bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional control of the S10 ribosomal protein operon of Escherichia coli after a shift to higher temperature
Authors: Zengel J M , Lindahl L
Published: January 2020

In the 5 to 10 min immediately following a shift from 30 to 42 degrees C, the differential synthesis rates of ribosomal proteins encoded by the 11-gene S10 operon are transiently decreased. This effect results largely from a two- to threefold decrease in the differential rate of transcription of the operon. The inhibition of mRNA synthesis is apparently due to two types of control: (i) initiation of transcription at the S10 promoter is inhibited and (ii) readthrough at the attenuator in the S10 leader is decreased. Both of these effects on transcription are independent of the heat shock regulatory gene, htpR. Furthermore, the inhibition of transcription is observed in both relA+ and relA cells, suggesting that the temperature-induced repression does not require the relA-dependent accumulation of guanosine tetraphosphate (ppGpp). However, recovery from the heat shock was slower in relA+ strains than in relA strains. None of the other ribosomal protein operons that we analyzed showed such a strong decrease in transcription after the heat shock.
Cloning and mapping of the genetic determinants for microcin B17 production and immunity
Authors: San Millan J L , Hernandez-Chico C , Pereda P , Moreno F
Published: January 2020

Plasmid pMccB17 (70 kilobases [kb]) codes for the production of microcin B17, a peptide that inhibits DNA synthesis, and for microcin B17 immunity. A BamHI-EcoRI fragment of 5.1 kb from pMccB17 was cloned into pBR322 in two steps. The resulting plasmid (pMM102) overproduced microcin B17 and expressed immunity against microcin. Mcc- and Mcc- Imm- mutants were isolated on plasmids pMccB17 and pMM102 by deleting various DNA fragments and by inserting different translocatable elements. Physical and phenotypic characterization of these mutants showed that a DNA region of 3.0 to 3.5 kb is required to produce microcin B17, whereas an adjacent region of about 1.0 kb is required to express microcin B17 immunity.
Host DNA replication forks are not preferred targets for bacteriophage Mu transposition
Authors: Nakai H , Taylor A L
Published: January 2020

Bacteriophage Mu DNA integration in Escherichia coli strains infected after alignment of chromosomal replication was analyzed by a sandwich hybridization assay. The results indicated that Mu integrated into chromosomal segments at various distances from oriC with similar kinetics. In an extension of these studies, various Hfr strains were infected after alignment of chromosomal replication, and Mu transposition was shut down early after infection. The positions of integrated Mu copies were inferred from the transfer kinetics of Mu to an F- strain. Our analysis indicated that the location of Mu DNA in the host chromosome was not dependent on the positions of host replication forks at the time of infection. However, the procedure for aligning chromosomal replication affected DNA transfer by various Hfr strains differently, and this effect could account for prior results suggesting preferential integration of Mu at host replication forks.
Growth substrate effects on acetate and methanol catabolism in Methanosarcina sp. strain TM-1
Authors: Zinder S H , Elias A F
Published: January 2020

When Methanosarcina sp. strain TM-1 is grown in medium in which both methanol and acetate are present, growth is biphasic, with methanol used as the primary catabolic substrate during the first phase. To better understand this phenomenon, we grew cells on methanol or on acetate or on both and examined the abilities of anaerobically washed cells to catabolize these substrates. Washed acetate-grown cells incubated with 10 mM acetate, 10 mM methanol, or both substrates together produced methane at initial rates of 325, 3, and 315 nmol min-1 mg of protein-1, respectively. Although the initial rate of methanogenesis from both substrates was nearly identical to the rate for acetate alone, after several hours of incubation the rate was greater for cells provided with both substrates. Studies with 14C-labeled methanol indicated that methanol was catabolized to methane at increasing rates by acetate-grown cells in a manner reminiscent of an induction curve, but only when cells were provided with acetate as a cosubstrate. Acetate was presumably providing energy and carbon for induction of methanol-catabolic enzymes. Methanol-grown cells showed a pattern of substrate utilization significantly different from that of acetate-grown cells, producing methane from 10 mM acetate, 10 mM methanol, or both substrates at initial rates of 10, 280, and 450 nmol min-1 mg of protein-1, respectively. There was significant oxidation of the methyl group of acetate during metabolism of both substrates. Cells grown on methanol-acetate and harvested before methanol depletion (methanol phase) showed catabolic patterns nearly identical to those of methanol-grown cells, including a low rate of methanogenesis from acetate. Cells harvested from methanol-acetate cultures in the acetate phase were capable of significant methanogenesis from either methanol or acetate alone, and the rate from both substrates together was nearly equal to the sum of the rates for the single substrates. When both 10 mM methanol and 10 mM acetate were presented to the acetate-phase cells, there was a preference for the methanol. These results are consistent with a model for regulation in Methanosarcina sp. strain TM-1 in which methanol represses acetate catabolism while methanol catabolism is inducible.
Regulation of ugp, the sn-glycerol-3-phosphate transport system of Escherichia coli K-12 that is part of the pho regulon
Authors: Schweizer H , Boos W
Published: January 2020

The expression of the ugp-dependent sn-glycerol-3-phosphate transport system that is part of the pho regulon was studied in mutants of Escherichia coli K-12 containing regulatory mutations of the pho regulon. The phoR and phoST gene products exerted a negative control on the expression of ugp. Induction of the system was positively controlled by the phoB, phoM, and phoR gene products. Using a ugp-lacZ operon fusion, we showed that the ugp and phoA genes were coordinately derepressed and repressed.
Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene
Authors: Nakajima R , Imanaka T , Aiba S
Published: January 2020

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.
Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity
Authors: Wang W B , Tessman E S
Published: January 2020

The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity.
Cryptic plasmid of Neisseria gonorrhoeae: complete nucleotide sequence and genetic organization
Authors: Korch C , Hagblom P , Ohman H , Göransson M , Normark S
Published: January 2020

The naturally occurring cryptic plasmid pJD1 of Neisseria gonorrhoeae is 4,207 base pairs long and is found in about 96% of gonococcal strains. The total probable coding capacity of pJD1 was determined from the complete nucleotide sequence by using computational probes to identify open reading frames with similar codon usage and by screening for the presence of ribosomal binding sites before the start codons. Candidates for promoters and terminators were also found in the sequence. Based on these findings, we propose a model for the genetic organization of the plasmid. The model predicts two transcriptional units, each composed of five compactly spaced genes. A promoter of one of the transcripts was shown to function in Escherichia coli, and the products of three of the five genes in this operon were identified in minicell expression experiments. Of these, the cppA gene encoded a 9-kilodalton protein, and the cppB and cppC genes both coded for 24-kilodalton proteins. No expression of the other transcriptional unit was detected, but two genes in this operon were expressed in minicells when transcribed from an E. coli promoter. The experimental data were consistent with the model.
Photoreactivation of Escherichia coli reverses umuC induction by UV light
Authors: Brash D E , Haseltine W A
Published: January 2020

UV mutagenesis in Escherichia coli depends on the presence of a premutagenic lesion in DNA and on the induction of the umuCD gene product as part of the SOS response. Using operon fusions between the E. coli lacZ gene and the SOS genes umuC, uvrB, and dinD, we have affirmed the expected role of the cyclobutane pyrimidine dimer in inducing SOS gene transcription. In addition, we found that photoreactivation of cyclobutane pyrimidine dimers reversed umuC induction to the same extent as it reversed mutagenesis. Therefore, the photoreactivability of UV mutagenesis does not itself identify the cyclobutane pyrimidine dimer as the UV mutagenic lesion in E. coli.
Characterization of a trithionate reductase system from Desulfovibrio vulgaris
Authors: Kim J H , Akagi J M
Published: January 2020

A trithionate reductase system was isolated and purified from extracts of Desulfovibrio vulgaris. This system reduced trithionate to thiosulfate and consisted of two proteins. One was bisulfite reductase, an enzyme that reduces bisulfite to trithionate, and the second component was designated TR-1. Both enzymes were required to reduce trithionate to thiosulfate. Flavodoxin and cytochrome c3 from D. vulgaris were tested for their ability to function as electron carriers during trithionate reduction. When molecular hydrogen was the source of electrons for the reduction, both flavodoxin and cytochrome c3 were required. In contrast, when the pyruvate phosphoroclastic system was the reductant, flavodoxin alone participated as the electron carrier. The results indicate that flavodoxin, but not cytochrome c3, interacted with the trithionate reductase system. The cytochrome in the hydrogenase-linked assay functioned as an electron carrier between hydrogenase and flavodoxin.
Biosynthetic arginine decarboxylase in Escherichia coli is synthesized as a precursor and located in the cell envelope
Authors: Buch J K , Boyle S M
Published: January 2020

The biosynthetic form of arginine decarboxylase (ADC) catalyzes the synthesis of agmatine, a precursor of putrescine, in Escherichia coli. Selective disruption of the cell envelope and an assessment of ADC activity or immunoprecipitable ADC in various fractions demonstrated its location between the cytoplasmic membrane and peptidoglycan layer. Expression in minicells of the speA gene encoding ADC resulted in the production of two immunoprecipitable species (74 and 70 kilodaltons). Studies in vivo with a pulse and chase of radiolabeled amino acid into the two species suggest a precursor-product relationship. This relationship was corroborated by demonstrating the accumulation of the 74-kilodalton species in a strain of E. coli unable to process signal sequences. Peptide mapping experiments with V8 protease, trypsin, and alpha-chymotrypsin demonstrated that the two species of ADC were very similar except for a minor difference. These data were used to substantiate the compartmentalization hypothesis as to how exogenous arginine can be channeled preferentially into putrescine.
Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10
Authors: Keuning S , Janssen D B , Witholt B
Published: January 2020

A new enzyme, haloalkane dehalogenase, was isolated from the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10. The purified enzyme catalyzed the hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes, including chlorinated, brominated, and iodinated compounds. The highest activity was found with 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dibromoethane. The enzyme followed Michaelis-Menten kinetics, and the Km for 1,2-dichloroethane was 1.1 mM. Maximum activity was found at pH 8.2 and 37 degrees C. Thiol reagents such as p-chloromercuribenzoate and iodoacetamide rapidly inhibited the enzyme. The protein consists of a single polypeptide chain of a molecular weight of 36,000, and its amino acid composition and N-terminal sequence are given.
Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations
Authors: Skarstad K , Steen H B , Boye E
Published: January 2020

A computer simulation routine has been made to calculate the DNA distributions of exponentially growing cultures of Escherichia coli. Calculations were based on a previously published model (S. Cooper and C.E. Helmstetter, J. Mol. Biol. 31:519-540, 1968). Simulated distributions were compared with experimental DNA distributions (histograms) recorded by flow cytometry. Cell cycle parameters were determined by varying the parameters to find the best fit of theoretical to experimental histograms. A culture of E. coli B/r A with a doubling time of 27 min was found to have a DNA replication period (C) of 43 min and an average postreplication period (D) of 22 to 23 min. Similar cell cycle parameters were found for a 60-min B/r A culture. Initiations of DNA replication at multiple origins in one and the same cell were shown to be essentially synchronous. A slowly growing B/r A culture (doubling time, 5.5 h) had an average prereplication period (B) of 2.3 h; C = 2.4 h and D = 0.8 h. It was concluded the the C period has a constant duration of 43 min (at 37 degrees C) at fast growth rates (doubling times, less than 1 h) but increases at slow growth rates. Thus, our results obtained with unperturbed exponential cultures in steady state support the model of Cooper and Helmstetter which was based on data obtained with synchronized cells.
Fimbriae from the oral anaerobe Bacteroides gingivalis: physical, chemical, and immunological properties
Authors: Yoshimura F , Takasawa T , Yoneyama M , Yamaguchi T , Shiokawa H , Suzuki T
Published: January 2020

Circular dichroism spectra indicated the predominance of beta-sheet structure in Bacteroides gingivalis fimbriae regardless of the presence of sodium dodecyl sulfate. By using a computer program, the alpha-helix, beta-sheet, and beta-turn contents and the remainder were estimated to be 0, 55, 18, and 27%, respectively, judging from the circular dichroism spectra of the fimbriae. Heating for 5 min at 100 degrees C in sodium dodecyl sulfate was necessary to denature the fimbriae into their constituent protein (fimbrilin) monomers with a reduced content of beta-sheet structure. The amino-terminal amino acid sequence of the fimbrilin was different from partial or complete amino acid sequences of fimbrilins so far determined from Bacteroides nodosus, which falls into the same nonfermentative species of the genus Bacteroides as B. gingivalis, and from various other bacteria. Fimbrilin monomers had an isoelectric point of 6.0. Examination of antibodies against fimbriae and sodium dodecyl sulfate-denatured fimbrilin by enzyme-linked immunosorbent assay reinforced a previous notion (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) that different sets of antigenic determinants seemed to be exposed on their surfaces.
N conjugative transfer system of plasmid pCU1
Authors: Thatte V , Bradley D E , Iyer V N
Published: January 2020

The mating and plasmid DNA transfer functions in Escherichia coli K-12 strains that are determined by the IncN group plasmid pCU1 are specified by a single 19.4-kilobase (kb) segment of the 39-kb plasmid. Analysis of this tra region by transposon Tn5 mutagenesis and by complementation tests indicated that a subset of tra comprising six complementation groups (presumably transcription units) determines sensitivity to the N pilus-specific bacteriophages IKe and PRD1. Deletion derivatives and molecular clones that contained only this part of tra conferred sensitivity to IKe and PRD1 and the ability to produce N pili without conferring the Tra phenotype. The entire tra region contained two other complementation groups. If the 19.4-kb tra region is interrupted by DNA that is irrelevant to the Tra phenotype, such DNA must be less than 2 kb long. There are two regions within tra where this is a possibility. These observations are compared with those obtained recently with a related plasmid.
Mol- mutants of Klebsiella pneumoniae requiring high levels of molybdate for nitrogenase activity
Authors: Imperial J , Ugalde R A , Shah V K , Brill W J
Published: January 2020

Mol- mutants of Klebsiella pneumoniae requiring high levels of molybdate for nitrogenase and nitrate reductase activity were characterized. The effects of mol mutations on nitrogenase activity were very similar to those caused by nifQ mutations. Mol- mutants of K. pneumoniae appear to be equivalent to ChlD- mutants of Escherichia coli.
Presence of DNA, encoding parts of bacteriophage tail fiber genes, in the chromosome of Escherichia coli K-12
Authors: Riede I , Eschbach M L , Henning U
Published: January 2020

The classical T-even bacteriophages recognize host cells with their long tail fibers. Gene products 35, 36, and 37 constitute the distal moiety of these fibers. The free ends of the tail fibers, which are formed by the CO2H terminus of gene product 37, possess the host range determinants. It was found that 4 out of 10 different strains of Escherichia coli K-12 contained regions of chromosomal DNA which hybridized with a probe consisting of genes 35, 36, and 37 of the T-even phage K3. From one strain this homologous DNA, which was associated with an EcoRI fragment of about 5 kilobases, was cloned into plasmid pUC8. Two independently recovered hybrid plasmids had undergone a peculiar rearrangement which resulted in the loss of about 3 kilobases of cloned DNA and a duplication of both the vector and the remaining chromosomal DNA. The mechanisms causing this duplication-deletion may be related to that of transposases. The cloned DNA was capable of recombination with phage T4 gene 36 and a phage T2 gene 37 amber mutant. DNA sequencing revealed the existence of regions of identity between the cloned DNA and genes 36 and 37 of phage T2. In addition, after growth of a derivative of phage K3 on a strain harboring T2 DNA, it was found that this phage contained the same parts of the T2 tail fiber genes which had been recovered from the bacterial chromosome. There appears to be little doubt that the phage had picked up this DNA from the host. The possibility is considered that a repertoire of parts of genes 36 and 37 of various T-even-type phages is present in their hosts, allowing the former to change their host ranges.
Nucleotide sequence analysis of the cat gene of Proteus mirabilis: comparison with the type I (Tn9) cat gene
Authors: Charles I G , Keyte J W , Shaw W V
Published: January 2020

In Proteus mirabilis PM13 chloramphenicol resistance is mediated by the cat gene, a single copy of which is present in both resistant and sensitive isolates and which reverts at a high frequency. RNA measurements show an about 8.5-fold increase in cat-specific mRNA in cells expressing the resistance phenotype as compared with those which are sensitive to chloramphenicol. DNA sequence analysis has revealed a high degree of homology between the P. mirabilis cat gene and the type I cat variant (Tn9), 76% at the amino acid level and 73% when nucleotides in the coding sequence are compared. Sequence homology between the strain PM13 cat variant and Tn9 cat was not apparent however in the 5' and 3' flanking regions. Segments of near identity were seen when the upstream sequence of the cat of P. mirabilis was compared with the 5' regions of the Salmonella typhimurium flagellin genes H1 and H2, which are alternately expressed by a flip-flop control mechanism involving an invertible promoter and a trans-acting product.
Induction by folate and folate analogs of extracellular and membrane-bound phosphodiesterase from Dictyostelium discoideum
Authors: van Ophem P , van Driel R
Published: January 2020

Folate stimulation is known to enhance Dictyostelium discoideum differentiation. During early differentiation, D. discoideum cells possess two classes of folate receptors which can be distinguished by their difference in specificity (R. J. W. de Wit, FEBS Lett. 150, 445-448, 1982). We investigated the type of receptor by which folate affects cell differentiation. Two independently regulated developmental markers were used: the extracellular phosphodiesterase-inhibitor system and cell-surface phosphodiesterase activity. Our results indicate that the major effect of folate on development is mediated by the folate-specific receptor. The nonspecific folate receptor was only involved in a minor, transient enhancement of the extracellular phosphodiesterase activity very early in development.
Characterization of a Rhizobium meliloti fixation gene (fixF) located near the common nodulation region
Authors: Aguilar O M , Kapp D , Pühler A
Published: January 2020

Rhizobium meliloti 2011 DNA from pRmSL26, a plasmid which is known to carry genes involved in the early stages of nodulation, was used to construct Tn5 mutations by site-directed Tn5 mutagenesis. Tn5 mutations located within an 8.7 kilobase EcoRI fragment defined two adjacent loci encoding functions for nodulation (nod) and symbiotic N2 fixation (fix). We investigated the organization and regulation of the fix locus and the characteristics of alfalfa nodules induced by these Fix- mutants. By monitoring expression in Escherichia coli minicells, we determined that the fix locus encoded a 36-kilodalton polypeptide. The gene corresponding to this locus was designated fixF. Morphological and ultrastructural studies of the ineffective nodules formed by R. meliloti fixF mutants showed infected host cells similar to those of the wild type. The ineffective nodules were able to accumulate leghemoglobin, but at lower levels than those found in the wild-type nodules. Expression of the nifHDK operon was unaffected by Tn5 insertions in the fixF gene. Expression of the fixF gene was monitored in E. coli by using translational lacZ fusions. It was shown that transcription of the fixF gene in E. coli could be activated by Klebsiella pneumoniae nifA and the R. meliloti nifA-like regulatory gene products. Expression of the fixF gene was also studied in free-living and symbiotic R. meliloti cells. It was found that the fixF gene was transcribed in the symbiotic state.
Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity
Authors: Morris E J , Ganeshkumar N , McBride B C
Published: January 2020

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.
Participation of cytochromes in some oxidation-reduction systems in Campylobacter fetus
Authors: Lascelles J , Calder K M
Published: January 2020

Campylobacter species are rich in c-type cytochromes, including forms which bind carbon monoxide. The role of the various forms of cytochromes in Campylobacter fetus has been examined in cell-free preparations by using physiological electron donor and acceptor systems. Under anaerobic conditions, NADPH reduced essentially all of the cytochrome c in crude cell extracts, whereas the reduction level with succinate was 50 to 60%. The carbon monoxide spectrum with NADPH was predominated by the cytochrome c complex; evidence of a cytochrome o type was seen in the succinate-reduced extracts and in membrane fractions. Succinate-reduced cytochrome c was oxidized by oxygen via a cyanide-sensitive, membrane-associated system. NADPH-reduced cytochrome c was oxidized by a cyanide-insensitive system. Partially purified carbon monoxide-binding cytochrome c, isolated from the cytoplasm, could serve as electron acceptor for NADPH-cytochrome c oxidoreductase; the reduced cytochrome was oxidized by oxygen by a cyanide-insensitive system present in the cytoplasmic fraction. Horse heart cytochrome c was also reducible by NADPH and by succinate; the reduced cytochrome was oxidized by a cyanide-sensitive system in the membrane fraction. NADPH and NADH oxidase activities were observed aerobically and under anaerobic conditions with fumarate. NADPH was more active than NADH. NADP was also more effective than NAD as an electron acceptor for the coenzyme A-dependent pyruvate and alpha-ketoglutarate dehydrogenase activities found in crude extracts. These dehydrogenases used methyl viologen and metronidazole as electron acceptors; they could be loci for oxygen inhibition of growth. It is proposed that energy provision via the high-potential cytochrome c oxidase system in the cytoplasmic membrane is limited by oxygen-sensitive primary dehydrogenases and that the carbon monoxide-binding cytochrome c may have a role as an oxygen scavenger.
Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria
Authors: Schmidhauser T J , Helinski D R
Published: January 2020

The replication and maintenance properties of the broad-host-range plasmid RK2 and its derivatives were examined in nine gram-negative bacterial species. Two regions of RK2, the origin of replication (oriV) and a segment that encodes for a replication protein (trfA delta kilD, designated trfA*), are sufficient for replication in all nine species tested. However, stable maintenance of this minimal replicon (less than 0.3% loss per generation under nonselection conditions) is observed only in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii. Maintenance of this minimal replicon is unstable in Rhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodopseudomonas sphaeroides. A maintenance function has been localized to a 3.1-kilobase (kb) region of RK2 encoding three previously described functions: korA (trfB korB1 korD), incP1-(II), and korB. The 3.1-kb maintenance region can increase or decrease the stability of maintenance of RK2 derivatives dependent on the host species and the presence or absence of the RK2 origin of conjugal transfer (oriT). In the case of A. calcoaceticus, stable maintenance requires an RK2 segment that includes the promoter and the kilD (kilB1) functions of the trfA operon in addition to the 3.1-kb maintenance region. The broad-host-range maintenance requirements of plasmid RK2, therefore, are encoded by multiple functions, and the requirement for one or more of these functions varies among gram-negative bacterial species.
Identification of the active site in penicillin-binding protein 3 of Escherichia coli
Authors: Nicholas R A , Strominger J L , Suzuki H , Hirota Y
Published: January 2020

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.
Isolation of a dihydrofolate reductase-deficient mutant of Escherichia coli
Authors: Singer S , Ferone R , Walton L , Elwell L
Published: January 2020

A strain of Escherichia coli was isolated in which dihydrofolate reductase was not detected by an enzyme assay or by competition for antibody. This strain requires methionine, glycine, a purine, and thymidine for growth in addition to the auxotrophic requirements of the parent strain. It was found to be useful as a recipient of plasmids harboring dihydrofolate reductase genes.
Molecular cloning of the Escherichia coli gene for diadenosine 5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase
Authors: Mechulam Y , Fromant M , Mellot P , Plateau P , Blanchin-Roland S , Fayat G , Blanquet S
Published: January 2020

A clone overproducing diadenosine tetraphosphatase (diadenosine 5', 5'''-P1, P4-tetraphosphate pyrophosphohydrolase) activity was isolated from an Escherichia coli cosmid library. Localization of the DNA region responsible for stimulation of this activity was achieved by deletion mapping and subcloning in various vectors. Maxicell experiments and immunological assays demonstrated that a 3.5-kilobase-pair DNA fragment carried the structural gene apaH encoding the E. coli diadenosine tetraphosphatase. The DNA coding strand was determined by cloning this fragment in both orientations in pUC plasmids. It was also shown that the overproduction of diadenosine tetraphosphatase decreased the dinucleoside tetraphosphate concentration in E. coli by a factor of 10.
Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5
Authors: Gentz R , Bujard H
Published: January 2020

Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be used for the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems.
Comparative studies of the amino acid and nucleotide sequences of pilin derived from Pseudomonas aeruginosa PAK and PAO
Authors: Sastry P A , Finlay B B , Pasloske B L , Paranchych W , Pearlstone J R , Smillie L B
Published: January 2020

The entire amino acid sequence for Pseudomonas aeruginosa PAO pilin was determined through peptide sequencing and from the complete nucleotide sequence encoding the pilin gene. The precursor PAO pilin is 149 amino acids in length which includes a 6-amino-acid positively charged leader sequence. Comparison of the amino acid sequences of pilin produced by P. aeruginosa PAO and PAK reveals a region of high homology corresponding to the leader peptide and residues 1 to 54 of the mature pilin. The amino acid sequence of the peptide encompassing the major antigenic determinant of PAK differs greatly from that of the equivalent region in PAO. The C-terminal regions of these proteins are semiconserved. Few major differences were found when the predicted secondary structures for PAO and PAK pilins were compared. Major nucleotide sequence variation between the equivalent restriction fragments from PAO and PAK occurred within the areas coding for the peptides containing the immunodominant site for PAK pilin and the C termini.
Streptococcus faecalis R plasmid pJH1 contains a pAM alpha 1 delta 1-like replicon
Authors: Banai M , Gonda M A , Ranhand J M , LeBlanc D J
Published: January 2020

Streptococcus faecalis R plasmid pJH1 did not transform competent strains of Streptococcus sanguis. A hybrid plasmid, pDL310, consisting of virtually all of the S. faecalis hemolysin-bacteriocin plasmid pJH2 and a segment of pJH1 DNA that included the tetracycline resistance determinant, yielded tetracycline-resistant transformants at a frequency of less than 10(-8) transformants per CFU, when it was added to a competent culture of S. sanguis Wicky. Four of the transformants contained a 4.7-kilobase plasmid (pDL316) that transformed strain Wicky at a frequency of 8.6 X 10(-8). Restriction endonuclease digests, agarose gel electrophoresis, and Southern blot hybridizations indicated that pDL316 consisted entirely of pJH1-derived DNA. Additional restriction studies, Southern blot hybridizations, and heteroduplex analyses indicated that pDL316 was very closely related to 4.6-kilobase tetracycline resistance plasmid pAM alpha 1 delta 1, a derivative of 9.0-kilobase S. faecalis plasmid pAM alpha 1.
In vivo and in vitro synthesis of Escherichia coli maltose-binding protein under regulatory control of the lacUV5 promoter-operator
Authors: Rasmussen B A , MacGregor C H , Ray P H , Bassford P J
Published: January 2020

It has not been possible to obtain in vitro expression of the positively regulated malE gene encoding the periplasmic maltose-binding protein (MBP) of Escherichia coli. To facilitate in vitro malE expression, we constructed plasmids that place the malE gene under transcriptional control of the lacUV5 promoter-operator. These plasmids could be grouped into three classes, based upon their ability to complement in vivo a chromosomal malE deletion in the presence or absence of isopropyl thiogalactoside. In the one class I plasmid analyzed, the lacUV5-malE junction was just 3' to the malE ATG initiation codon, and this plasmid did not complement the malE deletion. Class II and class III plasmids retained various amounts of the malE promoter. MBP synthesis was solely under control of the lacUV5 promoter in the class II plasmids, and MBP synthesis was under control of both the lacUV5 and malE promoters in the class III plasmids. A malE mutation that renders the MBP signal peptide export defective was genetically recombined onto one of the class II plasmids. The in vivo synthesis and export of plasmid-encoded MBP were studied in the presence and absence of isopropyl thiogalactoside and maltose and in a strain harboring a prlA mutation that suppresses the malE signal sequence mutation and is thought to alter the export machinery of cells. In addition, both class II and class III plasmids programmed the synthesis of precursor MBP in an in vitro-coupled transcription-translation system. When precursor MBP was synthesized in vitro in the presence of E. coli membrane vesicles, a significant portion of wild-type precursor MBP, but not export-defective precursor MBP, was converted to a form that migrated on sodium dodecyl sulfate-polyacrylamide gels identically to mature MBP synthesized in vivo.
Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile)
Authors: Renosto F , Schultz T , Re E , Mazer J , Chandler C J , Barron A , Segel I H
Published: January 2020

ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.
Cell cycle parameters of Proteus mirabilis: interdependence of the biosynthetic cell cycle and the interdivision cycle
Authors: Gmeiner J , Sarnow E , Milde K
Published: January 2020

We investigated the time periods of DNA replication, lateral cell wall extension, and septum formation within the cell cycle of Proteus mirabilis. Cells were cultivated under three different conditions, yielding interdivision times of approximately 55, 57, and 160 min, respectively. Synchrony was achieved by sucrose density gradient centrifugation. The time periods were estimated by division inhibition studies with cephalexin, mecillinam, and nalidixic acid. In addition, DNA replication was measured by thymidine incorporation, and murein biosynthesis was measured by incorporation of N-acetylglucosamine into sodium dodecyl sulfate-insoluble murein sacculi. At interdivision times of 55 to 57 min murein biosynthesis for reproduction of a unit cell lasted longer than the interdivision time itself, whereas DNA replication finished within 40 min. Surprisingly, inhibition of DNA replication by nalidixic acid did not inhibit the subsequent cell division but rather the one after that. Because P. mirabilis fails to express several reactions of the recA-dependent SOS functions known from Escherichia coli, the drug allowed us to determine which DNA replication period actually governed which cell division. Taken together, the results indicate that at an interdivision time of 55 to 57 min, the biosynthetic cell cycle of P. mirabilis lasts approximately 120 min. To achieve the observed interdivision time, it is necessary that two subsequent biosynthetic cell cycles be tightly interlocked. The implications of these findings for the regulation of the cell cycle are discussed.
Phenotypic properties of a unique rpoA mutation (phs) of Escherichia coli
Authors: Giffard P M , Rowland G C , Kroll R G , Stewart L M , Bakker E P , Booth I R
Published: January 2020

The phs mutation of Escherichia coli has been suggested to affect the Na+/H+ antiport (D. Zilberstein, E. Padan, and S. Schuldiner, FEBS Lett. 168:327-330, 1980). We have recently shown that the mutation affects the rpoA gene and thus affects transcription. The extent of the pleiotropy of the phs mutation was investigated. In addition to the previously reported growth defect on L-glutamate and melibiose, the mutation also affects at least two other metabolic systems. The transport and metabolism of arabinose is impaired and the transport of sulfate is reduced. The extent to which the effects of the phs mutation on metabolism are due to a defect in the Na+/H+ antiport was investigated, and no causal role for this transport system in the metabolic defects was found.
Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae
Authors: Tamai Y , Toh-e A , Oshima Y
Published: January 2020

A kinetic study of Pi transport with 32Pi revealed that Saccharomyces cerevisiae has two systems of Pi transport, one with a low Km value (8.2 microM) for external Pi and the other with a high Km value (770 microM). The low-Km system was derepressed by Pi starvation, and the activity was expressed under the control of a genetic system which regulates the repressible acid and alkaline phosphatases. The function of the PHO2 gene, which is essential for the derepression of repressible acid phosphatase but not for the derepression of repressible alkaline phosphatase, was also indispensable for the derepression of the low-Km system.
Fatty aldehyde dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanol metabolism
Authors: Singer M E , Finnerty W R
Published: January 2020

The role of fatty aldehyde dehydrogenases (FALDHs) in hexadecane and hexadecanol metabolism was studied in Acinetobacter sp. strain HO1-N. Two distinct FALDHs were demonstrated in Acinetobacter sp. strain HO1-N: a membrane-bound, NADP-dependent FALDH activity induced 5-, 15-, and 9-fold by growth on hexadecanol, dodecyl aldehyde, and hexadecane, respectively, and a constitutive, NAD-dependent, membrane-localized FALDH. The NADP-dependent FALDH exhibited apparent Km and Vmax values for decyl aldehyde of 5.0, 13.0, 18.0, and 18.3 microM and 537.0, 500.0, 25.0, and 38.0 nmol/min in hexadecane-, hexadecanol-, ethanol-, palmitate-grown cells, respectively. FALDH isozymes ald-a, ald-b, and ald-c were demonstrated by gel electrophoresis in extracts of hexadecane- and hexadecanol-grown cells. ald-a, ald-b, and ald-d were present in dodecyl aldehyde-grown cells, while palmitate-grown control cells contained ald-b and ald-d. Dodecyl aldehyde-negative mutants were isolated and grouped into two phenotypic classes based on growth: class 1 mutants were hexadecane and hexadecanol negative and class 2 mutants were hexadecane and hexadecanol positive. Specific activity of NADP-dependent FALDH in Ald21 (class 1 mutant) was 85% lower than that of wild-type FALDH, while the specific activity of Ald24 (class 2 mutant) was 55% greater than that of wild-type FALDH. Ald21R, a dodecyl aldehyde-positive revertant able to grow on hexadecane, hexadecanol, and dodecyl aldehyde, exhibited a 100% increase in the specific activity of the NADP-dependent FALDH. The oxidation of [3H]hexadecane byAld21 yielded the accumulation of 61% more fatty aldehyde than the wild type, while Ald24 accumulated 27% more fatty aldehyde, 95% more fatty alcohol, and 65% more wax ester than the wild type. This study provides genetic and physiological evidence for the role of fatty aldehyde as an essential metabolic intermediate and NADP-dependent FALDH as a key enzyme in the dissimilation of hexadecane, hexadecanol, and dodecyl aldehyde in Acinetobactor sp. strain HO1-N.
Alcohol dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecane and hexadecanol metabolism
Authors: Singer M E , Finnerty W R
Published: January 2020

Multiple alcohol dehydrogenases (ADH) were demonstrated in Acinetobacter sp. strain HO1-N. ADH-A and ADH-B were distinguished on the basis of electrophoretic mobility, pyridine nucleotide cofactor requirement, and substrate specificity. ADH-A is a soluble, NAD-linked, inducible ethanol dehydrogenase (EDH) exhibiting an apparent Km for ethanol of 512 microM and a Vmax of 138 nmol/min. An ethanol-negative mutant (Eth1) was isolated which contained 6.5% of wild-type EDH activity and was deficient in ADH-A. Eth1 exhibited normal growth on hexadecane and hexadecanol. A second ethanol-negative mutant (Eth3) was acetaldehyde dehydrogenase (ALDH) deficient, having 12.5% of wild-type ALDH activity. Eth3 had threefold-higher EDH activity than the wild-type strain. ALDH is a soluble, NAD-linked, ethanol-inducible enzyme which exhibited an apparent Km for acetaldehyde of 50 microM and a Vmax of 183 nmol/min. Eth3 exhibited normal growth on hexadecane, hexadecanol, and fatty aldehyde. ADH-B is a soluble, constitutive, NADP-linked ADH which was active with medium-chain-length alcohols. Hexadecanol dehydrogenase (HDH), a soluble and membrane-bound, NAD-linked ADH, was induced 5- to 11-fold by growth on hexadecane or hexadecanol. HDH exhibited apparent Kms for hexadecanol of 1.6 and 2.8 microM in crude extracts derived from hexadecane- and hexadecanol-grown cells, respectively. HDH was distinct from ADH-A and ADH-B, since HDH and ADH-A were not coinduced; Eth1 had wild-type levels of HDH; and HDH requires NAD, while ADH-B requires NADP. NAD- and NADP-independent HDH activity was not detected in the soluble or membrane fraction of extracts derived from hexadecane- or hexadecanol-grown cells. NAD-linked HDH appears to possess a functional role in hexadecane and hexadecanol dissimilation.
Characterization of a gamma-glutamyl kinase from Escherichia coli that confers proline overproduction and osmotic tolerance
Authors: Smith L T
Published: January 2020

Mutation(s) in the proBA operon of Escherichia coli confers proline overproduction and enhanced osmotic tolerance in enteric bacteria (L. N. Csonka, Mol. Gen. Genet. 182:82-86, 1981; M. J. Mahan and L. N. Csonka, J. Bacteriol. 156:1249-1262, 1983). A glutamate-dependent ATPase assay was developed and used to determine proB-encoded gamma-glutamyl kinase activity in the absence of glutamate-gamma-semialdehyde dehydrogenase. This assay indicated that the feedback insensitivity of mutant gamma-glutamyl kinase was independent of glutamate-gamma-semialdehyde dehydrogenase. However, the capacity of glutamate-gamma-semialdehyde dehydrogenase from the osmotolerant mutant to interact with the kinase was altered in thermal stability, suggesting that mutations in both proB and proA may be required for osmotolerance.