binds iron and is algaecidal against the algal host Emiliania huxleyi. It appears to be produced by a rarely observed combination of nonenzymatic and enzymatic transformations.

Phaeobacter porticola sp. nov., an antibiotic-producing bacterium isolated from a sea harbour.

Three heterotrophic, aerobic, brown-pigmented strains, designated P97T, P100 and P104, were isolated from a harbour in the southern North Sea. Phylogenetic analysis of 16S rRNA gene sequences revealed that the isolates are affiliated to the genus Phaeobacter. In silico DNA-DNA hybridization of the genome of strain P97T against those of existing type species indicated that P97T represents a novel species within the genus Phaeobacter, with Phaeobacter inhibens T5T as the closest described organism (29.6 % DNA-DNA relatedness) followed by P. gallaeciensis CIP 105210T (26.4 %). DNA-DNA hybridization demonstrated that the three new strains belong to the same species. The new isolates inhibited Pseudoalteromonas tunicata DSM 14096T, and were Gram-stain-negative, catalase- and oxidase-positive, chemo-organoheterotrophic and motile. Growth occurred at pH 6.5-9.5 (optimum 7.0-8.0) and at 4-30 °C (optimum 20-28 °C). The strains required NaCl for growth. The salinity range was 0.5-6.0 % (w/v) NaCl for P97T and P100, and 0.5-5.0 % for P104, lower than values described for Phaeobacter gallaeciensis and Phaeobacter inhibens. The optimum NaCl concentration for strains P97T and P104 was 2.0-4.0 %, and for P100 was 2.0-3.0 %. Fatty acids (>1 %) comprised 18 : 1ω7c, 16 : 0, 18 : 1 ω7c 11-methyl, 18 : 0, 12 :1, 18 : 2ω7c,12, 10 : 0 3-OH and 12 : 0 3-OH. Polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an aminolipid, one unknown lipid and one additional unknown lipid in strain P97T. The major respiratory quinone was Q10. Based on phylogenetic and phenotypic differences, the strains represent a novel species in the genus Phaeobacter, for which the name Phaeobacter porticola sp. nov. is proposed. The type strain is P97T (=DSM 103148T=LMG 29594T).

The limits to growth - energetic burden of the endogenous antibiotic tropodithietic acid in Phaeobacter inhibens DSM 17395.

Phaeobacter inhibens DSM 17395, a model organism for marine Roseobacter group, was studied for its response to its own antimicrobial compound tropodithietic acid (TDA). TDA biosynthesis is encoded on the largest extrachromosomal element of P. inhibens, the 262 kb plasmid, whose curation leads to an increased growth and biomass yield. In this study, the plasmid-cured strain was compared to the wild-type strain and to transposon mutants lacking single genes of the TDA biosynthesis. The data show that the growth inhibition of the wild-type strain can be mainly attributed to the TDA produced by P. inhibens itself. Oxygen uptake rates remained constant in all strains but the growth rate dropped in the wild-type which supports the recently proposed mode of TDA action. Metabolome analysis showed no metabolic alterations that could be attributed directly to TDA. Taken together, the growth of P. inhibens is limited by its own antibacterial compound due to a partial destruction of the proton gradient which leads to a higher energetic demand. The universal presence of TDA biosynthesis in genome-sequenced isolates of the genus Phaeobacter shows that there must be a high benefit of TDA for P. inhibens in its ecological niche despite the drawback on its metabolism.

Non-Redfield, nutrient synergy and flexible internal elemental stoichiometry in a marine bacterium.

The stoichiometric constraints of algal growth are well understood, whereas there is less knowledge for heterotrophic bacterioplankton. Growth of the marine bacterium Phaeobacter inhibens DSM 17395, belonging to the globally distributed Roseobacter group, was studied across a wide concentration range of NH4+ and PO43−. The unique dataset covers 415 different concentration pairs, corresponding to 207 different molar N:P ratios (from 10−2 to 105). Maximal growth (by growth rate and biomass yield) was observed within a restricted concentration range at N:P ratios (∼50−120) markedly above Redfield. Experimentally determined growth parameters deviated to a large part from model predictions based on Liebig's law of the minimum, thus implicating synergistic co-limitation due to biochemical dependence of resources. Internal elemental ratios of P. inhibens varied with external nutrient supply within physiological constraints, thus adding to the growing evidence that aquatic bacteria can be flexible in their internal elemental composition. Taken together, the findings reported here revealed that P. inhibens is well adapted to fluctuating availability of inorganic N and P, expected to occur in its natural habitat (e.g. colonized algae, coastal areas). Moreover, this study suggests that elemental variability in bacterioplankton needs to be considered in the ecological stoichiometry of the oceans.

Dual function of tropodithietic acid as antibiotic and signaling molecule in global gene regulation of the probiotic bacterium Phaeobacter inhibens

Antibiotics are typically regarded as microbial weapons, but whereas their function at concentrations lethal for bacteria is often well characterized, the role of antibiotics at much lower concentrations as possibly found under natural conditions remains poorly understood. By using whole-transcriptome analyses and phenotypic screenings of the marine bacterium Phaeobacter inhibens we found that the broad-spectrum antibiotic tropodithietic acid (TDA) causes the same regulatory effects in quorum sensing (QS) as the common signaling molecule N-acyl-homoserine lactone (AHL) at concentrations 100-fold lower than the minimal inhibitory concentration against bacteria. Our results show that TDA has a significant impact on the expression of ~10% of the total genes of P. inhibens, in the same manner as the AHL. Furthermore, TDA needs the AHL associated LuxR-type transcriptional regulator, just as the AHL molecule. Low concentrations of antibiotics can obviously have a strong influence on the global gene expression of the bacterium that produces it and drastically change the metabolism and behaviour of the bacterium. For P. inhibens this includes motility, biofilm formation and antibiotic production, all important for settlement on new host-associated surfaces. Our results demonstrate that bacteria can produce antibiotics not only to antagonise other bacteria, but also to mediate QS like endogenous AHL molecules.

Jindonia aestuariivivens gen. nov., sp. nov., isolated from a tidal flat on the south-western sea in Republic of Korea.

A Gram-stain-negative, aerobic, non-flagellated, and coccoid, ovoid or rod-shaped bacterium, designated JDTF-65T, was isolated from a tidal flat on the south-western sea in Republic of Korea. Strain JDTF-65T grew optimally at 25°C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Strain JDTF-65T exhibited 16S rRNA gene sequence similarities of 97.1-97.6% to the type strains of 'Aliisedimentitalea scapharcae', Phaeobacter gallaeciensis, Phaeobacter inhibens, Leisingera aquimarina, Tropicibacter litoreus, Sulfitobacter pseudonitzschiae, and Pseudoseohaeicola caenipelagi. Phylogenetic trees based on 16S rRNA gene sequences showed that strain JDTF-65T forms an independent lineage within the radiation enclosed by the family Rhodobacteraceae. Strain JDTF-65T contained Q-10 as the predominant ubiquinone and C18:1 ω7c as the major fatty acid. The major polar lipids of strain JDTF-65T were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminolipid, and one unidentified lipid. The DNA G+C content of strain JDTF-65T was 56.8 mol% and its DNA-DNA relatedness values with the type strains of the phylogenetically related species were 13-27%. Differential phenotypic properties revealed that strain JDTF-65T is separated from representatives of some phylogenetically related taxa. On the basis of the data presented, strain JDTF-65T represents a new genus and species within the family Rhodobacteraceae, for which the name Jindonia aestuariivivens gen. nov., sp. nov. is proposed. The type strain of Jindonia aestuariivivens is JDTF-65T (=KCTC 52564T =NBRC 112534T).

Photometric Determination of Ammonium and Phosphate in Seawater Medium Using a Microplate Reader

To more efficiently process the large sample numbers for quantitative determination of ammonium (NH₄⁺) and phosphate (orthophosphate, PO₄^3-) generated during comprehensive growth experiments with the marine Roseobacter group member Phaeobacter inhibens DSM 17395, specific colorimetric assays employing a microplate reader (MPR) were established. The NH₄⁺ assay is based on the reaction of NH₄⁺ with hypochlorite and salicylate, yielding a limit of detection of 14 µ<smlcap>M</smlcap>, a limit of quantitation of 36 µ<smlcap>M,</smlcap> and a linear range for quantitative determination up to 200 µ<smlcap>M</smlcap>. The PO₄^3-assay is based on the complex formation of PO₄^3- with ammonium molybdate in the presence of ascorbate and zinc acetate, yielding a limit of detection of 13 µ<smlcap>M</smlcap>, a limit of quantitation of 50 µ<smlcap>M,</smlcap> and a linear range for quantitative determination up to 1 m<smlcap>M</smlcap>. Both MPR-based assays allowed for fast (significantly lower than 1 h) analysis of 21 samples plus standards for calibration (all measured in triplicates) and showed only low variation across a large collection of biological samples.