bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation
Authors: Burlingame R P , Wyman L , Chapman P J
Published: November 2016

Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities. Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium. Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate. The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer. Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme. One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways.
Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans"
Authors: Kieser T , Moss M T , Dale J W , Hopwood D A
Published: November 2016

The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.
Association of transformation of xenosomes from nonkiller to killer with extrachromosomal DNA
Authors: Soldo A T , Brickson S A , Castiglione G A , Freytag A F
Published: November 2016

Extrachromosomal DNA in the form of covalently closed circular DNA molecules was isolated from killer and nonkiller xenosomes, bacterial endosymbionts of the marine protozoan Parauronema acutum. Restriction endonuclease digests of these molecules derived from 12 isolates revealed consistent, readily identifiable, differences in the pattern of fragments of the killer as compared with those present in the nonkiller. Transformation of the nonkiller to killer by infection is also accompanied by a change from the nonkiller to killer pattern. Based on analysis of fragments resulting from restriction endonuclease digests, two circular duplex DNA molecules, each 63 kilobase pairs (kbp) in length, were identified in the 263-20 nonkiller stock and mapped. The maps revealed that each possesses a single BamHI site and multiple BglI, BstIIE, PstI, and SalI sites. A distinguishing feature of these maps is that the two molecules share a region about 17 kbp in length in which multiple restriction sites are in register with each other. Allowing for a 0.5-kbp insertion or deletion and the introduction or removal of only a few restriction sites, an additional stretch extending approximately 31 kbp beyond this sequence could also be considered to be homologous. The structure of the killer plasmid appears to be more complex, and we have been unable, as yet, to construct physical maps for this DNA. We postulate that the killer plasmid DNA is composed of three, perhaps four, circular 63-kbp duplexes, at least one which contains a single BamHI site and another which contains two BamHI sites. The remaining molecules may represent copies of either or both of the other two, modified to contain additional restriction sites. Transformation from the nonkiller to the killer is visualized as the insertion of restriction sites at various points along parent nonkiller plasmid DNA molecules. The mechanism by which these sites are introduced is unknown.
Purification of extracellular lipase from Pseudomonas aeruginosa
Authors: Stuer W , Jaeger K E , Winkler U K
Published: November 2016

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.
Molecular cloning and expression of Rhizobium fredii USDA 193 nodulation genes: extension of host range for nodulation
Authors: Ramakrishnan N , Prakash R K , Shantharam S , Duteau N M , Atherly A G
Published: November 2016

DNA hybridization with the cloned nodulation region of Rhizobium meliloti as a probe revealed DNA homology with four HindIII fragments, 12.5, 6.8, 5.2, and 0.3 kilobases (kb) in size, of the symbiotic plasmid pRjaUSDA193. Both hybridization and complementation studies suggest that the common nodulation genes nodABC and nodD of R. fredii USDA 193 are present on the 5.2-kb HindIII and 2.8-kb EcoRI fragments, respectively, of the Sym plasmid. Both fragments together could confer nodulation ability on soybeans when present in Sym plasmid-cured (Sym-) and wild-type (Sym+) Rhizobium strains or in a Ti plasmid-cured Agrobacterium tumefaciens strain. Furthermore, the 2.8-kb EcoRI fragment alone was able to form nodulelike structures on Glycine max L. cv. "Peking" (soybean). Microscopic examination of these nodules revealed bacterial invasion of the cells, probably via root hair penetration. Bacterial strains harboring plasmids carrying the 5.2- and 2.8-kb nod fragments elicited root-hair-curling responses on infection. These data suggest that the genes responsible for host range determination and some of the early events of nodulation may be coded for by the 5.2-kb HindIII and 2.8-kb EcoRI fragments.
Ferrous iron transport in Streptococcus mutans
Authors: Evans S L , Arceneaux J E , Byers B R , Martin M E , Aranha H
Published: November 2016

Radioiron uptake from 59FeCl3 by Streptococcus mutans OMZ176 was increased by anaerobiosis, sodium ascorbate, and phenazine methosulfate (PMS), although there was a 10-min lag before PMS stimulation was evident. The reductant ascorbate may have provided ferrous iron. The PMS was reduced by the cells, and the reduced PMS then may have generated ferrous iron for transport; reduced PMS also may have depleted dissolved oxygen. We conclude that S. mutans transports only ferrous iron, utilizing reductants furnished by glucose metabolism to reduce iron prior to its uptake.
Transport of trehalose in Salmonella typhimurium
Authors: Postma P W , Keizer H G , Koolwijk P
Published: November 2016

We have studied trehalose uptake in Salmonella typhimurium and the possible involvement of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in this process. Two transport systems could recognize and transport trehalose, the mannose PTS and the galactose permease. Uptake of trehalose via the latter system required that it be expressed constitutively (due to a galR or galC mutation). Introduction of a ptsM mutation, resulting in a defective IIMan/IIIMan system, in S. typhimurium strains that grew on trehalose abolished growth on trehalose. A ptsG mutation, eliminating IIGlc of the glucose PTS, had no effect. In contrast, a crr mutation that resulted in the absence of IIIGlc of the glucose PTS prevented growth on trehalose. The inability of crr and also cya mutants to grow on trehalose was due to lowered intracellular cyclic AMP synthesis, since addition of extracellular cyclic AMP restored growth. Subsequent trehalose metabolism could be via a trehalose phosphate hydrolase, if trehalose phosphate was formed via the PTS, or trehalase. Trehalose-grown cells contained trehalase activity, but we could not detect phosphoenolpyruvate-dependent phosphorylation of trehalose in toluenized cells.
cis-acting sites required for osmoregulation of ompF expression in Escherichia coli K-12
Authors: Ostrow K S , Silhavy T J , Garrett S
Published: November 2016

OmpF and OmpC are major outer membrane proteins which form passive diffusion pores in Escherichia coli K-12. The expression of the structural genes for these proteins, ompF and ompC, is influenced by medium osmotic strength and requires the products of two regulatory genes, ompR and envZ. We have constructed a series of ompF-lacZ fusions containing different regions of ompF to determine sites involved with osmoregulation. These fusions were crossed onto a specialized transducing phage and integrated into the bacterial chromosome in unit copy. By measuring the fluctuations of beta-galactosidase activity in lysogens grown in high versus low osmolarity, we have identified three regions which are necessary. Furthermore, we have determined that, although the OmpR activation site is not sufficient, OmpR is probably essential for ompF osmoregulation.
Oxidative phosphorylation and energy buffering in cyanobacteria
Authors: Nitschmann W H , Peschek G A
Published: November 2016

The onset of respiration in the cyanobacteria Anacystis nidulans and Nostoc sp. strain Mac upon a shift from dark anaerobic to aerobic conditions was accompanied by rapid energization of the adenylate pool (owing to the combined action of ATP synthase and adenylate kinase) and also the guanylate, uridylate, and cytidylate pools (owing to nucleoside diphosphate and nucleoside monophosphate kinases). Rates of the various transphosphorylation reactions were comparable to the rate of oxidative phosphorylation, thus explaining, in part, low approximately P/O ratios which incorporate adenylates only. The increase of ATP, GTP, UTP, and CTP levels (nanomoles per minute per milligram [dry weight]) in oxygen-pulsed cells of A. nidulans and Nostoc species was calculated to be, on average, 2.3, 1.05, 0.8, and 0.57, respectively. Together with aerobic steady-state pool sizes of 1.35, 0.57, 0.5, and 0.4 nmol/mg (dry weight) for these nucleotides, a fairly uniform turnover of 1.3 to 1.5 min-1 was derived. All types of nucleotides, therefore, may be conceived of as being in equilibrium with each other, reflecting the energetic homeostasis or energy buffering of the (respiring) cyanobacterial cell. For the calculation of net efficiencies of oxidative phosphorylation in terms of approximately P/O ratios, this energy buffering was taken into account. Moreover, in A. nidulans an additional 30% of the energy initially conserved in ATP by oxidative phosphorylation was immediately used up by a plasma membrane-bound reversible H+-ATPase for H+ extrusion. Consequently, by allowing for energy buffering and ATPase-linked H+ extrusion, maximum P/O ratios of 2.6 to 3.3 were calculated. By contrast, in Nostoc sp. all the H+ extrusion, appeared to be linked to a plasma membrane-bound respiratory chain, thus bypassing any ATP formation and leading to P/O ratios of only 1.3 to 1.5 despite the correction for energy buffering.
Germination conditions that require mitochondrial function in Saccharomyces cerevisiae: utilization of acetate and galactose
Authors: Donnini C , Artoni N , Marmiroli N
Published: November 2016

Ascospores of Saccharomyces cerevisiae inherited at least one functioning mitochondrion as shown by their ability to germinate on nonfermentable carbon sources. After transfer to germination medium, the optical density of the culture at 600 nm decreased (phase-dark), reaching a minimum within 60 min in the presence of glucose and within 180 min after transfer to acetate medium; thereafter, the optical density increased. Budding cells first appeared 90 min after transfer to glucose and 150 min after transfer to acetate. Augmentation of respiratory components, respiratory activity, and macromolecular synthesis (except for DNA synthesis) started at about the same time on glucose and on acetate, although the highest values for all these processes were reached in the presence of glucose. Mitochondrial inhibitors which affected germination on acetate did not arrest germination on glucose. However, mitochondrial activity was required for germination on galactose in a strain carrying the mutated allele imp1 of the nucleomitochondrion-connecting gene IMP1.
The Saccharomyces cerevisiae start mutant carrying the cdc25 mutation is defective in activation of plasma membrane ATPase by glucose
Authors: Portillo F , Mazón M J
Published: November 2016

Activation of plasma membrane ATPase by the addition of glucose was examined in several cell division cycle mutants of Saccharomyces cerevisiae. The start mutant carrying the cdc25 mutation was shown to be defective in ATPase activation at the restrictive temperature. Genetic analysis showed that lack of growth and defective activation of ATPase at the restrictive temperature were caused by the same mutation. It was also found that CDC25 does not map at the same locus as the structural gene of plasma membrane ATPase (PMA1). We conclude that the product of CDC25 controls the activation of ATPase.
Isolation and sequence of the gene for ferredoxin I from the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Alam J , Whitaker R A , Krogmann D W , Curtis S E
Published: November 2016

The structural gene for ferredoxin I, petF, from the cyanobacterium Anabaena sp. strain PCC 7120 has been isolated from a recombinant lambda library. Mixtures of tetradecanucleotides and heptadecanucleotides, each containing all possible DNA sequences corresponding to two separate regions of the ferredoxin amino acid sequence, were synthesized and used as hybridization probes to identify a genomic clone containing the coding sequence for the petF gene. The sequence of the entire petF coding region and portions of the 3'- and 5'-flanking regions was determined. The DNA sequence of petF suggests that, in contrast to the nucleus-encoded plant protein, cyanobacterial apoferredoxin is not synthesized as a higher-molecular-weight precursor. The Anabaena petF gene is a single-copy gene. During growth on complete medium it was transcribed into a monocistronic mRNA species of approximately 500 bases that initiated 100 base pairs upstream from the petF coding region.
Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications
Authors: Thompson J F , Waechter-Brulla D , Gumport R I , Gardner J F , Moitoso de Vargas L , Landy A
Published: November 2016

The manner in which integration host factor (IHF) regulates lambda site-specific recombination has been analyzed by examining the behavior of both wild-type and mutant DNAs in integrative and excisive recombination as well as in protein binding. While integrative recombination of an attP with two base changes in the H1 site required 8-fold more IHF than did wild type, binding to this site was lowered at least 500-fold, suggestive of cooperative interactions. A mutant attP with nine base changes did not integrate at all in vitro, with the defect being less severe in vivo. IHF inhibition of excisive recombination was relieved by both mutations in vitro and in vivo. These results imply that occupancy of the H1 site is critical for determining the direction of recombination. It is proposed that IHF inhibition of excision provides a monitor of the strength of the induction stimulus and the nutritional state of the cell; this would allow the prophage to excise selectively in conditions which favor successful completion of the lytic cycle.
Purification and properties of the 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum
Authors: DiMarco A A , Donnelly M I , Wolfe R S
Published: November 2016

The 5,10-methenyltetrahydromethanopterin cyclohydrolase of Methanobacterium thermoautotrophicum was purified 128-fold to homogeneity. The enzyme had a subunit Mr of 41,000 as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From high-performance size exclusion chromatography of the native protein, an Mr of 82,000 was determined, suggesting a dimer of identical subunits. The enzyme was inhibited by 10-formyltetrahydromethanopterin and stimulated by Mg2+. Evaluation of the reaction equilibrium indicated that the methenyl derivative was favored over 5-formyltetrahydromethanopterin, with a much higher equilibrium constant than for the analogous reaction of tetrahydrofolate derivatives. Folate derivatives did not serve as substrates for this enzyme.
Malate transport in Schizosaccharomyces pombe
Authors: Osothsilp C , Subden R E
Published: November 2016

The transport of malate was studied in a Schizosaccharomyces pombe wild-type strain and in mutant strains unable to utilize malic acid. Two groups of such mutants, i.e., malic enzyme-deficient and malate transport-defective mutants, were differentiated by a 14C-labeled L-malate transport assay and by starch gel electrophoresis followed by activity staining for malic enzyme (malate dehydrogenase [oxaloacetate decarboxylating] [NAD+]; 1.1.1.38) and malate dehydrogenase (1.1.1.37). Transport of malate in S. pombe was constitutive and strongly inhibited by inhibitors of oxidative phosphorylation and of the formulation of proton gradients. Transport was a saturable function of the malate concentration. The apparent Km and Vmax values for transport by the parent were 3.7 mM and 40 nmol/min per mg of protein, respectively, while those of the malic enzyme-deficient mutant were 5.7 mM and 33 nmol/min per mg of protein, respectively. Malate transport was pH and temperature dependent. The specificity of transport was studied with various substrates, including mono- and dicarboxylic acids, and the possibility of a common transport system for dicarboxylic acids is discussed.
Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene
Authors: Hmiel S P , Snavely M D , Miller C G , Maguire M E
Published: November 2016

The influx of Mg2+ in Salmonella typhimurium LT-2 was studied by both kinetic and genetic techniques. Wild-type cells grown in a high MgSO4 concentration (10 mM) exhibited a Km of 15 microM for Mg2+ influx, with a Vmax of 0.25 nmol of Mg2+ per min per 10(8) cells. The apparent Km decreased to 3 microM, and the Vmax increased 60% after growth in a low MgSO4 concentration (10 microM). Co2+ was a simple competitive inhibitor (Ki = 30 microM) of Mg2+ influx in cells grown in high Mg2+ concentrations but blocked only a portion of the Mg2+ influx in cells grown in low Mg2+ concentrations. Co2+ influx exhibited kinetics similar to those of Mg2+ influx (Km = 30 microM; Vmax = 0.5 nmol of Co2+ per min per 10(8) cells) but was not affected by growth conditions. Co2+ influx was competitively inhibited by both Mg2+ and Mn2+. Mutations affecting Mg2+ uptake were isolated by selection for spontaneous resistance to toxic levels of Co2+. One class of mutants designated corA mapped at 84 min near metE with the following gene order: corA, metE, zie-3161::Tn10, pepQ. A second class designated corB mapped at 98 min near pyrB. Mg2+ influx was decreased in a corA mutant strain (relative to that of the wild type) when grown in high Mg2+ concentrations but was restored when grown in low Mg2+ concentrations. Co2+ transport was completely abolished by the corA mutation under all growth conditions. Recombinant plasmids carrying the corA region from either Escherichia coli K-12 or S. typhimurium complemented the corA mutation in S. typhimurium, restoring uptake of both Co2+ and Mg2+ and conferring sensitivity to Co2+. The S. typhimurium corA gene was localized to a restriction fragment of approximately 1.5 kilobases.
Effect of iron on accumulation of exotoxin A-specific mRNA in Pseudomonas aeruginosa
Authors: Lory S
Published: November 2016

A DNA probe from an internal fragment of the exotoxin A structural gene was used to study the effects of selected culture conditions on steady-state levels of exotoxin-specific mRNA in Pseudomonas aeruginosa. Cells grown under conditions of iron deprivation began to synthesize and excrete the exotoxin A polypeptide during the late exponential phase of growth and throughout the stationary phase of growth, concomitant with a sharp increase in exotoxin A mRNA pools in P. aeruginosa cells. The addition of iron to the medium resulted in the failure of these cells to synthesize exotoxin A mRNA, despite significantly enhanced growth. The inhibition of the production of exotoxin A and the accumulation of its mRNA by iron was dose dependent, with a half-maximal inhibitory concentration of FeSO4 of 5 to 10 microM. A blockade of the initiation of transcription by rifampin resulted in the decay of exotoxin A mRNA, with a half-life of approximately 8 to 10 min, depending on the media used for growth. The addition of iron to cells actively engaged in exotoxin A synthesis also resulted in a gradual decrease in the amount of this mRNA in bacteria. However, the rate of decline of mRNA induced by iron was relatively slow (half-life, 90 min), with a considerable lag time between the iron addition and the first detectable effect on mRNA. While iron clearly appears to influence the production of exotoxin A at the transcriptional level, the molecular basis of this effect may involve several interacting factors affecting the initiation of transcription and perhaps mRNA turnover.
Biological activity of the Asn-5,Arg-7 tridecapeptide encoded by MF alpha 2 of Saccharomyces cerevisiae
Authors: Raths S , Shenbagamurthi P , Naider F , Becker J M
Published: November 2016

The precursor predicted by the nucleotide sequence of the MF alpha 2 gene of Saccharomyces cerevisiae contains one copy of the tridecapeptide alpha-factor previously characterized (H2N-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-COOH) and one copy of a peptide that contains two conservative amino acid substitutions (H2N-Trp-His-Trp-Leu-Asn-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr-COOH). To determine whether the novel molecule possesses biological activity, the Asn-5,Arg-7 tridecapeptide was prepared chemically by solid-phase peptide synthesis. Growth arrest and morphogenesis assays gave identical activity profiles for the Asn-5,Arg-7 peptide and the other gene product, the Gln-5,Lys-7 peptide. The activities of the two peptides were additive and indistinguishable for S. cerevisiae X2180-1A. When present in fourfold molar excess, the biologically inactive desTrp-1,Ala-3 dodecapeptide reversed activity of the Asn-5,Arg-7 and Gln-5,Lys-7 tridecapeptides. Furthermore, neither peptide caused growth arrest of a MATa ste2(Ts) mutant when assayed at the restrictive temperature. These studies suggest that both pheromones interact with the alpha-factor receptor in a similar manner.
Agglutination and mating activity of the MF alpha 2-encoded alpha-factor analog in Saccharomyces cerevisiae
Authors: Kurjan J , Lipke P N
Published: November 2016

The MF alpha 2-encoded Asn-5,Arg-7 alpha-factor-like peptide has been shown shown to have similar activity to Gln-5,Lys-7 alpha-factor in morphogenesis and growth arrest studies (S. Raths, P. Shenbagamurthi, F. Naider, and J. M. Becker, J. Bacteriol. 168:1468-1471, 1986). We tested the Asn-5,Arg-7 peptide in agglutination and mating assays and found that its activity was similar to or slightly less than that of the Gln-5,Lys-7 alpha-factor. The Asn-5,Arg-7 alpha-factor-like peptide is thus the most active analog of the Gln-5,Lys-7 alpha-factor known.
Biological role of DNA methylation: sequence-specific single-strand breaks associated with hypomethylation of GATC sites in Escherichia coli DNA
Authors: Szyf M , Meisels E , Razin A
Published: November 2016

The effect of methylation of GATC sites in Escherichia coli DNA on the formation of single-strand breaks was studied with dam+, dam mutant, and Dam-overproducer strains. Single-strand breaks have been observed in dam mutant cells predominantly at TpT and, to a lesser extent, at CpC. In dam mutant cells harboring pTP166 (a plasmid containing the dam gene), no such nicks were observed.
Involvement of chlA, E, M, and N loci in Escherichia coli molybdopterin biosynthesis
Authors: Johnson M E , Rajagopalan K V
Published: November 2016

All molybdenum enzymes except nitrogenase contain a common molybdenum cofactor, whose organic moiety is a novel pterin called molybdopterin (MPT). To assist in elucidating the biosynthetic pathway of MPT, two MPT-deficient mutants of Escherichia coli K-12 were isolated. They lacked activities of the molybdenum enzymes nitrate reductase and formate dehydrogenase, did not reconstitute apo nitrate reductase from a Neurospora crassa nit-1 strain, and did not yield form A, a derivative of MPT. By P1 mapping, these two mutations mapped to chlA and chlE, loci previously postulated but never definitely shown to be involved in MPT biosynthesis. The two new mutations are in different genetic complementation groups from previously isolated chlA and chlE mutations and have been designated as chlM and chlN (closely linked to chlA and chlE, respectively). The reported presence of Mo cofactor activity in the chlA1 strain is shown to be due to in vitro synthesis of MPT through complementation between a trypsin-sensitive macromolecule from the chlA1 strain and a low-molecular-weight compound from the nit-l strain.
Mutations affecting the regulation of the metB gene of Salmonella typhimurium LT2
Authors: Urbanowski M L , Plamann L S , Stauffer G V
Published: November 2016

We isolated and characterized cis-acting mutations that affect the regulation of the metB gene of Salmonella typhimurium LT2. The mutations were isolated in an Escherichia coli lac deletion strain lysogenized with lambda bacteriophage carrying a metB-lacZ gene fusion (lambda JBlac) in which beta-galactosidase production is dependent upon metB gene expression. The mutant lysogens show elevated, poorly regulated beta-galactosidase production. The altered regulation is a result of disruption of the methionine control system mediated by the metJ repressor. The mutations are located in a region of dyad symmetry centered near the -35 sequence of the metB promoter. We propose that these mutations alter the repressor binding site and define the metB operator sequence. In addition, we discuss a highly conserved, nonsymmetric DNA sequence of unknown function which occurs in the control regions of the metA, metC, metE, metF, metG, and metJB genes of both S. typhimurium and E. coli.
Characterization of the ftsB gene as an allele of the nrdB gene in Escherichia coli
Authors: Kren B , Fuchs J A
Published: November 2016

A temperature-sensitive, salt-rescuable ftsB cell division mutant, MFT84, was found to be hydroxyurea sensitive on low-salt medium. Complementation studies with plasmids and a marker rescue study with bacteriophage M13 nrd indicated that ftsB is an allele of nrdB and that the mutation occurs in the region corresponding to nucleotides 6729 to 7032 of the nrdB gene. Enzymatic characterization demonstrated that the B2 subunit of ribonucleoside-diphosphate reductase encoded by ftsB was responsible for the decreased activity and the thermolability of the enzyme. The ftsB-encoded B2 subunit was activated by the addition of 0.1 M NaCl to an in vitro assay, corroborating the in vivo temperature-dependent salt requirement was a result of a defective B2 subunit.
Immunoelectron microscopic analysis of elongation of type 1 fimbriae in Escherichia coli
Authors: Lowe M A , Holt S C , Eisenstein B I
Published: November 2016

Using 10- and 20-nm-diameter gold particles conjugated to an antifimbrial monoclonal antibody, we analyzed the location of assembly of newly formed subunits on growing type 1 fimbriae of Escherichia coli. Fimbriae were removed from an E. coli K-12-derived strain, CSH50, by blending. Blended cells were allowed to regenerate their fimbriae in growth medium for approximately 25 min, after which they were labeled with a 20-nm-gold-monoclonal antibody probe. Continued outgrowth of these labeled fimbriae was allowed for additional time intervals, after which they were labeled with a 10-nm-gold-monoclonal antibody probe. The resulting fimbriae, double labeled with 10- and 20-nm-diameter gold particles, were examined in an electron microscope. The pattern of labeling on individual fimbrial organelles indicated morphologically that newly synthesized subunits are added to a growing organelle at its base.
Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia
Authors: Kropinski A M , Parr T R , Angus B L , Hancock R E , Ghiorse W C , Greenberg E P
Published: November 2016

The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl.
Genetic and morphological characterization of ftsB and nrdB mutants of Escherichia coli
Authors: Taschner P E , Verest J G , Woldringh C L
Published: November 2016

The ftsB gene of Escherichia coli is believed to be involved in cell division. In this report, we show that plasmids containing the nrdB gene could complement the ftsB mutation, suggesting that ftsB is an allele of nrdB. We compared changes in the cell shape of isogenic nrdA, nrdB, ftsB, and pbpB strains at permissive and restrictive temperatures. Although in rich medium all strains produced filaments at the restrictive temperature, in minimal medium only a 50 to 100% increase in mean cell mass occurred in the nrdA, nrdB, and ftsB strains. The typical pbpB cell division mutant also formed long filaments at low growth rates. Visualization of nucleoid structure by fluorescence microscopy demonstrated that nucleoid segregation was affected by nrdA, nrdB, and ftsB mutations at the restrictive temperature. Measurements of beta-galactosidase activity in lambda p(sfiA::lac) lysogenic nrdA, nrdB, and ftsB mutants in rich medium at the restrictive temperature showed that filamentation in the nrdA mutant was caused by sfiA (sulA) induction, while filamentation in nrdB and ftsB mutants was sfiA independent, suggesting an SOS-independent inhibition of cell division.
Reconstitution of pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase
Authors: Matsushita K , Nonobe M , Shinagawa E , Adachi O , Ameyama M
Published: November 2016

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.
Insertion-sequence-dependent rearrangements of Pseudomonas cepacia plasmid pTGL1
Authors: Gaffney T D , Lessie T G
Published: November 2016

Pseudomonas cepacia 249 (ATCC 17616) harbors a 170-kilobase (kb) plasmid designated pTGL1. We identified three insertion sequences, IS405, IS408, and IS411, on this plasmid. Various prototrophic and auxotrophic derivatives in our collection contained variants of pTGL1 formed by accretion and deletion of other elements. Plasmid pTGL6, the variant in one prototroph, evolved from pTGL1 by the addition of three copies of IS401 (1.3 kb) and one of IS402 (1 kb), to generate pTGL5, and recombination between two of the copies of IS401 on pTGL5 to form pTGL6. The latter event entailed loss of one copy of IS401 and an additional 5.4 kb of plasmid DNA. Derivatives of the broad-host-range plasmid pRP1 carrying the above insertion sequences and recombinant plasmids carrying fragments of plasmids pTGL6 and pTGL5 were used as probes to ascertain the extent of reiteration of the various elements in the P. cepacia genome. The data indicate a high frequency of genomic rearrangements which presumably contributes to the extraordinary adaptability of this bacterium.
Role of the response oscillator in inverse responses of Halobacterium halobium to weak light stimuli
Authors: Hildebrand E , Schimz A
Published: November 2016

Under certain conditions Halobacterium halobium organisms respond to a weak attractant light stimulus with a repellent response and to a weak repellent stimulus with an attractant response. The appearance of inverse responses depends on the stimulus strength, on the interval length between spontaneous reversals, and on the moment of stimulation during the interval. Although the cells are absolutely refractory to repellent stimuli for 500 ms after a reversal, repellent responses can be evoked even during that period if they are inverse responses to weak attractant stimuli. Simultaneous attractant and repellent stimuli cancel each other even when one of them leads to an inverse response, indicating that normal cellular signals occur at the site of signal integration. We postulate that the inverse responses are caused by certain properties of a cellular oscillator for which we previously postulated a role in response regulation and sensory control in halobacteria (A. Schimz and E. Hildebrand, Nature [London] 317:641-643, 1985).
Complementation of nitrogen-regulatory (ntr-like) mutations in Rhodobacter capsulatus by an Escherichia coli gene: cloning and sequencing of the gene and characterization of the gene product
Authors: Allibert P , Willison J C , Vignais P M
Published: November 2016

In vivo genetic engineering by R' plasmid formation was used to isolate an Escherichia coli gene that restored the Ntr+ phenotype to Ntr- mutants of the photosynthetic bacterium Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata; J. F. Imhoff, H. G. Trüper, and N. Pfenning, Int. J. Syst. Bacteriol. 34:340-343, 1984). Nucleotide sequencing of the gene revealed no homology to the ntr genes of Klebsiella pneumoniae. Furthermore, hybridization experiments between the cloned gene and different F' plasmids indicated that the gene is located between 34 and 39 min on the E. coli genetic map and is therefore unlinked to the known ntr genes. The molecular weight of the gene product, deduced from the nucleotide sequence, was 30,563. After the gene was cloned in an expression vector, the gene product was purified. It was shown to have a pI of 5.8 and to behave as a dimer during gel filtration and on sucrose density gradients. Antibodies raised against the purified protein revealed the presence of this protein in R. capsulatus strains containing the E. coli gene, but not in other strains. Moreover, elimination of the plasmid carrying the E. coli gene from complemented strains resulted in the loss of the Ntr+ phenotype. Complementation of the R. capsulatus mutations by the E. coli gene therefore occurs in trans and results from the synthesis of a functional gene product.
Mechanism and regulation of phosphate transport in Streptococcus pyogenes
Authors: Reizer J , Saier M H
Published: November 2016

In contrast to results reported with other bacteria, uptake of 32Pi in Streptococcus pyogenes was found to occur rapidly in starved cultures and to be strongly and immediately inhibited by addition of exogenous glycolytic energy sources (such as glucose) and nonglycolytic sources of ATP (such as arginine). Preincubation of starved cells with NaF, iodoacetate, or arsenate eliminated the inhibiting effect of glucose but not that of arginine. In accordance with the hypothesis that transport was attributable to Pi-Pi exchange, uptake and efflux of 32Pi in the presence of trans unlabeled Pi exhibited similar characteristics and were largely eliminated by reduction of the trans Pi concentration. Neither process was inhibited appreciably by pretreatment of cells with ionophores or metabolic inhibitors, but both processes were abolished by exposure to p-chloromercuribenzoate. Inhibition by both exogenous energy sources resulted in a reduction in the maximal velocity of transport (Vmax). Whereas arginine also caused a shift in the apparent Michaelis-Menten constant (Km) to larger values, glucose did not alter the Km. On the basis of the results reported, we propose that the rate of Pi exchange is determined positively by the intracellular and extracellular concentrations of Pi and negatively by ATP or metabolites thereof. The mechanism of ATP action is unknown but could involve either covalent or noncovalent modification of the carrier protein.
Cloning of the genes involved in synthesis of coenzyme pyrrolo-quinoline-quinone from Acinetobacter calcoaceticus
Authors: Goosen N , Vermaas D A , van de Putte P
Published: November 2016

Mutants of Acinetobacter calcoaceticus LMD79.41 were isolated that are defective in the synthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ). A gene bank of the wild-type. A. calcoaceticus genome was constructed with the binary plasmid system pLV21-RP4 delta Km. The DNA of A. calcoaceticus LMD79.41 was partially digested with Sau3A, and fragments of about 15 kilobases were inserted into the BamHI site of pLV21. The hybrid plasmids maintained in Escherichia coli were transferred by conjugation to the PQQ- mutants of A. calcoaceticus. One hybrid plasmid was isolated that complements all isolated PQQ- mutants. Subcloning of this plasmid in the vector pRK290 resulted in an insert of 5 kilobases on which at least four different genes involved in PQQ synthesis could be indicated. With Tn5 insertions the four PQQ genes were mapped, and it was shown that these genes are most probably located in three operons.
Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis
Authors: Amory A , Kunst F , Aubert E , Klier A , Rapoport G
Published: November 2016

The sacQ gene from Bacillus licheniformis was cloned and expressed in Bacillus subtilis. Deletion analysis shows that it encodes a 46-amino-acid polypeptide homologous to the B. subtilis sacQ gene product. The polypeptide, when it is overexpressed, activates the expression of a number of target genes in B. subtilis, all encoding secreted enzymes: alkaline protease, levansucrase, beta-glucanase(s), xylanase, and alpha-amylase. The maximum stimulations measured for alkaline protease and levansucrase were by a factor of 70 and 50, respectively, when the sacQ gene from B. licheniformis was present on a multicopy plasmid in B. subtilis. The sacQ genes from B. subtilis and B. licheniformis, cloned in the same multicopy plasmid, were compared under the same conditions. The sacQ gene from B. licheniformis was more efficient than the sacQ gene from B. subtilis in producing the hypersecretion phenotype. The sacQ structural genes from B. subtilis and B. licheniformis were placed under the control of the same inducible promoter. Hypersecretion was specifically obtained under conditions of full induction of the promoter. The target site of levansucrase regulation by sacQ was identified as a 440-base-pair fragment located in the 5' noncoding region of sacB, suggesting transcriptional control.
Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa
Authors: Mattick J S , Bills M M , Anderson B J , Dalrymple B , Mott M R , Egerton J R
Published: November 2016

Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.
Expression of the gene for NAD-dependent glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides cloned in Escherichia coli K-12
Authors: Murphy N B , McConnell D J , Schwarz T F
Published: November 2016

The structural gene (zwf) for Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase has been cloned into Escherichia coli on pBR322 by selecting for complementation of a zwf mutation of E. coli which eliminates glucose-6-phosphate dehydrogenase. The gene has been subcloned on a 3.4-kilobase DNA segment. The encoded glucose-6-phosphate dehydrogenase displays the dual nucleotide specificity (reacting with NAD and NADP) of the L. mesenteroides enzyme and has a subunit Mr of 55,000. A second gene encoding protein of subunit Mr 24,000 is also encoded on the 3.4-kilobase DNA segment. The genes have been located by the analysis of deletions and insertions generated in vitro and by transcriptional mapping with a promoterless chloramphenicol acetyl transferase cartridge inserted at different sites in the 3.4-kilobase fragment.
Cloning and analysis of ermG, a new macrolide-lincosamide-streptogramin B resistance element from Bacillus sphaericus
Authors: Monod M , Mohan S , Dubnau D
Published: November 2016

To analyze the regulation of a newly discovered macrolide-lincosamide-streptogramin B resistance element (ermG) found in a soil isolate of Bacillus sphaericus, we cloned this determinant and obtained its DNA sequence. Minicell analysis revealed that ermG specifies a 29,000-dalton protein, the synthesis of which is induced by erythromycin. S1 nuclease mapping was used to identify the transcriptional start site. These experiments demonstrated the presence on the ermG mRNA of a 197 to 198-base leader. Within the leader are two small open reading frames (ORFs) capable of encoding 11- and 19-amino-acid peptides. Each ORF is preceded by a suitably spaced Shine-Dalgarno sequence. The ermG protein is encoded by a large ORF that encodes a 244-amino-acid protein, in agreement with the minicell results. This protein and the 19-amino-acid peptide are highly homologous to the equivalent products of ermC and ermA. We conclude, on the basis of this homology, that ermG encodes an rRNA transmethylase. The leader of ermG can be folded into a structure that sequesters the Shine-Dalgarno sequence and start codon for the large ORF (SD3). On the basis of these data and on the observed greater responsiveness of the ermG system than of the ermC system to low concentrations of erythromycin, we propose a model for the regulation of this gene in which the stalling of a ribosome under the influence of an inducer, while reading either peptide, suffices to uncover SD3 and allow translation of the rRNA transmethylase. The evolution of ermG is discussed.
Transcription control of the aroP gene in Escherichia coli K-12: analysis of operator mutants
Authors: Chye M L , Pittard J
Published: November 2016

The nucleotide sequence of the region containing the promoter-operator for the aroP gene was determined. The start site of aroP transcription was identified by using S1 nuclease mapping and primer extension techniques. Examination of the nucleotide sequence revealed the presence of two "TYR R" boxes which are similar to those identified in the regulatory regions of other genes in the tyrR regulon. Bisulfite-induced aroP operator-constitutive mutants were analyzed, and the base-pair changes responsible for alterations in aroP regulation were located within these boxes.
Purification and properties of saccharopine dehydrogenase (glutamate forming) in the Saccharomyces cerevisiae lysine biosynthetic pathway
Authors: Storts D R , Bhattacharjee J K
Published: November 2016

Saccharopine dehydrogenase (glutamate forming) of the biosynthetic pathway of lysine in Saccharomyces cerevisiae was purified 1,122-fold by using acid precipitation, ammonium sulfate precipitation, DEAE-Sepharose, gel filtration, and Reactive Red-120 agarose chromatography. The enzyme exhibited a native molecular size of 69,000 daltons by gel filtration and consisted of a single 50,000-dalton polypeptide based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was readily denatured by exposures to temperatures exceeding 46 degrees C. The pH optimum for the reverse reaction was 9.5. The apparent Kms for L-saccharopine and NAD+ were 2.32 and 0.054 mM, respectively. The enzyme was inhibited by mercuric chloride but not by carbonyl or metal complexing agents.