bluuebunny/crossref_metadata_2025 huggingface dataset:
Haptenic oligosaccharides in antigenic variants of mycobacterial C-mycosides antagonize lipid receptor activity for mycobacteriophage D4 by masking a methylated rhamnose
Authors: Dhariwal K R , Liav A , Vatter A E , Dhariwal G , Goren M B
Published: November 2016

The simple apolar C-mycosides, i.e., structurally well-defined hydrophobic glycopeptidolipids of several Mycobacterium species (see diagram below), were earlier shown to behave as receptors for adsorption of mycobacteriophage D4. This phage is usually virulent for Mycobacterium smegmatis. More complex, polar C-mycosides with additional carbohydrate substituents attached solely to the deoxytalose have recently been described. They are the highly specific serotyping antigens discovered by W. B. Schaefer--lipids which characterize members of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex. Both kinds are depicted in the structure below: (Formula: see text) where X equals H (for simple, apolar C-mycosides) and X equals small oligosaccharides (for antigenic forms; more complex, polar C-mycosides). The present investigations showed that the purified polar antigenic lipids exhibit considerably less adsorptive activity for D4 than do the apolar C-mycosides. Thus, the haptenic oligosaccharides are believed to shield the site in the molecule that the phage recognizes, and the blocking is reinforced by the specific antibodies that the antigens elicit. Although the MAIS serovars usually also produce the phage-reactive apolar C-mycosides, they are not permissive hosts for D4, nor do whole cells adsorb the phage. We suggest that in these species the apolar forms are probably "covered" at the cell surface by the antigenic lipids. Therefore, these antigenic mycosides may play a putative role in virulence of the MAIS members by protecting these mycobacteria from their own potential pathogen. The results of chemical transformations at specific sites of the mycoside core coupled with studies of simple synthetic lipid glycosides indicated that the principal phage receptor activity resides in the terminal methylated rhamnose (see diagram). It is this sugar which is evidently masked by the (seemingly remote) haptenic oligosaccharides.
Nucleotide sequence of the phoM region of Escherichia coli: four open reading frames may constitute an operon
Authors: Amemura M , Makino K , Shinagawa H , Nakata A
Published: November 2016

The phoM gene is one of the positive regulatory genes for the phosphate regulon of Escherichia coli. We analyzed the nucleotide sequence of a 4.7-kilobase chromosomal DNA segment that encompasses the phoM gene and its flanking regions. Four open reading frames (ORFs) were identified in the order ORF1-ORF2-ORF3 (phoM)-ORF4-dye clockwise on the standard E. coli genetic map. Since these ORFs are preceded by a putative promotor sequence upstream of ORF1 and followed by a putative terminator distal to ORF4, they seem to constitute an operon. The 157-amino-acid ORF1 protein contains highly hydrophobic amino acids in the amino-terminal portion, which is a characteristic of a signal peptide. The 229-amino-acid ORF2 protein is highly homologous to the PhoB protein, a positive regulatory protein for the phosphate regulon. The ORF3 (phoM gene) protein contains two stretches of highly hydrophobic residues in the amino-terminal and central regions and, therefore, may be a membrane protein. The 450-amino-acid ORF4 protein contains long hydrophobic regions and is likely to be a membrane protein.
Three-dimensional structure of the T-layer of Bacillus sphaericus P-1
Authors: Lepault J , Martin N , Leonard K
Published: November 2016

The three-dimensional structure of the regular surface layer of Bacillus sphaericus P-1 (T-layer) was determined to a resolution of ca. 2.5 nm by electron microscopy and image analysis. The T-layer has P4 symmetry, a lattice constant of 13 +/- 0.2 nm, and a thickness of ca. 8 nm. The reconstruction revealed three distinct domains: a major, a minor, and an arm domain. In the z-direction, the domains are arranged in two planes creating two different surface reliefs.
Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus
Authors: Verhoeven J A , Schenck K M , Meyer R R , Trela J M
Published: November 2016

An inorganic pyrophosphatase was purified over 600-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme is a tetramer of Mr = 84,000, has a sedimentation coefficient of 5.8S, a Stokes radius of 3.5 nm, and an isoelectric point of 5.7. Like the enzyme of Escherichia coli, the pyrophosphatase appears to be made constitutively. The pH and temperature optima are 8.3 and 80 degrees C, respectively. The Km for PPi is 0.6 mM. A divalent cation is essential, with Mg2+ preferred. The enzyme uses only PPi as a substrate.
Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp
Authors: Clejan S , Krulwich T A , Mondrus K R , Seto-Young D
Published: November 2016

The membrane lipids from two obligately and two facultatively alkalophilic strains of Bacillus spp. were characterized in a comparative study that included B. subtilis. Preparations of membrane lipids were made from pH 10.5-grown cells of all of the alkalophiles and from pH 7.5- or 7.0-grown cells of the two facultative strains and B. subtilis. The two obligate alkalophiles contained high ratios of membrane lipid to membrane protein, and the lipid fraction contained a high proportion of neutral lipid. These characteristics are probably not prerequisites for growth at very high pH since one or another of the facultative strains failed to show these properties at high pH. All of the alkalophiles contained appreciable amounts of squalene and C40 isoprenoids. Among the polar lipids, the alkalophiles all contained high concentrations of anionic phospholipids, including phosphatidylglycerol and especially large amounts of cardiolipin; phosphatidylethanolamine was the other major phospholipid. Small amounts of bis(monoacylglycero)phosphate were found in most, but not all, of the alkalophile preparations. Glycolipids and phosphoglycolipids were absent. The fatty acid composition of the total phospholipid and individual fractions revealed two features that distinguished between the obligate and facultative strains. Membranes from the obligately alkalophilic species contained a high concentration of branched-chain fatty acids, comparable to that in membranes from B. subtilis, as well as a relatively high content of unsaturated fatty acids. By contrast, the facultatively alkalophilic strains contained almost no unsaturated fatty acids and a lower concentration of branched-chain fatty acids than either the obligate alkalophiles or B. subtilis.
Control of developmental gene expression by cell-to-cell interactions in Myxococcus xanthus
Authors: Gill R E , Cull M G
Published: November 2016

The ssbA mutants of Myxococcus xanthus behave as if they are unable to produce a cell-to-cell signal required for normal development. They are unable to form fruiting bodies or spores on developmental medium. They do sporulate, however, if allowed to develop in mixtures with wild-type cells. Fusions of developmentally induced promoters of M. xanthus to the Escherichia coli lacZ gene were used to characterize the effect of the ssbA mutations on developmental gene expression. Each of the five independent fusions tested was found to be dependent upon the ssbA+ allele for full expression. The ssbA mutants were able to express each of these fusions if the mutants were allowed to develop in mixtures with wild-type (Lac-) cells. These results cannot be explained on the basis of genetic exchange. The data are consistent with regulation of gene expression mediated by cell-to-cell interactions.
Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing
Authors: Groisman E A , Casadaban M J
Published: November 2016

New mini-Mu transposons with plasmid replicons were constructed with additional features for in vivo DNA cloning and lac gene fusing in Escherichia coli. These mini-Mu replicons can be used to clone DNA by growing them with a complementing Mu bacteriophage and by using the resulting lysate to transduce Mu-lysogenic cells. These mini-Mu phage have selectable genes for resistance to kanamycin, chloramphenicol, and spectinomycin-streptomycin, and replicons from the high-copy-number plasmids pMB1 and P15A and the low-copy, broad-host-range plasmid pSa. The most efficient of these elements can be used to clone genes 100 times more frequently than with the previously described mini-Mu replicon Mu dII4042, such that complete gene banks can be made with as little as 1 microliter of a lysate containing 10(6) helper phage. The 39-kilobase-pair Mu headful DNA packaging mechanism limits the size of the clones formed. The smallest of the mini-Mu elements is only 7.9 kilobase pairs long, allowing the cloning of DNA fragments of up to 31.1 kilobase pairs, and the largest of them is 21.7 kilobase pairs, requiring that clones carry insertions of less than 17.3 kilobase pairs. Elements have been constructed to form both transcriptional and translational types of lac gene fusions to promoters present in the cloned fragment. Two of these elements also contain the origin-of-transfer sequence oriT from the plasmid RK2, so that clones obtained with these mini-Mu bacteriophage can be efficiently mobilized by conjugation.
Origin of small beta-lactamase-specifying plasmids in Haemophilus species and Neisseria gonorrhoeae
Authors: Brunton J , Meier M , Erhman N , Clare D , Almawy R
Published: November 2016

Fifty-nine percent of unselected strains of Haemophilus parainfluenzae were found to carry small, phenotypically cryptic plasmid DNA species. Using filter blot hybridization, we found several plasmids which were homologous to the small beta-lactamase-specifying plasmids pJB1 and pFA7, which were originally isolated from Haemophilus ducreyi and Neisseria gonorrhoeae, respectively. Detailed filter hybridization studies combined with electron microscope heteroduplex analysis suggested that three cryptic plasmids are completely homologous to the non-TnA sequences of pJB1. One cryptic plasmid was found to be highly homologous to pJB603, a small beta-lactamase plasmid previously found in two isolates of H. influenzae. A second group of plasmids were found to carry sequences homologous to pJB1 and other sequences homologous to pJB603. These results strongly suggest that small beta-lactamase plasmids found in Haemophilus species and N. gonorrhoeae may have arisen by insertion of the transposable beta-lactamase-specifying element TnA into small, phenotypically cryptic replicons resident in H. parainfluenzae. Attempts to reproduce such a recombination event in the laboratory were not successful.
Analysis of spontaneous base substitutions generated in mismatch-repair-deficient strains of Escherichia coli
Authors: Leong P M , Hsia H C , Miller J H
Published: November 2016

We used the lacI system of Escherichia coli to examine the distribution of base substitution mutations occurring spontaneously in different mismatch-repair-deficient strains. The examination of almost 1,200 nonsense mutations generated in strains carrying the mutS, mutH, and mutU alleles confirmed that transitions are highly favored over transversions. The detailed analysis of relative mutation rates at different sites revealed that the pattern of hot spots and cold spots is strikingly similar in each of the three strain backgrounds, strongly supporting the notions that the products of the three genes are part of the same system and that in the absence of any of the components the entire system fails to function. The distribution of mutations occurring in the absence of mismatch repair defined a pronounced topography of the lacI gene. There was no obvious correlation of the hot spots or cold spots with either nearest-neighbor sequences or A X T richness of the immediate surrounding sequence.
Cloning in Saccharomyces cerevisiae of a cycloheximide resistance gene from the Candida maltosa genome which modifies ribosomes
Authors: Takagi M , Kawai S , Shibuya I , Miyazaki M , Yano K
Published: November 2016

We have previously shown that cycloheximide resistance can be induced in a strain of Candida maltosa by modifying ribosomes (M. Takagi, S. Kawai, Y. Takata, N. Tanaka, M. Sunairi, M. Miyazaki, and K. Yano, J. Gen. Appl. Microbiol. 31:267-275, 1985). The present paper describes the cloning of the gene involved in this resistance (designated RIM-C for ribosome modification by cycloheximide) by using a host-vector system of Saccharomyces cerevisiae.
Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli
Authors: Daumy G O , Williams J A , McColl A S , Zuzel T J , Danley D
Published: November 2016

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.
Multicopy derivative of pock-forming plasmid pSA1 in Streptomyces azureus
Authors: Miyoshi Y K , Ogata S , Hayashida S
Published: November 2016

Streptomyces azureus carried one copy or less of plasmid pSA1, which elicited pocks at 0.1 to 1.0%. Strain PK100 was isolated from the wild-type strain after UV irradiation. PK100 carried approximately 20 to 30 copies of pSA1.1, a derivative of pSA1. Plasmid pSA1.1 elicited pocks at 100% and inhibited spore and thiostrepton production.
Repair response of Escherichia coli to hydrogen peroxide DNA damage
Authors: Hagensee M E , Moses R E
Published: November 2016

The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43 degrees C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.
Mapping and complementation studies of the gene for release factor 1
Authors: Ryden M , Murphy J , Martin R , Isaksson L , Gallant J
Published: November 2016

In Escherichia coli the release factor 1 protein (RF1) recognizes and terminates translation at UAG and UAA codons. Using the technique of ColE1 plasmid integration in polA strains, we have mapped the cloned gene for RF1 to 27 min on the E. coli chromosome. This is the same location as that of the uar gene in which temperature-sensitive mutations increase the suppression of UAG and UAA alleles. In this study we proved that the uar mutation lies in the gene for RF1 by complementation of the uar phenotype with plasmids carrying the RF1 gene and by cloning the uar allele onto the RF1 plasmid by means of homologous recombination. In addition, complementation and P1 mapping data suggest that sueB is also a mutation in the same position as the RF1 gene. We propose that the gene for RF1 be named prfA after protein release factor.
Heat shock proteins of vegetative and fruiting Myxococcus xanthus cells
Authors: Nelson D R , Killeen K P
Published: November 2016

The heat shock response of Myxococcus xanthus was investigated and characterized. When shifted from 28 to 40 degrees C, log-phase cells rapidly ceased growth, exhibited a 50% reduction in CFU, and initiated the synthesis of heat shock proteins (HTPs). Heat-shocked log-phase M. xanthus cells labeled with [35S]methionine were found to produce 18 major HTPs. The HTPs, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, were characterized with regard to molecular mass, subcellular location (periplasm, membrane, or cytoplasm), and temperature required for expression. Most HTPs were expressed at 36 degrees C, the optimum growth temperature of M. xanthus. Cells preincubated at 36 degrees C for 1 h before being shifted to 40 degrees C demonstrated increased thermotolerance compared with cells shifted directly from 28 to 40 degrees C. The HTPs produced by heat-shocked starvation-induced fruiting cells and glycerol-induced sporulating cells were also analyzed and characterized. Thirteen HTPs were detected in fruiting cells shifted from 28 to 40 degrees C. Six of these HTPs were not seen in vegetative M. xanthus cells. Log-phase cells induced to sporulate by the addition of glycerol produced 17 HTPs after being shifted to 40 degrees C. These HTPs were found to be a mixture of HTPs detected in heat-shocked log-phase cells and heat-shocked fruiting cells.
Photophosphorylation and oxidative phosphorylation in intact cells and chromatophores of an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114
Authors: Okamura K , Mitsumori F , Ito O , Takamiya K , Nishimura M
Published: November 2016

Light-induced ATP synthesis was studied in intact cells and chromatophores of Erythrobacter sp. strain OCh114. ATP synthesis was measured by both the pH method and the luciferin-luciferase luminescence method. The rate of ATP synthesis was moderate (a typical value of 0.65 mol of ATP per mol of bacteriochlorophyll per min), and synthesis was inhibited by antimycin A. ATP was synthesized under illumination only under aerobic conditions and not under anaerobic conditions. This characteristic was similar to that of other light-induced energy transduction processes in this bacterial species, such as oxidation of reaction center, oxidation of cytochrome c551, and translocation of H+, which were not observed under anaerobic conditions. This phenomenon was reconciled with the fact that the Erythrobacter sp. could not grow anaerobically even in the light. The characteristics of oxidative phosphorylation and ATP hydrolysis were also investigated. The respiratory ratio of chromatophores was 2.3. Typical rates of oxidative phosphorylation by NADH and by succinate were 2.9 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.22) and 1.1 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.19), respectively. A typical rate of ATP hydrolysis was 0.25 mol of ATP per mol of bacteriochlorophyll per min in chromatophores. ATPase and adenylate kinase are also involved in the metabolism of adenine nucleotides in this bacterium.
Heat shock regulatory gene rpoH mRNA level increases after heat shock in Escherichia coli
Authors: Tilly K , Erickson J , Sharma S , Georgopoulos C
Published: November 2016

The Escherichia coli rpoH gene product sigma 32 is essential for the increase in heat shock gene transcription found after exposure of the bacteria to a sudden temperature increase. It is not known how the concentration of active sigma 32 is modulated. We showed that rpoH transcript levels increased after heat shock and that the magnitude of the increase in the level of mRNA was correlated with the magnitude of the temperature shift. The increase in the level of rpoH mRNA was still found in rpoH mutants so the mechanism of induction differed from that of the set of previously identified heat shock genes. The increased concentration of rpoH mRNA should result in a higher level of sigma 32, which is likely to be important for increasing heat shock gene transcription.
Isolation of intact chains of polyphosphate from "Propionibacterium shermanii" grown on glucose or lactate
Authors: Clark J E , Beegen H , Wood H G
Published: November 2016

A procedure is presented for the isolation of intact polyphosphate (poly P) from "Propionibacterium shermanii." It is demonstrated, by including [32P]poly P during the extraction, that this procedure does not hydrolyze the poly P, and it is shown that two other widely used procedures do cause breakdown of the poly P. The procedure presented allows isolation of three fractions, short-chain poly P which is soluble in trichloroacetic acid, long-chain poly P which is soluble at neutral pH, and long-chain poly P which is present in volutin granules. Cells which had been grown on lactate did not contain short-chain poly P but did contain a high amount of long-chain poly P, which accumulated to 3% of the cell dry weight. At least 70% of this poly P was present in volutin granules. The poly P ranged in length from 250 to 725 phosphate residues and was the same average size as that synthesized in vitro by the poly P kinase from "P. shermanii". This indicates that the poly P kinase is responsible for catalyzing the synthesis of the poly P. In contrast to cells grown on lactate, those which had been grown on glucose did not contain volutin granules, did contain short-chain poly P and had 100-fold less long-chain poly P than lactate-grown cells. We propose that during the fermentation of glucose, the amount of poly P is lower than during growth on lactate because it is continuously utilized as a substrate in the phosphorylation of glucose.
Comparison of the malA regions of Escherichia coli and Klebsiella pneumoniae
Authors: Bloch M A , Raibaud O
Published: November 2016

Using the mini-Mu-duction technique, we cloned the malA regions from Escherichia coli K-12 and Klebsiella pneumoniae. A comparison of the structures of the cloned DNAs indicated that the malT, malP, and malQ genes, encoding the transcriptional activator of the maltose regulon, maltodextrin phosphorylase, and amylomaltase, respectively, are similarly organized in both species; malP and malQ constitute an operon divergent from the malT gene. We sequenced 1,200 nucleotides encompassing the beginnings of the malT and malP genes, their promoters, and the intergenic region. The DNA sequences from the two species were very different; the levels of homology ranged from 28 to 80%, depending on the region. The sequences of the coding regions and of elements known to be important for the functions of these two promoters in E. coli were well conserved between the two bacteria, whereas the sequence of the malT-malP intergenic region had totally diverged.
Cloning and expression in Escherichia coli of the gene for an Arthrobacter beta-(1----3)-glucanase
Authors: Doi K , Doi A
Published: November 2016

When inserted in the correct orientation at the BamHI site of plasmid YRp7, an 8.6-kilobase BamHI fragment of Arthrobacter sp. strain YCWD3 DNA gave Escherichia coli HB101 cells harboring the recombinant plasmid pBX20 the ability to lyse bakers' yeast cell walls or bakers' yeast glucan in agar medium. An extract of the transformed E. coli cells contained an endo-beta-(1----3)-glucanase with the same activity pattern as that of glucanase I produced by Arthrobacter sp. strain YCWD3. Although part of the glucanase activity was contributed by apparently defective molecules, two protein species were found which had high lytic activity on yeast cell walls and adsorbed to microcrystalline cellulose, and both had a single constituent polypeptide with a molecular weight of about 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these properties the protein species were indistinguishable from those glucanase I protein species of Arthrobacter sp. strain YCWD3 which we believe are nearly the intact molecule. We conclude that the cloned fragment of Arthrobacter sp. strain YCWD3 DNA contains the structural gene for glucanase I. A recombinant plasmid obtained by subcloning a PstI fragment of pBX20 into pBR322 caused the transformed E. coli cells to produce apparently defective glucanase molecules only. This observation serves as additional supporting evidence for our conclusion.
The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA
Authors: Hood E E , Helmer G L , Fraley R T , Chilton M D
Published: November 2016

We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.
Interaction between two regulatory proteins in osmoregulatory expression of ompF and ompC genes in Escherichia coli: a novel ompR mutation suppresses pleiotropic defects caused by an envZ mutation
Authors: Matsuyama S , Mizuno T , Mizushima S
Published: November 2016

The ompR and envZ genes, which together constitute the ompB operon, are involved in osmoregulatory expression of the OmpF and OmpC proteins, major outer membrane proteins of Escherichia coli. The envZ11 mutation results in the OmpF- OmpC-constitutive phenotype. A mutant which suppressed defects caused by the envZ11 mutation was isolated. The suppressor mutation also suppressed the LamB- PhoA- phenotype caused by the envZ11 mutation. The mutation occurred in the ompR gene and hence was termed ompR77. The ompR77 mutation alone produced no obvious phenotype. Functioning of the ompR77 allele remained envZ gene dependent. Although the ompR77 mutation suppressed the envZ11 mutation, it did not suppress a mutation that occurred in another position within the envZ gene (envZ160). These results indicate that OmpR and EnvZ, two regulatory proteins, functionally interact with each other.
Isolation and nucleotide sequence analysis of the ferredoxin I gene from the cyanobacterium Anacystis nidulans R2
Authors: Reith M E , Laudenbach D E , Straus N A
Published: November 2016

Two mixed oligonucleotide probes derived from conserved regions of the Synechocystis sp. strain PCC 6714 ferredoxin amino acid sequence were utilized to isolate an Anacystis nidulans R2 clone containing the ferredoxin I gene. Nucleotide sequence analysis revealed a 297-base-pair (bp) open reading frame with a deduced amino acid sequence having high homology to other cyanobacterial ferredoxins. Assuming proteolytic cleavage of the initial methionine residue, the molecular weight of the mature A. nidulans R2 ferredoxin was 10,370. The initial methionine residue was preceded by a probable ribosome-binding site sequence, AGGA. Northern hybridization analysis with the cloned ferredoxin gene indicated an RNA transcript of approximately 450 bp. S1 nuclease mapping localized the transcription start site to a position 64 bases upstream from the initial methionine residue. The nucleotide sequence 14 to 8 bp preceding the transcription start site resembled a typical Escherichia coli promoter, but the sequence in the -35 region did not. Southern hybridization detected only a single copy of the ferredoxin sequence in the A. nidulans R2 genome.
gamma-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties
Authors: Suzuki H , Kumagai H , Tochikura T
Published: November 2016

gamma-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) was purified from the periplasmic fraction of Escherichia coli K-12 to electrophoretic homogeneity. The final purification step, chromatofocusing, gave two protein peaks showing GGT activity (fractions A and B). The major heavy fraction (fraction A) consisted of two different subunits, with molecular weights of 39,200 and 22,000. The minor light fraction (fraction B) consisted of those with molecular weights of 38,600 and 22,000. Fraction A catalyzes the hydrolysis and transpeptidation of all gamma-glutamyl compounds tested, but it prefers basic amino acids and aromatic amino acids as acceptors. The apparent Km values for glutathione and gamma-glutamyl-p-nitroanilide as gamma-glutamyl donors in the transpeptidation reaction were both 35 microM, and those for glycylglycine and L-arginine as acceptors were 0.59 and 0.21 M, respectively. The enzyme was inhibited by some amino acids and by protease inhibitors and affinity-labeling reagents for GGT. The temperature stability of the purified GGT supports our hypothesis that E. coli GGT is synthesized only at lower temperature rather than that the synthesized GGT is degraded or inactivated at higher temperature.
gamma-Glutamyltranspeptidase from Escherichia coli K-12: formation and localization
Authors: Suzuki H , Kumagai H , Tochikura T
Published: November 2016

Escherichia coli cells showed maximum activity of gamma-glutamyltranspeptidase (EC 2.3.2.2) when they were grown at 20 degrees C, 14% of maximum activity at 37 degrees C, and none at 43 degrees C. The enzyme activity of intact cells grown at 20 degrees C was stably maintained after the temperature was changed to 45 degrees C. The activity increased during the exponential phase, and maximum activity was found at stationary phase. Its intracellular localization in the periplasmic space was confirmed.
Growth-rate-dependent regulation of the expression and inactivation of thymidylate synthase in Saccharomyces cerevisiae
Authors: Greenwood M T , Calmels E M , Storms R K
Published: November 2016

Thymidylate synthase activity fluctuated dramatically as cultures of Saccharomyces cerevisiae progressed through the different stages of batch culture growth. During logarithmic growth these yeast cultures each contained about 40 microU (1 microU is 1 pmol of 3H released per min) of thymidylate synthase activity per 10(8) haploid cells, but as cultures entered the stationary phase and during the stationary phase, activity dropped dramatically, eventually reaching undetectable levels. Stimulation of stationary-phase cells with fresh medium resulted in rapid reestablishment of log phase levels. Two mechanisms, the regulation of thymidylate synthase-specific transcripts and the irreversible inactivation of thymidylate synthase activity, acted in concert to regulate activity levels. These results suggested that thymidylate synthase represents a special subset of yeast proteins whose levels per cell vary quickly and dramatically in response to changes in proliferation rates.
Accumulation of a murein-membrane attachment site fraction when cell division is blocked in lkyD and cha mutants of Salmonella typhimurium and Escherichia coli
Authors: Chakraborti A S , Ishidate K , Cook W R , Zrike J , Rothfield L I
Published: November 2016

Membrane fractionation studies were performed on Salmonella typhimurium lkyD(Ts) and E. coli cha(Ts) mutants that appeared to be blocked at a late stage of the cell division cycle. In both cases growth of the mutant strains at nonpermissive temperatures was associated with accumulation of a characteristic cell envelope fraction (fraction OML) that contained inner membrane, murein, and outer membrane components. The isolated fraction corresponded in composition and bouyant density to a fraction from wild-type strains that had previously been suggested (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-767, 1982; K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, B. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) to contain adhesion sites between inner membrane, murein, and outer membrane. The accumulation of OML in LkyD- and Cha- cells was prevented by treatments that blocked DNA synthesis. The effects of interference with DNA synthesis did not appear to involve the SOS response.
Compartmentalization of the periplasmic space at division sites in gram-negative bacteria
Authors: Cook W R , MacAlister T J , Rothfield L I
Published: November 2016

Phase-contrast and serial-section electron microscopy were used to study the patterns of localized plasmolysis that occur when cells of Salmonella typhimurium and Escherichia coli are exposed to hypertonic solutions of sucrose. In dividing cells the nascent septum was flanked by localized regions of periseptal plasmolysis. In randomly growing populations, plasmolysis bays that were not associated with septal ingrowth were clustered at the midpoint of the cell and at 1/4 and 3/4 cell lengths. The localized regions of plasmolysis were limited by continuous zones of adhesion that resembled the periseptal annular adhesion zones described previously in lkyD mutants of S. typhimurium (T. J. MacAlister, B. MacDonald, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 80:1372-1376, 1983). When cell division was blocked by growing divC(Ts) cells at elevated temperatures, the localized regions of plasmolysis were clustered along the aseptate filaments at positions that corresponded to sites where septum formation occurred when cell division was permitted to resume by a shift back to the permissive temperature. Taken together the results are consistent with a model in which extended zones of adhesion define localized compartments within the periplasmic space, predominantly located at future sites of cell division.
Biosynthesis of Rhizobium trifolii capsular polysaccharide: enzymatic transfer of pyruvate substitutions into lipid-bound saccharide intermediates
Authors: Gardiol A E , Dazzo F B
Published: November 2016

The activity of capsular polysaccharide pyruvyltransferase catalyzing the pyruvylation of acidic heteropolysaccharide was measured in Rhizobium trifolii 843 and 0403 rif. This enzyme activity was determined with EDTA-treated cells, uridine diphosphate-sugar precursors, and phosphoenol [1-14C]pyruvate. Activity was measured by the incorporation of radioactivity into organic solvent-soluble glycoconjugates. Enzymatic pyruvylation of capsular polysaccharide occurred from phosphoenolpyruvate at the lipid-bound saccharide stage.
Spiral conformation of Vibrio cholerae as determined by scanning electron microscopy of elongated cells induced by cephalexin treatment
Authors: Konishi H , Katayama A , Ito T , Tanaka S , Yoshii Z
Published: November 2016

The elongated cells of Vibrio spp. induced by cephalexin treatment were examined by scanning electron microscopy. The results showed that Vibrio cholerae has a twisted cell body and a right-handed spiral conformation and that the cell bodies of V. parahaemolyticus and V. alginolyticus are straight rather than curved.
Inhibition of biosynthesis of Saccharomyces cerevisiae sugar transport system by tunicamycin
Authors: Lagunas R , DeJuan C , Benito B
Published: November 2016

Tunicamycin apparently inhibited the biosynthesis of glucose, galactose, and maltose transport systems in Saccharomyces cerevisiae. Under the conditions used, the antibiotic also blocked the biosynthesis of invertase, a well-known yeast glycoprotein, as well as the glycosylation of a marker mannoprotein of the yeast cell wall. However, the antibiotic did not affect certain proteins which did not contain carbohydrate. It seems, therefore, that these sugar carriers are glycoproteins.
Apurinic endonucleases from Saccharomyces cerevisiae also recognize urea residues in oxidized DNA
Authors: Chang C C , Kow Y W , Wallace S S
Published: November 2016

Saccharomyces cerevisiae apurinic endonucleases E cochromatographed with activity against a DNA substrate containing urea residues. The urea-recognizing activity of endonuclease E was competitively inhibited by apurinic DNA, and the heat labilities of both activities were the same. The apparent VmaxS of endonuclease E for both substrates were about the same, while the apparent Km for urea-containing DNA was about threefold greater than that for apurinic DNA. These results were similar to those obtained previously with Escherichia coli exonuclease III (Y. Kow and S. Wallace, Proc. Natl. Acad. Sci. USA 82:8354-8358, 1985) and suggest that the ability to recognize urea residues may be a general property of apurinic endonucleases.
Negative modulation of Escherichia coli NAD kinase by NADPH and NADH
Authors: Zerez C R , Moul D E , Gomez E G , Lopez V M , Andreoli A J
Published: November 2016

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2+ of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyme was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme. These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2+ and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.
"White-opaque transition": a second high-frequency switching system in Candida albicans
Authors: Slutsky B , Staebell M , Anderson J , Risen L , Pfaller M , Soll D R
Published: November 2016

A second high-frequency switching system was identified in selected pathogenic strains in the dimorphic yeast Candida albicans. In the characterized strain WO-1, cells switched heritably, reversibly, and at a high frequency (approximately 10(-2] between two phenotypes readily distinguishable by the size, shape, and color of colonies formed on agar at 25 degrees C. In this system, referred to as the "white-opaque transition," cells formed either "white" hemispherical colonies, which were similar to the ones formed by standard laboratory strains of C. albicans, or "opaque" colonies, which were larger, flatter, and grey. At least three other heritable colony phenotypes were generated by WO-1 and included one irregular-wrinkle and two fuzzy colony phenotypes. The basis of the white-opaque transition appears to be a fundamental difference in cellular morphology. White cells were similar in shape, size, and budding pattern to cells of common laboratory strains. In dramatic contrast, opaque cells were bean shaped and exhibited three times the volume and twice the mass of white cells, even though these alternative phenotypes contained the same amount of DNA and a single nucleus in the log phase. In addition to differences in morphology, white and opaque cells differed in their generation time, in their sensitivity to low and high temperatures, and in their capacity to form hypae. The possible molecular mechanisms involved in high-frequency switching in the white-opaque transition are considered.
Characterization of the motile hormogonia of Mastigocladus laminosus
Authors: Hernández-Muñiz W , Stevens S E
Published: November 2016

The cyanobacterium Mastigocladus laminosus produces motile hormogonia which move by gliding motility. These hormogonia were characterized in terms of their morphology, state of differentiation of the cells, optimal temperature for production and motility, minimal nutritional requirements to sustain motility, liberation of the hormogonium from its parental trichome, average surface velocity, and maximal concentration of agar through which the hormogonium may move. We found that an average hormogonium consisted of 13.6 cells of only the narrow-cell-type morphology. Gliding motility and the production of hormogonia were maximal at 45 degrees C. Agarose plus 0.20 mM Ca2+ was sufficient to sustain gliding motility. Hormogonia were liberated from the parental trichome by formation and lysis of a necridium. The average surface velocity of a hormogonium was 1.7 micron/s with a maximal velocity of 3 micron/s. Hormogonia were motile through 7% agar. Motile hormogonia leave a record of their passage in the form of easily visible tracks on the surface of solid media. Three types of tracks were observed: straight, sinusoidal, and circular. Normal, forward-directed motion involves screwlike rotation that describes a right-handed helix. However, observations are presented which suggest that rotational motion is not a prerequisite for gliding motility in this cyanobacterium.
Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa
Authors: Deretic V , Gill J F , Chakrabarty A M
Published: November 2016

Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 35S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglII-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability. With the algD-xylE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor.
Analysis of mutational lesions of acetate metabolism in Neurospora crassa by 13C nuclear magnetic resonance
Authors: Thomas G H , Baxter R L
Published: November 2016

The adaptation of Neurospora crassa mycelium to growth on acetate as the sole carbon source was examined by using 13C nuclear magnetic resonance. Extracts were examined by nuclear magnetic resonance at various times after transfer of the mycelium from medium containing sucrose to medium containing [2-13C]acetate as the sole carbon source. The label was initially seen to enter the alanine, glutamate, and glutamine pools, and after 6 h 13C-enriched trehalose was evident, indicating that gluconeogenesis was occurring. Analysis of the isotopomer ratios in the alanine and glutamate-glutamine pools indicated that substantial glyoxylate cycle activity became evident between 2 and 4 h after transfer. Immediately after transfer of the mycelium to acetate medium, the alanine pool increased to about four times its previous level, only a small fraction of which was enriched with 13C. The quantity of 13C-enriched alanine remained almost constant between 2 and 7.5 h after the transfer, whereas the overall alanine pool decreased to its original level. The selective catabolism of the unenriched alanine leads us to suggest that the alanine pool is partitioned into two compartments during adaptation. Two acetate-nonutilizing mutants were also studied by this technique. An acu-3 strain, deficient for isocitrate lyase (EC 4.1.3.1) activity, showed metabolic changes consistent with this lesion. An acp strain, previously thought to be deficient in an inducible acetate permease, took up [2-13C]acetate but showed no evidence of glyoxylate cycle activity despite synthesizing the necessary enzymes; the lesion was therefore reinterpreted.
Phenol: a complex chemoeffector in bacterial chemotaxis
Authors: Imae Y , Oosawa K , Mizuno T , Kihara M , Macnab R M
Published: November 2016

Earlier observations that phenol is a repellent for Salmonella typhimurium but an attractant for Escherichia coli were confirmed. This behavioral difference was found to correlate with a difference in the effect phenol had on receptor methylation levels; it caused net demethylation in S. typhimurium but net methylation in E. coli. On the basis of mutant behavior and measurement of phenol-stimulated methylation, the attractant response of E. coli was shown to be mediated principally by the Tar receptor. In S. typhimurium, two receptors were found to be sensitive to phenol, namely, an unidentified receptor, which mediated the repellent response and showed phenol-stimulated demethylation; and the Tar receptor, which (as with E. coli) mediated the attractant response and showed phenol-stimulated methylation. In wild-type S. typhimurium, the former receptor dominated the Tar receptor, with respect to both behavior and methylation changes. However, when the amount of Tar receptor was artificially increased by the use of Tar-encoding plasmids, S. typhimurium cells exhibited an attractant response to phenol. No protein analogous to the phenol-specific repellent receptor was evident in E. coli, explaining the different behavioral responses of the two species toward phenol.
Characterization of the specific pyruvate transport system in Escherichia coli K-12
Authors: Lang V J , Leystra-Lantz C , Cook R A
Published: November 2016

A mutant of Escherichia coli K-12 lacking pyruvate dehydrogenase and phosphoenolpyruvate synthase was used to study the transport of pyruvate by whole cells. Uptake of pyruvate was maximal in mid-log phase cells, with a Michaelis constant for transport of 20 microM. Pretreatment of the cells with respiratory chain poisons or uncouplers, except for arsenate, inhibited transport up to 95%. Lactate and alanine were competitive inhibitors, but at nonphysiological concentrations. The synthetic analogs 3-bromopyruvate and pyruvic acid methyl ester inhibited competitively. The uptake of pyruvate was also characterized in membrane vesicles from wild-type E. coli K-12. Transport required an artificial electron donor system, phenazine methosulfate and sodium ascorbate. Pyruvate was concentrated in vesicles 7- to 10-fold over the external concentration, with a Michaelis constant of 15 microM. Energy poisons, except arsenate, inhibited the transport of pyruvate. Synthetic analogs such as 3-bromopyruvate were competitive inhibitors of transport. Lactate initially appeared to be a competitive inhibitor of pyruvate transport in vesicles, but this was a result of oxidation of lactate to pyruvate. The results indicate that uptake of pyruvate in E. coli is via a specific active transport system.