scholarly journals Benzylsuccinate Synthase of Azoarcussp. Strain T: Cloning, Sequencing, Transcriptional Organization, and Its Role in Anaerobic Toluene and m-Xylene Mineralization

2001 ◽  
Vol 183 (23) ◽  
pp. 6763-6770 ◽  
Author(s):  
Gypsy R. Achong ◽  
Ana M. Rodriguez ◽  
Alfred M. Spormann
Keyword(s):  
Response Regulator ◽  
Transcriptional Start Site ◽  
Amino Acid Sequences ◽  
Α Subunit ◽  
Reverse Transcription Pcr ◽  
Thauera Aromatica ◽  
Regulatory Systems ◽  
Site Mapping ◽  
Genes Encoding ◽  
Benzylsuccinate Synthase

ABSTRACT Biochemical studies in Azoarcus sp. strain T have demonstrated that anaerobic oxidation of both toluene andm-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from Azoarcus sp. strain T and examined the role of this enzyme in both anaerobic toluene and m-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase,bssCAB, together with two additional genes,bssD and bssE, were organized in an operon in the order bssDCABE. bssD is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase. bssE shows homology to tutHfrom Thauera aromatica strain T1, whose function is currently unknown. A second operon that is upstream ofbssDCABE and divergently transcribed contains two genes,tdiS and tdiR. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null bssA mutant was constructed (the bssAgene encodes the α-subunit of benzylsuccinate synthase). ThisbssA null mutant strain was unable to grow under denitrifying conditions on either toluene or m-xylene, while growth on benzoate was unaffected. The growth phenotype of the ΔbssA mutant could be rescued by reintroducingbssA in trans. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and m-xylene.

scholarly journals The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits

1996 ◽  
Vol 317 (1) ◽  
pp. 187-194 ◽  
Author(s):  
Stanislaw ZOLNIEROWICZ ◽  
Christine VAN HOOF ◽  
Nataša ANDJELKOVIĆ ◽  
Peter CRON ◽  
Ilse STEVENS ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Phosphatase ◽  
Amino Acid Sequences ◽  
Regulatory Subunits ◽  
Consensus Sequences ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Phosphatase 2A ◽  
Molecular Masses ◽  
Specific Mrnas

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein microsequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription–PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either α or β isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the ϵ isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.

Expression of growth hormone genes in Atlantic salmon

1993 ◽  
Vol 11 (2) ◽  
pp. 167-179 ◽  
Author(s):  
J B Lorens ◽  
A H Nerland ◽  
R Aasland ◽  
I Lossius ◽  
R Male
Keyword(s):  
Atlantic Salmon ◽  
Transcriptional Start Site ◽  
Northern Analysis ◽  
Rnase Protection ◽  
Reverse Transcription Pcr ◽  
Transcript Length ◽  
Genes Encoding ◽  
Polymerase Chain ◽  
Pcr Assays

ABSTRACT Atlantic salmon (Salmo salar) possess two genes encoding GH. We have investigated the expression of these two genes in the salmon pituitary. The transcriptional start site was localized 64 nucleotides upstream of the first methionyl codon using primer extension and 5′ specific polymerase chain reaction (PCR) assays. Northern analysis revealed a major Atlantic salmon GH (salGH) transcript band of approximately 1400 nucleotides. As coexpression of the salGH genes is not discernible by transcript length, other techniques were used to assess gene activity; RNase protection analysis revealed GH transcript heterogeneity, while reverse transcription-PCR assays detected transcripts from both genes at approximately equivalent amounts. The encoded salGH protein, generated in vitro and by Escherichia coli, shares electrophoretic and immunoreactive identity with native pituitary salGH.

scholarly journals Characterization of Novel Carbazole Catabolism Genes from Gram-Positive Carbazole Degrader Nocardioides aromaticivorans IC177

2006 ◽  
Vol 72 (5) ◽  
pp. 3321-3329 ◽  
Author(s):  
Kengo Inoue ◽  
Hiroshi Habe ◽  
Hisakazu Yamane ◽  
Hideaki Nojiri
Keyword(s):  
Amino Acid ◽  
Gene Clusters ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Class Iii ◽  
Rt Pcr ◽  
Gram Positive ◽  
Mononuclear Iron ◽  
Reverse Transcription Pcr ◽  
Genes Encoding

ABSTRACT Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.

scholarly journals The Alkene Monooxygenase from Xanthobacter Strain Py2 Is Closely Related to Aromatic Monooxygenases and Catalyzes Aromatic Monohydroxylation of Benzene, Toluene, and Phenol

1999 ◽  
Vol 65 (4) ◽  
pp. 1589-1595 ◽  
Author(s):  
Ning-Yi Zhou ◽  
Alister Jenkins ◽  
Chan K. N. Chan Kwo Chion ◽  
David J. Leak
Keyword(s):  
Consensus Sequence ◽  
Detailed Comparison ◽  
Amino Acid Sequences ◽  
Effector Protein ◽  
Chromosomal Dna ◽  
Α Subunit ◽  
Γ Subunit ◽  
Genes Encoding ◽  
Benzene Toluene ◽  
Α Subunits

ABSTRACT The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase α-subunit (XamoA); aamB, encoding the 88-residue oxygenase γ-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase β-subunit (XamoE); andaamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of ς54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase α-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase fromRhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacterstrain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture ofo-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studiesXanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of theaam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.

scholarly journals Functional organization of a singlenifcluster in the mesophilic archaeonMethanosarcina mazeistrain Gö1

Archaea ◽  
2002 ◽  
Vol 1 (2) ◽  
pp. 143-150 ◽  
Author(s):  
Claudia Ehlers ◽  
Katharina Veit ◽  
Gerhard Gottschalk ◽  
Ruth A. Schmitz
Keyword(s):  
Sequence Analysis ◽  
Functional Organization ◽  
Transcriptional Start Site ◽  
Molecular Nitrogen ◽  
Methanosarcina Barkeri ◽  
Amino Acid Sequences ◽  
Pcr Analysis ◽  
Methanosarcina Mazei ◽  
Reverse Transcription Pcr ◽  
Sequence Similarities

The mesophilic methanogenic archaeonMethanosarcina mazeistrain Gö1 is able to utilize molecular nitrogen (N2) as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif) gene cluster inM. mazeiGö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities tonifH, nifI1,nifI2,nifD,nifK,nifEandnifN, similar to other diazotrophic methanogens and certain bacteria such asClostridium acetobutylicum, with the twoglnB-like genes (nifI1andnifI2) located betweennifHandnifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes ofM. mazeiGö1 showed that they are most closely related toMethanosarcina barkeri nif2genes, and also closely resemble those for the correspondingnifproducts of the gram-positive bacteriumC. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that theM. mazei nifgenes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of theM. mazei nifHgene, which may have a function in transcriptional regulation of thenifoperon.

scholarly journals Analysis of the Activity and Regulon of the Two-Component Regulatory System Composed by Cjj81176_1484 and Cjj81176_1483 of Campylobacter jejuni

2015 ◽  
Vol 197 (9) ◽  
pp. 1592-1605 ◽  
Author(s):  
Paul M. Luethy ◽  
Steven Huynh ◽  
Craig T. Parker ◽  
David R. Hendrixson
Keyword(s):  
Campylobacter Jejuni ◽  
Histidine Kinase ◽  
Intestinal Tract ◽  
Diarrheal Disease ◽  
Response Regulator ◽  
Optimal Growth ◽  
Content Type ◽  
Regulatory Systems ◽  
Two Component ◽  
Genes Encoding

ABSTRACTCampylobacter jejuniis a leading cause of bacterial diarrheal disease and a frequent commensal of the intestinal tract in poultry and other animals. For optimal growth and colonization of hosts,C. jejuniemploys two-component regulatory systems (TCSs) to monitor environmental conditions and promote proper expression of specific genes. We analyzed the potential ofC. jejuniCjj81176_1484(Cjj1484) andCjj81176_1483(Cjj1483) to encode proteins of a cognate TCS that influences expression of genes possibly important forC. jejunigrowth and colonization. Transcriptome analysis revealed that the regulons of the Cjj81176_1484 (Cjj1484) histidine kinase and the Cjj81176_1483 (Cjj1483) response regulator contain many common genes, suggesting that these proteins likely form a cognate TCS. We found that this TCS generally functions to repress expression of specific proteins with roles in metabolism, iron/heme acquisition, and respiration. Furthermore, the TCS repressed expression ofCjj81176_0438andCjj81176_0439, which had previously been found to encode a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract. However, the TCS and other specific genes whose expression is repressed by the TCS were not required for colonization of chicks. We observed that the Cjj1483 response regulator binds target promoters in both unphosphorylated and phosphorylated forms and influences expression of some specific genes independently of the Cjj1484 histidine kinase. This work further expands the signaling mechanisms ofC. jejuniand provides additional insights regarding the complex and multifactorial regulation of many genes involved in basic metabolism, respiration, and nutrient acquisition that the bacterium requires for optimal growth in different environments.IMPORTANCEBacterial two-component regulatory systems (TCSs) link environmental cues to expression of specific genes that enable optimal bacterial growth or colonization of hosts. We found that theCampylobacter jejuniCjj1484 histidine kinase and Cjj1483 response regulator function as a cognate TCS to largely repress expression of target genes encoding a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract, as well as other genes encoding proteins for heme or iron acquisition, metabolism, and respiration. We also discovered different modes by which Cjj1483 may mediate repression with and without Cjj1484. This work provides insight into the signal transduction mechanisms of a leading cause of bacterial diarrheal disease and emphasizes the multifactorial and complex regulation of specific biological processes inC. jejuni.

scholarly journals Nucleotide sequence of the Klebsiella pneumoniae nifD gene and predicted amino acid sequence of the α-subunit of nitrogenase MoFe protein

1987 ◽  
Vol 247 (2) ◽  
pp. 287-291 ◽  
Author(s):  
I Ioannidis ◽  
M Buck
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Klebsiella Pneumoniae ◽  
Amino Acid Sequences ◽  
Alpha Subunit ◽  
Predicted Amino Acid Sequence ◽  
Structural Homology ◽  
Α Subunit ◽  
Mofe Protein ◽  
Genes Encoding

The nucleotide sequence of the Klebsiella pneumoniae nifD gene is presented and together with the accompanying paper [Holland, Zilberstein, Zamir & Sussman (1987) Biochem. J. 247, 277-285] completes the sequence of the nifHDK genes encoding the nitrogenase polypeptides. The K. pneumoniae nifD gene encodes the 483-amino acid-residue nitrogenase alpha-subunit polypeptide of Mr 54156. The alpha-subunit has five strongly conserved cysteine residues at positions 63, 89, 155, 184 and 275, some occurring in a region showing both primary sequence and potential structural homology to the K. pneumoniae nitrogenase beta-subunit. A comparison with six other alpha-subunit amino acid sequences has been made, which indicates a number of potentially important domains within alpha-subunits.

scholarly journals Genetic Analysis and Functional Characterization of the Streptococcus pneumoniae vic Operon

2002 ◽  
Vol 70 (11) ◽  
pp. 6121-6128 ◽  
Author(s):  
Christian Wagner ◽  
Antoine de Saizieu ◽  
Hans-Joachim Schönfeld ◽  
Markus Kamber ◽  
Roland Lange ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Functional Characterization ◽  
Reading Frame ◽  
Aryl Hydrocarbon ◽  
Intracellular Signals ◽  
Genes Encoding

ABSTRACT The vic two-component signal transduction system of Streptococcus pneumoniae is essential for growth. The vic operon comprises three genes encoding the following: VicR, a response regulator of the OmpR family; VicK, its cognate histidine kinase; and VicX, a putative protein sharing 55% identity to the predicted product (YycJ) of an open reading frame in the Bacillus subtilis genome. We show that not only is vic essential for viability but it also influences virulence and competence. A putative transcriptional start site for the vic operon was mapped 16 bp upstream of the ATG codon of vicR. Only one transcript of 2.9 kb, encoding all three genes, was detected by Northern blot analysis. VicK, an atypical PAS domain-containing histidine kinase, can be autophosphorylated in vitro, and VicR functions in vitro as a phospho-acceptor protein. (PAS is an acronym formed from the names of the proteins in which the domains were first recognized: the Drosophila period clock protein [PER], vertebrate aryl hydrocarbon receptor nuclear translocator [ARNT], and Drosophila single-minded protein [SIM].) PAS domains are commonly involved in sensing intracellular signals such as redox potential, which suggests that the signal for vic might also originate in the cytoplasm. Growth rate, competence, and virulence were monitored in strains with mutations in the vic operon. Overexpression of the histidine kinase, VicK, resulted in decreased virulence, whereas the transformability of a null mutant decreased by 3 orders of magnitude.

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