bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae
Authors: Boucherie H
Published: January 2020

Metabolic changes have been investigated during continuous growth of yeast cells inoculated in glucose-containing medium until the cells entered the stationary phase in response to glucose exhaustion. Well in advance of glucose exhaustion, a transition phase was observed, characterized by a decrease in the growth rate and a progressive reduction of protein and RNA accumulation. Two-dimensional gel analysis of the proteins synthesized during this stage showed that the pattern of proteins remained similar to that of log-phase cells. When the cells entered the stationary phase, protein accumulation was 10% of that in log-phase cells, and incorporation of labeled RNA precursor was undetectable. Analysis of protein synthesis gave evidence that the synthesis of 95% of the proteins present in log-phase cells was arrested in stationary-phase cells. Among the 20 proteins whose synthesis continues throughout the stationary phase were identified actin, aldehyde dehydrogenase, enolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and five heat shock proteins. In addition, the synthesis of six new proteins was observed. The occurrence of these new proteins in stationary-phase cells is presumed to result from the release of carbon catabolite repression due to glucose exhaustion.
Expression of cytochrome o in hydrogen uptake constitutive mutants of Rhizobium japonicum
Authors: O'Brian M R , Maier R J
Published: January 2020

Mutant strains of Rhizobium japonicum constitutive for H2 uptake activity (Hupc) contained significantly more membrane-bound b-type cytochrome than did the wild type when grown heterotrophically. The Hupc strains contained approximately three times more dithionite- and NADH-reducible CO-reactive b-type cytochrome than did the wild type; the absorption features of the CO spectra were characteristic of cytochrome o. This component, designated cytochrome b', was not reduced by NADH in the presence of cyanide. Cytochrome o from the wild type (SR) and cytochrome b' from mutants SR476 and SR481 bound to CO with similar dissociation constants of 5.4, 7.4, and 5.6 microM, respectively. NADH-dependent reduction of cytochrome b' from SR476 and SR481 and the cytochrome o from SR followed pseudo-first-order kinetics with similar rate constants. Based on these spectral, ligand-binding, and kinetic measurements, it was concluded that cytochrome b' expressed by the Hupc mutants is equivalent to cytochrome o found in the wild type. H2, NADH, and succinate each reduced the same amount of total b-type cytochrome in membranes from SR481, and the rate of H2-dependent cytochrome o reduction was significantly less than with succinate or NADH as the reductants. It was concluded that neither cytochrome o nor any b-type cytochrome expressed by the Hupc mutants was unique to the H2 oxidation system. At low O2 concentrations, the inhibition of H2 and NADH oxidase activities by CO closely paralleled the binding of CO to cytochrome o rather than cytochromes a3 or c'. This suggested that NADH and H2 oxidation involved primarily cytochrome o as the terminal oxidase at low O2 tensions.
Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome
Authors: Breton A M , Jaoua S , Guespin-Michel J
Published: January 2020

The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains.
Identification of the transcriptional suppressor sof-1 as an alteration in the spo0A protein
Authors: Hoch J A , Trach K , Kawamura F , Saito H
Published: January 2020

The mutation sof-1 suppresses the sporulation defect of mutations in either the spo0B, spo0E, or spo0F stage 0 sporulation genes. Through the use of integrative plasmids carrying the portion of the chromosome including the spo0A locus and flanking regions, the sof-1 mutation was localized near the spo0A locus. A plasmid carrying a fragment of DNA with sof genetic activity was constructed. Nucleic acid sequence analysis of this fragment revealed a single base change that resulted in a substitution of lysine for asparagine in the 12th codon of the spo0A gene. The results indicate that certain missense mutations in the spo0A gene bypass the necessity for the spo0B, spo0E, and spo0F gene products in sporulation. Several models for the interaction of these gene products may be imagined.
Sequence analysis of the spo0B locus reveals a polycistronic transcription unit
Authors: Ferrari F A , Trach K , Hoch J A
Published: January 2020

A 2.3-kilobase pair EcoRI fragment containing the spo0B locus has been sequenced. The spo0B locus was shown to code for a protein of 22,542 daltons. Promoter distal to the spo0B locus, an open reading frame was uncovered which was preceded by a strong ribosome-binding site. S1 nuclease protection experiments revealed that both the spo0B locus and this open reading frame were part of the same transcript. A portion of the middle of the open reading frame was cloned in the integrative vector pJH101. Transformation of this plasmid into Bacillus subtilis 168 was only rarely successful, and those few colonies that arose consisted of cells that had lost the plasmid. The results suggested that the product of this open reading frame is essential for the growth of the bacterium. The regulation of the spo0B locus was studied by using translational spo0B-lacZ fusions in an integrative vector. These studies revealed that the spo0B locus was maximally expressed during vegetative growth. It was estimated that 50 to 100 copies of the protein are present during this period. Sequence analysis of the region upstream from the spo0B locus revealed another operon that contained a gene coding for a protein homologous to ribosomal protein L27 of Escherichia coli.
Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions
Authors: Hays J B , Martin S J , Bhatia K
Published: January 2020

The system previously used to study recombination of nonreplicating UV-irradiated phage lambda DNA was adapted to study UV repair. Irradiated phages infected undamaged homoimmune lysogens. Pyrimidine dimer content (by treatment with Micrococcus luteus UV endonuclease and alkaline sucrose sedimentation) and a biological activity endpoint (infectivity in transfection of uvrB recA recB spheroplasts) were followed. Unless room light was excluded during DNA extraction procedures, photoreactivation (Phr function) was significant. In uvr delta phr bacteria, repair, by both assays, was very low but not zero. Even when light was totally excluded, Phr function appeared to play a role in Uvr-mediated excision repair: both dimer removal and restoration of infectivity were two to five times as efficient in uvr+ phr+ bacteria as in uvr+ delta phr bacteria. Similarly, UV-irradiated phages plated with higher efficiencies on phr+ than delta phr bacteria even under totally dark conditions. In uvr phr+ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr+ infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system.
Correlation between degradation and ultrastructure of peptidoglycan during autolysis of Escherichia coli
Authors: Leduc M , Frehel C , van Heijenoort J
Published: January 2020

The kinetics of peptidoglycan degradation were examined under different conditions of autolysis of Escherichia coli. With cephaloridine- or moenomycin-induced autolysis, degradation did not exceed 25 to 35%, whereas in EDTA-induced autolysis it rapidly reached 65 to 70%. When nonautolyzing cells were fixed overnight with glutaraldehyde, followed by an osmium fixation, and thin sections were stained by the phosphotungstic acid method, a dark, 15-nm-thick layer of uniform appearance and constant width occupied the whole area between the inner and outer membranes of the envelope. The stained material was tentatively identified with peptidoglycan. Ultrastructural changes in this phosphotungstic acid-stained periplasmic space were investigated at different time intervals after induction of autolysis. In all cases, breakdown proceeded over the whole cell surface. During antibiotic-induced autolysis a progressive thinning down limited to the inner side of the layer was observed. During EDTA-induced autolysis, the rapid decrease in thickness correlated well with the important loss of material labeled with [3H]diaminopimelic acid. Considering these changes and the insufficient amounts of peptidoglycan (1.3 U/nm2) necessary to account for a regularly structured polymer occupying the whole 15-nm layer, it was speculated that peptidoglycan might be unevenly distributed throughout the periplasmic space.
Characterization of a tetraploid derivative of Candida albicans ATCC 10261
Authors: Hubbard M J , Poulter R T , Sullivan P A , Shepherd M G
Published: January 2020

A morphometric analysis of Candida albicans yeast cells utilizing scanning electron microscopy showed that the cell volume and the DNA content of a tetraploid strain (derived by cell fusion) were 2.4 to 3.0 and 2.0 times, respectively, those of the progenitor diploid strain, ATCC 10261. The pathogenicities of both strains were similar.
Salmonella typhimurium contains an anion-selective outer membrane porin induced by phosphate starvation
Authors: Bauer K , Benz R , Brass J , Boos W
Published: January 2020

A mutant of Salmonella typhimurium was selected that is constitutive for the pho regulon. It exhibited constitutive glycerol-3-phosphate transport activity and synthesized a new outer membrane porin. Upon measurement of porin activity in black lipid films, it exhibited anion selectivity. It therefore appears analogous to the Escherichia coli PhoE porin.
Escherichia coli mutant with altered respiratory control of the frd operon
Authors: Iuchi S , Kuritzkes D R , Lin E C
Published: January 2020

In wild-type Escherichia coli, fumarate reductase encoded by the frd operon is inducible by its substrate in the absence of molecular oxygen and nitrate. Synthesis of this enzyme under permissive conditions requires the fnr+ gene product, which is believed to be a pleiotropic regulatory protein that activates transcription. A spontaneous mutant was isolated in which the expression of the frd operon no longer depended on the presence of fumarate or the fnr+ gene product. Aerobic repression of the operon was abolished, but nitrate repression remained intact. Transductional analysis showed that the mutation was closely linked to the frd locus. The mutant phenotype strongly suggests that repression by molecular oxygen and nitrate is mediated by different mechanisms.
Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region
Authors: Smith C J
Published: January 2020

Genetic and physical analyses were used to characterize the Bacteroides ovatus R plasmid pBI136. Results from restriction endonuclease cleavage studies were used to construct a physical map of the plasmid for the enzymes EcoRI, BamHI, ClaI, XbaI, SalI, and SmaI. Based on the sizes of restriction fragments generated in these studies, the plasmid was estimated to be 80.6 kilobase pairs (kb). A 7.2-kb region of the plasmid required for resistance to lincosamide and macrolide (LM) antibiotics was mapped by analysis of spontaneously occurring LM-sensitive deletion derivatives. Hybridization studies showed that this region and an adjoining 2.9-kb EcoRI fragment were responsible for the previously reported homology among Bacteroides plasmids pBF4, pBFTM10, and pBI136. Within this region of homology, 0.5 kb was attributed to a directly repeated sequence thought to bound the LM resistance determinant on pBF4 and pBFTM10. Two pBI136 EcoRI fragments spanning the putative LM resistance region were cloned in Escherichia coli, and heteroduplex analysis of these recombinant plasmids revealed the presence of a 1.2-kb directly repeated sequence. These results suggested that the pBI136 LM resistance determinant resides on an 8.4-kb segment of DNA containing 6.0 kb of intervening DNA sequences bounded by a 1.2-kb directly repeated sequence.
Essential and nonessential sequences in malPp, a positively controlled promoter in Escherichia coli
Authors: Raibaud O , Gutierrez C , Schwartz M
Published: January 2020

A plasmid bearing the malPp promoter was digested with Bal31 to obtain a set of deletions with closely spaced endpoints in the upstream region of this promoter. Some of these deletions were sequenced, and their effect on malPQ expression was determined after having transferred them onto the chromosome. We found that a site which binds the cyclic AMP receptor protein in vitro and which is centered at position -93 with respect to the site of transcription initiation could be deleted without affecting malPQ expression. In contrast, the activity of the malPp promoter decreased abruptly when the deletions reached position -72. The downstream region of the promoter was analyzed by using a technique of "sequence replacement" which involved the selection of Mal+ pseudorevertants from strains which carried small deletions in the -25 region. The pseudorevertants, which expressed the malPQ operon in a manner indistinguishable from wild type, had grossly different sequences downstream from position -38, except for a few positions, some of which must be important for promoter function. By combining all presently available information, it is suggested that the malPp promoter contains three binding sites for its activator, the product of gene malT. These sites are defined by three quasi-identical hexanucleotides present in one orientation around position -37 and twice in the other orientation around positions -60 and -73.
Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae
Authors: McEwen J E , Cameron V L , Poyton R O
Published: January 2020

We describe here a new method for the specific isolation of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae. One unique feature of the method is the use of tetramethyl-p-phenylenediamine as a cytochrome c oxidase activity stain for yeast colonies. The staining of yeast colonies by tetramethyl-p-phenylenediamine is dependent upon a functional cytochrome c oxidase and is unaffected by other lesions in respiration. Since the tetramethyl-p-phenylenediamine colony staining reaction is rapid and simple, it greatly facilitates both the identification and characterization of cytochrome c oxidase-deficient mutants. Another feature of the method, which is made possible by the tetramethyl-p-phenylenediamine colony stain, is the use of an op1 parent strain for the isolation of nuclear pet or mitochondrial mit mutants in specific protein-coding genes. A parent strain that carries this marker selects against rho0 or rho- classes of pleiotropic respiratory-deficient mutants, since these are lethal in op1 strains. We have used this method to isolate 123 independently derived cytochrome c oxidase-deficient pet mutants and 300 independently derived mit mutants.
Regulation of phenylalanine ammonia lyase in Rhodotorula glutinis
Authors: Kane J F , Fiske M J
Published: January 2020

In the red yeast Rhodotorula glutinis, phenylalanine ammonia lyase (PAL) was induced 10-fold during carbon starvation even in the absence of exogenous phenylalanine, although maximal induction occurred when phenylalanine was the nitrogen (40-fold) or carbon (100-fold) source. Apparent regulatory mutations that affected the expression of PAL were isolated by selecting mutants resistant to the analog p-fluoro-D,L-phenylalanine (PFP). One such mutant, designated FP1, could use phenylalanine as a nitrogen source but not as a carbon source. Similarly, FP1 failed to utilize intermediates of the phenylalanine degradative pathway, namely, benzoate, p-hydroxybenzoate, or 3,4-dihydroxybenzoate, as carbon sources. Although the PFP-resistant mutant contained a low level of PAL, no increase was found when it was grown with phenylalanine as the nitrogen source. A derivative of FP1, FP1a, was isolated that simultaneously regained an inducible PAL and the ability to use phenylalanine, benzoate, p-hydroxybenzoate, and 3,4-dihydroxybenzoate as carbon sources. In addition, when p-hydroxybenzoate was the carbon source, PAL was induced in the mutant FP1a but not in the PFP-sensitive parental strain. We propose that the mutation to PFP resistance occurred in a regulatory gene that controls the entire phenylalanine degradative pathway. Secondary mutations at this locus, as found in strain FP1a, not only restored expression of this pathway, but also altered the induction of PAL by metabolites of this pathway.
Mutations in bacteriophage lambda repressor that prevent RecA-mediated cleavage
Authors: Gimble F S , Sauer R T
Published: January 2020

In this paper, we report on the isolation and sequence analysis of mutations that confer an induction-deficient phenotype to lambda repressor. A total of 16 different mutations, which occur at 13 different sites in the repressor gene, have been characterized. For most of the mutant lysogens, frequencies of spontaneous induction in a recA+ strain were reduced dramatically in comparison with those for a wild-type phage, and these mutant lysogens showed little or no prophage induction after UV irradiation. The immunity properties of cells containing the mutant repressors show that all of the mutants but one exhibit operator-binding properties indistinguishable from wild-type repressor.
Identity of Escherichia coli D-1-amino-2-propanol:NAD+ oxidoreductase with E. coli glycerol dehydrogenase but not with Neisseria gonorrhoeae 1,2-propanediol:NAD+ oxidoreductase
Authors: Kelley J J , Dekker E E
Published: January 2020

The properties of D-1-amino-2-propanol oxidoreductase from wild-type Escherichia coli have been compared with those of a glycerol dehydrogenase from mutant E. coli 424 and of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae. Several independent lines of evidence indicate that the former two enzymes are identical. (i) Both enzymatic activities purified to virtual homogeneity in an identical manner, and the ratio of specific activities (glycerol/aminopropanol) remained constant at all stages. (ii) When electrophoresed, both purified enzymes showed a major as well as a minor band of protein coincident with activity, and these two bands from each enzyme had the same mobility. (iii) The subunit molecular weights and isoelectric points were identical for each enzyme, and (iv) kinetic constants (Km and Vmax values) determined with three different substrates were the same. The somewhat greater stability of the glycerol dehydrogenase to controlled heat denaturation at 74 degrees C was the only difference observed between these two enzymes. In contrast, D-1-amino-2-propanol oxidoreductase was found to be immunochemically and kinetically distinct from the 1,2-propanediol oxidoreductase from N. gonorrhoeae.
Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector
Authors: Ulmanen I , Lundström K , Lehtovaara P , Sarvas M , Ruohonen M , Palva I
Published: January 2020

Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis. All three model genes were expressed by using a secretion vector, constructed by joining the B. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I. Palva, M. Sarvas, P. Lehtovaara, M. Sibakov, and L.Kääriäinen, Proc. Natl. Acad. Sci. U.S.A. 79:5582-5586, 1982). When transformed B. subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca. 10% and that of E1 was ca. 0.01% of the amount of alpha-amylase. The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal. The half-lives of these transcripts in B. subtilis were also similar. Pulse-chase experiments with [35S]methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods. In transformed minicells from B. subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates. We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B. subtilis. In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B. subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both.
Mechanisms of iron regulation of luminescence in Vibrio fischeri
Authors: Haygood M G , Nealson K H
Published: January 2020

Synthesis of luciferase (an autoinducible enzyme) is repressed by iron in the symbiotic bioluminescent bacterium Vibrio fischeri. Possible mechanisms of iron regulation of luciferase synthesis were tested with V. fischeri and with Escherichia coli clones containing plasmids carrying V. fischeri luminescence genes. Experiments were conducted in complete medium with and without the synthetic iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid). Comparison of the effect of ethylenediamine-di(o-hydroxyphenyl acetic acid) and another growth inhibitor, (2-n-heptyl-4-hydroxyquinoline-N-oxide), showed that iron repression is not due to inhibition of growth. A quantitative bioassay for autoinducer was developed with E. coli HB101 containing pJE411, a plasmid carrying V. fischeri luminescence genes with a transcriptional fusion between luxI and E. coli lacZ. Bioassay experiments showed no effect of iron on either autoinducer activity or production (before induction) or transcription of the lux operon. Ethylenediamine-di(o-hydroxyphenyl acetic acid) did not affect luciferase induction in E. coli strains with wild-type iron assimilation (ED8654) or impaired iron assimilation (RW193) bearing pJE202 (a plasmid with functional V. fischeri lux genes), suggesting that the genes responsible for the iron effect are missing or substituted in these clones. Two models are consistent with the data: (i) iron represses autoinducer transport, and (ii) iron acts through an autoinduction-independent regulatory system (e.g., an iron repressor).
Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities
Authors: Thompson J , Chassy B M , Egan W
Published: January 2020

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose-phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of [14C]lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution 31P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM. We conclude from our data that phosphorylation of glucose by S. lactis 133 can be mediated by only two mechanisms: (i) via ATP-dependent glucokinase, and (ii) by the phosphoenolpyruvate-dependent mannose-PTS system.
Lactose metabolism in Erwinia chrysanthemi
Authors: Hugouvieux-Cotte-Pattat N , Robert-Baudouy J
Published: January 2020

Wild-type strains of the phytopathogenic enterobacterium Erwinia chrysanthemi are unable to use lactose as a carbon source for growth although they possess a beta-galactosidase activity. Lactose-fermenting derivatives from some wild types, however, can be obtained spontaneously at a frequency of about 5 X 10(-7). All Lac+ derivatives isolated had acquired a constitutive lactose transport system and most contained an inducible beta-galactosidase. The transport system, product of the lmrT gene, mediates uptake of lactose in the Lac+ derivatives and also appears to be able to mediate uptake of melibiose, raffinose, and galactose. Two genes encoding beta-galactosidase enzymes were detected in E. chrysanthemi strains. That mainly expressed in the wild-type strains was the lacZ product. The other, the lacB product, is very weakly expressed in these strains. These enzymes showed different affinities for the substrates o-nitrophenyl-beta-D-galactopyranoside and lactose and for the inhibitors isopropyl-beta-D-thiogalactopyranoside and galactose. The lmrT and lacZ genes of E. chrysanthemi, together with the lacI gene coding for the regulatory protein controlling lacZ expression, were cloned by using an RP4::miniMu vector. When these plasmids were transferred into Lac- Escherichia coli strains, their expression was similar to that in E. chrysanthemi. The cloning of the lmrT gene alone suggested that the lacZ or lacB gene is not linked to the lmrT gene on the E. chrysanthemi chromosome. One Lac+ E. chrysanthemi derivative showed a constitutive synthesis of the beta-galactosidase encoded by the lacB gene. This mutation was dominant toward the lacI lacZ cloned genes. Besides these mutations affecting the regulation of the lmrT or lacB gene, the isolation of structural mutants unable to grow on lactose was achieved by mutagenic treatment. These mutants showed no expression of the lactose transport system, the lmrT mutants, or the mainly expressed beta-galactosidase, lacZ mutants. The lacZ mutants retained a very low beta-galactosidase level, due to the lacB product, but this level was low enough to permit use of the lacZ mutants for the construction of gene fusions with the Escherichia coli lac genes.
Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli
Authors: Richarme G
Published: January 2020

We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply. The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone. The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers.
General recombination in Escherichia coli K-12: in vivo role of RecBC enzyme
Authors: Yancey S D , Porter R D
Published: January 2020

Heterozygous lacZ- merodiploids of Escherichia coli K-12 have been used to study the role of the RecBC enzyme in general recombination. The transcribable intermediate assay detects the product of early steps in recombination without requiring the formation of viable recombinant colonies. Recombination is initiated by infection with lambda precA+. We have found that transcribable intermediate formation in crosses between F42 lac and chromosomal lac is dependent on F fertility functions and an active RecBC enzyme. Thus, the products of the recB and recC genes are required in early steps of recombination between these two substrates. Introduction of the F42 lac donor DNA by conjugation immediately after infection with lambda precA+ abolishes the requirement for an active RecBC enzyme.
Identification of a bacterial sensing protein and effects of its elevated expression
Authors: Clegg D O , Koshland D E
Published: January 2020

The Escherichia coli flaA gene product (also called cheC) plays a crucial role in switching flagellar rotational direction during chemotactic responses. Wild-type and mutant alleles have been cloned onto plasmid vectors, and the gene product has been identified as a 37,000-dalton protein. The flaA product appeared as a soluble protein in the cytoplasm when overproduced in minicells and maxicells. The protein could not be detected in flagellar basal structures purified from a wild-type strain. To assess the effects of altered flaA expression, the gene was fused to a synthetic tac promoter that could be regulated by the addition of an inducer. Overproduction resulted in strong counterclockwise flagellar rotational bias and partial paralysis of flagellar motors. These results suggest that the flaA protein provides the interface between the flagellar machinery and the chemotaxis signaling system in a motor structure external to the basal body.
Acylated proteins in Acholeplasma laidlawii
Authors: Dahl C E , Sacktor N C , Dahl J S
Published: January 2020

The covalent modification of membrane proteins by long-chain fatty acids was determined in two strains of Acholeplasma laidlawii by one-dimensional gel electrophoresis of radiolabeled membranes. Of the more than 50 membrane polypeptides detected, approximately 30 were labeled with [3H]palmitate, whereas covalent binding of [3H]oleate to membrane proteins could not be demonstrated. We suggest that in these wall-less bacteria, membrane protein acylation with saturated fatty acids may serve to ensure the structural integrity of the membrane.
Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin
Authors: Gabay J E , Blake M , Niles W D , Horwitz M A
Published: January 2020

We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins.
Cloning vector system for Corynebacterium glutamicum
Authors: Yoshihama M , Higashiro K , Rao E A , Akedo M , Shanabruch W G , Follettie M T , Walker G C , Sinskey A J
Published: January 2020

A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD10. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was not. The various parameters in the transformation protocol were analyzed separately and optimized. The resulting transformation system is simple and routinely yields 10(4) transformants per microgram of plasmid DNA.
Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element
Authors: Shoemaker N B , Guthrie E P , Salyers A A , Gardner J F
Published: January 2020

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.
Purification and subunit characterization of Rhizobium meliloti RNA polymerase
Authors: Nielsen B L , Brown L R
Published: January 2020

The RNA polymerase of the symbiotic, nitrogen-fixing bacterium Rhizobium meliloti was purified, and its subunit composition was determined. The cells were disrupted in the presence of protease inhibitors, and holoenzyme fractions were purified by fractionation by using DEAE-cellulose and DNA-agarose chromatography. The core polymerase was purified by additional chromatography on phosphocellulose. The subunit structure is beta prime (155,000 molecular weight), beta (151,000), alpha (43,000), and sigma (93,000), with an additional polypeptide of 29,000 molecular weight, which we have designated tau, found associated with both core and holoenzyme fractions. The measured stoichiometry of the holoenzyme complex was found to be 2 alpha:1 beta':1 beta:0.7 sigma:1 tau. The 93,000 molecular-weight protein subunit was identified as the sigma subunit based upon stimulation of specific transcription in assays with reconstituted polymerase.
Evidence for two nonidentical subunits of bacterioferritin from Azotobacter vinelandii
Authors: Harker A R , Wullstein L H
Published: January 2020

The bacterioferritin from Azotobacter vinelandii exhibits properties which in ferritins from other sources are attributed to the heteropolymeric nature of the holoprotein. The native bacterioferritin displayed multiple bands on isoelectric focusing gels. On discontinuous sodium dodecyl sulfate-polyacrylamide gels, there were two subunit polypeptides of approximate Mr 21,000 and 23,000. These molecular weights were corroborated by gel filtration experiments. Peptide maps produced by partial trypsin digestion and electrophoresis showed no detectable differences between the subunits. Similarities to well-characterized mammalian ferritins and apparent anomalies in two commonly applied electrophoretic procedures are discussed.
Identification of three complementation units in the gerA spore germination locus of Bacillus subtilis
Authors: Zuberi A R , Feavers I M , Moir A
Published: January 2020

The gerA locus, mutations in which affect the germination response of spores to L-alanine and related amino acids, is contained within a 6-kilobase region of DNA cloned in phage and plasmid vectors. Fragments from this region, subcloned in the shuttle vector pHV33, were introduced into Bacillus subtilis, and their ability to complement chromosomal gerA mutations in a recE4 background was examined. Although the plasmids were somewhat unstable, it was possible to score complementation within spore-containing colonies on nutrient agar by their ability to reduce 2,3,5-triphenyltetrazolium chloride in an overlay. These studies have assigned the 10 gerA mutations tested to three complementation groups. An analysis of Tn1000 insertions into the cloned DNA of two relatively stable plasmids that together encompass the entire gerA region has identified more precisely the location and extent of the complementation units; recombination studies and in vitro mutagenesis were used to further delineate the extents of two of the units. The evidence suggests that the three complementation units are adjacent and that they are probably capable of separate transcription.
Formation of septum-like structures at locations remote from the budding sites in cytokinesis-defective mutants of Saccharomyces cerevisiae
Authors: Slater M L , Bowers B , Cabib E
Published: January 2020

Cell wall structures that partition membrane-bound portions of cytoplasm were formed at sites along the peripheral wall when a cytokinesis-defective cell division cycle mutant (cdc3) of Saccharomyces cerevisiae was grown at a restrictive temperature. The appearance of these structures, as observed in electron micrographs, was similar to that of normal septa. Aberrant septa were also detected in cytokinesis mutants harboring mutations cdc10, cdc11, and cdc12, after growth at 37 degrees C. Formation of the abnormal septa was abolished by the introduction, in a cdc3-containing strain, of additional cell cycle mutations that precluded events leading to cytokinesis and cell division. These results showed that septum formation can occur in the absence of cytokinesis. Formation of the abnormal structures was controlled by the same sequences of cell cycle events as formation of normal septa but was not subject to the spatial controls that ensure association of the septum with the budding site.
Cadmium uptake in Escherichia coli K-12
Authors: Laddaga R A , Silver S
Published: January 2020

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.
Plasmid transduction by Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and bacteriophage
Authors: Deichelbohrer I , Alonso J C , Lüder G , Trautner T A
Published: January 2020

Any SPP1 DNA restriction fragment cloned into Bacillus subtilis plasmid pC194 or pUB110 increased the transduction frequency of the plasmid by SPP1 100- to 1,000-fold over the transduction level of the plasmid alone. This increment was observed irrespective of whether a fragment contained the SPP1 packaging origin (pac). Furthermore, an SPP1 derivative into whose genome pC194 DNA had been integrated transduced pC194 DNA with a greatly enhanced frequency. Transduction enhancement mediated by DNA-DNA homology between plasmid and SPP1 was independent of the extent of homology (size range analyzed, 0.5 to 3.9 kilobases) and the recombination proficiency of donor or recipient.
Oxygen does not directly regulate carotenoid biosynthesis in Rhodopseudomonas capsulata
Authors: Biel A J , Marrs B L
Published: January 2020

We examined the role of bacteriochlorophyll synthesis on the regulation of carotenoid synthesis in Rhodopseudomonas capsulata. Strains capable of making bacteriochlorophyll accumulated greater amounts of carotenoids under low oxygen than they did under high oxygen. However, strains unable to produce bacteriochlorophyll did not regulate their carotenoid production in response to changes in oxygen tension. This indicates that oxygen does not directly regulate carotenoid production.
Broad-host-range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome
Authors: Keen M G , Street E D , Hoffman P S
Published: January 2020

Transposon Tn5 was introduced into Legionella pneumophila on plasmid pRK340, which is temperature sensitive for plasmid maintenance. The presence of plasmid DNA was confirmed by agarose gel electrophoresis and by conjugal transfer of the plasmid to Escherichia coli. Tn5 insertions were obtained by culturing L. pneumophila at the nonpermissive temperature (43 degrees C) on buffered charcoal-yeast extract agar containing kanamycin. Of the 260 kanamycin-resistant colonies picked, 220 failed to conjugate pRK340 to E. coli. Plasmid DNA was not visualized from eight randomly picked Tn5-containing strains, and Southern hybridizations indicated that Tn5, but not pRK340, inserted into multiple sites in the Legionella chromosome. In addition, the streptomycin resistance determinant on Tn5 was expressed in L. pneumophila.
Genetic heterogeneity in the cysA-fus region of the Bacillus subtilis chromosome: identification of the hos gene
Authors: Matsuzaki S , Kobayashi Y
Published: January 2020

We identified a new gene, hos, which exerts different sporulation phenotypes in Bacillus subtilis strains with different genetic backgrounds. The hos+ gene showed normal sporulation in the genetic background of JH642 but showed temperature-sensitive sporulation in that of the Tano-oka W. The hos gene was mapped between cysA and rpoB.
Chromosomal mapping of mutations affecting glycerol and glucose catabolism in Pseudomonas aeruginosa PAO
Authors: Cuskey S M , Phibbs P V
Published: January 2020

Mutations causing deficiencies in the inducible, membrane-associated sn-glycerol-3-phosphate dehydrogenase (glpD) and in inducible glucose transport (glcT) were mapped on the Pseudomonas aeruginosa PAO1 chromosome by using the generalized transducing phages F116L and G101. These mutations, in separate catabolic regulatory units, were cotransducible with a previously described cluster of carbohydrate catabolic gene loci (zwf-1 eda-9001 edd-1) that maps at ca. 50 to 53 min on the chromosome. Mutant strain PFB362 (glcT1) did not transport glucose and did not produce a functional, periplasmic, glucose-binding protein that is required for glucose transport. This mutation was cotransducible with zwf-1 (70%), nalA (29%), and phe-2 (19%) but not with glpD1 or leu-10. The glpD1 mutation in strain PRP408 was cotransducible with zwf-1 (5%), eda-9001 (4%), and edd-1 (1%) and also with ami-151 (17%) and phe-2 (33%). These results expand the number of known carbohydrate catabolism genes that are clustered in the 50- to 55-min region of the PAO1 chromosome and allow us to propose the following relative gene order: ami-151 glpD1 phe-2 nalA zwf-1 eda-9001 edd-1 glcT1 leu-10. Three independently obtained nal determinants for high-level resistance to nalidixic acid, which were employed in these studies, exhibited similar cotransduction frequencies with several flanking marker mutations.
Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: cloning and expression of the hexA gene
Authors: Balganesh T S , Lacks S A
Published: January 2020

Mutations affecting heteroduplex DNA mismatch repair in Streptococcus pneumoniae were localized in two genes, hexA and hexB, by fractionation of restriction fragments carrying mutant alleles. A fragment containing the hexA4 allele was cloned in the S. pneumoniae cloning system, and the hexA+ allele was introduced into the recombinant plasmid by chromosomal facilitation of plasmid transfer. Subcloning localized the functional hexA gene to a 3.5-kilobase segment of the cloned pneumococcal DNA. The product of this gene was shown in Bacillus subtilis minicells to be a polypeptide with an Mr of 86,000. Two mutant alleles of hexA showed partial expression of the repair system when present in multicopy plasmids. A model for mismatch repair, which depends on the interaction of two protein components to recognize the mismatched base pair and excise a segment of DNA between strand breaks surrounding the mismatch, is proposed.
Chemotactic transducer proteins of Escherichia coli exhibit homology with methyl-accepting proteins from distantly related bacteria
Authors: Nowlin D M , Nettleton D O , Ordal G W , Hazelbauer G L
Published: January 2020

Transducers are transmembrane, methyl-accepting proteins central to the chemotactic systems of the enteric bacteria Escherichia coli and Salmonella typhimurium. Methyl-accepting proteins have been reported in a number of species in addition to these enteric bacteria. Those species include Bacillus subtilis and Spirochaeta aurantia, representatives of groups that diverged from ancestral enteric bacteria and from each other very early in bacterial evolution. An antiserum that reacts with all transducers of E. coli precipitated specifically methyl-accepting proteins from B. subtilis and S. aurantia, indicating that these proteins share antigenic determinants with transducers of E. coli. In addition, analysis of tryptic peptides by high-pressure liquid chromatography revealed similarities in the regions of methyl-accepting sites for proteins from all three species. These observations imply that structural features have been preserved in the three species from transducers contained in a common ancestor of eubacteria. It is thus reasonable to predict that other flagellated, chemotactic bacteria will be found to contain methyl-accepting proteins homologous to transducers of enteric bacteria.