bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron supply to Escherichia coli by synthetic analogs of enterochelin
Authors: Heidinger S , Braun V , Pecoraro V L , Raymond K N
Published: January 2020

Synthetic analogs of enterochelin (enterobactin) were tested for their ability to support the growth of Escherichia coli K-12 under iron-limiting conditions. The cyclic compound MECAM [1,3,5-N.N'; N"-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene] and its N-methyl derivative Me3MECAM promoted growth, whereas the 2,3-dihydroxy-5-sulfonyl derivatives MECAMS and Me3MECAMS were inactive. The same results were obtained with TRIMCAM [1,3,5-tris(2,3-dihydroxybenzoylcarbamido)-benzene] and TRIMCAMS (the 2,3-dihydroxy-5-sulfonyl derivative of TRIMCAM). However, the sulfonic acid-containing linear compound LICAMS [1,5,10-N,N', N"-tris(5-sulfo-2,3-dihydroxybenzoyl)-triaza-decane] supported growth. In contrast, LIMCAMC, in which the sulfonyl groups at the five position of LICAMS are replaced by carboxyl groups at the four position, was inactive. The uptake of the active analogs required the functions specified by the fepB, fesB, and tonB genes. Surprisingly, growth promotion of mutants lacking the enterochelin receptor protein in the outer membrane was observed. Only MECAM protected cells against colicin B (which kills cells after entering at the enterochelin uptake sites) and transported Fe3+ at about half the enterochelin rate.
Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli
Authors: Boronat A , Caballero E , Aguilar J
Published: January 2020

Spontaneous mutants of Escherichia coli able to grow on ethylene glycol as a sole source of carbon and energy were obtained from mutants that could grow on propylene glycol. Attempts to obtain ethylene glycol-utilizing mutants from wild-type E. coli were unsuccessful. The two major characteristics of the ethylene glycol-utilizing mutants were (i) increased activities of propanediol oxidoreductase, an enzyme present in the parental strain (a propylene glycol-positive strain), which also converted ethylene glycol into glycolaldehyde; and (ii) constitutive synthesis of high activities of glycolaldehyde dehydrogenase, which converted glycolaldehyde to glycolate. Glycolate was metabolized via the glycolate pathway, which was present in the wild-type cells; this was indicated by the induction in ethylene glycol-grown cells of glycolate oxidase, the first enzyme in the pathway. Glycolaldehyde dehydrogenase was partially characterized as an enzyme of this new metabolic pathway in E. coli, and glycolate was identified as the product of the reaction. This enzyme used NAD and NADP as coenzymes, although the NADP-dependent activity was about 10 times lower than the NAD-dependent activity. Uptake of [14C]ethylene glycol was dependent on the presence of the enzymes capable of metabolism of ethylene glycol. Glycolaldehyde and glycolate were identified as intermediate metabolites in the pathway.
Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins
Authors: Nikaido H , Rosenberg E Y
Published: January 2020

Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels.
Nutritional regulation of degradation of aspartate transcarbamylase and of bulk protein in exponentially growing Bacillus subtilis cells
Authors: Bond R W , Field A S , Switzer R L
Published: January 2020

The rate of degradation of aspartate transcarbamylase in exponentially growing Bacillus subtilis cells was determined by measurement of enzyme activity after the addition of uridine to repress further enzyme synthesis and by specific immunoprecipitation of the enzyme from cells grown in the presence of [3H]leucine. Aspartate transcarbamylase was degraded with a half-life of about 1.5 h in cells growing on a glucose-salts medium with NH4+ ions as the sole source of nitrogen. Replacement of NH4+ in this medium with a combination of the amino acids aspartate, glutamate, isoleucine, proline, and threonine reduced the degradation rate to an undetectable level. Various other amino acids and amino acid mixtures had smaller effects on the rate of degradation. The carbon source also influenced the degradation rate, but to a smaller extent than the nitrogen source. The effects of these nutritional variables on the rate of bulk protein turnover in growing cells were generally similar to their effects on degradation of aspartate transcarbamylase. Since the degradation of aspartate transcarbamylase has been shown to be 10 to 20 times faster than bulk protein turnover, the results suggest that a substantial portion of protein turnover in growing cells represents regulable, rapid degradation of a number of normal proteins, of which aspartate transcarbamylase is an example.
Contributing carbohydrate catabolic pathways in Cyclobacterium marinus
Authors: Raj H D , Paveglio K A
Published: January 2020

The primary and secondary pathways of carbohydrate metabolism were determined in a nonfermentative gram-negative ring-forming marine bacterium, Cyclobacterium marinus, by radiorespirometric studies. Whereas glucose is oxidized mainly via the Embden-Meyerhof pathway, gluconate is catabolized mainly via the Entner-Doudoroff pathway, both in conjunction with the tricarboxylic acid cycle as a secondary pathway and with some participation of the pentose phosphate pathway. The operation of these contributing catabolic pathways in this unique marine bacterium was substantiated by assaying the activities of the key enzymes specific to each pathway.
Absence of Significant Light-Induced Changes in cAMP Levels in Sporangiophores of
Phycomyces blakesleeanus
Authors: Leutwiler Leslie S. , Brandt Michaela
Published: January 2020

We were unable to find transient changes in the amount of cAMP or cGMP that had been proposed to mediate the light-growth response in sporangiophores of
Phycomyces blakesleeanus
.
Pleiotropic mutations regulating resistance to glucose repression in Saccharomyces carlsbergensis are allelic to the structural gene for hexokinase B
Authors: Michels C A , Hahnenberger K M , Sylvestre Y
Published: January 2020

Previously, we described a mutation glr1-1 in Saccharomyces carlsbergensis which pleiotropically relieves the synthesis of the following enzymes from glucose repression: maltase, galactokinase, alpha-galactosidase, NADH:cytochrome c reductase, and cytochrome c oxidase (C. A. Michels and A. Romanowski, J. Bacteriol, 143:674-679, 1980.) In this report, we demonstrate that glr1-1 and two other alleles, glr1-3 and glr1-16, are also insensitive to the glucose repression of invertase synthesis. Determinations of the levels of hexokinase activity and the rate of glucose transport in these mutants show that both are reduced as compared with the parent strain. Complementation tests and genetic analysis indicate that the glr1 mutations are allelic to HXK2, the structural gene for hexokinase B. The significance of this result is discussed with regard to the mechanism of glucose repression in S. carlsbergensis.
Bacteriophage Lambda Receptor Protein in
Escherichia coli
K-12: Lowered Affinity of Some Mutant Proteins for Maltose-Binding Protein In Vitro
Authors: Luckey Mary , Nikaido Hiroshi
Published: January 2020

Mutant and wild-type LamB proteins (phage λ receptor proteins) were purified by affinity chromatography with immobilized maltose-binding protein, and their transport functions were tested in reconstituted liposomes. Two mutant proteins exhibited a marked decrease in affinity for immobilized maltose-binding protein, as well as altered transport rates.
Genetic Mapping of the
minB
Locus in
Escherichia coli
K-12
Authors: Schaumberg Thomas H. , Kuempel Peter L.
Published: January 2020

The
minB
(minicell production) locus of
Escherichia coli
K-12 was mapped by transduction using bacteriophage P1.
minB
is located at min 25.6, between
purB
(min 25.2) and
dadR
(min 25.8). The mapping was facilitated by the use of insertion
zcf-236
::Tn
10
, which is inserted at min 25.4.
Role of the sfiA-dependent cell division regulation system in Escherichia coli
Authors: Huisman O , Jacques M , D'ari R , Caro L
Published: January 2020

Several authors have suggested that the SOS-associated (sfiA-dependent) system of division inhibition, normally induced by perturbations of DNA replication, also regulates steady-state (unperturbed) cell division. The present work shows that mean cell mass is identical in sfiA+ and sfiA mutant cultures during steady-state growth, that mass adjustment is identical after shift up, that sfiA expression is not induced by shift up, and that a sfiA mutation does not cause aberrant chromosome segregation.
Apparent bacteriophage-binding region of an Escherichia coli K-12 outer membrane protein
Authors: Cole S T , Chen-Schmeisser U , Hindennach I , Henning U
Published: January 2020

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli. It serves as the receptor for several T-even-like phages and is required for the action of certain colicins and for the stabilization of mating aggregates in conjugation. We have isolated two mutant alleles of the cloned ompA gene which produce a protein that no longer functions as a phage receptor. Bacteria possessing the mutant proteins were unable to bind the phages, either reversibly or irreversibly. However, both proteins still functioned in conjugation, and one of them conferred colicin L sensitivity. DNA sequence analysis showed that the phage-resistant, colicin-sensitive phenotype exhibited by one mutant was due to the amino acid substitution Gly leads to Arg at position 70. The second mutant, which contained a tandem duplication, encodes a larger product with 8 additional amino acid residues, 7 of which are a repeat of the sequence between residues 57 and 63. In contrast to the wild-type OmpA protein, this derivative was partially digested by pronase when intact cells were treated with the enzyme. The protease removed 64 NH2-terminal residues, thereby indicating that this part of the protein is exposed to the outside. It is argued that the phage receptor site is most likely situated around residues 60 to 70 of the OmpA protein and that the alterations characterized have directly affected this site.
Thiamine pyrophosphate requirement for o-succinylbenzoic acid synthesis in Escherichia coli and evidence for an intermediate
Authors: Meganathan R , Bentley R
Published: January 2020

Cell-free extracts of various strains of Escherichia coli synthesize the menaquinone biosynthetic intermediate o-succinylbenzoic acid (OSB) when supplied with chorismic acid, 2-ketoglutaric acid, and thiamine pyrophosphate (TPP). To assay for OSB synthesis, 2-[U-14C]ketoglutaric acid was used as substrate, and the synthesized OSB was examined by radiogas chromatography (as the dimethyl ester). [U-14C]Shikimic acid also gave rise to radioactive OSB if the cofactors necessary for enzymatic conversion to chorismic acid were added. Use of 2-[1-14C]ketoglutaric acid does not give rise to labeled OSB. In the absence of TPP during the incubations, OSB synthesis was much reduced; these observations are consistent with the proposed role for the succinic semialdehyde-TPP anion as the reagent adding to chorismic acid. Extracts of cells from menC and menD mutants did not form OSB separately, but did so in combination. There was evidence for formation of a product, X, by extracts of a menC mutant incubated with chorismic acid, TPP, and 2-ketoglutaric acid; X was converted to OSB by extracts of a menD mutant. It appears that the intermediate, X, is formed by one gene product and converted to OSB by the second gene product.
Characterization of lipid-linked octa- through undecasaccharides implicated in the biosynthesis of Saccharomyces cerevisiae mannoproteins
Authors: Prakash C , Katial A , Vijay I K
Published: January 2020

The lipid-linked oligosaccharide Glc3-Man9(GlcNAc)2 (Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine) serves as a precursor for the biosynthesis of the inner core portion of the asparagine-linked polysaccharide of Saccharomyces cerevisiae mannoproteins. It has been shown previously that incubation of a microsomal preparation from this organism with UDP-N-acetylglucosamine and GDP-[14C]mannose gives rise to a series of lipid-linked oligosaccharides of the general structure Mann(GlcNAc)2, with n from 1 to 9. A structural characterization of Man1- to Man5(GlcNAc)2 oligosaccharides indicated that the major structures among these were identical to the intermediates proposed for the biosynthesis of animal glycoproteins (C. Prakash and I. K. Vijay, Biochemistry 21:4810-4818, 1982). In the present study, the structural characterization of the Man6- through Man9(GlcNAc)2 species was conducted. The Man6- through Man8(GlcNAc)2 species have two isomers, whereas Man9(GlcNAc)2 is monoisomeric. One isomer each of Man6- through Man8(GlcNAc)2 and the monoisomeric Man9(GlcNAc)2 are identical to the intermediates for the biosynthesis of asparagine-linked glycoproteins in animal systems. It is proposed that the steps of the lipid-linked assembly of the carbohydrate precursor for S. cerevisiae mannoproteins are identical to those of the major pathway in animal systems. A lack of acceptor substrate specificity by the mannosyltransferases, as observed with in vitro studies with animal systems, also might be responsible for the biosynthesis of multiple isomers reported here.
Digestion of proteoglycan by Bacteroides thetaiotaomicron
Authors: Kuritza A P , Salyers A A
Published: January 2020

It has been shown previously that Bacteroides thetaiotaomicron, a human colonic anaerobe, can utilize the tissue mucopolysaccharide chondroitin sulfate as a source of carbon and energy and that the enzymes involved in this utilization are all cell associated (A. A. Salyers and M. B. O'Brien, J. Bacteriol. 143:772-780, 1980). Since chondroitin sulfate does not generally occur in isolated form in tissue, but rather is bound covalently in proteoglycan, we investigated the extent to which chondroitin sulfate which is bound in such a sterically hindered complex can be utilized by intact bacteria. Intact cells of B. thetaiotaomicron were able to digest chondroitin sulfate in proteoglycan, although at a slightly slower rate than free chondroitin sulfate. Prior digestion of proteoglycan with trypsin to produce small fragments of protein with several chondroitin sulfate chains attached did not increase the rate at which the bound chondroitin sulfate was digested. Accordingly, the slower rate of digestion was probably due to attachment of chondroitin sulfate chains to the protein backbone rather than to steric hindrance by other components of the proteoglycan. When proteoglycan which had been incubated with intact bacteria was treated with sodium borohydride to release the undigested fragments of chondroitin sulfate from the protein backbone, the size and composition of the fragments indicated that intact bacteria were able to digest all but three monosaccharides of the chondroitin sulfate chains. Thus, despite steric hindrance due to attachment of the chondroitin sulfate chains to the protein backbone, digestion of bound chondroitin sulfate by intact bacteria was nearly complete.
Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter
Authors: Débarbouillé M , Raibaud O
Published: January 2020

The malPQ operon, one of the three operons of the maltose regulon, is positively controlled by the product of gene malT. The starting point for malPQ transcription was deduced from experiments which involved a hybridization of in vivo-synthesized malPQ mRNA with adequate DNA probes, followed either by a digestion of nonhybridized DNA (S1 nuclease mapping) or by an extension of the hybridized probe (reverse transcriptase mapping). In the wild-type strain, this starting point was 37 nucleotides upstream from the initiation codon for malP. This analysis was also performed on a double mutant which contained both a 13-base pair deletion and a 3-base pair insertion in the promoter region. This double mutant expressed the malPQ operon exactly as the wild-type strain did, in a maltose-inducible manner. In this strain, the starting point for malPQ transcription was shifted 11 nucleotides downstream from the wild-type location. An analysis of these results suggests that (i) the binding site for the malT product is located upstream from the region which is severely altered in the double mutant, i.e., upstream from position -31; and (ii) the 30-base pair sequence which precedes the transcription starting point contains very few positions which are essential for promoter activity.
Occurrence of diphthamide in archaebacteria
Authors: Pappenheimer A M , Dunlop P C , Adolph K W , Bodley J W
Published: January 2020

We examined the nature of the diphtheria toxin fragment A recognition site in the protein synthesis translocating factor present in cell-free preparations from the archaebacteria Thermoplasma acidophilum and Halobacterium halobium. In agreement with earlier work (M. Kessel and F. Klink, Nature (London) 287:250-251, 1980), we found that extracts from these organisms contain a protein factor which is a substrate for the ADP-ribosylation reaction catalyzed by diphtheria toxin fragment A. However, the rate of the reaction was approximately 1,000 times slower than that typically observed with eucaryotic elongation factor 2. We also demonstrated the presence of diphthine (the deamidated form of diphthamide, i.e., 2-[3-carboxyamide-3-(trimethylammonio)propyl]histidine) in acid hydrolysates of H. halobium protein in amounts comparable to those found in hydrolysates of similar preparations from eucaryotic cells (Saccharomyces cerevisiae and HeLa). Diphthine could not be detected in hydrolysates of protein from the eubacterium Escherichia coli. Whereas both archaebacterial and eucaryotic elongation factors contain diphthamide, they differ importantly in other respects.
Occurrence of dialkyl ether phospholipids in Stigmatella aurantiaca DW4
Authors: Caillon E , Lubochinsky B , Rigomier D
Published: January 2020

We investigated the lipid composition of vegetative cells of Stigmatella aurantiaca. Four phospholipids were isolated and identified: phosphatidylethanolamine as the main component, phosphatidylglycerol, lysophosphatidylethanolamine in an exceptionally large amount (17%), and phosphatidylinositol (18 to 25%), rare in procaryotic cells. This composition did not change significantly during growth. The fatty acids of total lipids were found to be rather similar to those of other strains of myxobacteria; the main fatty acids found were unsaturated and branched. We noted a different fatty acid pattern for each phospholipid. The presence of unusual alkyl ether linkages, established by chemical hydrolysis and infrared spectroscopy, was unexpected in these bacteria. Diacyl ester, dialkyl ether, and monoacyl-monoalkyl structures were shown in phosphatidylethanolamine and phosphatidylglycerol. Lysophosphatidylethanolamine was essentially a monoacyl form, whereas phosphatidylinositol was a unique dialkyl ether phospholipid.
Isolation of conditional lethal mutator mutants of Escherichia coli by localized mutagenesis
Authors: Maki H , Horiuchi T , Sekiguchi M
Published: January 2020

By using localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, we isolated 39 temperature-sensitive growth mutants that exhibited high mutability when the bacteria were grown at the permissive temperature. Two of the mutations, dnaQ186 and dnaQ231, were shown to be new alleles of the dnaQ gene by genetic mapping and complementation tests with the dnaQ49 mutation previously isolated. They shared common properties with the dnaQ49 strain, but their mutator activity was not temperature dependent. The dnaQ mutants exhibited increased sensitivity to inhibitors of DNA gyrase and to DNA intercalating and alkylating agents.
Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli
Authors: Gay P , Le Coq D , Steinmetz M , Ferrari E , Hoch J A
Published: January 2020

A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA. This lambda clone also was found to contain the sacR and smo loci. Subcloning the sacB-sacR region in plasmid pBR325 resulted in a clone which directed levansucrase synthesis in Escherichia coli. The nucleotide sequence coding for the secreted protein was localized on the physical map of the cloned DNA.
Molecular cloning and characterization of the genes (pbpA and rodA) responsible for the rod shape of Escherichia coli K-12: analysis of gene expression with transposon Tn5 mutagenesis and protein synthesis directed by constructed plasmids
Authors: Asoh S , Matsuzawa H , Matsuhashi M , Ohta T
Published: January 2020

Two cell shape-determining genes of Escherichia coli K-12, pbpA, the structural gene for penicillin-binding protein 2, and rodA, whose protein is unknown, were subcloned into plasmid vectors from the transducing phage lambda MAd lip24, which carries the lip-leuS region of the E. coli chromosome. Plasmids with restriction enzyme-created deletions or transposon Tn5 insertions were isolated, and studies of genetic complementation of these plasmids with chromosomal mutations were carried out. Thus, a physical and genetic map of the rodA-pbpA region was established. The genes rodA and pbpA lie side by side within a 4.4-kilobase-pair region. The size of the rodA gene has been shown to be between 0.86 and 1.6 kilobase pairs; such DNA would encode a protein with a molecular weight between 32,000 and 59,000. Since Tn5 mutagenesis of the rodA gene did not affect the expression of the pbpA gene and vice versa, the genes rodA and pbpA seem to have independent promoters. Analysis of the proteins synthesized from the constructed plasmids in maxicells revealed that the plasmid carrying the pbpA gene encoded penicillin-binding protein 2 and amplification of the protein occurred. The product of the rodA gene was not identified.
Chromosomal location of the gene encoding phosphoribosylpyrophosphate synthetase in Escherichia coli
Authors: Hove-Jensen B
Published: January 2020

A mutant of Escherichia coli with a partially defective phosphoribosylpyrophosphate synthetase (ribosephosphate pyrophosphokinase) has been characterized genetically. The genetic lesion causing the altered phosphoribosylpyrophosphate synthetase, prs, was mapped at 26 min on the linkage map by conjugation. Transductional analysis of the prs region established the gene order as purB-fadR-dadR-tre-pth-prs-hemA-trp. Two additional mutations were identified in the mutant: one in gsk, the gene encoding guanosine kinase, and one in lon, conferring a mucoid colony morphology. The contribution of each mutation to the phenotype of the mutant has been evaluated.
Mutations in a new gene, secB, cause defective protein localization in Escherichia coli
Authors: Kumamoto C A , Beckwith J
Published: January 2020

We isolated a new class of Escherichia coli mutants with pleiotropic defects in protein secretion. Using a previously described selection procedure (Oliver et al., Ann. Microbiol. [Paris] 133A:105-110, 1982), we obtained a large collection of strains containing mutations that affect protein localization. In many cases, the lesions causing the secretion defects were mapped in or near the previously identified gene, secA (Oliver and Beckwith, Cell 25:765-772, 1981). However, the selection also yielded mutants with mutations in a new locus, which was designated secB. These secB mutants were defective in the localization of maltose-binding protein and, in at least one case, OmpF protein. Double mutants with lesions in both secA and secB had strong defects in the secretion of maltose-binding protein and OmpF protein. The secB locus mapped near cysE at min 80.5 on the E. coli genetic map. The properties of secB mutants suggest that the secB product could be a component of the E. coli secretory apparatus.
Comparison of cytochromes from anaerobically and aerobically grown cells of Pseudomonas perfectomarinus
Authors: Liu M C , Payne W J , Peck H D , LeGall J
Published: January 2020

Pseudomonas perfectomarinus (ATCC 14405) is a facultative anaerobe capable of either oxygen respiration or anaerobic nitrate respiration, i.e., denitrification. A comparative study of the electron transfer components of cells revealed five c-type cytochromes and cytochrome cd in the soluble fraction from anaerobically grown cells and four c-type cytochromes in the soluble fraction from aerobically grown cells. Purification procedures yielded three c-type cytochromes (designated c-551, c-554, and acidic c-type) from both kinds of cells as indicated by similarities in absorption spectra, molecular weight, and electrophoretic mobility. Cytochrome cd, a diheme c-type cytochrome (cytochrome c-552), and a split-alpha c-type cytochrome were recovered only from anaerobically grown cells. A c-type cytochrome with a low ratio of alpha to beta absorption peak heights was uniquely present in the aerobically grown cells. Liquid N2 temperature absorption spectroscopy on the membrane fraction from anaerobically grown cells revealed residual cytochrome cd as well as differences in the relative amounts of c-type and b-type cytochromes in membranes prepared from cells grown under the two different conditions.
Diversity of locations for Bacillus thuringiensis crystal protein genes
Authors: Kronstad J W , Schnepf H E , Whiteley H R
Published: January 2020

The location of crystal protein genes in 22 crystalliferous Bacillus thuringiensis strains representing 14 subspecies was investigated by hybridization of an intragenic restriction fragment from a cloned crystal protein gene to whole plasmid preparations. Hybridization was found to a single plasmid in eight strains, to more than one plasmid in seven strains, and to one or both of two large, unresolved plasmids in two strains. The sizes of the hybridized plasmids ranged from 33 to over 150 megadaltons. In one additional subspecies, hybridization was only to linear DNA fragments, suggesting a chromosomal crystal protein gene, and for four other subspecies, not reported to be toxic to lepidopteran insects, no hybridization was found to either plasmids or to total cell DNA. Hybridization to restriction digests of plasmids and total cell DNA of several strains of subspecies thuringiensis and kurstaki revealed that all homology to the cloned crystal protein gene was plasmid associated and that several of these strains contained multiple regions of homology, implying the presence of multiple crystal protein genes.
Replication origin region of Bacillus subtilis chromosome contains two rRNA operons
Authors: Ogasawara N , Seiki M , Yoshikawa H
Published: January 2020

The first replicating DNA fragment (BamHI-7) of the Bacillus subtilis chromosome contains two promoters for a rRNA operon. A map of restriction enzyme cleavage sites of the region of replication origin suggests the presence of a second rRNA operon in this region. Hybridization of rRNA genes (rDNA) with DNA fragments derived from the origin region by treatment with various enzymes clearly revealed two rRNA operons in this region, one at the B7-B3 junction and the other at the B5-B6 junction. The restriction enzyme cleavage sites surrounding the rRNA operons show that the operon at the B5-B6 junction corresponds to the rrnA operon. A novel operon at the B7-B3 junction was termed rrnO. Transformation by density-labeled fragments of the origin region showed that the first replicating marker, guaA, is located in the B3 fragment. From these results, a map was constructed for the first time to correlate the genetic markers with the physical structure of the replication origin region of the B. subtilis chromosome. The role of the rrnO operon in regulating the initiation of chromosomal replication is discussed, based on the fact that the promoter of the rrnO operon suppresses the replication of the plasmid carrying the promoter.
Demonstration by a Novel Genetic Technique That Leader Peptidase Is an Essential Enzyme of
Escherichia coli
Authors: Date Takayasu
Published: January 2020

It was previously shown that two separate regions of DNA are required for expression of the cloned leader peptidase gene on plasmid pTD101 (T. Date and W. Wickner, Proc. Natl. Acad. Sci. U.S.A.
78:
6106-6110, 1981). Both loci have been mapped in detail, and their roles have been established. A 1.3-kilobase region, termed the L region, encodes the 37,000-dalton leader peptidase protein. Another region, termed the P region, is about 1.5 kilobases away from the L region and is less than 350 base pairs long. The P region acts in
cis
to the L region, suggesting that it plays a role as a promoter. A technique for inactivation of the leader peptidase gene on the
Escherichia coli
chromosome has been developed to examine whether the leader peptidase which we had cloned is essential for cell growth. A specific plasmid (P
−
L
−
) which deletes both the P region and a substantial portion of the L region was constructed and transformed into a
polA
mutant strain. The plasmid cannot replicate in this strain; thus, the plasmid-borne ampicillin resistance is lost unless the plasmid DNA recombines into the chromosome. Integration of the P
−
L
−
plasmid did not yield any viable ampicillin-resistant cells, whereas the three control plasmids, P
+
L
+
, P
+
L
−
, and P
−
L
+
, did. When the P
−
L
−
plasmid was transformed into the
polA
(Ts) strain, the strain could only grow in the presence of ampicillin at a permissive temperature, suggesting that integration of the plasmid into the host chromosome leads to inactivation of the chromosomal leader peptidase gene. Southern hybridization analysis demonstrated that the integration of plasmids into the chromosome occurred at the homologous site. This study demonstrates that expression of the leader peptidase gene is critical for cell growth.
Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli
Authors: Nunn W D , Giffin K , Clark D , Cronan J E
Published: January 2020

Escherichia coli K-12 mutants constitutive for the synthesis of the enzymes of fatty acid degradation (fad) synthesize significantly less unsaturated fatty acid (UFA) than do wild-type (fadR+) strains. The constitutive fadR mutants synthesize less UFA than do fadR+) strains both in vivo and in vitro. The inability of fadR strains to synthesize UFAs at rates comparable to those of fadR+ strains is phenotypically asymptomatic unless the fadR strain also carries a lesion in fabA, the structural gene for beta-hydroxydecanoyl-thioester dehydrase. Unlike fadR+ fabA(Ts) mutants, fadR fabA(Ts) strains synthesize insufficient UFA to support their growth even at low temperatures and, therefore, must be supplemented with UFA at both low and high temperatures. The low levels of UFA in fadR strains are not due to the constitutive level of fatty acid-degrading enzymes in these strains. These results suggest that a functional fadR gene is required for the maximal expression of UFA biosynthesis in E. coli.
Membrane cytochromes of Escherichia coli chl mutants
Authors: Hackett N R , Bragg P D
Published: January 2020

The cytochromes present in the membranes of Escherichia coli cells having defects in the formate dehydrogenase-nitrate reductase system have been analyzed by spectroscopic, redox titration, and enzyme fractionation techniques. Four phenotypic classes differing in cytochrome composition were recognized. Class I is represented by strains with defects in the synthesis or insertion of molybdenum cofactor. Cytochromes of the formate dehydrogenase-nitrate reductase pathway are present. Class II strains map in the chlC-chlI region. The cytochrome associated with nitrate reductase (cytochrome bnr) is absent in these strains, whereas that associated with formate dehydrogenase (cytochrome bfdh) is the major cytochrome in the membranes. Class III strains lack both cytochromes bfdh and bnr but overproduce cytochrome d of the aerobic pathway even under anaerobic conditions in the presence of nitrate. Class III strains have defects in the regulation of cytochrome synthesis. An fdhA mutant produced cytochrome bnr but lacked cytochrome bfdh. These results support the view that chlI (narI) is the structural gene for cytochrome bnr and that chlC (narG) and chlI(narI) are in the same operon, and they provide evidence of the complexity of the regulation of cytochrome synthesis.
Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus
Authors: Im D S , Friedrich C G
Published: January 2020

Alcaligenes eutrophus formed ribulosebisphosphate carboxylase (RuBPCase; EC 4.1.1.39) when grown on fructose. Addition of sodium fluoride (NaF) to fructose minimal medium resulted in a slightly decreased growth rate and a rapid fivefold increase in RuBPCase specific activity. With citrate, a glucogenic carbon source, RuBPCase was also formed, However, addition of NaF to cells growing on citrate resulted in a 50% decrease in RuBPCase specific activity. Among the enzymes of fructose catabolism, NaF (10 mM) inhibited enolase in vitro by 98% and gluconate 6-phosphate dehydratase by 87%. Inhibition of the dehydratase by NaF was insignificant in vivo, as determined with a mutant defective in phosphoglycerate mutase activity. Growth of this mutant on fructose was not inhibited by NaF, and only a minor increase in RuBPCase activity was observed. From these results, we concluded that the product of the enolase reaction, phosphoenolpyruvate, played a role in RuBPCase formation. Addition of H2 or formate to the wild type growing on fructose or citrate did not affect the growth rate but resulted in rapid formation of RuBPCase activity. Mutants impaired in H2 metabolism formed RuBPCase at a low rate during growth on fructose plus H2 but at a high rate on formate. Apparently, additional reductant from H2 or formate metabolism induced RuBPCase formation in A. eutrophus.
Cloning of sporulation genes spo0A and spo0C of Bacillus subtilis onto rho 11 temperate bacteriophage
Authors: Ikeuchi T , Kudoh J , Kurahashi K
Published: January 2020

A HindIII fragment harboring the intact spo0C gene of Bacillus subtilis was cloned with rho 11 temperate bacteriophage as a vector. Transformation experiments with the DNA from rho 11 dspo0C+ specialized transducing phage showed that the spo0C gene resides on a 5.3-megadalton fragment generated by HindIII digestion. The 5.3-megadalton fragment also contains the intact spo0A gene, but not spoIIIA, spoIIIB, or spoIVB.
Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli
Authors: Lambert J M , Boileau G , Howe J G , Traut R R
Published: January 2020

The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis. Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension. The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides. The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase. There was no accumulation of S1 in the stationary phase. In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase. The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase. The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.
Nonsense mutations in the can1 locus of Saccharomyces cerevisiae
Authors: Ono B I , Ishino Y , Shinoda S
Published: January 2020

Yeast mutants resistant to L-canavanine were selected. All were recessive and fell into the can1 complementation group. Nonsense mutations were identified among them by using a set of different suppressors. Frequencies of UAA, UAG, and presumed UGA mutations were 14.8, 0.8, and 0.4%, respectively. A high incidence of nonsense mutations having discriminatory suppression patterns was characteristic of the locus.
Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168
Authors: Roitsch C A , Hageman J H
Published: January 2020

Bacillopeptidase F is a serine endopeptidase excreted by Bacillus subtilis 168 after the end of exponential growth. As a step toward discovering a physiological function for this protease, an enzymological and immunological study was undertaken. When bacillopeptidase F was purified at pH 10, a number of enzymically active, rapidly moving electrophoretic forms were observed, as had been previously reported. Rabbit antiserum was prepared against one form. When the enzyme was purified at pH 6.0 in the presence of the covalent inhibitor phenylmethylsulfonyl fluoride, using the rabbit antiserum to detect the bacillopeptidase F protein, no fast-moving electrophoretic forms were observed. Instead, only two forms of the enzyme were isolated. One form had a molecular weight of 33,000, and the other had a molecular weight of 50,000, as determined by equilibrium sedimentation methods. Both forms appeared to be glycoproteins, both contained compounds, released on acid hydrolysis, which cochromatographed with phosphoserine and galactosamine, and the two gave identical immunoprecipitin lines in Ouchterlony double-diffusion tests. The smaller form had a pI of 4.4, whereas the larger had a pI of 5.4. The data suggest that bacillopeptidase F is distinct from all other proteases of B. subtilis.
Cloning of the pif region of the F sex factor and identification of a pif protein product
Authors: Rotman G S , Cooney R , Malamy M H
Published: January 2020

This paper reports a detailed investigation of the pif region of the F factor responsible for inhibition of development of T7 and related "female-specific" phages. We have mapped a series of pif::Tn5 insertions to a region between 39.6 and 42.8 kilobases on the physical map of F. All pif::Tn5 insertions plated T7 at full efficiency; most were clustered in a 1.8-kilobase interval on both sides of the EcoRI site located at F coordinate 40.3 kilobases. A 5.2-kilobase Pst-I fragment with F coordinates 38.9 to 44.1 has been cloned into a pSC101 vector to create the Pif+ plasmid pGS103. A series of Pif- deletion mutants and nonsense mutants were isolated from pGS103. Using minicells carrying pGS103 or its derivatives, we have identified a 70,000-dalton pif protein.
High endogenous nitrogenase activity in isolated heterocysts of Anabaena sp. strain CA after nitrogen starvation
Authors: Kumar A , Tabita F R , Van Baalen C
Published: January 2020

Metabolically active heterocysts were isolated from a mutant of Anabaena sp. strain CA with fragile vegetative cells. Heterocysts isolated from cultures grown in 1% CO2 in air reduced C2H2 at 57 and 10 nmol of C2H2 per mg (dry weight) per min under H2 and Ar, respectively. However, if whole filaments were sparged with 1% CO2 in 99% Ar for 12 h before heterocyst isolation, these heterocysts showed C2H2 reduction rates of 83 nmol of C2H4 per mg (dry weight) per min under either H2 or Ar, or 40% the activity of whole filaments grown in 1% CO2 in air. Heterocysts isolated from cultures sparged with 100% Ar or 1% CO2 in 99% N2 had the same C2H2 reduction pattern as heterocysts from cultures grown in 1% CO2 in air, i.e., low activity under Ar and high activity under H2. Labeling of whole filaments incubated with NaH14CO3 for 12 h under 1% CO2 in air or 1% CO2 in 99% Ar resulted in a twofold higher accumulation of 14C-labeled compounds in vegetative cells and heterocysts of Ar-incubated cells. Our results suggest that during incubation under 1% CO2 in 99% Ar, presumably a nitrogen starvation condition, continuing photosynthetic fixation of CO2 leads to accumulation of material(s) in the heterocysts that supports a high, persistent endogenous rate of C2H2 reduction. This material appears to be, in part, glycogen.
Cloning and physical mapping of the cysB region of Salmonella typhimurium
Authors: Jagura-Burdzy G , Kredich N M
Published: January 2020

The cysB region of Salmonella typhimurium was cloned in pBR322 and localized to a 1.75-kilobase HincII fragment. Two-dimensional protein electropherograms showed levels of the cysB polypeptide chain that were several fold higher in plasmid-bearing strains than in the wild type. Fully derepressed levels of sulfite reductase and O-acetylserine sulfhydrylase in cysB plasmid-bearing strains were only 25% higher than in the wild type, suggesting that the product of this regulatory gene ordinarily is not a limiting factor in the expression of the cysteine regulon. The mapping of cysB deletions by Southern blots showed a good correlation between the genetic and the physical maps of this gene. The supX gene was initially cloned with cysB and is within 0.7 kilobase of cysB.
14C-labeled metabolites in heterocysts and vegetative cells of Anabaena cylindrica filaments and their presumptive function as transport vehicles of organic carbon and nitrogen
Authors: Jüttner F
Published: January 2020

To investigate the transport of primary metabolites in Anabaena cylindrica from vegetative cells into heterocysts, intact filaments were labeled with the heterocysts were separated from the vegetative cells after different time intervals, and the labeling patterns were determined. After a 20-s fixation time, a high percentage of labeling of alanine, glutamate and glutamine, and, to a lesser extent, glucose 6-phosphate was found in heterocysts as compared with whole filaments. The results can be explained if transport of alanine, glutamate, and sugars from vegetative cells into heterocysts is assumed. Alanine can serve as a precursor for reducing equivalents if it is oxidized to glutamine which flows back to the vegetative cells. This idea is supported by an experiment in which exogenous alanine is readily converted by isolated heterocysts to glutamate and glutamine under a N2-H2 atmosphere. The incorporation of [14C]carbonate in isolated heterocysts demonstrated the absence of the reductive pentose phosphate pathway; however, it revealed marked activity of an acid fixation reaction.
Peptidoglycan synthesis by partly autolyzed cells of Bacillus subtilis W23
Authors: Harrington C R , Baddiley J
Published: January 2020

Partly autolyzed, osmotically stabilized cells of Bacillus subtilis W23 synthesized peptidoglycan from the exogenously supplied nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramyl pentapeptide. Freshly harvested cells did not synthesize peptidoglycan. The peptidoglycan formed was entirely hydrolyzed by N-acetylmuramoylhydrolase, and its synthesis was inhibited by the antibiotics bacitracin, vancomycin, and tunicamycin. Peptidoglycan formation was optimal at 37 degrees C and pH 8.5, and the specific activity of 7.0 nmol of N-acetylglucosamine incorporated per mg of membrane protein per h at pH 7.5 was probably decreased by the action of endogenous wall autolysins. No cross-linked peptidoglycan was formed. In addition, a lysozyme-resistant polymer was also formed from UDP-N-acetylglucosamine alone. Peptidoglycan synthesis was inhibited by trypsin and p-chloromercuribenzenesulfonic acid, and we conclude that it occurred at the outer surface of the membrane. Although phospho-N-acetylmuramyl pentapeptide translocase activity was detected on the outside surface of the membrane, no transphosphorylation mechanism was observed for the translocation of UDP-N-acetylglucosamine. Peptidoglycan was similarly formed with partly autolyzed preparations of B. subtilis NCIB 3610, B. subtilis 168, B. megaterium KM, and B. licheniformis ATCC 9945. Intact protoplasts of B. subtilis W23 did not synthesize peptidoglycan from externally supplied nucleotides although the lipid intermediate was formed which was inhibited by tunicamycin and bacitracin. It was therefore considered that the lipid cycle had been completed, and the absence of peptidoglycan synthesis was believed to be due to the presence of lysozyme adhering to the protoplast membrane. The significance of these results and similar observations for teichoic acid synthesis (Bertram et al., J. Bacteriol. 148:406-412, 1981) is discussed in relation to the translocation of bacterial cell wall polymers.
Bacteriocin from Bacillus megaterium ATCC 19213: comparative studies with megacin A-216
Authors: Von Tersch M A , Carlton B C
Published: January 2020

A bacteriocin produced by Bacillus megaterium ATCC 19213 was identified, purified, and compared with megacin A from B. megaterium 216. The ATCC 19213 bacteriocin was inducible with mitomycin C and showed phospholipase A activity. Both megacin A-216 and megacin A-19213 contained two dissimilar polypeptide subunits. Megacin A-216 contains a 30,000-dalton alpha subunit and a 15,000-dalton beta subunit. Megacin A-19213 is composed of an alpha subunit 18,000 daltons in mass and a beta subunit about 7,500 daltons in mass. No sequence similarities between alpha and beta subunits of either megacin were detected. The two megacins were further distinguished by quantitative differences in activity spectra and by immunodiffusion analyses.
A mutant form of maltose-binding protein of Escherichia coli deficient in its interaction with the bacteriophage lambda receptor protein
Authors: Bavoil P , Wandersman C , Schwartz M , Nikaido H
Published: January 2020

In one malE mutant known to be deficient in the transport of maltose and maltodextrins across the outer membrane, the altered MalE protein was shown to be defective in its interaction with the phage lambda receptor, or LamB protein, of the outer membrane.