bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity and origin of Desulfovibrio species: phylogenetic definition of a family
Authors: Devereux R , He S H , Doyle C L , Orkland S , Stahl D A , LeGall J , Whitman W B
Published: November 2016

The different nutritional properties of several Desulfovibrio desulfuricans strains suggest that either the strains are misclassified or there is a high degree of phenotypic diversity within the genus Desulfovibrio. The results of partial 16S rRNA and 23S rRNA sequence determinations demonstrated that Desulfovibrio desulfuricans ATCC 27774 and "Desulfovibrio multispirans" are closely related to the type strain (strain Essex 6) and that strains ATCC 7757, Norway 4, and El Agheila Z are not. Therefore, these latter three strains of Desulfovibrio desulfuricans are apparently misclassified. A comparative analysis of nearly complete 16S rRNA sequences in which we used a least-squares analysis method for evolutionary distances, an unweighted pair group method, a signature analysis method, and maximum parsimony was undertaken to further investigate the phylogeny of Desulfovibrio species. The species analyzed were resolved into two branches with origins deep within the delta subdivision of the purple photosynthetic bacteria. One branch contained five deep lineages, which were represented by (i) Desulfovibrio salexigens and Desulfovibrio desulfuricans El Agheila Z; (ii) Desulfovibrio africanus; (iii) Desulfovibrio desulfuricans ATCC 27774, Desulfomonas pigra, and Desulfovibrio vulgaris; (iv) Desulfovibrio gigas; and (v) Desulfomicrobium baculatus (Desulfovibrio baculatus) and Desulfovibrio desulfuricans Norway 4. A correlation between 16S rRNA sequence similarity and percentage of DNA relatedness showed that these five deep lineages are related at levels below the minimum genus level suggested by Johnson (in Bergey's Manual of Systematic Bacteriology, vol. 1, 1984). We propose that this branch should be grouped into a single family, the Desulfovibrionaceae. The other branch includes other genera of sulfate-reducing bacteria (e.g., Desulfobacter and Desulfococcus) and contains Desulfovibrio sapovorans and Desulfovibrio baarsii as separate, distantly related lineages.
Regulation of trehalose metabolism by Streptomyces griseus spores
Authors: McBride M J , Ensign J C
Published: November 2016

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.
An operon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from a Streptomyces sp
Authors: Horii M , Ishizaki T , Paik S Y , Manome T , Murooka Y
Published: November 2016

The nucleotide sequence of the promoter region of the gene for cholesterol oxidase (choA) from Streptomyces sp. strain SA-COO was determined. We found an open reading frame (choP) that is located between a potential promoter sequence and the structural gene for the ChoA protein. Deletion analysis showed that the promoter region for choP is essential for expression of the choA gene. Mappings of S1 nuclease and primer extension of transcripts generated in vivo suggested that the synthesis of mRNA starts at a site 41 bases upstream from the ATG initiation codon of the choP gene. By Northern (RNA) blot analysis of the transcripts, we found a 2.9-kilobase transcript that is identical in size to the total sequence of the choP and choA genes. These results suggest that the two genes, choP and choA, are transcribed polycistronically under the control of the promoter that is upstream from the structural gene for choP. The choP gene encodes a protein of 381 amino acids with a calculated Mr of 41,668. The nucleotide sequence of the choP gene has a high degree of similarity to the sequence of the genes for cytochrome P-450s from humans and Pseudomonas species. A region of homology with the cytochrome P-450s from various organisms was identified in the choP protein and may represent a region associated with a binding site for heme iron. Analysis of the CO difference spectrum of an extract of Streptomyces lividans cells that carry a plasmid which includes the choP gene revealed a unique peak, characteristic of cytochrome P-450, which is identical to that obtained with the parent strain.
Characterization of ompF domains involved in Escherichia coli K-12 sensitivity to colicins A and N
Authors: Fourel D , Hikita C , Bolla J M , Mizushima S , Pagès J M
Published: November 2016

Various ompF-ompC, ompC-ompF, and ompF-ompC-ompF chimeric genes were used to locate the domains of the OmpF protein involved in cellular sensitivity to colicins. Various parts of the porin participate in the entry of colicins. Colicin N receptor activity was found to require three regions: RN1, located between residues 1 and 63; RN2, located between residues 115 and 262; and RN3, located between residues 279 and 297. The central domain from residues 143 to 262 is involved during the translocation step after the binding step. A large region, including residues 1 to 262, is necessary during colicin A entry. The locations and interactions between these domains specifically required for the uptake of colicins to occur are described and discussed with regard to the homology and topology of the OmpC, OmpF, and PhoE porins.
Properties of cell wall-associated DD-carboxypeptidase of Enterococcus hirae (Streptococcus faecium) ATCC 9790 extracted with alkali
Authors: Kariyama R , Massidda O , Daneo-Moore L , Shockman G D
Published: November 2016

DD-Carboxypeptidase (DD-CPase) activity of Enterococcus hirae (Streptococcus faecium) ATCC 9790 was extracted from intact bacteria and from the insoluble residue (crude cell wall fraction) of mechanically disrupted bacteria by a brief treatment at pH 10.0 (10 mM glycine-NaOH) at 0 degrees C or by extraction with any of several detergents. Extractions with high salt concentrations failed to remove DD-CPase activity from the crude wall fraction. In contrast to N-acetylmuramoylhydrolase (both muramidase 2 and muramidase 1) activities, DD-CPase activity failed to bind to insoluble cell walls or peptidoglycan matrices. Thus, whereas muramidase 1 and muramidase 2 activities can be considered to be cell wall proteins, the bulk of the data are consistent with the interpretation that the DD-CPase of this species is a membrane protein that is sometimes found in the cell wall fraction, presumably because of hydrophobic interactions with other proteins and cell wall polymers. The binding of [14C]penicillin to penicillin-binding protein 6 (43 kilodaltons) was proportional to DD-CPase activity. Kinetic parameters were also consistent with the presence of only one DD-CPase (penicillin-binding protein 6) in E. hirae.
Bacterial cell shape regulation: testing of additional predictions unique to the two-competing-sites model for peptidoglycan assembly and isolation of conditional rod-shaped mutants from some wild-type cocci
Authors: Lleo M M , Canepari P , Satta G
Published: November 2016

The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsiella pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42 degrees C were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.
Site-specific transposition of insertion sequence IS630
Authors: Tenzen T , Matsutani S , Ohtsubo E
Published: November 2016

IS630 is a 1.15-kilobase sequence in Shigella sonnei that, unlike many mobile elements, seems not to mediate cointegration between different replicons. To assess its transposition, we constructed composite elements containing inverted copies of IS630 flanking a drug resistance gene. We found that these composite elements transposed to plasmid ColE1 in Escherichia coli. DNA sequencing showed that transposition was, in all cases, to the dinucleotide sequence 5'-TA-3'. There were two preferred insertion sites which corresponded to the TA sequences in the inverted repeats of a 13-base-pair stem region of the [rho]-dependent transcription terminator. IS630 is flanked by TA, and nucleotide substitution by in vitro mutagenesis at these ends did not affect transposition activity of a composite element or its ability to insert preferentially into TA within the 13-base-pair inverted repeat sequences or to duplicate the target sequence.
DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene
Authors: Teather R M , Erfle J D
Published: November 2016

The DNA sequence of a mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined. The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes. The gene product was processed for export in E. coli. There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage beta-glucanase from Bacillus subtilis. The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence Pro-Xxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonas fluorescens cellulase and the serine- and/or threonine-rich regions observed in Cellulomonas fimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I.
Cloning, sequence, and expression of the pantothenate permease (panF) gene of Escherichia coli
Authors: Jackowski S , Alix J H
Published: November 2016

Pantothenate permease, the product of the panF gene, catalyzes the sodium-dependent uptake of extracellular pantothenate. The panF gene was isolated from an Escherichia coli genomic DNA library and subcloned into multicopy plasmids. Increased copy number of the panF+ allele resulted in increased rates of pantothenate uptake and a significant increase in the steady-state intracellular pantothenate concentration. Despite the higher levels of pantothenate, the utilization of pantothenate for coenzyme A formation was not elevated, indicating that pantothenate kinase activity is the dominant regulator of coenzyme A biosynthesis. DNA sequencing of the panF gene revealed the presence of a single open reading frame that encoded a hydrophobic protein with a molecular weight of 51,992. Sequence analysis predicts that pantothenate permease is an integral membrane protein possessing 12 hydrophobic membrane-spanning domains connected by short hydrophilic sequences. The predicted topological profile of pantothenate permease is similar to that of other membrane carriers that catalyze cation-dependent symport.
Molecular cloning and characterization of an extracellular protease gene from Aeromonas hydrophila
Authors: Rivero O , Anguita J , Paniagua C , Naharro G
Published: November 2016

A structural gene which codes for an extracellular protease in Aeromonas hydrophilia SO2/2 and D13 was cloned in Escherichia coli C600-1 by using pBR322 as a vector. The gene codes for a temperature-stable protease with a molecular mass of approximately 38,000 daltons. The protein was secreted to the periplasm of E. coli C600-1 and purified by osmotic shock. Cloned protease (P3) was identical in molecular mass and properties to the one purified from A. hydrophila SO2/2 culture supernatant as an extracellular product.
Co-overproduction and localization of the Escherichia coli motility proteins motA and motB
Authors: Wilson M L , Macnab R M
Published: November 2016

The motility genes motA and motB of Escherichia coli were placed under control of the Serratia marcescens trp promoter. After induction with beta-indoleacrylic acid, the levels of MotA and MotB rose over about a 3-h period, reaching plateau levels approximately 50-fold higher than wild-type levels. Both overproduced proteins inserted into the cytoplasmic membrane. Growth and motility were essentially normal, suggesting that although the motor is a proton-conducting device, MotA and MotB together do not constitute a major proton leak. Derivative plasmids which maintained an intact version of motB but had the motA coding region deleted in various ways were constructed. With these, the levels of MotB were much lower, reaching a peak within 30 min after induction and declining thereafter; pulse-chase measurements indicated that a contributing factor was MotB degradation. The low levels of MotB occurred even with an in-frame internal deletion of motA, whose translational initiation and termination sites were intact, suggesting that it is the MotA protein, rather than the process of MotA synthesis, that is important for MotB stability. Termination at the usual site of overlap with the start of motB (ATGA) was not an absolute requirement for MotB synthesis but did result in higher rates of synthesis than when translation of motA information terminated prematurely. Even in the total absence of MotA, the MotB that was synthesized was found exclusively in the cytoplasmic membrane fraction. In wild-type cells, MotA was estimated by immunoprecipitation to be in about fourfold excess over MotB; a previous estimate of 600 +/- 250 copies of MotA per cell then yielded an estimate of 150 +/- 70 copies of MotB per cell.
Hyperthermus butylicus, a hyperthermophilic sulfur-reducing archaebacterium that ferments peptides
Authors: Zillig W , Holz I , Janekovic D , Klenk H P , Imsel E , Trent J , Wunderl S , Forjaz V H , Coutinho R , Ferreira T
Published: November 2016

The hyperthermophilic peptide-fermenting sulfur archaebacterium Hyperthermus butylicus was isolated from the sea floor of a solfataric habitat with temperatures of up to 112 degrees C on the coast of the island of São Miguel, Azores. The organism grows at up to 108 degrees C, grows optimally between 95 and 106 degrees C at 17 g of NaCl per liter and pH 7.0, utilizes peptide mixtures as carbon and energy sources, and forms H2S from elemental sulfur and molecular hydrogen as a growth-stimulating accessory energy source but not by sulfur respiration. The same fermentation products, CO2, 1-butanol, acetic acid, phenylacetic acid, and a trace of hydroxyphenylacetic acid, are formed both with and without of S0 and H2. Its ether lipids, the absence of a mureine sacculus, the nature of the DNA-dependent RNA polymerase, and phylogenetic classification by DNA-rRNA cross-hybridization characterize H. butylicus as part of a novel genus of the major branch of archaebacteria comprising the orders Thermoproteales and Sulfolobales, representing a particularly long lineage bifurcating with the order Sulfolobales above the branching off of the genus Thermoproteus and distinct from the genera Desulfurococcus and Pyrodictium.
Use of regulated cell lysis in a lethal genetic selection in Escherichia coli: identification of the autoinducer-binding region of the LuxR protein from Vibrio fischeri ATCC 7744
Authors: Shadel G S , Young R , Baldwin T O
Published: November 2016

A lethal genetic selection utilizing the bacteriophage lambda lysis genes (S, R, RZ) has been developed and used in conjunction with a luminescence screen to allow the isolation and characterization of six missense mutations and two nonsense mutations in the luxR gene from Vibrio fischeri ATCC 7744. A transcriptional fusion of the lysis genes in operonR downstream of a truncated luxI gene allows control of cell lysis by the addition of synthetic autoinducer to the growth medium. The six missense mutations isolated resulted in changes in the LuxR protein of Asp at position 79 to Asn (hereafter designated as D79N), V82I, V109L, L118F, S123I, and H217Y. Variant LuxR proteins with amino acid changes of D79N, V82I, V82L, and H127Y were shown to require higher concentrations of autoinducer to elicit a certain amplitude response than is required by the wild-type protein. We believe that the clustering of a total of seven randomly generated missense mutations in a 49-amino-acid region of the LuxR primary sequence defines a critical portion of the LuxR protein. The observation that proteins with lesions in this region responded to elevated levels of autoinducer suggests that the autoinducer-binding site is constructed, at least in part, from several amino acid residues within the 79-to-127 region of the LuxR protein.
Streptomyces relC mutants with an altered ribosomal protein ST-L11 and genetic analysis of a Streptomyces griseus relC mutant
Authors: Ochi K
Published: November 2016

Several relaxed (rel) mutants have been obtained from Streptomyces species by selecting colonies resistant to thiopeptin, an analogue of thiostrepton. Using two-dimensional gel electrophoresis, I compared the ribosomal proteins from rel and rel+ pairs of S. antibioticus, S. lavendulae, S. griseoflavus, and S. griseus. It was found that all of the Streptomyces rel mutants thus examined had an altered or missing ribosomal protein, designated tentatively ST-L11. These rel mutants therefore could be classified as relC mutants and were highly sensitive to erythromycin or high temperature. A relC mutant of S. griseus was defective in streptomycin production, but phenotypic reversion of this defect to normal productivity was found at high incidence among progeny of the relC mutant. This phenotypic reversion did not accompany a reappearance of ribosomal protein ST-L11, and furthermore the ability of accumulating ppGpp still remained at a low level, thus suggesting existence of a mutation (named sup) which suppresses the streptomycin deficiency phenotype exhibited by the relC mutant. Genetic analysis revealed that there is a correlation between the rel mutation and the inability to produce streptomycin or aerial mycelia. The sup mutation was found to lie at a chromosomal locus distinct from that of the relC mutation. It was therefore concluded that the dependence of streptomycin production on the normal function of the relC gene could be entirely bypassed by a mutation at the suppressor locus (sup). The suppressing effect of the sup mutation on the relC mutation was blocked when the afs mutation (defective in A-factor synthesis) was introduced into a relC sup double mutant. It is proposed that the sup gene or its product can be direct or indirect target for ppGpp.
Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium
Authors: Huang C J , Barrett E L
Published: November 2016

Transposon Tn5 insertions causing anaerobic cysteine auxotrophy were isolated from a Salmonella typhimurium cysI parent (auxotrophic under aerobic but not anaerobic conditions). Insertions in one mutant group appeared to be in cysG. A second group of insertions, designated asr (anaerobic sulfite reduction), were located near map unit 53 on the S. typhimurium chromosome. They did not cause aerobic or anaerobic auxotrophy in a cys1+ background but did prevent dissimilatory sulfite reduction. Plasmids containing asr DNA cloned from wild-type S. typhimurium conferred anaerobic prototrophy and the ability to produce hydrogen sulfide from sulfite on an Escherichia coli cys1 mutant.
In vitro characterization of two different Phycomyces blakesleeanus mutants with impaired phytoene desaturation
Authors: Schmidt A , Sandmann G
Published: November 2016

In vitro phytoene desaturation was investigated in two Phycomyces blakesleeanus mutants, C5 and S442, in which phytoene is accumulated instead of beta-carotene. For strain C5 but not strain S442 the phenotypic block of phytoene conversion could be overcome in vitro by the addition of Tween 40. Immunodetection of phytoene desaturase revealed in all cases the presence of a 40-kilodalton protein.
Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways
Authors: Keszenman D J , Salvo V A , Nunes E
Published: November 2016

In order to analyze the roles of some repair genes in the processing of bleomycin-induced DNA damage and, especially, the interrelationships among the involved repair pathways, we investigated the potentially lethal effect of bleomycin on radiosensitive mutants of Saccharomyces cerevisiae defective in recombination, excision, and RAD6-dependent DNA repair. Using single, double, and triple rad mutants, we analyzed growth kinetics and survival curves as a function of bleomycin concentration. Our results indicate that genes belonging to the three epistasis groups interact in the repair of bleomycin-induced DNA damage to different degrees depending on the concentration of bleomycin. The most important mechanisms involved are recombination and postreplication repair. The initial action of a potentially inducible excision repair gene could provide intermediate substrates for the RAD6- and RAD52-dependent repair processes. Interaction between RAD6 and RAD52 genes was epistatic for low bleomycin concentrations. RAD3 and RAD52 genes act independently in processing DNA damage induced by high concentrations of bleomycin. The synergistic interaction observed at high concentrations in the triple mutant rad2-6 rad6-1 rad52-1 indicates partial independence of the involved repair pathways, with possible common substrates. On the basis of the present results, we propose a heuristic model of bleomycin-induced DNA damage repair.
Coexpression of UmuD' with UmuC suppresses the UV mutagenesis deficiency of groE mutants
Authors: Donnelly C E , Walker G C
Published: November 2016

The GroE proteins of Escherichia coli are heat shock proteins which have also been shown to be molecular chaperone proteins. Our previous work has shown that the GroE proteins of E. coli are required for UV mutagenesis. This process requires the umuDC genes which are regulated by the SOS regulon. As part of the UV mutagenesis pathway, the product of the umuD gene, UmuD, is posttranslationally cleaved to yield the active form, UmuD'. In order to investigate what role the groE gene products play in UV mutagenesis, we measured UV mutagenesis in groE+ and groE strains which were expressing either the umuDC or umuD'C genes. We found that expression of umuD' instead of umuD will suppress the nonmutability conferred by the groE mutations. However, cleavage of UmuD to UmuD' is unaffected by mutations at the groE locus. Instead we found that the presence of UmuD' increased the stability of UmuC in groE strains. In addition, we obtained evidence which indicates that GroEL interacts directly with UmuC.
Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity
Authors: Arnaud M , Vary P , Zagorec M , Klier A , Debarbouille M , Postma P , Rapoport G
Published: November 2016

The sacT gene which controls the sacPA operon of Bacillus subtilis encodes a polypeptide homologous to the B. subtilis SacY and the Escherichia coli BglG antiterminators. Expression of the sacT gene is shown to be constitutive. The DNA sequence upstream from sacP contains a palindromic sequence which functions as a transcriptional terminator. We have previously proposed that SacT acts as a transcriptional antiterminator, allowing transcription of the sacPA operon. In strains containing mutations inactivating ptsH or ptsI, the expression of sacPA and sacB is constitutive. In this work, we show that this constitutivity is due to a fully active SacY antiterminator. In the wild-type sacT+ strain or in the sacT30 mutant, SacT requires both enzyme I and HPr of the phosphotransferase system (PTS) for antitermination. It appears that the PTS exerts different effects on the sacB gene and the sacPA operon. The general proteins of the PTS are not required for the activity of SacY while they are necessary for SacT activity.
Characterization of bofA, a gene involved in intercompartmental regulation of pro-sigma K processing during sporulation in Bacillus subtilis
Authors: Ricca E , Cutting S , Losick R
Published: November 2016

Sporulating cells of the gram-positive bacterium Bacillus subtilis are partitioned into two cellular compartments called the mother cell and the forespore. Gene expression in the mother cell and the forespore is regulated differentially by the compartment-specific transcription factors sigma K and sigma G, respectively. Gene expression between the two compartments is also coordinated by a signal transduction pathway that couples the activation of sigma K (by processing of its inactive precursor pro-sigma K) in the mother cell to sigma G-directed gene expression in the forespore. To dissect the signal transduction pathway genetically, we previously isolated bypass of forespore mutations at loci called bofA and bofB that relieve the dependence of pro-sigma K processing on the action of sigma G. bofB mutations were previously shown to be allelic to the two-cistron sporulation operon spoIVF, which encodes the pro-sigma K-processing enzyme or its regulator. We now report that bofA mutations are located in a small open reading frame of 87 codons that encodes a putative integral membrane protein with three potential membrane-spanning domains. The possibility is discussed that BofA and the SpoIVF proteins form a heteromeric complex in the mother cell membrane that surrounds the forespore and that this complex mediates the intercompartmental coupling of pro-sigma K processing to events in the forespore.
Conserved gene arrangement in the origin region of the Streptomyces coelicolor chromosome
Authors: Calcutt M J , Schmidt F J
Published: November 2016

A 23-kb fragment of the Streptomyces coelicolor chromosome spanning the dnaA region has been isolated as a cosmid clone. Nucleotide sequence analysis of a 5-kb portion shows that the genes for the RNase P protein (rnpA), ribosomal protein L34 (rpmH), the replication initiator protein (dnaA), and the beta subunit of DNA polymerase III (dnaN) are present in the highly conserved gene arrangement found in all eubacterial genomes studied so far. The dnaA-dnaN intergenic region is approximately 1 kb and contains a cluster of at least 12 DnaA boxes with a consensus sequence of TTGTCCACA matching the consensus DnaA box in the phylogenetically related Micrococcus luteus. Two DnaA boxes precede the dnaA sequence. We propose that the chromosomal origin (oriC) of S. coelicolor lies between dnaA and dnaN. In related work, J. Zakrzewska-Czerwinska and H. Schrempf (J. Bacteriol. 174:2688-2693, 1992) have identified the homologous sequence from the closely-related Streptomyces lividans as capable of self-replication.
Sucrose fermentation by Fusobacterium mortiferum ATCC 25557: transport, catabolism, and products
Authors: Thompson J , Nguyen N Y , Robrish S A
Published: November 2016

Studies of sucrose utilization by Fusobacterium mortiferum ATCC 25557 have provided the first definitive evidence for phosphoenolpyruvate-dependent sugar:phosphotransferase activity in the family Bacteroidaceae. The phosphoenolpyruvate-dependent sucrose:phosphotransferase system and the two enzymes required for the dissimilation of sucrose 6-phosphate are induced specifically by growth of F. mortiferum on the disaccharide. Monomeric sucrose 6-phosphate hydrolase (M(r), 52,000) and a dimeric ATP-dependent fructokinase (subunit M(r), 32,000) have been purified to electrophoretic homogeneity. The physicochemical and catalytic properties of these enzymes have been examined, and the N-terminal amino acid sequences for both proteins are reported. The characteristics of sucrose 6-phosphate hydrolase and fructokinase from F. mortiferum are compared with the same enzymes from both gram-positive and gram-negative species. Butyric, acetic, and D-lactic acids are the end products of sucrose fermentation by F. mortiferum. A pathway is proposed for the translocation, phosphorylation, and metabolism of sucrose by this anaerobic pathogen.
The lysP gene encodes the lysine-specific permease
Authors: Steffes C , Ellis J , Wu J , Rosen B P
Published: November 2016

Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine. The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine. It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine). Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA. A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport. This lysP mutation was mapped to about 46.5 min on the E. coli chromosome. The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene. The nucleotide sequence of the 2.7-kb BamHI fragment was determined. An open reading frame from nucleotides 522 to 1989 was observed. The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues. The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E. coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1). Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels. These results demonstrate that the lysP gene encodes the lysine-specific permease.
Deletion of an additional domain located between SXXK and SXN active-site fingerprints in penicillin-binding protein 4 from Escherichia coli
Authors: Mottl H , Nieland P , de Kort G , Wierenga J J , Keck W
Published: November 2016

It was suggested previously that the primary structure of penicillin-binding protein 4 (PBP4) is new and unique among proteins that interact with penicillin. Our proposal that PBP4 carries an additional domain, located between the active-site fingerprints SXXK and SXN, was investigated by mutational deletion analysis. A clustered set of internal deletions was created in this region by exonuclease treatment of the dacB coding DNA, starting from two internal restriction sites. PBP4 mutants carrying internal deletions were selected by screening for immunoreactive forms of PBP4 with reduced molecular weight that were still active with respect to penicillin binding. DNA sequencing revealed 24 distinct PBP4 mutants with internal deletions ranging from 37 to 113 amino acids. The amino- and carboxy-terminal end points of the deletions were not randomly distributed but tended to cluster in certain areas. Overproduction of the individual mutated forms of PBP4 resulted in accumulation of the major portion of the proteins in the particulate cell fraction. The yield of soluble and active mutated forms of the protein was reduced from below 1% to 79% of the level obtained for the native protein. The deletions that were introduced had minor effects on the deacylation rate of bound benzylpenicillin. Two pairs of cysteine residues (Cys-139-Cys-153 and Cys-197-Cys-214) that are located in the deletable region may form disulfide bridges.
Identification of the vibriobactin receptor of Vibrio cholerae
Authors: Stoebner J A , Butterton J R , Calderwood S B , Payne S M
Published: November 2016

Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.
Mutational activation of CheA, the protein kinase in the chemotaxis system of Escherichia coli
Authors: Tawa P , Stewart R C
Published: November 2016

In Escherichia coli and Salmonella typhimurium, appropriate changes of cell swimming patterns are mediated by CheA, an autophosphorylating histidine protein kinase whose activity is regulated by receptor/transducer proteins. The molecular mechanism underlying this regulation remains unelucidated but may involve CheA shifting between high-activity and low-activity conformations. We devised an in vivo screen to search for potential hyperkinase variants of CheA and used this screen to identify two cheA point mutations that cause the CheA protein to have elevated autokinase activity. Each point mutation resulted in alteration of proline 337. In vitro, CheA337PL and CheA337PS autophosphorylated significantly more rapidly than did wild-type CheA. This rate enhancement reflected the higher affinities of the mutant proteins for ATP and an increased rate constant for acquisition by CheA of the gamma-phosphoryl group of ATP within a kinetically defined CheA.ATP complex. In addition, the mutant proteins reacted with ADP more rapidly than did wild-type CheA. We considered the possibility that the mutations served to lock CheA into an activated signaling conformation; however, we found that both mutant proteins were regulated in a normal fashion by the transducer Tsr in the presence of CheW. We exploited the activated properties of one of these mutants to investigate whether the CheA subunits within a CheA dimer make equivalent contributions to the mechanism of trans phosphorylation. Our results indicate that CheA trans phosphorylation may involve active-site residues that are located both in cis and in trans to the autophosphorylation site and that the two protomers of a CheA dimer make nonequivalent contributions in determining the affinity of the ATP-binding site(s) of CheA.
Effects of reduced levels of GroE chaperones on protein metabolism: enhanced synthesis of heat shock proteins during steady-state growth of Escherichia coli
Authors: Kanemori M , Mori H , Yura T
Published: November 2016

The GroE heat shock proteins (GroEL and GroES) of Escherichia coli represent major molecular chaperones that participate in folding (and assembly) of a variety of proteins and are essential for cell growth at all temperatures. We have examined the effects of reducing the cellular content of GroE on the synthesis and stability of proteins during steady-state growth with near-normal rates. The GroE protein level was manipulated by placing groE under the control of lacUV5 promoter on a multicopy plasmid in a strain lacking the chromosomal groE operon. When this strain was grown with a limited concentration (40 microM) of inducer (IPTG [isopropyl-beta-D-thiogalactopyranoside]) at 37 degrees C, the GroE level and growth rate were comparable to those of the wild type. When cells were depleted of IPTG, they continued to grow at or below 37 degrees C albeit at reduced rates, despite the much-reduced GroE level (ca. 25% of that of wild type). Under these conditions, the cellular contents of at least 13 polypeptides were affected. Among the most striking effects was the enhanced synthesis of a set of heat shock proteins which resulted from the increased level of sigma 32 which is required for transcription of heat shock genes. This increase in the sigma 32 level was brought about by both stabilization and increased synthesis of sigma 32. Other proteins affected by the reduced GroE level included two proteins (enzymes of the Entner-Doudoroff pathway) encoded by the edd-eda operon and the ribosomal protein S6, suggesting that the GroE chaperones are involved in regulating expression of genes for carbohydrate metabolism and in modulating biogenesis or function of the ribosome.
Evidence for two NAD kinases in Salmonella typhimurium
Authors: Cheng W , Roth J R
Published: November 2016

The electron-carrying cofactor NADP is formed by phosphorylation of NAD. A strategy for the isolation of NAD kinase mutants revealed two classes of temperature-sensitive mutations, nadF and nadG, mapping at min 13 and 72 of the Salmonella chromosome. Both mutant types grew on nutrient broth at both 30 and 42 degrees C but on minimal medium showed a temperature-sensitive growth defect which was not corrected by any of the single nutritional supplements tested. A nadF deletion mutant grew on nutrient broth but not on minimal medium. A double mutant with the nadF deletion and a nadG(Ts) mutation showed temperature-sensitive growth on all media. We propose that Salmonella typhimurium has two NAD kinases, one encoded by the nadF and one by the nadG gene. This is supported by the fact that temperature-sensitive mutants of both genes produce kinase activity with altered heat stability. Results suggest that either one of two NAD kinases is sufficient for growth on rich medium, but that both are needed for growth on minimal media. Enzyme assays show that the nadF gene is responsible for about 70% of total NAD kinase activity, and that the nadG gene dictates the remaining 30%. While testing nutritional phenotypes of nadF and nadG mutants, we found that the biosynthetic intermediate, quinolinic acid (QA) inhibited growth of nadF mutants on nutrient broth. This suggested that the NadG enzyme might be inhibited by QA. Enzyme assays demonstrated that QA inhibits the NadG but not the NadF enzyme. This suggests the existence of a regulatory mechanism which controls NADP levels.
Nucleotide sequence and functional analysis of the meta-cleavage pathway involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102
Authors: Kikuchi Y , Yasukochi Y , Nagata Y , Fukuda M , Takagi M
Published: November 2016

Pseudomonas sp. strain KKS102 is able to degrade biphenyl and polychlorinated biphenyls via the meta-cleavage pathway. We sequenced the upstream region of the bphA1A2A3BCD (open reading frame 1 [ORF1]) A4 and found four ORFs in this region. As the deduced amino acid sequences of the first, second, and third ORFs are homologous to the meta-cleavage enzymes from Pseudomonas sp. strain CF600 (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), these ORFs have been named bphE, bphG, and bphF, respectively. The fourth ORF (ORF4) showed homology with ORF3 from Pseudomonas pseudoalcaligenes KF707 (K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), whose function is unknown. The functions of meta-cleavage enzymes (BphE, BphG, and BphF) were analyzed by using crude extracts of Escherichia coli which expressed the encoding genes. The results showed that bphE, bphG, and bphF encode 2-hydroxypenta-2,4-dienoate hydratase, acetaldehyde dehydrogenase (acylating), and 4-hydroxy-2-oxovalerate aldolase, respectively. The biphenyl and polychlorinated biphenyl degradation pathway of KKS102 is encoded by 12 genes in the order bphEGF (ORF4)A1A2A3BCD (ORF1)A4. The functions of ORF1 and ORF4 are unknown. The features of this bph gene cluster are discussed.
Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae
Authors: Morona J K , Guidolin A , Morona R , Hansman D , Paton J C
Published: November 2016

A comparative hybridization protocol was used to isolate a small segment of DNA present in the Streptococcus pneumoniae type 19F strain SSZ but absent from strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp imperfect inverted repeats and containing a single open reading frame sufficient to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either end of the element. IS1202 is not related to any of the currently known insertion elements and is the first reported for S. pneumoniae. Although found predominantly in type 19F strains in up to five copies, it has also been shown to be present in the chromosomes of pneumococci belonging to other serotypes. One of the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp downstream of the dexB gene, and transformation studies reveal that it is also closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.
Cloning and analysis of a locus (mcr) involved in mitomycin C resistance in Streptomyces lavendulae
Authors: August P R , Flickinger M C , Sherman D H
Published: November 2016

Two genes (mcrA and mcrB) from Streptomyces lavendulae that together confer resistance to mitomycin C were identified. This DNA appears to comprise a polycistronic operon with a drug-inducible leaderless mRNA. The deduced amino acid sequence of mcrA shows similarity to sequences of a special class of bacterial, plant, and animal oxygen oxidoreductases.
Differential reduction in soluble and membrane-bound c-type cytochrome contents in a Paracoccus denitrificans mutant partially deficient in 5-aminolevulinate synthase activity
Authors: Page M D , Ferguson S J
Published: November 2016

A mutant of Paracoccus denitrificans, DP104, unable to grow anaerobically with nitrate as the terminal electron acceptor or aerobically with methanol as the electron donor and staining negatively in the dimethylphenylene diamine oxidation (Nadi) test, was isolated by transposon Tn5::phoA mutagenesis. P. denitrificans DP104 grown aerobically with succinate or choline had very low levels (2 to 3% of the wild-type levels) of spectroscopically detectable soluble c-type cytochromes. In contrast, membrane cytochromes of the a, b, and c types were present at 50% of the levels found in the wild type. The apo form of cytochrome c550, at an approximately 1:1 molar ratio with the holo form, was found in the periplasm of DP104. The TnphoA element was shown to be inserted immediately upstream of the translational start of hemA, the gene coding for 5-aminolevulinate synthase, which was sequenced. Low-level expression of this gene, driven off an incidental promoter provided by TnphoA-cointegrated suicide vector DNA, is the basis of the phenotype which could be complemented by the addition of 5-aminolevulinate to growth media. Disruption of the hemA gene generated a P. denitrificans strain auxotrophic for 5-aminolevulinate, establishing that there is no hemA-independent pathway of heme synthesis in this organism. The differential deficiency in periplasmic c-type cytochromes relative to membrane cytochromes in DP104 is suggested to arise from unequal competition for the restricted supply of heme which results from the effects of the transposon insertion.
Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB
Authors: Samuels D S , Mach K E , Garon C F
Published: November 2016

No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the physiology and pathogenesis of the Lyme disease agent on a molecular genetic level.
Effect of amino acid substitutions in a potential metal-binding site of AnfA on expression from the anfH promoter in Azotobacter vinelandii
Authors: Premakumar R , Loveless T M , Bishop P E
Published: November 2016

AnfA, an activator required for transcription of the structural genes encoding nitrogenase 3 (anfHDGK) in Azotobacter vinelandii, has a potential metal-binding site [(S19)H(C21)FTGE(C26)R] in its N terminus. Growth studies and expression of an anfH-lacZ fusion in mutants containing amino acid substitutions in this site indicate that Ser-19 is not required for AnfA activity whereas Cys-21 and Cys-26 are required. Residual expression of the anfH-lacZ fusion in AnfA- mutants was found to be due to activation by VnfA, the activator required for expression of genes encoding nitrogenase 2.