bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction in vitro of a cloned nar operon from Escherichia coli
Authors: Rondeau S S , Hsu P Y , DeMoss J A
Published: January 2020

To clone the nar operon of Escherichia coli without an effective selection procedure for the nar+ phenotype, a strategy utilizing nar::Tn5 mutants was employed. Partial segments of the nar operon containing Tn5 insertions were cloned into plasmid pBR322 by using the transposon resistance character for selection. A hybrid plasmid was constructed in vitro from two of these plasmids and isolated by a procedure that involved screening a population of transformed nar(Ts) mutant TS9A for expression of thermal stable nitrate reductase activity. A detailed restriction site map of the resulting plasmid, pSR95, corresponded closely to the composite restriction endonuclease map deduced for the nar region from maps of the cloned nar::Tn5 fragments. When transformed with pSR95, wild-type strain PK27 overproduced the alpha, beta, and gamma subunits of nitrate reductase, although nitrate reductase activity was only slightly increased. The alpha and beta subunits were overproduced about 5- to 10-fold and accumulated mostly as an inactive aggregate in the cytoplasm; the gamma subunit overproduction was detected as a threefold increase in the specific content of cytochrome b555 in the membrane fraction. Functional nitrate reductase and the cytochrome spectrum associated with functional nitrate reductase were restored in the nar::Tn5 mutant EE1 after transformation with pSR95. Although the specific activity of nitrate reductase in this case was less than that of the wild type, both the alpha and beta subunits appeared to be overproduced in an inactive form. In both strains PK27(pSR95) and EE1(pSR95), the formation of nitrate reductase activity and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the demonstration that pSR95 contains a functional nor operon that encodes the alpha, beta, gamma subunits of nitrate reductase.
Purification and properties of the myo-inositol-binding protein from a Pseudomonas sp
Authors: Deshusses J , Belet M
Published: January 2020

A myo-inositol-binding protein was isolated from a Pseudomonas sp. soil isolate and was purified to homogeneity. Its molecular weight is 30,000, and it has a single binding site. The amino acid analysis showed that the protein contains three tryptophan residues and no cysteine. Tryptophan residues seem to be involved in the binding of the ligand, as shown by the modification of the fluorescence spectra and by the fact that oxidation of tryptophan residues with N-bromosuccinimide abolished the binding of myo-inositol. Sequence analysis of the N-terminal segment of 37 amino acids showed that 13 are conserved when compared with the galactose-binding protein of Escherichia coli.
Porin from Rhodopseudomonas sphaeroides
Authors: Weckesser J , Zalman L S , Nikaido H
Published: January 2020

A protein homooligomer was purified from both the cell envelope fractions and the saline extracts of Rhodopseudomonas sphaeroides cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with egg phosphatidylcholine. In the saline extracts of both chemotrophically and phototrophically grown cells, the porin oligomer was the most predominant polypeptide, which produced pores whose behavior toward various sugars could be approximated by hollow cylinders of 0.62 nm in radius. The oligomer was dissociated, in the presence of EDTA, into monomers that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as though their molecular weight was about 47,000. The monomer was active in the reconstitution assay and produced pores with sizes comparable to those produced by the oligomer. Circular dichroism spectra indicated the predominance of beta-sheet structure in both the oligomeric and EDTA-dissociated monomeric forms. Drastic conditions, for example, precipitation with 10% trichloroacetic acid or heating for a few hours at 100 degrees C in sodium dodecyl sulfate, were necessary to denature the protein into a form with a reduced content of beta-sheet structure.
Purification and characterization of a dUTPase from Acholeplasma laidlawii B-PG9
Authors: Williams M V , Pollack J D
Published: January 2020

dUTP was purified 120-fold from extracts of Acholeplasma laidlawii B-PG9 by Blue-Sepharose, Phenyl-Sepharose, hydroxyapatite, and DEAE-Sephacel chromatography techniques. The only substrate for the enzyme was dUTP with an apparent Km of 4.5 microM. The only reaction products were dUMP and PPi. The dUTPase did not exhibit any specific divalent cation requirement, but it was inhibited by EDTA. The enzyme was not inhibited by Pi or p-hydroxymercuribenzoate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be 48,000, and its isoelectric point was 5.3. The enzyme was thermostable at 55 degrees C for 1 h. A. laidlawii dUTPase was distinguishable from KB (human epidermoid carcinoma) dUTPase by differences in electrophoretic migration, isoelectric point, and thermostability. The enzyme is important in preventing dUTP from being incorporated into DNA and may have a significant role in both the synthesis of thymidine- and PPi-dependent phosphorylations.
Distribution and evolutionary significance of mitochondrial plasmids in Neurospora spp
Authors: Natvig D O , May G , Taylor J W
Published: January 2020

A mitochondrial plasmid line in the fungal genus Neurospora is geographically widely distributed and occurs in isolates of at least two species. On the basis of characterization with restriction endonucleases, it is apparent that plasmids from isolates of Neurospora tetrasperma are more closely related to one another than to an evolutionarily homologous plasmid from Neurospora intermedia; N. tetrasperma plasmids from Surinam and Hawaii differed from one another only slightly by our analysis, whereas the plasmid from N. intermedia (Fiji) exhibited substantial restriction site divergence from all N. tetrasperma plasmids. We believe these observations strengthen the presumption that four-spored isolates of Neurospora spp. represent a natural taxonomic grouping (N. tetrasperma). The plasmids from N. tetrasperma and N. intermedia (Fiji), although clearly related to each other as shown by hybridization studies, exhibited no detectable homology with either of two additional plasmid lines from isolates of Neurospora spp. Nor did they exhibit homology with the mitochondrial genome. Despite this lack of homology among three distinct plasmid lines, all the plasmids may possess a common mode of replication.
Uptake of D-xylose and D-glucose by Spirochaeta aurantia
Authors: Roberts S , Paden C A , Greenberg E P
Published: January 2020

Uptake of D-[14C]glucose and D-[14C]xylose by Spirochaeta aurantia was demonstrated to be osmotic shock sensitive and to require a high-energy phosphorylated compound rather than a proton motive force. These features are similar to those of binding protein-mediated transport systems in other gram-negative bacteria.
Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase
Authors: Goncharoff P , Nichols B P
Published: January 2020

Biochemical and immunological experiments have suggested that the Escherichia coli enzyme p-aminobenzoate synthetase and anthranilate synthetase are structurally related. Both enzymes are composed of two nonidentical subunits. Anthranilate synthetase is composed of proteins encoded by the genes trp(G)D and trpE, whereas p-aminobenzoate synthetase is composed of proteins encoded by pabA and pabB. These two enzymes catalyze similar reactions and produce similar products. The nucleotide sequences of pabA and trp(G)D have been determined and indicate a common evolutionary origin of these two genes. Here we present the nucleotide sequence of pabB and compare it with that of trpE. Similarities are 26% at the amino acid level and 40% at the nucleotide level. We propose that pabB and trpE arose from a common ancestor and hence that there is a common ancestry of genes encoding p-aminobenzoate synthetase and anthranilate synthetase.
Shuttle plasmids for Escherichia coli and Clostridium perfringens
Authors: Squires C H , Heefner D L , Evans R J , Kopp B J , Yarus M J
Published: January 2020

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform L-phase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.
Hydrolysis of soluble, linear, un-cross-linked peptidoglycans by endogenous bacterial N-acetylmuramoylhydrolases
Authors: Barrett J F , Schramm V L , Shockman G D
Published: January 2020

Soluble, linear, uncross-linked peptidoglycans, prepared from two autolysis-defective mutants of Streptococcus faecium ATCC 9790 and from Micrococcus leuteus, were used as substrates for studies of hydrolysis by an N-acetylmuramoylhydrolase (muramidase). The kinetics of hydrolysis of these substrates and the ability of the muramidases isolated from S. faecium ATCC 9790 and from two autolysis-defective mutants, Lyt-14 and Aut-3, to carry out transglycosylation reactions were compared with the action of hen egg white lysozyme (EC 3.2.1.17). Hydrolysis of these substrates by the endogenous streptococcal muramidases resulted in the production of disaccharide-peptide monomers with the structure (formula; see text) as nearly the sole product. As estimated from increases in reducing groups, hydrolysis proceeded at a linear rate for extended intervals, with consumption of up to 75% of the substrate, even at substrate concentrations well below the Km value. Apparent Km and relative Vmax values for the three streptococcal enzymes were indistinguishable from each other or from those for hen egg white lysozyme. These results indicate that the autolysis-defective phenotype of these mutants cannot be attributed to differences in their muramidases. In contrast to the action of hen egg white lysozyme, the streptococcal muramidase failed to catalyze transglycosylations. The extended periods of hydrolysis at constant rates are consistent with the occurrence of multiple catalytic events after the formation of the enzyme-substrate complex.
Gene responsible for protecting Escherichia coli from sodium dodecyl sulfate and toluidine blue plus light
Authors: Wakayama Y , Takagi M , Yano K
Published: January 2020

Escherichia coli cells were killed by visible light irradiation in the presence of the photosensitizing dye, toluidine blue. Two uvrB mutant strains of E. coli K-12 (AB1885 and N3-1) were much more sensitive than the isogenic uvrA and uvrC strains to treatment with toluidine blue plus light, suggesting that the uvrB+ gene product was involved in repair of DNA damage induced by the treatment. The uvrB+ gene cloned in a high- or low-copy-number plasmid was transformed into the uvrB strain (AB1885). Although all the transformants showed the same resistance as its wild-type strain (AB1157) to UV irradiation, they were as sensitive as AB1885 was to treatment with toluidine blue plus light. The two uvrB strains were more sensitive to sodium dodecyl sulfate than the other strains, suggesting that these strains had a defect in the cell surface. A sodium dodecyl sulfate-resistant revertant obtained from AB1885 was more resistant than AB1885 was to treatment with toluidine blue plus light. The two uvrB strains (AB1885 and N3-1) appear to have a defective gene (tentatively called dvl) different from uvrB. Its map position was around 7 min on the E. coli map.
Regulation of outer membrane protein synthesis in Escherichia coli K-12: deletion of ompC affects expression of the OmpF protein
Authors: Schnaitman C A , McDonald G A
Published: January 2020

A chromosomal deletion beginning at a Tn10 located ca. 8 kilobases upstream from the ompC structural gene and extending through the 2.6-kilobase HindIII fragment carrying the ompC was isolated. The 2.6-kilobase ompC fragment was cloned into lambda 540 to obtain phage lambda 540C1. When the deletion mutant was lysogenized with lambda 540C1, the resulting strain produced normal levels of OmpC protein, and expression of this protein was regulated by osmolarity, carbon source, and the lc gene of phage PA-2, indicating that the cloned fragment contained all of the information required for regulated expression of ompC. The strain carrying the deletion was partially constitutive for expression of OmpF protein, whereas the lambda 540C1 lysogen of this strain and other strains with mutations in ompC repressed OmpF synthesis under conditions which lead to high-level expression of OmpC protein. Strains which are diploid or triploid for ompC show strong inhibition of synthesis of OmpF protein. We conclude that a regulatory element located upstream from the ompC coding sequence inhibits translation of OmpF protein under conditions which favor OmpC expression. Since ompF is known to repress transcription of ompC, we propose that these two genes constitute a closed regulatory loop which acts to amplify regulatory signals which control expression of these proteins.
Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions
Authors: Kuhn M , Steinbüchel A , Schlegel H G
Published: January 2020

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.
Novel system for recognizing and eliminating foreign DNA in Pseudomonas putida
Authors: Kim K , Meyer R J
Published: January 2020

Derivatives of pQSR49 (R1162::Tn1) containing cloned fragments of Escherichia coli chromosomal DNA are stable in E. coli but unstable in Pseudomonas putida. Similar derivatives containing P. putida chromosomal DNA are stable in both species. Instability is a consequence of plasmid loss during growth and is not due to death or inhibition of growth of plasmid-containing cells. Average copy numbers per cell of the unstable hybrid plasmids are similar to that of pQSR49, indicating that instability is not the result of reduced replication of plasmid DNA. A plasmid unrelated to pQSR49, RK2, becomes unstable in the presence of the unstable hybrids but only when it also contains foreign DNA. The data suggest an inducible mechanism in P. putida active in the elimination of plasmid-borne foreign DNA from the cell.
Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci
Authors: Uhlén M , Guss B , Nilsson B , Götz F , Lindberg M
Published: January 2020

Two shuttle vectors containing the gene for protein A (spa) from Staphylococcus aureus have been constructed to study expression of the gene in various strains of S. aureus and in the coagulase-negative species Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus xylosus. One plasmid, pSPA15, contains the complete structural gene for protein A, which binds to the cell wall in various Staphylococcus species. The other plasmid, pSPA16, codes for a truncated protein A lacking the C-terminal part called region X. The latter is exclusively extracellular in all Staphylococcus species tested, which confirms the importance of region X for cell wall binding. The expression of the plasmid-coded protein A in various strains of S. aureus is strongly correlated to the expression of the chromosomal spa gene. The coagulase-negative species expressing plasmid-encoded protein A produce 12 to 30% of the amount coded by the chromosomal spa gene in S. aureus strains Cowan I and A676.
Cloning of Bordetella pertussis outer membrane proteins in Escherichia coli
Authors: Shareck F , Cameron J
Published: January 2020

We constructed and screened a gene bank of phase I chromosomal DNA of Bordetella pertussis in Escherichia coli. A single immunopositive clone was recovered, and the hybrid plasmid obtained, designated pFSH200, had a molecular size of 46.6 kilobases. Smaller derivatives were generated by partial digestion of plasmid pFSH200 and were further characterized. One such derivative, plasmid pFSH201, contained a 4.5-kilobase chromosomal DNA fragment of B. pertussis which coded for the synthesis of the two outer membrane proteins of 33 and 30 kilodaltons specific to B. pertussis.
Use of the beta-lactamase inhibitor clavulanic acid in the isolation of auxotrophic mutants of Bacillus licheniformis
Authors: Fields P I , Schreier H J , Saha A L , Bernlohr R W
Published: January 2020

The isolation of auxotrophic mutants of Bacillus licheniformis, a microbe containing constitutive beta-lactamase activity, was found to be facilitated by the addition of clavulanic acid and cefotaxime during enrichment.
Conidia induce the formation of protoperithecia in Neurospora crassa: further characterization of white collar mutants
Authors: Degli-Innocenti F , Chambers J A , Russo V E
Published: January 2020

The treatment of undifferentiated mycelia with heavy suspensions of their own conidia triggers protoperithecial development. This effect was also observed with white collar (wc) mutants and suggests that the wc genes are not structural genes necessary for morphogenesis of protoperithecia but that they are probably involved in regulation.
Isolation and expression of the Bradyrhizobium japonicum adenylate cyclase gene (cya) in Escherichia coli
Authors: Guerinot M L , Chelm B K
Published: January 2020

A 5.0-kilobase-pair HindIII fragment of Bradyrhizobium japonicum DNA containing the cya gene which encodes adenylate cyclase was isolated as an insert in pBR322, using marker rescue of the maltose-negative phenotype of an Escherichia coli cya mutant for identification. The isolated B. japonicum DNA fragment was capable of reversing the pleiotropic phenotype of cya mutations when inserted in either orientation in the HindIII site of pBR322. The complemented E. coli strains produced high levels of cyclic AMP. No sequence homology between the B. japonicum cya gene and that of E. coli was detected by hybridization analysis.
Intergeneric homology of the speC gene encoding biosynthetic ornithine decarboxylase in Escherichia coli
Authors: Wright J M , Boyle S M
Published: January 2020

A 32P-labeled fragment of DNA containing the speC gene, which encodes the biosynthetic enzyme ornithine decarboxylase of Escherichia coli, was used as a hybridization probe for homologous sequences in the genomes of gram-negative and gram-positive bacteria. The speC probe detected homologous sequences in the DNA of only four members of the Enterobacteriaceae (Citrobacter freundii, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes); no homology was detected with the DNA of other representative members of the Enterobacteriaceae and gram-negative and gram-positive bacteria.
Map locations and functions of Salmonella typhimurium men genes
Authors: Kwan H S , Barrett E L
Published: January 2020

Menaquinone (men) mutants of Salmonella typhimurium isolated on the basis of their inability to produce trimethylamine were characterized with respect to mutation site, the ability to cross-feed each other and be cross-fed by known Escherichia coli men mutants, and response to intermediates of the menaquinone biosynthetic pathway. Cross-feeding tests were based on the requirement of menaquinone for hydrogen sulfide production. Genotypes corresponding to the menA, B, C, D, and possibly E genes described in E. coli were all identified. Additional studies of deletions in the menBCD area revealed that this cluster lies between ack/pta and glpT, as in E. coli. The ack and pta mutants were also defective in the production of trimethylamine and failed to produce gas in the absence of added formate.
Plasmidless, photosynthetically incompetent mutants of Rhodospirillum rubrum
Authors: Kuhl S A , Wimer L T , Yoch D C
Published: January 2020

Ethyl methanesulfonate rendered a high percentage of Rhodospirillum rubrum cells plasmidless and photosynthetically incompetent (Kuhl et al., J. Bacteriol. 156:737-742, 1983). By probing restriction endonuclease-digested chromosomal DNA from these plasmidless strains with 32P-labeled R. rubrum plasmid DNA, we showed that no homology exists between the plasmid and the chromosomal DNA of the mutant. Loss of the plasmid in all the nonphotosynthetic isolates was accompanied by the synthesis of spirilloxanthin under aerobic growth conditions, resistance to cycloserine and HgCl2, and loss of ability to grow fermentatively on fructose. Changes in both the protein and lipid composition of the membranes and the impaired uptake of 203HgCl2 in the plasmidless strains (compared with the wild type) suggest either that membrane modification occurs as a result of plasmid loss, accounting for several of the acquired phenotype characteristics of the cured strains, or that both membrane modification and plasmid loss are part of the same pleiotropic mutation.
Differential gene expression during aerobic germination of Mucor racemosus sporangiospores
Authors: Linz J E , Orlowski M
Published: January 2020

Evidence is provided suggesting that several modes of differential gene expression operate concomitantly during the first 60 min of germination of Mucor racemosus sporangiospores under air. Protein synthesis was initiated immediately upon exposure of the spores to nutrient medium and accelerated exponentially throughout the period of observation. All translation during the first 30 min of germination occurred using only preformed stable mRNAs as a template. Analysis of the protein products synthesized in vivo was performed by L-[35S]methionine labeling, one- and two-dimensional polyacrylamide gel electrophoresis, and autoradiography. The population of proteins accumulated during spore formation and present at the time of harvest differed significantly from those proteins synthesized during spore germination. Autoradiographs displayed several proteins synthesized during the former but not the latter morphogenetic process. Conversely, other proteins were synthesized during the first 30 min of germination but not during spore formation, even though the mRNA specifying these proteins must have been synthesized and stored in the dormant spore. A posttranscriptional regulatory mechanism that directs selective translation thus appears to exist in the developing spore. In addition, autoradiographs showed that many proteins, although made throughout the intervals examined, displayed significant changes in their relative rates of synthesis. One gene product exemplified a possible case of post-translational modification during the first hour of sporangiospore germination.
Monoclonal antibodies to Rhizobium meliloti and surface mutants insensitive to them
Authors: Johansen E , Finan T M , Gefter M L , Signer E R
Published: January 2020

Monoclonal antibodies were produced to the surface of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti. Bacterial lysis in the presence of complement or cycles of agglutination and growth were used to select mutants no longer recognized by the antibodies. The mutants were used to produce new antibodies with different specificities. Several mutants had altered sensitivity to one or more bacteriophages. R. meliloti strains from different sources had distinct patterns of sensitivity to monoclonal antibodies and phages, which together can be used for discriminative typing.
Characterization of interspecific plasmid transfer mediated by Bacillus subtilis temperate bacteriophage SP02
Authors: Marrero R , Young F E , Yasbin R E
Published: January 2020

Plasmid pPL1010 is a 7.0-kilobase derivative of plasmid pUB110 that harbors the cohesive end site of the bacteriophage SP02 genome. Plasmid pPL1017 is a 6.8-kilobase derivative of plasmid pC194 that contains the immunity region of bacteriophage phi 105 and the cohesive end site of bacteriophage SP02. These plasmids are transducible by bacteriophage SP02 at a frequency of 10(-2) transductants per PFU among mutant derivatives of Bacillus subtilis 168 and have been transferred to other strains of B. subtilis and B. amyloliquefaciens by means of bacteriophage SP02-mediated transduction, with frequencies ranging from 10(-5) to 10(-7) transductants per PFU. The introduced plasmids were stably maintained in nearly all new hosts in the absence of selective pressure. An exception was found in B. subtilis DSM704, which also harbored three cryptic plasmids. Plasmids pPL1010 and pPL1017 were incompatible with a 7.9-kilobase replicon native to strain DSM704. Furthermore, plasmid pPL1017 was processed by strain DSM704 into a approximately 5.3-kilobase replicon that was compatible with the resident plasmid content of strain DSM704. The use of bacteriophage SP02-mediated plasmid transduction has allowed the identification of Bacillus strains that are susceptible to bacteriophage SP02-mediated genetic transfer but cannot support bacteriophage SP02 lytic infection.
Possible relationship between cyclic AMP and idiophasic metabolism in the white rot fungus Phanerochaete chrysosporium
Authors: MacDonald M J , Paterson A , Broda P
Published: January 2020

In the hymenomycete Phanerochaete chrysosporium, a 10-fold increase in intracellular cyclic AMP preceded the onset of the two idiophasic activities, lignin degradation and veratryl alcohol production. Further, addition of L-glutamate during this period reduced cyclic AMP levels and suppressed ligninolytic activity.
Transposon Tn10 contains two structural genes with opposite polarity between tetA and IS10R
Authors: Schollmeier K , Hillen W
Published: January 2020

The nucleotide sequence of the central part of Tn10 has been determined from the rightmost HindIII site to IS10R. This sequence contains two open reading frames with opposite polarity. The in vivo transcription start points in this sequence have been determined by S1 mapping. These results define one minor and two major promoters. The transcription starts of the two major promoters are only 18 base pairs apart, and the transcripts show different polarity and overlap by 18 base pairs. The nucleotide sequence reveals two regions with palindromic symmetry which may serve as operators. Their possible involvement in the regulation of transcription of both genes is discussed. Taken together these results allow for a maximal coding capacity of 138 amino acids directed toward IS10R and 197 amino acids directed toward tetA. The possible function of these gene products is discussed. The accompanying article (Braus et al., J. Bacteriol. 160:504-509, 1984) presents evidence that these genes are expressed.
Export and processing of MalE-LacZ hybrid proteins in Escherichia coli
Authors: Rasmussen B A , Bankaitis V A , Bassford P J
Published: January 2020

Five classes of MalE-LacZ hybrid proteins have previously been characterized. These proteins differ in the amount of the maltose-binding protein (MBP) that is attached to beta-galactosidase. Although none of these proteins is secreted into the periplasm, the four larger classes of hybrid proteins, those that include an intact MBP signal peptide, are inserted into the cytoplasmic membrane, suggesting that the secretion process has at least been initiated. In this study, we demonstrated that some portion of the four larger hybrid proteins can be translocated across the cytoplasmic membrane, thus permitting processing of the signal peptide. We have found that hybrid proteins that include only a small portion of the mature MBP are inefficiently recognized as exported proteins, and translocation and processing of these appear to be relatively slow, posttranslational events. In marked contrast, hybrid proteins that include a substantial portion of the mature MBP are efficiently recognized, and translocation and processing of these occur very rapidly, possibly cotranslationally. Our results complement other studies and very strongly suggest a role for the mature MBP in the export process.
[14C]methylammonium transport by Frankia sp. strain CpI1
Authors: Mazzucco C E , Benson D R
Published: January 2020

We describe an NH4+-specific transport system in the N2-fixing symbiotic actinomycete Frankia sp. strain CpI1. [14C]methylammonium was used as an NH4+ analog. No specific transport process was detected when cells were grown on high concentrations of NH4+. A transport system with a high affinity for CH3NH3+ was synthesized after 3 to 4 h of nitrogen starvation. Methylammonium transport was not significantly inhibited by a variety of amino acids, primary amines, and polyamines. Ammonium completely eliminated CH3NH3+ transport. The Km for CH3NH3+ transport was around 2 +/- 1.8 microM with a Vmax of 4 to 5 nmol/min per mg of protein. The electron transport inhibitors cyanide and azide eliminated uptake, as did the uncoupler carbonyl cyanide-m-chlorophenylhydrazone. The sulfydryl reagent p-chloromercuribenzoic acid and the heavy metal thallium also inhibited uptake, suggesting the presence of an NH4+-specific permease. Concentration of CH3NH3+ across the membrane was demonstrated by conducting uptakes at low temperature to slow the metabolism of CH3NH3+ by glutamine synthetase. At 7 degrees C most of the label was concentrated inside the cells in a form that could be chased from the cells by adding excess NH4+ to the medium. At 30 degrees C most of the label was present as an impermeant metabolite. Thin-layer chromatography of cell extracts confirmed that the radioactivity inside the cells was mainly in the form of CH3NH3+ at 7 degrees C but was present as an unidentified metabolite at 30 degrees C. These studies demonstrate that Frankia sp. strain CpI1 has a high-affinity NH4+ transport system that is synthesized in response to NH4+ starvation.
Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences
Authors: Ishiguro N , Sato G
Published: January 2020

The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.
Mechanism of acquisition of chromosomal markers by plasmids in Haemophilus influenzae
Authors: Setlow J K , Cabrera-Juárez E , Griffin K
Published: January 2020

The hybrid plasmid pNov1 readily acquired genetic information from the chromosome of wild-type, but not rec-2, cells. Most of the recombination had taken place 1 h after entrance of the plasmid into the cell, as judged by transformation of rec-2 by lysates made from wild-type cells exposed to pNov1. Measurement of physical transfer from radioactively labeled cellular DNA to plasmids recombining in wild-type cells failed, since there was little more radioactivity in plasmids from such cells than from labeled rec-2 recipients, in which no recombination took place. EcoRI digestion of pNov1 divided the DNA into a 1.7-kilobase-pair fragment containing the novobiocin resistance marker and a 13-kilobase-pair fragment containing all of the original vector and considerable portions homologous to the chromosome. Transformation by the large fragment alone resulted in a plasmid the size of the original pNov1. Our hypothesis to explain the data is that genetic transfer from chromosome to plasmid took place by a copy choice mechanism.
TOL plasmid can prevent induction of chemotactic responses to aromatic acids
Authors: Harwood C S , Ornston L N
Published: January 2020

Growth conditions that elicited positive chemotaxis to benzoate and m-toluate in TOL- Pseudomonas putida cells failed to elicit taxis to these compounds in TOL+ cells. The inability of TOL+ cells to respond to these aromatic acids appears to be due to the preferential expression of TOL-encoded genes for aromatic degradation over chromosomally encoded genes. Expression of chromosomal genes for aromatic degradation is required for cells to form beta-ketoadipate, the inducer of benzoate and m-toluate taxis.
Trapping of the substrate-derived acyl enzyme intermediate of purified penicillin-binding protein 1a of Escherichia coli
Authors: Amanuma H , Strominger J L
Published: January 2020

Purified penicillin-binding protein 1a of Escherichia coli formed an acyl enzyme intermediate with the highly reactive synthetic substrate diacetyl-L-lysyl-D-alanyl-D-lactate at acid pH, although in extremely low yields.
Cyclic AMP phosphodiesterase in Salmonella typhimurium: characteristics and physiological function
Authors: Botsford J L
Published: January 2020

The physiological function of cyclic AMP (cAMP) phosphodiesterase in Salmonella typhimurium was investigated with strains which were isogenic except for the cpd locus. In crude broken-cell extracts the properties of the enzyme were found to be similar to those reported for Escherichia coli. The specific activity in the mutant was less than 1% that in the wild type. Rates of cAMP production in the mutant were as much as twice those observed in the wild type. The amount of cAMP accumulated when cells grew overnight with limiting glucose was 4.5-fold greater in the mutant than in the wild type. The intracellular concentration of cAMP in the two strains was measured directly, using four different techniques to wash the cells to remove extracellular cAMP. The cAMP level in the cpd strain was only 25% greater than in the wild type. The functional concentration of the cAMP receptor protein-cAMP complex was estimated indirectly from the specific activity of beta-galactosidase in the two strains after introducing F'lac. When cells were grown with carbon sources permitting synthesis of different levels of cAMP, the specific activity of the enzyme was at most 25% greater in the cpd strain. The cpd strain was more sensitive to the effects of exogenous cAMP. Exogenous cAMP relieved both permanent and transient catabolite repression of the lac operon at lower concentrations in the cpd strain than in the wild type. When cells grew with glucose, glycerol, or ribose, exogenous cAMP inhibited growth of the mutant strain more than the wild type.
Level of rRNA, not tRNA, synthesis controls transcription of rRNA and tRNA operons in Escherichia coli
Authors: Gourse R L , Nomura M
Published: January 2020

We have recently proposed a model for the negative feedback control of rRNA and tRNA synthesis in Escherichia coli by products of rRNA operons or their derivatives (e.g., nontranslating ribosomes) (S. Jinks-Robertson, R.L. Gourse, and M. Nomura, Cell 33:865-876, 1983). In this paper, we examined the following questions. (i) Are the spacer tRNAs carried within rRNA operons the products responsible for the regulation of rRNA and tRNA transcription? (ii) Are tRNAs capable of regulating their own syntheses? We measured tRNA accumulations in cells containing plasmids with intact or defective rRNA operons or with tRNA operons. From the results obtained, we conclude that neither the tRNAs encoded within rRNA operons nor the tRNAs encoded in non-rRNA operons are capable of controlling rRNA or tRNA transcription. Therefore, the products responsible for the initial step leading to rRNA and tRNA regulation are rRNAs (or their derivatives).
Purine-mediated growth inhibition caused by a pyrE mutation in Escherichia coli K-12
Authors: Shimosaka M , Fukuda Y , Murata K , Kimura A
Published: January 2020

A purine-sensitive phenotype results from a previously described mutation in the structural gene (pyrE) for orotate phosphoribosyltransferase (OPT) in Escherichia coli K-12. OPT from both the mutant and the wild-type was partially inhibited by adenine and adenosine, although other purine derivatives were not effective for this inhibition. The Km values of the mutant OPT were 580 and 760 microM for orotate and 5'-phosphoribosyl-1'-pyrophosphate (PRib-PP), respectively, whereas the corresponding values for the wild-type OPT were 40 and 60 microM. The intracellular level of PRib-PP was decreased to less than 15% of the normal level when purine derivatives were added to exponentially growing cultures of both the parent and mutant strains. However, this decrease of the PRib-PP level was not found in strains derived from the mutant, in which the purine-sensitive phenotype was suppressed by a secondary mutation. The purine-sensitive phenotype was caused by retardation of the pyrimidine de novo pathway, when the intracellular level of PRib-PP was diminished by exogenously supplied purine derivatives.
Cloning of the major protein of the Caulobacter crescentus periodic surface layer: detection and characterization of the cloned peptide by protein expression assays
Authors: Smit J , Agabian N
Published: January 2020

A precisely ordered crystalline array is found on the surface of the bacterium Caulobacter crescentus CB15. Using an immunological assay, we identified recombinant bacteriophage clones expressing the predominant protein of this structure from a lambda 1059 library of C. crescentus CB15 DNA. A single 4.4-kilobase HindIII fragment encoded a polypeptide whose antigenic determinants, molecular weight, and peculiar solubilization properties were identical with those of the authentic predominant polypeptide (130K) of the surface array. The 130K protein was produced as a discrete product as a result of gene transcription initiated from a lambda promoter; several experiments suggested that the Caulobacter promoter for this gene is not efficiently recognized by the Escherichia coli transcription machinery. Genomic Southern analysis revealed a single copy of the 130K protein gene per genome. The 130K protein gene was hybridized with DNA of two closely related laboratory strains of C. crescentus which have lost their ability to produce a surface array. One of these strains, CB2, possesses an homologous copy of the 130K gene, whereas DNA from the other strain, CB13B1a, showed a lesser degree of hybridization to the 130K gene probe; genomic fragments which did hybridize were of different sizes in CB13 as compared with those of CB15. These findings are discussed in relation to studies of the surface array function and its role in cellular morphogenesis in this stalk-forming bacterium.
Escherichia coli strains containing mutations in the structural gene for topoisomerase I are recombination deficient
Authors: Fishel R A , Kolodner R
Published: January 2020

Mutations in the gene encoding topoisomerase I of Escherichia coli were tested for their effect on plasmid recombination. Recombination was decreased 1,000-fold at 30 and 37 degrees C and occurred at approximately wild-type frequencies at 42 degrees C. The suppression of topA mutations at 42 degrees C did not appear to be a result of increased topoisomerase I activity at 42 degrees C.
Molecular cloning of structural and immunity genes for megacins A-216 and A-19213 in Bacillus megaterium
Authors: Von Tersch M A , Carlton B C
Published: January 2020

A host-vector system was developed for molecular cloning in Bacillus megaterium and used to clone the structural and immunity genes for megacins A-216 and A-19213. Recombinant clones that expressed immunity only or both immunity to and production of each megacin were obtained. Restriction mapping of native megacinogenic plasmids and recombinant clones was used to construct physical and genetic maps of megacinogenic plasmids pBM309 and pBM113. Limited sequence homology between pBM309 and pBM113 was detected by Southern blot hybridization and was mapped to, at most, a 6.4-kilobase-pair region of pBM309 and a 6.1-kilobase-pair region of pBM113.
Biochemical and immunological properties of Coxiella burnetii cell wall and peptidoglycan-protein complex fractions
Authors: Amano K , Williams J C , McCaul T F , Peacock M G
Published: January 2020

Coxiella burnetii morphological cell types were fractionated into large-cell variant cell walls, two fractions of small-cell variant cell walls, and one fraction of small-cell variant whole cells. Based on the contents of peptidoglycan (PG)-constituents and the yields of the sodium dodecyl sulfate-insoluble PG-protein complex (PG-PC) from cell walls, the fraction of large-cell variant cell walls contained significantly less PG than did the fraction of small-cell variant cell walls. The yields of PG-PC from the fractions of large-cell variant cell walls and small-cell variant cell walls were 2 and 32%, respectively. These results indicated that the PG of the large-cell variant cell walls may be partially digested by PG-lytic enzymes or incompletely synthesized, whereas the small-cell variant cell walls appeared to have intact PG. Proteins associated with PG-PC were resistant to proteolysis by trypsin, protease VI, and proteinase K. Saturated and unsaturated fatty acids were detected in whole cells and cell walls but not in PG-PC, which contained a 3-deoxy-D-mannooctulosonic acid-like component that is also present in phase I lipopolysaccharide. Immunogenicity of the fractions was tested by measuring the temporal sequence of phase II and phase I antibody responses in vaccinated rabbits. Both phase II and phase I antibody responses were demonstrated with all fractions except the sodium dodecyl sulfate supernatant of the small-cell variant cell walls, whereas PG-PC elicited a pure phase II antibody response up to 29 days postvaccination. The immunogenicity of these fractions may reflect a quantitative difference in antigen concentration or may be due to a qualitative difference in phase II and I determinants.
Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae
Authors: Balganesh M , Setlow J K
Published: January 2020

Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec+ and Rec- strains. The frequency of plasmid establishment in Rec+ cells by transformation increased exponentially with increasing insert size, but in Rec- cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec+ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.
A 2.2-kilobase repeated DNA segment is associated with DNA amplification in Streptomyces fradiae
Authors: Fishman S E , Rosteck P R , Hershberger C L
Published: January 2020

We have previously identified a 10.5-kilobase DNA sequence which is highly amplified and tandemly repeated in the mutant Streptomyces fradiae JS85. A library of DNA was prepared from S. fradiae T776, which does not contain amplified DNA. The library was screened by plaque hybridization to identify phage clones containing the unamplified 10.5-kilobase DNA sequence. Four phage isolates were identified which contained DNA homology to the amplified DNA sequence. This sequence was designated the amplifiable unit of DNA. None of the clones carried an entire amplifiable unit of DNA, and so overlapping regions were aligned to create a map of the entire region. Detailed restriction mapping identified a 2.2-kilobase direct repeat at the ends of the amplifiable unit of DNA. Analysis by Southern hybridization confirmed that the direct repeats were homologous to each other. The DNA of S. fradiae contained at least two additional copies of DNA that was homologous to the repeat sequence.
Escherichia coli supH suppressor: temperature-sensitive missense suppression caused by an anticodon change in tRNASer2
Authors: Thorbjarnardóttir S , Uemura H , Dingermann T , Rafnar T , Thorsteinsdóttir S , Söll D , Eggertsson G
Published: January 2020

We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes. supH is a mutant form of serU which codes for tRNASer2. The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon. The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon. We propose that the supH suppressor causes the insertion of serine in response to this codon. The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species.