scholarly journals A Chemogenomic Screening of Sulfanilamide-Hypersensitive Saccharomyces cerevisiae Mutants Uncovers ABZ2, the Gene Encoding a Fungal Aminodeoxychorismate Lyase

2007 ◽  
Vol 6 (11) ◽  
pp. 2102-2111 ◽  
Author(s):  
Javier Botet ◽  
Laura Mateos ◽  
José L. Revuelta ◽  
María A. Santos
Keyword(s):  
Large Scale ◽  
Phylogenetic Analyses ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Cellular Processes ◽  
Founder Member ◽  
Yeast Genes ◽  
Vacuole Biogenesis ◽  
Reading Frames

ABSTRACT Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.

scholarly journals The Type II Pullulanase of Thermococcus hydrothermalis: Molecular Characterization of the Gene and Expression of the Catalytic Domain

1999 ◽  
Vol 181 (10) ◽  
pp. 3284-3287 ◽  
Author(s):  
Marta Erra-Pujada ◽  
Philippe Debeire ◽  
Francis Duchiron ◽  
Michael J. O’Donohue
Keyword(s):  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Open Reading Frames ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Rich Region ◽  
Gene Encoding ◽  
Multidomain Structure ◽  
Reading Frames

ABSTRACT The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated.

scholarly journals A Gene Encoding l-Methionine γ-Lyase Is Present in Enterobacteriaceae Family Genomes: Identification and Characterization of Citrobacter freundiil-Methionine γ-Lyase

2005 ◽  
Vol 187 (11) ◽  
pp. 3889-3893 ◽  
Author(s):  
Ilya V. Manukhov ◽  
Daria V. Mamaeva ◽  
Sergei M. Rastorguev ◽  
Nicolai G. Faleev ◽  
Elena A. Morozova ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
High Sequence Identity ◽  
E Coli ◽  
The Family ◽  
Serovar Typhimurium ◽  
Identification And Characterization ◽  
Reading Frames

ABSTRACT Citrobacter freundii cells produce l-methionine γ-lyase when grown on a medium containing l-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded l-methionine γ-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3′-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found.

scholarly journals Characterization of the Pasteurella multocida hgbA Gene Encoding a Hemoglobin-Binding Protein

2002 ◽  
Vol 70 (11) ◽  
pp. 5955-5964 ◽  
Author(s):  
Montserrat Bosch ◽  
M. Elena Garrido ◽  
Montserrat Llagostera ◽  
Ana M. Pérez de Rozas ◽  
Ignacio Badiola ◽  
...  
Keyword(s):  
Pasteurella Multocida ◽  
Bacterial Pathogen ◽  
Open Reading Frames ◽  
Transcriptional Unit ◽  
Gene Encoding ◽  
Hemoglobin Binding ◽  
Reading Frames

ABSTRACT Reverse transcriptase PCR analyses have demonstrated that open reading frames (ORFs) PM0298, PM0299, and PM0300 of the animal pathogen Pasteurella multocida constitute a single transcriptional unit. By cloning and overexpression studies in Escherichia coli cells, the product of ORF PM0300 was shown to bind hemoglobin in vitro; this ORF was therefore designated hgbA. In vitro and in vivo quantitative assays demonstrated that the P. multocida hgbA mutant bound hemoglobin to the same extent as the wild-type strain, although the adsorption kinetics was slightly slower for the hgbA cells. In agreement with this, the virulence of P. multocida hgbA cells was not affected, suggesting that other functional hemoglobin receptor proteins must be present in this organism. On the other hand, P. multocida mutants defective in PM0298 and PM0299 could be isolated only when a plasmid containing an intact copy of the gene was present in the cells, suggesting that these genes are essential for the viability of this bacterial pathogen. By adapting the recombinase-based expression technology in vivo to P. multocida, we also demonstrated that the transcriptional PM0298-PM0299-hgbA unit is iron regulated and that its expression is triggered in the first 2 h following infection in a mouse model. Furthermore, hybridization experiments showed that the hgbA gene is widespread in P. multocida strains regardless of their serotype or the animal from which they were isolated.

scholarly journals Isolation and Characterization of a Novel Klebsiella pneumoniae N4-like Bacteriophage KP8

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1115 ◽  
Author(s):  
Vera Morozova ◽  
Igor Babkin ◽  
Yuliya Kozlova ◽  
Ivan Baykov ◽  
Olga Bokovaya ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
New Genus ◽  
Wide Spectrum ◽  
Biological Properties ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Distinct Branch ◽  
Reading Frames

Klebsiella pneumoniae is a common pathogen, associated with a wide spectrum of infections, and clinical isolates of K. pneumoniae often possess multiple antibiotic resistances. Here, we describe a novel lytic N4-like bacteriophage KP8, specific to K. pneumoniae, including its genome, partial structural proteome, biological properties, and proposed taxonomy. Electron microscopy revealed that KP8 belongs to the Podoviridae family. The size of the KP8 genome was 73,679 bp, and it comprised 97 putative open reading frames. Comparative genome analysis revealed that the KP8 genome possessed the highest similarity to the genomes of Enquatrovirus and Gamaleyavirus phages, which are N4-like podoviruses. In addition, the KP8 genome showed gene synteny typical of the N4-like podoviruses and contained the gene encoding a large virion-encapsulated RNA polymerase. Phylogenetic analysis of the KP8 genome revealed that the KP8 genome formed a distinct branch within the clade, which included the members of Enquatrovirus and Gamaleyavirus genera besides KP8. The average evolutionary divergences KP8/Enquatrovirus and KP8/Gamaleyavirus were 0.466 and 0.447 substitutions per site (substitutes/site), respectively, similar to that between Enquatrovirus and Gamaleyavirus genera (0.468 substitutes/site). The obtained data suggested that Klebsiella phage KP8 differs from other similar phages and may represent a new genus within the N4-like phages.

scholarly journals The ggpS Gene from Synechocystis sp. Strain PCC 6803 Encoding Glucosyl-Glycerol-Phosphate Synthase Is Involved in Osmolyte Synthesis

1998 ◽  
Vol 180 (18) ◽  
pp. 4843-4849 ◽  
Author(s):  
Kay Marin ◽  
Ellen Zuther ◽  
Thomas Kerstan ◽  
Anja Kunert ◽  
Martin Hagemann
Keyword(s):  
Crude Protein ◽  
Open Reading Frames ◽  
Glycerol Phosphate ◽  
Gene Encoding ◽  
Type Region ◽  
Key Enzyme ◽  
Reading Frames ◽  
Pcc 6803 ◽  
Phosphate Synthase

ABSTRACT A salt-sensitive mutant of Synechocystis sp. strain PCC 6803 defective in the synthesis of the compatible solute glucosylglycerol (GG) was used to search for the gene encoding GG-phosphate synthase (GGPS), the key enzyme in GG synthesis. Cloning and sequencing of the mutated region and the corresponding wild-type region revealed that a deletion of about 13 kb occurred in the genome of mutant 11. This deletion affected at least 10 open reading frames, among them regions coding for proteins showing similarities to trehalose (otsA homolog)- and glycerol-3-phosphate-synthesizing enzymes. After construction and characterization of mutants defective in these genes, it became obvious that an otsA homolog (sll1566) (T. Kaneko et al., DNA Res. 3:109–136, 1996) encodes GGPS, since only the mutant affected in sll1566 showed salt sensitivity combined with a complete absence of GG accumulation. Furthermore, the overexpression ofsll1566 in Escherichia coli led to the appearance of GGPS activity in the heterologous host. The overexpressed protein did not show the salt dependence that is characteristic for the GGPS in crude protein extracts of Synechocystis.

scholarly journals Molecular Characterization of Brucella abortus Chromosome II Recombination

2003 ◽  
Vol 185 (20) ◽  
pp. 6130-6136 ◽  
Author(s):  
Georgios Tsoktouridis ◽  
Christian A. Merz ◽  
Simon P. Manning ◽  
Renée Giovagnoli-Kurtz ◽  
Leanne E. Williams ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Large Scale ◽  
Brucella Melitensis ◽  
Subtractive Hybridization ◽  
Open Reading Frames ◽  
Suppressive Subtractive Hybridization ◽  
Recombination Mechanism ◽  
Chromosome Ii ◽  
Reading Frames

ABSTRACT Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed.

scholarly journals Genetic Characterization of 2,4,6-Trichlorophenol Degradation in Cupriavidus necator JMP134

2007 ◽  
Vol 73 (9) ◽  
pp. 2769-2776 ◽  
Author(s):  
M. A. Sánchez ◽  
B. González
Keyword(s):  
Ralstonia Eutropha ◽  
Degradation Pathway ◽  
Cupriavidus Necator ◽  
Open Reading Frames ◽  
Aerobic Bacterium ◽  
Gene Encoding ◽  
Encoding Genes ◽  
Genetic Context ◽  
Reading Frames

ABSTRACT The degradation pathway of 2,4,6-trichlorophenol (2,4,6-TCP), a hazardous pollutant, in the aerobic bacterium Cupriavidus necator JMP134(pJP4) (formerly Ralstonia eutropha JMP134) is encoded by the tcp genes. These genes are located in a genetic context, tcpRXABCYD, which resembles a putative catabolic operon. In this work, these gene sequences were individually disrupted and mutant strains were evaluated for their ability to grow on or degrade 2,4,6-TCP. The tcpX and tcpA mutants completely failed to degrade this compound. Although the tcpC mutant was also unable to grow on 2,4,6-TCP, it still transformed this chlorophenol to 6-chlorohydroquinol. In contrast, the tcpD mutant grew on 2,4,6-TCP, suggesting the presence of additional maleylacetate reductase-encoding genes. Five other open reading frames encoding maleylacetate reductases, in addition to the tcpD gene, were found in the genome of C. necator, and two of them provide this function in the tcpD mutant. The tcpR gene, encoding a putative LysR-type transcriptional regulator, was disrupted, and this mutant strain completely failed to grow on 2,4,6-TCP. Transcriptional fusion studies demonstrated that TcpR activates the expression of the tcp genes, responding specifically to 2,4,6-TCP. The transcriptional start of the tcp operon was mapped, and a putative σ70-type promoter was identified.

scholarly journals DeORFanizing Candida albicans Genes using Co-Expression

2020 ◽  
Author(s):  
Teresa R. O’Meara ◽  
Matthew J. O’Meara
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Gene Function ◽  
Fungal Pathogen ◽  
Large Scale ◽  
Functional Characterization ◽  
Open Reading Frames ◽  
Model Organisms ◽  
Reading Frames

AbstractFunctional characterization of open reading frames in non-model organisms, such as the common opportunistic fungal pathogen Candida albicans, can be labor intensive. To meet this challenge, we built a comprehensive and unbiased co-expression network for C. albicans, which we call CalCEN, from data collected from 853 RNA sequencing runs from 18 large scale studies deposited in the NCBI Sequence Read Archive. Retrospectively, CalCEN is highly predictive of known gene function annotations and can be synergistically combined with sequence similarity and interaction networks in Saccharomyces cerevisiae through orthology for additional accuracy in gene function prediction. To prospectively demonstrate the utility of the co-expression network in C. albicans, we predicted the function of under-annotated open reading frames (ORF)s and identified CCJ1 as a novel cell cycle regulator in C. albicans. This study provides a tool for future systems biology analyses of gene function in C. albicans. We provide a computational pipeline for building and analyzing the co-expression network and CalCEN itself at (http://github.com/momeara/CalCEN).ImportanceCandida albicans is a common and deadly fungal pathogen of humans, yet the genome of this organism contains many genes of unknown function. By determining gene function, we can help identify essential genes, new virulence factors, or new regulators of drug resistance, and thereby give new targets for antifungal development. Here, we use information from large scale RNAseq studies and generate a C. albicans co-expression network (CalCEN) that is robust and able to predict gene function. We demonstrate the utility of this network in both retrospective and prospective testing, and use CalCEN to predict a role for C4_06590W/CCJ1 in cell cycle. This tool will allow for a better characterization of under-annotated genes in pathogenic yeasts.

scholarly journals Molecular Cloning of ATR5Emoy2 from Hyaloperonospora arabidopsidis, an Avirulence Determinant That Triggers RPP5-Mediated Defense in Arabidopsis

2011 ◽  
Vol 24 (7) ◽  
pp. 827-838 ◽  
Author(s):  
Kate Bailey ◽  
Volkan Çevik ◽  
Nicholas Holton ◽  
Jane Byrne-Richardson ◽  
Kee Hoon Sohn ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Signal Peptide ◽  
Copy Number ◽  
Open Reading Frames ◽  
Downy Mildew Resistance ◽  
Gene Duplications ◽  
Gene Encoding ◽  
Hyaloperonospora Arabidopsidis ◽  
Reading Frames

RPP5 is the seminal example of a cytoplasmic NB-LRR receptor-like protein that confers downy mildew resistance in Arabidopsis thaliana. In this study, we describe the cloning and molecular characterization of the gene encoding ATR5Emoy2, an avirulence protein from the downy mildew pathogen Hyaloperonospora arabidopsidis isolate Emoy2. ATR5Emoy2 triggers defense response in host lines expressing the functional RPP5 allele from Landsberg erecta (Ler-0). ATR5Emoy2 is embedded in a cluster with two additional ATR5-like (ATR5L) genes, most likely resulting from gene duplications. ATR5L proteins do not trigger RPP5-mediated resistance and the copy number of ATR5L genes varies among H. arabidopsidis isolates. ATR5Emoy2 and ATR5L proteins contain a signal peptide, canonical EER motif, and an RGD motif. However, they lack the canonical translocation motif RXLR, which characterizes most oomycete effectors identified so far. The signal peptide and the N-terminal regions including the EER motif of ATR5Emoy2 are not required to trigger an RPP5-dependent immune response. Bioinformatics screen of H. arabidopsidis Emoy2 genome revealed the presence of 173 open reading frames that potentially encode for secreted proteins similar to ATR5Emoy2, in which they share some motifs such as EER but there is no canonical RXLR motif.

scholarly journals DeORFanizing Candida albicans Genes using Coexpression

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Teresa R. O’Meara ◽  
Matthew J. O’Meara
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Gene Function ◽  
Fungal Pathogen ◽  
Large Scale ◽  
Open Reading Frames ◽  
Coexpression Network ◽  
Content Type ◽  
Reading Frames

ABSTRACT Functional characterization of open reading frames in nonmodel organisms, such as the common opportunistic fungal pathogen Candida albicans, can be labor-intensive. To meet this challenge, we built a comprehensive and unbiased coexpression network for C. albicans, which we call CalCEN, from data collected from 853 RNA sequencing runs from 18 large-scale studies deposited in the NCBI Sequence Read Archive. Retrospectively, CalCEN is highly predictive of known gene function annotations and can be synergistically combined with sequence similarity and interaction networks in Saccharomyces cerevisiae through orthology for additional accuracy in gene function prediction. To prospectively demonstrate the utility of the coexpression network in C. albicans, we predicted the function of underannotated open reading frames (ORFs) and identified CCJ1 as a novel cell cycle regulator in C. albicans. This study provides a tool for future systems biology analyses of gene function in C. albicans. We provide a computational pipeline for building and analyzing the coexpression network and CalCEN itself at http://github.com/momeara/CalCEN. IMPORTANCE Candida albicans is a common and deadly fungal pathogen of humans, yet the genome of this organism contains many genes of unknown function. By determining gene function, we can help identify essential genes, new virulence factors, or new regulators of drug resistance, and thereby give new targets for antifungal development. Here, we use information from large-scale RNA sequencing (RNAseq) studies and generate a C. albicans coexpression network (CalCEN) that is robust and able to predict gene function. We demonstrate the utility of this network in both retrospective and prospective testing and use CalCEN to predict a role for C4_06590W/CCJ1 in cell cycle. This tool will allow for a better characterization of underannotated genes in pathogenic yeasts.

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