Crosstalk of Highly Purified Microglia and Astrocytes in the Frame of Toll-like Receptor (TLR)2/1 Activation.

The major immune cells of the central nervous systems (CNS) are microglia and astrocytes, subsets of the glial cell population. The crosstalk between glia via soluble signaling molecules plays an indispensable role for neuropathologies, brain development as well as homeostasis. However, the investigation of the microglia-astrocyte crosstalk has been hampered due to the lack of suitable glial isolation methods. In this study, we investigated for the first time the crosstalk between highly purified Toll-like receptor (TLR)2-knock out (TLR2-KO) and wild-type (WT) microglia and astrocytes. We examined the crosstalk of TLR2-KO microglia and astrocytes in the presence of WT supernatants of the respective other glial cell type. Interestingly, we observed a significant TNF release by TLR2-KO astrocytes, which were activated with Pam3CSK4-stimulated WT microglial supernatants, strongly indicating a crosstalk between microglia and astrocytes after TLR2/1 activation. Furthermore, transcriptome analysis using RNA-seq revealed a wide range of significant up- and down-regulated genes such as Cd300, Tnfrsf9 or Lcn2, which might be involved in the molecular conversation between microglia and astrocytes. Finally, co-culturing microglia and astrocytes confirmed the prior results by demonstrating a significant TNF release by WT microglia co-cultured with TLR2-KO astrocytes. Our findings suggest a molecular TLR2/1-dependent conversation between highly pure activated microglia and astrocytes via signaling molecules. Furthermore, we demonstrate the first crosstalk experiments using ∼100% pure microglia and astrocyte mono-/co-cultures derived from mice with different genotypes highlighting the urgent need of efficient glial isolation protocols, which particularly holds true for astrocytes.

Genetic mechanisms for Se(VI) reduction and synthesis of trigonal 1-D nanostructures in Stenotrophomonas bentonitica: Perspectives in eco-friendly nanomaterial production and bioremediation.

Selenate (Se(VI)) is one of the most soluble and toxic species of Se. Microbial Se(VI) reduction is an efficient tool for bioremediation strategies. However, this process is limited to a few microorganisms, and its molecular basis remains unknown. We present detailed Se(VI)-resistance mechanisms under 50 and 200 mM, in Stenotrophomonas bentonitica BII-R7, coupling enzymatic reduction of Se(VI) to formation of less toxic trigonal Se (t-Se). The results reveal a concentration-dependent response. Despite the lack of evidence of Se(VI)-reduction to Se(0) under 50 mM Se(VI), many genes were highly induced, indicating that Se(VI)-resistance could be based on intracellular reduction to Se(IV), mainly through molybdenum-dependent enzymes (e.g. respiratory nitrate reductase), and antioxidant activity by enzymes like glutathione peroxidase. Although exposure to 200 mM provoked a sharp drop in gene expression, a time-dependent process of reduction and formation of amorphous (a), monoclinic (m) and t-Se nanostructures was unravelled: a-Se nanospheres were initially synthesized intracellularly, which would transform into m-Se and finally into t-Se nanostructures during the following phases. This is the first work describing an intracellular Se(VI) reduction and biotransformation process to long-term stable and insoluble t-Se nanomaterials. These results expand the fundamental understanding of Se biogeochemical cycling, and the effectiveness of BII-R7 for bioremediation purposes.

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."

Corallococcus soli sp. Nov., a Soil Myxobacterium Isolated from Subtropical Climate, Chalus County, Iran, and Its Potential to Produce Secondary Metabolites.

A novel myxobacterial strain ZKHCc1 1396T was isolated in 2017 from a soil sample collected along Chalus Road connecting Tehran and Mazandaran, Iran. It was a Gram-negative, rod-shaped bacterial strain that displayed the general features of Corallococcus, including gliding and fruiting body formation on agar and microbial lytic activity. Strain ZKHCc1 1396T was characterized as an aerobic, mesophilic, and chemoheterotrophic bacterium resistant to many antibiotics. The major cellular fatty acids were branched-chain iso-C17:0 2-OH, iso-C15:0, iso-C17:1, and iso-C17:0. The strain showed the highest 16S rRNA gene sequence similarity to Corallococcusterminator CA054AT (99.67%) and C. praedator CA031BT (99.17%), and formed a novel branch both in the 16S rRNA gene sequence and phylogenomic tree. The genome size was 9,437,609 bp, with a DNA G + C content of 69.8 mol%. The strain had an average nucleotide identity (ANI) value lower than the species cut-off (95%), and with the digital DNA-DNA hybridization (dDDH) below the 70% threshold compared to the closest type strains. Secondary metabolite and biosynthetic gene cluster analyses revealed the strain's potential to produce novel compounds. Based on polyphasic taxonomic characterization, we propose that strain ZKHCc1 1396T represents a novel species, Corallococcus soli sp. nov. (NCCB 100659T = CIP 111634T).

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.

Complete Genome Sequencing of Leptospira interrogans Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes.

The ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical L. interrogans isolates from Malaysia. All six contained the larger (4.28-4.56 Mb) and smaller (0.34-0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0-7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49-179 coding sequences. These sequences enabled detailed genomic strain typing (Genome BLAST Distance Phylogeny, DNA-DNA hybridization, and multi locus sequence typing) and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence.

A fetal wave of human type 3 effector γδ cells with restricted TCR diversity persists into adulthood.

Accumulating evidence suggests that the mouse embryonic thymus produces distinct waves of innate effector γδ T cells. However, it is unclear whether this process occurs similarly in humans and whether it comprises a dedicated subset of innate-like type 3 effector γδ T cells. Here, we present a protocol for high-throughput sequencing of TRG and TRD pairs that comprise the clonal γδTCR. In combination with single-cell RNA sequencing, multiparameter flow cytometry, and TCR sequencing, we reveal a high heterogeneity of γδ T cells sorted from neonatal and adult blood that correlated with TCR usage. Immature γδ T cell clusters displayed mixed and diverse TCRs, but effector cell types segregated according to the expression of either highly expanded individual Vδ1+ TCRs or moderately expanded semi-invariant Vγ9Vδ2+ TCRs. The Vγ9Vδ2+ T cells shared expression of genes that mark innate-like T cells, including ZBTB16 (encoding PLZF), KLRB1, and KLRC1, but consisted of distinct clusters with unrelated Vγ9Vδ2+ TCR clones characterized either by TBX21, FCGR3A, and cytotoxicity-associated gene expression (type 1) or by CCR6, RORC, IL23R, and DPP4 expression (type 3). Effector γδ T cells with type 1 and type 3 innate T cell signatures were detected in a public dataset of early embryonic thymus organogenesis. Together, this study suggests that functionally distinct waves of human innate-like effector γδ T cells with semi-invariant Vγ9Vδ2+ TCR develop in the early fetal thymus and persist into adulthood.

Filling the Gaps in the Cyanobacterial Tree of Life-Metagenome Analysis of Stigonema ocellatum DSM 106950, Chlorogloea purpurea SAG 13.99 and Gomphosphaeria aponina DSM 107014.

Cyanobacteria represent one of the most important and diverse lineages of prokaryotes with an unparalleled morphological diversity ranging from unicellular cocci and characteristic colony-formers to multicellular filamentous strains with different cell types. Sequencing of more than 1200 available reference genomes was mainly driven by their ecological relevance (Prochlorococcus, Synechococcus), toxicity (Microcystis) and the availability of axenic strains. In the current study three slowly growing non-axenic cyanobacteria with a distant phylogenetic positioning were selected for metagenome sequencing in order to (i) investigate their genomes and to (ii) uncover the diversity of associated heterotrophs. High-throughput Illumina sequencing, metagenomic assembly and binning allowed us to establish nearly complete high-quality draft genomes of all three cyanobacteria and to determine their phylogenetic position. The cyanosphere of the limnic isolates comprises up to 40 heterotrophic bacteria that likely coexisted for several decades, and it is dominated by Alphaproteobacteria and Bacteriodetes. The diagnostic marker protein RpoB ensured in combination with our novel taxonomic assessment via BLASTN-dependent text-mining a reliable classification of the metagenome assembled genomes (MAGs). The detection of one new family and more than a dozen genera of uncultivated heterotrophic bacteria illustrates that non-axenic cyanobacteria are treasure troves of hidden microbial diversity.

No impact of a short-term climatic "El Niño" fluctuation on gut microbial diversity in populations of the Galápagos marine iguana (Amblyrhynchus cristatus).

Gut microorganisms are crucial for many biological functions playing a pivotal role in the host's well-being. We studied gut bacterial community structure of marine iguana populations across the Galápagos archipelago. Marine iguanas depend heavily on their specialized gut microbiome for the digestion of dietary algae, a resource whose growth was strongly reduced by severe "El Niño"-related climatic fluctuations in 2015/2016. As a consequence, marine iguana populations showed signs of starvation as expressed by a poor body condition. Body condition indices (BCI) varied between island populations indicating that food resources (i.e., algae) are affected differently across the archipelago during 'El Niño' events. Though this event impacted food availability for marine iguanas, we found that reductions in body condition due to "El Niño"-related starvation did not result in differences in bacterial gut community structure. Species richness of gut microorganisms was instead correlated with levels of neutral genetic diversity in the distinct host populations. Our data suggest that marine iguana populations with a higher level of gene diversity and allelic richness may harbor a more diverse gut microbiome than those populations with lower genetic diversity. Since low values of these diversity parameters usually correlate with small census and effective population sizes, we use our results to propose a novel hypothesis according to which small and genetically less diverse host populations might be characterized by less diverse microbiomes. Whether such genetically depauperate populations may experience additional threats from reduced dietary flexibility due to a limited intestinal microbiome is currently unclear and calls for further investigation.

Complete Genome Sequences of Streptococcus suis Pig-Pathogenic Strains 10, 13-00283-02, and 16085/3b.

Streptococcus suis is an important pathogen of pigs that, as a zoonotic agent, can also cause severe disease in humans, including meningitis, endocarditis, and septicemia. We report complete and annotated genomes of S. suis strains 10, 13-00283-02, and 16085/3b, which represent the highly prevalent serotypes cps2, cps7, and cps9, respectively.

T cell receptor repertoires within liver allografts are different to those in the peripheral blood.

BACKGROUND & AIMS: T cells are the main mediators of allogeneic immune responses. Specific T cell clones can be tracked by their unique T cell receptor (TCR), but specificity and function remain elusive and have not been investigated in human liver biopsies thus far. METHODS: TCR repertoire analysis of CD4+, CD8+, and regulatory T cells of the peripheral blood and liver graft was performed in 7 liver transplant recipients with either stable course (non-rejector, NR), subclinical cellular rejection (SCR), or acute cellular rejection (ACR) during an observation period from pre-transplant to 6 years post-transplant. Furthermore, donor-reactive T cells, identified by their expression of CD154 and glycoprotein A repetitions predominant (GARP) after allogeneic activation, were tracked longitudinally in peripheral blood and within the liver allograft. RESULTS: Although overall clonality of the TCR repertoire did not increase in peripheral blood after liver transplantation, clonality of donor-reactive CD4+ and regulatory T cells increased and these clones accumulated within the liver graft. Surprisingly, the TCR repertoires between the liver graft and the periphery were distinct and showed only limited overlap. Notably, during ACR, TCR repertoires aligned suggesting either graft-specific homing or release of activated T cells from the graft. CONCLUSIONS: This is the first study comparing TCR repertoires between liver grafts and blood in patients with NR, SCR, and ACR. Moreover, we attribute specificity and function to a subgroup of intragraft T cell populations. Given the limited overlap between peripheral blood and intragraft repertoires, future studies investigating function and specificities of T cells after liver transplantation should focus on the intragraft immune response. LAY SUMMARY: In solid organ transplantation, T cells are key mediators of the recipient's immune response directed at the transplanted organ. In our study, we characterised the T cell repertoire in a cohort of 7 liver transplant recipients. We demonstrate that donor-specific T cells expand clonally and accumulate in the transplanted liver. Moreover, we show that the composition of T cells in peripheral blood differs from the T cells in the liver allograft, only aligning in the context of acute cellular rejection but not in normal graft or subclinical cellular rejection. This indicates that the intragraft immune response is not mirrored in the peripheral blood. Our findings clarify the importance of protocol liver biopsies in identifying intragraft immune responses for future investigations of allo-directed immune responses.

Complete Genome Sequence and Manual Reannotation of Mycobacterium avium subsp. paratuberculosis Strain DSM 44135.

Here, we report the complete genome sequence of the Mycobacterium avium subsp. paratuberculosis reference strain DSM 44135, amended with a manual genome reannotation. The strain was originally described as M. paratuberculosis strain 6783. It was isolated from feces from a dairy cow in northern Germany.

Crypt residing bacteria and proximal colonic carcinogenesis in a mouse model of Lynch syndrome.

Colorectal cancer is a multifactorial disease involving inherited DNA mutations, environmental factors, gut inflammation and intestinal microbiota. Certain germline mutations within the DNA mismatch repair system are associated with Lynch syndrome tumors including right-sided colorectal cancer with mucinous phenotype and presence of an inflammatory infiltrate. Such tumors are more often associated with bacterial biofilms, which may contribute to disease onset and progression. Inflammatory bowel diseases are also associated with colorectal cancer and intestinal dysbiosis. Herein we addressed the question, whether inflammation can aggravate colorectal cancer development under mismatch repair deficiency. MSH2loxP/loxP Vill-cre mice were crossed into the IL-10-/- background to study the importance of inflammation and mucosal bacteria as a driver of tumorigenesis in a Lynch syndrome mouse model. An increase in large bowel tumorigenesis was found in double knockout mice both under conventional housing and under specific pathogen-free conditions. This increase was mostly due to the development of proximal tumors, a hotspot for tumorigenesis in Lynch syndrome, and was associated with a higher degree of inflammation. Additionally, bacterial invasion into the mucus of tumor crypts was observed in the proximal tumors. Inflammation shifted fecal and mucosal microbiota composition and was associated with enrichment in Escherichia-Shigella as well as Akkermansia, Bacteroides and Parabacteroides genera in fecal samples. Tumor-bearing double knockout mice showed a similar enrichment for Escherichia-Shigella and Parabacteroides. Lactobacilli, Lachnospiraceae and Muribaculaceae family members were depleted upon inflammation. In summary, chronic inflammation aggravates colonic tumorigenesis under mismatch repair deficiency and is associated with a shift in microbiota composition.

Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

Zinc homeostasis is crucial for bacterial cells, since imbalances affect viability. However, in mycobacteria, knowledge of zinc metabolism is incomplete. Mycobacterium smegmatis (MSMEG) is an environmental, nonpathogenic Mycobacterium that is widely used as a model organism to study mycobacterial metabolism and pathogenicity. How MSMEG maintains zinc homeostasis is largely unknown. SmtB and Zur are important regulators of bacterial zinc metabolism. In mycobacteria, these regulators are encoded by an operon, whereas in other bacterial species, SmtB and Zur are encoded on separate loci. Here, we show that the smtB-zur operon is consistently present within the genus Mycobacterium but otherwise found only in Nocardia, Saccharothrix, and Corynebacterium diphtheriae By RNA deep sequencing, we determined the Zur and SmtB regulons of MSMEG and compared them with transcriptional responses after zinc starvation or excess. We found an exceptional genomic clustering of genes whose expression was strongly induced by zur deletion and zinc starvation. These genes encoded zinc importers such as ZnuABC and three additional putative zinc transporters, including the porin MspD, as well as alternative ribosomal proteins. In contrast, only a few genes were affected by deletion of smtB and zinc excess. The zinc exporter ZitA was most prominently regulated by SmtB. Moreover, transcriptional analyses in combination with promoter and chromatin immunoprecipitation assays revealed a special regulation of the smtB-zur operon itself: an apparently zinc-independent, constitutive expression of smtB-zur resulted from sensitive coregulation by both SmtB and Zur. Overall, our data revealed yet unknown peculiarities of mycobacterial zinc homeostasis.IMPORTANCE Zinc is crucial for many biological processes, as it is an essential cofactor of enzymes and a structural component of regulatory and DNA binding proteins. Hence, all living cells require zinc to maintain constant intracellular levels. However, in excess, zinc is toxic. Therefore, cellular zinc homeostasis needs to be tightly controlled. In bacteria, this is achieved by transcriptional regulators whose activity is mediated via zinc-dependent conformational changes promoting or preventing their binding to DNA. SmtB and Zur are important antagonistically acting bacterial regulators in mycobacteria. They sense changes in zinc concentrations in the femtomolar range and regulate transcription of genes for zinc acquisition, storage, and export. Here, we analyzed the role of SmtB and Zur in zinc homeostasis in Mycobacterium smegmatis Our results revealed novel insights into the transcriptional processes of zinc homeostasis in mycobacteria and their regulation.

Donor-derived IL-17A and IL-17F deficiency triggers Th1 allo-responses and increases gut leakage during acute GVHD.

IL-17A and IL-17F cytokines are important regulators of acute graft-versus-host-disease (GVHD). However, contrary effects of these cytokines in inflammatory diseases have been reported. To investigate the effects of donor-derived IL-17A and IL-17F on GVHD, we made use of single (Il17a-/- or Il17f-/-) and double deficient (Il17af-/-) allogeneic donor CD4+ T cells. We could demonstrate that transplantation of Il17af-/- CD4+ donor T cells led to aggravated GVHD. However, this phenotype was not observed after transplantation of single, Il17a-/- or Il17f-/-, deficient CD4+ T cells, suggesting redundant effects of IL-17A and IL-17F. Moreover, Il17af-/- cell recipients showed an increase of systemic IFNγ, indicating a heightened pro-inflammatory state, as well as infiltration of IFNγ-secreting CD4+ T cells in the recipients' intestinal tract. These recipients exhibited significant gut leakage, and markedly macrophage infiltration in the gastrointestinal epithelial layer. Moreover, we saw evidence of impaired recovery of gut epithelial cells in recipients of Il17af-/- CD4+ T cells. In this study, we show that IL-17A/F double deficiency of donor CD4+ T cells leads to accelerated GVHD and therefore highlight the importance of these cytokines. Together, IL-17 cytokines might serve as a brake to an intensified Th1 response, leading to the exacerbated gut damage in acute GVHD.

Low-load pathogen spillover predicts shifts in skin microbiome and survival of a terrestrial-breeding amphibian.

Wildlife disease dynamics are strongly influenced by the structure of host communities and their symbiotic microbiota. Conspicuous amphibian declines associated with the waterborne fungal pathogen Batrachochytrium dendrobatidis (Bd) have been observed in aquatic-breeding frogs globally. However, less attention has been given to cryptic terrestrial-breeding amphibians that have also been declining in tropical regions. By experimentally manipulating multiple tropical amphibian assemblages harbouring natural microbial communities, we tested whether Bd spillover from naturally infected aquatic-breeding frogs could lead to Bd amplification and mortality in our focal terrestrial-breeding host: the pumpkin toadlet Brachycephalus pitanga. We also tested whether the strength of spillover could vary depending on skin bacterial transmission within host assemblages. Terrestrial-breeding toadlets acquired lethal spillover infections from neighbouring aquatic hosts and experienced dramatic but generally non-protective shifts in skin bacterial composition primarily attributable to their Bd infections. By contrast, aquatic-breeding amphibians maintained mild Bd infections and higher survival, with shifts in bacterial microbiomes that were unrelated to Bd infections. Our results indicate that Bd spillover from even mildly infected aquatic-breeding hosts may lead to dysbiosis and mortality in terrestrial-breeding species, underscoring the need to further investigate recent population declines of terrestrial-breeding amphibians in the tropics.

Community richness of amphibian skin bacteria correlates with bioclimate at the global scale.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.

Day and Night: Metabolic Profiles and Evolutionary Relationships of Six Axenic Non-Marine Cyanobacteria.

Cyanobacteria are dominant primary producers of various ecosystems and they colonize marine as well as freshwater and terrestrial habitats. On the basis of their oxygenic photosynthesis they are known to synthesize a high number of secondary metabolites, which makes them promising for biotechnological applications. State-of-the-art sequencing and analytical techniques and the availability of several axenic strains offer new opportunities for the understanding of the hidden metabolic potential of cyanobacteria beyond those of single model organisms. Here, we report comprehensive genomic and metabolic analyses of five non-marine cyanobacteria, that is, Nostoc sp. DSM 107007, Anabaena variabilis DSM 107003, Calothrix desertica DSM 106972, Chroococcidiopsis cubana DSM 107010, Chlorogloeopsis sp. PCC 6912, and the reference strain Synechocystis sp. PCC 6803. Five strains that are prevalently belonging to the order Nostocales represent the phylogenetic depth of clade B1, a morphologically highly diverse sister lineage of clade B2 that includes strain PCC 6803. Genome sequencing, light and scanning electron microscopy revealed the characteristics and axenicity of the analyzed strains. Phylogenetic comparisons showed the limits of the 16S rRNA gene for the classification of cyanobacteria, but documented the applicability of a multilocus sequence alignment analysis based on 43 conserved protein markers. The analysis of metabolites of the core carbon metabolism showed parts of highly conserved metabolic pathways as well as lineage specific pathways such as the glyoxylate shunt, which was acquired by cyanobacteria at least twice via horizontal gene transfer. Major metabolic changes were observed when we compared alterations between day and night samples. Furthermore, our results showed metabolic potential of cyanobacteria beyond Synechocystis sp. PCC 6803 as model organism and may encourage the cyanobacterial community to broaden their research to related organisms with higher metabolic activity in the desired pathways.

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.