bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae
Authors: Letts V A , Henry S A
Published: January 2020

chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. As expected, when chol mutants were starved for ethanolamine, the rates of synthesis of the phospholipids phosphatidylethanolamine and PC declined rapidly. Surprisingly, however, coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. The results obtained suggest that the slowing of PC biosynthesis in ethanolamine-starved chol cells leads to a coordinated decrease in the synthesis of all phospholipids. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.
Inhibitory effect of high-level transcription of the bacteriophage lambda nutL region on transcription of rRNA in Escherichia coli
Authors: Sharrock R A , Gourse R L , Nomura M
Published: January 2020

Transcription of the bacteriophage lambda nutL region from the PL promoter on a multicopy plasmid in Escherichia coli causes a reduction in growth rate and in transcription of rRNA relative both to total transcription and to transcription of tRNAs that are not encoded in rRNA operons. These observations support the hypothesis, previously based on nut site DNA sequence homology, that the phage lambda and rRNA antitermination systems are related.
Flocculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation
Authors: Sadasivan L , Neyra C A
Published: January 2020

The phenomena of flocculation and floc formation by Azospirillum brasilense Sp7 (ATCC 29145) and Azospirillum lipoferum Sp59b (ATCC 29707) were studied in aerobic liquid cultures. Carbon sources representative of various entry pathways in combination with various nitrogen sources induced flocculation in both species of azospirilla. Noticeably, the combination of fructose and nitrate was the most effective in terms of floc yields. Phase-contrast microscopic observations revealed a transition in cell morphology from freely motile, vibrioid cells to nonmotile, highly refractile encysting forms during the formation of flocs. The nonmotile forms in flocs appeared to be entangled within a fibrillar matrix, and the cells were highly resistant to desiccation. Dried flocs kept for almost 6 months still maintained the highly refractile encysting forms, and their viability was confirmed by pellicle formation and acetylene reduction in semisolid malate medium. Electron microscopic observations of the desiccated flocs revealed the presence of cell forms containing abundant poly beta-hydroxybutyrate granules within a central body and surrounded by a thick layer of exopolysaccharides. The latter were characterized by alkali and acid digestion, crude cellulase hydrolysis, and calcofluor staining. It was concluded that the overproduction of exocellular polymers induces the flocculent growth and is associated with the concomitant transformation of vegetative cells to the desiccation-resistant encysting forms under limiting cultural conditions.
Direct selection of mutations reducing transcription or translation of the recA gene of Escherichia coli with a recA-lacZ protein fusion
Authors: Weisemann J M , Weinstock G M
Published: January 2020

When a recA-lacZ protein fusion was cloned into phage lambda, the resulting transducing phage grew normally on wild-type Escherichia coli, but its growth was severely inhibited in lexA(Def) mutant strains that express recA constitutively at high levels. Mutants of the transducing phage that grew on the lexA(Def) strains were isolated and were found to affect production of the RecA-beta-galactosidase hybrid protein. Most mutants, including a number of nonsense mutants, were phenotypically LacZ-. LacZ+ mutants were also isolated; most of these expressed lower basal and induced levels of beta-galactosidase activity. DNA sequence analysis revealed that some of the LacZ+ mutations were in the recA promoter. One of these was found to prevent induction. Unexpectedly, three of the mutations that reduced expression were located in the recA structural gene, at codons 10, 11, and 12. Further analysis of the codon 10 mutant showed that it most likely affected translation since it had little effect on transcription as measured by beta-galactosidase synthesis from a recA-lacZ operon fusion. This expression defect was not limited to the protein fusion, since the codon 10 mutation also reduced synthesis of RecA protein when present in a complete recA gene. Analysis of the recA DNA sequence in the fusion revealed that each of the mutations at codons 10, 11, and 12 increases the homology between this region of the mRNA and a sequence found at codons 1 to 4. Thus, the secondary structure of the mutant recA mRNAs may be affecting translation.
Cloning of GLN4: an essential gene that encodes glutaminyl-tRNA synthetase in Saccharomyces cerevisiae
Authors: Ludmerer S W , Schimmel P
Published: January 2020

The structural gene for glutaminyl-tRNA synthetase has been isolated from a gene bank of Saccharomyces cerevisiae chromosomal DNA. Cloning was achieved by complementation of a recently described yeast strain that is auxotrophic for glutamine. A multicopy recombinant plasmid with a 5-kilobase-pair genomic insert conferred sixfold elevation in glutaminyl-tRNA synthetase activity and restored a Gln+ phenotype to strains that were Gln- by virtue of a mutant gln4 allele. Subfragments of the 5-kilobase insert directed integration of URA3 to GLN4. Further experiments established that GLN4 is an essential gene that is located on chromosome XV. RNA blots with a GLN4-specific probe detected a single transcript of approximately 2,900 nucleotides.
Succinate dehydrogenase in Rhodopseudomonas sphaeroides: subunit composition and immunocross-reactivity with other related bacteria
Authors: Barassi C A , Kranz R G , Gennis R B
Published: January 2020

Antibodies were raised against the succinate dehydrogenase (SDH) present in the chromatophores of phototrophically grown Rhodopseudomonas sphaeroides. Crossed immunoelectrophoresis experiments indicated that the SDH present in the cytoplasmic membranes of heterotrophically grown R. sphaeroides is probably the same enzyme observed in the chromatophores. The enzyme was extracted by Triton X-100 in a form which consisted of only two subunits (molecular weight, 68,000 and 30,000) and was not associated with a cytochrome b. The antibodies directed against SDH from R. sphaeroides showed no immunocross-reactivity with SDH from phylogenetically related bacterial species, including Rhodopseudomonas capsulata, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodospirillum rubrum, and Rhodospirillum fulvum.
Cloning and location of the dgsA gene of Escherichia coli
Authors: Morris P W , Binkley J P , Henson J M , Kuempel P L
Published: January 2020

The dgsA locus of Escherichia coli was isolated on plasmids obtained from the library of L. Clarke and J. Carbon (Cell 9:91-99, 1976). Restriction fragment analysis and further subcloning demonstrated that the gene is located at kilobase 425 on the Bouché physical map of the terminus region (J. P. Bouché, J. Mol. Biol., 154:1-20, 1982). This corresponds to 35.2 min on the Bachmann genetic map (B. J. Bachmann, Microbiol. Rev. 47:180-230, 1983).
Thermoinducible transcription system for Bacillus subtilis that utilizes control elements from temperate phage phi 105
Authors: Osburne M S , Craig R J , Rothstein D M
Published: January 2020

We describe a thermoinducible-expression system for Bacillus subtilis which utilized an early promoter-operator sequence from temperate phage phi 105 and the thermolabile prophage repressor from the phage variant phi 105 cts23. The system operated at the transcriptional level to control expression in B. subtilis of the cat-86 gene derived from Bacillus pumilis. Details of the strategies used to isolate the early phage promoter are described. This promoter lay in close proximity to the prophage repressor gene on the phi 105 genome. The sequence of the early promoter differed from that of the vegetative B. subtilis consensus promoter by 1 base pair in both the -10 and -35 regions. We also present evidence that our phage-derived expression system could function in Escherichia coli to effect thermoinducible expression of the galK gene.
Kinetic evidence for two interconvertible forms of the folate transport protein from Lactobacillus casei
Authors: Henderson G B , Kojima J M , Kumar H P
Published: January 2020

Lactobacillus casei cells contain a folate transport protein which exhibits a high affinity for folate. The dissociation constant (KD) for folate derived from binding parameters at the steady state (at 0 degree C) is 0.4 nM at pH 7.5 and 0.1 nM at pH 6.0. In the present study, folate binding to this protein at pH 7.5 (and 0 degree C) was shown to follow second-order kinetics and to proceed with an association constant (k+1) of 4.9 X 10(7) liter/mol per min. K+1 was not affected by preincubation conditions which alter the energetic state of the cell. Measurements on the extent of binding showed further that (at 0 degree C) essentially all unoccupied folate-binding sites reside at or are readily accessible to the outer surface of the membrane. In contrast, after saturating the binding site with [3H]folate, the first-order rate constant (k-1) for dissociation of the bound substrate (at 0 degree C) was found to vary substantially with the conditions employed. k-1 was 0.028/min in freshly harvested cells, but it increased by 2.8-fold in cells preincubated at 23 degrees C for 60 min and by 5.4-fold in isolated membranes. In addition, the faster rate observed in preincubated cells (k-1 = 0.077/min) returned to a slower rate after brief exposure of the cells to pH 6.0 (k-1 = 0.041/min), glucose (k-1 = 0.050/min), or both (k-1 = 0.012/min). k-1 was twofold lower at pH 6.0 than at pH 7.5 and was less dependent on the preincubation conditions, although it also increased substantially (5.5-fold) when the cells were converted to plasma membranes. The proposed explanation for these results is that folate transport protein of L. casei exists in two forms which can be distinguished by the accessibility of the binding site to the external medium and whose amounts are dependent upon the presence of bound folate, the pH, and the energetic state of the cell. It is suggested that these forms are transport proteins with binding sites oriented towards the inner and outer surfaces of the membrane.
Sucrose uptake is driven by the Na+ electrochemical potential in the marine bacterium Vibrio alginolyticus
Authors: Kakinuma Y , Unemoto T
Published: January 2020

Na+ was found to be essential for the accumulation of sucrose by Vibrio alginolyticus. Sucrose uptake was completely inhibited by the addition of proton conductor at neutral pH, but not at alkaline pH, where the primary electrogenic Na+ pump generates the Na+ electrochemical gradient. We therefore conclude that sucrose transport is driven by the electrochemical potential of Na+ in this organism.
Plasmid-mediated mineralization of 4-chlorobiphenyl
Authors: Shields M S , Hooper S W , Sayler G S
Published: January 2020

Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 X 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB.
DNA sequence and complementation analysis of a mutation in the rplX gene from Escherichia coli leading to loss of ribosomal protein L24
Authors: Nishi K , Dabbs E R , Schnier J
Published: January 2020

A mutation in Escherichia coli leads to the loss of ribosomal protein L24, severely impaired growth, and a temperature-sensitive phenotype. The mutation was shown to be in rplX, the gene for protein L24, and was due to the alteration of an AAA codon to a TAA stop codon at position 61 in rplX that resulted in a 20-amino acid peptide instead of the 104 amino acids of wild-type L24 protein. rplX genes from three temperature-resistant and fast growing pseudorevertants of the mutant were cloned and sequenced. They were found to have different base substitutions in the TAA codon, resulting in the reappearance of a full-sized protein L24 moiety. Complementation of the slow growth in trans could be achieved with several plasmids containing at least the spc promoter and intact L14 and L24 genes. Plasmids containing genes distal to rplX could further stimulate growth, and the wild type arose when the entire spc operon and the alpha operon were present. In all cases, protein L24 was expressed by the plasmids. Therefore, slow growth could be explained by polarity extending to the alpha operon. However, temperature sensitivity could not be complemented by any of the plasmids in trans, although we found that this phenotype was caused by the mutation in the rplX gene.
Regulation of Bacillus subtilis glutamate synthase genes by the nitrogen source
Authors: Bohannon D E , Rosenkrantz M S , Sonenshein A L
Published: January 2020

The wild-type alleles of the gltA292 and gltB1 mutations of Bacillus subtilis have been identified in banks of B. subtilis DNA cloned in phage lambda. These mutations are thought to define the genes for the two subunits of glutamate synthase. Sequences having transforming activity for each allele were subcloned in plasmids and used as hybridization probes for measurements of the rates of synthesis and steady-state levels of glt mRNAs under different growth conditions. For both gltA and gltB, the level of mRNA varied according to the nitrogen source in the growth medium, to an extent sufficient to explain the variation in glutamate synthase activity under the same conditions. Two start points for mRNA synthesis were detected within the cloned DNA, one of which corresponded to the gltA locus. The other start point appears to define a transcription unit, separate from gltA and gltB, within which mutations cause loss of glutamate synthase activity.
Cell wall and DNA cosegregation in Bacillus subtilis studied by electron microscope autoradiography
Authors: Schlaeppi J M , Schaefer O , Karamata D
Published: January 2020

Cells of a Bacillus subtilis mutant deficient in both major autolytic enzyme activities were continuously labeled in either cell wall or DNA or both cell wall and DNA. After appropriate periods of chase in minimal as well as in rich medium, thin sections of cells were autoradiographed and examined by electron microscopy. The resolution of the method was adequate to distinguish labeled DNA units from cell wall units. The latter, which could be easily identified, were shown to segregate symmetrically, suggesting a zonal mode of new wall insertion. DNA units could also be clearly recognized despite a limited fragmentation; they segregated asymmetrically with respect to the nearest septum. Analysis of cells simultaneously labeled in cell wall and DNA provided clear visual evidence of their regular but asymmetrical cosegregation, confirming a previous report obtained by light microscope autoradiography (J.-M. Schlaeppi and D. Karamata, J. Bacteriol. 152:1231-1240, 1982). In addition to labeled wall units, electron microscopy of thin sections of aligned cells has revealed fibrillar networks of wall material which are frequently associated with the cell surface. Most likely, these structures correspond to wall sloughed off by the turnover mechanism but not yet degraded to filterable or acid-soluble components.
Isolation and characterization of Escherichia coli pantothenate permease (panF) mutants
Authors: Vallari D S , Rock C O
Published: January 2020

Mutants of Escherichia coli K-12 defective in the pantothenate permease (panF) were isolated and characterized. The panF mutation resulted in the complete loss of pantothenate uptake and of the ability to use extracellular vitamin for growth. The growth phenotypes of panF panD, panF panB, and panF panC double mutants showed that the cytoplasmic membrane was impermeable to external pantothenate. Analysis of the intracellular and extracellular metabolites from strain DV1 (panF panD) labeled with beta-[3-3H]alanine demonstrated that a carrier-mediated mechanism for efficient pantothenate efflux remained in the panF mutant. Genetic mapping of this nonselectable allele was facilitated by the isolation of three independent Tn10 insertions close to panF. Two- and three-factor crosses located panF at minute 72 of the E. coli chromosome and established the gene order fabE panF aroE.
Restoration of bacterioopsin gene expression in a revertant of Halobacterium halobium
Authors: Pfeifer F A , Boyer H W , Betlach M C
Published: January 2020

Restoration of bacterioopsin (bop) gene expression in a revertant of Halobacterium halobium was investigated. The phenotype of the revertant is the result of a translocation of the 588-base-pair (bp) sequence "ISH25", adjacent to an ISH24 insertion found in the parental mutant IV-4. These insertions are located about 1,400 bp upstream of the bop gene within the coding region of the putative brp (bacterioopsin-related protein) gene. The level at which the brp gene affects bop gene expression is unknown. Analysis of bop and brp gene transcription in the wild type, mutant IV-4, and the revertant supports the hypothesis that transcription of the putative brp gene is necessary for bop gene expression in the revertant. Eight insertion mutants of the Bop revertant were analyzed to further elucidate restoration of bop gene expression in the revertant. Bop mutants of the revertant were recovered with a frequency of about 10(-4) and were found to contain insertion elements in addition to ISH24 and "ISH25". Six-eighths of these mutants have the insertion element ISH2, and two mutants have previously uncharacterized insertion elements (ISH27 [1,400 bp] and ISH28 [1,000 bp]). ISH27 and ISH28 are confined to the more A + T-rich fraction of the H. halobium genome, as are most copies of other halobacterial insertion elements. The insertion sites in the Bop mutants of the revertant mapped within the coding region of the bop gene (three mutants), immediately upstream of the bop gene presumably in the bop promoter region (two mutants), or within a region from 241 to 449 bp upstream of the bop gene (three mutants). This distribution of insertion sites suggests that the integrity of the 526-bp region between the bop and the brp genes is important for bop gene expression in the revertant.
Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis-Escherichia coli gene fusion
Authors: Zaghloul T I , Kawamura F , Doi R H
Published: January 2020

Translational coupling was demonstrated in a gene fusion in which the promoter and the N-terminal region of the Bacillus subtilis subtilisin (aprA) gene were fused to a promoterless Tn9-derived chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. Expression of this gene fusion results in the production of a native-sized CAT product, whereas the Tn9-derived CAT gene is usually not translated from its own ribosome binding site in B. subtilis (D. S. Goldfarb, R. L. Rodriguez, and R. H. Doi, Proc. Natl. Acad. Sci. USA 79:5886-5890, 1982). A 178-base-pair deletion, which removed part of the signal peptide and the propeptide of the aprA gene and created a translational stop codon 230 base pairs upstream of the CAT gene ribosome binding site, reduced expression of the CAT gene. A BamHI 10-mer linker insertion into this deletion site, which restored the reading frame and simultaneously removed the translation stop codon, restored CAT gene expression. The data indicate that expression of the CAT gene was dependent on translation of the truncated aprA gene into the ribosome binding site of the CAT gene.
Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins
Authors: Comeau D E , Ikenaka K , Tsung K L , Inouye M
Published: January 2020

The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC. By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype. In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift. The identity of the envZ gene product was confirmed by immunoprecipitation. M13 dideoxy sequencing of the DNA around the wild-type ompR-envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented. A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence. The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products.
Identification of the structural gene encoding maltase within the MAL6 locus of Saccharomyces carlsbergensis
Authors: Dubin R A , Needleman R B , Gossett D , Michels C A
Published: January 2020

Saccharomyces yeast strains able to ferment maltose carry at least one member of a family of MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. The MAL6 locus has been cloned and shown to be a cluster of at least three transcribed regions, all of which are required for maltose fermentation. Transcription at two of these genes, MAL61 and MAL62, is both induced by maltose and repressed by glucose. The third gene, MAL63, appears to encode a regulatory product controlling maltose fermentation. In this report, we demonstrate that the MAL62 gene is the structural gene coding for the enzyme maltase. Strain 332-5A is a maltose fermenter of the genotype MAL6 mal1(0). Integrative disruption of the MAL62 gene of the MAL6 locus produces a strain which is still capable of fermenting maltose, but which synthesizes a more heat-labile form of maltase than the undisrupted strain. Synthesis of this more heat-labile maltase was shown to be linked to the mal1(0) locus present in the strain. Integrative disruption of both the MAL62 gene and the MAL62-homologous sequence present at the mal1(0) locus produces a nonfermenter which is unable to synthesize maltase. These results identify MAL62 as the maltase structural gene.
Isolation and characterization of Ca2+-blind mutants of Yersinia pestis
Authors: Yother J , Goguen J D
Published: January 2020

The plasmid pCD1 is required for expression of the low-calcium response (LCR), virulence, and production of V antigen in Yersinia pestis KIM. Five independent mutants constitutive for the LCR at 37 degrees C (Lcrc) were obtained through ethyl methanesulfonate mutagenesis followed by ampicillin enrichment. A sixth, spontaneous mutant was obtained directly through ampicillin enrichment. These mutants failed to grow at 37 degrees C regardless of calcium concentration and produced V antigen constitutively at this temperature. All six mutations were located on pCD1. One mutation was mapped to a 1-kilobase region of lcrA. Based on complementation mapping of this mutation, the lcrA locus was divided into two new loci, lcrD and lcrE. This mutation, lcrE1, did not alter the transcription of other genes in the LCR region and was cis-recessive to lcr mutations. Several lower-molecular-weight outer membrane proteins which were observed in the parent strain grown at 37 degrees C in the presence of 2.5 mM calcium were reduced in quantity or absent from the mutant strain.
Control of STA1 gene expression by the mating-type locus in yeasts
Authors: Yamashita I , Takano Y , Fukui S
Published: January 2020

Extracellular production of glucoamylase from the yeast Saccharomyces diastaticus carrying an STA1 gene (a structural gene for the enzyme) is repressed by the mating-type constitution a/alpha. We obtained mutants that were relieved from the mating-type control of the STA1 gene expression. Tetrad analysis showed that the mutation was closely linked to MATa. The mutation was complemented by a MATa2 gene; hence, it was designated mata2. Northern blot analysis revealed that the MATa2-mediated repression occurs at the posttranscriptional level.
Complementation and characterization of chemotaxis mutants of Bacillus subtilis
Authors: Ordal G W , Parker H M , Kirby J R
Published: January 2020

A set of chemotaxis mutants of Bacillus subtilis was complemented by using SP beta c2 transducing bacteriophage either containing cloned segments of DNA or derived from abnormal excision of SP beta c2 dl2::Tn917 inserted into the chemotaxis region. Representative mutants were characterized in capillary assays for chemotaxis toward four amino acids and mannitol and in tethered-cell experiments for addition and removal of two attractants and two repellents. Twenty complementation groups were identified, in addition to the cheR previously characterized. All were found to be defective in chemotaxis toward all chemoeffectors. They were assigned the names cheA through cheU. The large number of general chemotaxis genes in B. subtilis, in contrast to the six in Escherichia coli, suggests fundamental differences in the mechanism of chemotaxis in the two species.
Enzymatic activities in cell fractions of mycoplasmalike organisms purified from aster yellows-infected plants
Authors: Arora Y K , Sinha R C
Published: January 2020

Mycoplasmalike organisms (MLOs), purified from aster yellows-infected plants were osmotically lysed, and the membranes were separated from the cytoplasmic fraction through differential centrifugation. Electron microscopic examinations of sections of the purified MLOs and the isolated membranes showed pleomorphic bodies and unit membranous empty vesicles, respectively. Cell fractions were tested for NADH oxidase, NADPH oxidase, ATPase, RNase, DNase, and p-nitrophenyl phosphatase activity. NADH oxidase and ATPase were confined to the membrane fraction and NADPH oxidase to the cytoplasmic fraction of the MLOs. para-Nitrophenyl phosphatase, RNase, and DNase activities were detected in both membrane and cytoplasmic fractions, but p-nitrophenyl phosphatase and RNase appeared to be associated with membranes and DNase with the cytoplasmic fraction. Glucose-6-phosphate dehydrogenase was found in the cytoplasmic fraction of the MLO cells. Our findings on the distribution of enzymes in MLO cells and cell fractions are the first basic documentation on nonhelical, nonculturable microbes parasitic to plants.
Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli
Authors: Bueno R , Pahel G , Magasanik B
Published: January 2020

We have isolated insertion and deletion mutants in glnB, the structural gene of PII, a member of the adenylylation system for glutamine synthetase of Escherichia coli, to study the role of PII in the regulation of the synthesis of glutamine synthetase and of histidase in response to nitrogen deprivation or excess. We have studied the effects of this mutation alone and combined with null mutations resulting from the insertion of transposons or from a deletion in the other genes affecting this regulation, glnD, glnF (ntrA), glnG (ntrC), and glnL (ntrB). Our results confirm that only the products of glnF and glnG are essential for this regulation. In cells of the wild type, the response is mediated by the products of glnD and glnB via the product of glnL. In the condition of nitrogen excess, PII, the product of glnB, appears to convert the product of glnL to a form that prevents the activation of transcription of the structural genes for glutamine synthetase and for histidase by the products of glnF and glnG. During nitrogen deprivation, uridylyltransferase, the product of glnD, is activated by the intracellular excess of 2-ketoglutarate over glutamine and converts PII to PII-UMP and changes the form of the glnL product to one that stimulates the activation of transcription of glutamine synthetase and histidase by the products of glnF and glnG.
Negative regulation of adenylate cyclase gene (cya) expression by cyclic AMP-cyclic AMP receptor protein in Escherichia coli: studies with cya-lac protein and operon fusion plasmids
Authors: Kawamukai M , Kishimoto J , Utsumi R , Himeno M , Komano T , Aiba H
Published: January 2020

We constructed cya-lac protein and operon fusion plasmids in vitro. The effect of cyclic AMP (cAMP) on cya expression was examined by measuring the synthesis of beta-galactosidase in Escherichia coli cells containing fused plasmids. In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenous cAMP. Functional cAMP receptor protein (CRP) was necessary for this effect. On the other hand, in a tet-lacZ fused protein as a control system, tet expression was not affected by cAMP. The inhibition of cya expression by cAMP was also observed in the cya-lac fused operon system, although it was necessary to increase the amount of cAMP or CRP in the cells to detect the effect. The results indicate that cAMP-CRP is a negative regulator of cya expression at the level of transcription.
Measurement of the proton motive force in Rhizobium meliloti with the Escherichia coli lacY gene product
Authors: Gober J W , Kashket E R
Published: January 2020

An Escherichia coli lac operon constitutive for lacY was subcloned into the EcoRI site of a wide-host-range plasmid of the Q incompatibility group, and the resulting recombinant plasmid was introduced into Tn5-generated Lac- mutants of Rhizobium meliloti. The R. meliloti transconjugants accumulated lactose about 1,000-fold, equivalent to a proton motive force of -170 to -180 mV, not significantly different from the values calculated from the distributions of weak acids and lipophilic cations.
Common evolutionary origin of the phage T4 dam and host Escherichia coli dam DNA-adenine methyltransferase genes
Authors: Hattman S , Wilkinson J , Swinton D , Schlagman S , Macdonald P M , Mosig G
Published: January 2020

We compared the known DNA nucleotide and encoded amino acid sequences of the Escherichia coli and bacteriophage T4 dam (DNA-adenine methyltransferase) genes. Despite the absence of any DNA sequence homology, there were four regions (11 to 33 residues long) of amino acid sequence homology containing 45 to 64% identity. These results suggest that the genes for these two enzymes have a common evolutionary origin.
Evidence for a sex factor in Streptomyces erythreus
Authors: DeWitt J P
Published: January 2020

A lethal zygosis-sensitive mutant of Streptomyces erythreus, ER720, was isolated. Pocks were formed when spores of the parental type were plated on a lawn of ER720, suggesting the loss of a transmissible plasmid, SEP1, from this strain. Recombination did not occur between derivatives of ER720 lacking SEP1, but it did occur if SEP1 was transferred to one of these strains or if these strains were crossed with other strains containing SEP1. SEP1 could also be transferred at high frequency between strains. This is consistent with SEP1 acting as a sex factor in S. erythreus.
Protein synthesis and proteolysis in immobilized cells of the cyanobacterium Nostoc commune UTEX 584 exposed to matric water stress
Authors: Potts M
Published: January 2020

Cells of the cyanobacterium Nostoc commune UTEX 584 in exponential growth were subjected to acute water stress by immobilizing them on solid supports and drying them at a matric water potential (psi m) of -99.5 MPa. Cells which had been grown in the presence of Na235SO4 before immobilization and rapid drying continued to incorporate 35S into protein for 90 min. This incorporation was inhibited by chloramphenicol. No unique proteins appeared to be synthesized during this time. Upon further drying, the level of incorporation of 35S in protein began to decrease. In contrast, there was an apparent increase in the level of certain phycobiliprotein subunits in solubilized protein extracts of these cells. Extensive proteolysis was detected after prolonged desiccation (17 days) of the cells in the light, although they still remained intact. Phycobilisomes became dissociated in both light- and dark-stored desiccated material.
Respiratory-competent conditional developmental mutant of Mucor racemosus
Authors: Borgia P T , Gokul N K , Phillips G J
Published: January 2020

A conditional developmental mutant of Mucor racemosus which is capable of oxidative energy metabolism is described. Unlike the wild-type strain the mutant was highly fermentative and exhibited the yeast morphology when grown aerobically in glucose-containing media. The high fermentative activity and yeast morphology under these conditions correlated well with maximal expression of glycolytic enzymes and with expression of some polypeptides characteristic of anaerobic growth. Aerobic growth of the mutant on amino acids as the sole carbon source resulted in growth in the mycelial morphology. The mutant was fully capable of oxidative metabolism as judged by its ability to grow on amino acids, respiratory capacity, and complement of tricarboxylic acid cycle enzymes. The results support the hypothesis that oxygen controls both the expression of glycolytic enzymes and the expression of proteins involved in morphogenesis. Moreover, they suggest that there are common regulatory elements in the control of these two classes of gene products. Abnormally high levels of aconitase and isocitrate dehydrogenase in the mutant are consistent with the proposal that pool sizes of citrate may act as a regulator of genes responsive to environmental oxygen concentration.
Chromosomal mapping in Erwinia carotovora subsp. carotovora with the IncP plasmid R68::Mu
Authors: Forbes K J , Pérombelon M C
Published: January 2020

Conjugational gene transfer was established in Erwinia carotovora subsp. carotovora SCRI193 by using plasmid R68::Mu c+ to mobilize the chromosome into multiply mutant recipients. It was observed that although the plasmid alone mobilized markers randomly at a frequency of ca. 10(-5) chromosomal recombinants per donor, the presence of a Mu prophage on the chromosome of the donor increased the frequency of mobilization of markers adjacent to the prophage by up to 10-fold. Using this system it was possible to order 17 chromosomal mutations. The behavior of Mu in E. carotovora subsp. carotovora was also studied.
Alterations in intracellular deoxyribonucleotide levels of mutationally altered ribonucleotide reductases in Escherichia coli
Authors: Platz A , Karlsson M , Hahne S , Eriksson S , Sjöberg B M
Published: January 2020

Four recombinant plasmid clones (pPS305, pPS308, pPS317, and pPS319) coding for Escherichia coli ribonucleotide reductase have been characterized in vivo and in vitro. Each clone carried a different missense mutation affecting the B1 subunit. Measurements were made of deoxyribonucleoside triphosphate pools. Cells carrying the wild-type plasmid, pPS2, overproduced ribonucleotide reductase 10 to 20 times. As a consequence of this elevated enzyme level, the deoxyribonucleotide pools were approximately three times higher. All four mutant clones showed disturbed deoxyribonucleotide pools. The in vitro studies involved chromatography on affinity media, measurements of enzyme activity and allosteric regulation with a variety of substrates and effector molecules, and direct photoaffinity labeling in the presence of dTTP. Clones pPS305 and pPS308 were shown to code for catalytically defective enzymes, whereas clones pPS317 and pPS319 were shown to code for allosterically altered enzymes. The characterized missense mutations can thus be localized to areas involved in regulation of the substrate specificity or to the active site of protein B1. The alteration of the deoxyribonucleotide pools found in cells containing the allosterically defective clones pPS317 and pPS319 clearly demonstrated in vivo significance for the allosteric control of protein B1 in E. coli cells.
Purification and properties of the nitrogenase of Azospirillum amazonense
Authors: Song S D , Hartmann A , Burris R H
Published: January 2020

The nitrogenase of the free-living, microaerobic, N2-fixing bacterium Azospirillum amazonense (strain Y1) was purified by chromatography on DEAE-52 cellulose, by heat treatment, and by preparative polyacrylamide gel electrophoresis. The specific nitrogenase activities were 2,400 nmol of C2H4 formed per min per mg of protein for dinitrogenase (MoFe protein) and 1,800 nmol of C2H4 formed per min per mg of protein for dinitrogenase reductase (Fe protein). The MoFe protein was composed of a minimum of 1,852 amino acid residues, had an isoelectric point of 5.2, and contained 2 atoms of Mo, 24 atoms of Fe, and 28 atoms of acid-labile sulfide per molecule. The Fe protein had 624 amino acid residues and an isoelectric point of 4.6 and contained four atoms of Fe and six atoms of acid-labile sulfide per molecule. The purified MoFe protein showed two subunits with molecular weights of 55,000 and 50,000. The purified Fe protein revealed two polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weights of 35,000 and 31,000. The two Fe protein polypeptides were demonstrated with immunological techniques in the purified, highly active enzyme as well as in extracts. Also, Azotobacter vinelandii Fe protein showed two closely migrating polypeptides that migrated differently from the Fe protein polypeptides of Azospirillum brasilense or Rhodospirillum rubrum. The nitrogenase activity of Azospirillum amazonense Y1 was independent of Mn2+, and the addition of activating enzyme had no effect. No activating enzyme could be found in Azospirillum amazonense. Obviously, the nitrogenase system of Azospirillum amazonense Y1 is different from that of Azospirillum brasilense Sp7 and resembles the Azotobacter system.
Calcium transport in membrane vesicles of Bacillus subtilis
Authors: de Vrij W , Bulthuis R , Postma E , Konings W N
Published: January 2020

Right-side-out membrane vesicles of Bacillus subtilis W23 grown on tryptone-citrate medium accumulated Ca2+ under aerobic conditions in the presence of a suitable electron donor. Ca2+ uptake was an electrogenic process which was completely inhibited by carbonyl cyanide m-chlorophenylhydrazone or valinomycin and not by nigericin. This electrogenic uptake of calcium was strongly dependent on the presence of phosphate and magnesium ions. The system had a low affinity for Ca2+. The kinetic constants in membrane vesicles were Km = 310 microM Ca2+ and Vmax = 16 nmol/mg of protein per min. B. subtilis also possesses a Ca2+ extrusion system. Right-side-out-oriented membrane vesicles accumulated Ca2+ upon the artificial imposition of a pH-gradient, inside acid. This system had a high affinity for Ca2+; Km = 17 microM Ca2+ and Vmax = 3.3 nmol/mg of protein per min. Also, a membrane potential, inside positive, drove Ca2+ transport via this Ca2+ extrusion system. Evidence for a Ca2+ extrusion system was also supplied by studies of inside-out-oriented membrane vesicles in which Ca2+ uptake was energized by respiratory chain-linked oxidation of NADH or ascorbate-phenazine methosulfate. Both components of the proton motive force, the pH gradient and the membrane potential, drove Ca2+ transport via the Ca2+ extrusion system, indicating a proton-calcium antiport system with a H+ to Ca2+ stoichiometry larger than 2. The kinetic parameters of this Ca2+ extrusion system in inside-out-oriented membranes were Km = 25 microM and Vmax = 0.7 nmol/mg of protein per min.
Purification and Characterization of Thin Pili of IncI1 Plasmids ColIb-P9 and R64: Formation of PilV-Specific Cell Aggregates by Type IV Pili
Authors: Yoshida Tetsu , Furuya Nobuhisa , Ishikura Masayuki , Isobe Toshiaki , Haino-Fukushima Kazu , Ogawa Toshio , Komano Teruya
Published: December 2019

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which
Escherichia coli
cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the
pilS
gene. Pilin was first synthesized as a 22-kDa prepilin from the
pilS
gene and subsequently processed to a 19-kDa protein by the function of the
pilU
product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the
pilV
products vary among six or seven different types, as a result of shufflon DNA rearrangements of the
pilV
gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.
Promoter Recognition by
Bacillus subtilis
ς
W
: Autoregulation and Partial Overlap with the ς
X
Regulon
Authors: Huang Xuejun , Fredrick Kurt L. , Helmann John D.
Published: December 2019

The
Bacillus subtilis
genome encodes at least 17 distinct sigma factors, including seven members of the extracytoplasmic function (ECF) subfamily. We have investigated the expression and regulation of the ECF ς factor encoded by the
sigW
gene. A ς
W
-dependent promoter (P
W
) precedes
sigW
, demonstrating that this transcription factor is positively autoregulated. Expression of
sigW
is regulated by both growth phase and medium composition. Maximal expression is attained in early-stationary-phase cells grown in rich medium. We previously reported that
sigW
mutants have elevated transcription of some ς
X
-controlled genes, and we now report that the converse is also true: in a
sigX
mutant, P
W
is derepressed during logarithmic growth. Thus, these two regulons are mutually antagonistic. Reconstituted ς
W
holoenzyme faithfully recognizes the P
W
preceding
sigW
but does not recognize the P
X
promoter preceding the
sigX
gene. Autoregulation of
sigX
is also highly specific: ς
X
holoenzyme initiates transcription from P
X
but recognizes P
W
poorly if at all. In contrast, several promoters that are at least partially under ς
X
control are active with both the ς
X
and ς
W
holoenzymes in vitro. This finding supports the suggestion that the ς
W
and ς
X
regulons overlap. Sequence comparisons suggest that promoters recognized by these two ς factors have similar −35 elements but are distinguished by different base preferences at two key positions within the −10 element.
Induced Levels of Heat Shock Proteins in a
dnaK
Mutant of
Lactococcus lactis
Authors: Koch Birgit , Kilstrup Mogens , Vogensen Finn K. , Hammer Karin
Published: December 2019

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases, including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes. In order to investigate the importance of the DnaK chaperone complex for growth and heat shock response regulation in
Lactococcus lactis
, we have constructed two
dnaK
mutants with C-terminal deletions in
dnaK
. The minor deletion of 65 amino acids in the
dnaKΔ2
mutant resulted in a slight temperature-sensitive phenotype. BK6, containing the larger deletion of 174 amino acids (
dnaKΔ1
), removing the major part of the inferred substrate binding site of the DnaK protein, exhibited a pronounced temperature-sensitive phenotype and showed altered regulation of the heat shock response. The expression of the heat shock proteins was increased at the normal growth temperature, measured as both protein synthesis rates and mRNA levels, indicating that DnaK could be involved in the regulation of the heat shock response in
L. lactis
. For
Bacillus subtilis
, it has been found (A. Mogk, G. Homuth, C. Scholz, L. Kim, F. X. Schmid, and W. Schumann, EMBO J. 16:4579–4590, 1997) that the activity of the heat shock repressor HrcA is dependent on the chaperone function of the GroELS complex and that a
dnaK
insertion mutant has no effect on the expression of the heat shock proteins. The present data from
L. lactis
suggest that the DnaK protein could be involved in the maturation of the homologous HrcA protein in this bacterium.
Identification of OmpT as the Protease That Hydrolyzes the Antimicrobial Peptide Protamine before It Enters Growing Cells of
Escherichia coli
Authors: Stumpe Stefan , Schmid Roland , Stephens Daren L. , Georgiou George , Bakker Evert P.
Published: December 2019

The influence of extracytoplasmic proteases on the resistance of
Escherichia coli
to the antimicrobial peptide protamine was investigated by testing strains with deletions in the protease genes
degP
,
ptr
, and
ompT
. Only Δ
ompT
strains were hypersusceptible to protamine. This effect was abolished by plasmids carrying
ompT
. Both at low and at high Mg
2+
concentrations,
ompT
+
strains cleared protamine from the medium within a few minutes. By contrast, at high Mg
2+
concentrations, protamine remained present for at least 1 h in the medium of an
ompT
strain. These data indicate that OmpT is the protease that degrades protamine and that it exerts this function at the external face of the outer membrane.