42 research outputs found
A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization
Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102) and sync_2523 (strain CC9311) are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins
Influence of a Guanidine Riboswitch in Bacterial Cells
Bacteria live in environments containing complex ecologies of other microbes that communicate and survive through the action of a variety of small metabolic compounds. One common yet relatively unstudied metabolite is guanidine. Although it can be toxic to cells, recent studies have revealed that guanidine may function as a cellular metabolite through a specialized RNA sequence known as a riboswitch. Within our overall project on improving algal productivity, the focus of this study is to: (a) describe various guanidine riboswitch sequences in bacteria that interact with biofuel producing algae; and (b) determine if guanidine has a positive or negative influence on the growth of the bacteria containing these riboswitches.
Over 2,000 species across four phyla of bacteria contain genes that help overcome guanidine toxicity. Recently it was discovered that guanidine, a small molecule with three nitrogen linked to a single carbon, regulates some of these genes by specific interactions with a segment of mRNA called a riboswitch. In this investigation, we used the largely uncharacterized cyanobacterium ESFC-1, and others across the four phyla, that contain the guanidine riboswitch, of which there are two subtypes. Both of these two subtypes regulate expression of proteins involved in the export and modification of guanidine inside the bacterial cell.
Genome sequence analysis of our guanidine riboswitches indicate that our test bacteria differ in four key highly conserved residues for a guanidine-binding pocket in the model riboswitch. However, structures of the riboswitches may be similar, indicating their functions and guanidine-binding capabilities may be similar
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Scarcity of fixed carbon transfer in a model microbial phototroph–heterotroph interaction
Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii's capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga's fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15NH4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide-a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12 and supported the growth of a B12-dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions
Reaction of O2 with a di-iron protein generates a mixed valent Fe2+/Fe3+ center and peroxide
The gene encoding the cyanobacterial ferritin SynFtn is up-regulated in response to copper stress. Here, we show that, while SynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2 with the di-Fe2+ center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+ form. Iron–O2 chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+ form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2 reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2 bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron–O2 chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not
Developing an intervention to facilitate family communication about inherited genetic conditions, and training genetic counsellors in its delivery.
Many families experience difficulty in talking about an inherited genetic condition that affects one or more of them. There have now been a number of studies identifying the issues in detail, however few have developed interventions to assist families. The SPRinG collaborative have used the UK Medical Research Council's guidance on Developing and Evaluating Complex Interventions, to work with families and genetic counsellors (GCs) to co-design a psycho-educational intervention to facilitate family communication and promote better coping and adaptation to living with an inherited genetic condition for parents and their children (<18 years). The intervention is modelled on multi-family discussion groups (MFDGs) used in psychiatric settings. The MFDG was developed and tested over three phases. First focus groups with parents, young people, children and health professionals discussed whether MFDG was acceptable and proposed a suitable design. Using evidence and focus group data, the intervention and a training manual were developed and three GCs were trained in its delivery. Finally, a prototype MFDG was led by a family therapist and co-facilitated by the three GCs. Data analysis showed that families attending the focus groups and intervention thought MFDG highly beneficial, and the pilot sessions had a significant impact on their family' functioning. We also demonstrated that it is possible to train GCs to deliver the MFDG intervention. Further studies are now required to test the feasibility of undertaking a definitive randomised controlled trial to evaluate its effectiveness in improving family outcomes before implementing into genetic counselling practice.The National Institute of Health Research funded the study but any views expressed do not necessarily reflect those of the Authority. Funded by NIHR reference number: RP-DG-1211-10015
Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.
Eukaryotic gene transcription is accompanied by acetylation and methylation of nucleosomes near promoters, but the locations and roles of histone modifications elsewhere in the genome remain unclear. We determined the chromatin modification states in high resolution along 30 Mb of the human genome and found that active promoters are marked by trimethylation of Lys4 of histone H3 (H3K4), whereas enhancers are marked by monomethylation, but not trimethylation, of H3K4. We developed computational algorithms using these distinct chromatin signatures to identify new regulatory elements, predicting over 200 promoters and 400 enhancers within the 30-Mb region. This approach accurately predicted the location and function of independently identified regulatory elements with high sensitivity and specificity and uncovered a novel functional enhancer for the carnitine transporter SLC22A5 (OCTN2). Our results give insight into the connections between chromatin modifications and transcriptional regulatory activity and provide a new tool for the functional annotation of the human genome. Activation of eukaryotic gene transcription involves the coordination of a multitude of transcription factors and cofactors on regulatory DNA sequences such as promoters and enhancers and on the chromatin structure containing these elements 1-3 . Promoters are located at the 5¢ ends of genes immediately surrounding the transcriptional start site (TSS) and serve as the point of assembly of the transcriptional machinery and initiation of transcription 4 . Enhancers contribute to the activation of their target genes from positions upstream, downstream or within a target or neighboring gene Recent investigations using chromatin immunoprecipitation (ChIP) and microarray (ChIP-chip) experiments have described the chromatin architecture of transcriptional promoters in yeast, fly and mammalian systems 9 . In a manner largely conserved across species, active promoters are marked by acetylation of various residues of histones H3 and H4 and methylation of H3K4, particularly trimethylation of this residue. Nucleosome depletion is also a general characteristic of active promoters in yeast and flies, although this feature remains to be thoroughly examined in mammalian systems. Although some studies suggest that distal regulatory elements like enhancers may be marked by similar histone modification patterns 10-13 , the distinguishing chromatin features of promoters and enhancers have yet to be determined, hindering our understanding of a predictive histone code for different classes of regulatory elements. Here, we present high-resolution maps of multiple histone modifications and transcriptional regulators in 30 Mb of the human genome, demonstrating that active promoters and enhancers are associated with distinct chromatin signatures that can be used to predict these regulatory elements in the human genome. RESULTS Chromatin architecture and transcription factor localization We performed ChIP-chip analysis 14 to determine the chromatin architecture along 44 human loci selected by the ENCODE consortium as common targets for genomic analysis 15 , totaling 30 Mb
Histone Modifications at Human Enhancers Reflect Global Cell-Type-Specific Gene Expression
The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene1, 2, 3, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome4, 5, 6. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast, enhancers are marked with highly cell-type-specific histone modification patterns, strongly correlate to cell-type-specific gene expression programs on a global scale, and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression
Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans
Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have
fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in
25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16
regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of
correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP,
while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in
Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium
(LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region.
Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant
enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the
refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa,
an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of
PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent
signals within the same regio