The ntrBC genes of Azospirillum brasilense are part of a nifR3-like–ntrB–ntrC operon and are negatively regulated

1995 ◽  
Vol 41 (8) ◽  
pp. 674-684 ◽  
Author(s):  
H. B. Machado ◽  
M. G. Yates ◽  
S. Funayama ◽  
L. U. Rigo ◽  
M. B. R. Steffens ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Rhizobium Leguminosarum ◽  
Rhodobacter Capsulatus ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Kanamycin Cassette ◽  
Nitrogenase Activities ◽  
Dependent Growth ◽  
Transcriptional Fusions ◽  
Reading Frames

A cosmid able to complement the Nif− and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. ΔORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.Key words: Azospirillum brasilense, ntrB, ntrC, nifR3-like, nitrogen fixation.

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.

scholarly journals Identification and Sequencing of β-Myrcene Catabolism Genes from Pseudomonas sp. Strain M1

1999 ◽  
Vol 65 (7) ◽  
pp. 2871-2876 ◽  
Author(s):  
Sandra Iurescia ◽  
Andrea M. Marconi ◽  
Daniela Tofani ◽  
Augusto Gambacorta ◽  
Annalisa Paternò ◽  
...  
Keyword(s):  
Genomic Library ◽  
Enrichment Culture ◽  
Open Reading Frames ◽  
Gas Chromatography Mass Spectrometry ◽  
Complete Agreement ◽  
Wild Type ◽  
Genomic Insert ◽  
The One ◽  
Acyl Coenzyme A ◽  
Reading Frames

ABSTRACT The M1 strain, able to grow on β-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One β-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique β-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on β-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA,myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, andmyrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. ThemyrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for β-myrcene catabolism in Pseudomonas sp. strain M1.

scholarly journals Nitrogen or Sulfur Starvation Differentially Affects Phycobilisome Degradation and Expression of the nblA Gene in Synechocystis Strain PCC 6803

2001 ◽  
Vol 183 (10) ◽  
pp. 2989-2994 ◽  
Author(s):  
Catherine Richaud ◽  
Gérald Zabulon ◽  
Annette Joder ◽  
Jean-Claude Thomas
Keyword(s):  
Sequence Data ◽  
Open Reading Frames ◽  
Northern Hybridization ◽  
Sulfur Starvation ◽  
P Limitation ◽  
Kanamycin Cassette ◽  
N Starvation ◽  
Pcc 7942 ◽  
Reading Frames ◽  
Pcc 6803

ABSTRACT Nitrogen (N) limitation in cyanobacteria is well documented: a reduced growth rate is observed, accompanied by a cessation of phycobiliprotein synthesis and an ordered degradation of phycobilisomes (PBS). This leads to a dramatic bleaching phenomenon known as chlorosis. In Synechococcus strain PCC 7942, bleaching due to PBS degradation is also observed under sulfur (S) or phosphorus (P) limitation, and all three are under the control of the nblAgene product, a 59-amino-acid polypeptide which is overexpressed under N, S, and P starvation (J. L. Collier, and A. R. Grossman, EMBO J. 13:1039–1047, 1994). Cyanobase sequence data forSynechocystis strain PCC 6803 indicate the presence of two tandem open reading frames (sll0452 and sll0453) homologous tonblA. We cloned the two genes, identified a unique 5′ mRNA end suggestive of a single transcription start site, and studiednblA expression under conditions of N or S starvation by Northern hybridization: transcripts were detected only under N starvation (no signal is detected in replete medium or with S starvation), whether nblA1 or nblA2 was used as a probe. Mutations in nblA1 and nblA2 were constructed by insertion of a kanamycin cassette; both mutations were nonbleaching under N starvation. Synechocystis strain PCC 6803 does not bleach under S starvation, consistent with the absence ofnblA induction in these conditions. These results were confirmed by analysis of the PBS components: sequential degradation of phycocyanin and associated linkers was observed only under conditions of N starvation. This indicates differences betweenSynechocystis strain PCC 6803 and Synechococcusstrain PCC 7942 in their regulatory and signaling pathways leading to N- and S-starved phenotypes.

Protection against bacteriocin 28b in Serratia matcescens is apparently not related to the expression of an immunity gene

1995 ◽  
Vol 41 (3) ◽  
pp. 217-226 ◽  
Author(s):  
Margarita Beatriz Viejo ◽  
Josefina Enfedaque ◽  
Joan Francesc Guasch ◽  
Santiago Ferrer ◽  
Miguel Regué
Keyword(s):  
Nucleotide Sequence ◽  
Serratia Marcescens ◽  
Structural Gene ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Genetic Determinants ◽  
Gene Encoding ◽  
Immunity Gene ◽  
Release Analysis ◽  
Reading Frames

The gene encoding bacteriocin 28b from Serratia marcescens N28b (bss gene) has been cloned in Escherichia coli and its nucleotide sequence has been determined. The genetic determinants coding for other well-characterized bacteriocins from enterobacteria (colicins) are located in plasmids and they have always been shown to contain a gene responsible for immunity located downstream from the bacteriocin structural gene. In some cases there is another gene located downstream from the immunity gene, which is responsible for bacteriocin release. Analysis of bacteriocin 28b release and the sensitivity to this bacteriocin of E. coli strains harbouring recombinant plasmids containing the bss gene showed that bacteriocin 28b is not released from the cell in these strains and that their phenotypic insensitivity is not associated with any region close to the structural gene. The nucleotide sequence of the region downstream from the bss gene contains two putative open reading frames transcribed in the opposite direction to the bss gene. These open reading frames apparently encode proteins that seem not to be involved in bacteriocin immunity or release. Moreover, a S. marcescens N28b genomic library was screened and no immunity gene was found. Therefore, bacteriocin 28b differs greatly from the bacteriocins from other enterobacteria, and in the following senses it is unique: firstly, the gene encoding bacteriocin 28b seems to be located on the chromosome, and secondly, insensitivity to this bacteriocin in S. marcescens N28b is not associated with the expression of an immunity gene.Key words: bacteriocin, pore-forming colicins, immunity, Serratia marcescens.

scholarly journals Sulfide-Quinone Reductase from Rhodobacter capsulatus: Requirement for Growth, Periplasmic Localization, and Extension of Gene Sequence Analysis

1999 ◽  
Vol 181 (20) ◽  
pp. 6516-6523 ◽  
Author(s):  
Michael Schütz ◽  
Iris Maldener ◽  
Christoph Griesbeck ◽  
Günter Hauska
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Primary Structure ◽  
Gene Sequence ◽  
Rhodobacter Capsulatus ◽  
Quinone Reductase ◽  
Open Reading Frames ◽  
Fusion Construct ◽  
Periplasmic Localization ◽  
Reading Frames

ABSTRACT The entire sequence of the 3.5-kb fragment of genomic DNA fromRhodobacter capsulatus which contains the sqrgene and a second complete and two further partial open reading frames has been determined. A correction of the previously publishedsqr gene sequence (M. Schütz, Y. Shahak, E. Padan, and G. Hauska, J. Biol. Chem. 272:9890–9894, 1997) which in the deduced primary structure of the sulfide-quinone reductase changes four positive into four negative charges and the number of amino acids from 425 to 427 was necessary. The correction has no further bearing on the former sequence analysis. Deletion and interruption strains document that sulfide-quinone reductase is essential for photoautotrophic growth on sulfide. The sulfide-oxidizing enzyme is involved in energy conversion, not in detoxification. Studies with an alkaline phosphatase fusion protein reveal a periplasmic localization of the enzyme. Exonuclease treatment of the fusion construct demonstrated that the C-terminal 38 amino acids of sulfide-quinone reductase were required for translocation. An N-terminal signal peptide for translocation was not found in the primary structure of the enzyme. The possibility that the neighboring open reading frame, which contains a double arginine motif, may be involved in translocation has been excluded by gene deletion (rather, the product of this gene functions in an ATP-binding cassette transporter system, together with the product of one of the other open reading frames). The results lead to the conclusion that the sulfide-quinone reductase of R. capsulatus functions at the periplasmic surface of the cytoplasmic membrane and that this flavoprotein is translocated by a hitherto-unknown mechanism.

scholarly journals Synthesis of a Klebsiella pneumoniae O-Antigen Heteropolysaccharide (O12) Requires an ABC 2 Transporter

2003 ◽  
Vol 185 (5) ◽  
pp. 1634-1641 ◽  
Author(s):  
Luis Izquierdo ◽  
Susana Merino ◽  
Miguel Regué ◽  
Florencia Rodriguez ◽  
Juan M. Tomás
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Library ◽  
Gene Clusters ◽  
Open Reading Frames ◽  
Complete Analysis ◽  
E Coli ◽  
O Antigen ◽  
High Level ◽  
K 12 ◽  
Reading Frames

ABSTRACT A recombinant clone encoding enzymes for Klebsiella pneumoniae O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of K. pneumoniae KT776 (O12:K80) introduced into Escherichia coli DH5α. A total of eight open reading frames (ORFs) (wb O12 gene cluster) were necessary to produce K. pneumoniae O12-antigen LPS in E. coli K-12. A complete analysis of the K. pneumoniae wb O12 cluster revealed an interesting coincidence with the wb O4 cluster of Serratia marcescens from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of K. pneumoniae strain KT776 (O12) and to study complementation between the two enterobacterial wb clusters using mutants of S. marcescens N28b (O4) obtained previously. Both wb gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The wzm-wzt genes and the wbbA or wbbB genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (wzm-wzt-wbbA or wzm-wzt-wbbB) into mutants unable to produce O antigen allowed production of the specific O antigen. The K. pneumoniae O12 WbbL protein performs the same function as WbbL from S. marcescens O4 in either the S. marcescens O4 or E. coli K-12 genetic background.

scholarly journals Synthesis of Catalytically Active Form III Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Archaea

2003 ◽  
Vol 185 (10) ◽  
pp. 3049-3059 ◽  
Author(s):  
Michael W. Finn ◽  
F. Robert Tabita
Keyword(s):  
Rhodobacter Capsulatus ◽  
Quaternary Structure ◽  
Active Form ◽  
Normal Growth ◽  
Growth Conditions ◽  
Methanosarcina Barkeri ◽  
Open Reading Frames ◽  
Bisphosphate Carboxylase ◽  
Biological Reduction ◽  
Reading Frames

ABSTRACT Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth. Until recently it was believed that there are only two forms of RubisCO, form I and form II. However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea. Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III. While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the rbcL genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms. In this report, it is shown not only that Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina acetivorans, and Methanosarcina barkeri possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions. In addition, the form III RubisCO gene (rbcL) from M. acetivorans was shown to complement RubisCO deletion strains of Rhodobacter capsulatus and Rhodobacter sphaeroides under both photoheterotrophic and photoautotrophic growth conditions. These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO2. Furthermore, recombinant M. jannaschii, M. acetivorans, and A. fulgidus RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described Thermococcus kodakaraensis and halophile proteins.

scholarly journals Identification of Campylobacter jejuni ATCC 43431-Specific Genes by Whole Microbial Genome Comparisons

2004 ◽  
Vol 186 (14) ◽  
pp. 4781-4795 ◽  
Author(s):  
Frédéric Poly ◽  
Deborah Threadgill ◽  
Alain Stintzi
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Dna Fragments ◽  
Helicobacter Hepaticus ◽  
Type Iv ◽  
Novel Approach ◽  
The Mean ◽  
Reading Frames

ABSTRACT This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.

scholarly journals The dar Genes of Pseudomonas chlororaphis PCL1606 Are Crucial for Biocontrol Activity via Production of the Antifungal Compound 2-Hexyl, 5-Propyl Resorcinol

2013 ◽  
Vol 26 (5) ◽  
pp. 554-565 ◽  
Author(s):  
Claudia E. Calderón ◽  
Alejandro Pérez-García ◽  
Antonio de Vicente ◽  
Francisco M. Cazorla
Keyword(s):  
Genomic Dna ◽  
Genetic Basis ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Antifungal Compound ◽  
Pseudomonas Chlororaphis ◽  
Phylogenetic Studies ◽  
Biocontrol Activity ◽  
Reading Frames

To determine the genetic basis by which 2-hexyl, 5-propyl resorcinol (HPR) is produced by the biocontrol rhizobacterium Pseudomonas chlororaphis (formerly known as P. fluorescens) PCL1606, the presence and role of dar genes were investigated. To accomplish this aim, the pCGNOV-1 plasmid was isolated from a PCL1606 genomic library and was shown to hybridize to various dar probes by Southern blot. An analysis of the pCGNOV-1 genomic DNA revealed the presence of five open reading frames that were homologous to dar genes and had an organization that resembled the arrangement of previously described P. chlororaphis strains. Phylogenetic studies resulted in the clustering of PCL1606 with the P. chlororaphis subgroup, which supported the renaming of this strain from P. fluorescens to P. chlororaphis PCL1606. The construction of insertional mutants for each homologous dar gene in P. chlororaphis PCL1606 along with their corresponding complemented derivative strains restored HPR production and confirmed the key role of the dar A and darB genes in HPR production and in the antagonistic phenotype. Finally, biocontrol assays were performed on avocado–Rosellinia and tomato–Fusarium test systems using the HPR-defective and -complemented derivative strains generated here and demonstrated the crucial role of the biosynthetic dar genes in the biocontrol phenotype of P. chlororaphis PCL1606. This biocontrol phenotype is dependent on the dar genes via their production of the HPR antibiotic. Some of the dar genes not directly involved in the biosynthesis of HPR, such as darS or darR, might contribute to regulatory features of HPR production.

scholarly journals Selection of Virulence-Associated Determinants ofStreptococcus suis Serotype 2 by In Vivo Complementation

2001 ◽  
Vol 69 (3) ◽  
pp. 1961-1966 ◽  
Author(s):  
Hilde E. Smith ◽  
Herma Buijs ◽  
Henk J. Wisselink ◽  
Norbert Stockhofe-Zurwieden ◽  
Mari A. Smits
Keyword(s):  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Pathogenic Strain ◽  
Nucleotide Sequence Analysis ◽  
Suis Serotype ◽  
Reading Frames ◽  
Selection Of

ABSTRACT Within Streptococcus suis serotype 2, pathogenic, weakly pathogenic, and nonpathogenic strains can be found. We introduced a genomic library of a pathogenic strain into a weakly pathogenic strain. After infection of the library into young piglets pathogenic transformants were selected. One specific transformant containing a 3-kb fragment of the pathogenic strain appeared to be dominantly enriched in diseased pigs. The observed enrichment was not tissue specific. The selected fragment, when introduced into two different weakly pathogenic strains, increased the virulence of these strains considerably. In contrast, introduction of the corresponding fragment of a weakly pathogenic strain had only minor effects on virulence. Nucleotide sequence analysis of the selected fragment of the pathogenic strain revealed the presence of two potential open reading frames, both of which were found to be mutated in the corresponding fragment of the weakly pathogenic strain. These data strongly suggest that the selected fragment contains determinants important for virulence.

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