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A Toukdarian

Publications and source records attributed to A Toukdarian.

11 recordsLinked to original sources
PubMed2001

A broad host range replicon with different requirements for replication initiation in three bacterial species.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.

Amino Acid Sequence↗
PubMed2001

Sequence analysis of a 101-kilobase plasmid required for agar degradation by a Microscilla isolate.

An agar-degrading marine bacterium identified as a Microscilla species was isolated from coastal California marine sediment. This organism harbored a single 101-kb circular DNA plasmid designated pSD15. The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization. The most striking feature was the occurrence of five putative agarase genes. Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.

Agar↗
PubMed1998

Isolation of broad-host-range replicons from marine sediment bacteria.

Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties. Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P. A. Sobecky, T. J. Mincer, M. C. Chang, and D. R. Helinski, Appl. Environ. Microbiol. 63:888-895, 1997). In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates. 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the alpha and gamma subclasses of the class Proteobacteria. Three of the marine sediment isolates are related to the gamma-3 subclass organisms Vibrio splendidus and Vibrio fischeri, while the fourth isolate may be related to Roseobacter litoralis. Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria. In addition to replication in Escherichia coli DH5alpha and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the genera Achromobacter, Pseudomonas, Serratia, and Vibrio. While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other. The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates. Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences. In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.

Bacteria↗
PubMed1994

The role of regulatory genes nifA, vnfA, anfA, nfrX, ntrC, and rpoN in expression of genes encoding the three nitrogenases of Azotobacter vinelandii.

Several regulatory gene mutants of Azotobacter vinelandii were tested for ability to synthesize functional nitrogenase-1 (Nif phenotype), nitrogenase-2 (Vnf), or nitrogenase-3 (Anf). While nifA mutants were Nif-, Vnf+, and Anf+/-, and ntrC mutants were Nif+, Vnf+, and Anf+, nifA ntrC double mutants were Nif-, Vnf-, and Anf-. A vnfA mutant was Nif+, Vnf+/-, and Anf+/-, and an anfA strain was Nif+, Vnf+, and Anf-. lacZ fusions in the nifH, vnfH, vnfD, anfH, and nifM genes of Azotobacter vinelandii were constructed and introduced into wild-type and regulatory mutants of A. vinelandii. Expression of these operons correlated with the growth phenotype of the regulatory mutants. Apparently either NifA or NtrC can activate expression of nifM. Also, expression of the anf operon required the NifA transcriptional activator, although there are no NifA binding sites at appropriate locations upstream of anfH (or anfA). The results confirm previous reports that VnfA and AnfA are required for expression of vnf and anf genes, respectively, and that VnfA is involved in repression of the nifHDK operon in the absence of molybdenum and of the anfHDGK operon in the presence of vanadium.

Amino Acid Sequence↗
PubMed1991

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2.

The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five iterons was identical for all three protein preparations and extended from the beginning of the first iteron to 5 base pairs upstream of the fifth iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five iterons were measured. This analysis showed that unlike complexes involving two or five iterons the TrfA/one iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.

Bacterial Proteins↗
PubMed1990

Molecular analysis of the Azotobacter vinelandii glnA gene encoding glutamine synthetase.

The gene encoding glutamine synthetase (GS), glnA, was cloned from Azotobacter vinelandii on a 6-kb EcoRI fragment that also carries the ntrBC genes. The DNA sequence of 1,952 bp including the GS-coding region was determined. An open reading frame of 467 amino acids indicated a gene product of Mr 51,747. Transcription of glnA occurred from a C residue located 32 bases upstream of an ATG considered to be the initiator codon because (i) it had a nearby potential ribosome-binding site and (ii) an open reading frame translated from this site indicated good N-terminal homology to 10 other procaryotic GSs. Sequences similar to the consensus RNA polymerase recognition sites at -10 and -35 were present at the appropriate distance upstream of the transcription initiation site. As expected from earlier genetic studies indicating that expression of A. vinelandii glnA did not depend on the rpoN (ntrA; sigma 54) gene product, no sigma 54 recognition sequences were present, nor was there significant regulation of glnA expression by fixed nitrogen. Repeated attempts to construct glutamine auxotrophs by recombination of glnA insertion mutations were unsuccessful, Although the mutated DNA could be found by hybridization experiments in drug-resistant A. vinelandii transformants, the wild-type glnA region was always present. These results suggest that glnA mutations are lethal in A. vinelandii. In [14C]glutamine uptake experiments, very little glutamine was incorporated into cells, suggesting that glutamine auxotrophs are nonviable because they cannot be supplied with sufficient glutamine to support growth.

Amino Acid Sequence↗
PubMed1990

Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers.

Mutated forms of trfA, the replication protein gene of plasmid RK2, that support a minimal RK2 origin plasmid in Escherichia coli at copy numbers up to 23-fold higher than normal have been isolated. Six such high-copy-number (copy-up) mutations were mapped and sequenced. In each case, a single base transition led to an amino acid substitution in the TrfA protein primary sequence. The six mutations affected different residues of the protein and were located within a 69-base-pair region encoding 24 amino acids. Dominance tests showed that each of the mutants can be suppressed by wild-type trfA in trans, but suppression is highly dependent on the amount of wild-type protein produced. Excess mutant TrfA protein provided in trans significantly increased the copy number of RK2 and other self-replicating derivatives of RK2 that contain a wild-type trfA gene. These observations suggest that the mutations affect a regulatory activity of the TrfA replication protein that is a key factor in the control of initiation of RK2 replication.

Alleles↗
PubMed1988

Identification and characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum.

Five Tn5-induced Nif- mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae. The mutants fell into two different classes on the basis of hybridization to a Rhizobium leguminosarum nifA gene probe and by complementation with cosmids isolated from pLAFRI gene banks of A. vinelandii and Azotobacter chroococcum. One mutant, MV3, was located in or near a nifA gene. The others, MV12, MV16, MV18 and MV26, defined a new regulatory gene, which has been called nfrX. The lack of expression of different nif-lacZ fusions confirmed the regulatory phenotype of all five mutant strains. The ability of both nifA and nfrX mutants to grow on nitrogen-free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V-containing nitrogenase of A. vinelandii. A fragment carrying Tn5 and flanking DNA from MV3 was used as a probe to isolate the nifA region of A. chroococcum. Ligation of two adjacent EcoRI fragments of A. chroococcum yielded an intact nifA gene that activated expression of nifH-lac fusions and also restored MV3 to Nif+. The four nfrX mutants were complemented by pLAFR1 cosmids pLV163 and pLC121. The nfrX gene was subcloned from pLV163 and located within a 3.2 kb fragment. To determine whether nfrX might be found in other nitrogen-fixing organisms, DNA from 13 different species was hybridized to an nfrX probe. The failure to observe hybridization suggests that nfrX may be specific to nif regulation in Azotobacter.

Azotobacter↗
PubMed1987

The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins.

The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined. It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA). NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors. Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing greater than 50% homology and an intervening sequence of less than 10% homology. The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors. The C-terminal region contains a region of homology to the beta' subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs. In A. vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K. pneumoniae and R. meliloti.

Amino Acid Sequence↗
PubMed1986

Regulation of nitrogen metabolism in Azotobacter vinelandii: isolation of ntr and glnA genes and construction of ntr mutants.

The ntrA, ntrB and ntrC products are responsible for regulating the transcription of many genes involved in the assimilation of poor nitrogen sources in enteric bacteria. The presence of a similar system in the non-enteric bacterium Azotobacter vinelandii is reported here. Genes analogous to ntrA and ntrC were isolated from an A. vinelandii gene library by complementation of Escherichia coli mutants. The gene encoding glutamine synthetase, glnA, was also isolated and found to be adjacent to ntrC but distant from ntrA, as it is in enteric organisms. The cloned Azotobacter genes also complemented Klebsiella pneumoniae mutants and hybridized to K. pneumoniae ntrA, ntrC and glnA gene probes. The role of ntrA and ntrC in A. vinelandii was established by using Tn5 insertions in the cloned genes to construct mutants by marker exchange. These mutants show that both ntrA and ntrC are required for the utilization of nitrate as a nitrogen source. However, ntrC is not required for nitrogen fixation by A. vinelandii, in contrast with K. pneumoniae where both ntrA and ntrC are essential.

Azotobacter↗