scholarly journals Comparison of novel GH 68 levansucrases of levan-overproducing Gluconobacter species

2012 ◽  
Vol 1 (1) ◽  
pp. 2 ◽  
Author(s):  
Frank Jakob ◽  
Daniel Meißner ◽  
Rudi F. Vogel
Keyword(s):  
Open Reading Frames ◽  
Ex Situ ◽  
Inverse Pcr ◽  
E Coli ◽  
Food Biotechnology ◽  
Food Applications ◽  
Pcr Techniques ◽  
Single Primer ◽  
Reading Frames

<em>Gluconobacter</em> species are capable of incomplete oxidations which are exploited in food biotechnology. Levans isolated from exopolysaccharide (EPS)-overproducing <em>Gluconobacter</em> species are promising functional compounds for food applications. Fructan production strongly depends on the corresponding fructosyltransferases (Ftfs), which catalyze the formation of these polymers from sucrose. Therefore, we characterized novel Ftfs from three EPS-overproducing food-grade strains, i.e. <em>Gluconobacter</em> sp. TMW 2.767 and <em>Gluconobacter</em> sp. TMW 2.1191 isolated from water kefir, and <em>Gluconobacter cerinus</em> DSM 9533T isolated from cherries. Several PCR techniques, including degenerate gradient temperature PCR, modified and standard inverse PCR, modified site-finding PCR and modified single primer PCR, were used to finally detect complete open reading frames coding for Ftfs. The prospective ftf-gene sequences were heterologously expressed in <em>Escherichia coli</em> Top 10. <em>E. coli</em> transformants harboring one of the three different ftf-genes produced polysaccharides from sucrose in contrast to the <em>E. coli</em> wildtype. Each of the heterologously expressed proteins encoded a levansucrase, catalyzing the formation of b-(2&rarr;6)-linked fructose polymers, which corresponded to our previous analyses about the chemical nature of the isolated polymers formed by these <em>Gluconobacter</em> strains. Structurally, these enzymes belong to the glycoside hydrolase 68 family (GH 68), sharing the typical modular topology of levansucrases from gram-negative bacteria. In conclusion, we could identify novel active levansucrases, which can be used for <em>ex situ</em> (enzymatic catalyses) or <em>in situ</em> (fermentation) production of functional fructan polymers by <em>Gluconobacter</em> strains in food and other applications.

scholarly journals Genome Sequence of a T4-like Phage, Escherichia Phage vB_EcoM-Sa45lw, Infecting Shiga Toxin-Producing Escherichia coli Strains

2019 ◽  
Vol 8 (32) ◽  
Author(s):  
Yen-Te Liao ◽  
Yujie Zhang ◽  
Alexandra Salvador ◽  
Vivian C. H. Wu
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Genome Size ◽  
Shiga Toxin ◽  
Open Reading Frames ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
A Genome ◽  
Reading Frames

Escherichia phage vB_EcoM-Sa45lw, a new member of the T4-like phages, was isolated from surface water in a produce-growing area. The phage, containing double-stranded DNA with a genome size of 167,353 bp and 282 predicted open reading frames (ORFs), is able to infect generic Escherichia coli and Shiga toxin-producing E. coli O45 and O157 strains.

scholarly journals Production by Clostridium acetobutylicum ATCC 824 of CelG, a Cellulosomal Glycoside Hydrolase Belonging to Family 9

2003 ◽  
Vol 69 (2) ◽  
pp. 869-877 ◽  
Author(s):  
Ana M. López-Contreras ◽  
Aernout A. Martens ◽  
Nora Szijarto ◽  
Hans Mooibroek ◽  
Pieternel A. M. Claassen ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Biochemical Properties ◽  
Open Reading Frames ◽  
Extracellular Medium ◽  
Endoglucanase Activity ◽  
E Coli ◽  
High Affinity ◽  
Clostridial Species ◽  
Reading Frames ◽  
Clostridium Acetobutylicum Atcc 824

ABSTRACT The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.

scholarly journals A Stress-Induced Bias in the Reading of the Genetic Code in Escherichia coli

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Adi Oron-Gottesman ◽  
Martina Sauert ◽  
Isabella Moll ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Genetic Code ◽  
Open Reading Frames ◽  
Translation Machinery ◽  
E Coli ◽  
Universal Characteristic ◽  
Living Organisms ◽  
Reading Frames

ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. IMPORTANCE The genetic code is a universal characteristic of all living organisms. It defines the set of rules by which nucleotide triplets specify which amino acid will be incorporated into a protein. Our results represent the first existing report on a stress-induced bias in the reading of the genetic code. We found that in E. coli , under stress, the amino acid threonine is encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. This is because under stress, MazF generates a stress-induced translation machinery (STM) in which MazF cleaves in-frame ACA sites of the processed mRNAs.

scholarly journals Two C—P Lyase Operons in Pseudomonas stutzeri and Their Roles in the Oxidation of Phosphonates, Phosphite, and Hypophosphite

2004 ◽  
Vol 186 (14) ◽  
pp. 4730-4739 ◽  
Author(s):  
Andrea K. White ◽  
William W. Metcalf
Keyword(s):  
Insertion Site ◽  
Pseudomonas Stutzeri ◽  
Gene Organization ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Transposon Insertion ◽  
Intergenic Sequences ◽  
Reading Frames

ABSTRACT DNA sequencing and analysis of two distinct C—P lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes C—P lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional C—P lyase pathway in this organism. To identify the genes comprising this pathway, a Δhtx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both C—P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

scholarly journals Complete DNA Sequence and Comparative Analysis of the 50-Kilobase Virulence Plasmid of Salmonella entericaSerovar Choleraesuis

2001 ◽  
Vol 69 (4) ◽  
pp. 2612-2620 ◽  
Author(s):  
Takeshi Haneda ◽  
Nobuhiko Okada ◽  
Noriko Nakazawa ◽  
Takatoshi Kawakami ◽  
Hirofumi Danbara
Keyword(s):  
Comparative Analysis ◽  
Systemic Infection ◽  
Open Reading Frames ◽  
Virulence Plasmid ◽  
Sequence Element ◽  
E Coli ◽  
Virulence Plasmids ◽  
Serovar Typhimurium ◽  
Insertion Sequence Element ◽  
Reading Frames

ABSTRACT The complete nucleotide sequence of pKDSC50, a large virulence plasmid from Salmonella enterica serovar Choleraesuis strain RF-1, has been determined. We identified 48 of the open reading frames (ORFs) encoded by the 49,503-bp molecule. pKDSC50 encodes a known virulence-associated operon, the spv operon, which is composed of genes essential for systemic infection by nontyphoidalSalmonella. Analysis of the genetic organization of pKDSC50 suggests that the plasmid is composed of several virulence-associated genes, which include the spvRABCD genes, plasmid replication and maintenance genes, and one insertion sequence element. A second virulence-associated region including the pef(plasmid-encoded fimbria) operon and rck (resistance to complement killing) gene, which has been identified on the virulence plasmid of S. enterica serovar Typhimurium, was absent. Two different replicon regions, similar to the RepFIIA and RepFIB replicons, were found. Both showed high similarity to those of the pO157 plasmid of enterohemorrhagic Escherichia coliO157:H7 and the enteropathogenic E. coli (EPEC) adherence factor plasmid harbored by EPEC strain B171 (O111:NM), as well as the virulence plasmids of Salmonella serovars Typhimurium and Enteritidis. Comparative analysis of the nucleotide sequences of the 50-kb virulence plasmid of serovar Choleraesuis and the 94-kb virulence plasmid of serovar Typhimurium revealed that 47 out of 48 ORFs of the virulence plasmid of serovar Choleraesuis are highly homologous to the corresponding ORFs of the virulence plasmid of serovar Typhimurium, suggesting a common ancestry.

scholarly journals An O-Phosphotransferase Catalyzes Phosphorylation of Hygromycin A in the Antibiotic-Producing Organism Streptomyces hygroscopicus

2008 ◽  
Vol 52 (10) ◽  
pp. 3580-3588 ◽  
Author(s):  
Vidya Dhote ◽  
Shuchi Gupta ◽  
Kevin A. Reynolds
Keyword(s):  
Ribosomal Subunit ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Streptomyces Hygroscopicus ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Naturally Occurring ◽  
Insertional Inactivation ◽  
Pathway Regulation ◽  
Reading Frames

ABSTRACT The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (Km = 30 ± 4 μM) at the C-2‴ position of the fucofuranose ring in the presence of ATP (Km = 200 ± 20 μM) or GTP (Km = 350 ± 60 μM) with a k cat of 2.2 ± 0.1 min−1. The phosphorylated HA is inactive against HA-sensitive ΔtolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with k cat and Km values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5‴-dihydrohygromycin A and 5‴-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.

scholarly journals Genome Structure and Thymic Expression of an Endogenous Retrovirus in Zebrafish

2004 ◽  
Vol 78 (2) ◽  
pp. 899-911 ◽  
Author(s):  
Ching-Hung Shen ◽  
Lisa A. Steiner
Keyword(s):  
Leukemia Virus ◽  
Genome Structure ◽  
Endogenous Retrovirus ◽  
Northern Blotting ◽  
Open Reading Frames ◽  
Genomic Locus ◽  
Dna Libraries ◽  
Adult Thymus ◽  
Reading Frames

ABSTRACT In a search for previously unknown genes that are required for lymphocyte development in zebrafish, a retroviral sequence was identified in a subtracted thymus cDNA library and in genomic DNA libraries. The provirus is 11.2 kb and contains intact open reading frames for the gag, pol, and env genes, as well as nearly identical flanking long terminal repeat sequences. As determined by in situ hybridization, the thymus appears to be a major tissue for retroviral expression in both larval and adult fish. Several viral transcripts were found by Northern blotting in the adult thymus. The provirus was found at the same genomic locus in sperm from four fish, suggesting that it is an endogenous retrovirus. Phylogenetic analysis indicates that it is closest to, yet distinct from, the cluster of murine leukemia virus-related retroviruses, suggesting that this virus represents a new group of retroviruses.

scholarly journals Cloning, sequencing and expression in Escherichia coli of the rubredoxin gene from Clostridium pasteurianum

1992 ◽  
Vol 285 (1) ◽  
pp. 255-262 ◽  
Author(s):  
I Mathieu ◽  
J Meyer ◽  
J M Moulis
Keyword(s):  
Escherichia Coli ◽  
Protein Sequence ◽  
Coli Strain ◽  
Open Reading Frames ◽  
Transcriptional Unit ◽  
Clostridium Pasteurianum ◽  
Visible Absorption ◽  
E Coli ◽  
Sequencing And Expression ◽  
Reading Frames

A 3.9 kb BglII-HindIII DNA fragment containing the rubredoxin gene from Clostridium pasteurianum has been cloned using oligonucleotide probes designed from the protein sequence. The 2675 bp SspI-HindIII portion of this fragment has been sequenced and found to contain three open reading frames in addition to the rubredoxin gene. The putative product of one of these open reading frames is similar to various thioredoxin reductases. The rubredoxin gene translates into a sequence that differs from the previously published protein sequence in three positions, D-14, D-22 and E-48 being replaced by the corresponding amides. These changes have been confirmed by partial resequencing of the protein. Promoter-like sequences and a transcription termination signal have been found near the sequence of the rubredoxin gene, which may therefore constitute an independent transcriptional unit. Expression of C. pasteurianum rubredoxin in Escherichia coli strain JM109 has been optimized by subcloning a 476 bp SspI-SspI fragment encompassing the rubredoxin gene. Under these conditions, the latter gene was partly under the control of the lac promoter of pUC18, and the level of rubredoxin production could be increased twofold on addition of a lactose analogue, thus reaching 2-3 mg of pure protein/l of culture. Recombinant rubredoxin was produced in E. coli cells as the holoprotein, and displayed a u.v.-visible-absorption spectrum identical with that of the rubredoxin purified from C. pasteurianum. M.s. and N-terminal sequencing showed that C. pasteurianum rubredoxin expressed in E. coli differs from its native counterpart by having an unblocked N-terminal methionine.

scholarly journals Analysis of the Organization of Multicopy Linear- and Circular-Plasmid-Carried Open Reading Frames inBorrelia burgdorferi Sensu Lato Isolates

1998 ◽  
Vol 66 (3) ◽  
pp. 1149-1158 ◽  
Author(s):  
Jason A. Carlyon ◽  
Crystal LaVoie ◽  
Shian-Ying Sung ◽  
Richard T. Marconi
Keyword(s):  
Southern Hybridization ◽  
Fragment Length Polymorphism ◽  
Pulsed Field Gel Electrophoresis ◽  
Open Reading Frames ◽  
Homogeneous Electric Field ◽  
Related Sequence ◽  
Circular Plasmid ◽  
Genomic Location ◽  
Single Primer ◽  
Reading Frames

ABSTRACT Plasmid cp8.3 of Borrelia afzelii IP21 carries several open reading frames (ORFs) and a 184-bp inverted repeat (IR) element. It has been speculated that this plasmid may encode factors involved in virulence or infectivity. In this report, we have characterized the distribution, molecular variability, and organization of ORFs 1, 2, and 4 and the IR elements among isolates of the Borrelia burgdorferi sensu lato complex. ORFs 1 and 2 are contained within a segment of cp8.3 that is bordered by the IR elements, while ORF 4 resides just outside of the IR-bordered region. By PCR, ORF 4 was amplified from most isolates while ORFs 1 and 2 were amplified from only some B. afzelii isolates. However, Southern hybridization analyses with ORF 1, 2, and 4 probes detected related sequences even in some isolates that were PCR negative. The ORF restriction fragment length polymorphism patterns varied widely even among isolates of the same species. Two-dimensional contour-clamped homogeneous electric field–pulsed-field gel electrophoresis and Southern hybridization detected ORF 1-, 2-, and 4-related sequences on linear and circular plasmids. In addition, an ORF 4-related sequence was detected on a previously uncharacterized, circular plasmid that is greater than 70 kb in size. The IR elements originally identified on plasmid cp8.3 of B. afzelii IP21 were also analyzed by Southern hybridization. Related sequences were detected in some but not all B. burgdorferi sensu lato isolates. These sequences are carried on plasmids in addition to cp8.3 in some isolates. Single-primer PCR analyses demonstrated that in some isolates these sequences exist with IR orientation. The data presented here demonstrate that the IR elements and the ORF 1-, 2-, and 4-related sequences are multicopy and are variable in organization and in genomic location among isolates of the B. burgdorferi sensu lato complex. These analyses provide additional evidence for the highly variable organization of the plasmid component of the B. burgdorferi sensu lato genome.

scholarly journals Cloning, Sequencing, and Characterization of thecgmB Gene of Sinorhizobium meliloti Involved in Cyclic β-Glucan Biosynthesis

1999 ◽  
Vol 181 (15) ◽  
pp. 4576-4583 ◽  
Author(s):  
Ping Wang ◽  
Cheryl Ingram-Smith ◽  
Jill A. Hadley ◽  
Karen J. Miller
Keyword(s):  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Genomic Library ◽  
Rhizobium Meliloti ◽  
Functional Expression ◽  
Open Reading Frames ◽  
Inverse Pcr ◽  
Insertion Mutant ◽  
Overlapping Open Reading Frames ◽  
Reading Frames

ABSTRACT Periplasmic cyclic β-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known asRhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic β-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346–6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transformRhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic β-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated thecgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.

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