Project description:ChIP-seq was performed to map the association of SPA-tagged DnaA across the Escherichia coli MG1655 chromosome during exponential phase growth in LB. As a control to remove background, ChIP-Seq was also performed on SPA-tagged AcpS, a protein that is not known to bind DNA.
Project description:We have identified proliferative cells in adipose tissue expressing adipocyte specific genes, which were named small proliferative adipocytes (SPA). Comparison of gene expression by microarray and real time PCR among stromal vascular cells (SVC), SPA and mature adipocyte (MA) revealed that adipocyte specific genes and several neuronal genes were increased in the order of SVC < SPA < MWA. However, proliferin 2 (PLF) was detected only in SPA. We found that SPA changed to lipid laden cells by incubation with differentiation medium containing insulin/dexamethasone/IBMX. In this study, we investigated the gene expression of differentiated SPA (D-SPA) comparing with differentiated SVC (D-SVC). Further study using microarrays and real time PCR revealed that uncoupling protein 1 (UCP1) and PLF were strongly expressed in D-SPA. These data suggested that SPA might be able to differentiate into beige cells.
Project description:The purpose of this experiment was to demonstrate that SymE-SPA binds DNA in vivo when overexpressed, and identify where in the genome SymE-SPA binds. To do this SymE-SPA and an untagged SymE control were expressed, and areas of the genome enriched for SymE-SPA binding were identified.
Project description:A total of 20 S. aureus spa type t437 isolates were used for exoproteome analyses in the present study. Ten of them were selected from MLVA type (MT) 621 group which has was shown to represent the most predominant class of S. aureus isolates with the spa type t437; the other ten isolates belonged to different MTs, namely MT1870, MT4125, MT1035, MT1297, MT2075, MT1875, MT2322, MT1831, MT4183 and MT1830.
Project description:Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase with reduced susceptibility to beta-lactam antibiotics. Here, we show that preventing the expression of wall teichoic acids (WTAs) genetically or with a TarO inhibitor sensitizes MRSA strains to beta-lactams although PBP2A is still expressed. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of S. aureus COL. in order to further understand the sensitization of strain COL to methicillin by tunicamycin we determined the tunicamycin and methicillin transcriptomes alone and in combination. Methicillin treatment of COL at 500 µg/mL had almost no effect on cell growth rate and, remarkably, the only gene in the transcriptome that showed a more than two-fold change in expression was lytM, which was downregulated. The tunicamycin transcriptome of COL, acquired at 0.4 µg/mL, shows modest changes compared to the untreated control both in terms of the total numbers of affected genes and in the degree of up- or downregulation. Several of the genes upregulated upon tunicamycin treatment are part of the cell wall stress stimulon.COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. transcriptome for COL growing in the presence of both agents showed extensive changes in gene expression. , the cell wall stress stimulon, which was not induced by methicillin when tunicamycin was absent, was clearly induced in its presence and the changes were far more dramatic than observed with tunicamycin alone. ). vraS and vraR, which encode a two component signaling system dedicated to the cell wall regulon, were upregulated 3.8 and 3.7 fold, respectively. Other upregulated cell wall stress stimulon genes include pbp2, fmtA, mvaD (mevalonate diphosphate decarboxylase), crtN (dehydrosqualene desaturase) mvak1 (mevalonate kinase), recU, SAV1424 (methionine sulfoxide reductase A), prsA (peptidyl-prolyl cis/trans isomerase), tcaA (Tca protein) and cwrA. A considerable number of genes were also downregulated upon challenge of COL with the combination of tunicamycin and methicillin. These included sspB, lrgA, dltA, capL, SAS0988, sspA, pflB, and spa. Several of these genes have been found to be downregulated in previous studies of cell wall-active antibiotic challenge of S. aureus.
Project description:The present study focuses on the use of a metaproteomic approach to analyse Black Extrinsic Tooth Stains, a specific type of pigmented extrinsic substance, in a cohort of 96 Children. Metaproteomics is a powerful emerging technology that successfully enabled human protein and bacterial identification of this specific dental biofilm using mass spectrometry. 1600 bacterial proteins were identified in black stains (BS) samples and 2058 proteins in dental plaque (DP) samples whereas 607 and 582 human proteins identified in (BS and DP, respectively). 132 genera bacteria in black stains and dental plaque were identified using phylopeptidomic analysis, showing prevalence of Rothia, Kingella, Nesseria and Pseudopropionibatcterium in black stains samples. We additionally confirmed the metaproteomic approach by performing 16S rRNA. In this work, we showed an interesting diversity of the microbiota and proteome including significant difference between Black stain and dental plaque samples.
Project description:Sequence-based epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) has recently been promoted because it results in unambiguous data sets that can be organized in local and global databases. The replacement of previous typing methods, such as the highly discriminatory pulsed-field gel electrophoresis (PFGE), has been attempted with various markers and typing schemes, including spa typing and multilocus sequence typing. However, despite a number of advantages, none of these methods showed convincing evidence for performance in epidemiological typing comparable to that of PFGE. By using three sets of 48 MRSA strains comprising isolates that were (i) genetically highly diverse, (ii) genetically related, and (iii) obtained from long-term carriers, we analyzed the performance of the four highly polymorphic S. aureus markers: clfA, clfB, fnbA, and spa. Typeability, discriminatory power, in vivo stability, and evolution of these markers were compared to those of PFGE. Clearly, none of the markers alone could match the discriminatory power of PFGE (63 genotypes; index of discrimination of 0.96). Instead, this could be achieved by combining markers in pairs. We showed that by using only 3' partial sequences of approximately 500 bp, the majority of each marker's discriminatory power was displayed, and using the partial sequences, the best performance was obtained with the combination of clfB and spa (57 genotypes; index of discrimination of 0.94). Genetic changes were not observed for any of the sequence markers over a period of 3 years and in the case of partial sequences for a period of more than 4 years. This is in contrast to PFGE where changes occurred after several months. The genetic differences found between isolate pairs of long-term carriers and among highly related isolates indicated clonal evolution. A typing scheme based on 500-bp 3' partial sequences of clfB and spa is proposed.
Project description:Background: Photomorphogenesis is repressed in the dark mainly by an E3 ubiquitin ligase complex comprising CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and four homologous proteins called SUPPRESSOR OF PHYA-105 (SPA1-SPA4) in Arabidopsis. This complex induces the ubiquitination and subsequent degradation of positively acting transcription factors (e.g., HY5, HFR1, PAP1 and others) in the dark to repress photomorphogenesis. Genomic evidence showed a large number of genes regulated by COP1 in the dark, of which many are direct targets of HY5. However, the genomic bases for the constitutive photomorphogenic phenotypes of spaQ remain unknown. Results: Here, we show that >7200 genes are differentially expressed in the spaQ background compared to wild type in the dark. Comparison of the RNAseq data between cop1 and spaQ revealed a large overlapping set of genes regulated by the COP1-SPA complex. In addition, many of the genes coordinately regulated by the COP1-SPA complex are also regulated by HY5 directly and indirectly. Moreover, a large number of genes (~2790) are predominantly regulated by SPA proteins and not by COP1, suggesting that SPA proteins might regulate many biological processes independently of COP1. Conclusions: Taken together, our data reveal that SPA proteins repress photomorphogenesis by controlling expression of a large number of genes in concert with COP1, likely through regulating the abundance of downstream transcription factors in light signaling pathways. Moreover, SPA proteins may function both in a COP1-dependent and –independent manner in regulating many biological processes and developmental pathways in Arabidopsis.