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W K Maas

Publications and source records attributed to W K Maas.

At least 19 recordsLinked to original sources
PubMed1992

Binding of the arginine repressor of Escherichia coli K12 to its operator sites.

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.

Arginine↗
PubMed1991

Isolation and properties of the RepA1 protein of the IncFII replicon, RepFIC.

The initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the lambda PL promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40,000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame.

Amino Acid Sequence↗
PubMed1990

Distribution of msDNAs among serotypes of enteropathogenic Escherichia coli strains.

A genetic element, called a retron, is present in certain Escherichia coli strains. It consists of genes for the production of a covalently linked DNA-RNA compound and a reverse transcriptase. The presence of a retron can be detected by testing for a satellite DNA band by polyacrylamide gel electrophoresis. This DNA band consists of the DNA portion of the DNA-RNA compound and is called msDNA (multicopy single-stranded DNA). In a survey of intestinal E. coli isolates we detected msDNAs in classical enteropathogenic (EPEC) strains and in strains with aggregative adherence to tissue-culture cells (AA), but not in enteroinvasive (EIEC) and enterotoxigenic (ETEC) strains. Among 76 EPEC strains belonging to 14 different serotypes, msDNA was found to be present in 7 serotypes. In total, five different types of msDNA were found, although within each serotype, the msDNAs were the same. These results suggest that different retrons are clonally inherited.

DNA, Bacterial↗
PubMed1990

Mapping of the msDNA operon in the chromosome of Escherichia coli B.

An msDNA operon, consisting of genes for msDNA and a reverse transcriptase, is present in Escherichia coliB and absent from E. coliK12. We have found that the msDNA operon is located on a DNA fragment, longer than 15kb, that is absent from E. coliK12. Using conjugation, P1 transduction, and nucleic acid hybridization between E. coliB and E. coliK12 strains, we have located the position of the msDNA operon on the E. coliB chromosome at a site that corresponds to minute 19 on the genetic map and to position 900 on the physical map of the E. coliK12 chromosome.

Blotting, Southern↗
PubMed1990

Expression of the cloned gene for enterotoxin STb of Escherichia coli.

This study involved the construction of hybrid plasmids to produce heat-stable enterotoxin type II of Escherichia coli (STb). The translation of the open reading frame for the STb gene estA was demonstrated in several ways. Studies using in vivo labeling with [35S]cysteine demonstrated a radiolabeled protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expected molecular weight of 5,000 for toxin STb. Insertion of translational or transcriptional termination signals into the BglII site of the estA gene blocked the expression of estA. The estA gene was cloned into high-expression vector pKC30 downstream from the strong pL promoter. Northern (RNA) blotting assays revealed a 10- to 20-fold increase in mRNA produced by strain C600F(pKC30STb) over other STb-producing strains, compared with little or no increase in enterotoxin activity demonstrated by bioassay. The estA gene, with its own promoter and Shine-Delgarno region and a portion of the sequence for the signal peptide deleted, was also inserted under the control of the tac promoter. Even after induction of the tac promoter by addition of isopropyl-beta-D-thiogalactopyranoside, no biologic enterotoxin activity could be identified. Neutralizing antibodies to STb were produced in rabbits by using either a purified OmpF-STb-beta-galactosidase fusion protein or a 19-amino-acid synthetic STb peptide coupled to keyhole limpet hemocyanin.

Animals↗
PubMed1989

Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA-RNA compound in E. coli B.

We have found a branched DNA-RNA compound in E. coli B, that is similar in its secondary structure, but not its nucleotide sequence, to the previously described branched DNA-RNA compounds in myxobacteria. This compound is not produced in E. coli K12. We have cloned a 3.5 kb chromosomal segment of E. coli B, which, when transferred into E. coli K12, leads to the production of the DNA-RNA compound. We describe the isolation of the DNA-RNA compound, the determination of its nucleotide sequence, and the nucleotide sequence of the genes required for its formation. The sequence contains the coding regions for the DNA component, the RNA component, and an open reading frame encoding a reverse transcriptase. This reverse transcriptase is shown to be required for the formation of the DNA-RNA compound in vivo and in vitro.

Amino Acid Sequence↗
PubMed1989

Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86.

Many plasmids belonging to the F incompatibility groups contain more than one basic replicon. The chimeric plasmid pCG86 is an example of such a multireplicon plasmid. The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied. The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication. In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility. This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated. We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.

Cloning, Molecular↗
PubMed1989

Reverse transcriptase in bacteria.

Reverse transcriptase, discovered in 1970 in retroviruses, has until recently been found only in eukaryotic organisms. Recently it was shown to occur in two groups of bacteria: myxobacteria and Escherichia coli. The gene for reverse transcriptase is part of a chromosomal genetic element that codes for the production of a branched DNA-RNA compound. In this compound a single-stranded DNA is connected to RNA at a specific G residue by a 2'-5' phosphodiester linkage. The precursor for the DNA-RNA compound is a folded messenger RNA, in which the specific G residue is the initiation point for reverse transcription. In the final DNA-RNA compound, the portion of the RNA transcribed by reverse transcriptase is eliminated by RNase H. The DNA-RNA compound is present in several hundred copies per cell. Its biological function is unknown at present.

Chromosomes, Bacterial↗
PubMed1988

A basic replicon of virulence-associated plasmids of Shigella spp. and enteroinvasive Escherichia coli is homologous with a basic replicon in plasmids of IncF groups.

Shigella species and enteroinvasive Escherichia coli strains carry a large (120- to 140-megadalton) plasmid called pINV, which contains genes essential for the invasiveness of these pathogens. Hybridization with specific probes derived from the RepFIC and RepFIB replicons of the IncF1 Ent plasmid P307 showed that pINVs present in 35 clinical isolates are homologous with RepFIC but not RepFIB, regardless of the serogroup of the Shigella or E. coli strain. RepFIC of P307, in turn, is very similar to RepFIIA replicons of IncFII R plasmids. These and other related replicons constitute the RepFIIA family. With one pINV, pWR110, a plasmid of Shigella flexneri 5, we demonstrated the existence of a functional replicon, RepINV, with a restriction map similar to that of RepFIIA of plasmid R1. We isolated the putative inc RNA coding region of RepINV, which is a major determinant of incompatibility. The nucleotide sequence of the RepINV-inc RNA-coding region was determined and compared with the corresponding sequences of RepFIC and RepFIIA. The differences were small, but apparently were sufficient to affect the target specificity of the inc RNAs, thus rendering the replicons compatible with each other. We conclude that pINVs present in Shigella spp. and enteroinvasive E. coli constitute a homogeneous group, containing one basic replicon that belongs to the RepFIIA family of replicons.

Base Sequence↗
PubMed1987

Nucleotide sequence of the argR gene of Escherichia coli K-12 and isolation of its product, the arginine repressor.

In Escherichia coli, the arginine repressor, the product of the argR gene, in conjunction with L-arginine controls the synthesis of the enzymes of arginine biosynthesis. We describe the nucleotide sequence of the argR gene, including its control region, and show that formation of the repressor is autoregulated. The argR control region contains two promoters, one of which overlaps the operator site and, as with other arg genes, consists of two adjacent palindromic sequences ("ARG boxes"). The arginine repressor protein and an arginine repressor-beta-galactosidase fusion protein were purified, and the amino acid sequence of the N-terminal end of the repressor protein portion of the fusion protein was determined. Antibodies prepared against the fusion protein react with the repressor. The repressor is precipitable by L-arginine, which facilitates its purification. The native repressor is a hexamer with a molecular weight of 98,000; its monomeric subunit has a molecular weight of 16,500. To verify its properties postulated from genetic studies, we show that in the presence of L-arginine, repressor inhibits transcription of argF and binds to the ARG boxes of argF and argR.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence analysis of RepFIC, a basic replicon present in IncFI plasmids P307 and F, and its relation to the RepA replicon of IncFII plasmids.

RepFIC is a basic replicon of IncFI plasmid P307 which is located within a 3.09-kilobase SmaI fragment. The nucleotide sequence of this region has been determined and shown to be homologous with the RepFIIA replicon of IncFII plasmids. The two replicons share three homologous regions, HRI, HRII, and HRIII, which are flanked by two nonhomologous regions, NHRI and NHRII. A comparison of coding regions reveals that the two replicons have several features in common. RepFIC, like RepFIIA, codes for a repA2 protein with its amino-terminal codons in HRI and its carboxy-terminal codons in NHRI. Although the codons for the repA1 proteins are located in NHRII, the DNA region containing a putative promoter, ribosomal binding site, and initiation codons is located in HRII. This region also codes for an inc RNA. There are nine base-pair differences between the inc RNA of RepFIIA and that of RepFIC, and as a result, RepFIC and RepFIIA replicons are compatible. An EcoRI fragment from the F plasmid which shows homology with RepFIC of P307 has also been sequenced. This fragment contains only a portion of RepFIC, including the genes for the putative repA2 protein and inc RNA. The region coding for a putative repA1 protein is interrupted by the transposon Tn1000 and shows no homology with the repA1 region of RepFIIA and RepFIC of P307. Our comparative and structural analyses suggest that RepFIC and RepFIIA, although different, have a similar replication mechanism and thus can be assigned to the same replicon family, which we designate the RepFIIA family.

Base Sequence↗
PubMed1986

Distribution of basic replicons having homology with RepFIA, RepFIB, and RepFIC among IncF group plasmids.

Plasmids encoding F-like pili have been divided into groups on the basis of their incompatibility behavior. Three basic replicons have been recognized previously in the IncFI plasmid group and we have now examined their distribution in representative plasmids from 22 of the currently recognized incompatibility groups. The occurrence of these basic replicons was found to be rare outside of the IncF group, and significant hybridization was shown only for RepFIA to IncH1 and I group plasmids. Homology to the RepFIC basic replicon was found in all but one of the IncF group plasmids examined but RepFIA and RepFIB have a more restricted distribution. It appears likely that some plasmids carry vestiges of replicons which still express incompatibility but are incapable of replication. We suggest that evolutionary divergence among the plasmids of the IncF group has resulted from various genetic rearrangements among these basic replicons.

Cloning, Molecular↗
PubMed1986

Increased survival from peritonitis after blockade of transdiaphragmatic absorption of bacteria.

Based upon the knowledge that bacteria in the peritoneal cavity gain access to circulating blood by way of transdiaphragmatic absorption into lymph, a study was carried out to determine whether this absorption protects or endangers the host. Blockade of absorption produced by intraperitoneal (IP) injection of platelet rich plasma (PRP) or by scarification of the peritoneal surface of the diaphragm increased survival time in rats with double colonic perforation from 20 per cent in control rats to 86 and 93 per cent, respectively. Each of these maneuvers significantly increased the number of negative blood culture findings.

Absorption↗
PubMed1985

Construction of a conjugative plasmid with potential use in vaccines against heat-labile enterotoxin.

A conjugative plasmid with potential usefulness for vaccine strains was constructed. In the first step, a 5.9-kilobase DNA segment containing the two loci for the A and B subunits of heat-labile enterotoxin with a mutation in the gene for the A subunit was joined to the cloning vehicle pGA22, generating the nonconjugative plasmid pPMC4 with genes for resistance to tetracycline and chloramphenicol. In the second step, a segment of pPMC4 containing the genes for the A and B subunits, the gene for chloramphenicol resistance, and the replication genes of pGA22 was ligated to the genes for conjugal transfer of the F plasmid, generating the 54.9-kb plasmid pPMC5. Eleven porcine Escherichia coli isolates were tested as recipients for pPMC4 and pPMC5. For pPMC4, transformation and mobilization with a conjugative R plasmid were used to effect plasmid transfer. Only 1 of the 11 strains acted as a recipient in transformation. Mobilization with the R plasmid occurred with two strains, but the plasmids were altered during transfer. In contrast, pPMC5 was transferred with high frequency and unaltered to 9 of the 11 E. coli strains. Transconjugants from these nine matings produced high titers of the B subunit and no active heat-labile enterotoxin. Plasmid pPMC5 was stable in three porcine E. coli strains tested; plasmid pPMC4 was somewhat less stable in these strains. The method we describe for the construction of conjugative chimeric plasmids offers an opportunity for introducing genes with potential for immunization into bacterial strains that are suitable for colonizing the appropriate host sites.

Animals↗
PubMed1985

Detection of genes for heat-stable enterotoxin I in Escherichia coli strains isolated in Brazil.

Heat-stable enterotoxin I (STI) can be assayed in intestinal loops of pigs and rabbits and in the gut of infant mice. To produce a simpler and more discriminating assay procedure, we used three gene probes corresponding to three forms of STI called STIa, STIb, and STIc. We tested 159 Brazilian isolates, of which 40 were positive in the infant mouse assay. The STIb and STIc probes are similar (93% DNA homology) and are both different from the STIa probe (70% DNA homology). Of 33 strains that were still active for STI 3 years after their isolation, 25 reacted with both the STIb and STIc probes, 4 reacted with the STIc probe only, and 7 reacted strongly with the STIa probe and weakly or not at all with the other probes. Two strains reacted with all three probes. Further analysis showed that each of these two strains contains a small plasmid that reacts with the STIa probe and a large plasmid that reacts with the STIc probe in one strain and weakly with both the STIa and STIc probes in the other strain. It was also shown that the STIa probe reacts with the cloning vehicle pACYC184 used for the cloning of STIc. We conclude that the gene probes used can identify most STI-producing strains and that in cases of positive responses with several probes careful scrutiny is necessary for analysis.

Bacterial Toxins↗