bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis
Authors: Zuber P , Losick R
Published: November 2016

Transcription of the Bacillus subtilis gene spoVG is induced at the onset of sporulation and is dependent on the products of the stage-0 regulatory genes spo0A, spo0B, and spo0H. We show here that the dependence of spoVG transcription on Spo0A and Spo0B (but not Spo0H) can be bypassed by a mutation at abrB, a previously identified locus at which mutations that suppress some of the phenotypes of spo0A are often located, or by a cis-acting mutation within the spoVG promoter. To explain the epistatis of abrB to spo0A and spo0B mutations, we propose that AbrB acts, directly or indirectly, to block transcription of spoVG and that Spo0A and Spo0B cause inactivation of the abrB gene product(s). Spo0A-Spo0B-dependent inactivation of AbrB could be a general explanation for the pleiotropic effects of spo0A and spo0B mutations on B. subtilis gene expression.
Regulation of the glutamate-glutamine transport system by intracellular pH in Streptococcus lactis
Authors: Poolman B , Hellingwerf K J , Konings W N
Published: November 2016

Various methods of manipulation of the intracellular pH in Streptococcus lactis result in a unique relationship between the rate of glutamate and glutamine transport and the cytoplasmic pH. The initial rate of glutamate uptake by S. lactis cells increases more than 30-fold when the intracellular pH is raised from 6.0 to 7.4. A further increase of the cytoplasmic pH to 8.0 was without effect on transport. The different levels of inhibition of glutamate and glutamine transport at various external pH values by uncouplers and ionophores, which dissipate the proton motive force, can be explained by the effects exerted on the intracellular pH. The dependence of glutamate transport on the accumulation of potassium ions in potassium-filled and -depleted cells is caused by the regulation of intracellular pH by potassium movement.
Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene
Authors: Gonzy-Tréboul G , Steinmetz M
Published: November 2016

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.
Efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmids
Authors: Matsushima P , McHenney M A , Baltz R H
Published: November 2016

Conditions for efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmid cloning vectors were identified. Three streptomycete plasmid origins of replication function in A. orientalis, as do the apramycin resistance gene from Escherichia coli, the thiostrepton resistance gene from Streptomyces azureus, and the tyrosinase gene from Streptomyces antibioticus. A. orientalis appears to express some restriction and modification, because highest transformation frequencies (10(6)/micrograms of DNA) were obtained when plasmid pIJ702 was modified by passage in A. orientalis.
Some properties of subunits of DNA gyrase from Pseudomonas aeruginosa PAO1 and its nalidixic acid-resistant mutant
Authors: Inoue Y , Sato K , Fujii T , Hirai K , Inoue M , Iyobe S , Mitsuhashi S
Published: November 2016

Subunits A and B of DNA gyrase were purified from Pseudomonas aeruginosa PAO1 and its mutant, which was resistant to nalidixic acid. Inhibition tests of DNA gyrases reconstituted with a combination of subunits from the two strains showed that an alteration of subunit A but not subunit B caused bacteria to resist fluoroquinolones.
Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide
Authors: Fikes J D , Bankaitis V A , Ryan J P , Bassford P J
Published: November 2016

The wild-type maltose-binding protein (MBP) signal peptide is 26 amino acids in length. A mutationally altered MBP signal peptide has been previously described that is missing one of the basic residues from the hydrophilic segment and seven residues from the hydrophobic core; however, it still facilitates MBP secretion to the periplasm at a rate and efficiency comparable to those of the wild-type structure. Thus, this truncated signal peptide (designated the R2 signal peptide) must retain all of the essential features required for proper export function. In this study, alterations were obtained in the R2 signal peptide that resulted in an export-defective MBP. For the first time, signal sequence mutations were obtained that resulted in the synthesis of a totally export-defective MBP. As was previously the case for the wild-type signal peptide, the introduction of either charged residues or helix-breaking proline residues adversely affected export function. Despite these similarities, the position of these alterations within the R2 signal peptide, their relative effects on MBP secretion and processing, and an analysis of the ability of various extragenic prl mutations to suppress the secretion defects provide additional insight into the minimal requirements for a functional MBP signal peptide.
2-Methylthio-1,4-naphthoquinone, a unique sulfur-containing quinone from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus
Authors: Ishii M , Kawasumi T , Igarashi Y , Kodama T , Minoda Y
Published: November 2016

A quinone was extracted and purified from the cells of an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695). Its chemical structure was determined as 2-methylthio-3-VI, VII-tetrahydromultiprenyl-1,4-naphthoquinone by elemental analysis, 1H nuclear magnetic resonance spectroscopy, mass spectroscopy, and infrared spectroscopy of the quinone and by gas-liquid chromatography and gas chromatography-mass spectroscopy analysis of the ozonolysis products of the quinone. It was shown that the other five strains of H. thermophilus have the same quinone system. We named the quinone with the 2-methylthio-1,4-naphthoquinone nucleus "methionaquinone." The abbreviation of MTK is recommended for this class of quinone.
Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii
Authors: Anderson B E , Regnery R L , Carlone G M , Tzianabos T , McDade J E , Fu Z Y , Bellini W J
Published: November 2016

DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19. The ligation mixture was used to transform Escherichia coli. A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum. One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R. rickettsii serum that comigrated with an antigen from R. rickettsii. A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen. The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences. The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840. The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E. coli. The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.
Regulation of expression of pectate lyase genes pelA, pelD, and pelE in Erwinia chrysanthemi
Authors: Reverchon S , Robert-Baudouy J
Published: November 2016

The regulation of pelA, pelD, and pelE genes encoding three of the five major pectate lyase isoenzymes (PLa, PLd, and PLe) in Erwinia chrysanthemi B374 was analyzed by using genetic fusions to lacZ. These three genes are clustered on a 5-kilobase DNA fragment in the order pelD-pelE-pelA and constitute three independent transcriptional units. We localized the pelDEA cluster near the pro-1 marker on the genetic map of B374 by chromosomal mobilization with RP4::mini-Mu plasmid pULB110. Three classes of regulatory mutations responsible for constitutive pectate lyase synthesis have been described (kdgR, gpiR, and cri). We studied the effects of each mutation on pelE, pelD, and pelA expression independently. The mutations kdgR and gpiR mainly affect the expression of pelE and pelD, although PLa synthesis is slightly increased. The cri mutation results in a low level of constitutive expression of the three pel genes, but it is a pleiotropic mutation since other genes not involved in pectinolysis are also affected. In addition, we demonstrated that exuR, a negative regulatory gene governing the catabolism of hexuronates, does not modify the expression of pel genes. The frequency of gpiR or cri mutations (about 10(-8)) and the resulting constitutivity of pectate lyase synthesis suggest that these genes act as negative regulatory genes in addition to kdgR, which is already known to encode a repressor. Moreover, we found that expression of pel-lac fusions carried on pBR322 derivatives was higher in E. chrysanthemi than in Escherichia coli; this fact suggests the existence of positive regulation of pectate lyase synthesis in E. chrysanthemi.
Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions
Authors: Ronson C W , Nixon B T , Albright L M , Ausubel F M
Published: November 2016

We report the identification and cloning of an ntrA-like (glnF rpoN) gene of Rhizobium meliloti and show that the R. meliloti ntrA product (NtrA) is required for C4-dicarboxylate transport as well as for nitrate assimilation and symbiotic nitrogen fixation. DNA sequence analysis showed that R. meliloti NtrA is 38% homologous with Klebsiella pneumoniae NtrA. Subcloning and complementation analysis suggested that the R. meliloti ntrA promoter lies within 125 base pairs of the initiation codon and may be constitutively expressed.
Cloning and characterization of Saccharomyces cerevisiae genes that confer L-methionine sulfoximine and tabtoxin resistance
Authors: Marek E T , Dickson R C
Published: November 2016

Pseudomonas tabaci produces a toxin, tabtoxin, that causes wildfire disease in tobacco. The primary target of tabtoxin is presumed to be glutamine synthetase. Some effects of tabtoxin in tobacco can be mimicked by methionine sulfoximine (MSO), a compound that is known to inactivate glutamine synthetase. To understand how organisms can be made resistant to tabtoxin and MSO, we used Saccharomyces cerevisiae. We demonstrate that yeast strains carrying the glutamine synthetase gene, GLN1, on a multicopy plasmid overproduced glutamine synthetase and showed increased drug resistance. These and other data indicate that glutamine synthetase is the primary target of tabtoxin and MSO in S. cerevisiae. We also isolated three S. cerevisiae DNA inserts of 2.1, 2.3, and 2.8 kilobases that conferred tabtoxin and MSO resistance when the inserts were present on a multicopy plasmid. These plasmids conferred resistance to MSO by blocking intracellular transport of the drug. Transport appeared to occur by one or more methionine permeases. Resistance to tabtoxin could also occur by blockage of intracellular transport, but the drug was transported by some permease other than a methionine permease. These drug resistance plasmids did not block transport of citrulline, indicating that they did not affect the general amino acid permease.
Regulation of expression of glutamine synthetase in a symbiotic Nostoc strain associated with Anthoceros punctatus
Authors: Joseph C M , Meeks J C
Published: November 2016

A characteristic of N2-fixing cyanobacteria in symbiotic associations appears to be release of N2-derived NH4+. The specific activity of the primary ammonium-assimilating enzyme, glutamine synthetase (GS), was found to be three- to fourfold lower in Nostoc sp. strain 7801 grown in symbiotic association with the bryophyte Anthoceros punctatus than in free-living Nostoc sp. strain 7801. Quantitative immunological assays with antisera against GS purified from Nostoc sp. strain 7801 and from Escherichia coli indicated that similar amounts of the GS protein were present in symbiotic (50 micrograms mg-1) and free-living (68 micrograms mg-1) cultures. The conclusion from these experiments is that GS is regulated by a posttranslational mechanism in Anthoceros-associated Nostoc sp. strain 7801. However, the results of comparative catalytic and immunological experiments between N2- and NH4+-grown free-living Nostoc sp. strain 7801 implied control of GS synthesis. A correlation was not observed between the level of GS expression and the extent of symbiotic heterocyst differentiation in Nostoc sp. strain 7801 associated with A. punctatus.
Characterization of the sppA gene coding for protease IV, a signal peptide peptidase of Escherichia coli
Authors: Suzuki T , Itoh A , Ichihara S , Mizushima S
Published: November 2016

The sppA gene codes for protease IV, a signal peptide peptidase of Escherichia coli. Using the gene cloned on a plasmid, we constructed an E. coli strain carrying the ampicillin resistance gene near the chromosomal sppA gene and an sppA deletion strain in which the deleted portion was replaced by the kanamycin resistance gene. Using these strains, we mapped the sppA gene at 38.5 min on the chromosome, the gene order being katE-xthA-sppA-pncA. Although digestion of the signal peptide that accumulated in the cell envelope fraction was considerably slower in the deletion mutant than in the sppA+ strain, it was still significant, suggesting the participation of another envelope protease(s) in signal peptide digestion.
Positive and negative regulation of the bgl operon in Escherichia coli
Authors: Mahadevan S , Reynolds A E , Wright A
Published: November 2016

We have analyzed the functions encoded by the bgl operon in Escherichia coli K-12. Based on the ability of cloned regions of the operon to complement a series of Bgl- point mutations, we show that the three bgl structural genes, bglC, bglS, and bglB, are located downstream of the regulatory locus bglR in the order indicated. Using a bgl-lacZ transcriptional fusion, we show that bglC and bglS are involved in regulating operon expression. The presence of the bglC gene in trans is absolutely required for the expression of the fusion, which is constitutive when only the bglC gene is present. When the bglC and the bglS genes are both present in the cell, expression of the fusion requires a beta-glucoside inducer. From these observations, we conclude that (i) the bglC gene encodes a positive regulatory of bgl operon expression and (ii) the bglS gene encodes a negative regulator of operon expression, causing the requirement for a beta-glucoside inducer. These conclusions are supported by our observations that (i) a majority of bglC mutants exhibits a Bgl- phenotype, whereas rare trans-dominant mutations in bglC result in constitutive expression of the bgl operon and the fusion, and (ii) mutations in the bglS gene lead to constitutive expression of the fusion. Based on several lines of evidence presented, we propose that the bglS gene product has an additional role as a component of the beta-glucoside transport system.
Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes
Authors: Schnetz K , Toloczyki C , Rak B
Published: November 2016

Wild-type Escherichia coli cells are unable to grow on beta-glucosides. Spontaneous mutants arise, however, which are able to utilize certain aromatic beta-glucosides such as salicin or arbutin as carbon sources, revealing the presence of a cryptic operon called bgl. Mutations activating the operon map within (or close to) the promoter region of the operon and are due to the transposition of an IS1 or IS5 insertion element into this region. This operon was reported to consist of three genes coding for a phospho-beta-glucosidase, a specific transport protein (enzyme IIBgl), and a positively regulating protein. We have defined the extent and location of three structural genes, bglC, bglS, and bglB, and have determined their DNA sequence. The amino acid sequences deduced from the open reading frames together with deletion and subcloning analyses suggest that the first gene, bglC, codes for the regulatory protein, the second, bglS, codes for the transport protein, and the third, bglB, for phospho-beta-glucosidase. A fourth gene may exist which codes for a product of unknown function. We discuss structural features of the DNA sequence which may bear on the regulation of the operon. Homologies to sequences preceding the gene for an excreted levansucrase of Bacillus subtilis, which are known to be involved in the regulation of this gene, and to sequences preceding the gene for an excreted beta-endoglucanase of B. subtilis, for which data pertaining to regulation are not yet available, suggest a close evolutionary relationship among the regulatory components of all three systems.
Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis
Authors: Conway T , Sewell G W , Osman Y A , Ingram L O
Published: November 2016

The gene which encodes alcohol dehydrogenase II (adhB) from Zymomonas mobilis was cloned in Escherichia coli as a 1.4-kilobase DNA fragment by using a novel indicator plate which directly detects the expression of this activity by recombinant colonies. The DNA sequence for this clone contained an open reading frame encoding a polypeptide of 383 amino acids, with a molecular weight of 40,141. Although this protein exhibited very little homology with other known alcohol dehydrogenases, the predicted amino acid composition was in excellent agreement with that reported for the purified alcohol dehydrogenase II protein from Z. mobilis. In Z. mobilis, the adhB gene was transcribed from tandem promoters which were separated by 100 base pairs and ended with a transcriptional terminator (13-base-pair palindrome). In Escherichia coli, only one of the Z. mobilis promoters was used, despite apparent similarity to the enteric consensus promoter. The adhB gene was transcribed at low levels in E. coli from the P2 promoter of Z. mobilis but was expressed well in E. coli under control of the lac promoter (approximately 0.25% of the total cell protein).
Ureidosuccinate is transported by the allantoate transport system in Saccharomyces cerevisiae
Authors: Turoscy V , Cooper T G
Published: November 2016

The regulatory characteristics exhibited by ureidosuccinate transport in Saccharomyces cerevisiae led us to hypothesize that this biosynthetic intermediate was transported via the degradative allantoate transport system. The hypothesis was verified by the finding that neither dal5 nor urep1 mutant strains could transport allantoate or ureidosuccinate. Mutations in the two loci were tightly linked and failed to complement one another, suggesting that they were allelic. The use of a common transport system for accumulation of both biosynthetic and degradative metabolites explains the paradoxical characteristics observed for control of ureidosuccinate and allantoate transport.
A site-specific recombination function in Staphylococcus aureus plasmids
Authors: Gennaro M L , Kornblum J , Novick R P
Published: November 2016

All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the "core," flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.
Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor
Authors: de Lorenzo V , Wee S , Herrero M , Neilands J B
Published: November 2016

The promoter region of the pColV-K30-encoded operon specifying biosynthesis and transport of the siderophore aerobactin was subjected to deletion analysis to determine the smallest DNA sequence affording iron regulation of a iucA'-'lacZ gene fusion. A 78-base-pair (bp) region containing the main (P1) promoter retained the character of inducibility under iron starvation. A 250-bp fragment carrying this sequence was examined for protection against DNase I by the Fur protein, the product of a gene (fur) required for negative control of several iron-regulated functions. The DNase I footprints, in the presence of various divalent heavy-metal ions added as corepressors, revealed two contiguous binding sites with different lengths and affinities for Fur. Increased concentrations of the protein appeared to elicit formation of repressor oligomers which bind to the upstream and downstream regions of the P1 promoter in a metal-dependent fashion, but with a presently undefined stoichiometry. The primary site for Fur binding spans 31 bp and contains two overlapping symmetry dyads which share the sequence 5'-TCATT-3'. It also contains extensive homology with a 19-bp consensus sequence for iron-regulated genes as deduced from comparison with the fhuA and fepA putative promoter sequences.
A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum
Authors: Nieuwkoop A J , Banfalvi Z , Deshmane N , Gerhold D , Schell M G , Sirotkin K M , Stacey G
Published: November 2016

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).
Association of thioredoxin with the inner membrane and adhesion sites in Escherichia coli
Authors: Bayer M E , Bayer M H , Lunn C A , Pigiet V
Published: November 2016

The intracellular localization of thioredoxin in Escherichia coli was determined by immunoelectron microscopy and correlated to previous biochemical data which had suggested that thioredoxin resides at inner-outer membrane adhesion sites. Since a considerable amount of thioredoxin was lost during preparation of cells for electron microscopy, we immobilized the protein with the heterobifunctional photoactivatable cross-linker p-azidophenacylbromide before the cells were fixed with aldehyde and embedded in Lowicryl K4M. Thin sections were labeled with affinity-purified antithioredoxin antiserum and protein A-gold complexes. Densities of immunolabel in a designated membrane-associated area and in the rest of the cytoplasm were compared and the data were statistically evaluated. Wild-type strain W3110 and strain SK3981, an overproducer of thioredoxin, exhibited increased labeling at the inner membrane and its adjacent cytoplasmic area. In contrast, the more centrally located cytoplasm of both strains showed much lower label density. This label distribution did not change with cell growth or in the stationary phase. Immunolabel was often found at bridges between the inner and outer membranes; this result is consistent with a model which places at least a portion of the thioredoxin at membrane adhesion sites, corresponding to an osmotically sensitive cytoplasmic compartment bounded by a hybrid inner-outer membrane (C.A. Lunn and V. Pigiet, J. Biol. Chem. 257:11424-11430, 1982; C.A. Lunn and V. Pigiet, J. Biol. Chem. 261:832-838, 1986). Specific label was absent in the periplasmic space.
Isolation and characterization of light-regulated phycobilisome linker polypeptide genes and their transcription as a polycistronic mRNA
Authors: Lomax T L , Conley P B , Schilling J , Grossman A R
Published: November 2016

Several cyanobacteria adjust both the phycobiliprotein and linker protein composition of the phycobilisome, a light-harvesting complex in cyanobacteria and some eucaryotic algae, to maximize absorption of prevalent wavelengths of light. This process is called complementary chromatic adaptation. We sequenced the amino terminus of a linker polypeptide which is associated with phycocyanin and accumulates to high levels during growth of the cyanobacterium Fremyella diplosiphon in red light. A mixed oligonucleotide encoding a region of this amino terminus was synthesized and used to identify a fragment of F. diplosiphon genomic DNA encoding the linker polypeptide. This linker gene was located between two other linker genes and contiguous to the red-light-induced phycocyanin gene set. Sequences of all three linker genes are presented. These genes were transcribed together onto a large polycistronic mRNA which also encoded the red-light-induced phycocyanin subunits. The relationship of this transcript to the biogenesis of the phycobilisome when F. diplosiphon is grown under different conditions of illumination is discussed.
Expression and regulation of a Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli
Authors: Scholle R R , Coyne V E , Maharaj R , Robb F T , Woods D R
Published: November 2016

A halotolerant collagenolytic Vibrio alginolyticus strain isolated from salted hides had intracellular sucrase activity and did not secret sucrase into the medium. The strain actively transported sucrose by a sucrose-inducible, Na+-independent process. A 10.4-kilobase DNA fragment of V. alginolyticus DNA was cloned into Escherichia coli. The recombinant E. coli(pVS100) could utilize sucrose as a sole carbon source. In contrast to V. alginolyticus, the recombinant E. coli produced both intra- and extracellular sucrase activities. Up to 20% of the total sucrase activity was in the supernatant. Sucrase synthesis in E. coli(pVS100) was inducible and was subject to glucose repression, which was relieved by cyclic AMP. Sucrose was actively transported by a sucrose-inducible, Na+-independent system in E. coli(pVS100). Sucrose uptake was inhibited by the addition of a proton conductor. The maximum velocity and apparent Km values of sucrose uptake for the V. alginolyticus strain and E. coli(pVS100) were 130 nmol/mg of protein per min and 50 microM and 6 nmol/mg of protein per min and 275 microM, respectively.
Cloning and characterization of elastase genes from Pseudomonas aeruginosa
Authors: Schad P A , Bever R A , Nicas T I , Leduc F , Hanne L F , Iglewski B H
Published: November 2016

A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.
An aminoacyl-tRNA synthetase complex in Escherichia coli
Authors: Harris C L
Published: November 2016

Aminoacyl-tRNA synthetases from several strains of Escherichia coli are shown to elute as a high-molecular-weight complex on 6% agarose columns (Bio-Gel A-5M). In contrast, very little synthetase activity was observed in such complexes on Sephadex G-200 columns, suggesting that these enzymes may interact with or are dissociated during chromatography on dextran. The size of the complex observed on Bio-Gel A-5M was influenced by the method of cell breakage and the salt concentrations present in buffers. The largest complexes (greater than 1,000,000 daltons) were seen with cells broken with a freeze press, whereas with sonicated preparations the average size of the complex was about 400,000 daltons. Extraction of synthetases at 0.15 M NaCl, to mimic physiological salt concentrations, also resulted in high-molecular-weight complexes, as demonstrated by both agarose gel filtration and ultracentrifugation analysis. Evidence is presented that dissociation of some synthetases does occur in the presence of higher salt levels (0.4 M NaCl). Partial purification of the synthetase complex on DEAE-Sephacel was accomplished with only minor dissociation of individual synthetases. These data suggest that a complex(es) of aminoacyl-tRNA synthetase does exist in bacterial cells, just as in eucaryotes, and that the complex may have escaped earlier detection due to its fragility during isolation.
Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris
Authors: Poolman B , Smid E J , Konings W N
Published: November 2016

In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force.
Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638
Authors: Durham D R , Stewart D B , Stellwag E J
Published: November 2016

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.
Tetrahydrothiophene 1-oxide as an electron acceptor for Escherichia coli
Authors: Meganathan R , Schrementi J
Published: November 2016

Escherichia coli used tetrahydrothiophene 1-oxide (THTO) as an electron acceptor for anaerobic growth with glycerol as a carbon source; the THTO was reduced to tetrahydrothiophene. Cell extracts also reduced THTO to tetrahydrothiophene in the presence of a variety of electron donors. Chlorate-resistant (chl) mutants (chlA, chlB, chlD, and chlE) were unable to grow with THTO as the electron acceptor. However, growth and THTO reduction by the chlD mutant were restored by high concentrations of molybdate. Similarly, mutants of E. coli that are blocked in the menaquinone (vitamin K2) biosynthetic pathway, i.e., menB, menC, and menD mutants, did not grow with THTO as an electron acceptor. Growth and THTO reduction were restored in these mutants by the presence of appropriate intermediates of the vitamin K biosynthetic pathway.
Characterization of transfer regions within the HII incompatibility group plasmid pHH1508a
Authors: Yan W , Taylor D E
Published: November 2016

A circular map of the 208-kilobase IncHII plasmid pHH1508a was constructed by using the restriction endonucleases XbaI, XhoI, and NotI. Tn5 insertion mutagenesis showed that transfer genes were distributed widely over the plasmid map. Analysis of the deletion mutant pDT1178 indicated that genes required for conjugative pilus production are located in a 96.0-kilobase region of the plasmid.
Evidence that colicin X is microcin B17
Authors: San Millán J L , Kolter R , Moreno F
Published: November 2016

The DNA replication inhibitor microcin B17 is a peptide antibiotic produced by Escherichia coli strains carrying plasmid pMccB17. Here we present evidence that antibiotic activities previously named colicin X are probably identical to microcin B17. Our results include comparison of the conditions of production of the antibiotics, their mode of action, cross-immunity of producer strains, and cross-resistance of resistant mutants. Plasmids encoding colicin X have been identified and shown to have a region of significant homology with the microcin B17-producing region of pMccB17 DNA.
Secretion of human serum albumin from Bacillus subtilis
Authors: Saunders C W , Schmidt B J , Mallonee R L , Guyer M S
Published: November 2016

We have fused the structural gene (hsa) for human serum albumin (HSA) to the expression elements and signal sequence coding region of each of two genes from Bacillus amyloliquefaciens P, an alpha-amylase gene (amyBamP) and a neutral protease gene (nprBamP). Bacillus subtilis strains harboring either of these gene fusions synthesized a protein with the antigenic characteristics and size (68 kilodaltons) of HSA. Results from pulse-labeling studies indicated that the bacterially produced HSA was secreted from cells which had been converted to protoplasts. Results from similar studies with intact cells suggested that the signal sequence was removed from the hybrid protein, providing further evidence that B. subtilis can translocate this foreign protein across the cell membrane. Signal sequence removal was efficient when the level of HSA synthesis was low. However, in strains which synthesized HSA at a high level, signal sequence removal was less efficient.
Hybrid Escherichia coli sensory transducers with altered stimulus detection and signaling properties
Authors: Slocum M K , Halden N F , Parkinson J S
Published: November 2016

The tar and tap loci of Escherichia coli encode methyl-accepting inner membrane proteins that mediate chemotactic responses to aspartate and maltose or to dipeptides. These genes lie adjacent to each other in the same orientation on the chromosome and have extensive sequence homology throughout the C-terminal portions of their coding regions. Many spontaneous deletions in the tar-tap region appear to be generated by recombination between these regions of homology, leading to gene fusions that produce hybrid transducer molecules in which the N terminus of Tar is joined to the C terminus of Tap. The properties of two such hybrids are described in this report. Although Tar and Tap molecules have homologous domain structures, these Tar-Tap hybrids exhibited defects in stimulus detection and flagellar signaling. Both hybrid transducers retained Tar receptor specificity, but had reduced detection sensitivity. This defect was correlated with the presence of the C-terminal methyl-accepting segment of Tap, which may have more methylation sites than its Tar counterpart, leading to elevated steady-state methylation levels in the hybrid molecules. One of the hybrids, which carried a more extensive segment from Tap, appeared to generate constitutive signals that locked the flagellar motors in a counterclockwise rotational mode. Changes in the methylation state of this transducer were ineffective in cancelling this aberrant signal. These findings implicate the conserved C-terminal domain of bacterial transducers in the generation or regulation of flagellar signals.
Lysis gene t of T-even bacteriophages: evidence that colicins and bacteriophage genes have common ancestors
Authors: Riede I
Published: November 2016

The lysis gene t of the T-even-like bacteriophage K3 has been cloned and sequenced. The gene codes for a protein with a predicted molecular weight of 25,200. Expression of the complete lysis protein was impossible, but peptides complementing T4 amber mutants in t are described. No known lysis protein of other phages is homologous to protein T. Also, the Escherichia coli phospholipase A is different from protein T. CelB, the lysis protein of the colicin E2 operon, shows a similarity to protein T. Sequences of colicins A, E1, and E2 are related to gene 38 sequences, the gene preceding t and coding for the phage adhesin. A common origin for colicin genes and phage genes is discussed, and a protein region in colicins that is responsible for receptor recognition is predicted.
Expression of degradative genes of Pseudomonas putida in Caulobacter crescentus
Authors: Chatterjee D K , Chatterjee P
Published: November 2016

The recombinant plasmid RP4-TOL was transferred into Caulobacter crescentus at a high frequency, and the plasmid was maintained for at least 50 generations. C. crescentus cells which contained RP4-TOL grew on all the aromatic compounds that the plasmid normally allowed Pseudomonas putida to grow on. Reciprocal transfers from C. crescentus donor to P. putida or Escherichia coli recipients were less efficient and occurred at frequencies of approximately 10(-3). Some representative TOL-specified enzymes in cell-free extracts of C. crescentus(RP4-TOL) were inducible, and their levels were similar to those of P. putida. The amounts of mRNA from induced cells of C. crescentus(RP4-TOL) and P. putida(RP4-TOL) were also similar. Moreover, the restriction enzyme digestion maps of RP4-TOL from both C. crescentus and P. putida were the same, indicating that the expression of the TOL genes occurred without any apparent alteration of the gene structure. This suggest that the degradative genes of Pseudomonas spp. can be transferred, maintained, and expressed efficiently in C. crescentus and that the mechanism of transcriptional activation of TOL genes observed in C. crescentus is similar to that of Pseudomonas spp.
Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs
Authors: Saxild H H , Nygaard P
Published: November 2016

Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively.
Energetics of the initial phase of adhesion of Streptococcus sanguis to hydroxylapatite
Authors: Cowan M M , Taylor K G , Doyle R J
Published: November 2016

The initial adhesion of Streptococcus sanguis 10556 to artificial salivary pellicle and to bare hydroxylapatite was studied at several temperatures between 18 and 37 degrees C. When the natural logarithms of rate constants for adsorption and desorption were plotted against reciprocal temperatures in Arrhenius plots, curved lines were obtained, indicating that the thermodynamic quantities of enthalpy and entropy of activation were temperature dependent. For the bare hydroxylapatite system, the heat capacity (delta Cp = dH/dT) was large and negative. delta Cp was also negative for adhesion to saliva-coated hydroxylapatite, although its value was lower. Negative heat capacities, when coupled with favorable entropy, are often indicative of either electrostatic or hydrophobic interactions. When electrolyte (100 mM ammonium sulfate) was added to the cell-hydroxylapatite bead mixture, the rate and extent of adhesion were decreased. Addition of nonpolar p-dioxane (10% [vol/vol], final concentration) to the mixture enhanced binding. This suggests that electrostatic linkages participate in the primary adhesion of streptococci to both substrata. The strongly positive entropy values and the lesser temperature dependence of the saliva-coated hydroxylapatite system suggest that another entropy-driven process is imposed on the electrostatic linkages. This supports a role for hydrophobicity, suggesting that a combination of electrostatic and hydrophobic forces mediate the initial adhesion of S. sanguis to the salivary pellicle.
Purification of aconitase from Bacillus subtilis and correlation of its N-terminal amino acid sequence with the sequence of the citB gene
Authors: Dingman D W , Sonenshein A L
Published: November 2016

The DNA sequence for the promoter region of the Bacillus subtilis citB gene has been determined. Presumed "-10" and "-35" regions of the promoter have been identified, and transcriptional and translational start points of citB have been located. To correlate the DNA sequence of citB with the amino acid sequence of its presumed product, aconitase, it was necessary to devise a scheme for purification of this labile enzyme. This procedure relies on the ability to restore enzyme activity at each stage of purification by incubation in a reducing buffer containing a source of ferrous ions. B. subtilis aconitase appears to be a monomer with a molecular weight of approximately 120,000. The amino-terminal amino acids of aconitase fit the sequence predicted by analysis of the citB gene. Thus, citB codes for aconitase.
Cloning and nucleotide sequencing of genes for a second type of small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus"
Authors: Sun D X , Setlow P
Published: November 2016

The nucleotide sequences of the single genes coding for the B-type small, acid-soluble spore proteins (SASP) of Bacillus cereus, B. stearothermophilus, and "Thermoactinomyces thalpophilus" were determined, and the amino acid sequences of all B-type SASP were compared. While this type of SASP showed significant sequence conservation around the two spore protease cleavage sites, alignment of these sequences required the introduction of gaps, and even then only 19 of the residues were conserved exactly in all five proteins. However, all five B-type SASP did contain a large (27 to 35-residue), rather well-conserved amino acid sequence repeat, and four of the five proteins had well-conserved regions of 14 to 17 amino acids which appeared three times.