The effect of site-specific recombinases XerCD on the removal of over-replicated chromosomal DNA through outer membrane vesicles in bacteria.

Outer membrane vesicles (OMVs) are universally produced by Gram-negative bacteria and play important roles in symbiotic and pathogenic interactions. The DNA from the lumen of OMVs from the Alphaproteobacterium Dinoroseobacter shibae was previously shown to be enriched for the region around the terminus of replication ter and specifically for the recognition sequence dif of the two site-specific recombinases XerCD. These enzymes are highly conserved in bacteria and play an important role in the last phase of cell division. Here, we show that a similar enrichment of ter and dif is found in the DNA inside OMVs from Prochlorococcus marinus, Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli. The deletion of xerC or xerD in E. coli reduced the enrichment peak directly at the dif sequence, while the enriched DNA region around ter became broader, demonstrating that either enzyme influences the DNA content inside the lumen of OMVs. We propose that the intra-vesicle DNA originated from over-replication repair and the XerCD enzymes might play a role in this process, providing them with a new function in addition to resolving chromosome dimers.IMPORTANCEImprecise termination of replication can lead to over-replicated parts of bacterial chromosomes that have to be excised and removed from the dividing cell. The underlying mechanism is poorly understood. Our data show that outer membrane vesicles (OMVs) from diverse Gram-negative bacteria are enriched for DNA around the terminus of replication ter and the site-specific XerCD recombinases influence this enrichment. Clearing the divisome from over-replicated parts of the bacterial chromosome might be a so far unrecognized and conserved function of OMVs.

Engraftment of essential functions through multiple fecal microbiota transplants in chronic antibiotic-resistant pouchitis-a case study using metatranscriptomics.

BACKGROUND: Ileal pouch-anal anastomosis (IPAA) is the standard of care after total proctocolectomy for ulcerative colitis (UC). Around 50% of patients will experience pouchitis, an idiopathic inflammatory condition. Antibiotics are the backbone of treatment of pouchitis; however, antibiotic-resistant pouchitis develops in 5-10% of those patients. It has been shown that fecal microbiota transplantation (FMT) is an effective treatment for UC, but results for FMT antibiotic-resistant pouchitis are inconsistent. METHODS: To uncover which metabolic activities were transferred to the recipients during FMT and helped the remission, we performed a longitudinal case study of the gut metatranscriptomes from three patients and their donors. The patients were treated by two to three FMTs, and stool samples were analyzed for up to 140 days. RESULTS: Reduced expression in pouchitis patients compared to healthy donors was observed for genes involved in biosynthesis of amino acids, cofactors, and B vitamins. An independent metatranscriptome dataset of UC patients showed a similar result. Other functions including biosynthesis of butyrate, metabolism of bile acids, and tryptophan were also much lower expressed in pouchitis. After FMT, these activities transiently increased, and the overall metatranscriptome profiles closely mirrored those of the respective donors with notable fluctuations during the subsequent weeks. The levels of the clinical marker fecal calprotectin were concordant with the metatranscriptome data. Faecalibacterium prausnitzii represented the most active species contributing to butyrate synthesis via the acetyl-CoA pathway. Remission occurred after the last FMT in all patients and was characterized by a microbiota activity profile distinct from donors in two of the patients. CONCLUSIONS: Our study demonstrates the clear but short-lived activity engraftment of donor microbiota, particularly the butyrate biosynthesis after each FMT. The data suggest that FMT triggers shifts in the activity of patient microbiota towards health which need to be repeated to reach critical thresholds. As a case study, these insights warrant cautious interpretation, and validation in larger cohorts is necessary for generalized applications. In the long run, probiotics with high taxonomic diversity consisting of well characterized strains could replace FMT to avoid the costly screening of donors and the risk of transferring unwanted genetic material. Video Abstract.

Multi-omics analysis reveals the molecular response to heat stress in a "red tide" dinoflagellate.

BACKGROUND: "Red tides" are harmful algal blooms caused by dinoflagellate microalgae that accumulate toxins lethal to other organisms, including humans via consumption of contaminated seafood. These algal blooms are driven by a combination of environmental factors including nutrient enrichment, particularly in warm waters, and are increasingly frequent. The molecular, regulatory, and evolutionary mechanisms that underlie the heat stress response in these harmful bloom-forming algal species remain little understood, due in part to the limited genomic resources from dinoflagellates, complicated by the large sizes of genomes, exhibiting features atypical of eukaryotes. RESULTS: We present the de novo assembled genome (~ 4.75 Gbp with 85,849 protein-coding genes), transcriptome, proteome, and metabolome from Prorocentrum cordatum, a globally abundant, bloom-forming dinoflagellate. Using axenic algal cultures, we study the molecular mechanisms that underpin the algal response to heat stress, which is relevant to current ocean warming trends. We present the first evidence of a complementary interplay between RNA editing and exon usage that regulates the expression and functional diversity of biomolecules, reflected by reduction in photosynthesis, central metabolism, and protein synthesis. These results reveal genomic signatures and post-transcriptional regulation for the first time in a pelagic dinoflagellate. CONCLUSIONS: Our multi-omics analyses uncover the molecular response to heat stress in an important bloom-forming algal species, which is driven by complex gene structures in a large, high-G+C genome, combined with multi-level transcriptional regulation. The dynamics and interplay of molecular regulatory mechanisms may explain in part how dinoflagellates diversified to become some of the most ecologically successful organisms on Earth.

The microbiome of the dinoflagellate Prorocentrum cordatum in laboratory culture and its changes at higher temperatures.

In the ocean, phytoplankton are dependent on communities of bacteria living in the phycosphere, a hot spot of metabolic and genetic exchange. Many types of interactions between phytoplankton and phycosphere bacteria have been shown, but it is unclear if the microbial communities associated with microalgae strains in culture collections are beneficial or harmful to the host strain. Here, we studied the microbial communities associated with four strains of the dinoflagellate Prorocentrum cordatum that had been isolated from distant geographical locations and maintained in culture collection for hundreds of generations. Community composition was determined by 16S rRNA gene amplicon sequencing. The dinoflagellate host strain was the strongest parameter separating communities, while growth phase, lifestyle (particle-attached versus free-living) and temperature had only a modulating effect. Although the strains had been isolated from distant locations in the Atlantic and Pacific Ocean, 14 ASVs were shared among all strains, the most abundant ones being Gilvibacter, Marivita, uncultivated Rhodobacteraceae, Marinobacter, Hyphomonadaceae, Cupriavidus, Variovorax, and Paucibacter. Adaptation to higher temperatures resulted in specific changes in each phycosphere microbiome, including increased abundance of rare community members. We then compared the growth of the four xenic cultures to that of the axenic P. cordatum CCMP1329. At 20°C, growth of the xenic cultures was similar or slower than that of CCMP1329. At 26°C, all four xenic cultures experienced a death phase, while the axenic culture stably remained in the stationary phase. At 30°C, only two of the xenic cultures were able to grow. A shift of dinoflagellate metabolism from autotrophy to mixotrophy and competition between dinoflagellate and bacteria for limiting nutrients, including essential vitamins, may contribute to these differences in growth patterns.

The Sixth Element: a 102-kb RepABC Plasmid of Xenologous Origin Modulates Chromosomal Gene Expression in Dinoroseobacter shibae.

The model organism Dinoroseobacter shibae and many other marine Rhodobacterales (Roseobacteraceae, Alphaproteobacteria) are characterized by a multipartite genome organization. Here, we show that the original isolate (Dshi-6) contained six extrachromosomal replicons (ECRs), whereas the strain deposited at the DSMZ (Dshi-5) lacked a 102-kb plasmid. To determine the role of the sixth plasmid, we investigated the genomic and physiological differences between the two strains. Therefore, both genomes were (re)sequenced, and gene expression, growth, and substrate utilization were examined. For comparison, we included additional plasmid-cured strains in the analysis. In the Dshi-6 population, the conjugative 102-kb RepABC-9 plasmid was present in only about 50% of the cells, irrespective of its experimentally validated stability. In the presence of the sixth plasmid, copy number changes of other ECRs, in particular, a decrease of the 86-kb plasmid, were observed. The most conspicuous finding was the strong influence of plasmids on chromosomal gene expression, especially the repression of the CtrA regulon and the activation of the denitrification gene cluster. Expression is inversely controlled by either the presence of the 102-kb plasmid or the absence of the 86-kb plasmid. We identified regulatory genes on both plasmids, i.e., a sigma 70 factor and a quorum sensing synthase, that might be responsible for these major changes. The tremendous effects that were probably even underestimated challenge the current understanding of the relevance of volatile plasmids not only for the original host but also for new recipients after conjugation. IMPORTANCE Plasmids are small DNA molecules that replicate independently of the bacterial chromosome. The common view of the role of plasmids is dominated by the accumulation of resistance genes, which is responsible for the antibiotic crisis in health care and livestock breeding. Beyond rapid adaptations to a changing environment, no general relevance for the host cell's regulome was attributed to these volatile ECRs. The current study shows for the model organism D. shibae that its chromosomal gene expression is strongly influenced by two plasmids. We provide evidence that the gain or loss of plasmids not only results in minor alterations of the genetic repertoire but also can have tremendous effects on bacterial physiology. The central role of some plasmids in the regulatory network of the host could also explain their persistence despite fitness costs, which has been described as the "plasmid paradox."

Composition and Biogeography of Planktonic Pro- and Eukaryotic Communities in the Atlantic Ocean: Primer Choice Matters.

Basin-scale biogeographic observations of marine pelagic pro- and eukaryotic communities are necessary to understand forces driving community composition and for providing a baseline to monitor global change. Deep sequencing of rRNA genes provides community composition at high resolution; yet, it is unclear how the choice of primers affects biogeographic patterns. Here, we re-amplified 16S rRNA genes from DNA sampled during R/V Polarstern Cruise ANT28-5 over a latitudinal transect across the Atlantic Ocean from 52°S to 47°N using universal V4-V5 primers and compared the results with those obtained previously with V5-V6 bacteria-specific primers. For validation of our results, we inferred community composition based on 16S rRNA genes of metagenomes from the same stations and single amplified genomes (SAGs) from the Global Ocean Reference Genome (GORG) database. We found that the universal V4-V5 primers retrieved SAR11 clades with similar relative proportions as those found in the GORG database while the V5-V6 primers recovered strongly diverging clade abundances. We confirmed an inverse bell-shaped distance-decay relationship and a latitudinal diversity gradient that did not decline linearly with absolute latitude in the Atlantic Ocean. Patterns were modified by sampling depth, sequencing depth, choice of primers, and abundance filtering. Especially richness patterns were not robust to methodological change. This study offers a detailed picture of the Atlantic Ocean microbiome using a universal set of PCR primers that allow for the conjunction of biogeographical patterns among organisms from different domains of life.

Identification of Volatiles of the Dinoflagellate Prorocentrum cordatum.

The dinoflagellate Prorocentrum cordatum, often called P. minimum, is a potentially toxic alga found in algal blooms. Volatile compounds released by the alga might carry important information, e.g., on its physiological state, and may act as chemical messengers. We report here the identification of volatile organic compounds emitted by two strains, xenic P. cordatum CCMP 1529 and axenic P. cordatum CCMP 1329. The volatiles released during culture were identified despite their low production rates, using sensitive methods such as open-system-stripping analysis (OSSA) on Tenax TA desorption tubes, thermodesorption, cryofocusing and GC/MS-analysis. The analyses revealed 16 compounds released from the xenic strain and 52 compounds from the axenic strain. The majority of compounds were apocarotenoids, aromatic compounds and small oxylipins, but new natural products such as 3,7-dimethyl-4-octanolide were also identified and synthesized. The large difference of compound composition between xenic and axenic algae will be discussed.

Fatal affairs - conjugational transfer of a dinoflagellate-killing plasmid between marine Rhodobacterales.

The roseobacter group of marine bacteria is characterized by a mosaic distribution of ecologically important phenotypes. These are often encoded on mobile extrachromosomal replicons. So far, conjugation had only been experimentally proven between the two model organisms Phaeobacter inhibens and Dinoroseobacter shibae. Here, we show that two large natural RepABC-type plasmids from D. shibae can be transferred into representatives of all known major Rhodobacterales lineages. Complete genome sequencing of the newly established Phaeobacter inhibens transconjugants confirmed their genomic integrity. The conjugated plasmids were stably maintained as single copy number replicons in the genuine as well as the new host. Co-cultivation of Phaeobacter inhibens and the transconjugants with the dinoflagellate Prorocentrum minimum demonstrated that Phaeobacter inhibens is a probiotic strain that improves the yield and stability of the dinoflagellate culture. The transconjugant carrying the 191 kb plasmid, but not the 126 kb sister plasmid, killed the dinoflagellate in co-culture.

Dinoroseobacter shibae Outer Membrane Vesicles Are Enriched for the Chromosome Dimer Resolution Site dif.

Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.

The Influence of Genes on the "Killer Plasmid" of Dinoroseobacter shibae on Its Symbiosis With the Dinoflagellate Prorocentrum minimum.

The marine bacterium Dinoroseobacter shibae shows a Jekyll-and-Hyde behavior in co-culture with the dinoflagellate Prorocentrum minimum: In the initial symbiotic phase it provides the essential vitamins B12 (cobalamin) and B1 (thiamine) to the algae. In the later pathogenic phase it kills the dinoflagellate. The killing phenotype is determined by the 191 kb plasmid and can be conjugated into other Roseobacters. From a transposon-library of D. shibae we retrieved 28 mutants whose insertion sites were located on the 191 kb plasmid. We co-cultivated each of them with P. minimum in L1 medium lacking vitamin B12. With 20 mutant strains no algal growth beyond the axenic control lacking B12 occurred. Several of these genes were predicted to encode proteins from the type IV secretion system (T4SS). They are apparently essential for establishing the symbiosis. With five transposon mutant strains, the initial symbiotic phase was intact but the later pathogenic phase was lost in co-culture. In three of them the insertion sites were located in an operon predicted to encode genes for biotin (B7) uptake. Both P. minimum and D. shibae are auxotrophic for biotin. We hypothesize that the bacterium depletes the medium from biotin resulting in apoptosis of the dinoflagellate.

Deep sequencing of biofilm microbiomes on dental composite materials.

Background: The microbiome on dental composites has not been studied in detail before. It has not been conclusively clarified whether restorative materials influence the oral microbiome. Methods: We used Illumina Miseq next-generation sequencing of the 16S V1-V2 region to compare the colonisation patterns of bovine enamel (BE) and the composite materials Grandio Flow (GF) and Grandio Blocs (GB) after 48 h in vivo in 14 volunteers. Applying a new method to maintain the oral microbiome ex vivo for 48 h also, we compared the microbiome on GF alone and with the new antimicrobial substance carolacton (GF+C). Results: All in vitro biofilm communities showed a higher diversity and richness than those grown in vivo but the very different atmospheric conditions must be considered. Contrary to expectations, there were only a few significant differences between BE and the composite materials GB and GF either in vivo or in vitro: Oribacterium, Peptostreptococcaceae [XI][G-1] and Streptococcus mutans were more prevalent and Megasphaera, Prevotella oulorum, Veillonella atypica, V. parvula, Gemella morbillorum, and Fusobacterium periodonticum were less prevalent on BE than on composites. In vivo, such preferences were only significant for Granulicatella adiacens (more prevalent on BE) and Fusobacterium nucleatum subsp. animalis (more prevalent on composites). On DNA sequence level, there were no significant differences between the biofilm communities on GF and GF+C. Conclusion: We found that the oral microbiome showed an increased richness when grown on various composites compared to BE in vitro, but otherwise changed only slightly independent of the in vivo or in vitro condition. Our new ex vivo biofilm model might be useful for pre-clinical testing of preventive strategies.

Integrated Transcriptional Regulatory Network of Quorum Sensing, Replication Control, and SOS Response in Dinoroseobacter shibae.

Quorum sensing (QS) coordinates population wide gene expression of bacterial species. Highly adaptive traits like gene transfer agents (GTA), morphological heterogeneity, type 4 secretion systems (T4SS), and flagella are QS controlled in Dinoroseobacter shibae, a Roseobacter model organism. Its QS regulatory network is integrated with the CtrA phosphorelay that controls cell division in alphaproteobacteria. To elucidate the network topology, we analyzed the transcriptional response of the QS-negative D. shibae strain ΔluxI1 toward externally added autoinducer (AI) over a time period of 3 h. The signaling cascade is initiated by the CtrA phosphorelay, followed by the QS genes and other target genes, including the second messenger c-di-GMP, competence, flagella and pili. Identification of transcription factor binding sites in promoters of QS induced genes revealed the integration of QS, CtrA phosphorelay and the SOS stress response mediated by LexA. The concentration of regulatory genes located close to the origin or terminus of replication suggests that gene regulation and replication are tightly coupled. Indeed, addition of AI first stimulates and then represses replication. The restart of replication comes along with increased c-di-GMP levels. We propose a model in which QS induces replication followed by differentiation into GTA producing and non-producing cells. CtrA-activity is controlled by the c-di-GMP level, allowing some of the daughter cells to replicate again. The size of the GTA producing subpopulation is tightly controlled by QS via the AI Synthase LuxI2. Finally, induction of the SOS response allows for integration of GTA DNA into the host chromosome.

The complete genome sequence of Rhodobaca barguzinensis alga05 (DSM 19920) documents its adaptation for life in soda lakes.

Soda lakes, with their high salinity and high pH, pose a very challenging environment for life. Microorganisms living in these harsh conditions have had to adapt their physiology and gene inventory. Therefore, we analyzed the complete genome of the haloalkaliphilic photoheterotrophic bacterium Rhodobaca barguzinensis strain alga05. It consists of a 3,899,419 bp circular chromosome with 3624 predicted coding sequences. In contrast to most of Rhodobacterales, this strain lacks any extrachromosomal elements. To identify the genes responsible for adaptation to high pH, we compared the gene inventory in the alga05 genome with genomes of 17 reference strains belonging to order Rhodobacterales. We found that all haloalkaliphilic strains contain the mrpB gene coding for the B subunit of the MRP Na+/H+ antiporter, while this gene is absent in all non-alkaliphilic strains, which indicates its importance for adaptation to high pH. Further analysis showed that alga05 requires organic carbon sources for growth, but it also contains genes encoding the ethylmalonyl-CoA pathway for CO2 fixation. Remarkable is the genetic potential to utilize organophosphorus compounds as a source of phosphorus. In summary, its genetic inventory indicates a large flexibility of the alga05 metabolism, which is advantageous in rapidly changing environmental conditions in soda lakes.

Metatranscriptome Analysis of the Vaginal Microbiota Reveals Potential Mechanisms for Protection against Metronidazole in Bacterial Vaginosis.

Bacterial vaginosis (BV) is a prevalent multifactorial disease of women in their reproductive years characterized by a shift from the Lactobacillus species-dominated microbial community toward a taxonomically diverse anaerobic community. For unknown reasons, some women do not respond to therapy. In our recent clinical study, among 37 women diagnosed with BV, 31 were successfully treated with metronidazole, while 6 still had BV after treatment. To discover possible reasons for the lack of response in those patients, we performed a metatranscriptome analysis of their vaginal microbiota, comparing them to the patients who responded. Seven of 8 clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genes of Gardnerella vaginalis were highly upregulated in nonresponding patients. Cas genes, in addition to protecting against phages, might be involved in DNA repair, thus mitigating the bactericidal effect of DNA-damaging agents such as metronidazole. In the second part of our study, we analyzed the vaginal metatranscriptomes of four patients over 3 months and showed high in vivo expression of genes for pore-forming toxins in L. iners and of genes encoding enzymes for the production of hydrogen peroxide and d-lactate in L. crispatusIMPORTANCE Bacterial vaginosis is a serious issue for women in their reproductive years. Although it can usually be cured by antibiotics, the recurrence rate is very high, and some women do not respond to antibiotic therapy. The reasons for that are not known. Therefore, we undertook a study to detect the activity of the complete microbiota in the vaginal fluid of women who responded to antibiotic therapy and compared it to the activity of the microbiota in women who did not respond. We found that one of the most important pathogens in bacterial vaginosis, Gardnerella vaginalis, has activated genes that can repair the DNA damage caused by the antibiotic in those women that do not respond to therapy. Suppressing these genes might be a possibility to improve the antibiotic therapy of bacterial vaginosis.

Horizontal operon transfer, plasmids, and the evolution of photosynthesis in Rhodobacteraceae.

The capacity for anoxygenic photosynthesis is scattered throughout the phylogeny of the Proteobacteria. Their photosynthesis genes are typically located in a so-called photosynthesis gene cluster (PGC). It is unclear (i) whether phototrophy is an ancestral trait that was frequently lost or (ii) whether it was acquired later by horizontal gene transfer. We investigated the evolution of phototrophy in 105 genome-sequenced Rhodobacteraceae and provide the first unequivocal evidence for the horizontal transfer of the PGC. The 33 concatenated core genes of the PGC formed a robust phylogenetic tree and the comparison with single-gene trees demonstrated the dominance of joint evolution. The PGC tree is, however, largely incongruent with the species tree and at least seven transfers of the PGC are required to reconcile both phylogenies. The origin of a derived branch containing the PGC of the model organism Rhodobacter capsulatus correlates with a diagnostic gene replacement of pufC by pufX. The PGC is located on plasmids in six of the analyzed genomes and its DnaA-like replication module was discovered at a conserved central position of the PGC. A scenario of plasmid-borne horizontal transfer of the PGC and its reintegration into the chromosome could explain the current distribution of phototrophy in Rhodobacteraceae.

Screening for inhibitors of mutacin synthesis in Streptococcus mutans using fluorescent reporter strains.

BACKGROUND: Within the polymicrobial dental plaque biofilm, bacteria kill competitors by excreting mixtures of bacteriocins, resulting in improved fitness and survival. Inhibiting their bacteriocin synthesis might therefore be a useful strategy to eliminate specific pathogens. We used Streptococcus mutans, a highly acidogenic inhabitant of dental plaque, as a model and searched for natural products that reduced mutacin synthesis. To this end we fused the promoter of mutacin VI to the GFP+ gene and integrated the construct into the genome of S. mutans UA159 by single homologous recombination. RESULTS: The resulting reporter strain 423p - gfp + was used to screen 297 secondary metabolites from different sources, mainly myxobacteria and fungi, for their ability to reduce the fluorescence of the fully induced reporter strain by > 50% while growth was almost unaffected (> 90% of control). Seven compounds with different chemical structures and different modes of action were identified. Erinacine C was subsequently validated and shown to inhibit transcription of all three mutacins of S. mutans. The areas of the inhibition zones of the sensor strains S. sanguinis and Lactococcus lactis were reduced by 35% to 61% in comparison to controls in the presence of erinacine C, demonstrating that the amount of active mutacins in the culture supernatants of S. mutans was reduced. Erinacines are cyathane diterpenes that were extracted from cultures of the edible mushroom Hericium erinaceus. They have anti-inflammatory, antimicrobial and neuroprotective effects. For erinacine C, a new biological activity was found here. CONCLUSIONS: We demonstrate the successful development of a whole-cell fluorescent reporter for the screening of natural compounds and report that erinacine C suppresses mutacin synthesis in S. mutans without affecting cell viability.

Worlds Apart - Transcriptome Profiles of Key Oral Microbes in the Periodontal Pocket Compared to Single Laboratory Culture Reflect Synergistic Interactions.

Periodontitis is a worldwide prevalent oral disease which results from dysbiosis of the periodontal microbiome. Some of the most active microbial players, e.g., Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum, have extensively been studied in the laboratory, but it is unclear to which extend these findings can be transferred to in vivo conditions. Here we show that the transcriptional profiles of P. gingivalis, T. denticola, and F. nucleatum in the periodontal niche are distinct from those in single laboratory culture and exhibit functional similarities. GO (gene ontology) term enrichment analysis showed up-regulation of transporters, pathogenicity related traits and hemin/heme uptake mechanisms for all three species in vivo. Differential gene expression analysis revealed that cysteine proteases, transporters and hemin/heme-binding proteins were highly up-regulated in the periodontal niche, while genes involved in DNA modification were down-regulated. The data suggest strong interactions between those three species regarding protein degradation, iron up-take, and mobility in vivo, explaining their enhanced synergistic pathogenicity. We discovered a strikingly high frequency of Single Nucleotide Polymorphisms (SNPs) in vivo. For F. nucleatum we discovered a total of 127,729 SNPs in periodontal niche transcripts, which were found in similar frequency in health and disease and covered the entire genome, suggesting continuous evolution in the host. We conclude that metabolic interactions shape gene expression in vivo. Great caution is required when inferring pathogenicity of microbes from laboratory data, and microdiversity is an important adaptive trait of natural communities.

Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random.

Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world's oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a "headful" type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated.

An Unprecedented Medium-Chain Diunsaturated N-acylhomoserine Lactone from Marine Roseobacter Group Bacteria.

N-acylhomoserine lactones (AHLs), bacterial signaling compounds involved in quorum-sensing, are a structurally diverse group of compounds. We describe here the identification, synthesis, occurrence and biological activity of a new AHL, N-((2E,5Z)-2,5-dodecadienoyl)homoserine lactone (11) and its isomer N-((3E,5Z)-3,5-dodecadienoyl)homoserine lactone (13), occurring in several Roseobacter group bacteria (Rhodobacteraceae). The analysis of 26 strains revealed the presence of 11 and 13 in six of them originating from the surface of the macroalgae Fucus spiralis or sediments from the North Sea. In addition, 18 other AHLs were detected in 12 strains. Compound identification was performed by GC/MS. Mass spectral analysis revealed a diunsaturated C12 homoserine lactone as structural element of the new AHL. Synthesis of three likely candidate compounds, 11, 13 and N-((2E,4E)-2,4-dodecadienoyl)homoserine lactone (5), revealed the former to be the natural AHLs. Bioactivity test with quorum-sensing reporter strains showed high activity of all three compounds. Therefore, the configuration and stereochemistry of the double bonds in the acyl chain seemed to be unimportant for the activity, although the chains have largely different shapes, solely the chain length determining activity. In combination with previous results with other Roseobacter group bacteria, we could show that there is wide variance between AHL composition within the strains. Furthermore, no association of certain AHLs with different habitats like macroalgal surfaces or sediment could be detected.

Oxygenated N-Acyl Alanine Methyl Esters (NAMEs) from the Marine Bacterium Roseovarius tolerans EL-164.

The marine bacterium Roseovarius tolerans EL-164 (Rhodobacteraceae) can produce unique N-acylalanine methyl esters (NAMEs) besides strucutrally related N-acylhomoserine lactones (AHLs), bacterial signaling compounds widespread in the Rhodobacteraceae. The structures of two unprecedented NAMEs carrying a rare terminally oxidized acyl chain are reported here. The compounds (Z)-N-16-hydroxyhexadec-9-enoyl-l-alanine methyl ester (Z9-16-OH-C16:1-NAME, 3) and (Z)-N-15-carboxypentadec-9-enoyl-l-alanine methyl ester (16COOH-C16:1-NAME, 4) were isolated, and the structures were determined by NMR and MS experiments. Both compounds were synthesized to prove assignments and to test their biological activity. Finally, non-natural, structurally related Z9-3-OH-C16:1-NAME (18) was synthesized to investigate the mass spectroscopy of structurally related NAMEs. Compound 3 showed moderate antibacterial activity against microorganisms such as Bacillus, Streptococcus, Micrococcus, or Mucor strains. In contrast to AHLs, quorum-sensing or quorum-quenching activity was not observed.