bluuebunny/crossref_metadata_2025 huggingface dataset:
New pleiotropic alkaline phosphatase-negative mutants of Escherichia coli K-12
Authors: Heyde M , Portalier R
Published: January 2020

Escherichia coli K-12 mutants showing reduced alkaline phosphatase activity were isolated as 5-fluorouracil-plus-adenosine-resistant derivatives of a upp pho (either phoS or phoT) strain. One class of these mutants displayed a temperature-sensitive alkaline phosphatase-negative phenotype, a pleiotropic defect for growth on some substrates, an increased sensitivity to toxic compounds (e.g., EDTA, mitomycin, and chloramphenicol), and alterations in the expression of some membrane proteins. It phenotypically differed from previously described mutants. The mutation was located at min 8.5 close to the phoA gene and defines a new genetic locus we called napA (for negative alkaline phosphatase pleiotropic phenotype). As these mutants have lost the ability to grow on lactose and galactose, Lac+ and Gal+ revertants were isolated that simultaneously recovered the parental phenotype.
Molecular cloning of four tricarboxylic acid cyclic genes of Escherichia coli
Authors: Spencer M E , Guest J R
Published: January 2020

A fragment of DNA (3.1 kilobases [kb]) from a ColE1 Escherichia coli DNA hybrid plasmid containing the bacterial citrate synthase gene (gltA) was subcloned in both orientations into phage lambda vectors by in vitro recombination. The resulting phages were able to transduce gltA and, as prophages, complemented the lesion of a gltA mutant, showing that a functional gltA gene is contained in the 3.1-kb fragment. The segment of E. coli DNA cloned in these lambda gltA phages was extended in vivo by prophage integration and aberrant excision in the gltA region. Plaque-forming derivatives, carrying up to three additional tricarboxylic acid cycle genes, succinate dehydrogenase (sdh), 2-oxoglutarate dehydrogenase (sucA), and dihydrolipoamide succinyltransferase (sucB), were isolated and characterized by their transducing and complementing activities with corresponding mutants, and the order of the genes was confirmed as gltA-sdh-sucA-sucB. Physical maps of a variety of the transducing phages showed that the four tricarboxylic acid cycle genes are contained in a 12.8-kb segment of bacterial DNA. The four gene products, plus a possible succinate dehydrogenase small subunit, were identified in postinfection labeling studies, and the polarities of gene expression were defined as counterclockwise for gltA and clockwise for sdh, sucA, and sucB, relative to the E. coli linkage map.
High-density lipoprotein that supports Ureaplasma urealyticum growth
Authors: Sayed I A , Sweat F W
Published: January 2020

A high-density lipoprotein with growth-promoting activity for Ureaplasma urealyticum was purified in high yield from equine serum by ammonium sulfate fractionation and molecular filtration. Fractions enriched in growth-promoting activity represented 5% of the total serum protein, and 30 micrograms of the purified protein per ml gave an activity equivalent to that from 100 micrograms of whole serum per ml. The serum was totally replaced by purified lipoprotein when tested in a soy peptone-yeast dialysate or when added to a chemically defined synthetic medium. Polyacrylamide gel electrophoresis indicated that one major protein with growth-promoting activity is present. A total of 10 proteins were distinguished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 75% of the total contributed by two proteins with molecular weights of 160,000 and 170,000. A total of 90% of the lipoprotein was an alpha-protein with a mobility of 0.67 in two-dimensional immunoelectrophoresis (albumin = 1.0). The active component was further characterized as high-density lipoprotein by density ultracentrifugation. Two components with S = 6.4 and S = 15.8 were distinguished by velocity sedimentation. The lipid was removed from lipoprotein during its precipitation with acetone. The growth-promoting activity of delipidized protein was dependent upon the addition of exogenous cholesterol, and [14C]cholesterol was transferred to urea-plasmic cells in cultures containing the delipidized protein. A major portion of the [14C]cholesterol remained associated with the protein during filtration on Sepharose 4B columns.
Molecular characterization of three chloramphenicol acetyltransferases isolated from Haemophilus influenzae
Authors: Roberts M , Corney A , Shaw W V
Published: January 2020

Three plasmid-mediated chloramphenicol acetyltransferases isolated from different Haemophilus influenzae strains were purified and characterized. All three enzymes had properties in common with the gram-negative family of chloramphenicol acetyltransferase. The Haemophilus enzymes and the enteric type II enzyme were sensitive to 5,5'-dithiobis(2-nitrobenzoic acid), gave the same elution patterns from a highly substituted resin containing a bound chloramphenicol base, and had similar reactions to antisera. All four differed from each other in subunit molecular weight, enzyme activity, and partial protein digestion patterns. The data suggest that the three Haemophilus enzymes belong to the less common type II group and are related, but is not identical, to each other and to the enteric type II enzyme.
Biosynthesis of bacterial glycogen: purification and structural and immunological properties of Rhodopseudomonas sphaeroides ADPglucose synthetase
Authors: Yung S G , Preiss J
Published: January 2020

ADPglucose synthetase from the photosynthetic bacterium Rhodopseudomonas sphaeroides was purified to greater than 95% purity. The molecular weight of the R. sphaeroides enzyme, as determined by sucrose density gradient ultracentrifugation, was approximately 204,000. The subunit molecular weight of the enzyme based on sodium dodecyl sulfate-gel electrophoresis was 46,000. Although the amino acid composition of the enzyme was similar to that found for the enzymes from Escherichia coli, Salmonella typhimurium, and Rhodospirillum tenue, no apparent homology has been observed between the N-terminal or C-terminal amino acid sequences. Antisera prepared against the ADPglucose synthetase could inhibit the activities of the enzyme from other photosynthetic bacteria. Therefore, some sequence homology may exist within the internal portion of their peptide chain.
L-serine degradation in Escherichia coli K-12: a combination of L-serine, glycine, and leucine used as a source of carbon
Authors: Newman E B , Walker C
Published: January 2020

Escherichia coli K-12 strain CU1008 cannot use L-serine as the sole carbon source, but it could use L-serine as an auxiliary carbon source with glucose, L-alanine, or pyruvate and could derive energy from L-serine to support oxygen uptake. CU1008 grew with L-serine if it was also provided with glycine and leucine. These may act by increasing the available activity of L-serine deaminase; other explanations are also explored.
Nitrate reductase in Escherichia coli K-12: involvement of chlC, chlE, and chlG loci
Authors: Stewart V , MacGregor C H
Published: January 2020

We examined the properties of mutants of E. coli which are defective with respect to nitrate reductase activity. chlE::Mu cts and chlG::Mu cts mutants were all chlorate resistant, and the strains that we examined all synthesized nitrate reductase apoenzyme. We concluded that the chlE and chlG loci, like the chlA, chlB, and chlD loci, are involved in the synthesis of insertion of molybdenum cofactor. We identified at least four distinct phenotypic classes of chlC::Tn10 mutants, all of which were fully or partially sensitive to chlorate. Two of these classes may represent lesions in the structural genes for nitrate reductase subunits A and C. Two other classes may be altered in the regulation of the expression of nitrate reductase or other anaerobic enzymes. We propose the mnemonic nar for naming individual genes within the chlC locus.
Nucleotide sequence analysis of the complement resistance gene from plasmid R100
Authors: Ogata R T , Winters C , Levine R P
Published: January 2020

The multiple antibiotic resistance plasmid R100 renders Escherichia coli resistant to the bactericidal action of serum complement. We constructed a plasmid (pOW3) consisting of a 1,900-base-pair-long restriction fragment from R100 joined to a 2,900-base-pair-long fragment of pBR322 carrying ampicillin resistance. E. coli strains carrying pOW3 or R100 were up to 10,000-fold less sensitive to killing by serum complement than were plasmid-free bacteria or bacteria carrying pBR322. Nucleotide sequencing revealed that 875 of the 1,900 bases from R100 correspond exactly to part of the bacterial insertion sequence IS2. The remaining 1,075 bases contained only one sizeable open reading frame; it covered 729 base pairs (243 amino acids) and was preceded by nucleotide sequences characteristic of bacterial promoters and ribosome binding sites. The first 20 amino acids of the predicted protein showed features characteristic of a signal sequence. The remainder of the predicted protein showed an amino acid composition almost identical with that determined for the traT protein from the E. coli F factor. Southern blot analysis showed that the resistance gene from R100 does not hybridize to the serum resistance gene from ColV,I-K94 isolated by Binns et al.; we concluded that these genes are distinct.
Altered regulation of isoleucine-valine biosynthesis in a hisW mutant of Salmonella typhimurium
Authors: Davis L , Williams L S
Published: January 2020

Control of isoleucine-valine biosynthesis was examined in the cold-sensitive hisW3333 mutant strain of Salmonella typhimurium. During growth at the permissive temperature (37 degrees C), the isoleucine-valine (ilv) biosynthetic enzyme levels of the hisW mutant were two- to fourfold below these levels in an isogenic hisW+ strain. Upon a reduction in growth temperature to partially permissive (30 degrees C), the synthesis of these enzymes in the hisW mutant was further reduced. However, synthesis of the ilv enzymes was responsive to the repression signal(s) caused by the addition of excess amounts of isoleucine, valine, and leucine to the hisW mutants. Such a "super-repressed" phenotype as that observed in this hisW mutant is similar to that previously shown for the hisU1820 mutant, but was different from the regulatory response of the hisT1504 mutant strain. Moreover, by the use of growth-rate-limiting amounts of the branched-chain amino acids, it was shown that this hisW mutant generally did not increase the synthesis of the ilv enzymes as did the hisW+ strain. Overall, these results are in agreement with the hypothesis that the hisW mutant is less responsive to ilv specific attenuation control than is the hisW+ strain and suggest that this limited regulatory response is due to an alteration in the amount or structure of an element essential to attenuation control of the ilv operons.
Uptake hydrogenase activity and ATP formation in Rhizobium leguminosarum bacteroids
Authors: Nelson L M , Salminen S O
Published: January 2020

The role of uptake hydrogenase was studied in Rhizobium leguminosarum bacteroids from the nodules of Pisum sativum L. cv. Homesteader. Uptake hydrogenase activity, measured by the 3H2 uptake method, was dependent on O-consumption and was similar to H2 uptake measured by gas chromatography. Km for O2 of 0.0007 atm (0.0709 kPa) and a Km for H2 of 0.0074 atm (0.7498, kPa) were determined. H2 increased the rate of endogenous respiration by isolates with uptake hydrogenase (Hup+) but had no effect on an isolate lacking uptake hydrogenase (Hup-). A survey of 14 Hup+ isolates indicated a wide range of H2 uptake activities. Four of the isolates tested had activities similar to or higher than those found in two Hup+ Rhizobium japonicum strains. H2 uptake was strongly coupled to ATP formation in only 5 of the 14 isolates. H2 increased the optimal O2 level of C2H2 reduction by 0.01 atm and permitted enhanced C2H2 reduction at O2 levels above the optimum in both a coupled and an uncoupled isolate. At suboptimal O2 concentrations a small enhancement of C2H2 reduction by H2 was seen in two out of three isolates in which H2 oxidation was coupled to ATP formation. Thus, the main function of uptake hydrogenase in R. leguminosarum appears to be in the protection of nitrogenase from O2 damage.
Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1
Authors: Riise E , Stougaard P , Bindslev B , Nordström K , Molin S
Published: January 2020

Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.
Insertion of bacteriophage SP beta into the citF gene of Bacillus subtilis and specialized transduction of the ilvBC-leu genes
Authors: Mackey C J , Zahler S A
Published: January 2020

We isolated a strain of Bacillus subtilis in which the SP beta c2 prophage is inserted into the citF (succinate dehydrogenase) gene. Defective specialized transducing particles for the ilvBC-leu genes were isolated from phage-induced lysates of this lysogen. We isolated a group of phages that differ in the amount of genetic material they carry from this region. Also, we incorporated mutant ilv and leu alleles into the genomes of several transducing phages. Our phage collection enables us to identify the cistron of new ilv and leu mutations by complementation analysis. In this process we discovered a fourth leu cistron, leuD. Characterization of the phages confirmed the published gene order: ilvB-ilvC-leuA-leuC-leuB; leuD lies to the right of leuB.
Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli
Authors: Goebel W , Hedgpeth J
Published: January 2020

We cloned the DNA containing the Escherichia coli hemolysin determinant on a small, high-copy plasmid. We generated plasmids containing fragments of this DNA and used them either alone or in two-plasmid complementation systems to define the limits of the structural genes. This system also allowed us to partially characterize the function of each of the gene products in the production and transport of hemolysin. Taken with previously published data, the present experiments indicate the following. (i) At least three cistrons, hlyC, hlyA, and hlyB (these were previously designated cisC, etc. [Noegel et al., Mol. Gen. Genet. 175:343-350, 1979]), contain the specific genetic information for the hemolytic phenotype, (ii) hlyA encodes a 107,000-kilodalton protein, which seems to be an inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity require only the products of hlyA and hlyC. This activity was found in the periplasm; very little hemolysin activity was found in the cytoplasm, suggesting that the hlyC product is required for transport or activation of the hlyA product or both. (iv) Active hemolysin remains in the periplasm in the absence of hlyB function, hence the hlyB product seems to be necessary for the transport of hemolysin to the exterior of the cell. We further show that overproduction of the hlyA product is lethal, probably causing lysis of the cell.
A third L-proline permease in Salmonella typhimurium which functions in media of elevated osmotic strength
Authors: Csonka L N
Published: January 2020

Exogenous proline specifically stimulates the growth rate of enteric bacteria in media of inhibitory osmotic strength (J. H. B. Christian, Aust. J. Biol. Sci. 8:490-497, 1955). I observed that Salmonella typhimurium mutants which lack both of the previously known proline permeases (putP proP) are stimulated by proline in media of inhibitory osmolarity. I propose that there is a third proline permease which functions only in media of elevated osmolarity. This conclusion is based on the observations that, in media of elevated osmolarity, (i) the sensitivity of putP proP mutants to toxic proline analogs increases, (ii) proline requirements for maximal growth of proline auxotrophic putP proP mutants decreases, and (iii) the specific rate of incorporation of radioactive proline into protein of growing cells increases. I obtained a Tn10-induced mutation in a gene (proU) required for the functioning of the third proline permease and determined the map location to be at 59 map units of the chromosome, between srlA and tct, 66% linked to nalB in P22 transduction. My results suggest that the function of the third, osmotically stimulated permease might be to accumulate high intracellular proline levels during osmotic stress. Possible mechanisms by which proline might cause growth stimulation are discussed.
Ferrous- or cobalt ion-dependent D-(-)-tartrate dehydratase of pseudomonads: purification and properties
Authors: Rode H , Giffhorn F
Published: January 2020

D-(-)-Tartrate dehydratases [D-(-)-tartrate hydro-lyase, EC 4.2.1...] were isolated from two Pseudomonas strains. The molecular weights of the native enzymes were determined to be 72,000 and 7 8,000, respectively, and each enzyme was composed of two subunits of identical size. The dehydratases had no requirements for thiol compounds, were insensitive to oxygen, and required Fe2+ (0.1 mM) or Co2+ (0.5 mM) ions for optimal activity.
Regulation of hydrogenase formation is temperature sensitive and plasmid coded in Alcaligenes eutrophus
Authors: Friedrich C G , Friedrich B
Published: January 2020

Alcaligenes eutrophus grew well autotrophically with molecular hydrogen at 30 degrees C, but failed to grow at 37 degrees C (Hox Ts). At this temperature the strain grew well heterotrophically with a variety of organic compounds and with formate as an autotrophic substrate, restricting the thermolabile character to hydrogen metabolism. The soluble hydrogenase activity was stable at 37 degrees C. The catalytic properties of the wild-type enzyme were identical to those of a mutant able to grow lithoautotrophically at 37 degrees C (Hox Tr). Soluble hydrogenase was not rapidly degraded at elevated temperatures since the preformed enzyme remained stable for at least 5 h in resting cells or was diluted by growth, as shown in temperature shift experiments. Immunochemical studies revealed that the formation of the hydrogenase proteins was temperature sensitive. No cross-reactivity was detected above temperatures of 34 degrees C. The genetic information of Hox resides on a self-transmissible plasmid in A. eutrophus. Using Hox Tr mutants as donors of hydrogen-oxidizing ability resulted in Hox+ transconjugants which not only had recovered plasmid pHG1 and both hydrogenase activities but also were temperature resistant. This is evidence that the Hox Tr phenotype is coded by plasmid pHG1.
Role of Acetohydroxy Acid Isomeroreductase in Biosynthesis of Pantothenic Acid in
Salmonella typhimurium
Authors: Primerano Donald A. , Burns R. O.
Published: January 2020

Structural genes have been identified for all of the enzymes involved in the biosynthesis of pantothenic acid in
Salmonella typhimurium
and
Escherichia coli
K-12, with the exception of ketopantoic acid reductase, which catalyzes the conversion of α-ketopantoate to pantoate. The acetohydroxy acid isomeroreductase from
S. typhimurium
efficiently bound α-ketopantoate (

K
m

= 0.25 mM) and catalyzed its reduction at 1/20 the rate at which α-acetolactate was reduced. Since two enzymes could apparently participate in the synthesis of pantoate, a
S. typhimurium ilvC8
strain was mutagenized to derive strains completely blocked in the conversion of α-ketopantoate to pantoate. Several isolates were obtained that grew in isoleucine-valine medium supplemented with either pantoate or pantothenate, but not in the same medium supplemented with α-ketopantoate or β-alanine. The mutations that conferred pantoate auxotrophy (designated
panE
) to these isolates appeared to be clustered, but were not linked to
panB
or
panC
. All
panE
strains tested had greatly reduced levels of ketopantoic acid reductase (3 to 12% of the activity present in DU201). The capacity of the isomeroreductase to synthesize pantoate in vivo was assessed by determining the growth requirements of
ilvC
+
derivatives of
panE ilvC8
strains. These strains required either α-ketopantoate, pantoate, or pantothenate when the isomeroreductase was present at low levels; when the synthesis of isomeroreductase was induced,
panE ilvC
+
strains grew in unsupplemented medium. These phenotypes indicate that a high level of isomeroreductase is sufficient for the synthesis of pantoate.
panE ilvC
+
strains also grew in medium supplemented with lysine and methionine. This phenotype resembles that of some
S. typhimurium ilvG
mutants (e.g., DU501) which are partially blocked in the biosynthesis of coenzyme A and are limited for succinyl coenzyme A.
panE ilvC
+
strains which lack the acetohydroxy acid synthases required only methionine for growth (in the presence of leucine, isoleucine, and valine). This and other evidence suggested that the synthesis of pantoic acid by isomeroreductase was blocked by the α-acetohydroxy acids and that pantoic acid synthesis was enhanced in the absence of these intermediates, even when the isomeroreductase was at low levels.
panE ilvC
+
strains reverted to pantothenate independence. Several of these revertants were shown to have elevated isomeroreductase levels under noninduced and induced conditions; the suppressing mutation in each revertant was shown to be closely linked to
ilvC
by P22 transduction. This procedure presents a means for obtaining mutants with altered regulation of isomeroreductase.
Transport and processing of staphylococcal enterotoxin B
Authors: Tweten R K , Iandolo J J
Published: January 2020

A larger, membrane-bound form of staphylococcal enterotoxin B was shown by in vivo pulse-chase analysis to be the kinetic precursor to extracellular enterotoxin B. Processing of the enterotoxin B precursor molecules can apparently occur either cotranslationally or posttranslationally. Subcellular fractionation of cells revealed that all of the precursor toxin was associated with the membrane fraction. Once processed and released from the membrane, it was transiently associated with the cell wall before being released into the extracellular environment. The cell-wall-associated enterotoxin B was completely resistant to protease treatment and to extraction by high- or low-salt solutions at 0 to 2 degrees C, although it could be easily released from the cell by removal of the cell wall with lysostaphin. These data imply that newly formed enterotoxin B may be temporarily sequestered in specialized regions that require cell wall integrity before being released into the extracellular environment.
Carbonyl cyanide-m-chlorophenyl hydrazone-resistant Escherichia coli mutant that exhibits a temperature-sensitive unc phenotype
Authors: Ito M , Ohnishi Y , Itoh S , Nishimura M
Published: January 2020

Two spontaneous Escherichia coli mutant strains which are resistant to an oxidative phosphorylation uncoupler, carbonyl cyanide-m-chlorophenyl hydrazone, were isolated. Strain CM22 (ccr-2) was resistant to another uncoupler, pentachlorophenol, and to the inhibitors of proton-translocating ATPase, namely tributyltin and sodium azide. Carbonyl cyanide-m-chlorophenyl hydrazone or pentachlorophenol administered to cell suspensions of strain CM22 did not cause a pH change induced by H+ influx, and a similar result was obtained with everted particles. The respiratory rate of strain CM22 with succinate was twice that of wild-type strain KH434. When carbonyl cyanide-m-chlorophenyl hydrazone was administered, a stimulation of O2 uptake was observed in wild-type strain KH434 but not in the mutant strain CM22. Strain CM22 did not grow on succinate at 42 degrees C. Isolation of a true revertant at a frequency of 10(-8) demonstrated that the pleiotropic phenotype was induced by a single mutation. P1 transduction indicated that the mutant allele, ccr-2, was cotransduced with the ilv genes at a frequency of about 55%.
Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim
Authors: Rosano C L , Bunce S C , Hurwitz C
Published: January 2020

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.
Interactions of DNA replication factors in vivo as detected by introduction of suppressor alleles of dnaA into other temperature-sensitive dna mutants
Authors: Blinkowa A , Walker J R
Published: January 2020

Suppressor mutations located within dnaA can suppress the temperature sensitivity of a dnaZ polymerization mutant, indicating in vivo interaction of the products of these genes. The suppressor allele of dnaA [designated dnaA(SUZ, Cs)] could not be introduced, even at the permissive temperature, by transduction into temperature-sensitive (Ts) dnaC or dnaG recipients; it was transduced into dnaB(Ts) and dnaE(Ts) strains but at very low frequency. Recipient cells which were dnaA+ dnaE(Ts) were killed by the incoming dnaA(SUZ, Cs) allele, and it is presumed that combinations of dnaA(SUZ, Cs) with dnaB(Ts), dnaC(Ts), or dnaG(Ts) are lethal also. In one specific case, the lethality required the presence of three alleles: the incoming dnaA suppressor mutation, the resident dnaA+ gene, and the dnaB(Ts) gene. This was shown by the fact that dnaB(Ts) could readily be introduced into a dnaA(SUZ, Cs) dnaB+ recipient. That is, in the absence of dnaA+, the dnaA suppressor and dnaB(Ts) double mutant was stable. One model to explain these results proposes that the dnaA protein functions not only in initiation but also in the replication complex which contains multiple copies of dnaA and other replication factors.
Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus
Authors: Wicken A J , Ayres A , Campbell L K , Knox K W
Published: January 2020

Strains of Lactobacillus casei subsp. rhamnosus possessing two cell wall polysaccharides, a hexosamine-containing H-polysaccharide and a rhamnose-containing R-polysaccharide, were examined for the effect of growth conditions on the production of these two components. In strain NCTC 6375, R- and H-polysaccharides accounted for an estimated 44 and 20%, respectively, of the cell wall for organisms grown in batch culture with glucose as the carbohydrate source. Growth on fructose-containing media reduced the amount of R-polysaccharide by approximately 50% without affecting the amount of H-polysaccharide. Subculture of fructose-grown organisms in glucose restored the original proportions of the two polysaccharides. Galactose- and sucrose-grown cells behaved similarly to glucose-grown cells with respect to polysaccharide production, whereas growth in rhamnose or ribose showed values close to those for fructose-grown cells. Continuous culture of strain NCTC 6375 for more than 100 generations showed a gradual and irreversible reduction of the R-polysaccharide to less than 5% of the cell wall and an increase of the H-polysaccharide to 40% of the cell wall. Other type culture strains of L. casei subsp. rhamnosus, NCIB 7473 and ATCC 7469, behaved similarly in batch and continuous culture. In contrast, strains of L. casei subsp. rhamnosus isolated at the Institute of Dental Research showed phenotypic stability with respect to the relative proportions of R- and H-polysaccharides in both batch and continuous culture. Changes in polysaccharide composition of type culture strains were also mirrored in changes in the immunogenicity of the two components and resistance to the rate of enzymic lysis of whole organisms. For L. casei subsp. rhamnosus strain NCTC 10302 the R-polysaccharide is present entirely as capsular material. The amount of R-polysaccharide produced was also markedly dependent on the carbohydrate component of the medium in batch culture and both dilution rate and nature of the limiting carbohydrate in continuous culture, varying over a 10-fold range, whereas the cell wall H-polysaccharide remained constant.
Transductional analysis of the flagellar genes in Pseudomonas aeruginosa
Authors: Tsuda M , Iino T
Published: January 2020

Complementation in bacteriophage E79 tv-l-mediated transduction and the phenotypic properties of the flagellar genes in Pseudomonas aeruginosa PAO were investigated by using 195 flagellar mutants of this organism. A total of 15 fla. 1 mot, and 2 che cistrons were identified. At least 5 fla cistrons (fla V to flaZ) and one mot cistron resided in one region, and at least 10 fla cistrons (flaA to flaJ) and two che cistrons (cheA and cheB) resided in another. The flaC mutants exhibited cistron-specific leakiness on motility agar plates. The flaE cistron may be the structural gene for the component protein of the flagellar filament. The cheA mutations, which resulted in pleiotropic phenotypes for flagellar formation, motility, and taxis, belonged to the same complementation group as the flaF mutations; that is, we inferred that cheA and flaF are synonymous.
Clustered Genes for Galactose Metabolism from
Streptococcus mutans
Cloned in
Escherichia coli
Authors: Smorawinska Maryla , Hsu J. Charles , Hansen Jeffrey B. , Jagusztyn-Krynicka E. Katarzyna , Abiko Yoshimitsu , Curtiss Roy
Published: January 2020

DNA cloned into
Escherichia coli
from a serotype
c
strain of
Streptococcus mutans
allowed a
galKTE
mutant to utilize galactose for growth. However, the DNA does not appear to encode enzymes of the Leloir pathway used by
E. coli
, but rather appears to encode enzymes of the tagatose phosphate pathway.
Transmembrane electrical potential in Rickettsia prowazekii and its relationship to lysine transport
Authors: Zahorchak R J , Winkler H H
Published: January 2020

The transmembrane electrical potential (delta psi) generated by Rickettsia prowazekii metabolizing glutamic acid or ATP was determined by flow dialysis with the lipophilic cation tetraphenylphosphonium and with lysine. At pH 7.0, the rickettsiae generated a delta psi as measured by tetraphenylphosphonium distribution of 90 mV. Under similar conditions, cells of R.prowazekii concentrated lysine to a gradient indicating a delta psi of 90 mV. Energy-starved cells of R. prowazekii were able to utilize exogenously supplied ATP as well as glutamic acid to generate a delta psi of 110 mV at pH 8.0. Lysine transport was markedly affected by environmental pH, the optimum pH ranging from 8.0 to 8.5. delta psi as measured with tetraphenyl-phosphonium was similarly affected in this system, with values ranging from 70 mV at pH 6.0 to 100 mV at pH 8.0. Respiration rates were also affected by the external pH, with a maximum rate of 28 nmol of O2 consumed per min per mg of rickettsial protein occurring at pH 8.0. The pH effects were readily reversible and with a rapid onset.
Cloning and expression of the cloacin DF13/aerobactin receptor of Escherichia coli (ColV-K30)
Authors: Krone W J , Oudega B , Stegehuis F , de Graaf F K
Published: January 2020

A DNA fragment derived from the ColV-K30 plasmid and coding for both sensitivity to cloacin DF13 and Fe3+-aerobactin uptake was cloned into pBR322. The cloned fragment coded for two polypeptides with molecular masses of 74,000 (the cloacin DF13/aerobactin receptor protein) and 50,000 daltons, respectively. When grown with sufficient iron, cells harboring pFS8 (with this fragment) possessed about 10 times as many receptor protein molecules as compared with cells of Escherichia coli (ColV-K30). The synthesis of the receptor protein specified by pFS8, however, was independent of the availability of iron, in contrast to strains harboring the intact ColV-K30 plasmid. Aerobactin was taken up but not synthesized by cells harboring pFS8. No growth occurred when iron-starved cultures of these cells were incubated with Fe3+-aerobactin, suggesting that expression of other ColV-K30-encoded genes is necessary to remove the iron from the Fe3+-aerobactin complex.
Identification and control of synthesis of the dsdC activator protein
Authors: McFall E , Heincz M C
Published: January 2020

Operon fusions between the D-serine deaminase regulatory and structural genes and lacZ were constructed and used to examine the control of expression of the positive regulatory gene, dsdC. Merodiploid strains containing both dsdCp::Mu d (lac Apr) and dsdC+A+ produced only one-fourth as much beta-galactosidase as did the haploid dsdCp::Mu d (lac Apr) strains, indicating that the dsdC+ product repressed its own synthesis. The repression was reversed by D-serine. dsdC expression was not depressed in a cya background. The basal level of D-serine deaminase was the same in wild-type and dsdCp fusion strains. The dsdC gene product was identified in maxicell strains harboring dsd plasmids as a 34,000-dalton protein. dsdC gene transcription proceeded clockwise; thus, its promoter is adjacent to that of dsdA.
Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid
Authors: Klee H J , White F F , Iyer V N , Gordon M P , Nester E W
Published: January 2020

Forty-nine Tn3 and Tn5 transposition insertion mutations were introduced into the virulence region of the pTiA6NC plasmid of Agrobacterium tumefaciens. Five Tn5 transposition mutations from an earlier study (D. Garfinkel and E. Nester, J. Bacteriol. 144:732-743, 1980) were also mapped more accurately. These mutations defined five separate loci within the virulence region. Two Tn3 insertions into one of these loci, virA, result in a strain which is only weakly virulent; however, a Tn5 insertion into this locus eliminates virulence. One Tn5 insertion into another locus, virC, results in a strain which is weakly virulent. Two additional Tn5 insertions into this locus eliminate virulence. Insertions into the remaining three loci eliminate virulence entirely.
Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae
Authors: Maurer K H , Pfeiffer F , Zehender H , Mecke D
Published: January 2020

Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 microM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PL-insensitive enzyme suggest that the protein is an octamer, whereas the PL-sensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.
Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli
Authors: Black R A , Hobson A C , Adler J
Published: January 2020

We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system. We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations. Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response. Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system. Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars. Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars.
Overproduction of nitrogenase by nitrogen-limited cultures of Rhodopseudomonas palustris
Authors: Arp D J , Zumft W G
Published: January 2020

Rhodopseudomonas palustris cells grown on limiting nitrogen produced four- to eightfold higher nitrogenase specific activity relative to cells sparged with N2. The high activity of N-limited cells was the result of overproduction of the nitrogenase proteins. This was shown by four independent techniques: (i) titration of the Mo-Fe protein in cell-free extracts with Fe protein from Azotobacter vinelandii; (ii) direct detection of the subunits of Mo-Fe protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (iii) monitoring of the electron paramagnetic resonance spectrum of Mo-Fe protein in whole cells; and (iv) immunological assay of the Fe protein level with an antiserum against the homologous protein of Rhodospirillum rubrum. The derepressed level of nitrogenase found in N2-grown cells was not due to an increased turnover of nitrogenase. The apparent half-lives of nitrogenase in N2-grown and N-limited cells were 58 and 98 h, respectively, but were too long to account for the difference in enzyme level. Half-lives were determined by measuring nitrogenase after repression of de novo synthesis by ammonia and subsequent release of nitrogenase switch-off by methionine sulfoximine. Observations were extended to R. rubrum, Rhodopseudomonas capsulata, and Rhodomicrobium vannielii and indicated that overproduction of nitrogenase under nitrogen limitation is not an exceptional property of R. palustris, but rather a general property of phototrophic bacteria.
Developmental cell interactions of Myxococcus xanthus: analysis of mutants
Authors: LaRossa R , Kuner J , Hagen D , Manoil C , Kaiser D
Published: January 2020

A set of developmental mutants have been examined that behave as if defective in cellular interactions necessary for the formation of myxospores during fruiting body development. Sporulation is rescued in these mutants if they are mixed with wild-type cells. Complementation experiments with whole cells divide the mutants into four groups (A, B, C, and D). Mutants of group A appear to be less responsive to starvation, a condition that normally initiates development. Mutants of group D respond to starvation but fail to synthesize myxobacterial hemagglutinin, a protein normally synthesized midway in development. Mutants of groups B and C respond to starvation and synthesize hemagglutinin, but they can be distinguished genetically. Group C mutations all map in a single cluster near insertion omega 1519 of transposon Tn5, which is distant from group B mutations. Thus, each group represents a different defect in development. All of the mutants are induced to sporulate by glycerol. Therefore, we argue that sporulation during fruiting body development depends on several prior interactions between cells.
Cell wall-DNA association in Bacillus subtilis
Authors: Doyle R J , Koch A L , Carstens P H
Published: January 2020

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.
Thermoactivation of a periplasmic heat-stable enterotoxin of Escherichia coli
Authors: Guzmán-Verduzco L M , Fonseca R , Kupersztoch-Portnoy Y M
Published: January 2020

Strains of Escherichia coli that host a plasmid that codes for the heat-stable (ST) enterotoxin showed 160 times more extracellular enterotoxin than intracellular activity. However, when washed bacteria were sonicated and incubated at between 50 and 85 degrees C, an activity similar to that of the ST enterotoxin was detected. No such effect was present in strains lacking the plasmid, in a plasmid ST- mutant, or in chromosomal mutants that lack a cyclic AMP-linked positive regulatory system which previously were shown to yield an ST- phenotype. The thermoactivation was inhibited by iodoacetamide and N-ethylmaleimide; chloramphenicol did not affect the phenomenon. The heat-activated ST-like enterotoxin was localized in the periplasmic space. The results are discussed in relation to the export of the toxin from the periplasm to the outside of the cell.
Transport of hemolysin across the outer membrane of Escherichia coli requires two functions
Authors: Wagner W , Vogel M , Goebel W
Published: January 2020

Among a large collection of hemolysis-negative mutants obtained by mutagenesis of the Hly plasmid pHly152 with Tn5, we have isolated two classes of mutants which are defective in the transport of hemolysin across the outer membrane. The two cistrons (hylBa and hlyBb) which are affected in these mutants are located adjacent to each other on the hly determinant but are transcribed from different promoters. Recombinant plasmids were constructed which carry the two functions as combined or separated cistrons. These were shown to complement the two types of transport mutants. Studies on the compartmentation of hemolysin in these two classes of mutants indicate that most hemolysin (greater than 70%) in hlyBa mutants is located in the periplasmic space, whereas in hlyBb mutants a larger portion of hemolysin is associated with the outer membrane fraction. The phenotypic appearance of colonies from hlyBb mutants is that of beta-hemolytic Escherichia coli strains, indicating that a substantial portion of hemolysin has already reached the outside of the outer membrane without being released into the medium. Release was achieved readily when hlyBb mutants were complemented with a recombinant plasmid carrying hlyBb.
Products of phospholipid metabolism in Bacillus stearothermophilus
Authors: Card G L , Finn D J
Published: January 2020

The products of phospholipid turnover in Bacillus stearothermophilus were determined in cultures labeled to equilibrium and with short pulses of [32P]phosphate and [2-3H]glycerol. Label lost from the cellular lipid pool was recovered in three fractions: low-molecular-weight extracellular products, extracellular lipid, and lipoteichoic acid (LTA). The low-molecular-weight turnover products were released from the cells during the first 10 to 20 min of a 60-min chase period and appeared to be derived primarily from phosphatidylglycerol turnover. Phosphatidylethanolamine, which appeared to be synthesized in part from the phosphatidyl group of phosphatidylglycerol, was released from the cell but was not degraded. The major product of phospholipid turnover was LTA. Essentially all of the label lost from the lipid pool during the final 40 min of the chase period was recovered as extracellular LTA. The LTA appeared to be derived primarily from the turnover of cardiolipin and the phosphatidyl group of phosphatidylglycerol. Three types of LTA were isolated; an extracellular LTA was recovered from the culture medium, and two types of LTA were extracted from membrane preparations or whole-cell lysates by the hot phenol-water procedure. Cells contained 1.5 to 2.5 mg of cellular LTA per g of cells (dry weight), over 50% of which remained associated with the membrane when cells were fractionated. Over 75% of the 3H label incorporated into the cellular LTA pool during a 90-min labeling period was released from the cells during the first cell doubling after the chase. Label lost from the lipid pool was incorporated into cellular LTA which was then modified and released into the culture medium.
Adenine nucleotide levels in and nitrogen fixation by the cyanobacterium Anabaena sp. strain 7120
Authors: Privalle L S , Burris R H
Published: January 2020

Adenine nucleotide levels were determined in whole filaments of Anabaena sp. 7120 grown under different N2-fixing or non-N2-fixing conditions. These were compared with levels in isolated heterocysts, Rhodospirillum rubrum, and Azotobacter vinelandii. Adenine nucleotides in whole filaments of Anabaena sp. do not reflect the energetic expense of N2 fixation as they do in R. rubrum and A. vinelandii. However, adenine nucleotide levels in heterocysts were similar to the levels found in N2-fixing R. rubrum, i.e., an ATP:ADP ratio near 1 and an energy charge between 0.5 and 0.7. Nitrogenase activity was only 50% of optimal in permeabilized heterocysts at an exogenous ATP:ADP ratio of 3.33. Hydrogen, which increases acetylene reduction activity, also causes a transient increase (2 to 5 min) in the ATP:ADP ratio. Hydrogen has little effect on energy charge.