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Biomedical subjects

M Bagdasarian

Publications and source records attributed to M Bagdasarian.

At least 37 records · Page 2Linked to original sources
PubMed1995

Functional distinction among structural subsections in the specific priming signal for DNA replication of the broad host-range plasmid RSF1010.

To analyze the functional contribution to the ssiA function of subsections of the ssiA-determinant sequence based on their dimensions, we constructed ssiA mutants carrying insertions and deletions. Results of the examination of the ssiA mutants told us that, in addition to the base sequence, the dimensions were crucial factors for the functional contribution of the subsections of ssiA.

Bacterial Proteins↗
PubMed1994

Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication.

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV). If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins. Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above. These were also true for the RSF1010 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site). Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation. We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication.

Bacterial Proteins↗
PubMed1994

Functional features of oriV of the broad host range plasmid RSF1010 in Pseudomonas aeruginosa.

The broad host range plasmid RSF1010 requires for its replication in Escherichia coli three plasmid-encoded proteins and specific nucleotide sequences ssiA, ssiB, and iterons in the oriVRSF1010. In Pseudomonas aeruginosa, a recombinant mini-RSF1010 plasmid lacking ssiB lost its replication ability, but a miniplasmid lacking ssiA or carrying a primosome assembly site in place of ssiA could replicate. Moreover, ssiA, as a sole ssi signal, in the orientation that ssiB had originally taken was sufficient for replication of the miniplasmid. These results indicated that only one RSF1010-specific ssi signal in the orientation that ssiB takes in wild-type oriVRSF1010 was essential for replication of RSF1010. Replication of the miniplasmids was dependent on the three plasmid-encoded proteins, RepA, B', and C, as in E. coli.

Bacteriophage phi X 174↗
PubMed1993

A base-paired hairpin structure essential for the functional priming signal for DNA replication of the broad host range plasmid RSF1010.

The two single-strand DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010) of the broad host-range plasmid RSF1010 contain proposed stem-loop structures. Nine single base-change mutations in the stem of the ssiA structure, each of which destroyed a relevant base pairing, damaged the ssiA activity. A second single-base change was introduced into each of the nine ssiA mutants in such a way that the base pairing was restored. Only three out of nine second base changes that restored the base pairing restored the ssiA activity up to the wild-type level. Thus, the three are intramolecular suppressors. The results strongly suggested that, in the area of the stem of ssiA where the suppressor mutations fell, base pairing was the most important structural parameter for the ssiA activity. By contrast, it is most probable that, in the other part of the stem of ssiA, both base-pairing and the intrinsic base sequence were the major determinants of the ssiA activity.

Base Sequence↗
PubMed1993

Genes required for extracellular secretion of enterotoxin are clustered in Vibrio cholerae.

Pleiotropic transposon insertion mutants of Vibrio cholerae that are unable to secrete enterotoxin, HA/protease and chitinase through the outer membrane have been isolated. The gene, epsM, responsible for complementation of two of the Tn5 insertion mutations was sequenced. It encodes a putative cytoplasmic membrane protein of 18.5 kDa that exhibits similarity to proteins required for extracellular secretion of pullulanase, pectate lyase or elastase in other Gram-bacteria. It is present on a 15-kb DNA fragment from the V. cholerae genome, containing the epsE gene that was previously shown to be required for secretion of cholera toxin [Sandkvist et al., Gene 123 (1993) 81-86]. Partial reading frames flanking epsM also demonstrated similarity to genes required for extracellular secretion of pullulanase in Klebsiella oxytoca.

Amino Acid Sequence↗
PubMed1993

The role of active-site aromatic and polar residues in catalysis and substrate discrimination by xylose isomerase.

The functions of individual amino acid residues in the active site of Thermoanaerobacterium thermosulfurigenes D-xylose ketol-isomerase (EC 5.3.1.5) were studied by site-directed substitution. The role of aromatic residues in the active-site pocket was not limited to the creation of a hydrophobic environment. For example, Trp-188 provided for substrate binding and Trp-139 allowed for the discrimination between D-xylose and D-glucose. Substrate discrimination was accomplished by steric hindrance caused by the side chain of Trp-139 toward the larger glucose molecule. Preference of the enzyme for the alpha-anomer of glucose depended on the His-101/Asp-104 pair. Wide differences observed in the catalytic constant (kcat) for alpha- versus beta-glucose in the wild-type enzyme and the fact that only the kcat for alpha-glucose was changed in the His-101-->Asn mutants strongly suggest that the substrate molecule entering the hydride-shift step is still in the cyclic form. On the basis of these results a revised hypothesis for the catalytic mechanism of D-xylose isomerase has been proposed that involves His-101, Asp-104, and Asp-339 functioning as a catalytic triad.

Aldose-Ketose Isomerases↗
PubMed1993

A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae.

A gene essential for the secretion of cholera toxin from the periplasm of Vibrio cholerae into the extracellular medium has been isolated and its nucleotide sequence determined. It encodes a cytoplasmic protein of 56 kDa that exhibits a high degree of similarity to gene products required for extracellular protein secretion in several other Gram- organisms. Sequence similarities in its potential ATP-binding site suggest that the protein may act as an energy provider or signal transducer in the process of extracellular secretion.

Amino Acid Sequence↗
PubMed1993

Suppression of temperature-sensitive assembly mutants of heat-labile enterotoxin B subunits.

Deletions or substitutions of amino acids at the carboxyl-terminus of the heat-labile enterotoxin B subunit (EtxB) affect its assembly into pentamers in a temperature-dependent manner. At 42 degrees C, the mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers. However, mutant B subunits produced at 30 degrees C assemble, in the periplasm, into pentamers that remain stable when transferred to 42 degrees C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited. The mutant pentamers are, similarly to wild-type pentamers, SDS-resistant and stable, in vitro, at temperatures up to 65 degrees C. This suggests that although the C-terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process. Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C-terminus. The Thr-->Ile replacement at position 75 in the alpha 2-helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met-->Leu substitution at position 101 in the beta 6-strand of EtxB191.5. Interaction between the alpha 2-helix and beta 6-strand which contains the C-terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.

Amino Acid Sequence↗
PubMed1992

Intermolecular interactions between the A and B subunits of heat-labile enterotoxin from Escherichia coli promote holotoxin assembly and stability in vivo.

Cholera toxin and the related heat-labile enterotoxin (LT) produced by Escherichia coli consist of a holotoxin of one A subunit and five B subunits (AB5). Here we investigate the domains of the A subunit (EtxA) of E. coli LT which influence the events of B-subunit (EtxB) oligomerization and the formation of a stable AB5 holotoxin complex. We show that the C-terminal 14 amino acids of the A subunit comprise two functional domains that differentially affect oligomerization and holotoxin stability. Deletion of the last 14 amino acids (-14) from the A subunit resulted in a molecule that was significantly impaired in its capacity to promote the assembly of a mutant B subunit, EtxB191.5. In contrast, deletion of the last four amino acids (-4) from the A subunit gave a molecule that retained such a capacity. This suggests that C-terminal residues within the -14 to -4 region of the A subunit are important for promoting the oligomerization of EtxB. In addition, we demonstrate that the truncated A subunit lacking the last 4 amino acids was unable to form a stable AB5 holotoxin complex even though it promoted B-subunit oligomerization. This suggests that the last 4 residues of the A subunit function as an "anchoring" sequence responsible for maintaining the stability of A/B subunit interaction during holotoxin assembly. These data represent an important example of how intermolecular interactions between polypeptides in vivo can modulate the folding and assembly of a macromolecular complex.

Amino Acid Sequence↗
PubMed1992

DnaG-dependent priming signals can substitute for the two essential DNA initiation signals in oriV of the broad host-range plasmid RSF1010.

Broad host-range plasmid RSF1010 contains in the oriV region two DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010), which are essential for plasmid replication. Each of ssiA and ssiB could be substituted functionally by either of the two G4-type (DnaG-dependent) priming signals, the oric of bacteriophage G4 and an ssi signal from plasmid pSY343 (an R1 plasmid derivative). Functions of the chimeric oriVs of RSF1010 thus constructed were dependent on the RSF1010-specific replication proteins, RepA, RepB' and RepC. When both of ssiA and ssiB were replaced by the G4-type ssi signals, functions of the chimeric oriVs were no longer dependent on RepB' (RSF1010-specific DNA primase). The replication activities of the chimeric oriVs of RSF1010 were not influenced markedly by the type of heterologous priming signals they contained. It is conceivable that DNA replication of RSF1010 does not need the priming mechanism for lagging strand synthesis and proceeds by the strand displacement mechanism.

Bacterial Proteins↗
PubMed1992

Replication and copy number control of the broad-host-range plasmid RSF1010.

Initiation of replication of the broad-host-range plasmid RSF1010, is accurately controlled by the plasmid-encoded proteins, RepB (MobA), RepB', RepA and RepC [Haring et al., Proc. Natl. Acad. Sci. USA 82 (1985) 6090-6094; Scherzinger et al., Nucleic Acids Res. 19 (1991) 1203-1211]. The genes encoding these proteins which are essential for replication and conjugative mobilization are transcribed from a cluster of promoters, P1/P3 and P2, which partly overlap with the origin of conjugal transfer, oriT. Three regions were found where deletion mutations affect the mobilization of RSF1010 and increase its copy number in Escherichia coli. A deletion in the mobC gene increased the copy number of RSF1010 four-fold. Another deletion, that removed oriT and part of the promoter believed to be responsible for the expression of mobC, results in a three-fold increase in copy number. The third type of deletions affect the N-terminal part of RepB (MobA). A deletion that created a frame-shift results in a three-fold increase in copy number. A smaller, in-frame deletion of this region only affected the mobilization of RSF1010, but not its copy number. The extent by which RSF1010 or its deletion derivatives could repress the P1/P3 and P2 promoters has indicated that these promoters are negatively regulated by MobC and RepB (MobA), presumably by their attachment to the oriT region of RSF1010. Both MobC and RepB are required for the maximal repression of the rep operon. Optimal function of RSF1010 thus involves not only overlapping genes, but also proteins that exert multiple functions, mobilization, replication and regulation.

Amino Acid Sequence↗
PubMed1992

PsiB, and anti-SOS protein, is transiently expressed by the F sex factor during its transmission to an Escherichia coli K-12 recipient.

PsiB, an anti-SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence-deduced molecular mass of 15741 daltons. Using an immuno-assay with anti-PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6-5 plasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn10 out-going promoter located upstream of psiB. When we inserted a Tn10 promoter upstream of F psiB, the F PsiB protein concentration reached the level of R6-5 PsiB. We describe here the physiological role that PsiB protein may have in the cell and how it causes an anti-SOS function. We observed that PsiB protein was transiently expressed by a wild-type F sex factor during its transmission to an Escherichia coli K-12 recipient. In an F+ x F- cross, PsiB concentration increased at least 10-fold in F- recipient bacteria after 90 minutes and declined thereafter; the psiB gene may be repressed when F plasmid replicates vegetatively. PsiB protein may be induced zygotically so as to protect F single-stranded DNA transferred upon conjugation. PsiB protein, when overproduced, may interfere with RecA protein at chromosomal single-stranded DNA sites generated by discontinuous DNA replication, thus causing an SOS inhibitory phenotype.

Bacterial Proteins↗
PubMed1991

Switching substrate preference of thermophilic xylose isomerase from D-xylose to D-glucose by redesigning the substrate binding pocket.

The substrate specificity of thermophilic xylose isomerase from Clostridium thermosulfurogenes was examined by using predictions from the known crystal structure of the Arthrobacter enzyme and site-directed mutagenesis of the thermophile xylA gene. The orientation of glucose as a substrate in the active site of the thermophilic enzyme was modeled to position the C-6 end of hexose toward His-101 in the substrate-binding pocket. The locations of Met-87, Thr-89, Val-134, and Glu-180, which contact the C-6-OH group of the substrate in the sorbitol-bound xylose isomerase from Arthrobacter [Collyer, C.A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. 212, 211-235], are equivalent to those of Trp-139, Thr-141, Val-186, and Glu-232 in the thermophilic enzyme. Replacement of Trp-139 with Phe reduced the Km and enhanced the kcat of the mutant thermophilic enzyme toward glucose, whereas this substitution reversed the effect toward xylose. Replacement of Val-186 with Thr also enhanced the catalytic efficiency of the enzyme toward glucose. Double mutants with replacements Trp-139----Phe/Val-186----Thr and Trp-139----Phe/Val-186----Ser had a higher catalytic efficiency (kcat/Km) for glucose than the wild-type enzyme of 5- and 2-fold, respectively. They also exhibited 1.5- and 3-fold higher catalytic efficiency for D-glucose than for D-xylose, respectively. These results provide evidence that alteration in substrate specificity of factitious thermophilic xylose isomerases can be achieved by designing reduced steric constraints and enhanced hydrogen-bonding capacity for glucose in the substrate-binding pocket of the active site.

Aldose-Ketose Isomerases↗
PubMed1991

A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants.

A series of controlled expression vectors was constructed based on the wide-host-range plasmid pMMB66EH. Some of these new vectors code for the alpha-peptide of beta-galactosidase and allow the direct screening of recombinant clones by inactivation of alpha-complementation. The bla gene was replaced in some plasmids by the cat gene of Tn9 coding for chloramphenicol resistance, extending the use into beta-lactam-resistant strains. They all feature either the tac or taclac (tac-lac UV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. These vectors were tested by subcloning the xylE gene coding for the Pseudomonas putida catechol 2,3-oxygenase and the Escherichia coli lamB gene coding for the lambda receptor. The expression of these genes in E. coli indicated that the tac promoter is five times stronger than the taclac promoter and that both were tightly regulated. The tac promoter in Pseudomonas syringae pv glycinea and Xanthomonas campestris pv vesicatoria had a strength similar to that in E. coli, while the taclac promoter was much weaker, reaching only 6.5 and 3% of the level of expression of the tac promoter, respectively. The taclac promoter, however, proved to be useful for the cloning in E. coli of DNA fragments that were unstable in vectors with stronger promoters and higher copy number. Expression of the lamB gene in Vibrio cholerae strain TRH7000 was not sufficient to permit cosmid transduction. Two subunits of the E. coli mannose permease, coded by the ptsP and ptsM genes, are also required for cosmid DNA penetration into the recipient cells.

Amino Acid Sequence↗
PubMed1991

Functional division and reconstruction of a plasmid replication origin: molecular dissection of the oriV of the broad-host-range plasmid RSF1010.

Two single-stranded DNA initiation signals (designated ssi) present in the origin of vegetative DNA replication (oriV) of the broad-host-range plasmid RSF1010 are essential for the priming of replication of each complementary DNA strand of this plasmid in Escherichia coli. Each of the RSF1010 ssi signals, ssiA and ssiB, could be replaced by a primosome assembly site from plasmid pACY184 or from bacteriophage phi X174. In these chimeric origins, replication of the strand complementary to that containing the primosome assembly site was no longer dependent on the RSF1010 primase, protein RepB', but required the E. coli primase, DnaG. If both ssiA and ssiB sites of RSF1010 were replaced by primosome assembly sites, protein RepB' was no longer essential for the replication at this origin, whereas proteins RepA and RepC of RSF1010 were still required. These results strongly suggest that the two ssi sites and the RepB' protein actually direct the priming of DNA synthesis in the replication of RSF1010, and the proteins RepA and RepC are involved in the prepriming events--i.e., the opening of the DNA duplex at oriV. It is evident that the origin of RSF1010 can be separated into three functional domains and reconstructed by replacing the ssi sites with heterologous elements.

Base Sequence↗
PubMed1990

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine.

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.

Aldose-Ketose Isomerases↗
PubMed1990

Minimal deletion of amino acids from the carboxyl terminus of the B subunit of heat-labile enterotoxin causes defects in its assembly and release from the cytoplasmic membrane of Escherichia coli.

Minimal alterations at the carboxyl terminus of the B subunit (EtxB) of heat-labile enterotoxin from Escherichia coli were found to have a marked effect on the assembly and release of this polypeptide into the periplasm. Nine mutant EtxB polypeptides were obtained by genetic manipulation of the 3'-end of the etxB gene using Bal31 nuclease digestion and codon substitution. A correlation was observed between the magnitude of the changes introduced at the carboxyl terminus and the extent to which the mutant polypeptides were defective in assembly and release. Some of the mutant B subunits, exemplified by those in which the last 2 amino acids had been deleted or in which the last 4 residues had been replaced by three different ones, were found to be only partially defective, with a proportion being associated with the periplasmic face of the cytoplasmic membrane and the remainder being exported to the periplasm. The portion associated with membranes was detected as monomers on sodium dodecyl sulfate-polyacrylamide gels, whereas the portion exported to the periplasm were detected as assembled oligomers. We conclude that the last few amino acids at the carboxyl terminus of EtxB exert a profound influence on the assembly and release of the B subunit from the cytoplasmic membrane during export in E. coli.

Amino Acid Sequence↗