bluuebunny/crossref_metadata_2025 huggingface dataset:
Promoter for the unc operon of Escherichia coli
Authors: Porter A C , Brusilow W S , Simoni R D
Published: January 2020

Fragments of DNA carrying possible promoters for the unc operon of Escherichia coli were cloned into a promoter detection plasmid (pRZ5255). Similar fragments were transcribed in vitro to produce transcripts whose sizes were used to determine the approximate start site for transcription. One strong promoter and at least two very much weaker ones were detected by these methods. The exact position of the strongest promoter, presumed to be the true unc promoter, was determined by S1 nuclease mapping and shown to lie 73 base pairs upstream from the open reading frame that precedes uncB. It therefore appears that this reading frame (uncI) is part of the unc operon. S1 mapping also revealed the presence of a third weak promoter 25 base pairs upstream of uncI. All of the weak promoters occur between the proposed unc promoter and uncB, but their role in vivo, if any, is unclear.
Comparison of the overlapping frd and ampC operons of Escherichia coli with the corresponding DNA sequences in other gram-negative bacteria
Authors: Bergström S , Lindberg F P , Olsson O , Normark S
Published: January 2020

Specific DNA probes from Escherichia coli K-12 were used to analyze the sequence divergence of the frd and ampC operons in various species of gram-negative bacteria. These operons code for the fumarate reductase complex and the chromosomal beta-lactamase, respectively. We demonstrate that the two operons show the same general pattern of divergence, although the frd operon is considerably more conserved than is the ampC operon. The major exception is Salmonella typhimurium LT2, which shows a strong homology to the E. coli frd probe but none to the E. coli ampC probe. The operons from Citrobacter freundii and Shigella sonnei were cloned and characterized by physical mapping, Southern hybridization, and protein synthesis in minicells. In S. sonnei, as in E. coli K-12, the frd and ampC operons overlap (T. Grundström and B. Jaurin, Proc. Natl. Acad. Sci. U.S.A. 79:1111-1115, 1982). Only minor discrepancies between the two operons were found over the entire frd-ampC region. In C. freundii, the ampC and frd operons do not overlap, being separated by about 1,100 base pairs. Presumably the inducible property of the C. freundii chromosomal beta-lactamase is encoded by this 1,100-base-pair DNA segment.
Monoclonal antibodies reveal lamB antigenic determinants on both faces of the Escherichia coli outer membrane
Authors: Schenkman S , Couture E , Schwartz M
Published: January 2020

LamB protein is involved in the transport of maltose across the outer membrane and constitutes the receptor for phage lambda. In this study we characterized six previously described anti-LamB monoclonal antibodies (mAbs). Four of these, the E-mAbs, recognized determinants that were exposed at the cell surface, whereas the other two, the I-mAbs, recognized determinants which were not exposed. Competition experiments demonstrated that the domains recognized by these two classes of mAbs were completely distinct. In addition, the E-mAbs prevented LamB from neutralizing phage lambda in vitro and protected LamB against proteolytic degradation, whereas the I-mAbs had no such effects. The E-mAbs have been shown previously to constitute two classes: some E-mAbs inhibit maltose transport in vivo, and others do not. Immunoelectron microscopy demonstrated that the I-mAbs also define at least two types of determinants. One of these, which is accessible in membrane fragments from a mutant (lpp) devoid of lipoprotein but not in membrane fragments from an lpp+ strain, probably corresponds to a region of LamB that is involved in the interactions with peptidoglycan. The other determinant, which is fully accessible in LamB-peptidoglycan complexes and in LamB-containing phospholipid vesicles but only slightly accessible in membrane fragments from an lpp mutant, is probably located very close to the inner surface of the outer membrane. LamB also contains at least one additional determinant, which (i) is exposed at the inner surface of the membrane, (ii) is accessible to antibodies in membrane fragments from an lpp+ strain, and (iii) may be involved in the interaction of LamB with the periplasmic maltose-binding protein.
Physical mapping of the exuT and uxaC operators by use of exu plasmids and generation of deletion mutants in vitro
Authors: Mata-Gilsinger M , Ritzenthaler P
Published: January 2020

Operons uxaCA and exuT of the hexuronate system are very closely linked on the Escherichia coli genetic map. Using plasmid vectors constructed by Casadaban et al. (J. Bacteriol. 143:971-980, 1980), we formed exuT-lacZ and uxaA-lacZ fusions in vitro. The phenotypic properties of the new plasmids allowed us to confirm that the exuT and uxaCA operons are divergently transcribed. An analysis of these fusion plasmids and derivatives in the presence of multiple copies of the exuR regulatory gene demonstrated that the two operons possess separate control regions. The precise location of the operator site relative to endonuclease restriction sites was determined. In addition, deletions of different lengths were generated on exu plasmids by restriction enzymes and were recombined into the chromosome. The expression of the exu regulon genes in the resulting deletion mutants is in agreement with the postulated location of the exuT and uxaCA operators in the fusion plasmids.
Genetic and physical map of the structural genes (nifH,D,K) coding for the nitrogenase complex of Rhodopseudomonas capsulata
Authors: Avtges P , Scolnik P A , Haselkorn R
Published: January 2020

Functional genes coding for the structural components of the nitrogenase complex (nifH,D,K) have been cloned on an 11.8-kilobase-pair HindIII fragment of DNA from the photosynthetic bacterium Rhodopseudomonas capsulata. The genes were physically mapped by hybridization of individual cloned nif genes from Klebsiella pneumoniae and Anabaena sp. strain 7120 to Southern blots of HindIII digests of the cloned R. capsulata fragment, after introduction of HindIII sites into the latter at specified locations by insertion of Tn5. Plasmids with the 11.8-kilobase-pair HindIII fragment containing the Tn5 insertions were also used for complementation tests with chromosomal Nif- mutations and for the generation of subfragments to locate those mutations by marker rescue. The R. capsulata nifH,D,K genes comprise a single unit of expression, with the same organization and polarity as found in K. pneumoniae. However, the R. capsulata nifH,D,K fragment did not complement Nif- point mutations in the corresponding Klebsiella genes, and the Klebsiella nif genes did not function in R. capsulata.
Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium
Authors: Braun V , Günthner K , Hantke K , Zimmermann L
Published: January 2020

The antibiotic albomycin is actively taken up by Escherichia coli via the transport system for the structurally similar iron complex ferrichrome. Albomycin is cleaved, and the antibiotically active moiety is released into the cytoplasm, whereas the iron carrier moiety appears in the medium. Besides transport-negative mutants, additional albomycin-resistant mutants were isolated. The mutations were mapped outside the transport genes close to the pyrD gene at 21 min. The mutants were devoid of peptidase N activity. The molecular weight, sensitivity to inhibitors, and cytoplasmic location of the enzyme hydrolyzing albomycin in vitro corresponded to the known properties of peptidase N. The aminoacyl thioribosyl pyrimidine moiety of albomycin apparently has to be cleaved off the iron chelate transport vehicle to inhibit growth. Peptidase N is the major hydrolyzing enzyme. In Salmonella typhimurium peptidase N and peptidase A were equally active in hydrolyzing and activating albomycin.
Biosynthetic pathways in Methanospirillum hungatei as determined by 13C nuclear magnetic resonance
Authors: Ekiel I , Smith I C , Sprott G D
Published: January 2020

The main metabolic pathways in Methanospirillum hungatei GP1 were followed by using 13C nuclear magnetic resonance, with 13C-labeled acetate and CO2 as carbon sources. The labeling patterns found in carbohydrates, amino acids, lipids, and nucleosides were consistent with the formation of pyruvate from acetate and CO2 as the first step in biosynthesis. Carbohydrates are formed by the glucogenic pathway, and no scrambling of label was observed, indicating that the oxidative or reductive pentose phosphate pathways are not functioning at significant rates. The pathways for amino acid biosynthesis are the usual ones, with the exception of that for isoleucine. The tricarboxylic acid pathway is incomplete and operates in a reductive direction to form alpha-ketoglutarate. The phytanyl chains of lipids are synthesized from acetate via mevalonic acid.
Isolation and characterization of the Neurospora crassa endoplasmic reticulum
Authors: Borgeson C E , Bowman B J
Published: January 2020

The endoplasmic reticulum from Neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. After differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane H+-ATPase. However, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. The lighter membrane fraction exhibited characteristics that have been associated with the endoplasmic reticulum in other organisms: (i) the inclusion of magnesium caused this light membrane fraction to shift to a higher density on the gradient; (ii) it was highly enriched in cytochrome c reductase, an endoplasmic reticulum marker in other systems; and (iii) the morphology of the light fraction with and without added magnesium was clearly distinguishable from that of the plasma membrane fraction by electron microscopy. A reinvestigation of the location of chitin synthetase confirmed its association with the plasma membrane fraction even after separation of the lighter fractions.
Effect of iron deficiency and iron restoration on ultrastructure of Anacystis nidulans
Authors: Sherman D M , Sherman L A
Published: January 2020

The effects of iron deficiency and iron reconstitution on the ultrastructure of the unicellular cyanobacterium Anacystis nidulans R2 were studied by electron microscopy. Low-iron cells, grown with different amounts of aeration, were analyzed at 6, 12, and 24 h after the addition of iron. Low-iron cells had a decrease in the quantities of membranes, phycobilisomes, and carboxysomes and a large increase in glycogen storage granules. In cells aerated with gentle shaking, the addition of iron caused the number of carboxysomes to increase rapidly within 6 h. This was paralleled by a decrease in the quantity of glycogen storage granules. Carboxysomes were associated with the nucleoplasmic face of the inner photosynthetic membrane in normal, but not low-iron, cells; they once more contacted the membrane by 6 h after iron addition. Phycobilisome assembly was apparent by 6 h, and the number of phycobilisomes increased throughout reconstitution. Membrane restoration was accomplished in two stages: (i) components were added to preexisting membranes until about 12 h, and (ii) new membranes were synthesized beginning at 12 to 18 h. Low-iron cells grown by bubbling with air had only one to two concentric layers of membrane per cell. The addition of iron led to a pattern of reconstitution that was similar to that described above with two important exceptions. Under these conditions, the number of carboxysomes remained low and the carboxysomes rarely contacted the photosynthetic membranes. New membranes were not synthesized until the culture had reached the late-logarithmic growth phase and after all other morphological features had returned to normal.
Intracellular hexose-6-phosphate:phosphohydrolase from Streptococcus lactis: purification, properties, and function
Authors: Thompson J , Chassy B M
Published: January 2020

An intracellular hexose 6-phosphate:phosphohydrolase (EC 3.1.3.2) has been purified from Streptococcus lactis K1. Polyacrylamide disc gel electrophoresis of the purified enzyme revealed one major activity staining protein and one minor inactive band. The Mr determined by gel permeation chromatography was 36,500, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single polypeptide of apparent Mr 60,000. The enzyme exhibited a marked preference for hexose 6-phosphates, and the rate of substrate hydrolysis (at 5 mM concentration) decreased in the order, galactose 6-phosphate greater than 2-deoxy-D-glucose 6-phosphate greater than fructose 6-phosphate greater than mannose 6-phosphate greater than glucose 6-phosphate. Hexose 1-phosphates, p-nitrophenylphosphate, pyrophosphate, and nucleotides were not hydrolyzed at a significant rate. In addition, the glycolytic intermediates comprising the intracellular phosphoenolpyruvate potential in the starved cells (phosphoenolpyruvate and 2- and 3-phosphoglyceric acids) were not substrates for the phosphatase. Throughout the isolation, the hexose 6-phosphate:phosphohydrolase was stabilized by Mn2+ ion, and the purified enzyme was dependent upon Mn2+, Mg2+, Fe2+, or Co2+ for activation. Other divalent metal ions including Pb2+, Cu2+, Zn2+, Cd2+, Ca2+, Ba2+, Sr2+, and Ni2+ were unable to activate the enzyme, and the first four cations were potent inhibitors. Enzymatic hydrolysis of 2-deoxy-D-glucose 6-phosphate was inhibited by fluoride when Mg2+ was included in the assay, but only slight inhibition occurred in the presence of Mn2+, Fe2+, or Co2+. The inhibitory effect of Mg2+ plus fluoride was specifically and completely reversed by Fe2+ ion. The hexose 6-phosphate:phosphohydrolase catalyzes the in vivo hydrolysis of 2-deoxy-D-glucose 6-phosphate in stage II of the phosphoenolpyruvate-dependent futile cycle in S. lactis (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982).
Evidence that thiosulfate assimilation by Salmonella typhimurium is catalyzed by cysteine synthase B
Authors: Nakamura T , Kon Y , Iwahashi H , Eguchi Y
Published: January 2020

Mutants carrying defects in cysteine synthase A or B or both were isolated from Salmonella typhimurium LT2. Parent strains were able to grow on minimal media containing sulfate, sulfite, sulfide, or thiosulfate as sulfur sources. Mutants lacking cysteine synthase B were unable to grow on thiosulfate, whereas mutants lacking cysteine synthase A grew on the four inorganic sulfur sources described above with little difference in their growth rates. Mutants lacking both cysteine synthases failed to grow on media containing any of the inorganic sulfur sources tested. Purification of cysteine synthase B resulted in the copurification of S-sulfocysteine synthase. In addition, the two activities were also cotransduced. These activities appear to be associated with the cysM gene, and this is able to be cotransducted with the cysK gene at a high frequency. From these results, it may be concluded that thiosulfate is assimilated via S-sulfocysteine exclusively with the aid of S-sulfocysteine synthase.
Heat modifiability and detergent solubility of outer membrane proteins of Rhodopseudomonas sphaeroides
Authors: Kent N E , Wisnieski B J
Published: January 2020

The outer membrane fraction from Rhodopseudomonas sphaeroides was isolated by isopycnic density centrifugation. The purity of this fraction was assayed by several methods. When the outer membrane fraction obtained after French press lysis of cells was compared with the outer membrane fragments released during spheroplast formation, the polypeptide profiles were identical. Detergent solubilization of membrane fractions showed that Triton X-100 nonselectively solubilizes both the cytoplasmic membrane and the outer membrane, whereas Deriphat 160 selectively solubilizes the cytoplasmic membrane. Several outer membrane polypeptides, including the major outer membrane protein, exhibited changes in electrophoretic mobility that depended upon the temperature of solubilization in sodium dodecyl sulfate. Solubilization at room temperature in the presence of ions reproduced the effect of thermal denaturation on the major outer membrane polypeptide.
Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: delta psi-dependent 22Na+ efflux from whole cells
Authors: Garcia M L , Guffanti A A , Krulwich T A
Published: January 2020

The Na+/H+ antiporter of Bacillus alcalophilus was studied by measuring 22Na+ efflux from starved, cyanide-inhibited cells which were energized by means of a valinomycin-induced potassium diffusion potential, positive out (delta psi). In the absence of a delta psi, 22Na+ efflux at pH 9.0 was slow and appreciably inhibited by N-ethylmaleimide. Upon imposition of a delta psi, a very rapid rate of 22Na+ efflux occurred. This rapid rate of 22Na+ efflux was competitively inhibited by Li+ and varied directly with the magnitude of the delta psi. Kinetic experiments with B. alcalophilus and alkalophilic Bacillus firmus RAB indicated that the delta psi caused a pronounced increase in the Vmax for 22Na+ efflux. The Km values for Na+ were unaffected by the delta psi. Upon imposition of a delta psi at pH 7.0, a retardation of the slow 22Na+ efflux rate at pH 7.0 was caused by the delta psi. This showed that inactivity of the Na+/H+ antiporter at pH 7.0 was not secondary to a low delta psi generated by respiration at this pH. Indeed, 22Na+ efflux activity appeared to be inhibited by a relatively high internal proton concentration. By contrast, at a constant internal pH, there was little variation in the activity at external pH values from 7.0 to 9.0; at an external pH of 10.0, the rate of 22Na+ efflux declined. This decline at typical pH values for growth may be due to an insufficiency of protons when a diffusion potential rather than respiration is the driving force. Non-alkalophilic mutant strains of B. alcalophilus and B. firmus RAB exhibited a slow rate of 22Na+ efflux which was not enhanced by a delta psi at either pH 7.0 or 9.0.
Characterization of the mRNA coding for ribonucleoside diphosphate reductase in Escherichia coli
Authors: Hanke P D , Fuchs J A
Published: January 2020

Total Escherichia coli RNA was separated by electrophoresis on methyl mercury agarose gels, transferred to diazobenzyloxymethyl-paper, and hybridized to various DNA probes containing different segments of the nrd genes to determine the organization of these genes. A 3.2-kilobase polycistronic mRNA transcript which hybridizes to both the nrdA and nrdB genes indicated that the nrdA and nrdB genes are organized in an operon. The polycistronic transcript contained the nrdA gene at the 5' end and the nrdB gene at the 3' end. The size of the polycistronic mRNA was sufficient to code for the 80,000-molecular-weight B1 protein and the 40,000-molecular-weight B2 protein. The results also indicated that the nrdA and nrdB genes are the only genes in E. coli that code for ribonucleoside diphosphate reductase. Two smaller RNA species that hybridized to nrd DNA were observed and probably overlap with the 3.2-kilobase nrd mRNA.
Molecular cloning of the ferrichrome-iron receptor of Escherichia coli K-12
Authors: Coulton J W , Mason P , DuBow M S
Published: January 2020

A receptor protein in the outer membrane of Escherichia coli K-12 is required for the binding of ferrichrome-iron at the cell surface and for the transport of iron from this complex into the cell. This protein of Mr 78,000 is the product of the fhuA (previously called tonA) gene, located at 3.5 min on the E. coli chromosome. We cloned the fhuA gene into plasmid p343, a high-copy-number cosmid derived from pBR322. An 8.5-kilobase pair fragment of E. coli chromosomal DNA, generated by hydrolysis with the restriction endonuclease HindIII, was found to have conferred the FhuA+ phenotype to E. coli P8, which lacks the ferrichrome-iron receptor. A partial physical map of this recombinant plasmid pPM18 was established by determining the restriction endonuclease sites for BglII, EcoRI, PstI, PvuII, SmaI, and XhoI. The fhuA gene was localized to a 3.5-kilobase pair fragment of DNA whose extremities were defined by the restriction sites PstI-PvuII. A 7.5-fold enhancement of the rate of transport of iron from the ferrichrome complex was measured for cells which contained pPM18 as compared to wild-type E. coli K-12. Overproduction of the FhuA protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of the recombinant plasmid-containing strain. Proteins encoded by the subcloned DNA fragments were identified by [35S]methionine labeling of maxicells of E. coli CSR603, which contained recombinant plasmids; only one polypeptide chain, the presumptive fhuA gene product, was detected.
Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium
Authors: Miller E S , Brenchley J E
Published: January 2020

Glutamic acid is synthesized in enteric bacteria by either glutamate dehydrogenase or by the coupled activities of glutamate synthase and glutamine synthetase. A hybrid plasmid containing a fragment of the Salmonella typhimurium chromosome cloned into pBR328 restores growth of glutamate auxotrophs of S. typhimurium and Escherichia coli strains which have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 2.2-kilobase pair region was shown by complementation analysis, enzyme activity measurements, and the maxicell protein synthesizing system to carry the entire glutamate dehydrogenase structural gene, gdhA. Glutamate dehydrogenase encoded by gdhA carried on recombinant plasmids was elevated 5- to over 100-fold in S. typhimurium or E. coli cells and was regulated in both organisms. The gdhA promoter was located by recombination studies and by the in vitro fusion to, and activation of, a promoter-deficient galK gene. Additionally, S. typhimurium gdhA DNA was shown to hybridize to single restriction fragments of chromosomes from other enteric bacteria and from Saccharomyces cerevisiae.
Genetic analysis of the recJ gene of Escherichia coli K-12
Authors: Lovett S T , Clark A J
Published: January 2020

An insertion of transposon Tn10 was isolated in a gene essential for the RecF pathway of recombination. This mutation and six other previously isolated rec mutations, including one in the recJ gene, were mapped to 62 min on the Escherichia coli chromosome. All seven mutations were examined for their effects on recombination and UV survival in several genetic backgrounds. Although all affected the RecF, and not the RecBC, pathway of recombination, they could be separated into two classes: four severely reduced UV survival and three did not. In addition, whereas the effects of the former class appeared to be nearly equivalent in recB recC sbcA and recB recC sbcB mutant backgrounds, the latter class affected recombination differentially. The latter class was found to be either "leaky" or temperature-sensitive mutations. Transductional complementation assays indicated that representative members of each class were mutations in the same gene, recJ. Possible explanations for the differences in phenotypic effects produced by these recJ mutations are discussed.
Physical and biochemical analysis of the cloned recB and recC genes of Escherichia coli K-12
Authors: Dykstra C C , Prasher D , Kushner S R
Published: January 2020

A 19-kilobase BamHI fragment encoding the recB (exonuclease V), recC (exonuclease V), ptr (protease III), thyA, and argA genes of Escherichia coli K-12 was cloned into a multicopy plasmid (pCDK3). In E. coli maxicells, the plasmid specified the synthesis of seven polypeptides of 140,000 (recC), 128,000 (recB), 110,000 (ptr), 53,000 (argA), 50,000, 33,000 (thyA), and 22,000 Mr, as well as beta-lactamase and chloramphenicol acetyltransferase. From analysis of subclones and Tn1000 insertions, it appears that the 110,000- and 50,000-Mr proteins originated from the ptr DNA coding sequence which is located between the recB and recC genes. Although recC, ptr, and recB were physically closely linked and transcribed in the same direction, they do not appear to constitute an operon. Cells carrying pCDK3 contained a 30- to 50-fold increase in exonuclease V activity, without affecting cell viability.
Induced plasmid-genome rearrangements in Rhizobium japonicum
Authors: Berry J O , Atherly A G
Published: January 2020

The P group resistance plasmids RP1 and RP4 were introduced into Rhizobium japonicum by polyethylene-glycol-induced transformation of spheroplasts. After cell wall regeneration, transformants were recovered by selecting for plasmid determinants. Plant nodulation, nitrogen fixation, serological, and bacterial genetics studies revealed that the transformants were derived from the parental strains and possessed the introduced plasmid genetic markers. Agarose gel electrophoresis, restriction enzyme analysis, and DNA hybridization studies showed that many of the transformant strains had undergone genome rearrangements. In the RP1 transformants, chromosomal DNA was found to have transposed into a large indigenous plasmid of R. japonicum, producing an even larger plasmid, and the introduced R plasmid DNA was found to be chromosomally integrated rather than replicating autonomously or integrated into the endogenous plasmid. Seemingly, a similar section of chromosomal DNA was involved in all the genomic rearrangements observed in the R. japonicum RP1 and RP4 transformant strains.
Nitrogen fixation and nitrogenase activities in members of the family Rhodospirillaceae
Authors: Madigan M , Cox S S , Stegeman R A
Published: January 2020

Strains of all 18 species of the family Rhodospirillaceae (nonsulfur photosynthetic bacteria) were studied for their comparative nitrogen-fixing abilities. All species, with the exception of Rhodocyclus purpureus, were capable of growth with N2 as the sole nitrogen source under photosynthetic (anaerobic) conditions. Most rapid growth on N2 was observed in strains of Rhodopseudomonas capsulata. Within the genus Rhodopseudomonas, the species R. capsulata, R. sphaeroides, R. viridis, R. gelatinosa, and R. blastica consistently showed the highest in vivo nitrogenase rates (with the acetylene reduction technique); nitrogenase rates in other species of Rhodopseudomonas and in most species of Rhodospirillum were notably lower. Chemotrophic (dark microaerobic) nitrogen fixation occurred in all species with the exception of one strain of Rhodospirillum fulvum; oxygen requirements for dark N2 fixation varied considerably among species and even within strains of the same species. We conclude that the capacity to fix molecular nitrogen is virtually universal among members of the Rhodospirillaceae but that the efficacy of the process varies considerably among species.
Synergistic effect of himA and gyrB mutations: evidence that him functions control expression of ilv and xyl genes
Authors: Friedman D I , Olson E J , Carver D , Gellert M
Published: January 2020

We have constructed Escherichia coli strains containing mutations at two different loci, both originally selected for failure to support lambda site-specific recombination: himA and gyrB-him(Ts). Although the gyrB-him(Ts) mutations by themselves reduce supercoiling at high temperature, the double mutants show a far greater effect on supercoiling. Our studies show that growth of phage lambda is severely inhibited and that maintenance of plasmid pBR322 is extremely unstable in the double mutants. Physiological studies also reveal that the double mutants are isoleucine auxotrophs at 42 degrees C. The fact that himA mutants are isoleucine auxotrophs at 42 degrees C in the presence of leucine suggests that a significant component of the isoleucine auxotrophy of the double mutants is a result of the himA mutation. The himA gene encodes the alpha subunit of a protein called the integration host factor. Since mutations in the hip or himD gene encoding beta, the other subunit of the integration host factor, also result in isoleucine auxotrophy in the presence of leucine, we suggest that the integration host factor regulates the synthesis of at least one of the enzymes in the ilv pathway, acetohydroxyacid synthase I, which is encoded by the ilvB gene. Studies of the utilization of various sugars as the sole carbon source suggest that the integration host factor controls expression of some gene(s) involved in the utilization of xylose.
Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins
Authors: Park W , Matsuhashi M
Published: January 2020

Major peptidoglycan transglycosylase activities, which synthesize uncross-linked peptidoglycan from lipid-linked precursors, were solubilized from the membranes of Staphylococcus aureus and Micrococcus luteus and were partially purified. The transglycosylase activities were separated from penicillin-binding proteins by solubilization and by purification steps. Therefore, we concluded that these activities were not activities of the penicillin-binding proteins, which are the presumptive peptidoglycan transpeptidases in these gram-positive cocci. Unlike Escherichia coli, in which the network structure of peptidoglycan is synthesized by multiple two-headed penicillin-binding proteins with both transpeptidase and transglycosylase activities, these gram-positive cocci have cell wall peptidoglycan which seems to be synthesized by penicillin-binding protein transpeptidases and a separate transglycosylase.
Standard reference strains of Escherichia coli from natural populations
Authors: Ochman H , Selander R K
Published: January 2020

A set of 72 reference strains of Escherichia coli isolated from a variety of hosts and geographical locations has been established for use in studies of variation and genetic structure in natural populations. The strains, which have been characterized by multilocus enzyme electrophoresis, are representative of the range of genotypic variation in the species as a whole.
Participation of Escherichia coli K-12 groE gene products in the synthesis of cellular DNA and RNA
Authors: Wada M , Itikawa H
Published: January 2020

Cells having the temperature-sensitive mutation groES131(Ts) were isolated from Escherichia coli K-12 strain C600T by thymineless death selection at 44 degrees C. This conditionally expressed mutation affected both cellular DNA and RNA syntheses at nonpermissive temperature, in addition to rendering cells unable to propagate phage lambda at permissive temperature.
Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci
Authors: Neve H , Geis A , Teuber M
Published: January 2020

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.
Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase
Authors: Matsuoka M , Himeno T , Aiba S
Published: January 2020

Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.
Effects of deletions in transposon Tn7 on its frequency of transposition
Authors: Smith G M , Jones P
Published: January 2020

Deletions in transposon Tn7 either abolished transposition or reduced transposition frequency. Except for a deletion in the right-hand terminus, these deletions could be complemented in trans. A 2.1-kilobase fragment of Tn7 encodes a diffusible gene product which stimulates transposition above the wild-type frequency. No cointegrate formation was detected.
Metabolism of 4'-phosphopantetheine in Escherichia coli
Authors: Jackowski S , Rock C O
Published: January 2020

Coenzyme A (CoA) and acyl carrier protein (ACP) contain 4'-phosphopantetheine moieties that are metabolically derived from the vitamin pantothenate. The utilization of metabolites in the biosynthetic pathway during growth was investigated by using an Escherichia coli beta-alanine auxotroph to specifically and uniformly label the pathway intermediates. Pantothenate and 4'-phosphopantetheine were the two intermediates detected in the highest concentration, both intracellularly and extracellularly. The specific cellular content of CoA and ACP was not constant during growth of strain SJ16 (panD) on 4 microM beta-[3-3H]alanine, and alterations in the utilization of 4'-phosphopantetheine and pantothenate correlated with the observed fluctuations of the intracellular pool sizes of CoA and ACP. Double-label experiments indicated that extracellular 4'-phosphopantetheine was derived from the degradation of ACP, and the extent that this intermediate was utilized by 4'-phosphopantetheine adenylyltransferase exerted control over the degradative aspect of the pathway. Control over the biosynthetic aspect of the biochemical pathway was exerted at the level of pantothenate utilization by pantothenate kinase. Reduction in the specific cellular content of CoA and ACP by 4'-phosphopantetheine excretion was irreversible since, in contrast to pantothenate, strain SJ16 was unable to assimilate exogenous 4'-phosphopantetheine into CoA or ACP.
Escherichia coli intracellular pH, membrane potential, and cell growth
Authors: Zilberstein D , Agmon V , Schuldiner S , Padan E
Published: January 2020

We studied the changes in various cell functions during the shift to alkaline extracellular pH in wild-type Escherichia coli and in strain DZ3, a mutant defective in pH homeostasis. A rapid increase in membrane potential (delta psi) was detected in both the wild type and the mutant immediately upon the shift, when both cell types failed to control intracellular pH. Upon reestablishment of intracellular pH - extracellular pH and growth in the wild type, delta psi decreased to a new steady-state value. The electrochemical proton gradient (delta muH+) was similar in magnitude to that observed before the pH shift. In the mutant DZ3, delta psi remained elevated, and even though delta muH+ was higher than in the wild type, growth was impaired. Cessation of growth in the mutant is not a result of cell death. Hence, the mutant affords an interesting system to explore the intracellular-pH-sensitive steps that arrest growth without affecting viability. In addition to delta muH+, we measured respiration rates, protein synthesis, cell viability, induction of beta-galactosidase, DNA synthesis, and cell elongation upon failure of pH homeostasis. Cell division was the only function arrested after the shift in extracellular pH. The cells formed long chains with no increase in colony-forming capacity.
Facile and gentle method for quantitative lysis of Escherichia coli and Salmonella typhimurium
Authors: Crabtree S , Cronan J E
Published: January 2020

Garrett et al. (Mol. Gen. Genet. 182:326-331, 1981) constructed strains of Escherichia coli harboring derivatives of plasmid pBR322 that carry the lysis genes (S, R, and Rz) of phage lambda. The plasmid construction placed the genes under control of the lactose operon operator-promotor (and thus of lac repressor). Induction of E. coli strains carrying these plasmids resulted in rapid lysis of the culture unless the S gene was defective, in which case the cells grew normally. A freeze-thaw treatment of induced cells carrying an S- plasmid gave quantitative lysis of either E. coli or Salmonella typhimurium cells under exceptionally gentle conditions. The method was equally effective on exponential phase cells and stationary phase cells and was readily extended to a large number of independent cultures.
Rapid turnover of mannitol-1-phosphate in Escherichia coli
Authors: Rosenberg H , Pearce S M , Hardy C M , Jacomb P A
Published: January 2020

The phosphate moiety of D-mannitol-1-phosphate in Escherichia coli is subject to rapid turnover and is in close equilibrium with Pi and the phosphorus of fructose-1,6-bisphosphate. These three compounds account for the bulk of 32P label found in cells after several minutes of uptake of 32Pi and mannitol-1-phosphate represents some 30% of this label. Mannitol-1-phosphate occurs in E. coli grown on a variety of carbon sources, in the absence of D-mannitol, and is synthesized de novo even in mutants lacking mannitol-1-phosphate dehydrogenase. The mannitol moiety of mannitol-1-phosphate was not affected during the total chase of the P moiety, which exchanged with a half-life of about 30 s. These findings suggest that the rapid equilibration of the phosphorus is a function of an enzyme, possibly a component of the phosphotransferase system, capable of forming a complex that allows the exchange of the phosphate without the equilibration of the mannitol moiety with free mannitol.
DNA supercoiling in gyrase mutants
Authors: Steck T R , Pruss G J , Manes S H , Burg L , Drlica K
Published: January 2020

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.
Purification and characterization of pro-TraTp, the signal sequence-containing precursor of a secreted protein encoded by the F sex factor
Authors: Minkley E G
Published: January 2020

The structural gene for the F sex factor outer membrane surface exclusion protein ( traT ) was cloned onto a high-copy-number plasmid where it is expressed from the phage lambda promoter pL. Conditional control over expression was provided by a temperature-sensitive lambda cI repressor. Induction of pL produced large quantities of the traT gene product ( TraTp ) and, in rich growth media, even larger amounts of a higher-molecular-weight form of TraTp . This polypeptide was purified and characterized as a pro- TraTp precursor, which contains at its amino terminus a typical signal-like sequence, which is not present in the mature form of TraTp as isolated from the outer membrane of F-containing cells. Accumulation of pro- TraTp seemed not to result from the jamming of export sites, as in another system for obtaining precursors of secreted proteins, but rather from overwhelming kinetically the ability of the cell to process exported proteins. Although pro- TraTp appeared to be successfully translocated to the outer membrane, it was defective in forming the oligomeric structure required for surface exclusion function. The procedure used is not a general method but can be applied to certain other secreted proteins.
Escherichia coli K-12 lysyl-tRNA synthetase mutant with a novel reversion pattern
Authors: Hirshfield I N , Tenreiro R , Vanbogelen R A , Neidhardt F C
Published: January 2020

Fast-growing revertants have been selected from a slow-growing lysyl-tRNA synthetase mutant. All of the revertants had increased lysyl-tRNA synthetase activity compared with the mutant (5- to 85-fold), and in some revertants this amounted to two to three times the wild-type synthetase activity. Two-dimensional gel electrophoresis of a whole-cell extract of revertant IH2018 (1.5- to 2-fold wild-type synthetase activity) showed that the increase in synthetase activity is due to the induction of cryptic lysyl-tRNA synthetase forms and not to a change in the constitutive lysyl-tRNA synthetase. Genetic studies have shown that a locus termed rlu (for regulation of lysU ) which is cotransducible with purF at 49.5 min influences the amount of the cryptic lysyl-tRNA synthetase.
Buoyant density variation during the cell cycle of Saccharomyces cerevisiae
Authors: Baldwin W W , Kubitschek H E
Published: January 2020

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines.
Autoplaquing in Myxococcus strains
Authors: Breyen S A , Dworkin M
Published: January 2020

Autoplaquing has been observed in Myxococcus strains freshly isolated from soil. Initial observations suggest that this phenomenon is not induced by elevated temperature or visible light; we suggest that it may be the result of a derangement in the developmental autolytic mechanism within the cell.
Genetic basis of minicell formation in Escherichia coli K-12
Authors: Davie E , Sydnor K , Rothfield L I
Published: January 2020

Hfr- and P1-mediated genetic transfer experiments failed to confirm the presence of a " minA " gene in Escherichia coli K-12, leading to the conclusion that mutation at a single locus, the minB locus, is sufficient to cause minicell production in this species.
Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli
Authors: Jakubowski H , Goldman E
Published: January 2020

The cellular content of all 20 aminoacyl-tRNA species was determined in small cultures of Escherichia coli by labeling cells with 3H-amino acids and extraction of 3H-amino acid-labeled nucleic acid by standard procedures. Of 3H-amino acid-labeled material, 25 to 90% was identified as 3H-aminoacyl-tRNA by the following criteria: sensitivity to base hydrolysis with expected kinetics; association of 3H counts released by base treatment of the 3H-amino acid-labeled nucleic acid with amino acid standards upon paper chromatography of the hydrolysate; and changes in the amount of 3H-amino acid-labeled nucleic acid recovered from cells as a function of time. Individual aminoacyl-tRNA content was determined with as few as 8 X 10(7) to 4 X 10(8) E. coli cells. Although the total number of aminoacyl-tRNA molecules per cell varied only by 10 to 20% among various strains of E. coli, some individual aminoacyl-tRNA families varied two- to threefold among strains. For a given amino acid, the number of aminoacyl-tRNA molecules per cell in E. coli strain K38 growing with a doubling time of 60 min varied from 730 (glutamyl-tRNA) to 7,910 (valyl-tRNA) with a mean value of 3,200. The total number of aminoacyl-tRNA molecules per cell (6.4 X 10(4)) in E. coli K38 was 5.5-fold higher than the number of ribosomes and was equal to 84% of the amount of elongation factor Tu molecules per cell. The ratio of aminoacyl-tRNA to synthetase for 10 amino acids varied from about 1 to 15 with a mean value of 4.7. The turnover of individual aminoacyl-tRNA families in E. coli cells was estimated to be in the range of 1.7 to 8.1 s-1 with a mean value of 3.7 s-1. An estimate of minimum in vivo molecular activity of aminoacyl-tRNA synthetases gives values of 2 to 48 s-1 for individual enzymes.
Genetic and physical characterization of recombinant plasmids associated with cell aggregation and high-frequency conjugal transfer in Streptococcus lactis ML3
Authors: Anderson D G , McKay L L
Published: January 2020

Restriction mapping was employed to characterize the 104-kilobase (kb) cointegrate lactose plasmids from 15 independent transconjugants derived from Streptococcus lactis ML3 as well as the 55-kb lactose plasmid ( pSK08 ) and a previously uncharacterized 48.4-kb plasmid ( pRS01 ) from S. lactis ML3. The data revealed that the 104-kb plasmids were cointegrates of pSK08 and pRS01 and were structurally distinct. The replicon fusion event occurred within adjacent 13.8- or 7.3-kb PvuII fragments of pSK08 and interrupted apparently random regions of pRS01 . Correlation of the transconjugants' clumping and conjugal transfer capabilities with the interrupted region of pRS01 identified pRS01 regions coding for these properties. In the 104-kb plasmids, the pRS01 region was present in both orientations with respect to the pSK08 region. The replicon fusion occurred in recombination-deficient (Rec-) strains and appeared to introduce a 0.8 to 1.0-kb segment of DNA within the junction fragments. The degeneration of the cointegrate plasmids was monitored by examining the lactose plasmids from nonclumping derivatives of clumping transconjugants. These plasmids displayed either precise or imprecise excision of pRS01 sequences or had dramatically reduced copy numbers. Both alterations occurred by rec-independent mechanisms. Alterations of a transconjugant 's clumping phenotype also occurred by rec-independent inversion of a 4.3-kb KpnI-PvuII fragment within the pRS01 sequences of the cointegrate plasmid.