scholarly journals Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

2005 ◽  
Vol 71 (11) ◽  
pp. 6736-6745 ◽  
Author(s):  
Andrea Jesenská ◽  
Martina Pavlová ◽  
Michal Strouhal ◽  
Radka Chaloupková ◽  
Iva Těšínská ◽  
...  
Keyword(s):  
Halogen Bond ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Maximal Activity ◽  
Biochemical Properties ◽  
Open Reading Frames ◽  
Haloalkane Dehalogenase ◽  
Ph Optimum ◽  
High Sequence Similarity ◽  
Pcr Screening

ABSTRACT Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45°C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47°C and DmbB is 57°C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.

scholarly journals Evolution of Chi motifs in Proteobacteria

2021 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif ◽  
And Staphylococcus Aureus

Abstract Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

scholarly journals Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina

2016 ◽  
Vol 198 (9) ◽  
pp. 1393-1400 ◽  
Author(s):  
Guangyu E. Chen ◽  
Andrew Hitchcock ◽  
Philip J. Jackson ◽  
Roy R. Chaudhuri ◽  
Mark J. Dickman ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
High Sequence Similarity ◽  
Content Type ◽  
Ethyl Group ◽  
Acaryochloris Marina ◽  
Vinyl Group ◽  
Vinyl Reductase ◽  
Reading Frames

ABSTRACTThe major photopigment of the cyanobacteriumAcaryochloris marinais chlorophylld, while its direct biosynthetic precursor, chlorophylla, is also present in the cell. These pigments, along with the majority of chlorophylls utilized by oxygenic phototrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosynthesis. Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB. The genome ofAcaryochloris marinacontains open reading frames (ORFs) encoding proteins displaying high sequence similarity to BciA or BciB, although they are annotated as genes involved in transcriptional control (nmrA) and methanogenesis (frhB), respectively. These genes were introduced into an 8-vinyl chlorophylla-producing ΔbciBstrain ofSynechocystissp. strain PCC 6803, and both were shown to restore synthesis of the pigment with an ethyl group at C-8, demonstrating their activities as 8-vinyl reductases. We propose thatnmrAandfrhBbe reassigned asbciAandbciB, respectively; transcript and proteomic analysis ofAcaryochloris marinareveal that bothbciAandbciBare expressed and their encoded proteins are present in the cell, possibly in order to ensure that all synthesized chlorophyll pigment carries an ethyl group at C-8. Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed.IMPORTANCEThe cyanobacteriumAcaryochloris marinais the best-studied phototrophic organism that uses chlorophylldfor photosynthesis. Unique among cyanobacteria sequenced to date, its genome contains ORFs encoding two unrelated enzymes that catalyze the reduction of the C-8 vinyl group of a precursor molecule to an ethyl group. Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophylld. Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained bothbciAandbciB.

scholarly journals Molecular Characteristics of Carnivore Protoparvovirus 1 With High Sequence Similarity Between Wild and Domestic Carnivores in Taiwan

2021 ◽  
Author(s):  
Ai-Mei Chang ◽  
Chen-Chih Chen
Keyword(s):  
Dna Sequences ◽  
Gastrointestinal Disease ◽  
Sequence Similarity ◽  
Domestic Dogs ◽  
Molecular Characteristics ◽  
High Sequence Similarity ◽  
Vp2 Gene ◽  
Pcr Screening ◽  
Wild Carnivores ◽  
Domestic Carnivores

Carnivore protoparvovirus 1 (CPPV-1) is a DNA virus causing gastrointestinal disease and immunosuppression in various terrestrial carnivores. Domestic dogs and cats are considered the primary CPPV-1 reservoirs. The habitat overlaps of wild carnivores and free-roaming dogs increases the threat of CPPV-1 transmission between them. This study explored the CPPV-1 distribution among wild carnivores through PCR screening and compared the DNA sequences of the partial capsid protein (VP2) between wild and domestic carnivores. In total, 181 samples were screened for the CPPV-1 VP2 gene, including 32 masked palm civets (Paguma larvata), 63 Chinese ferret badgers (Melogale moschata), and 86 crab-eating mongooses (Herpestes urva), from 2015 to 2019 in Taiwan. The average prevalence of CPPV-1 was 17.7% (32/181), with the highest prevalence in masked palm civets (37.5%). In addition, a masked palm civet was coinfected with two CPPV-1 strains. Among the 33 partial VP2 gene sequences, 23 were identical to sequences amplified from domestic dogs and cats in Asia and the remaining 10 were identified for the first time. This study demonstrated that CPPV-1 has circulated between domestic and wild carnivores in rural Taiwan. Therefore, further population control and health management of free-roaming domestic carnivores are recommended.

scholarly journals Evolution of Chi motifs in Proteobacteria

2020 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Escherichia Coli ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif

AbstractHomologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

scholarly journals Characterization of Novel Erwinia amylovora Jumbo Bacteriophages from Eneladusvirus Genus

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1373
Author(s):  
Sang Guen Kim ◽  
Sung Bin Lee ◽  
Sib Sankar Giri ◽  
Hyoun Joong Kim ◽  
Sang Wha Kim ◽  
...  
Keyword(s):  
Genome Size ◽  
Sequence Similarity ◽  
Phylogenetic Analyses ◽  
Gc Content ◽  
Genomic Analysis ◽  
Open Reading Frames ◽  
Comparative Genomic ◽  
Limited Information ◽  
High Sequence Similarity ◽  
A Genome

Jumbo phages, which have a genome size of more than 200 kb, have recently been reported for the first time. However, limited information is available regarding their characteristics because few jumbo phages have been isolated. Therefore, in this study, we aimed to isolate and characterize other jumbo phages. We performed comparative genomic analysis of three Erwinia phages (pEa_SNUABM_12, pEa_SNUABM_47, and pEa_SNUABM_50), each of which had a genome size of approximately 360 kb (32.5% GC content). These phages were predicted to harbor 546, 540, and 540 open reading frames with 32, 34, and 35 tRNAs, respectively. Almost all of the genes in these phages could not be functionally annotated but showed high sequence similarity with genes encoded in Serratia phage BF, a member of Eneladusvirus. The detailed comparative and phylogenetic analyses presented in this study contribute to our understanding of the diversity and evolution of Erwinia phage and the genus Eneladusvirus.

scholarly journals Cloning and Characterization of the DNA Region Responsible for Megacin A-216 Production in Bacillus megaterium 216

2008 ◽  
Vol 190 (19) ◽  
pp. 6448-6457 ◽  
Author(s):  
Antal Kiss ◽  
Gabriella Balikó ◽  
Attila Csorba ◽  
Tungalag Chuluunbaatar ◽  
Katalin F. Medzihradszky ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bacillus Megaterium ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Biologically Active ◽  
Mass Spectrometry Analysis ◽  
High Sequence Similarity ◽  
Spectrometry Analysis ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT Upon induction, Bacillus megaterium 216 produces the bacteriocin megacin A-216, which leads to lysis of the producer cell and kills B. megaterium and a few other bacterial species. The DNA region responsible for megacinogeny was cloned in B. megaterium. The nucleotide sequence of a 5,494-bp-long subfragment was determined, and the function of the genes on this fragment was studied by generating deletions and analyzing their effects on MegA phenotypes. An open reading frame (ORF) encoding a 293-amino-acid protein was identified as the gene (megA) coding for megacin A-216. BLAST searches detected sequence similarity between megacin A-216 and proteins with phospholipase A2 activity. Purified biologically active megacin A-216 preparations contained three proteins. Mass spectrometry analysis showed that the largest protein is the full-length translation product of the megA gene, whereas the two shorter proteins are fragments of the long protein created by cleavage between Gln-185 and Val-186. The molecular masses of the three polypeptides are 32,855, 21,018, and 11,855 Da, respectively. Comparison of different megacin preparations suggests that the intact chain as well as the two combined fragments can form biologically active megacin. An ORF located next to the megA gene and encoding a 91-amino-acid protein was shown to be responsible for the relative immunity displayed by the producer strain against megacin A-216. Besides the megA gene, at least two other genes, including a gene encoding a 188-amino-acid protein sharing high sequence similarity with RNA polymerase sigma factors, were shown to be required for induction of megacin A-216 expression.

scholarly journals An Alternative Succinate (2-Oxoglutarate) Transport System in Rhizobium tropici Is Induced in Nodules of Phaseolus vulgaris

2009 ◽  
Vol 191 (16) ◽  
pp. 5057-5067 ◽  
Author(s):  
Silvia Batista ◽  
Eduardo J. Patriarca ◽  
Rosarita Tatè ◽  
Gloria Martínez-Drets ◽  
Paul R. Gill
Keyword(s):  
Phaseolus Vulgaris ◽  
Carbon Source ◽  
Sequence Similarity ◽  
Optimal Growth ◽  
Open Reading Frames ◽  
Uptake System ◽  
Rhizobium Tropici ◽  
High Sequence Similarity ◽  
Encoding Gene ◽  
Reading Frames

ABSTRACT The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C4-dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 (dctA), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici. The dctBD-like genes in this cluster were found to be required for kgtP expression and were designated kgtSR. Analysis of a kgtP::lacZ transcriptional fusion indicated that a kgtSR-dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris.

scholarly journals Identification of GutQ from Escherichia coli as a d-Arabinose 5-Phosphate Isomerase

2005 ◽  
Vol 187 (20) ◽  
pp. 6936-6942 ◽  
Author(s):  
Timothy C. Meredith ◽  
Ronald W. Woodard
Keyword(s):  
Escherichia Coli ◽  
Sequence Similarity ◽  
Biochemical Properties ◽  
Phosphotransferase System ◽  
High Sequence Similarity ◽  
E Coli ◽  
Regulatory Molecule ◽  
Conditional Mutant ◽  
K 12 ◽  
Functional Copy

ABSTRACT The glucitol operon (gutAEBDMRQ) of Escherichia coli encodes a phosphoenolpyruvate:sugar phosphotransferase system that metabolizes the hexitol d-glucitol (sorbitol). The functions for all but the last gene, gutQ, have been previously assigned. The high sequence similarity between GutQ and KdsD, a d-arabinose 5-phosphate isomerase (API) from the 3-deoxy-d-manno-octulosonate (KDO)-lipopolysaccharide (LPS) biosynthetic pathway, suggested a putative activity, but its role within the context of the gut operon remained unclear. Accordingly, the enzyme was cloned, overexpressed, and characterized. Recombinant GutQ was shown to indeed be a second copy of API from the E. coli K-12 genome with biochemical properties similar to those of KdsD, catalyzing the reversible aldol-ketol isomerization between d-ribulose 5-phosphate (Ru5P) and d-arabinose 5-phosphate (A5P). Genomic disruptions of each API gene were constructed in E. coli K-12. TCM11[(ΔkdsD)] was capable of sustaining essential LPS synthesis at wild-type levels, indicating that GutQ functions as an API inside the cell. The gut operon remained inducible in TCM7[(ΔgutQ)], suggesting that GutQ is not directly involved in d-glucitol catabolism. The conditional mutant TCM15[(ΔgutQΔkdsD)] was dependent on exogenous A5P both for LPS synthesis/growth and for upregulation of the gut operon. The phenotype was suppressed by complementation in trans with a plasmid encoding a functional copy of GutQ or by increasing the amount of A5P in the medium. As there is no obvious obligatory role for GutQ in the metabolism of d-glucitol and there is no readily apparent link between d-glucitol metabolism and LPS biosynthesis, it is suggested that A5P is not only a building block for KDO biosynthesis but also may be a regulatory molecule involved in expression of the gut operon.

scholarly journals A Carbohydrate Hydrolysis Enzymes encoding Genes in Neurospora crassa

2015 ◽  
Vol 5 (3) ◽  
pp. 711-727
Author(s):  
Ravi Gedela
Keyword(s):  
Neurospora Crassa ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
High Sequence Similarity ◽  
Physiochemical Properties ◽  
Carbohydrate Hydrolysis ◽  
Genes Encoding ◽  
Multiple Sequences ◽  
Encoding Genes ◽  
Sequences Alignment

Neurospora crassa, NCU05882.7 (423aa) and NCU09774.7 (303 aa) (NCU, Neurospora 7 crassa unit) genes encoding a Cellulase, which hydrolysis the Cellulose. In addition to that, 8 reporting here other 35 Carbohydrate hydrolysis enzymes encoding genes in N.crassa. A 9 metagenomic analysis for multiple sequences alignment and Phylogenetics analysis, the evaluated 10 result showed high sequence similarity and 99% homology to the other class of fungi; in the 11 bacterial species showed extremely very less sequence similarities and 100 % homology. 12 Where as in inter species between fungi and bacteria, the results showed extremely less sequence 13 similarities and 97 % homology. The studies on physiochemical properties of Cellulase using 14 GeneDoc, the evaluated results showed Cellulase was an amphoteric (polor), aromatic, aliphatic 15 and highly repeated amino acids of glycine and proline. These metagenomic studies could help 16 to straightforward isolation of Cellulase enzymes from NCU05882.7 (Chromosome/Linkage 17 Group-VII), NCU09774.7 (Chromosome Linkage Group- II) and other 35 Carbohydrate 18 hydrolysis enzymes encoding genes in N.crassa.

scholarly journals Long-read cDNA sequencing identifies functional pseudogenes in the human transcriptome

2021 ◽  
Author(s):  
Robin-Lee Troskie ◽  
Yohaann Jafrani ◽  
Tim R Mercer ◽  
Adam D Ewing ◽  
Geoffrey J Faulkner ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Cdna Sequencing ◽  
High Sequence Similarity ◽  
Protein Coding ◽  
Dynamic Component ◽  
Gene Copies ◽  
Sequencing Platforms ◽  
The Impact

Pseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. When transcribed, pseudogenes may encode proteins or enact RNA-intrinsic regulatory mechanisms. However, the extent, characteristics and functional relevance of the human pseudogene transcriptome are unclear. Short-read sequencing platforms have limited power to resolve and accurately quantify pseudogene transcripts owing to the high sequence similarity of pseudogenes and their parent genes. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes. Pseudogene transcripts are expressed in tissue-specific patterns, exhibit complex splicing patterns and contribute to the coding sequences of known genes. We survey pseudogene transcripts encoding intact open reading frames (ORFs), representing potential unannotated protein-coding genes, and demonstrate their efficient translation in cultured cells. To assess the impact of noncoding pseudogenes on the cellular transcriptome, we delete the nucleus- enriched pseudogene PDCL3P4 transcript from HAP1 cells and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the transcriptional landscape underpinning human biology and disease.

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