bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida
Authors: Minton N P , Atkinson T , Sherwood R F
Published: January 2020

The gene coding for carboxypeptidase G2 was cloned from Pseudomonas sp. strain RS-16 into Escherichia coli W5445 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322. The plasmid isolated, pNM1, was restriction mapped, and the position of the gene on the 5.8-megadalton insert was pinpointed by subcloning. The expression of carboxypeptidase in E. coli was 100-fold lower than in the Pseudomonas sp. strain. When the cloned gene was subcloned into the Pseudomonas vector pKT230 and introduced into Pseudomonas putida 2440, a 30-fold increase in expression over that obtained in E. coli was observed. High expression (up to 5% soluble protein) was obtained in E. coli by subcloning a 3.1-megadalton Bg/II fragment into the BamHI site of pAT153. The increased expression was orientation dependent and is presumed to be due to transcriptional readthrough from the Tc promoter of the vector. Production of carboxypeptidase was shown to be induced (two-fold) by the presence of folic acid, and the mature protein was shown to be located in the periplasmic space of E. coli.
Genetic analysis of the proBA genes of Salmonella typhimurium: physical and genetic analyses of the cloned proB+ A+ genes of Escherichia coli and of a mutant allele that confers proline overproduction and enhanced osmotolerance
Authors: Mahan M J , Csonka L N
Published: January 2020

Because of the fact that proline overproduction relieves the inhibitory effects of high external osmotic strength in a number of procaryotes, we wished to clone a mutant allele, pro-74, that confers proline overproduction and enhanced osmotolerance on Salmonella typhimurium and Escherichia coli. Therefore, the pro-74 allele, originally located on an E. coli episome F'128, was cloned into pBR322. In a parallel experiment, the wild type proB+ A+ genes of E. coli were also cloned from F'128 into pBR322. Both the pro-74 and the proB+ A+ alleles were obtained on a 10.4-kilobase-pair fragment that also contained the unrelated phoE gene. Strains carrying either the wild-type proB+ A+ or the pro-74 alleles on pBR322 grew more slowly, both in minimal medium and media of elevated osmotic strength, than strains carrying the same alleles on the low-copy plasmid, F'128, indicating that some gene in the cloned region is deleterious in high copy. We constructed Tn5 insertion mutations in the proB and the proA genes of E. coli, carried on F'128 in S. typhimurium. Using P22 transduction in S. typhimurium, we transferred these proB and proA::Tn5 insertions from F'128 into the cloned proBA genes on pBR322. From the restriction maps of the plasmids thus generated, we determined the approximate locations of the proB and the proA genes. We also performed complementation tests of S. typhimurium and E. coli proB and proA mutants by using the F'128 proB and proA::Tn5 insertions. These tests revealed that the proBA genes of S. typhimurium form an operon, whose direction of transcription is from proB to proA. They also indicated that in S. typhimurium, as in E. coli, the proB+ gene encodes gamma-glutamyl kinase, and the proA+ gene encodes gamma-glutamyl phosphate reductase. Complementation tests also indicated that the pro-74 mutation is either in the proB structural gene, or its promoter-operator.
Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12
Authors: Brouwer J , Adhin M R , van de Putte P
Published: January 2020

Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101. The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound. Both effects were shown to be dependent upon excision repair. Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E. coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid. With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101. These results suggest that pKM101-induced repair differs from normal SOS repair.
Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria
Authors: Selvaraj G , Iyer V N
Published: January 2020

We describe the construction and use of a set of plasmid vectors of the transposons Tn1, Tn5, and Tn9 that are suicidal in Rhizobium species and therefore suitable for mutagenesis with these three transposons. The vectors are composed of the p15A replicon which functions in Escherichia coli but not in Rhizobium species and a region encoding the N type of bacterial conjugation system which is very efficient in matings between E. coli and Rhizobium species. The usefulness of the vectors has been most extensively assessed in Rhizobium meliloti. It is likely that they will be useful for mutagenesis and genome manipulation in other bacteria as well.
Transfer of pRD1 to Pseudomonas syringae and evidence for its integration into the chromosome
Authors: Vincent J R , Fulbright D W
Published: January 2020

Plasmid pRD1 was conjugatively transferred from Escherichia coli to Pseudomonas syringae. Subculturing the transconjugate on a medium that selected for pRD1-determined His+ Kmr resulted in the loss of pRD1 as an extrachromosomal element as detected by agarose gel electrophoresis. DNA hybridization provided evidence for the integration of pRD1 into the P. syringae chromosome.
Cytoplasmic pH homeostasis in an acidophilic bacterium, Thiobacillus acidophilus
Authors: Zychlinsky E , Matin A
Published: January 2020

The cytoplasmic buffering capacity of Thiobacillus acidophilus (along with membrane properties) is responsible for the cytoplasmic pH homeostasis in metabolically compromised cells. When a large influx of H+ occurs, the cytoplasmic buffering capacity prevents drastic changes in pH; in addition, this influx, by increasing the positive membrane potential, eventually leads to a cessation of further H+ influx.
The chromosomal 3',5"-aminoglycoside phosphotransferase in Streptococcus pneumoniae is closely related to its plasmid-coded homologs in Streptococcus faecalis and Staphylococcus aureus
Authors: Collatz E , Carlier C , Courvalin P
Published: January 2020

The apparently chromosomally encoded 3',5"-aminoglycoside phosphotransferase (type III), from the high-level aminoglycoside-resistant Streptococcus pneumoniae BM4200, was compared with homologous enzymes coded for by the plasmids pJH1 and pSH2, originally isolated from Streptococcus faecalis and Staphylococcus aureus, respectively, and also found in a wild strain of S. aureus, BM4600. The enzymes appeared to be indistinguishable, and we conclude that the gene encoding 3',5"-aminoglycoside phosphotransferase (type III) can cross generic barriers within gram-positive cocci.
Genetic and biochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs and their revertants
Authors: Dávila G , Brom S , Mora Y , Palacios R , Mora J
Published: January 2020

In this paper we present the isolation and characterization of glutamine auxotrophs of Neurospora crassa and their revertants. The results show that although various enrichment procedures were used, we found only two types of auxotrophs. Genetic crosses performed between the different mutants showed that the mutations responsible for their phenotypes were highly linked and probably affected the same gene. The biochemical characterization of the glutamine synthetase polypeptides of the different mutants showed that both types contained the alpha monomer. However, in place of the normal beta monomer, each type had a new polypeptide differing from normal beta either in its molecular weight or in its isoelectric point. On the other hand, the revertants had only the alpha monomer and were capable of growing without glutamine. On the basis of these data, we propose that the lack of glutamine synthetase activity in the auxotrophs is due to the interaction of the altered beta with the alpha monomer, and as a consequence the alpha monomer of the revertants regains its activity because of the absence of the altered beta.
Role of quinones in the branch of the Escherichia coli respiratory chain that terminates in cytochrome o
Authors: Au D C , Green G N , Gennis R B
Published: January 2020

The role of quinones in the cytochrome o branch of the Escherichia coli respiratory chain was investigated by using mutant strains lacking the cytochrome d terminal oxidase complex. The only cytochromes present were cytochrome b556 and the cytochrome o complex, consisting of cytochrome b555-b562. Mutant strains missing ubiquinone, menaquinone, or both were constructed in the cytochrome d-minus (cyd) background. The steady-state levels of cytochrome b reduction were examined and compared in these strains to assess the effects of the quinone deficiencies. The data clearly show that a ubiquinone deficiency results in a lower level of cytochrome b reduction in the steady state. The data are consistent with a simple model in which ubiquinone is placed on the dehydrogenase side of all the cytochromes in this branch of the respiratory chain. There is no evidence from these experiments for a role of quinones in the respiratory chain at any site besides this one.
Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12
Authors: Eoyang L , Silverman P M
Published: January 2020

Acetohydroxyacid synthase I from Escherichia coli K-12 has been purified to near homogeneity. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two polypeptides, one with a molecular weight of 60,000 and one with a molecular weight of 9,500. These two polypeptides were present in constant proportion to each other and to enzyme activity. The molar ratio of the two polypeptides (Mr 9,500:60,000), estimated from stained polyacrylamide gels, was 1. Antisera raised against the 60,000 Mr polypeptide precipitated both the 60,000 and the 9,500 Mr polypeptides from extracts of cells labeled with [35S]methionine. The addition of sodium dodecyl sulfate before immunoprecipitation eliminated the smaller polypeptide, and only the larger one was recovered. The hydrodynamic properties of the native enzyme confirmed a previous report that the largest enzymatically active species has a molecular weight of about 200,000; this species contains both the 60,000- and 9,500-molecular-weight polypeptides.
Potassium permeability of Rickettsia prowazekii
Authors: Winkler H H
Published: January 2020

The potassium permeability of Rickettsia prowazekii was characterized by chemical measurement of the intracellular sodium and potassium pools and isotopic flux measurements with 86Rb+ as a tracer. R. prowazekii, in contrast to Escherichia coli, did not maintain a high potassium-to-sodium ratio in their cytoplasm except when the potassium-to-sodium ratio in the extracellular medium was high or when the extracellular concentrations of both cations were low (ca. 1 mM). Both influx and efflux assays with 86Rb+ demonstrated that the rickettsial membrane had limited permeability to potassium and that incorporation of valinomycin into these cells increased these fluxes at least 10-fold. The transport of potassium showed specificity and dependence on rickettsial metabolism. The increased flux of potassium which results from the incorporation of valinomycin into the rickettsial membrane was detrimental to both lysine transport and lysis of erythrocytes by the rickettsiae.
Disulfide reduction and sulfhydryl uptake by Streptococcus mutans
Authors: Thomas E L
Published: January 2020

Incubation of Streptococcus mutans cells with certain disulfide compounds resulted in accumulation of reduced sulfhydryl compounds in the extracellular medium or in both the medium and the cells. Oxidized lipoic acid and lipoamide competed for reduction. At high concentrations, these compounds were reduced at rates comparable to that of glucose metabolism, and all of the increase in sulfhydryls was in the medium. Cystamine did not compete with these compounds for reduction but was also reduced at high rates and low apparent affinity, and all of the cysteamine produced from cystamine accumulated in the medium. In contrast, glutathione disulfide (GSSG) and L-cystine were reduced slowly but with high apparent affinity, and 60 to 80% of the increase in sulfhydryls was intracellular. NADH-dependent lipoic acid or lipoamide reductase activity was present in the particulate (wall-plus-membrane) fraction, whereas NADPH-dependent GSSG reductase activity was present in the soluble (cytoplasmic) fraction. Two transport systems for disulfide and sulfhydryl compounds were distinguished. GSSG, L-cystine, and reduced glutathione competed for uptake. L-Cysteine was taken up by a separate system that also accepted L-penicillamine and D-cysteine as substrates. Uptake of glutathione or L-cysteine, or the uptake and reduction of GSSG or L-cystine, resulted in up to a 10-fold increase in cell sulfhydryl content that raised intracellular concentrations to between 30 and 40 mM. These reductase and transport systems enable S. mutans cells to create a reducing environment in both the extracellular medium and the cytoplasm.
Genetic mapping of Vibrio cholerae enterotoxin structural genes
Authors: Sporecke I , Castro D , Mekalanos J J
Published: January 2020

The structural genes which constitute the cholera toxin operon, ctxAB, were genetically mapped in the Vibrio cholerae El Tor strain RV79. This strain of V. cholerae contains two copies of the ctx operon located on a 7-kilobase-pair tandemly duplicated region. We began by isolating a vibriophage VcA1 insertion mutation in one of the two ctxA genes located in this region. The mutant carrying this ctxA::VcA1 insertion, DC24, was converted to a VcA1-facilitated donor by introduction of the conjugal plasmid pSJ15, which carries an inserted copy of a defective VcA1-like prophage. The donor characteristics of DC24(pSJ15) indicated that the ctxA::VcA1 insertion mutation was near the trp region of the V. cholerae chromosome. Subsequent RV79 three-factor crosses were performed between VcA1-facilitated donors and recipient strains carrying one of two structural gene mutations in ctx, either delta ctxA23P Kmr or delta ctx-7922. The former was constructed by an in vivo marker exchange procedure and could be scored either by its kanamycin resistance phenotype or by its lack of DNA sequences homologous to the ctxA region. The delta ctx-7922 mutation is a total deletion of both ctx copies of strain RV79. The three-factor cross data strongly suggest that the two ctx loci of RV79 map between the nal and his genes of V. cholerae in the trp nal his linkage group. Physical analysis and heterologous crosses between an RV79 El Tor donor and a 569B classical recipient indicates that one of the two 569B ctx operon copies maps in the same region as the RV79 ctx loci (i.e., linked to nal). Together with previously published observations, these data show that the ctx structural genes are not closely linked to other genes known to affect toxin production in V. cholerae.
Calcofluor staining of cellulose during microcyst differentiation in wild-type and mutant strains of Polysphondylium pallidum
Authors: Choi A H , O'Day D H
Published: January 2020

Calcofluor White ST was used to monitor the morphological events in the biogenesis of cellulose in the microcyst wall of the wild-type strain (WS-320) and two developmental mutants (mic-1 and mic-2) of Polysphondylium pallidum. During encystment, the cell surface acquires a Calcofluor-specific material which appears to be cellulose because of its sensitivity to purified cellulase. Cellulose-containing vesicles appear distributed throughout the cytoplasm of encysting cells of the three strains. Later, the cellulose-rich vesicles appear near the cell surface. Subsequently, the cell surface stains with Calcofluor, and the vesicles are no longer detectable. Intracellular vesicles resembling the cellulose-rich vesicles in size, in the timing of appearance, and in cellular location are also seen in thin sections. These vesicles are surrounded by a single unit membrane, and their amorphous matrix, which contains a dense irregular core, further implicates them as the basis for the bilayered microcyst wall.
Isolation of an aroF-lac plasmid by recombination in vivo
Authors: Cobbett C S , Pittard J
Published: January 2020

An aroF-lac operon fusion was transferred from a lambda aroF-lac prophage onto a plasmid carrying a 'Mu cts trp-lac fusion by recombination in vivo.
Shuttle vectors for cloning recombinant DNA in Escherichia coli and Streptomyces griseofuscus C581
Authors: Larson J L , Hershberger C L
Published: January 2020

The replicon of the Streptomyces plasmid SCP2 was located on a 5.9-kilobase EcoRI-SalI restriction fragment. The SCP2 replicon was combined with Escherichia coli plasmid pBR322 and genes specifying neomycin resistance and thiostrepton resistance in streptomycetes to construct shuttle vectors that are useful for cloning in E. coli and streptomycetes.
Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes
Authors: Stapleton M J , Jagger K S , Warren R L
Published: January 2020

Transposon Tn5 was used to generate protease-deficient insertion mutants of Pseudomonas aeruginosa. The presence of Tn5 in the chromosome of P. aeruginosa was demonstrated by transduction and DNA-DNA hybridization. The altered protease production and kanamycin resistance were cotransduced into a wild-type P. aeruginosa strain. A radiolabeled probe of Tn5 DNA hybridized to specific BamHI fragments isolated from the insertion mutants. Two independently isolated Tn5 insertion mutants had reduced protease production, partially impaired elastase activity, and no immunologically reactive alkaline protease.
Transmethylation inhibitors decrease chemotactic sensitivity and delay cell aggregation in Dictyostelium discoideum
Authors: van Waarde A , van Haastert P J
Published: January 2020

In Dictyostelium discoideum, extracellular cyclic AMP (cAMP) induces chemotaxis and cell aggregation. Suspensions of cAMP-sensitive cells respond to a cAMP pulse with a rapid, transient increase of protein carboxyl methylation. The transmethylation inhibitors cycloleucine, L-homocysteine thiolactone, and coformycin decrease chemotactic sensitivity and delay cell aggregation when administered in concentrations which do not influence cAMP binding to cell surface receptors or the activity of total phosphodiesterase. The ability of the drugs to inhibit chemotaxis could be correlated with their capacity to convert the initial transient positive response of carboxyl methylation to cAMP into a negative one. This suggests that both protein O-methyltransferase and protein methylesterase are activated after stimulation of aggregative cells with cAMP, the net effect being a transient, positive response of methylation. In the presence of a sufficiently large dose of inhibitor, methyltransferase is inhibited, whereas methylesterase activity is much less affected, so that a transient negative response of methylation to cAMP is observed. The slow, positive response of carboxyl methylation to cAMP which occurs ca. 2.5 to 5 min after stimulus administration is not affected by inhibitors of transmethylation. These results suggest that methylation reactions are involved in the chemotactic response of D. discoideum cells to cAMP.
Regulation of a Neurospora crassa extracellular RNase by phosphorus, nitrogen, and carbon derepressions
Authors: Lindberg R A , Drucker H
Published: January 2020

A new extracellular RNase, designated N4, was detected in culture filtrates from Neurospora crassa and its regulation was studied. Limitation of a nutrient obtainable from RNA alone was not sufficient to cause enzyme derepression. The addition of RNA to the medium had no inductive effect, but the addition of exogenous protein caused enzyme production. With protein in the medium, N4 was derepressible for all three elemental nutrients obtainable from RNA: carbon, nitrogen, and phosphorus. Successful carbon derepression required the addition of a small amount of proteolytic activity to the cultures, as has been reported for the carbon-derepressible proteases of N. crassa. Exogenous protein affected RNase production before translation. Effects of the exogenous protein appeared similar to those previously reported for N. crassa protease induction. N4 was under the control of the nit-2 and nuc-1 gene products. nit-2 and nuc-1 mutants were unable to derepress enzyme synthesis for nitrogen and phosphorus limitation, respectively; however, these mutants responded like wild types to the other two states of derepression. Enzyme synthesis was constitutive in the preg mutant. Results indicate that the transcription of the N4 structural gene responds to multiple regulatory gene products from different regulatory circuits and that external protein affects the synthesis of classes of hydrolases other than proteases.
Mutations in the DNA gyrB gene that are temperature sensitive for lambda site-specific recombination, Mu growth, and plasmid maintenance
Authors: Friedman D I , Plantefaber L C , Olson E J , Carver D , O'Dea M H , Gellert M
Published: January 2020

We report the isolation of two mutations in the gyrB gene of Escherichia coli K12 obtained from an initial selection for resistance to coumermycin A1 and a subsequent screening for bacteria that fail to support site-specific recombination of phage lambda, i.e., Him-. These two mutations have a temperature-sensitive Him- phenotype, supporting site-specific recombination efficiently at low temperature, but inefficiently at high temperatures. Like other Him mutants, the gyrB-him mutants fail to plate phage Mu; again this defect is observed only at high temperatures. Additional thermally sensitive characteristics have also been observed; growth of lambda as well as maintenance of the plasmids pBR322 and F' gal are reduced at high temperature. Restriction of foreign DNA imposed by a P1 prophage is also reduced in these mutants. The temperature-sensitive phenotypic characteristics imposed by both the gyrB-him-230(Ts) and gyrB-him-231(Ts) mutations correlate with in vitro studies that show decreased gyrase activity, especially at higher temperatures, and in vivo studies showing reduced supercoiling of lambda DNA in the mutants at high temperature.
Transduction analysis of transposon Tn551 insertions in the trp-thy region of the Staphylococcus aureus chromosome
Authors: Schroeder C J , Pattee P A
Published: January 2020

Previous studies have shown that Tn551, a 5.2-kilobase-pair transposon that determines constitutive resistance to erythromycin, can occupy a variety of chromosomal sites between thy-101 and trp-103 in Staphylococcus aureus 8325. Although many of these insertions were "silent," many others, including lys, thr, met, tyr, and trp, resulted in auxotrophic mutations. The close proximity and erythromycin-resistant phenotypes of the insertions in this region have made their mapping by transformation difficult. Analysis of these sites and similar chemically induced mutations by generalized transduction with phage 80 alpha have defined the order and relationship of these insertion sites and provided a detailed map of this region of the chromosome, including the orientation of the trp operon. The results of this study and a limited phenotypic characterization of the mutants have shown that the divergent pathway from aspartate to lysine, threonine, and methionine, several reactions in tyrosine biosynthesis, and the entire tryptophan operon are determined by this region of the chromosome. The linkage results obtained by transduction have been compared with similar data obtained previously by transformation; this comparison suggests the existence, between thy and lys, of a preferred headful cutting site for transducing phage DNA morphogenesis from the host chromosome.
Rhizobium meliloti competitiveness and the alfalfa agglutinin
Authors: Handelsman J , Ugalde R A , Brill W J
Published: January 2020

We have isolated two types of isolates having identical colony morphologies from stock cultures of two different Rhizobium meliloti strains. One isolate was agglutinated at a high-dilution titer (HA, highly agglutinable) of the alfalfa agglutinin and was sensitive to phage F20, and the other was agglutinated at a lower agglutinin titer (LA) and was sensitive to phage 16B. All LA isolates from the original slant produced nodules on alfalfa earlier than did HA strains from the original slant. When these HA and LA strains were mixed and used as the inoculum in both vermiculite and field soil in the laboratory, LA strains were always the predominant strains recovered from the nodules. LA strains were obtained from HA cells by selection for resistance to phage F20, and HA strains were obtained from LA cells by selection for resistance to phage 16B. All of the strains with the HA phenotype that were derived from LA strains by phage selection had the nodulation properties of the HA strains from the original slant. Two classes of strains with the LA phenotype were obtained from HA cells by phage selection. One was identical to the original LA strains from the slant, and the other had the nodulation properties of the HA strains. Thus, we have shown that some cell surface properties change the nodulation abilities of R. meliloti strains and, furthermore, that specific phages can be used to enrich for more competitive rhizobia.
Use of chromosomal integration in the establishment and expression of blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis
Authors: Saunders C W , Schmidt B J , Mirot M S , Thompson L D , Guyer M S
Published: January 2020

With several different vectors, attempts were made to establish blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis. Stable establishment of blaZ in B. subtilis was achieved by use of a vector that allowed the integration of a single copy of the gene into the chromosome of that host. blaZ was expressed in the heterologous host since B. subtilis strains carrying integrated blaZ produced beta-lactamase and were more resistant to ampicillin than was wild-type B. subtilis. blaZ was stably inherited in such strains, as no loss of the ability to produce beta-lactamase was observed after growth in nonselective liquid medium or on solid medium. In contrast, a blaZ-containing restriction fragment could not be established in B. subtilis with either pUB110- or pC194-based vectors. Similarly, a pC194-based shuttle vector (pGX318) containing the 5' end of blaZ (including the promoter and the coding region for the signal sequence and the first few amino acids of the mature protein) was unable to transform B. subtilis. Two derivatives of pGX318 that could be stably established in B. subtilis were isolated. The structures of these derivatives suggested that inactivation of the blaZ promoter was associated with the acquisition of the ability to be established.
Uptake of heterologous DNA by Haemophilus influenzae
Authors: Goodgal S H , Mitchell M
Published: January 2020

With the use of highly competent Haemophilus influenzae cells, it was possible to demonstrate the uptake of heterologous DNAs. However, these DNAs, as expected, were only 1% or less as effective when competing for uptake with Haemophilus DNA. Escherichia coli DNA was removed from solution by competent cells to the extent expected if all the E. coli DNA particles contained at least one uptake recognition signal. The data were consistent with a model in which there was one uptake signal per 20 X 10(6) to 30 X 10(6) daltons of E. coli DNA. Since H. influenzae DNA has many more recognition signals, approximately one per 2 X 10(6) daltons (Danner et al., Gene 77:311-318, 1980; K. Vogt and S. H. Goodgal, submitted for publication), it has been suggested that the slower rate of E. coli DNA binding and the so-called specificity of Haemophilus DNA binding are due to the number of recognition signals per molecule of DNA as well as the nature of the DNA receptor (Vogt and Goodgal, submitted for publication). The specificity of native H. influenzae DNA binding does not apply to the uptake of denatured DNA in the transforming system (low pH) for denatured DNA.
Isolation of uvrA mutation on a multicopy plasmid: preliminary characterization of the mutant protein
Authors: Lorensen E , Masker W E , Chase J W
Published: January 2020

A new uvrA mutation (uvrA276) has been isolated on a multicopy plasmid and shown to reside within the region of the uvrA gene defined by the KpnI to SalI endonuclease sites. The protein produced by the uvrA276 mutant gene is identical in size to the wild-type protein and binds to single-stranded DNA under the same conditions as the wild-type protein. However, extracts prepared from strains containing this mutant are deficient at incision of DNA that has been irradiated with UV light.
Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase
Authors: Lacks S A , Springhorn S S
Published: January 2020

The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene.
Expression of hydrogenase in Alcaligenes spp. is altered by interspecific plasmid exchange
Authors: Friedrich B , Friedrich C G , Meyer M , Schlegel H G
Published: January 2020

Alcaligenes hydrogenophilus was found to contain a soluble and a particulate hydrogenase whose control and structure differed in part from that in Alcaligenes eutrophus. One of at least two plasmids indigenous to A. hydrogenophilus determines hydrogenase genes (Hox). The interspecific exchange of Hox-encoding plasmids generated transconjugants which expressed the structural and regulatory Hox phenotype of the donor.
Genetic screen for cloned release factor genes
Authors: Weiss R B , Murphy J P , Gallant J A
Published: January 2020

Nonsense suppression reflects competition between a nonsense suppressor tRNA and a translational release factor. This provides a simple way to screen for release factor genes cloned into a multicopy plasmid. We have confirmed that the expected competition occurs with the gene for release factor 2, cloned by C.T. Caskey et al. (C.T. Caskey, W.C. Forrester, W. Tate, and C.D. Ward, J. Bacteriol. 158:365-368, 1984), and used it to clone the gene for release factor 1.
Origin and direction of in vitro replication of Haemophilus ducreyi and Neisseria gonorrhoeae ampicillin resistance plasmids
Authors: McNicol P J , Albritton W L , Ronald A R
Published: January 2020

The origin of replication of Haemophilus ducreyi and Neisseria gonorrhoeae ampicillin resistance plasmids was located by cloning BamHI restriction fragments into vector plasmid pAT153 and a derivative plasmid, pAT2. Selection was made for plasmid maintenance in a polA mutant. Direction of replication was determined by in vitro replication of plasmid DNA in the presence of radiolabeled deoxynucleotide.
DNA-DNA hybridization analysis of nylon oligomer-degradative plasmid pOAD2: identification of the DNA region analogous to the nylon oligomer degradation gene
Authors: Negoro S , Nakamura S , Okada H
Published: January 2020

Fine structure of the gene of 6-aminohexanoic acid cyclic dimer hydrolase, one of the enzymes responsible for the degradation of the nylon oligomer (6-aminohexanoic acid cyclic dimer), on the plasmid pOAD2 harbored in Flavobacterium sp. KI72 was determined by constructing miniplasmids from plasmid pNDH5 (a hybrid plasmid consisting of pBR322 and a 9.1-kilobase-pair HindIII fragment of pOAD2 ). The 6-aminohexanoic acid cyclic dimer hydrolase produced by cells of Escherichia coli C600 harboring pNDH5 or its miniplasmid was examined immunologically and electrophoretically and was found to be identical to that of Flavobacterium sp. KI72 . A fragment of pOAD2 (17.2- to 19.1-kilobase-pair region on pOAD2 ) was detected as hybridized fragment by Southern blotting experiments, indicating the presence of the DNA region analogous to the 6-aminohexanoic acid cyclic dimer hydrolase gene on the plasmid.
Regulation of protoxin synthesis in Bacillus thuringiensis
Authors: Minnich S A , Aronson A I
Published: January 2020

A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponential growth. The conditional phenotype could be transferred by cell mating to naturally acrystalliferous Bacillus cereus. In all such cases, a 29-Md protoxin -encoding plasmid was transferred, but this plasmid alone was barely sufficient for protoxin synthesis. Protoxin production increased to detectable levels, but well below those of the parental donor strain, by simultaneous transfer of a 44-Md protoxin -encoding plasmid. Transfer of a 5-Md plasmid with the two larger protoxin -coding plasmids resulted in a protoxin synthesis level approaching that of the donor strain. A role for some of the cryptic plasmids of kurstaki in parasporal body formation was implied. In contrast, a closely related B. thuringiensis strain, HD73 , produced crystals at both 25 and 32 degrees C even when the capacity was transferred on a 50-Md plasmid to B. cereus. The amount of protoxin produced in these B. cereus transcipients , however, was somewhat less than that produced in the parental strain HD73 , implying that catabolic differences, gene dosage, or the presence of a chromosomal gene (or a combination of these) may be necessary for maximum production. A regulatory component of the 29-Md plasmid appeared to be trans-acting and dominant since B. cereus transcipients containing the 29-Md plasmid from kurstaki and the 50-Md plasmid from HD73 produced more protoxin at 25 degrees C than at 30 degrees C. Similar results were obtained when protoxin synthetic capacity was transferred from B. thuringiensis subsp. israelensis to the conditional B. thuringiensis subsp. kurstaki strain.
Determination of the precise location and orientation of the Escherichia coli dnaE gene
Authors: Shepard D , Oberfelder R W , Welch M M , McHenry C S
Published: January 2020

The minimal region required for expression of the dnaE gene of Escherichia coli has been determined relative to a detailed restriction endonuclease map. This has been accomplished by analysis of Bal 31 exonuclease-generated deletions from the termini of the E. coli DNA contained in plasmid pMWE303 , a plasmid that we have previously demonstrated to contain the dnaE gene (M. M. Welch and C. S. McHenry , J. Bacteriol . 152:351-356, 1982). The competence of these deletion-containing plasmids in expressing the alpha subunit of DNA polymerase III holoenzyme has been determined by their ability both to complement a dnaE mutant and to direct the synthesis of a complete alpha subunit. The carboxyl-terminal coding region of dnaE has been identified through the detection of partial alpha polypeptides encoded by plasmids containing deletions from one end of the gene. This approach has permitted the precise determination of both termini of the dnaE gene and the determination of the orientation of the gene within the E. coli chromosome.
Proline transport and metabolism in Rickettsia prowazekii
Authors: Winkler H H , Daugherty R M
Published: January 2020

Purified Rickettsia prowazekii cells were able to transport L-proline. The influx of this amino acid had a Kt of 14 microM and a Vmax of about 64 pmol/min per mg of protein. Proline could not be transported by heat-killed or metabolically poisoned rickettsiae or at 0 degrees C. The uptake of proline was linear for almost 2 h. More than 90% of the accumulated intracellular radioactivity was proline. This intracellular pool could not be chased out of the cell by excess non-radioactive proline and did not exit into a proline-free medium. These results indicate that intracellular proline was bound or that the cell had a very limited efflux component for proline transport. The influx of proline was specific: among various analogs tested, only 3,4-dehydro-D,L-proline was effective in inhibiting proline uptake. R. prowazekii cells were unable to utilize proline as an energy source to drive hemolysis, and no measurable evolution from the rickettsiae of CO2 derived from proline occurred. The activities of the enzymes pyrroline-5-carboxylate-reductase and pyrroline-5-carboxylate dehydrogenase were not detectable. These enzymes are important in anabolism and catabolism of proline, respectively, and, if present in R. prowazekii have activities less than 1% of those in Escherichia coli.
Cloning of sporulation gene spoIIG in Bacillus subtilis
Authors: Ayaki H , Kobayashi Y
Published: January 2020

Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B. subtilis temperate phages p11 and phi 105 by the "prophage transformation" method. Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes. Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment. The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed. The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml.
Genetic determinant of pyocin AP41 as an insert in the Pseudomonas aeruginosa chromosome
Authors: Sano Y , Kageyama M
Published: January 2020

The genetic determinant for pyocin AP41 , a bacteriocin produced by Pseudomonas aeruginosa PAF, was transferred to P. aeruginosa PAO and analyzed. By conjugation experiments, the pyocin determinant was found to be located on the chromosome, being closely linked to argG at about 45 min on the genetic map. Cloning of the pyocin AP41 gene into the plasmid R68.45 was attempted in vivo by taking advantage of its linkage at argG. R' argG+ plasmids were isolated by interspecific conjugation between P. aeruginosa and Escherichia coli recA argG strains. Some of the R' argG+ plasmids did contain the pyocin AP41 determinant. Genetic and physical analyses of these R' plasmids indicated that the pyocin AP41 determinant was located within a 2.9-kilobase extra segment found at a certain position of the chromosome of various pyocin AP41 producer strains.
Transposon Tn5 specifies streptomycin resistance in Rhizobium spp
Authors: Selvaraj G , Iyer V N
Published: January 2020

Transposon Tn5 conferred streptomycin resistance on different strains of Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium trifolii but not on Escherichia coli. A gene (str) specifying this phenotype has been identified and localized on the physical and genetic map of Tn5. It is transcribed from the promoter of neo, the gene that encodes neomycin phosphotransferase. The str gene is downstream from neo in a single transcriptional unit, as revealed by molecular cloning of different segments of Tn5 and by cloning of the neo-str region of Tn5 downstream from a lac promoter. Fusion of the SalI-generated rightward segment of Tn5 (devoid of neo) to a part of a tetracycline resistance gene, tet, in a plasmid or downstream from a lac promoter in a plasmid resulted in significant levels of streptomycin resistance in an R. meliloti host, suggesting that the str gene product can function independent of neomycin phosphotransferase. A natural isolate of R. meliloti that does not express Tn5-associated streptomycin resistance has been identified. We have used the str of Tn5 as a genetic marker in Rhizobium spp.
Bacterial plasmids that carry two functional centromere analogs are stable and are partitioned faithfully
Authors: Austin S J
Published: January 2020

The par genes of unit-copy plasmids P1 and F promote equitable distribution of plasmid copies to daughter cells and can be considered to be functional analogs of eucaryotic centromeres. Composite plasmids were constructed which carry either two functional P1 par regions or one F and one P1 region. Unlike dicentric chromosomes, such plasmids are stably maintained.
Mutagenesis of Erwinia carotovora subsp. carotovora with bacteriophage Mu d1 (Apr lac cts62): construction of his-lac gene fusions
Authors: Jayaswal R K , Bressan R A , Handa A K
Published: January 2020

The bacteriophage Mu d1(Apr lac cts62 ) obtained from an Escherichia coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid phage was utilized as a vector for phage mutagenesis in Erwinia carotovora subsp. carotovora. Among ampicillin-resistant transductants. 1.4% were auxotrophs. The synthesis of beta-galactosidase was derepressed upon starvation for histidine in two different his-lac fusion strains.
Genetic control of a structural polymer of the Neurospora crassa cell wall
Authors: Silva M , Pincheira G
Published: January 2020

The heteropolysaccharide present in fraction 1 of the Neurospora crassa cell wall has been characterized in wild-type and morphological mutant strains of this fungus. Single and double mutations have been studied to determine possible genetic interactions controlling the chemical composition of such heteropolysaccharides . Single mutations studied were peak-2, scumbo ( FGSC 49), ragged ( FGSC 296), and crisp -1 ( FGSC 488). Double mutations studied were peak-2, scumbo ( FGSC 419), and ragged crisp -1. In all these strains, the main constituents of the heteropolysaccharide were glucose, mannose and galactose. Glycosidic linkages binding these neutral sugars have been identified by gas-liquid chromatography. A chemical structure of fraction I heteropolysaccharide is proposed. The results obtained with double mutants suggest the existence of genetic interactions, such as complementation or additive effects of lesions of different genes, to control the chemical composition and structure of the cell wall and the morphology of N. crassa mycelium.
Incorporation of isotope from specifically labeled glucose into alginates of Pseudomonas aeruginosa and Azotobacter vinelandii
Authors: Lynn A R , Sokatch J R
Published: January 2020

The incorporation of isotope from [6-14C]glucose into alginate by both Pseudomonas aeruginosa and Azotobacter vinelandii was 10-fold greater than that from either [1-14C]- or [2-14C]glucose, indicating preferential utilization of the bottom half of the glucose molecule for alginate synthesis. These data strongly suggest that the Entner - Doudoroff pathway plays a major role in alginate synthesis in both P. aeruginosa and A. vinelandii.
Transformation of Kluyveromyces fragilis
Authors: Das S , Kellermann E , Hollenberg C P
Published: January 2020

For the transformation of the yeast species Kluyveromyces fragilis, we have constructed a vector containing a bacterial kanamycin resistance (Kmr) gene, the TRP1 gene of Saccharomyces cerevisiae, and an autonomously replicating sequence of Kluyveromyces lactis called KARS2 . By utilizing the method based on treatment by alkali cations and with the Kmr gene as the selective marker, a wild-type strain of K. fragilis was transformed to resistance against the antibiotic G418 . In the transformed cell the plasmid replicates autonomously. The same plasmid could also be used to transform S. cerevisiae trp1 mutant to Trp+. Thus, KARS2 of K. lactis enables the vector to replicate in K. fragilis, K. lactis, and S. cerevisiae, whereas ARS1 of S. cerevisiae allows autonomous replication only in S. cerevisiae.
Effects of deletion of the gene for the development-specific protein S on differentiation in Myxococcus xanthus
Authors: Komano T , Furuichi T , Teintze M , Inouye M , Inouye S
Published: January 2020

A deletion mutation of the gene for protein S (tps), a development-specific protein of Myxococcus xanthus, was constructed. No significant differences in the process of fruiting body formation or the yield of myxospores were observed between mutant and wild-type cells. On the other hand, when the tps gene was deleted together with a 2.0-kilobase sequence including the ops gene immediately upstream of the tps gene, fruiting body formation was substantially delayed, and the yield of myxospores was reduced. These results indicate that protein S is not essential for differentiation of M. xanthus, whereas a gene product(s) coded from the sequence upstream of the tps gene appears to be required for normal fruiting body formation.
Sodium-stimulated ATPase in Streptococcus faecalis
Authors: Kinoshita N , Unemoto T , Kobayashi H
Published: January 2020

We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force.