Supplementary data for "Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose (original) (raw)
Related papers
2015
Background: Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a. Results: In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn2Cys6 transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be expressed over the basal constitutive level. Conclusion: The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype. Keywords: Single nucleotide polymorphism, SNP, Indel, Comparative genomics, Classical mutant, XYR1, Transcription factor shuttling, Cellulases, Trichoderma reesei, QM9136
Plant physiology, 2018
Piriformospora indica, an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. The fungal cell wall extract induces cytoplasmic calcium [Ca2+]cyt elevation in host plant roots. Here, we show that an elici-tor-active cell wall moiety, released by P. indica into the medium, is cellotriose (CT). CT in-duces a mild defense-like response including the production of reactive oxygen species, changes in membrane potentials and the expression of genes involved in growth regulation and root development. CT based [Ca2+]cyt elevation in Arabidopsis roots does not require BAK1 coreceptor, or the putative Ca2+ channels TPC1, GLR3.3, -2.4 and -2.5 and operates synergistically with the elicitor chitin. We identified an ethylmethane-sulfonate-induced mu-tant ([Ca2+]cyt elevation mutant, cycam) impaired in response to CT, cellooligomers (n = 2, 4-7), but not to chitooligomers (n = 4-8) in roots. The mutant contains a single...
Journal of Bacteriology, 2006
The OxyR transcription factor is a key regulator of the Escherichia coli response to oxidative stress. Previous studies showed that OxyR binding to a target promoter enhances RNA polymerase binding and vice versa, suggesting a direct interaction between OxyR and RNA polymerase. To identify the region of OxyR that might contact RNA polymerase, we carried out alanine scanning and random mutagenesis of oxyR. The combination of these approaches led to the identification of several mutants defective in the activation of an OxyR target gene. A subset of the mutations map to the DNA-binding domain, other mutations appear to affect dimerization of the regulatory domain, while another group is suggested to affect disulfide bond formation. The two mutations, D142A and R273H, giving the most dramatic phenotype are located in a patch on the surface of the oxidized OxyR protein and possibly define an activating region on OxyR.
A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway. Citation: Perez-Gil J, Uros EM, Sauret-Gü eto S, Lois LM, Kirby J, et al. (2012) Mutations in Escherichia coli aceE and ribB Genes Allow Survival of Strains Defective in the First Step of the Isoprenoid Biosynthesis Pathway. PLoS ONE 7(8): e43775.
Molecular Microbiology, 2008
Contact-dependent growth inhibition (CDI) is a phenomenon by which bacterial cell growth is regulated by direct cell-to-cell contact via the CdiA/CdiB two-partner secretion system. Characterization of mutants resistant to CDI allowed us to identify BamA (YaeT) as the outer membrane receptor for CDI and AcrB as a potential downstream target. Notably, both BamA and AcrB are part of distinct multi-component machines. The Bam machine assembles outer membrane β-barrel proteins (OMPs) into the outer membrane and the Acr machine exports small molecules into the extracellular milieu. We discovered that a mutation that reduces expression of BamA decreased binding of CDI + inhibitor cells, measured by flow cytometry with fluorescentlylabeled bacteria. In addition, α-BamA antibodies, which recognized extracellular epitopes of BamA based on immunofluorescence, specifically blocked inhibitor-target cell binding and CDI. A second class of CDI-resistant mutants identified carried null mutations in the acrB gene. AcrB is an inner membrane component of a multidrug efflux pump that normally forms a cell envelopespanning complex with the membrane-fusion protein AcrA and the outer membrane protein TolC. Strikingly, the requirement for the BamA and AcrB proteins in CDI is independent of their multicomponent machines, and thus their role in the CDI pathway may reflect novel, import-related functions.
Plant physiology, 2017
The plant cell wall, often the site of initial encounters between plants and their microbial pathogens, is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides as well as proteins. The concept of damage-associated molecular patterns (DAMPs) was proposed to describe plant elicitors like oligogalacturonides (OGs), which can be derived by the breakdown of the pectin homogalacturon by pectinases. OGs act via many of the same signaling steps as pathogen- or microbe-associated molecular patterns (PAMPs) to elicit defenses and provide protection against pathogens. Given both the complexity of the plant cell wall and the fact that many pathogens secrete a wide range of cell wall-degrading enzymes, we reasoned that the breakdown products of other cell wall polymers may be similarly biologically active as elicitors and may help to reinforce the perception of danger by plant cells. Our results indicate that oligomers derived from cellulose are perceived as sign...
Development of a Premature Stop Codon-detection method based on a bacterial two-hybrid system
BMC biotechnology, 2006
The detection of Premature Stop Codons (PSCs) in human genes is very useful for the genetic diagnosis of different hereditary cancers, e.g. Familial Breast Cancer and Hereditary Non-Polyposis Colorectal Cancer (HNPCC). The products of these PSCs are truncated proteins, detectable in vitro by the Protein Truncation Test and in vivo by using the living translation machinery of yeast or bacteria. These living strategies are based on the construction of recombinant plasmids where the human sequence of interest is inserted upstream of a reporter gene. Although simple, these assays have their limitations. The yeast system requires extensive work to enhance its specificity, and the bacterial systems yield many false results due to translation re-initiation events occurring post PSCs. Our aim was to design a recombinant plasmid useful for detecting PSCs in human genes and resistant to bacterial translation re-initiation interferences. A functional recombinant plasmid (pREAL) was designed ba...
Bioengineered bugs
Specific colonization of solid tumors by bacteria opens the way to novel approaches in both tumor diagnosis and therapy. However, even non-pathogenic bacteria induce responses by the immune system, which could be devastating for a tumor bearing patient. As such effects are caused e.g., by the lipid A moiety of the lipopolysaccharide, a msbB-mutant of the probiotic E. coli Nissle 1917 strain was investigated. Bacteria of the mutant strain did not show any growth defects in culture media when compared to wild-type E. coli Nissle 1917 but were unable to myristoylate lipid A, had less toxic effects on immunocompetent BALB/c mice, and were still able to specifically colonize tumors. Therefore, the modification of lipid A could result in bacterial strains that might be better suited for diagnosis and therapy of tumors than the corresponding wild-type strains, even if those are not considered pathogenic or are of probiotic background.