Identification of the regulatory circuit of PhoB in Bacteroides fragilis strain YCH46
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ABSTRACT: Comparison of transcriptional profiles of the wild-type (WT) and the phoB-mutant strain in B. fragilis strain YCH46 during phosphate (Pi) starvation. A four chip study using total RNA isolated from the WT culture and the phoB mutant culture under Pi-excess (6.6 mM) or Pi-limiting (0.0066mM) condition. Each sample contains duplicated data.
Project description:The human gut microbe Bacteroides fragilis can alter the expression of its surface molecules such as capsular polysaccharides and SusC/SusD family outer membrane proteins through the reversible DNA inversions.ã??We have revealed that the outer membrane vesicle (OMV) formation in B. fragilis is regulated by BF2766 (tyrosine recombinase)-mediated DNA inversions at two distantly located promoter regions previously designated as class IV regions. In this study, we aimed to identify the genetic loci associating with the OMV formation by means of transcriptome analysis on the isogenic BF2766 mutants. By comparing the transciptomes of four BF2766 deletion mutants, in which the promoter orientations in class IV-1 and IV-2 regions were locked ON/ON, OFF/ON, OFF/OFF, or ON/OFF, we found that the transcription of the genes downstream of class IV-2 markedly elevated in a hyper-vesiculating ON/ON strain. A four chip study using total RNA isolated from four BF2766 deletion mutants culture. Each sample contains duplicated data.
Project description:Investigation of whole genome gene expression level changes in a Bacteroides fragilis NCTC 9343 delta-ungD1 delta-ungD2 double mutant compared to the wild-type strain. Keywords: expression analysis A six chip study using total RNA recovered from three separate wild-type cultures of Bacteroides fragilis NCTC 9343 and three separate cultures of a double mutant strain, Bacteroides fragilis NCTC 9343 delta-ungD1 delta-ungD2, in which ungD1 (BF1706) and ungD2 (BF2848) are deleted. Each chip measures the expression level of 4,302 genes from Bacteroides fragilis NCTC 9343 and the associated plasmid pBF9343 with fourteen 24-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Investigation of whole genome gene expression level changes in a Bacteroides fragilis NCTC 9343 delta-ungD1 delta-ungD2 delta-PSH triple mutant, compared to the wild-type strain. The mutations engineered into this strain render it acapsular. The mutants analyzed in this study are further described in Coyne, M. J., M. Chatzidaki-Livanis, L. C. Paoletti, and L. E. Comstock. 2008. Role of glycan synthesis in colonization of the mammalian gut by the bacterial symbiont Bacteroides fragilis. PNAS 105(35):13098-13103 (PID 18723678). A six chip study using total RNA recovered from three separate wild-type cultures of Bacteroides fragilis NCTC 9343 and three separate cultures of a triple mutant strain, Bacteroides fragilis NCTC 9343 delta-ungD1 delta-ungD2 delta-PSH, in which ungD1 (BF1706), ungD2 (BF2848), and six genes (BF3454 through BF3459) of the PSH capsular polysaccharide locus are truncated or deleted entirely. Each chip measures the expression level of 4,302 genes from Bacteroides fragilis NCTC 9343 and the associated plasmid pBF9343 with fourteen 24-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Comparison of transcriptional profiles of the wild-type (WT) and the phoB-mutant strain in B. fragilis strain YCH46 during phosphate (Pi) starvation.
Project description:Phosphate is an essential ion for fungal growth, required for proper DNA and phospholipids biosyntesis, energetic metabolism and signal transduction. The systems for inorganic phosphate (Pi) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of Pi at normal or high external Pi concentrations) and a high-affinity (activated in response to Pi starvation). Here, as an initial step to understand the PHO pathway in A. fumigatus, we characterized the PHO80 homologue, phoB(PHO80). We showed that the delta phoB(PHO80) mutant has a severe polar growth defect and there is an interaction between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild type and delta phoB(PHO80) mutant cells showed that Afu4g03610 [phoD(PHO84)], Afu7g06350 [phoE9(PHO89)], Afu4g06020 [phoCPHO81)], and Afu2g09040 (vacuolar transporter Vtc4) were more expressed either in the delta phoB(PHO80) mutant background or in 0.1 mM Pi. Keywords: gene expression array-based (log2 ratio)
Project description:Phosphate is an essential ion for fungal growth, required for proper DNA and phospholipids biosyntesis, energetic metabolism and signal transduction. The systems for inorganic phosphate (Pi) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of Pi at normal or high external Pi concentrations) and a high-affinity (activated in response to Pi starvation). Here, as an initial step to understand the PHO pathway in A. fumigatus, we characterized the PHO80 homologue, phoB(PHO80). We showed that the delta phoB(PHO80) mutant has a severe polar growth defect and there is an interaction between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild type and delta phoB(PHO80) mutant cells showed that Afu4g03610 [phoD(PHO84)], Afu7g06350 [phoE9(PHO89)], Afu4g06020 [phoCPHO81)], and Afu2g09040 (vacuolar transporter Vtc4) were more expressed either in the delta phoB(PHO80) mutant background or in 11 mM Pi. Keywords: gene expression array-based (log2 ratio)
Project description:Bacteria acquire P primarily as inorganic orthophosphate (Pi, PO43-). Once internalized, Pi is rapidly assimilated into biomass during the synthesis of ATP. Because Pi is essential, but excessive ATP is toxic, the acquisition of environmental Pi is tightly regulated. In the bacterium Salmonella enterica (Salmonella), growth in Pi-limiting environments activates the membrane sensor histidine kinase PhoR, leading to the phosphorylation of its cognate transcriptional regulator PhoB and subsequent transcription of genes involved in adaptations to low Pi. Pi limitation is thought to promote PhoR kinase activity by altering the conformation of a membrane signaling complex comprised by PhoR, the multicomponent Pi transporter system PstSACB and the regulatory protein PhoU. The identity of the low Pi signal and how it controls PhoR activity remain unknown. Here we characterize the PhoB-dependent and independent transcriptional changes elicited by Salmonella in response to P starvation, and identify PhoB-independent genes that are required for the utilization of several organic-P sources. We use this knowledge to identify the cellular compartment where the PhoR signaling complex senses the low Pi signal. We demonstrate that the PhoB and PhoR signal transduction proteins can be maintained in an inactive state even when Salmonella is grown in media lacking Pi. Our results establish that PhoR activity is controlled by by an intracellular signal resulting from P insufficiency.
Project description:The phylum Bacteroidetes is a major component of the human gut microbiota which has a broad impact on the development and physiology of its host, and a potential role in a wide range of disease syndromes1-3. The predominance of Bacteroidetes and the genus Bacteroides in the distal gut is due in large part to the expansion of paralogous gene clusters, termed Polysaccharide Utilization Loci (PULs), dedicated to the uptake and catabolism of host derived and dietary polysaccharides4,5. It is generally thought that the diversity of PULs is key to Bacteroides successful competition for nutrients in the gut environment6. The nutritive value of the available polysaccharides varies greatly and thus their utilization is hierarchical and strictly controlled. A typical PUL includes regulatory genes that control expression in response to the presence of specific glycan substrates. However the existence of additional regulatory mechanisms has been predicted to explain phenomena such as the hierarchical control, catabolite repression, and the fine tuning of gene expression to match catabolic activity7-9. Using Bacteroides fragilis as a model organism, this report describes a previously unknown layer of regulatory control in which cis-encoded antisense small RNAs (sRNA) act as repressors of the PULsâ catabolic genes. Nearly 30% of B. fragilis PULs are subject to this type of sRNA control and these PULs tend to be more closely linked to the utilization of host-derived glycans than dietary polysaccharides. The findings described here indicate the presence of a global control mechanism that underlies the known regulatory circuits which modulate PUL expression in response to substrate availability, and hence provide novel insight into regulation of the gut Bacteroidetes physiology. This is a 4 chip study with 8 technical replicates on each chip. This was an in vitro, exploratory study to determine if mutation or overexpression of a sRNA associated with the Don locus would affect gene expression. In vitro cultures were grown in defined media with mucin glycans as the sole carbon source. The two chips representing growth of the wild type strain (638R) on mucin glycans were also used in a related study GSE53883 (GSM1303101 and GSM1303102).
Project description:In E. coli the phosphate homeostasis is regulated by the Pst system and the two-component system PhoB/R. Pathogens like E. coli O157:H7 are responsible for many outbreaks and can be found and survive in poor inorganic phosphate (Pi) environments. To understand global EHEC O157:H7 EDL933 strain responses to Pi-starvation, we compared the transcriptomes of EDL933 the WT strain grown in MOPS Pi rich medium and that grown in MOPS Pi poor medium, using the Affymetrix GeneChip® E. coli Genome 2.0 Array. Also we investigated the EDL933 global response to the absence of PhoB by comparing the transcriptomes of the WT strain the ΔphoB mutant both grown in Low Pi
Project description:Compare BF638R gene expression during growth in vivo in a rat tissue cage/artificial abscess model, to cells grown in vitro in minimal defined media with either glucose or mucin glycans as sole carbon/energy source 11 chip study with 8 technical replicates on each chip comparing gene expression in vivo in a rat tissue cage model over an 8 day period to in vitro cultures grown to mid-log phase in defined media