bluuebunny/crossref_metadata_2025 huggingface dataset:
Integration and excision of pMC7105 in Pseudomonas syringae pv. phaseolicola: involvement of repetitive sequences
Authors: Szabo L J , Mills D
Published: January 2020

The site for integration of pMC7105 into the chromosome of Pseudomonas syringae pv. phaseolicola has been mapped to a 2.6-kilobase-pair (kb) Bg/II-EcoRI fragment on this 150-kb indigenous plasmid. Selected excision plasmids resulting from imprecise excision of pMC7105 were used to identify one of the plasmid-chromosome juncture fragments and to characterize the mechanism of recombination from the chromosome. A 14.2-kb BamHI plasmid-chromosome juncture fragment has been identified in pEX8060 (234 kb), an excision plasmid which carries approximately 90 kb of chromosomal sequences to the left of the site of integration. This fragment contains a portion of the 2.6-kb Bg/II-EcoRI fragment as well as chromosomal sequences. Blot hybridization with a probe made from selected fragments of pMC7105 revealed three distinct repetitive sequences, RS-I, RS-II, and RS-III, on this plasmid. The 2.6-kb fragment, to which the site of integration maps, also contains RS-II. Five copies of RS-II are present in pMC7105, and more than 20 copies are present in the chromosome. Eight small excision plasmids were shown to result from recombination among fragments of pMC7105 that contain common repetitive sequences. The results indicate that integration and excision of pMC7105 occur through general recombination at homologous repetitive sequences.
Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus
Authors: Eisenberg E S , Mandel L J , Kaback H R , Miller M H
Published: January 2020

The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth. The electrical potential (delta psi) and the pH gradient across the cell membrane were determined by measuring the equilibrium distribution of [3H]tetraphenyl-phosphonium and [14C]acetylsalicylic acid, respectively. Incubation in the presence of the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) led to an increase in delta psi with no measurable effect on the pH gradient at external pHs ranging from 5.0 to 6.5, and the effect on delta psi was DCCD concentration dependent. In separate experiments, gentamicin uptake and killing were studied in the same cells under identical conditions. At pH 5.0 (delta psi = -140 mV), no gentamicin uptake occurred. In the presence of 40 and 100 microM DCCD, delta psi was increased to -162 and -184 mV, respectively, and gentamicin uptake was observed in a manner that was also dependent on the DCCD concentration. At pH 6.0 (delta psi = -164 mV), gentamicin uptake occurred in the absence of the carbodiimide but was enhanced in a concentration-dependent fashion by 40 and 100 microM DCCD (delta psi = -174 and -216 mV, respectively). In all cases increased gentamicin uptake was associated with an enhanced bactericidal effect. The results indicate that initiation of gentamicin uptake requires a threshold level of delta psi (-155 mV) and that above this level drug uptake is directly dependent on the magnitude of delta psi.
Genetic and biochemical characterization of the Escherichia coli K-12 fhuB mutation
Authors: Prody C A , Neilands J B
Published: January 2020

The fhuB region of Escherichia coli K-12 was subcloned from pLC4-44 into pP lac to obtain pCPN1. Deletions of this recombinant plasmid were made, and a 1.4-kilobase PstI fragment was further subcloned into the vector plasmid pKK177-2 to obtain pCPN12. The response of tonA and tonB strains and fhuB strains containing the plasmids to 15 hydroxamate siderophores were assayed. Results showed that tonA strains were deficient only in the utilization of ferrichrome-type siderophores, whereas fhuB strains were deficient in the utilization of all hydroxamate-type siderophores. The response of the plasmid-containing fhuB strains to the siderophores showed that the fhuB gene resides on a 1.4-kilobase PstI fragment of DNA. The proteins synthesized by these plasmids were examined in maxicells of strain CSR603. Plasmid pCPN1 expressed five proteins of molecular weights 78,000, 40,000, 30,000, 24,000, and 13,700. By the use of deletions of pCPN1, the approximate order of the genes for these proteins was determined. Plasmid pCPN12 expressed no proteins other than the beta-lactamase proteins in maxicell strain CSR603. However, in maxicell strain BN660, a lon mutant, it expressed a 20,000-molecular-weight protein. Inner membrane vesicles made from tonB and fhuB strains were able to transport [55Fe]ferrichrome and [55Fe]rhodotorulate at rates similar to those obtained in vesicles from tonB+ and fhuB+ strains.
Use of ureidopenicillins for selection of plasmid vector transformants in Pseudomonas aeruginosa and Pseudomonas putida
Authors: Day D L , Yasinow D , McDonough J , Shuster C W
Published: January 2020

Broad-host-range plasmids coding for beta-lactamase were successfully selected after transformation of Pseudomonas strains. Transformants of both Pseudomonas aeruginosa and Pseudomonas putida containing plasmid pRO1614 were isolated in media containing low concentrations of piperacillin. These strains were also susceptible to other ureidopenicillins. Similar selections of transformants with carbenicillin, ampicillin, or ticarcillin required high concentrations of antibiotics and yielded backgrounds of spontaneous resistant mutants.
Direction of flagellar rotation in bacterial cell envelopes
Authors: Ravid S , Eisenbach M
Published: January 2020

Cell envelopes with functional flagella, isolated from wild-type strains of Escherichia coli and Salmonella typhimurium by formation of spheroplasts with penicillin and subsequent osmotic lysis, demonstrate counterclockwise (CCW)-biased rotation when energized with an electron donor for respiration, DL-lactate. Since the direction of flagellar rotation in bacteria is central to the expression of chemotaxis, we studied the cause of this bias. Our main observations were: (i) spheroplasts acquired a clockwise (CW) bias if instead of being lysed they were further incubated with penicillin; (ii) repellents temporarily caused CW rotation of tethered bacteria and spheroplasts but not of their derived cell envelopes; (iii) deenergizing CW-rotating cheV bacteria by KCN or arsenate treatment caused CCW bias; (iv) cell envelopes isolated from CW-rotating cheC and cheV mutants retained the CW bias, unlike envelopes isolated from cheB and cheZ mutants, which upon cytoplasmic release lost this bias and acquired CCW bias; and (v) an inwardly directed, artificially induced proton current rotated tethered envelopes in CCW direction, but an outwardly directed current was unable to rotate the envelopes. It is concluded that (i) a cytoplasmic constituent is required for the expression of CW rotation (or repression of CCW rotation) in strains which are not defective in the switch; (ii) in the absence of this cytoplasmic constituent, the motor is not reversible in such strains, and it probably is mechanically constricted so as to permit CCW sense of rotation only; (iii) the requirement of CW rotation for ATP is not at the level of the motor or the switch but at one of the preceding functional steps of the chemotaxis machinery; (iv) the cheC and cheV gene products are associated with the cytoplasmic membrane; and (v) direct interaction between the switch-motor system and the repellent sensors is improbable.
Complete nucleotide sequence of mini-Rts1 and its copy mutant
Authors: Kamio Y , Tabuchi A , Itoh Y , Katagiri H , Terawaki Y
Published: January 2020

The nucleotide sequence of the whole mini-Rts1 genome consisting of 1,855 base pairs was determined. In addition to the cluster of five 24-base-pair direct repeats previously detected (Y. Kamio and Y. Terawaki, J. Bacteriol. 155:1185-1191, 1983), another cluster of three 21-base-pair direct repeats was found. All eight repeats were located in one direction and contained a consensus sequence TTCCCCPyPyPuPuCACACACC. Between the two clusters, a large open reading frame that could encode a 32,980-dalton polypeptide consisting of 288 amino acids was assigned. The molecular size predicted from the amino acid composition was close to the value of a unique mini-Rts1 product, the RepA protein, newly determined in minicells. A copy mutant of mini-Rts1 obtained in vitro by hydroxylamine treatment contained two-base-pair substitutions, one of which was located in the RepA protein coding region, and the other was close to the region where the oriC homologous sequences exist.
Terminal steps of bacteriochlorophyll a phytol formation in purple photosynthetic bacteria
Authors: Shioi Y , Sasa T
Published: January 2020

Four chemically different bacteriochlorophylls (Bchls) a esterified with geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol, and phytol have been detected by high-pressure liquid chromatography in cell extracts from Rhodopseudomonas sphaeroides and Chromatium vinosum. Bchl a containing phytol is the principal component, and the other three Bchls a comprise about 4% of the total Bchls a in stationary-phase cells of R. sphaeroides and C. vinosum. The high levels of the minor pigments occur in the beginning of Bchl a phytol formation, indicating that they are not degradation products, but intermediates of Bchl a phytol formation.
Metabolism of dihydrouracil in Rhodosporidium toruloides
Authors: Davis C H , Putnam M D , Thwaites W M
Published: January 2020

Previous studies, including those done with a similar species, have indicated that dihydrouracil is formed by the breakdown of uracil and is degraded into N-carbamyl-beta-alanine. (Fink et al., J. Biol. Chem. 201:349-355, 1953; S. R. Vilks and M. Y. Vitols, Mikrobiologiya 42:567-583, 1973; O. A. Milstein and M. L. Bekker, J. Bacteriol. 127:1-6, 1976). In the present work the conversion of dihydrouracil to uracil is studied in Rhodosporidium toruloides, and the growth characteristics of mutants that have lost the ability to use dihydrouracil as a source of nitrogen are examined. It is concluded that dihydrouracil must be converted to uracil before catabolism of the pyrimidine ring can take place.
Cloning of the Escherichia coli release factor 2 gene
Authors: Caskey C T , Forrester W C , Tate W , Ward C D
Published: January 2020

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.
Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway
Authors: Hughes E J , Bayly R C , Skurray R A
Published: January 2020

Alcaligenes eutrophus wild-type strain 345 metabolizes m- and p-toluate via a catechol meta-cleavage pathway. DNA analysis, curing studies, and transfer of this phenotype by conjugation and transformation showed that the degradative genes are encoded on a self-transmissible 85-kilobase plasmid, pRA1000. HindIII and XhoI restriction endonuclease analysis of pRA1000 showed it to be similar to the archetypal TOL plasmid, pWWO, differing in the case of HindIII only by the absence of fragments B and D present in pWWO. In strain 345, the presence of pRA1000 prevented the expression of chromosomally encoded enzymes required for the degradation of p-cresol, whereas these enzymes were expressed in strains cured of pRA1000. On the basis of studies with an R68.45-pRA1000 cointegrate plasmid, pRA1001, we conclude that the gene(s) responsible for the effect of p-cresol degradation resides within or near the m- and p-toluate degradative region on pRA1000.
Mapping of Rhodopseudomonas capsulata nif genes
Authors: Wall J D , Braddock K
Published: January 2020

The endogenous gene transfer system of Rhodopseudomonas capsulata was used to analyze mutations which block the ability to use molecular nitrogen as the sole nitrogen source (nif). With this fine-structure mapping tool, linkage of nif mutations could be reliably established if separated by 2,700 base pairs or less. Eleven independent mutations were analyzed, and five linkage groups were found. The overall chromosomal arrangement of these groups awaits conjugational or physical analysis. A candidate for the inactive subunit of R. capsulata Fe protein was located in gels at a position of about 38,000 molecular weight, 5,000 more than that of the presumed active subunit.
Intracellular localization of photosynthetic membrane growth initiation sites in Rhodopseudomonas sphaeroides
Authors: Inamine G S , Van Houten J , Niederman R A
Published: January 2020

Putative membrane invagination sites at which intracytoplasmic photosynthetic membrane growth is initiated in Rhodopseudomonas sphaeroides can be isolated in an upper pigmented fraction by rate-zone sedimentation. The intracellular localization of membranes present in the isolated fraction was investigated with the impermeant surface-labeling reagent pyridoxal 5'-phosphate, which has been shown to diffuse into the periplasmic space and to label proteins of both the peripheral cytoplasmic membrane and the mature intracytoplasmic membrane. A comparison of the extent of labeling at 25 and 0 degrees C was consistent with the possibility that membranes present in the upper pigmented fraction arise from sites near the cell periphery. Pronase digestion of the surface-labeled membranes suggested further that the purified upper fraction consisted largely of open membrane fragments and that the majority of the intracytoplasmic membrane is labeled by this procedure. The pigmented membrane growth initiation sites were separated partially from undifferentiated respiratory cytoplasmic membrane also present in the upper fraction.
Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death
Authors: Schoemaker J M , Gayda R C , Markovitz A
Published: January 2020

Mutations in sulA (sfiA) block the filamentation and death of capR (lon) mutants that occur after treatments that either damage DNA or inhibit DNA replication and thereby induce the SOS response. Previous sulA-lacZ gene fusion studies showed that sulA is transcriptionally regulated by the SOS response system (lexA/recA). SulA protein has been hypothesized to be additionally regulated proteolytically through the capR (lon) protease, i.e., in lon mutants lacking a functional ATP-dependent protease there would be more SulA protein. A hypothesized function for SulA protein is an inhibitor of cell septation. To investigate aspects of this model, we attempted to construct lon, lon sulA, and lon sulB strains containing multicopy plasmids specifying the sulA+ gene. Multicopy sulA+ plasmids could not be established in lon strains because more SulA protein accumulates than in a lon+ strain. When the sulA gene was mutated by a mini Mu transposon the plasmid could be established in the lon strains. In contrast, sulA+ plasmids could be established in lon+, lon sulA, and lon sulB strains. The sulA+ plasmids caused lon sulA and lon sulB cells to exist as filaments without SOS induction and to be sensitive to UV light and nitrofurantoin. Evidence implicated higher basal levels of SulA protein in these lon plasmid sulA+ strains as the cause of filamentation. We confirmed that the SulA protein is an 18-kilodalton polypeptide and demonstrated that it was induced by treatment with nalidixic acid. The SulA protein was rapidly degraded in a lon+ strain, but was comparatively more stable in vivo in a lon sulB mutant. Furthermore, the SulA protein was localized to the membrane by several techniques.
Gratuitous repression of avtA in Escherichia coli and Salmonella typhimurium
Authors: Whalen W A , Berg C M
Published: January 2020

avtA , which encodes transaminase C (alanine-valine transaminase), is repressed by excess-L-alanine or L-leucine, and also by limitation for any of a number of amino acids in Escherichia coli and Salmonella typhimurium. Amino acid limitation causes repression by promoting the accumulation of L-alanine or L-leucine or both. avtA is also repressed by L-alpha-aminobutyric acid and other nonprotein amino acids which are structurally similar to L-alanine. We hypothesize that L-alanine and L-alpha-aminobutyric acid, whose syntheses are catalyzed by transaminase C, are the true corepressors of avtA . Repression by structural analogs of the true corepressors is termed gratuitous repression.
Formaldehyde oxidation and methanogenesis
Authors: Escalante-Semerena J C , Wolfe R S
Published: January 2020

Formaldehyde oxidation by cell-free extracts of Methanobacterium thermoautotrophicum was shown to drive methanogenesis from CH3-S-coenzyme M or HCHO under a nonreductive atmosphere of N2. Under N2 when HCHO was the sole source of carbon and reducing equivalents in the reaction, it underwent oxidation and reduction events (disproportionation), the sum of the reactions being 3 HCHO + H2O----CH4 + 2 HCOO - + 2H+. This reaction predicts a CH4/HCHO ratio of 1/3, which is in agreement with the experimental finding of 1/2.9. In extracts of the mesophilic methanogen Methanococcus voltae and the extreme thermophile Methanococcus jannaschii , which exhibited formate dehydrogenase activity, the CH4/HCHO ratio was 1/2. NADPH stimulated methane formation from HCHO under N2. An unidentified, oxygen-labile cofactor, the formaldehyde activation factor, present in boiled-cell extract was discovered. Methanopterin , an oxygen-stable molecule, also substituted for boiled-cell extract.
Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe
Authors: Yanofsky C , Kelley R L , Horn V
Published: January 2020

Expression of the tryptophan operon of Escherichia coli is regulated over about a 500- to 600-fold range by the combined action of repression and attenuation. Repression regulates transcription initiation in response to variation in the intracellular concentration of tryptophan. Attenuation regulates transcription termination at a site in the leader region of the operon in response to changes in the extent of charging of tRNATrp. We measured repression independently of attenuation to ascertain whether these regulatory mechanisms were used differentially by the bacterium as the severity of tryptophan starvation was increased. We found that repression regulated transcription of the operon over the range from growth with excess tryptophan to growth under moderate tryptophan starvation. By contrast, attenuation (termination control) was not relaxed until tryptophan starvation was in the moderate-to-severe range. Thus, attenuation and repression were used to regulate transcription in response to different degrees of tryptophan deprivation. Consistent with this conclusion is the observation that when tryptophan starvation was sufficient to relieve repression 50 to 60%, 65% of the tRNATrp of the bacterium was charged. These findings provide a possible explanation for the existence of only two tryptophan codons in the coding region for the trp leader peptide of Enterobacteriaceae.
Construction and characterization of an Escherichia coli strain with a uncI mutation
Authors: Gay N J
Published: January 2020

A strain of Escherichia coli with a mutation in the promoter proximal gene ( uncI ) of the unc operon has been constructed by using a new gene replacement method. The mutation is a deletion of a defined sequence of 196 base pairs. It was constructed by homologous integration and segregation of a ColE1-derived recombinant plasmid containing the mutation, in a temperature-sensitive polA strain. The mutant strain is phenotypically unc+ but has a reduced growth yield compared to a normal sibling strain.
Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli
Authors: Kiino D R , Silhavy T J
Published: January 2020

The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity. A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion. This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion. prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE. The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins. When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold. Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.
Structure of an Escherichia coli tRNA operon containing linked genes for arginine, histidine, leucine, and proline tRNAs
Authors: Hsu L M , Klee H J , Zagorski J , Fournier M J
Published: January 2020

A plasmid containing a gene for the most abundant Escherichia coli leucine isoacceptor tRNA, tRNALeu1 (anticodon CAG) was isolated from the Clarke-Carbon bank of cloned E. coli DNA. The clone contains a 12.3-kilobase DNA insert which was mapped by F' DNA hybridization analysis to the region 82 to 89 min on the chromosome. The cloned tDNALeu corresponds to the minor of two chromosomal regions containing different amounts of DNA complementary to tRNALeuCAG . Sequencing of the tDNA region revealed it to contain a multimeric transcription unit consisting of four different tRNA genes. The genes are in the arrangement 5'-leader- tRNAArgCCG -57 base pairs- tRNAHisGUG -20 base pairs- tRNALeuCAG -42 base pairs- tRNAProUGG -3'. Coordinate expression of the component tRNAs in vivo and the absence of intercistronic promoters indicated that all four tDNAs reside in the same operon. The tDNA sequence is bounded by a promoter element showing good agreement with the procaryotic consensus sequence and a GC-rich stem-loop element that corresponds to a rho-independent terminator. The promoter region contains a GC-rich sequence that agrees with a suggested consensus stringency control element and two domains possessing dyad symmetry which flank the Pribnow box and include the putative stringency control region.
Physical and genetic analyses of the Inc-I alpha plasmid R64
Authors: Furuichi T , Komano T , Nisioka T
Published: January 2020

A 126-kilobase (kb) physical and genetic map of the Inc-I alpha plasmid R64 was constructed by using the restriction enzymes, BamHI, SalI, XhoI, HindIII, and EcoRI. The replication (Rep) and incompatability (Inc) functions of this plasmid were located in a 1.75-kb segment of an EcoRI fragment, E10 (3.3 kb). In addition, the genes determining growth inhibition of phage BF23 (Ibf), suppression of dnaG ( Sog ), resistance to tetracycline (Tetr), and resistance to streptomycin ( Strr ) were located on the 5.5-kb HindIII-XhoI fragment, the 8.1-kb EcoRI fragment (E5), the 4.6-kb HindIII fragment (H8), and the 4.1-kb HindIII fragment (H10), respectively. The map of R64 was compared with that of ColIb, which belongs to the Inc-I alpha group.
Isolation and characterization of the Legionella pneumophila outer membrane
Authors: Hindahl M S , Iglewski B H
Published: January 2020

A whole cell lysate of Legionella pneumophila was fractionated into five membrane fractions by sucrose gradient centrifugation. Membranes were characterized by enzymatic, chemical, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Two forms of cytoplasmic membrane (CM-1, CM-2), a band of intermediate density (IM), and two forms of outer membrane (OM-1, OM-2) were detected. The CM-1 fraction was the purest form of cytoplasmic membrane, and fraction CM-2 was primarily cytoplasmic membrane associated with small amounts of peptidoglycan. The IM, CM-1, and CM-2 fractions were enriched in peptidoglycan, and the amount of carbohydrate and 2-keto-3-deoxyoctonic acid was not appreciably greater in outer membrane relative to cytoplasmic membrane. Phosphatidylethanolamine and phosphatidylcholine were found to be the major phospholipids in the membrane fractions. The major outer membrane proteins had molecular sizes of 29,000 and 33,000 daltons and were both modified by heating. The 29,000-dalton protein was tightly associated with the peptidoglycan and was equally distributed in the IM, OM-1, and OM-2.
Regulatory changes in the formation of chromosomal dihydrofolate reductase causing resistance to trimethoprim
Authors: Flensburg J , Sköld O
Published: January 2020

High resistance to trimethoprim mediated by the several hundredfold overproduction of the drug target enzyme, dihyrofolate reductase, in a clinically isolated Escherichia coli strain, 1810, was cloned onto several vector plasmids and seemed to be comprised of a single dihydrofolate reductase gene, which by DNA-DNA hybridization and restriction enzyme digestion mapping was very similar to the corresponding gene of E. coli K-12. Determination of mRNA formation in the originally isolated resistant strain and strains with cloned trimethoprim resistance determinant demonstrated an about 15-fold increase in production of dihydrofolate reductase mRNA compared with that in E. coli K-12. This was explained by the occurrence of a promoter up mutation in the resistant isolate accompanied by changes in the restriction enzyme digestion pattern found by comparison with the corresponding pattern from E. coli K-12.
lon gene product of Escherichia coli is a heat-shock protein
Authors: Phillips T A , VanBogelen R A , Neidhardt F C
Published: January 2020

The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins. Purified Lon protease migrated with the prominent cellular protein H94.0 in E. coli K-12 strains. Peptide map patterns of Lon protease and H94.0 were identical. A mutant form of the protease had altered mobility during gel electrophoresis. An E. coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions. Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r. Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E. coli.
Extracellular proteins secreted by the basidiomycete Schizophyllum commune in response to carbon source
Authors: Willick G E , Morosoli R , Seligy V L , Yaguchi M , Desrochers M
Published: January 2020

The secretion of 1,4-beta-D-glucanases by the basidiomycete Schizophyllum commune in response to cellulose or cellobiose has been studied. The proteins were labeled with 35S, and the secretion of enzymes was measured by beta-glucosidase and carboxymethyl cellulase activities and by immunoprecipitation with specific antibodies. The antigen proteins used were a beta-glucosidase (Mr, 93,000), an avicelase (avicelase II; Mr, 64,000), and a carboxymethyl cellulose (carboxymethyl cellulase I; Mr 41,000). The beta-glucosidase was initially secreted as an Mr 110,000 form, which was followed later by lower-molecular-weight (88,000 to 93,000) forms. The avicelase II, which accounted for about 50% of the secreted labeled protein, had an Mr of 64,000. Secretion of the related avicelase I (Mr 61,000) followed later. The carboxymethyl cellulose I was secreted in two molecular weight forms, Mr 44,000 and 41,000. The evidence is consistent with the idea that three genes account for the secreted glucanase activities. Other species result from different glycosylation or proteolytic cleavage processing, which may occur during or after secretion. The beta-glucosidase secretion appears to be regulated differently than that of avicelase II or carboxymethyl cellulase I; the latter two were regulated coordinately under the conditions used in this work. No common immune determinants between the three antigens were observed.
Arrangement of pili in colonies of Neisseria gonorrhoeae
Authors: Todd W J , Wray G P , Hitchcock P J
Published: January 2020

The morphology and arrangement of pili in the P++ colony phenotype of Neisseria gonorrhoeae were examined by a variety of electron microscopic techniques. The apparent structure and organization of gonococcal pili varied depending upon the method of specimen preparation. Pili as thin, individual, unbranched structures were demonstrated by negative staining and in sections of epoxy-embedded specimens. Pili forming thick structures which branch, subdivide, and rejoin to form an irregular lattice were demonstrated in specimens processed by the critical-point drying method and by rapid freezing and low temperature sublimination. We propose that in gonococcal colonies of the P++ phenotype, pili exist as individual threadlike structures only on the bacterial surfaces; as the pili leave the bacterial surfaces, they form thick bundles which branch, subdivide, and rejoin to form a supporting framework interconnecting the colony members. This arrangement of pili is usually disrupted by the commonly used method of negative staining and cannot be clearly detected within epoxy-embedded specimens. These data are summarized in a model depicting the organization of pili in the P++ colony phenotype of N. gonorrhoeae.
Catabolism of carbohydrates and organic acids and expression of nitrogenase by azospirilla
Authors: Martinez-Drets G , Del Gallo M , Burpee C , Burris R H
Published: January 2020

Fructose, galactose, L-arabinose, gluconate, and several organic acids support rapid growth and N2 fixation of Azospirillum brasiliense ATCC 29145 (strain Sp7) as a sole source of carbon and energy. Growth of Azospirillum lipoferum ATCC 29707 (strain Sp59b) is also supported by glucose, mannose, mannitol, and alpha-ketoglutarate. Oxidation of fructose and gluconate by A. brasiliense Sp7 and of glucose, gluconate, and fructose by A. lipoferum Sp59b was achieved through inducible enzymatic mechanisms. Both strains exhibited all of the enzymes of the Embden-Meyerhof-Parnas pathway, and strain Sp59b also possesses all the enzymes of the Entner-Doudoroff pathway. Fluoride inhibited growth on fructose (strains Sp7 and Sp59b) or on glucose (strain Sp59b) but not on malate. There was no activity via the oxidative hexose monophosphate pathway in either strain. There was greater activity with 1-phosphofructokinase than with 6-phosphofructokinase in both strains. Strain Sp59b formed fructose-6-phosphate via hexokinase, an enzyme that is lacking in strain Sp7. A. brasiliense and A. lipoferum exhibited the enzymes both of the tricarboxylic acid cycle and of the glyoxylate shunt; iodoacetate, fluoropyruvate, and malonate were inhibitory. A. brasiliense Sp7 could not transport [14C]glucose and alpha-[14C]ketoglutarate into its cells.
Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii
Authors: Kow Y W , Burris R H
Published: January 2020

Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors.
Host range mutants of bacteriophage Ox2 can use two different outer membrane proteins of Escherichia coli K-12 as receptors
Authors: Morona R , Henning U
Published: January 2020

The Escherichia coli K-12 outer membrane protein OmpA functions as the receptor for bacteriophage Ox2. We isolated a host range mutant of this phage which was able to grow on an Ox2-resistant ompA mutant producing an altered OmpA protein. From this mutant, Ox2h5, a second-step host range mutant was recovered which formed turbid plaques on a strain completely lacking the OmpA protein. From one of these mutants, Ox2h10, a third-step host range mutant, Ox2h12, was isolated which formed clear plaques on a strain missing the OmpA protein. Ox2h10 and Ox2h12 apparently were able to use both outer membrane proteins OmpA and OmpC as receptors. Whereas there two proteins are very different with respect to primary structures and functions, the OmpC protein is very closely related to another outer membrane protein, OmpF, which was not recognized by Ox2h10 or Ox2h12. An examination of the OmpC amino acid sequence, in the regions where it differs from that of OmpF, revealed that one region shares considerable homology with a region of the OmpA protein which most likely is required for phage Ox2 receptor activity.
Voltage clamp effects on bacterial chemotaxis
Authors: Margolin Y , Eisenbach M
Published: January 2020

To examine whether or not sensory signaling in bacteria is by way of fluctuations in membrane potential, we studied the effect of clamping the potential on bacterial chemotaxis. The potential was clamped by valinomycin, a K+ -specific ionophore, in the presence of K+. Despite the clamped potential, sensory signaling did occur: both Escherichia coli and Bacillus subtilis cells were still excitable and adaptable under these conditions. It is concluded that signaling in the excitation and adaptation steps of chemotaxis is not by way of fluctuations in the membrane potential.
Regulation and coupling of argECBH mRNA and enzyme synthesis in cell extracts of Escherichia coli
Authors: Zidwick M J , Keller G , Rogers P
Published: January 2020

Cell extracts from Escherichia coli were used to study both transcription and coupled translation of the argECBH gene cluster. Argininosuccinase (the argH enzyme) and N-acetylornithinase (the argE enzyme) were synthesized for 90 to 120 min, and hybridizable argECBH mRNA was synthesized for 60 min after the addition of a lambda or phi 80 dargECBH DNA template. L-Arginine (2.5 mM) repressed synthesis by argR+ extracts of argECBH mRNA 2-, to 3-fold, argE enzyme 5- to 8-fold, and argH enzyme 20- to 60-fold. Repression was specific for L-arginine, and argR extracts were insensitive to added L-arginine. The argECBH mRNA made under conditions of restricted protein synthesis had reduced ability to function in the formation of the argE and argH enzymes and was found to be predominantly 6 to 8S in sucrose density gradients. When protein synthesis was allowed, the mRNA formed was functional, and large amounts of 14 to 23S argECBH mRNA appeared on sucrose gradients. An S-100 supernatant freed of ribosomes was capable of producing hybridizable arg mRNA, but significant functional message was only produced when ribosomes were present. When purified RNA polymerase was used, the formation of short 6 to 8S argECBH mRNA was dependent upon added rho protein. The data suggest that rho-dependent sites in the argECBH operon allow early termination of mRNA synthesis when transcription is not coupled to active enzyme synthesis.
Cloning and expression of a gene segment encoding the enzymatic moiety of Pseudomonas aeruginosa exotoxin A
Authors: Mozola M A , Wilson R B , Jordan E M , Draper R K , Clowes R C
Published: January 2020

Using the broad-host-range plasmid vector pRO1614, we cloned a segment of the gene from Pseudomonas aeruginosa PA103 encoding the enzymatically active part of the exotoxin A protein. Expression of the cloned gene segment has been achieved both in Escherichia coli and in a nontoxigenic P. aeruginosa host, as assayed by the production of exotoxin A-related antigen and by the ability of the gene product to ADP-ribosylate elongation factor 2. Western blot hybridization analysis revealed a series of polypeptides antigenically related to exotoxin A, the largest of which had a molecular weight of ca. 50,000.
Escherichia coli fimbriae recognizing sialyl galactosides
Authors: Korhonen T K , Väisänen-Rhen V , Rhen M , Pere A , Parkkinen J , Finne J
Published: January 2020

Fimbriae recognizing sialyl galactosides (S fimbriae) were purified from an Escherichia coli strain. The S fimbriae were morphologically identical to type 1 and P fimbriae of E. coli and showed a hemagglutination that was abolished when erythrocytes were treated with neuraminidase. Hemagglutination by the purified fimbriae was inhibited by orosomucoid but not by its desialylated derivative. Of the oligosaccharides tested, sialyl-(alpha 2-3)-lactose and sialyl-(alpha 2-3)-N-acetyllactosamine had the strongest inhibitory activities. It was concluded that S fimbriae have the strongest affinity for (alpha 2-3)-linked sialyl galactosides. In the enzyme-linked immunosorbent assay, the hyperimmune serum to the S fimbriae reacted strongly with the homologous antigen but not with type 1, P, or nonhemagglutinating KS71C fimbriae of E. coli. Analogously, the hyperimmune sera to the other E. coli fimbriae did not react with the purified S fimbriae. The immunoprecipitation assay showed that S fimbriae on different E. coli serotypes shared immunological cross-reactivity.
Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments
Authors: Jasin M , Schimmel P
Published: January 2020

Deletion of an essential gene in Escherichia coli was accomplished by transformation of linear DNA fragments that have a Kanr gene segment flanked by sequences homologous to closely spaced regions on the E. coli chromosome. Selection for a double crossover within homologous sequences can effectively delete an entire gene. Cell viability is maintained by provision of the essential gene on a plasmid with a temperature-sensitive replicon, resulting in cells which have a temperature-sensitive phenotype.
Influence of cyclic AMP on photosynthetic development in Rhodospirillum rubrum
Authors: Solaiman D , Uffen R L
Published: January 2020

During O2-free growth in the light and in medium with pyruvate, Rhodospirillum rubrum exhibits diauxic growth. The cells first fermented pyruvate and afterwards photometabolized. Exogenous cyclic AMP acted to prolong the lag period between fermentative and photosynthetic development, as well as to slow the light-dependent growth rate. This observation, and in situ changes in the cyclic AMP levels in cells undergoing biphasic growth, suggested that the cyclic nucleotide was involved in photosynthetic differentiation, perhaps by repressing the formation of the bacteriochlorophyll needed to support growth in the light.
Physiological function of hydrogen metabolism during growth of sulfidogenic bacteria on organic substrates
Authors: Lupton F S , Conrad R , Zeikus J G
Published: January 2020

Desulfovibrio vulgaris Madison and Thermodesulfobacterium commune contained functionally distinct hydrogenase activities, one which exchanged 3H2 into 3H2O and was inhibited by carbon monoxide and a second activity which produced H2 in the presence of CO. Cell suspensions of D. vulgaris used either lactate, pyruvate, or CO as the electron donor for H2 production in the absence of sulfate. Both sulfidogenic species produced and consumed hydrogen as a trace gas during growth on lactate or pyruvate as electron donors and on thiosulfate or sulfate as electron acceptors. Higher initial levels of hydrogen were detected during growth on lactate-sulfate than on pyruvate-sulfate. D. vulgaris but not T. commune also produced and then consumed CO during growth on organic electron donors and sulfate or thiosulfate. High partial pressures of exogenous H2 inhibited growth and substrate consumption when D. vulgaris was cultured on pyruvate alone but not when it was metabolizing pyruvate plus sulfate or lactate plus sulfate. The data are discussed in relation to supporting two different models for the physiological function of H2 metabolism during growth of sulfidogenic bacteria on organic electron donors plus sulfate. A trace H2 transformation model is proposed for control of redox processes during growth on either pyruvate or lactate plus sulfate, and an obligate H2 cycling model is proposed for chemiosmotic energy coupling during growth on CO plus sulfate.
Shrinkage of growing Escherichia coli cells by osmotic challenge
Authors: Koch A L
Published: January 2020

The immediate response of growing Escherichia coli to changing external osmotic pressure was studied with stopped-flow turbidimetric measurements with a narrow-beam spectrophotometer. It is shown theoretically that in such a photometer rod-shaped bacteria have an apparent absorbance which is proportional to the inverse of the surface area. The apparent optical density, corrected for effects of alteration of the index of refraction of the medium, increased continuously as the external osmotic pressure was raised. Because of the short time scale of the measurements, the turbidity increases could result either from shrinkage of the cells or from plasmolysis, or both, but not from growth or metabolic adaptation. With low concentrations of pentose such that the external osmotic pressure was not greater than that inside the cells, plasmolysis would not occur and, consequently, only shrinkage of the previously stretched sacculus remains to account for the observed optical effects. Taking the osmotic pressure of the growing cells as 5 atmospheres (506 kPa), the turbidity changes correspond to the murein fabric having been stretched 20% beyond its unstressed equilibrium area during growth under the conditions used.
Regulation of Salmonella typhimurium ilvYC genes
Authors: Blazey D L , Burns R O
Published: January 2020

The Salmonella typhimurium LT2 ilvYC genes were studied by fusion of each gene to the Escherichia coli K-12 galK gene. The expression of ilvY and ilvC could then be determined by measurement of the galK-encoded galactokinase enzyme. The promoter for ilvC, pC, was located by this technique to a 0.42-kilobase BglII-EcoRI fragment of the S. typhimurium ilvGEDAYC gene cluster. This sequence was completely sufficient for alpha-acetohydroxyacid-inducible galK expression. The ilvY gene was located within a 1.0-kilobase XhoI-SalI fragment. ilvY gene expression was constitutive with respect to ilv-specific control signals. The ilvY gene was transcribed in the same direction as the other two transcriptional units in the ilvGEDAYC gene cluster, ilvGEDA and ilvC. Transcription of the ilvC gene was completely dependent upon the activity of its own promoter, pC, and independent from transcription of the ilvY gene. The role of the intervening region between ilvY and ilvC in regulation of ilvC expression was explored.
Isolation and characterization of an alginase from mucoid strains of Pseudomonas aeruginosa
Authors: Linker A , Evans L R
Published: January 2020

Strains of Pseudomonas aeruginosa which produce an alginate-like slime polysaccharide were shown to also synthesize an intracellular enzyme which can degrade these polysaccharides and the seaweed alginic acids. The enzyme acts as an eliminase introducing delta 4,5 unsaturation into the uronic acid moiety. It appears to be a polymannuronide lyase which degrades the polysaccharides, depending on their uronic acid composition, to a series of oligosaccharides, the smallest of which is a disaccharide. L-Guluronic acid linkages are not split. The Pseudomonas alginase resembles other bacterial alginases and enzymes from molluscs but differs in some important properties, such as extent of degradation and linkage preference. Nonmucoid forms of the organism produce detectable but much lower amounts of enzyme.
Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli
Authors: Dean G E , Macnab R M , Stader J , Matsumura P , Burks C
Published: January 2020

The motA and motB gene products of Escherichia coli are integral membrane proteins necessary for flagellar rotation. We determined the DNA sequence of the region containing the motA gene and its promoter. Within this sequence, there is an open reading frame of 885 nucleotides, which with high probability (98% confidence level) meets criteria for a coding sequence. The 295-residue amino acid translation product had a molecular weight of 31,974, in good agreement with the value determined experimentally by gel electrophoresis. The amino acid sequence, which was quite hydrophobic, was subjected to a theoretical analysis designed to predict membrane-spanning alpha-helical segments of integral membrane proteins; four such hydrophobic helices were predicted by this treatment. Additional amphipathic helices may also be present. A remarkable feature of the sequence is the existence of two segments of high uncompensated charge density, one positive and the other negative. Possible organization of the protein in the membrane is discussed. Asymmetry in the amino acid composition of translated DNA sequences was used to distinguish between two possible initiation codons. The use of this method as a criterion for authentication of coding regions is described briefly in an Appendix.
Induction of superoxide dismutases in Escherichia coli by manganese and iron
Authors: Pugh S Y , DiGuiseppi J L , Fridovich I
Published: January 2020

Growth of Escherichia coli B in simple media enriched with Mn(II) resulted in the elevation of the manganese-containing superoxide dismutase, whereas growth in such medium enriched with iron caused increased content of the iron-containing superoxide dismutase. Enrichment of the medium with Co(II), Cu(II), Mo(VI), Zn(II), or Ni(II) had no effect. The inductions of superoxide dismutase by Mn(II) or by Fe(II) were dioxygen dependent, but these metals did not affect the CN- -resistant respiration of E. coli B and did not influence the increase in the CN- -resistant respiration caused by paraquat. Mn(II) and paraquat acted synergistically in elevating the superoxide dismutase content, and Mn(II) reduced the growth inhibition imposed by paraquat, E. coli grown in the complex 3% Trypticase soy broth (BBL Microbiology Systems)-0.5% yeast extract-0.2% glucose medium contained more superoxide dismutase than did cells grown in the simple media and were less responsive to enrichment of the medium with Mn(II) or Fe(II). Nevertheless, in the presence of paraquat, inductions of superoxide dismutase by these metals could be seen even in the Trypticase-yeast extract-glucose medium. On the basis of these observations we propose that the apo-superoxide dismutases may act as autogenous repressors and that Mn(II) and Fe(II) increase the cell content of the corresponding enzymes by speeding the conversion of the apo- to the holoenzymes.