scholarly journals Prospecting for Novel Biocatalysts in a Soil Metagenome

2003 ◽  
Vol 69 (10) ◽  
pp. 6235-6242 ◽  
Author(s):  
S. Voget ◽  
C. Leggewie ◽  
A. Uesbeck ◽  
C. Raasch ◽  
K.-E. Jaeger ◽  
...  
Keyword(s):  
Type I ◽  
The Novel ◽  
Gene Duplications ◽  
16S Rrna Sequence ◽  
Pectate Lyases ◽  
Dna Libraries ◽  
Genes Encoding ◽  
16S Rrna Sequence Analysis ◽  
Encoding Genes

ABSTRACT The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an α-amylase (amyA), a 1,4-α-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

scholarly journals Identification and Characterization of Hemolysin-Like Proteins Similar to RTX Toxin in Pasteurella pneumotropica

2009 ◽  
Vol 191 (11) ◽  
pp. 3698-3705 ◽  
Author(s):  
Hiraku Sasaki ◽  
Eiichi Kawamoto ◽  
Yoshikazu Tanaka ◽  
Takuo Sawada ◽  
Satoshi Kunita ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Opportunistic Pathogen ◽  
Pcr Analysis ◽  
Type I ◽  
Secretion Systems ◽  
Rtx Toxin ◽  
Rtx Toxins ◽  
Genes Encoding ◽  
Encoding Genes

ABSTRACT Pasteurella pneumotropica is an opportunistic pathogen that causes lethal pneumonia in immunodeficient rodents. The virulence factors of this bacterium remain unknown. In this study, we identified the genes encoding two RTX toxins, designated as pnxI and pnxII, from the genomic DNA of P. pneumotropica ATCC 35149 and characterized with respect to hemolysis. The pnxI operon was organized according to the manner in which the genes encoded the structural RTX toxin (pnxIA), the type I secretion systems (pnxIB and pnxID), and the unknown orf. The pnxII gene was involved only with the pnxIIA that coded for a structural RTX toxin. Both the structural RTX toxins of deduced PnxIA and PnxIIA were involved in seven of the RTX repeat and repeat-like sequences. By quantitative PCR analysis of the structural RTX toxin-encoding genes in P. pneumotropica ATCC 35149, the gene expression of pnxIA was found to have increased from the early log phase, while that of pnxIIA increased from the late log to the early stationary phase. As expressed in Escherichia coli, both the recombinant proteins of PnxIA and PnxIIA showed weak hemolytic activity in both sheep and murine erythrocytes. On the basis of the results of the Southern blotting analysis, the pnxIA gene was detected in 82% of the isolates, while the pnxIIA gene was detected in 39%. These results indicate that the products of both pnxIA and pnxIIA were putative associations of virulence factors in the rodent pathogen P. pneumotropica.

scholarly journals Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

2021 ◽  
Author(s):  
Fatma Ben Abid ◽  
Clement K. M. Tsui ◽  
Yohei Doi ◽  
Anand Deshmukh ◽  
Christi L. McElheny ◽  
...  
Keyword(s):  
Common Species ◽  
Clinical Samples ◽  
Whole Genome ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Sequence Types ◽  
Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

Isolation and characterization of phosphate solubilizing bacteria from the rhizospheric soil of Ponthenpuzha forest, Pathanamthitta (District), Kerala

2021 ◽  
Vol 16 (8) ◽  
pp. 110-117
Author(s):  
Kannan Abhirami ◽  
K. Jayakumar
Keyword(s):  
Bacterial Isolate ◽  
Phosphate Solubilizing Bacteria ◽  
Cymbopogon Citratus ◽  
16S Rrna Sequence ◽  
Isolation And Characterization ◽  
16S Rrna Sequence Analysis ◽  
Phosphate Solubilizing ◽  
Level Identification

Phosphorous is considered as a major parameter for crop yield. Its availability to plant is independent of its abundance. For the plants to utilize phosphorous, it is to be converted to absorbable form. Here, the part rendered by phosphate solubilizing bacteria is significant for it plays a crucial role in the formation of plant usable phosphate from organic forms. In the present work, an effort had been made to isolate and identify phosphate solubilising bacterial isolate from the rhizhospheric soils of various plants in Ponthenpuzha forest. One of the isolate from Cymbopogon citrates responded positively to Pikovskaya’s medium by producing a halo zone during in vitro culture. Colony features and 16S rRNA sequence analysis identified the isolate as Burkholderia sps. We have reported the presence of genus Burkholderia in the rhizospheric zone of Cymbopogon citratus. Further studies are warranted for species level identification of the isolate.

scholarly journals Characterization of the Arginine Deiminase Operon of Streptococcus rattus FA-1

2004 ◽  
Vol 70 (3) ◽  
pp. 1321-1327 ◽  
Author(s):  
Ann Griswold ◽  
Yi-Ywan M. Chen ◽  
Jennifer A. Snyder ◽  
Robert A. Burne
Keyword(s):  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Arginine Deiminase ◽  
Mutans Streptococci ◽  
Ph Homeostasis ◽  
Atp Production ◽  
Dna Libraries ◽  
Genes Encoding ◽  
Streptococcus Rattus

ABSTRACT The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology. The ADS consists of three enzymes. Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB). Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP. Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells. The purpose of this study was to initiate a characterization of the arc operon of Streptococcus rattus, the least cariogenic and sole ADS-positive member of the mutans streptococci. Using an arcB gene fragment obtained by degenerate PCRs, the FA-1 arc operon was identified in subgenomic DNA libraries and sequence analysis was performed. Results showed that the genes encoding the AD pathway in S. rattus FA-1 are organized as an arcABCDT-adiR operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR). The arcA transcriptional start site was identified by primer extension, and a σ70-like promoter was mapped 5′ to arcA. Reverse transcriptase PCR was used to establish that arcABCDT could be cotranscribed. Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.

scholarly journals Identification of Novel Benzoylformate Decarboxylases by Growth Selection

2006 ◽  
Vol 72 (12) ◽  
pp. 7510-7517 ◽  
Author(s):  
Helge Henning ◽  
Christian Leggewie ◽  
Martina Pohl ◽  
Michael Müller ◽  
Thorsten Eggert ◽  
...  
Keyword(s):  
Environmental Dna ◽  
Selective Medium ◽  
The Novel ◽  
Selection System ◽  
Dna Library ◽  
Benzoylformate Decarboxylase ◽  
Growth Deficiency ◽  
Growth Selection ◽  
Genes Encoding ◽  
Encoding Genes

ABSTRACT A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the β-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.

scholarly journals Identification and Characterization of the Asperthecin Gene Cluster of Aspergillus nidulans

2008 ◽  
Vol 74 (24) ◽  
pp. 7607-7612 ◽  
Author(s):  
Edyta Szewczyk ◽  
Yi-Ming Chiang ◽  
C. Elizabeth Oakley ◽  
Ashley D. Davidson ◽  
Clay C. C. Wang ◽  
...  
Keyword(s):  
Secondary Metabolite ◽  
Polyketide Synthase ◽  
Gene Clusters ◽  
Laboratory Culture ◽  
Culture Conditions ◽  
Type I ◽  
Genes Encoding ◽  
Identification And Characterization ◽  
Normal Laboratory

ABSTRACT The sequencing of Aspergillus genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of sumO, the gene that encodes the small ubiquitin-like protein SUMO in A. nidulans, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the sumO deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.

scholarly journals ALK7, a Receptor for Nodal, Is Dispensable for Embryogenesis and Left-Right Patterning in the Mouse

2004 ◽  
Vol 24 (21) ◽  
pp. 9383-9389 ◽  
Author(s):  
Henrik Jörnvall ◽  
Eva Reissmann ◽  
Olov Andersson ◽  
Mehrnaz Mehrkash ◽  
Carlos F. Ibáñez
Keyword(s):  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Limb Bud ◽  
Mutant Mice ◽  
Type I ◽  
Vertebrate Development ◽  
Serine Threonine Kinase ◽  
Genes Encoding ◽  
Limb Malformations

ABSTRACT Mesendoderm formation and left-right patterning during vertebrate development depend upon selected members of the transforming growth factor β superfamily, particularly Nodal and Nodal-related ligands. Two type I serine/threonine kinase receptors have been identified for Nodal, ALK4 and ALK7. Mouse embryos lacking ALK4 fail to produce mesendoderm and die shortly after gastrulation, resembling the phenotype of Nodal knockout mice. Whether ALK4 contributes to left-right patterning is still unknown. Here we report the generation and initial characterization of mice lacking ALK7. Homozygous mutant mice were born at the expected frequency and remained viable and fertile. Viability at weaning was not different from that of the wild type in ALK7 −/−; Nodal +/− and ALK7 −/−; ALK4 +/− compound mutants. ALK7 and ALK4 were highly expressed in interdigital regions of the developing limb bud. However, ALK7 mutant mice displayed no skeletal abnormalities or limb malformations. None of the left-right patterning abnormalities and organogenesis defects identified in mice carrying mutations in Nodal or in genes encoding ActRIIA and ActRIIB coreceptors, including heart malformations, pulmonary isomerism, right-sided gut, and spleen hypoplasia, were observed in mice lacking ALK7. Finally, the histological organization of the cerebellum, cortex, and hippocampus, all sites of significant ALK7 expression in the rodent brain, appeared normal in ALK7 mutant mice. We conclude that ALK7 is not an essential mediator of Nodal signaling during mesendoderm formation and left-right patterning in the mouse but may instead mediate other activities of Nodal and related ligands in the development or function of particular tissues and organs.

scholarly journals Phylogenomics reveals dynamic evolution of fungal nitric oxide reductases and their relationship to secondary metabolism

2018 ◽  
Author(s):  
Steven A. Higgins ◽  
Christopher W. Schadt ◽  
Patrick B. Matheny ◽  
Frank E. Löffler
Keyword(s):  
Nitric Oxide ◽  
Secondary Metabolism ◽  
Genomic Analysis ◽  
Primary Role ◽  
Ancestral State ◽  
The Novel ◽  
Gene Duplications ◽  
Genes Encoding ◽  
Denitrification Genes ◽  
Genomic Regions

AbstractFungi expressing P450nor, an unconventional nitric oxide (NO) reducing cytochrome P450, are thought to be significant contributors to soil nitrous oxide (N2O) emissions. However, fungal contributions to N2O emissions remain uncertain due to inconsistencies in measurements of N2O formation by fungi. Much of the N2O emitted from antibiotic-amended soil microcosms is attributed to fungal activity, yet fungal isolates examined in pure culture are poor N2O producers. To assist in reconciling these conflicting observations and produce a benchmark genomic analysis of fungal denitrifiers, genes underlying fungal denitrification were examined in >700 fungal genomes. Of 167p450nor–containing genomes identified, 0, 30, and 48 also harbored the denitrification genesnarG,napAornirK, respectively. Compared tonapAandnirK,p450norwas twice as abundant and exhibited two to five-fold more gene duplications, losses, and transfers, indicating a disconnect betweenp450norpresence and denitrification potential. Furthermore, co-occurrence ofp450norwith genes encoding NO-detoxifying flavohemoglobins (Spearman r = 0.87,p= 1.6e−10) confounds hypotheses regarding P450nor’s primary role in NO detoxification. Instead, ancestral state reconstruction united P450nor with actinobacterial cytochrome P450s (CYP105) involved in secondary metabolism (SM) and 19 (11 %)p450nor-containing genomic regions were predicted to be SM clusters. Another 40 (24 %) genomes harbored genes nearbyp450norpredicted to encode hallmark SM functions, providing additional contextual evidence linkingp450norto SM. These findings underscore the potential physiological implications of widespreadp450norgene transfer, support the novel affiliation ofp450norwith fungal SM, and challenge the hypothesis ofp450nor’s primary role in denitrification.

Molecular characterization of two clusters of genes encoding the Type I CAB polypeptides of PSII in Nicotiana plumbaginifolia

1987 ◽  
Vol 10 (2) ◽  
pp. 117-126 ◽  
Author(s):  
Carmen Castresana ◽  
Roberto Staneloni ◽  
Vedpal S. Malik ◽  
Anthony R. Cashmore
Keyword(s):  
Molecular Characterization ◽  
Nicotiana Plumbaginifolia ◽  
Type I ◽  
Genes Encoding
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