The mraZ and mraW genes are highly conserved in bacteria, both in sequence and in their position at the head of the division and cell wall (dcw) gene cluster. Located directly upstream of the mraZ ...gene, the Pmra promoter drives the transcription of mraZ and mraW, as well as many essential cell division and cell wall genes, but no regulator of Pmra has been found to date. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin and MraW is known to methylate the 16S rRNA, mraZ and mraW null mutants have no detectable phenotypes. Here we show that overproduction of Escherichia coli MraZ inhibited cell division and was lethal in rich medium at high induction levels and in minimal medium at low induction levels. Co-overproduction of MraW suppressed MraZ toxicity, and loss of MraW enhanced MraZ toxicity, suggesting that MraZ and MraW have antagonistic functions. MraZ-green fluorescent protein localized to the nucleoid, suggesting that it binds DNA. Consistent with this idea, purified MraZ directly bound a region of DNA containing three direct repeats between Pmra and the mraZ gene. Excess MraZ reduced the expression of an mraZ-lacZ reporter, suggesting that MraZ acts as a repressor of Pmra, whereas a DNA-binding mutant form of MraZ failed to repress expression. Transcriptome sequencing (RNA-seq) analysis suggested that MraZ also regulates the expression of genes outside the dcw cluster. In support of this, purified MraZ could directly bind to a putative operator site upstream of mioC, one of the repressed genes identified by RNA-seq.
Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. ...Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling.
Bacteria impact humans, industry and nature, but do so under viral constraints. Problematically, knowledge of viral infection efficiencies and outcomes derives from few model systems that ...over-represent efficient lytic infections and under-represent virus-host natural diversity. Here we sought to understand infection efficiency regulation in an emerging environmental Bacteroidetes-virus model system with markedly different outcomes on two genetically and physiologically nearly identical host strains. For this, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout both infections. While phage transcriptomes were similar, transcriptional differences between hosts suggested host-derived regulation of infection efficiency. Specifically, the alternative host overexpressed DNA degradation genes and underexpressed translation genes, which seemingly targeted phage DNA particle production, as experiments revealed they were both significantly delayed (by >30 min) and reduced (by >50%) in the inefficient infection. This suggests phage failure to repress early alternative host expression and stress response allowed the host to respond against infection by delaying phage DNA replication and protein translation. Given that this phage type is ubiquitous and abundant in the global oceans and that variable viral infection efficiencies are central to dynamic ecosystems, these data provide a critically needed foundation for understanding and modeling viral infections in nature.
We used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella ...putrefaciens W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph-heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic-heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.
A method for measuring mRNA copies in intact bacterial cells by fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) is presented. Unlike conventional single-molecule ...FISH, where the presence of a transcript is determined by fluorescence intensity, fliFISH relies on On-Off duty cycles of photo-switching dyes to set a predetermined threshold for distinguishing true signals from background noise. The method provides a quantitative approach for detecting and counting true mRNA copies and rejecting false signals with high accuracy.
The thromboxane (TX) A 2 receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type
receptors. Experimental evidence suggests that TP ...activity may be modulated by novel ligands, termed the isoprostanes, that
paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether
prototypical isoprostanes 8-iso-prostaglandin (PG)F 2α and 8-iso-PGE 2 regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase
activity as a reporter. AP-1âluciferase activity was increased by a TP agonist 9,11-dideoxy-9α,11α-methanoepoxy PGF 2α ( U46619 ) in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists
1 S -1α,2β( Z ),3α,5α-7-3-4-iodophenyl)sulfonylamino-6,6-dimethylbicyclo3.1.1hept-2-yl-5-heptenoic acid (I-SAP) and 1S-1α,2α( Z ),3α,4α-7-2-(phenylamino) carbonylhydrazinomethyl-7-oxabicyclo2.2.1 hept-2-yl-5-heptenoic acid (SQ 29,548). AP-1âluciferase
activity was potently (nanomolar concentrations) increased by 8-iso-PGE 2 in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, whereas
8-iso-PGF 2α was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE 2 mediated AP-1âluciferase activity, indicating that this response is not dependent on de novo TXA 2 biosynthesis. Interestingly, 8-iso-PGE 2 -mediated AP-1âluciferase activity was near maximal in naive cells between 1 and 10 nM concentrations, and this response was
not inhibited by TP antagonist or reproduced by agonists for TP or EP 1 /EP 3 receptors. These observations 1) support a role for novel ligands in the regulation of TP-dependent signaling, 2) indicate
that TP-P and TP-E couple to AP-1, 3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific
fashion, and 4) indicate that additional targets regulated by 8-iso-PGE 2 couple to AP-1.
Abstract Correction to: The ISME Journal (2017) 11, 284–295; doi:10.1038/ismej.2016.81 In the title of the article, ‘Bacteriodetes’ is misspelled. The proper spelling is ‘Bacteroidetes’.
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