bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteins induced by aerobiosis in Escherichia coli
Authors: Smith M W , Neidhardt F C
Published: January 2020

The role of protein induction and repression in the adaptation of Escherichia coli to changes in the supply of oxygen and other electron acceptors is only poorly understood. We have studied the changes in cellular protein composition associated with this adaptation by measuring the levels of 170 individual polypeptides produced during aerobic or anaerobic growth of E. coli, with and without nitrate. Nineteen polypeptides had levels highest during aerobic growth. These proteins include the enzymes of the pyruvate dehydrogenase complex, several tricarboxylic acid cycle enzymes, superoxide dismutase, and tetrahydropteroyltriglutamate transmethylase. The other aerobiosis-induced proteins have not been identified. These polypeptides are major cellular proteins during aerobic growth and display several different patterns of regulation in response to medium composition. Induction ratios for oxygen ranged from 2.2 to 11.2, with one exceptional member, superoxide dismutase, increasing 71-fold with aeration. Most of the proteins were also induced by nitrate during anaerobic growth. The time course of induction after shifts in oxygen supply revealed similarities in response among proteins of related function or metabolic regulation class. These results are discussed in relation to previously reported information on the identified aerobiosis-induced proteins.
Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli
Authors: Michaelis S , Inouye H , Oliver D , Beckwith J
Published: January 2020

A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal sequence. This was done by selecting Lac+ mutants from a phoA-lacZ fusion strain that produces a membrane-bound hybrid protein and is unable to grow on lactose. Two such mutant derivatives were characterized. The mutations lie within the phoA portion of the fused gene and cause internalization of the hybrid protein. When the mutations were genetically recombined into an otherwise wild-type phoA gene, they interfered with export of alkaline phosphatase to the periplasm. The mutant alkaline phosphatase protein was found instead in the cytoplasm in precursor form. DNA sequence analysis demonstrated that both mutations lead to amino acid alterations in the signal sequence of alkaline phosphatase.
Computer-assisted chromosome mapping by protoplast fusion in Staphylococcus aureus
Authors: Stahl M L , Pattee P A
Published: January 2020

Protoplasts of genetically marked derivatives of Staphylococcus aureus NCTC 8325 were fused with polyethylene glycol and regenerated without selection. Recombinants possessing one specific resistance marker from each parent were selected from the regenerated population and scored for seven or eight unselected markers. The results of these 9- and 10-factor crosses were entered directly into a programmed microcomputer from prescored replica plates. The data then were condensed into an array of phenotypes, together with the frequency with which each occurred. Further analyses by computer included the calculation of coinheritance frequencies for all possible pairs of markers; after entering a proposed order for the markers being analyzed, the minimum number of crossover events required to generate each phenotypic class was calculated. The linkage relationships of markers, based on the protoplast fusion data, were entirely consistent with the linkage relationships of markers already known to exist within each of the three linkage groups previously defined by transformation. The fusion data defined an arrangement of the three linkage groups into a circular chromosome map and predicted the approximate location of four previously unmapped markers (tet-3490, fus-149, purC193::Tn551, and omega [Chr::Tn551]42) on this map.
Myxococcus xanthusDoes Not Respond Chemotactically to Moderate Concentration Gradients
Authors: Dworkin Martin , Eide David
Published: January 2020

Using a number of approaches we were unable to demonstrate a chemotactic response ofMyxococcus xanthusto a variety of defined and complex materials. These data in addition to a number of prima facie arguments considerably reduce the likelihood thatM. xanthuspossesses a mechanism for chemotactic behavior.
Cytoplasmic
l
-Asparaginase: Isolation of a Defective Strain and Mapping of
ansA
Authors: Casale Thomas Del , Sollitti Paul , Chesney Robert H.
Published: January 2020

An
Escherichia coli
mutant defective in the production of cytoplasmic
l
-asparaginase I has been isolated. The mutation (
ansA
) has been mapped between the
gap
and
pncA
loci at 39 min on the
E. coli
map.
Mapping of the gene for cytidine deaminase (cdd) in Escherichia coli K-12
Authors: Josephsen J , Hammer-Jespersen K , Hansen T D
Published: January 2020

The structural gene encoding cytidine deaminase (cdd) has been mapped in Escherichia coli K-12. It is located counterclockwise to ptsF between 46 and 47 min. The gene order in this region of the E. coli chromosome was found to be his-udk-gat-dld-cdd-ptsF.
Timing and other features of the action of the ts1 division initiation gene product of Bacillus subtilis
Authors: Callister H , McGinness T , Wake R G
Published: January 2020

The ts1 division initiation mutation of Bacillus subtilis 160 was transferred into a thymine-requiring strain of B. subtilis 168. Aspects of the role and timing of the action of the ts1 gene product in relation to septum formation were studied by comparing the behavior of this new strain with that of the isogenic wild type after outgrowth of germinated spores. The ts1 gene product was shown to be required for the asymmetric division which occurs in the absence of chromosome replication, in addition to normal division septation. The time interval between completion of the action of the ts1 gene product and initiation of the first central division septum was estimated to be less than 4 min at 34 degrees C, and it is possible that an active ts1 gene product is required until the commencement of septal growth. Recovery of septa after transfer of outgrown spores (filaments) from the nonpermissive to the permissive temperature was also examined. During recovery, septa formed at sites which were discrete fractional lengths of the filaments, with the first septum located at the most polar of these sites. The data have been interpreted in terms of the formation of potential division sites at the nonpermissive temperature and the preferred utilization, upon recovery, of the most recently formed site. Recovery of septa at the permissive temperature occurred in the absence of DNA synthesis but was blocked completely by inhibitors of RNA and protein synthesis. It is possible that the only protein synthesis required for recovery of septa is that of the ts1 gene product itself.
Transport and localization of protein S, a spore coat protein, during fruiting body formation by Myxococcus xanthus
Authors: Nelson D R , Zusman D R
Published: January 2020

Protein S, the most abundant soluble protein synthesized by Myxococcus xanthus FB during early fruiting body formation, accumulates in the soluble fraction of developing cells, reaching a peak at about 24 h; at late stages of fruiting body formation, protein S is found on the surface of spores (M. Inouye et al. Proc. Natl. Acad. Sci. U.S.A. 76:209-213, 1979). In this study, the transport and localization of protein S were investigated. Cells were fractionated to give osmotic shock, membrane, cytoplasmic, and spore fractions. The various fractions were then analyzed for protein S. Protein S was first detected in the cytoplasmic fraction at about 3 to 6 h of development. However, transport of protein S through the cytoplasmic membrane was not observed until 15 to 18 h of development. Thus, protein S is unusual among translocated proteins in that it accumulates as a soluble cytoplasmic protein before translocation. Biosynthesis of protein S ceased after 48 h; by 72 h, protein S was only found on the surface of spores. Pulse-chase experiments were performed to determine the transport kinetics of protein S. The results showed that in 24-h developing cells, the transport of protein S across the cytoplasmic membrane was rapid, occurring in less than 2 min. However, transport across the outer membrane was slow, requiring 10 to 15 min. Pulses of 15 s with [35S]methionine failed to reveal any short-lived precursor form in immunoprecipitated material separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing also failed to detect any precursor form of protein S. Thus, protein S appears to be translocated in the absence of a cleaved signal peptide.
pH dependence of the Coxiella burnetii glutamate transport system
Authors: Hackstadt T , Williams J C
Published: January 2020

The transport of glutamate, apparently a primary energy source for Coxiella burnetii, has been examined. C. burnetii is shown to possess a pH-dependent active transport system for L-glutamate with an apparent Kt of 61.1 microM and Vmax of 8.33 pmol/s per mg at pH 3.5. Both L-glutamine and L-asparagine competitively inhibited transport of glutamate, but D-glutamate, L-aspartate, L-glutamate-gamma-methyl ester, methionine sulfoximine, or alpha-ketoglutarate did not compete. This transport system is both temperature and energy dependent. Uptake of glutamate is highly sensitive to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol and carbonyl cyanide-m-chlorophenyl hydrazone that decrease the proton motive force across the cytoplasmic membrane. ATPase inhibitors such as dicyclohexylcarbodiimide or metabolic poisons such as KCN, NaF, or arsenite were much less effective as inhibitors of glutamate transport. Uptake of glutamate did not appear to be coupled to Na+ symport as in Escherichia coli since no monovalent cation requirement could be demonstrated. Instead, the Vmax of glutamate transport showed good correlation with the transmembrane pH gradient (delta pH). From these results, we propose that L-glutamate transport by C. burnetii is energized via a proton motive force.
Isolation and characterization Salmonella typhimurium mutants lacking a tripeptidase (peptidase T)
Authors: Strauch K L , Miller C G
Published: January 2020

Salmonella typhimurium contains an enzyme, peptidase T, that hydrolyzes a variety of tripeptides. Specificity studies with a peptidase activity stain after gel electrophoresis of crude cell extracts showed that peptidase T hydrolyzes tripeptides containing N-terminal methionine, leucine, or phenylalanine. Little or no activity could be detected against dipeptides, N-blocked or C-blocked tripeptides, and tetrapeptides. Analysis of reaction products by high-pressure liquid chromatography showed that peptidase T removes the N-terminal amino acid from tripeptides. Mutants lacking peptidase T were isolated by screening microcultures grown in the wells of plastic microtitration plates for hydrolysis of Met-Ala-Ser or Met-Gly-Gly. Mutations (pepT) that eliminate this enzyme were found to be phage P22 cotransducible with purB at approximately 25 map units on the S. typhimurium map. Comparison of the growth properties of mutant and wild-type strains suggests that peptidase T does not function in utilization of tripeptides to provide amino acids during growth.
rpoB mutation in Escherichia coli alters control of ribosome synthesis by guanosine tetraphosphate
Authors: Little R , Ryals J , Bremer H
Published: January 2020

An isogenic pair of relA+ and relA strains of Escherichia coli B/r with a mutation in the RNA polymerase subunit gene rpoB (Rifr) was isolated in which the relationship between guanosine tetraphosphate (ppGpp) concentration and stable RNA (rRNA, tRNA) gene activity was altered. The RNA polymerase in the rpoB strains was found to be about 20-fold more sensitive to ppGpp with respect to its stable RNA promoter activity than was the wild-type enzyme. The existence of such mutants is consistent with the idea that ppGpp interacts with the RNA polymerase enzyme and thereby alters its promoter selectivity, i.e., reduces its affinity for the stable RNA promoters. Under most conditions, the rpoB mutants had a reduced rate of growth and about a 10-fold-reduced intracellular concentration of ppGpp compared with the rpoB wild-type strains. The reduction of the level of ppGpp in the rpoB mutants during exponential growth was presumably a reflection of an indirect effect of the rpoB mutation on the control of relA-independent ppGpp metabolism.
Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis
Authors: Fujii M , Takagi M , Imanaka T , Aiba S
Published: January 2020

The structural gene for a thermostable protease from Bacillus stearothermophilus was cloned in plasmid pTB90. It is expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about 15-fold more protease (310 U/mg of cell dry weight) than did the wild-type strain of B. stearothermophilus. Some properties of the proteases that have been purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant difference was observed among the enzyme properties studied here despite the difference in host cells. We found that the protease, neutral in pH characteristics and with a molecular weight of 36,000, retained about 80% of its activity even after treatment of 65 degrees C for 30 min.
Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection
Authors: Matthysse A G
Published: January 2020

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.
Disulfide-Linked Oligomers of the Major Outer Membrane Protein of Chlamydiae
Authors: Newhall V Wilbert J. , Jones Robert B.
Published: January 2020

The major outer membrane protein of chlamydial elementary bodies was identified in dimer, trimer, and other multimeric forms. These natural multimers were stabilized by disulfide-mediated cross-linking. Such cross-linking of outer membrane proteins may play an important role in the formation and evolution of chlamydial cell wall structure.
Construction of a new yeast cloning vector containing autonomous replication sequences from Candida utilis
Authors: Hsu W H , Magee P T , Magee B B , Reddy C A
Published: January 2020

DNA sequences from the Candida utilis genome which, when cloned into a yeast integration plasmid (YIp5), confer on YIp5 the ability to replicate autonomously in Saccharomyces cerevisiae are described. Several recombinant plasmids which transform S. cerevisiae YNN27 to Ura3+ with an efficiency of 2 X 10(3) transformants per microgram of DNA were obtained. One of the recombinant plasmids, pHMR22 (6.6 kilobases) contains ars (autonomous replication sequence), which is homologous with two different DNA fragments of the C. utilis genome but has no detectable homology to total DNA from Candida albicans, Pachysolen tannophilus, or S. cerevisiae. Restriction and subcloning analyses of pHMR22 showed that Sau3A destroys the functions of cloned ars whereas there are no BamHI, PstI, SalI, HindIII, EcoRI, or PvuII sites in the region of ars which is required for its functional integrity. Thus, pHMR22 appears to be a useful vector for cloning desired genes in S. cerevisiae.
Genetics of Bacillus subtilis chemotaxis: isolation and mapping of mutations and cloning of chemotaxis genes
Authors: Ordal G W , Nettleton D O , Hoch J A
Published: January 2020

We isolated a collection of chemotaxis mutants and characterized them for chemotactic phenotype and genotype. The mutations of most of these mutants mapped in the region between pyrD and thyA. However, the mutation in the gene specifying the chemotactic methyltransferase mapped very close to aroF. From a bank of phages containing Bacillus subtilis DNA we identified two lambda charon4 phages that contained genes specifying chemotactic functions. The inserted DNAs were removed by digestion with restriction endonuclease EcoRI and were found to share a 4.0-kilobase (kb) fragment. One of these DNAs also contained a 7.7-kb fragment, and the other also contained a 10.9-kb fragment. We identified mutants that were complemented by each fragment. The fragments were each ligated into plasmid pFH7 and were incorporated into lysogenic SP beta c2 or a deletion mutant of SP beta c2 in order to form transducing phages. The mutants in the collection containing mutations that mapped in the region between pyrD and thyA were tested for complementation by transducing phages containing the 4.0-kb fragment, the 7.7-kb fragment, the 4.0-kb fragment plus the 7.7-kb fragment, and the 10.9-kb fragment. A total of 24 mutants were complemented by the 4.0-kb fragment, 7 were complemented by the 7.7-kb fragment, 9 were complemented by the 4.0-kb fragment plus the 7.7-kb fragment, 15 were complemented by the 10.9-kb fragment, and 25 were complemented by none of the fragments.
Genetic localization and characterization of a pKM101-coded endonuclease
Authors: Winans S C , Walker G C
Published: January 2020

The genetic and biochemical properties of an endonuclease mediated by the mutagenesis-enhancing plasmid pKM101 have been investigated. Taking advantage of the observation that this endonuclease, unlike host-coded DNases, is active in the presence of EDTA, we have developed an assay with nondenaturing acrylamide gels containing DNA. We have localized the plasmid DNA sufficient for nuclease expression to a 0.8-kilobase sequence that is near regions of DNA necessary for conjugal transfer, and we have determined that this gene is transcribed clockwise on the pKM101 map. The pKM101 gene mediating this activity codes for a 16,000-dalton protein, which is the same molecular mass as the nuclease monomer, leading us to conclude that this gene codes for the nuclease itself rather than for an activator of some host-coded enzyme. Cellular fractionation experiments have shown that the enzyme is localized in the periplasm. We have not been able to demonstrate any physiological role for the enzyme, but we have ruled out a direct involvement of the nuclease in any of the following known plasmid-associated phenotypes: (i) mutagenesis enhancement, (ii) conjugal transfer, (iii) entry exclusion, (iv) fertility inhibition of coresident P-group plasmids, (v) killing of Klebsiella pneumoniae used as conjugal recipients, and (vi) plasmid curing induced by treatment of cells with fluorodeoxyuridine. In addition, we have shown that the enzyme does not restrict bacteriophage or affect the ability of the host to utilize DNA as a source of thymine. Finally, we have shown that 11 of the 26 other plasmids tested also elaborated EDTA-resistant DNases.
Base excision repair in the thermophile Thermus sp. strain X-1
Authors: Warner H R
Published: January 2020

The thermophile Thermus sp. strain X-1, grown at 70 degrees C, contains uracil-DNA glycosylase and apurinic endonuclease activities, both of which are known to have roles in the repair of DNA damaged by heat. Both of these activities have temperature optima of about 70 degrees C. However, neither of these activities is present in quantities significantly greater than that found in Escherichia coli grown at 37 degrees C. Therefore, it appears that thermophilic organisms may not contain greatly elevated levels of the enzymes thought to be involved in the repair of DNA damaged by heat.
Cotransductional Mapping of the
trp-his
Region of
Bacillus megaterium
Authors: Callahan James P. , Crawford Irving P. , Hess Gerard F. , Vary Patricia S.
Published: January 2020

Eight
trp
mutations (four
trpE
, two
trpB
, one
trpC
, and one
trpD
) have been mapped in
Bacillus megaterium
QM B1551 and were found to be linked to two
hisH
mutations and unlinked to several other
his
mutations.
Ti plasmid and chromosomal ornithine catabolism genes of Agrobacterium tumefaciens C58
Authors: Schardl C L , Kado C I
Published: January 2020

The pTiC58 plasmid noc genes of Agrobacterium tumefaciens C58 code for nopaline oxidase (nocC), nopaline permease (nocP), the inducible periplasmic protein n1 (nocB), and a function(s) required for ornithine catabolism (nocA). In addition, strains C58 and Ach-5 of A. tumefaciens have chromosomal ornithine catabolism genes. The chromosomal orc gene codes for ornithine dehydrogenase. Strain C58 is normally orc, but orc+ mutants can be selected. We have characterized both chromosomal orc and pTiC58 nocA plasmid genes. Complementation of most chromosomal orc mutants by pTiC58 restored growth on both nopaline and L-ornithine but did not restore ornithine dehydrogenase activity. We conclude that ornithine is an intermediate of nopaline degradation and that the Ti plasmid and chromosome both code for ornithine-degradative enzymes. A model for nopaline catabolism is presented.
Purification and characterization of a bifunctional L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) from Rhodopseudomonas sphaeroides Y
Authors: Giffhorn F , Kuhn A
Published: January 2020

A bifunctional enzyme, L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and EC 1.1.1. . . , respectively), was discovered in cells of Rhodopseudomonas sphaeroides Y, which accounts for the ability of this organism to grow on L-(+)-malate. The enzyme was purified 110-fold to homogeneity with a yield of 51%. During the course of purification, including ion-exchange chromatography and preparative gel electrophoresis, both enzyme activities appeared to be in association. The ratio of their activities remained almost constant [1:10, L-(+)-tartrate dehydrogenase/D-(+)-malate dehydrogenase (decarboxylating)] throughout all steps of purification. Analysis by polyacrylamide gel electrophoresis revealed the presence of a single protein band, the position of which was coincident with both L-(+)-tartrate dehydrogenase and D-(+)-malate dehydrogenase (decarboxylating) activities. The apparent molecular weight of the enzyme was determined to be 158,000 by gel filtration and 162,000 by ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 38,500, indicating that the enzyme consisted of four subunits of identical size. The isoelectric point of the enzyme was between pH 5.0 and 5.2. The enzyme catalyzed the NAD-linked oxidation of L-(+)-tartrate as well as the oxidative decarboxylation of D-(+)-malate. For both reactions, the optimal pH was in a range from 8.4 to 9.0. The activation energy of the reaction (delta Ho) was 71.8 kJ/mol for L-(+)-tartrate and 54.6 kJ/mol for D-(+)-malate. NAD was required as a cosubstrate, and optimal activity depended on the presence of both Mn2+ and NH4+ ions. The reactions followed Michaelis-Menten kinetics, and the apparent Km values of the individual reactants were determined to be: L-(+)-tartrate, 2.3 X 10(-3) M; NAD, 2.8 X 10(-4) M; and Mn2+, 1.6 X 10(-5) M with respect to L-(+)-tartrate; and D-(+)-malate, 1.7 X 10(-4) M; NAD, 1.3 X 10(-4); and Mn2+, 1.6 X 10(-5) M with respect to D-(+)-malate. Of a variety of compounds tested, only meso-tartrate, oxaloacetate, and dihydroxyfumarate were effective inhibitors. meso-Tartrate and oxaloacetate caused competitive inhibition, whereas dihydroxyfumarate caused noncompetitive inhibition. The Ki values determined for the inhibitors were, in the above sequence, 1.0, 0.014, and 0.06 mM with respect to L-(+)-tartrate and 0.28, 0.012, and 0.027 mM with respect to D-(+)-malate.
Cell wall assembly during inhibition of DNA synthesis in Streptococcus faecium
Authors: Gibson C W , Daneo-Moore L , Higgins M L
Published: January 2020

Growth sites which are bounded by raised wall bands can be observed in electron micrographs of replicas of Streptococcus faecium. When mitomycin C was added to an exponential-phase culture doubling in mass every 64 min, DNA synthesis was inhibited, and eventually cell division stopped. The growth sites formed before and after inhibition of DNA synthesis enlarged until they contained about 0.25 micron3 of cell volume, at which point they ceased to increase in size. When these sites approached this 0.25-micron3 limit, new sites were initiated; this result had also been observed in untreated cells undergoing a large range of exponential-phase mass doubling times. Thus, regardless of whether chromosome replication is inhibited or uninhibited, sites have the same finite capacity to enlarge to about 0.25 micron3, and when this capacity is reached, new sites are initiated. Although initiation of new growth sites seems to be independent of normal chromosome replication, these results confirm previous studies showing that chromosome replication is necessary for the terminal events of growth site development which result in the division of a site into two separate poles. Two classes of models for the regulation of growth site initiation are discussed.
Lactoperoxidase and Iodo-Gen-catalyzed iodination labels inner and outer membrane proteins of Haemophilus influenzae
Authors: Loeb M R , Smith D H
Published: January 2020

Both inner and outer membrane proteins of Haemophilus influenzae type b were labeled by iodination procedures believed to be specific for exposed surface proteins only. It is suggested that this is due to specific properties of the outer membrane of H. influenzae and that use of these procedures with other gram-negative bacteria be evaluated carefully.
DNA sequence adjacent to flagellar genes and evolution of flagellar-phase variation
Authors: Szekely E , Simon M
Published: January 2020

A variety of factors, including phase variation, are involved in the regulation of flagellin gene expression in Salmonella sp. Flagellar-phase variation refers to the alternate expression of two different flagellin genes, H1 and H2. Site-specific inversion of a DNA segment adjacent to the H2 gene is responsible for switching expression. The segment includes the H2 promoter as well as the hin gene, which is required to mediate the inversion. Sequences in this region have homology with the corresponding sequences adjacent to the H1 flagellin gene in Salmonella sp. and the hag flagellin gene in Escherichia coli. The hin gene has also been shown to be homologous to the gin gene, which is found on bacteriophage Mu. To understand gene expression and the origin of these relationships, we have compared the DNA sequence adjacent to all three flagellin genes. The sequence data suggest a mechanism for the evolution of the hin-H2 locus.
Nucleotide sequence of the control regions for the glnA and glnL genes of Salmonella typhimurium
Authors: Hanau R , Koduri R K , Ho N , Brenchley J E
Published: January 2020

We have partially characterized a DNA fragment encoding glutamine synthetase in Salmonella typhimurium. Restriction mapping and RNA polymerase binding studies identified two regions within the fragment which exhibit promoter activity when fused to lacZ in pMC1403, a plasmid used to detect transcriptional and translational control signals. DNA sequence analysis revealed that one region encodes amino acids corresponding to the amino terminus of the glutamine synthetase protein. The second region codes for the amino acids corresponding to the carboxy terminus of glutamine synthetase followed by a 330-nucleotide sequence containing an ideal Pribnow heptamer and a possible translation initiation signal. The location of this region is analogous to the position of the beginning of the glnL gene identified in Escherichia coli, and it is likely that the Pribnow heptamer is the RNA polymerase binding site for the glnL gene.
FAD requirement for the reduction of coenzyme F420 by formate dehydrogenase from Methanobacterium formicicum
Authors: Schauer N L , Ferry J G
Published: January 2020

The partial purification of the formate dehydrogenase from cell-free extracts of Methanobacterium formicicum decreased the rate of coenzyme F420 reduction 175-fold relative to the rate of methyl viologen reduction. FAD, isolated from this organism, reactivated the coenzyme F420-dependent activity of purified formate dehydrogenase and restored the activity ratio (coenzyme F420/methyl viologen) to near that in cell-free extracts. Neither flavin mononucleotide nor FADH2 replaced FAD. The reduced form of FAD inhibited the reactivation of coenzyme F420-dependent formate dehydrogenase activity by the oxidized form. The results suggest that native formate dehydrogenase from Methanobacterium formicicum contains noncovalently bound FAD that is required for coenzyme F420-dependent activity.
Mutations in multicopy Tn10 tet plasmids that confer resistance to inhibitory effects of inducers of tet gene expression
Authors: Moyed H S , Bertrand K P
Published: January 2020

Escherichia coli K-12 strains that carry the Tn10 tetracycline resistance determinant (tet) on multicopy plasmids are hypersensitive to 5a,6-anhydrotetracycline and heated chlortetracycline, two tetracycline derivatives that are relatively more effective as inducers of tet gene expression than as inhibitors of bacterial growth. Twenty spontaneous mutations that confer resistance to anhydrotetracycline (Atr) and resistance to heated chlortetracycline (Ctr) were isolated and characterized. All of these Atr mutations are located in the Tn10 tet region; the majority (18 of 20) have no effect on tetR repressor function. Atr mutations can increase, reduce, or eliminate the phenotypic expression of plasmid tetracycline resistance (Tcr). IS insertions that result in an Atr Tcs phenotype are clustered in a 150-base-pair promoter-proximal region of the tetA resistance gene. Some Atr mutations reduce expression of the tetA gene by altering either the tetR repressor or the tetA promoter. In addition, it appears that E. coli cannot tolerate constitutive expression of the wild-type tetA gene from a multicopy plasmid containing a tetR deletion. These observations support the proposal that high level expression of the 36-kilodalton tetA gene product inhibits the growth of E. coli. We speculate that this inhibition is related to the interaction of the tetA gene product with the cytoplasmic membrane.
Biosynthesis and degradation of storage protein in spores of the fungus Botryodiplodia theobromae
Authors: Petersen G R , Dahlberg K R , Van Etten J L
Published: January 2020

Muiridin, a spore-specific protein of the fungus Botryodiplodia theobromae, comprises about 25% of the mature pycnidiospore protein. It has an apparent molecular weight of 16,000 to 17,000 and is rich in glutamine, asparagine, and arginine. Muiridin is synthesized in developing spores via a precursor with an apparent molecular weight of 24,000. Two other polypeptides present in young developing spores with apparent molecular weights of 18,000 and 15,000 are immunologically related to muiridin. We propose a pathway for muiridin synthesis. Muiridin is actively degraded during the germination of spores from 30-day-old cultures. This degradation is independent of exogenous amino acids in the germination medium. In contrast, glutamine and, to a lesser extent, asparagine partially inhibit the degradation of muiridin during germination of spores from 7-day-old cultures.
The penicillinase of Bacillus licheniformis is an outer membrane protein in Escherichia coli
Authors: Sarvas M O , Palva I A
Published: January 2020

The cloned gene coding for Bacillus licheniformis penicillinase (penP) was introduced into Escherichia coli in a heat-inducible lambda Qam vector. After induction, significant amounts of penicillinase were synthesized in the new host. The cellular location of the penicillinase was found to be almost exclusively the outer membrane fraction of E. coli, and virtually no soluble penicillinase was found. According to sodium dodecyl sulfate-gel electrophoresis, the size of the penicillinase from E. coli was identical to that of the membrane-bound form of the B. licheniformis penicillinase. Gel filtration in the presence of Triton X-100 suggested that the penicillinase from E. coli had amphiphilic properties, as does B. licheniformis membrane penicillinase. These results show that the export of the penicillinase to the outer membrane of E. coli involves the cleavage of the signal peptide from the prepenicillinase, giving an outer membrane component indistinguishable from the membrane penicillinase of B. licheniformis.
Construction of a mobilizable Yersinia enterocolitica virulence plasmid
Authors: Heesemann J , Laufs R
Published: January 2020

Virulence of Yersinia enterocolitica O:8 is associated with pO:8, a 42-megadalton plasmid. We constructed a mobilizable pO:8 derivative by successive in vitro and in vivo genetic manipulations. The in vitro constructed hybrid molecule pRK290B8-5 consisting of the mobilizable vector pRK290B and a 2.9-megadalton BamHI fragment of pO:8 was conjugally transferred to a Y. enterocolitica strain of serotype O:8 which harbored the virulence plasmid pO:8. From Yersinia transconjugants, a cointegrate was isolated which apparently formed by homologous recombination between the two component plasmids. The cointegrate was mobilized into plasmidless Y. enterocolitica strains of different serotypes. The transconjugants of serotype O:8 were found to express all four plasmid-associated phenotypes: (i) mouse lethality (Ml), (ii) conjunctivitis provocation in the guinea pig eye (Con), (iii) calcium requirement for growth at 37 degrees C (Mox), and (iv) agglutinogens (Ag8). The transconjugants of serotype O:3 expressed the phenotypes Con, Mox, and Ag8 but were nonlethal for mice (Ml-). The transconjugants of serotype O:5 remained avirulent for mice (Ml-) and for the guinea pig eye (Con-) but expressed the phenotypes Mox and Ag8. These data show that the virulence plasmid is probably not functionally interchangeable within different serotypes of Y. enterocolitica.
Light-mediated regulation of phospholipid synthesis in Rhodopseudomonas sphaeroides
Authors: Campbell T B , Lueking D R
Published: January 2020

The relationship between the culture levels of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and the rates of synthesis and accumulation of cellular phospholipids was examined in cultures of Rhodopseudomonas sphaeroides that had been subjected to immediate decreases in incident light intensity. After a high-to-low light transition of high-light-adapted cells, an immediate inhibition of total cellular phospholipid production occurred coincident with a rapid accumulation of culture ppGpp. The inhibition of phospholipid accumulation occurred at the level of phospholipid synthesis rather than turnover, and both the extent of ppGpp accumulation and the degree of inhibition of phospholipid synthesis were directly dependent upon the magnitude of the light transition. Maximum inhibition (greater than 90%) of the rate of cellular phospholipid synthesis occurred after transitions from 5,350 to 268 1x and lower, including transitions to the dark, with comparable inhibition being exerted upon the rates of synthesis of individual species of phospholipids. Reinitiation of culture phospholipid accumulation in cultures shifted from 5,350 to 1,070 1x and lower occurred 65 to 70 min subsequent to the downshift in light intensity, apparently irrespective of the culture level of ppGpp.
In Rhizobium japonicum the nitrogenase genes nifH and nifDK are separated
Authors: Kaluza K , Fuhrmann M , Hahn M , Regensburger B , Hennecke H
Published: January 2020

In contrast to Klebsiella pneumoniae or fast-growing Rhizobium species, such as R. meliloti, where the nitrogenase structural genes are clustered in one operon (nifHDK), in slow-growing Rhizobium japonicum 110, nifH and nifDK are on separate operons.
Nif- Hup- mutants of Rhizobium japonicum
Authors: Moshiri F , Stults L , Novak P , Maier R J
Published: January 2020

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.
Isolation of a recombination-deficient mutant of Streptococcus lactis ML3
Authors: Anderson D G , McKay L L
Published: January 2020

A recombination-deficient mutant of Streptococcus lactis ML3 designated MMS36 was isolated on the basis of its sensitivity to methyl methanesulfonate. This mutant also displayed sensitivity to UV irradiation. The inability of MMS36 to mediate homologous recombination was demonstrated by transduction of plasmid-linked lactose fermenting ability but not chromosomally mediated streptomycin resistance.
Outer membrane porin proteins F, P, and D1 of Pseudomonas aeruginosa and PhoE of Escherichia coli: chemical cross-linking to reveal native oligomers
Authors: Angus B L , Hancock R E
Published: January 2020

Native oligomers of three Pseudomonas aeruginosa outer membrane porin proteins and one Escherichia coli porin were demonstrated by using a chemical cross-linking technique. P. aeruginosa protein F, the major constitutive outer membrane porin, was cross-linked to dimers in outer membrane and whole-cell cross-linking experiments. Purified preparations of P. aeruginosa proteins F, D1 (glucose induced), and P (phosphate starvation induced) and E. coli protein PhoE (Ic) were also cross-linked to reveal dimers and trimers upon two-dimensional sodium dodecyl sulfate-polyacrylamide electrophoretic analysis. Cross-linking of protein F was abolished by pretreatment of the protein with sodium dodecyl sulfate, indicating that the cross-linked products were due to native associations in the outer membrane.
Two distinct classes of polyuronide from the cell walls of a dimorphic fungus, Mucor rouxii
Authors: Dow J M , Darnall D W , Villa V D
Published: January 2020

Polyuronides were extracted from purified yeast and mycelial walls of Mucor rouxii by sequential treatments with lithium chloride and potassium hydroxide and were fractionated by ion-exchange chromatography on DEAE-Sephadex. Two polymers (I and II) of different acidity were found in both wall types. Polymer I contained D-glucuronic acid, L-fucose, D-mannose, and much smaller amounts of D-galactose. Yeast and mycelial polymer I had similar uronic acid contents but differed in their neutral sugar compositions and molecular weights. Polymer II from both cell types contained largely D-glucuronic acid and had similar molecular weights. On partial acid hydrolysis, both polymers I and II gave rise to insoluble glucuronans which appeared to be homopolymeric. One-third of the total uronosyl residues of polymer I, and almost all of the uronosyl residues of polymer II, were present in homopolymeric segments. However, homopolymers derived from polymers I and II may not be identical.
Genetic, molecular, and functional analysis of Streptococcus faecalis R plasmid pJH1
Authors: Banai M , LeBlanc D J
Published: January 2020

Streptococcus faecalis JH1 contains two conjugative plasmids, pJH1, an R plasmid that codes for resistance to kanamycin, streptomycin, erythromycin, and tetracycline, and pJH2, a hemolysin-bacteriocin plasmid. Strain JH1 was used as an antibiotic resistance donor in conjugation experiments with two plasmid-free S. faecalis recipient strains, JH2-2 and OG1-RF1. Plasmid pJH1 was purified from one transconjugant, DL77, and subjected to restriction endonuclease analyses. Five restriction enzymes, EcoRI, XbaI, BamHI, SalI, and XhoI, yielding 10, 9, 3, 2, and 2 fragments, respectively, were used to determine the size (80.7 kilobases) of pJH1 and to construct a restriction endonuclease map of the plasmid. Twenty-eight percent of the antibiotic-resistant transconjugants examined expressed only part of the resistance pattern (Kmr Smr Emr Tcr) associated with pJH1, that is, they were resistant to kanamycin, streptomycin, and erythromycin; to erythromycin and tetracycline; or to erythromycin or to tetracycline only. Most of these strains also produced hemolysin and bacteriocin, and several contained a hybrid plasmid consisting of pJH2 and specific segments of pJH1 DNA. Several of these hybrid plasmids, as well as a deletion derivative of pJH1 that coded for resistance to tetracycline but not to kanamycin, streptomycin, or erythromycin, were purified and used to confirm the arrangement of restriction endonuclease fragments on the pJH1 map and to locate the resistance determinants on this map.
Single-carbon catabolism in acetogens: analysis of carbon flow in Acetobacterium woodii and Butyribacterium methylotrophicum by fermentation and 13C nuclear magnetic resonance measurement
Authors: Kerby R , Niemczura W , Zeikus J G
Published: January 2020

The catabolism of methanol, formate, or carbon monoxide to acetate or butyrate or both was examined in two acetogenic bacteria. Butyribacterium methylotrophicum simultaneously transformed methanol and formate mainly to butyrate with concomitant H2 and CO2 production and consumption. In contrast, methanol plus CO was primarily converted to acetate, and only slight amounts of CO2 were produced. In vivo 13C nuclear magnetic resonance analysis of [13C]methanol transformation by B. methylotrophicum indicated that methanol was predominantly incorporated into the methyl of acetate. 13CO2 was produced and then consumed, and butyrate was formed from the condensation of two acetate precursors. The analysis of the position of acetate labeled by a given 13C single-carbon substrate when B. methylotrophicum or Acetobacterium woodii was grown in the presence of a second one-carbon substrate indicated two trends: when methanol was consumed, CO, CO2, or formate predominantly labeled the acetate carboxyl; when CO was consumed, CO2 and formate were principally funneled into the acetate methyl group, and CO remained a better carboxyl precursor. These data suggest a model of acetate synthesis via the combined operation of two readily reversible single-carbon pathways which are linked by CO2.
Isolation of carbon monoxide dehydrogenase from Acetobacterium woodii and comparison of its properties with those of the Clostridium thermoaceticum enzyme
Authors: Ragsdale S W , Ljungdahl L G , DerVartanian D V
Published: January 2020

An oxygen-labile carbon monoxide dehydrogenase was purified to at least 98% homogeneity from fructose-grown cells of Acetobacterium woodii. Gel filtration and electrophoresis experiments gave molecular weights of 480,000 and 153,000, respectively, of the active enzyme. The molecular weights for the subunits are 80,000 and 68,000; the subunits occur in equal proportion. The small subunit of the A. woodii enzyme differs in size from that of the Clostridium thermoaceticum enzyme; however, the large subunits are similar. The specific activity of the A. woodii enzyme, measured at 30 degrees C and pH 7.6, is 500 mumol of CO oxidized min-1 mg-1 with 20 mM methyl viologen as the electron acceptor. Analysis revealed (number per dimer) iron (9), acid-labile sulfide (12), nickel (1.4), and magnesium or zinc (1). This metal content is quite similar to that of the C. thermoaceticum enzyme (Ragsdale et al., J. Biol. Chem. 258:2364-2369, 1983). The nickel as well as the iron-sulfur clusters are redox-active, as was found for the C. thermoaceticum enzyme (Ragsdale et al., Biochem. Biophys. Res. Commun. 108:658-663, 1982). CO can reduce and CO2 can oxidize the iron-sulfur clusters. The enzyme is inhibited by cyanide, but CO2 in the presence of reduced methyl viologen or CO alone can reverse or prevent this inhibition. Several ferredoxins, flavodoxin, and rubredoxin and some artificial electron carriers were tested for their relative rates of reaction with the CO dehydrogenases from A. woodii, C. thermoaceticum, and Clostridium formicoaceticum. Rubredoxin was by far the most reactive acceptor and is proposed to be the primary natural electron carrier for the acetogenic CO dehydrogenases.
Structure and function of the internal promoter (hisBp) of the Escherichia coli K-12 histidine operon
Authors: Grisolia V , Riccio A , Bruni C B
Published: January 2020

The entire histidine operon of Escherichia coli K-12 was cloned in the vector plasmid pBR313, and a complete restriction map of the operon was determined. By using subclones, complementation tests, and enzyme assays, we were able to make a correlation between the physical map and the genetic map of the operon. We determined the sequence of a fragment of DNA 665 base pairs long, comprising the distal portion of the hisC gene, the proximal portion of the hisB gene, and the internal transcription initiation site hisBp. The efficiency of this promoter was assessed under different physiological conditions by cloning the DNA fragment in a recombinant vector system used to study transcriptional regulatory signals. The precise point at which transcription initiates was determined by S1 nuclease mapping.
Characterization of Tn2411 and Tn2410, two transposons derived from R-plasmid R1767 and related to Tn2603 and Tn21
Authors: Kratz J , Schmidt F , Wiedemann B
Published: January 2020

Two transposable elements, Tn2410 and Tn2411, were isolated from Salmonella typhimurium R-factor R1767. They have sizes of 18.5 and 18.0 kilobases, respectively. Tn2411 mediates resistance to streptomycin, sulfonamides, and mercury. In Tn2410, the streptomycin resistance gene was replaced by a gene coding for the production of the beta-lactamase OXA-2, which is responsible for ampicillin resistance. Physical and functional maps of both transposons were compared with those of Tn21, Tn4, and Tn2603. From these data it appeared that Tn21 could be an ancestral transposon from which Tn2411, Tn2410, Tn2603, and Tn4 were evolved by the addition or deletion of small DNA segments.