bluuebunny/crossref_metadata_2025 huggingface dataset:
Integration host factor and sequences downstream of the Pseudomonas aeruginosa algD transcription start site are required for expression
Authors: Wozniak D J
Published: November 2016

Pseudomonas aeruginosa is an extremely important opportunistic pathogen in immunocompromised individuals. Strains of P. aeruginosa isolated from chronic lung infections in patients with the genetic disease cystic fibrosis have a mucoid colony morphology. This phenotype is due to overproduction of the exopolysaccharide alginate, which is believed to confer a selective advantage on P. aeruginosa in cystic fibrosis lungs. Alginate biosynthesis is controlled by a complex regulatory mechanism. Genes located in the 34-min region of the P. aeruginosa chromosome form an operon which encodes most of the biosynthetic enzymes necessary for alginate production. algD, the first gene in the operon and a critical point for the transcriptional regulation of alginate biosynthesis, is controlled by several trans, cis, and environmental factors. In this study, the involvement of the histone-like protein integration host factor (IHF) in algD expression was examined. Sequences with similarity to consensus IHF-binding sites of Escherichia coli were identified 75 bp upstream (site 1) and 90 bp downstream (site 2) of the start of algD transcription. In gel band mobility shift assays, DNA fragments containing either site bind IHF but site 2 has an approximately 90-fold higher affinity for IHF. Mutations in each of the elements were generated, and they resulted in the reduction or loss of in vitro IHF binding and a three- to fourfold decrease in algD-cat expression. This indicates that IHF binding is necessary for high-level algD transcription. The presence of a high-affinity IHF-binding site located 3' of the algD transcription start site suggested that sequences further downstream of this element are involved in algD expression. When a fragment located downstream of site 2 and upstream of the promoterless cat gene (+110 to +835) was deleted, algD-cat expression was reduced 10-fold supporting the notion that 3' enhancer elements are required for algD transcription. This is the first direct evidence of a 3' element involved in the control of a P. aeruginosa gene. It is postulated that IHF mediates the formation of a higher-order looped structure which is necessary for efficient algD transcription.
Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli
Authors: Zaman M M , Boles T C
Published: November 2016

Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.
Cloning, expression, and nucleotide sequence of the alpha-amylase gene from the haloalkaliphilic archaeon Natronococcus sp. strain Ah-36
Authors: Kobayashi T , Kanai H , Aono R , Horikoshi K , Kudo T
Published: November 2016

The alpha-amylase gene of a Natronococcus sp. (1,512 bp) contained a signal peptide of 43 amino acids. Haloferax volcanii expressed the gene and cleaved the signal peptide accurately. The signal peptide shared an extremely high amino acid sequence identity with that of a protease from the halophilic archaeon 172P1.
Mutations in the Bordetella pertussis bvgS gene that confer altered expression of the fhaB gene in Escherichia coli
Authors: Goyard S , Bellalou J , Mireau H , Ullmann A
Published: November 2016

The bvg locus of Bordetella pertussis, required for coordinate regulation of virulence genes in response to environmental signals, encodes two proteins, BvgS and BvgA, that belong to the bacterial two-component signal transduction systems. We have isolated spontaneous mutations of the bvg locus in Escherichia coli and analyzed their effects on the expression of fhaB::lacZY transcriptional fusions. The mutations, localized in the linker and transmitter domain of BvgS, result in increased activation of fhaB and/or in insensitivity to a modulating agent, nicotinic acid.
The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters
Authors: Foster-Hartnett D , Kranz R G
Published: November 2016

The protein encoded by glnB of Rhodobacter capsulatus is part of a nitrogen-sensing cascade which regulates the expression of nitrogen fixation genes (nif). The expression of glnB was studied by using lacZ fusions, primer extension analysis, and in vitro DNase I footprinting. Our results suggest that glnB is transcribed from two promoters, one of which requires the R. capsulatus ntrC gene but is rpoN independent. Another promoter upstream of glnB is repressed by NtrC; purified R. capsulatus NtrC binds to sites that overlap this distal promoter region.
The flagellar bundle of Halobacterium salinarium is inserted into a distinct polar cap structure
Authors: Kupper J , Marwan W , Typke D , Grünberg H , Uwer U , Gluch M , Oesterhelt D
Published: November 2016

Flagellated envelopes of Halobacterium salinarium cells were prepared by lysis with taurodeoxycholate. After solubilization of the envelopes with Triton X-100 at high ionic strength, flagella and round patches from which numerous flagella emerged were isolated by gel filtration chromatography. We conclude that the flagellar bundle of H. salinarium is inserted into a differentiated polar cap structure.
The sequence of the single 16S rRNA gene of the thermophilic eubacterium Rhodothermus marinus reveals a distant relationship to the group containing Flexibacter, Bacteroides, and Cytophaga species
Authors: Andrésson O S , Fridjónsson O H
Published: November 2016

Rhodothermus marinus, a gram-negative heterotrophic marine thermophile, has been the subject of several recent studies. Isolation, sequencing, and analyses of a 16S rRNA gene have shown that R. marinus diverges sharply from major bacterial phyla and is most closely allied to the Flexibacter-Cytophaga-Bacteroides group. Further analyses revealed that the R. marinus chromosome contains a single rRNA operon with a 16S-23S intergenic region coding for tRNA(Ile) and tRNA(Ala).
Functional analysis of the tdcABC promoter of Escherichia coli: roles of TdcA and TdcR
Authors: Hagewood B T , Ganduri Y L , Datta P
Published: November 2016

The efficient expression of the tdc operon of Escherichia coli requires the products of two regulatory genes, tdcR and tdcA. We have identified the transcription site of tdcR by primer extension mapping and established the translation start site of TdcR by mutational analysis of its reading frame. In a tdcR tdcABC deletion strain, tdcR+ promoted high-level LacZ expression from a lambda tdcAB-lacZ lysogen and mutations introduced in tdcR resulted in a greater than sixfold decrease in LacZ level. In-frame deletions of tdcA also reduced LacZ expression, and chromosomal and plasmid-borne tdcA+ increased the LacZ level in tdcA mutant lysogens. Interestingly, multicopy tdcA+ plasmids introduced into tdcR mutant strains completely restored tdc expression. In separate experiments we found that mutations in the tdc promoter DNA around positions -70, -140, and -175 greatly reduced tdc expression relative to that for the wild-type promoter and the tdcP mutation around -175 prevented multicopy tdcA+ from rescuing tdcR mutants. Furthermore, competition experiments revealed that a wild-type promoter fragment encompassing the -175 region cloned into a plasmid reduced tdc expression by titrating TdcA in vivo, and this effect was reversed with excess TdcA. These results suggest that in tdcR+ cells TdcR interacts with tdcP and/or TdcA to enhance tdc transcription whereas in tdcR mutant cells a new tdcP-TdcA complex around -175 in the native promoter bypasses the requirement for TdcR. On the basis of the accumulated data summarized here and elsewhere we propose that multiple transcription factors enhance tdc operon expression by bending and looping of the promoter DNA to form an active transcription complex.
Isolation and sequence analysis of polyketide synthase genes from the daunomycin-producing Streptomyces sp. strain C5
Authors: Ye J , Dickens M L , Plater R , Li Y , Lawrence J , Strohl W R
Published: November 2016

A contiguous region of about 30 kbp of DNA putatively encoding reactions in daunomycin biosynthesis was isolated from Streptomyces sp. strain C5 DNA. The DNA sequence of an 8.1-kbp EcoRI fragment, which hybridized with actI polyketide synthase (PKS) and actIII polyketide reductase (PKR) gene probes, was determined, revealing seven complete open reading frames (ORFs), two in one cluster and five in a divergently transcribed cluster. The former two genes are likely to encode PKR and a bifunctional cyclase/dehydrase. The five latter genes encode: (i) a homolog of TcmH, an oxygenase of the tetracenomycin biosynthesis pathway; (ii) a PKS Orf1 homolog; (iii) a PKS Orf2 homolog (chain length factor); (iv) a product having moderate sequence identity with Escherichia coli beta-ketoacyl acyl carrier protein synthase III but lacking the conserved active site; and (v) a protein highly similar to several acyltransferases. The DNA within the 8.1-kbp EcoRI fragment restored daunomycin production to two dauA non-daunomycin-producing mutants of Streptomyces sp. strain C5 and restored wild-type antibiotic production to Streptomyces coelicolor B40 (act VII; nonfunctional cyclase/dehydrase), and to S. coelicolor B41 (actIII) and Streptomyces galilaeus ATCC 31671, strains defective in PKR activity.
Biochemical and structural analyses of the extracellular matrix fibrils of Myxococcus xanthus
Authors: Behmlander R M , Dworkin M
Published: November 2016

It is characteristic of myxobacteria to produce large amounts of extracellular material. This report demonstrates that this material in Myxococcus xanthus is fibrillar and describes the structure and chemical composition of the fibrils. The extracellular matrix fibrils are the mediators of cell-cell cohesion in M. xanthus. As such, the fibrils play an important role in the cell-cell interactions that form the basis for the social and developmental lifestyle of this organism. The fibrils are composed of protein and carbohydrate in a 1.0:1.2 ratio. Combined, the two fractions accounted for greater than 85% of the mass of isolated fibrils, and the fibrils were found to compose up to 10% of the dry weight of cells grown at high density on a solid surface. The polysaccharide portion of the fibrils was shown to be composed of five different monosaccharides: galactose, glucosamine, glucose, rhamnose, and xylose. Glucosamine, one of the component monosaccharides of the fibrils and a known morphogen for M. xanthus, inhibited cohesion to a level near that of Congo red (the positive control for cohesion inhibition). Glucose and xylose also inhibited cohesion but less than did glucosamine. Analysis of the morphology of the fibrils, the periodicities within the distribution of fibril diameters observed by field emission scanning electron microscopy, and the observation of fibrils on hydrated cells strongly suggested that the extracellular matrix of M. xanthus was indeed arranged as fibrils. Furthermore, results suggested that the fibrils were constructed as carbohydrate structures with associated proteins.
In vivo and in vitro characterization of the light-regulated cpcB2A2 promoter of Fremyella diplosiphon
Authors: Casey E S , Grossman A
Published: November 2016

When exposed to different spectral qualities of light, many cyanobacteria dramatically alter their phycobilisome rod composition in a process termed complementary chromatic adaptation. In the cyanobacterium Fremyella diplosiphon, this response is associated with differential expression of the cpcB2A2, cpeBA, and cpeCDE operons, which code for the phycobiliproteins phycocyanin and phycoerythrin and the phycoerythrin linker polypeptides, respectively. To define components of the signal transduction pathway involved in light-regulated expression of genes encoding phycobilisome polypeptides, we have used in vivo and in vitro techniques to identify cis-acting sequences and trans-acting factors necessary for the regulation of the red-light-inducible cpcB2A2 operon. Deletion of the cpcB2A2 upstream sequences to -76 bp with respect to the transcription start site had no effect on red-light induction of a cpcB2A2-beta-glucuronidase (GUS) chimeric gene, while deletion to -37 bp abolished GUS expression. Furthermore, a fragment of the cpcB2A2 gene from -76 to +25 bp linked to the untranslated leader of cpcB1A1 (a constitutively expressed operon encoding phycocyanin) is sufficient to drive high-level GUS expression in red light. Therefore, the sequence between positions -76 and -37 is necessary for the expression of cpcB2A2, and the region extending from -76 to +25 is sufficient for red-light induction of the operon. Attempts were made to correlate the in vivo data with protein binding in the region upstream of the transcription start site of cpcB2A2. Using in vitro analysis, we detected two protein-binding sites in the cpcB2A2 promoter which were localized to positions -162 to -122 and -37 to +25. Proteins from both red- and green-light-grown cells interacted with the former site, while only proteins present in extracts from red-light-grown cells interacted with the latter site. The data from both the in vivo and in vitro analyses suggest that while two regions upstream of the cpcB2A2 transcription initiation site specifically bind proteins, only the binding site bordering the transcription start site is important for complementary chromatic adaptation.
Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions
Authors: Prinz W A , Beckwith J
Published: November 2016

To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm.
4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation
Authors: Gibson J , Dispensa M , Fogg G C , Evans D T , Harwood C S
Published: November 2016

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.
Isolation of the hemF operon containing the gene for the Escherichia coli aerobic coproporphyrinogen III oxidase by in vivo complementation of a yeast HEM13 mutant
Authors: Troup B , Jahn M , Hungerer C , Jahn D
Published: November 2016

Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic and one for anaerobic conditions. Here we report the cloning of the hemF gene, encoding the aerobic coproporphyrinogen III oxidase from Escherichia coli, by functional complementation of a Saccharomyces cerevisiae HEM13 mutant. An open reading frame of 897 bp encoding a protein of 299 amino acids with a calculated molecular mass of 34.3 kDa was identified. Sequence comparisons revealed 43% amino acid sequence identity with the product of the S. cerevisiae HEM13 gene and 90% identity with the product of the recently cloned Salmonella typhimurium hemF gene, while a structural relationship to the proposed anaerobic enzyme from Rhodobacter sphaeroides was not obvious. The hemF gene is in an operon with an upstream open reading frame (orf1) encoding a 31.7-kDa protein with homology to an amidase involved in cell wall metabolism. The hemF gene was mapped to 52.6 min of the E. coli chromosome. Primer extension experiments revealed a strong transcription initiation site upstream of orf1. A weak signal, possibly indicative of a second promoter, was also identified just upstream of the hemF gene. A region containing bent DNA (Bent 111), previously mapped to 52.6 min of the E. coli chromosome, was discovered in the 5' region of orf1. Two potential integration host factor binding sites were found, one close to each transcription start site. An open reading frame (orf3) transcribed in a direction opposite that of the hemF gene was found downstream of the hemF gene. It encodes a protein of 40.2 kDa that showed significant homology to proteins of the XylS/AraC family of transcriptional regulators.
Role of the D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid
Authors: Heaton M P , Neuhaus F C
Published: November 2016

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) is a widespread macroamphiphile which plays a vital role in the growth and development of gram-positive organisms. The biosynthesis of this polymer requires the enzymic activation of D-alanine for its transfer to the membrane-associated LTA (mLTA). A small, heat-stable, and acidic protein that is required for this transfer was purified to greater than 98% homogeneity from Lactobacillus casei ATCC 7469. This protein, previously named the D-alanine-membrane acceptor ligase (V. M. Reusch, Jr., and F. C. Neuhaus, J. Biol. Chem. 246:6136-6143, 1971), functions as the D-alanyl carrier protein (Dcp). The amino acid composition, beta-alanine content, and N-terminal sequence of this protein are similar to those of the acyl carrier proteins (ACPs) of fatty acid biosynthesis. The isolation of Dcp and its derivative, D-alanyl approximately Dcp, has allowed the characterization of two novel reactions in the pathway for D-alanyl-mLTA biosynthesis: (i) the ligation of Dcp with D-alanine and (ii) the transfer of D-alanine from D-alanyl approximately Dcp to a membrane acceptor. It has not been established whether the membrane acceptor is mLTA or another intermediate in the pathway for D-alanyl-mLTA biosynthesis. Since the D-alanine-activating enzyme (EC 6.1.1.13) catalyzes the ligation reaction, this enzyme functions as the D-alanine-Dcp ligase (Dcl). Dcl also ligated the ACPs from Escherichia coli, Vibrio harveyi, and Saccharopolyspora erythraea with D-alanine. In contrast to the relaxed specificity of Dcl in the ligation reaction, the transfer of D-alanine to the membrane acceptor was highly specific for Dcp and did not occur with other ACPs. This transfer was observed by using only D-[14C]alanyl approximately Dcp and purified L. casei membranes. Thus, D-alanyl approximately Dcp is an essential intermediate in the transfer of D-alanine from Dcl to the membrane acceptor. The formation of D-alanine esters of mLTA provides a mechanism for modulating the net anionic charge in the cell wall.
Electric fields induce curved growth of Enterobacter cloacae, Escherichia coli, and Bacillus subtilis cells: implications for mechanisms of galvanotropism and bacterial growth
Authors: Rajnicek A M , McCaig C D , Gow N A
Published: November 2016

Directional growth in response to electric fields (galvanotropism) is known for eukaryotic cells as diverse as fibroblasts, neurons, algae, and fungal hyphae. The mechanism is not understood, but all proposals invoke actin either directly or indirectly. We applied electric fields to bacteria (which are inherently free of actin) to determine whether actin was essential for galvanotropism. Field-treated (but not control) Enterobacter cloacae and Escherichia coli cells curved rapidly toward the anode. The response was both field strength and pH dependent. The direction of curvature was reversed upon reversal of field polarity. The directional growth was not due to passive bending of the cells or to field-induced gradients of tropic substances in the medium. Field-treated Bacillus subtilis cells also curved, but the threshold was much higher than for E. cloacae or E. coli. Since the curved morphology must reflect spatial differences in the rates of cell wall synthesis and degradation, we looked for regions of active wall growth. Experiments in which the cells were decorated with latex beads revealed that the anode-facing ends of cells grew faster than the cathode-facing ends of the same cells. Inhibitors of cell wall synthesis caused spheroplasts to form on the convex regions of field-treated cells, suggesting that the initial curvature resulted from enhanced growth of cathode-facing regions. Our results indicate that an electric field modulates wall growth spatially and that the mechanism may involve differential stimulation of wall growth in both anode- and cathode-facing regions. Electric fields may therefore serve as valuable tools for studies of bacterial wall growth. Use of specific E. coli mutants may allow dissection of the galvanotropic mechanism at the molecular level.
Induced chromosome rearrangements and morphologic variation in Candida albicans
Authors: Barton R C , Scherer S
Published: November 2016

We have isolated a mutant of Candida albicans that switches between colony morphologies at high frequencies in a strain with several genetic markers. This strain, 1183, has an altered karyotype with two extra chromosomes. The 1183 karyotype is unstable upon passage. Using DNA transformation with the URA3 gene flanked by sequences from the C. albicans repeat sequence 27A, we have marked individual chromosomes of 1183 and 1161, a related smooth, stable strain. Many transformants contained one or more extra chromosomes, ranging in size from 150 kb to 2.1 Mb. Most were less than 800 kb and appeared to be fragments of a single chromosome. All fragments tested derive from one of the two smallest chromosomes. Six of 13 fragments contained the URA3 gene. In some cases, URA3 was located at the end of a fragment with adjacent telomere repeats. The integrated copy of URA3 was unstable in some 1183 transformants. Our results suggest that 1183 has a mutation affecting genomic stability. A connection between karyotypic changes and morphologic variation has been suggested from studies of several C. albicans strains; however, we find that gross karyotypic and morphological changes are separable processes.
DnaK mutants defective in ATPase activity are defective in negative regulation of the heat shock response: expression of mutant DnaK proteins results in filamentation
Authors: McCarty J S , Walker G C
Published: November 2016

Site-directed mutagenesis has previously been used to construct Escherichia coli dnaK mutants encoding proteins that are altered at the site of in vitro phosphorylation (J. S. McCarty and G. C. Walker, Proc. Natl. Acad. Sci. USA 88:9513-9517, 1991). These mutants are unable to autophosphorylate and are severely defective in ATP hydrolysis. These mutant dnaK genes were placed under the control of the lac promoter and were found not to complement the deficiencies of a delta dnaK mutant in negative regulation of the heat shock response. A decrease in the expression of DnaK and DnaJ below their normal levels at 30 degrees C was found to result in increased expression of GroEL. The implications of these results for DnaK's role in the negative regulation of the heat shock response are discussed. Evidence is also presented indicating the existence of a 70-kDa protein present in a delta dnaK52 mutant that cross-reacts with antibodies raised against DnaK. Derivatives of the dnaK+ E. coli strain MC4100 expressing the mutant DnaK proteins filamented severely at temperatures equal to or greater than 34 degrees C. In the dnaK+ E. coli strain W3110, expression of these mutant proteins caused extreme filamentation even at 30 degrees C. Together with other observations, these results suggest that DnaK may play a direct role in the septation pathway, perhaps via an interaction with FtsZ. Although delta dnaK52 derivatives of strain MC4100 filament extensively, a level of underexpression of DnaK and DnaJ that results in increased expression of the other heat shock proteins did not result in filamentation. The delta dnaK52 allele could be transduced successfully, at temperatures of up to 45 degrees C, into strains carrying a plasmid expressing dnaK+ dnaJ+, although the yield of transductants decreased above 37 degrees C. In contrast, with a strain that did not carry a plasmid expressing dnaK+ dnaJ+, the yield of delta dnaK52 transductants decreased extremely sharply between 39 and 40 degrees C, suggesting that DnaK and DnaJ play one or more roles critical for growth at temperatures of 40 degrees C or greater.
Molecular characterization of the P1-like adhesin gene from Mycoplasma pirum
Authors: Tham T N , Ferris S , Bahraoui E , Canarelli S , Montagnier L , Blanchard A
Published: November 2016

A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.
Degradation of fluorene by Brevibacterium sp. strain DPO 1361: a novel C-C bond cleavage mechanism via 1,10-dihydro-1,10-dihydroxyfluoren-9-one
Authors: Trenz S P , Engesser K H , Fischer P , Knackmuss H J
Published: November 2016

Angular dioxygenation has been established as the crucial step in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361 (V. Strubel, K. H. Engesser, P. Fischer, and H.-J. Knackmuss, J. Bacteriol. 173:1932-1937, 1991). The same strain utilizes biphenyl and fluorene as sole sources of carbon and energy. The fluorene degradation sequence is proposed to be initiated by oxidation of the fluorene methylene group to 9-fluorenol. Cells grown on fluorene exhibit pronounced 9-fluorenol dehydrogenase activity. Angular dioxygenation of the 9-fluorenone thus formed yields 1,10-dihydro-1,10-dihydroxyfluoren-9-one (DDF). A mechanistic model is presented for the subsequent C-C bond cleavage by an NAD(+)-dependent DDF dehydrogenase, acting on the angular dihydrodiol. This enzyme was purified and characterized as a tetramer of four identical 40-kDa subunits. The following Km values were determined: 13 microM for DDF and 65 microM for 2,3-dihydro-2,3-dihydroxybiphenyl. The enzyme also catalyzes the production of 3-(2'-carboxyphenyl)catechol, which was isolated, and structurally characterized, in the form of the corresponding lactone, 4-hydroxydibenzo-(b,d)-pyran-6-one. Stoichiometry analysis unequivocally demonstrates that angular dioxygenation constitutes the principal pathway in Brevibacterium sp. strain DPO 1361.
Genetic and molecular characterization of the Escherichia coli secD operon and its products
Authors: Pogliano K J , Beckwith J
Published: November 2016

The secD operon of Escherichia coli is required for the efficient export of proteins. We have characterized this operon, and found that, in addition to secD and secF, it contains the upstream gene yajC, but not the genes queA or tgt, in contrast to previous reports. An analysis of yajC mutations constructed in vitro and recombined onto the chromosome indicates that yajC is neither essential nor a sec gene. The secD operon is not induced in response to either secretion defects or temperature changes. TnphoA fusions have been used to analyze the topology of SecD in the inner membrane; the protein contains six transmembrane stretches and a large periplasmic domain. TnphoA fusions to SecD and SecF have also been recombined onto the chromosome and used to determine the level of these proteins within the cell. Our results indicate that there are fewer than 30 SecD and SecF molecules per cell.
Structure, composition, and assembly of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 and of phycobilisomes in the derivative strain BO 9201
Authors: Reuter W , Westermann M , Brass S , Ernst A , Böger P , Wehrmeyer W
Published: November 2016

The phycobiliproteins of the unicellular cyanobacterium Synechocystis sp. strain BO 8402 and its derivative strain BO 9201 are compared. The biliproteins of strain BO 8402 are organized in paracrystalline inclusion bodies showing an intense autofluorescence in vivo. These protein-pigment aggregates have been isolated. The highly purified complexes contain phycocyanin with traces of phycoerythrin, corresponding linker polypeptides LR35PC and LR33PE (the latter in a small amount), and a unique colored polypeptide with an M(r) of 55,000, designated L55. Allophycocyanin and the core linker polypeptides are absent. The substructure of the aggregates has been studied by electron microscopy. Repetitive subcomplexes of hexameric stacks of biliproteins form extraordinary long rods associated side by side in a highly condensed arrangement. Evidence that the linker polypeptides LR35PC and LR33PE stabilize the biliprotein hexamers is presented, while the location and function of the colored linker L55 remain uncertain. The derivative strain BO 9201 contains established hemidiscoidal phycobilisomes comprising phycoerythrin, phycocyanin, and allophycocyanin as well as the corresponding linker polypeptides. The core-membrane linker protein (LCM), and two polypeptides with M(r)s of 40,000 and 45,000 which are present in small amounts, exhibit strong cross-reactivity in Western blot (immunoblot) analysis using an antibody directed against the colored LCM of a Nostoc sp. In contrast, strain BO 8402 exhibits no polypeptide with a significant immunological cross-reactivity in Western blot analysis. Physiological and genetic implications of the unusual pigment compositions of both strains are discussed.
Cytoplasmic axial filaments in Escherichia coli cells: possible function in the mechanism of chromosome segregation and cell division
Authors: Okada Y , Wachi M , Hirata A , Suzuki K , Nagai K , Matsuhashi M
Published: November 2016

Overproduction of CafA caused formation of chained cells and minicells. The cafA gene is located downstream from the mre region at 71 min on the Escherichia coli chromosome map and was previously called orfF. A long axial structure running through the chained cells, consisting of bundles of filaments assembled in a long hexagonal pillar several micrometers long and about 0.1 to 0.2 micron in diameter, was visible in both phase-contrast micrographs of the lysozyme-treated cells and electron micrographs of ultrathin sections. The CafA protein displays 34% amino acid similarity with the N terminus of the Ams protein of E. coli, which cross-reacts with antibody to a nonmuscle myosin heavy chain.
Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis
Authors: Lupas A , Engelhardt H , Peters J , Santarius U , Volker S , Baumeister W
Published: November 2016

The three-dimensional structure of the Acetogenium kivui surface layer (S-layer) has been determined to a resolution of 1.7 nm by electron crystallographic techniques. Two independent reconstructions were made from layers negatively stained with uranyl acetate and Na-phosphotungstate. The S-layer has p6 symmetry with a center-to-center spacing of approximately 19 nm. Within the layer, six monomers combine to form a ring-shaped core surrounded by a fenestrated rim and six spokes that point towards the axis of threefold symmetry and provide lateral connectivity to other hexamers in the layer. The structure of the A. kivui S-layer protein is very similar to that of the Bacillus brevis middle wall protein, with which it shares an N-terminal domain of homology. This domain is found in several other extracellular proteins, including the S-layer proteins from Bacillus sphaericus and Thermus thermophilus, Omp alpha from Thermotoga maritima, an alkaline cellulase from Bacillus strain KSM-635, and xylanases from Clostridium thermocellum and Thermoanaerobacter saccharolyticum, and may serve to anchor these proteins to the peptidoglycan. To our knowledge, this is the first example of a domain conserved in several S-layer proteins.
RocR, a novel regulatory protein controlling arginine utilization in Bacillus subtilis, belongs to the NtrC/NifA family of transcriptional activators
Authors: Calogero S , Gardan R , Glaser P , Schweizer J , Rapoport G , Debarbouille M
Published: November 2016

Bacillus subtilis can use ammonium and various amino acids as sole nitrogen sources. The utilization of arginine or ornithine is abolished in a sigma L-deficient strain of B. subtilis, indicating that one or several genes involved in this pathway are transcribed by a sigma L-RNA polymerase holoenzyme. Three B. subtilis genes, called rocA, rocB, and rocC, which seem to form an operon, were found near the sacTPA locus (P. Glaser, F. Kunst, M. Arnaud, M.-P. Coudart, W. Gonzales, M.-F. Hullo, M. Ionescu, B. Lubochinsky, L. Marcelino, I. Moszer, E. Presecan, M. Santana, E. Schneider, J. Schweizer, A. Vertes, G. Rapport, and A. Danchin, Mol. Microbiol. 10:371-384, 1993). The expression of this putative operon is induced by arginine and is sigma L dependent. Mutants impaired in the transcription of rocA were obtained. One of these mutants was used as recipient to clone and sequence a new regulatory gene, called rocR. This gene encodes a polypeptide of 52 kDa which belongs to the NtrC/NifA family of transcriptional activators. Upstream activating sequences highly similar to those of NtrC in Escherichia coli were also identified upstream from the rocABC genes. A B. subtilis strain containing a rocR null mutation is unable to use arginine as the sole nitrogen source, indicating that RocR is a positive regulator of arginine catabolism. After LevR, RocR is the second example of an activator stimulating sigma 54-dependent promoters in gram-positive bacteria.
Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli
Authors: Dallmann H G , Dunn S D
Published: November 2016

Escherichia coli expresses the beta and epsilon subunits of F1F0-ATP synthase at relative levels consistent with the 3:1 (beta/epsilon) stoichiometry in the holoenzyme. The mechanism of translational control of expression of the uncC gene (epsilon subunit) relative to the immediately 5' uncD gene (beta subunit) was examined. Previous expression studies and a computer analysis suggested the presence of an RNA secondary structure including the 3' end of uncD, the uncDC intergenic region, and the uncC Shine-Dalgarno sequence (S. D. Dunn and H. G. Dallmann, J. Bacteriol. 172:2782-2784, 1990). Analysis of in vitro-transcribed RNA by cleavage with RNases T1, V1, and CL3 and by chemical modification with dimethyl sulfate and diethyl pyrocarbonate confirmed a predicted structure. Introduction of premature uncD stop codons inserted 5' of the secondary structure strongly reduced epsilon expression, whereas stop codons inserted at positions within the secondary structure showed smaller effects, indicating that translational control of epsilon synthesis involves partial coupling to beta synthesis. Possible mechanisms by which the RNA secondary structure and the unfolding of this structure by translation of uncD may govern the level of uncC expression are discussed.
Identification and characterization of the ilvR gene encoding a LysR-type regulator of Caulobacter crescentus
Authors: Malakooti J , Ely B
Published: November 2016

The ilvR gene was located upstream of and transcribed divergently from the ilvD gene of Caulobacter crescentus. DNA nucleotide analysis determined that the ilvR and ilvD translation initiation codons are 98 bp apart. The promoter activity of the DNA region containing the divergent promoters was analyzed by using transcriptional fusions to promoterless reporter genes and immunoblot assays. The results indicate that the ilvR gene product positively regulates the expression of the ilvD gene while negatively autoregulating its own expression. The ilvR gene codes for a protein of 296 amino acid residues (M(r), 37,212). The N-terminal amino acid sequence of the IlvR protein contains a helix-turn-helix motif, suggesting that it is involved in protein-DNA interactions. Protein extracts from both wild-type and merodiploid strains showed specific DNA binding to a 227-bp DNA fragment spanning the ilvD-ilvR promoter region, while no protein-DNA complexes were observed in cell extracts from an ilvR mutant strain. Amino acid sequence comparison revealed that the IlvR protein is a member of the LysR family of transcriptional regulators.
Nucleotide sequence of the rpoN gene and characterization of two downstream open reading frames in Pseudomonas aeruginosa
Authors: Jin S , Ishimoto K , Lory S
Published: November 2016

The rpoN gene of Pseudomonas aeruginosa is required for the expression of a number of diverse genes, ranging from several classes of bacterial adhesins to enzymes for amino acid biosynthesis. The nucleotide sequence of the rpoN gene and its flanking region has been determined. The deduced amino acid sequence of the rpoN product is highly homologous to sequences of RpoN proteins of other microorganisms. Moreover, two open reading frames (ORF1 and ORF2) encoding peptides of 103 and 154 amino acids long, respectively, were found downstream of the rpoN gene. These two ORF products have a high degree of amino acid sequence homology with products of similar ORFs located adjacent to the rpoN genes in other microorganisms. Mutations in either ORF lead to a significant increase in P. aeruginosa generation time when propagated on minimal medium. These mutations had no effect on the expression of pilin or flagellin genes, whose expression depends on RpoN. Complementation analysis showed that the two ORFs are in the same transcriptional unit and the growth defects of the two ORF mutants on minimal medium are due to mutational effects on ORF2. The adverse effect of the ORF mutations on the growth of P. aeruginosa in minimal media can be suppressed by the addition of glutamine but not arginine, glutamate, histidine, or proline. Since rpoN mutants of P. aeruginosa display this same amino acid requirement for growth, the ORF2 product very likely functions as a coinducer of some but not all of the RpoN-controlled genes.
Determinants of affinity and mode of DNA binding at the carboxy terminus of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1
Authors: Andera L , Geiduschek E P
Published: November 2016

The role of the carboxy-terminal amino acids of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1, in DNA binding was analyzed. Chain-terminating mutations truncating the normally 99-amino-acid TF1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. The binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethyl)uracil DNA, to binding sites in larger SPO1 [5-(hydroxymethyl)uracil-containing] DNA fragments, and to thymine-containing homologous DNA was analyzed by gel retardation and also by DNase I and hydroxy radical footprinting. We conclude that the C tail up to and including phenylalanine at amino acid 97 is essential for DNA binding and that the two C-terminal amino acids, 98 and 99, are involved in protein-protein interactions between TF1 dimers bound to DNA.
A low-pH-inducible, stationary-phase acid tolerance response in Salmonella typhimurium
Authors: Lee I S , Slonczewski J L , Foster J W
Published: November 2016

Acid is an important environmental condition encountered by Salmonella typhimurium during its pathogenesis. Our studies have shown that the organism can actively adapt to survive potentially lethal acid exposures by way of at least three possibly overlapping systems. The first is a two-stage system induced in response to low pH by logarithmic-phase cells called the log-phase acid tolerance response (ATR). It involves a major molecular realignment of the cell including the induction of over 40 proteins. The present data reveal that two additional systems of acid resistance occur in stationary-phase cells. One is a pH-dependent system distinct from log-phase ATR called stationary-phase ATR. It was shown to provide a higher level of acid resistance than log-phase ATR but involved the synthesis of fewer proteins. Maximum induction of stationary-phase ATR occurred at pH 4.3. A third system of acid resistance is not induced by low pH but appears to be part of a general stress resistance induced by stationary phase. This last system requires the alternative sigma factor, RpoS. Regulation of log-phase ATR and stationary-phase ATR remains RpoS independent. Although the three systems are for the most part distinct from each other, together they afford maximum acid resistance for S. typhimurium.
Characterization of a cell division gene from Bacillus subtilis that is required for vegetative and sporulation septum formation
Authors: Levin P A , Losick R
Published: November 2016

We report the cloning and characterization of a cell division gene, herein designated divIC, from the gram-positive, spore-forming bacterium Bacillus subtilis. This gene was previously identified on the basis of a temperature-sensitive mutation, div-355, that blocks septum formation at restrictive temperatures. We show that the divIC gene is a 125-codon open reading frame that is capable of encoding a protein of 14.7 kDa and that div-355 is a 5-bp duplication near the 3' end of the open reading frame. We also show that divIC is an essential gene by use of an in vitro-constructed null mutation. In confirmation and extension of earlier results, we show that divIC is necessary for both vegetative and sporulation septum formation, and we demonstrate that it is required for the activation of genes expressed under the control of the sporulation transcription factors sigma F and sigma E. The divIC gene is located 1.3 kb upstream of the coding sequence for the sporulation gene spoIIE. Between divIC and spoIIE is a 128-codon open reading frame whose predicted product contains a region of similarity to the RNA-binding domains of polynucleotide phosphorylase and ribosomal protein S1 from Escherichia coli and two putative tRNA genes for methionyl-tRNA and glutamyl-tRNA, the gene order being divIC orf128 tRNA(Met) tRNA(Glu) spoIIE.
Activation of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) in Streptomyces antibioticus
Authors: Jones G H
Published: November 2016

The activity of the ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase I [GPSI]) from Streptomyces antibioticus is stimulated maximally by methanol at 20% (vol/vol) in assay mixtures. Although the enzyme is not activated by ribosomes, its activity is stimulated by tRNA (uncharged or charged) and by synthetic mRNA [e.g., poly(U)]. The level of stimulation is greater in the presence of tRNA and poly(U) together than with either RNA alone. Incubation of GPSI with low levels of trypsin also leads to activation of the enzyme. Analysis of the products of mild trypsin digestion revealed the presence of two intermediates whose M(r)s are identical to those of species produced by incubation of purified GPSI with crude extracts of S. antibioticus mycelium. GPSI can be activated by incubation with crude mycelial extracts, and this activation is partially inhibited by the inclusion of trypsin inhibitor in reaction mixtures.
Sensory adaptation during negative chemotaxis in Myxococcus xanthus
Authors: Shi W , Zusman D R
Published: November 2016

Myxococcus xanthus exhibits many tactic movements that require the frz signal transduction system, such as colony swarming and cellular aggregation during fruiting body formation. Previously we demonstrated that the Frz proteins control the chemotactic movements of M. xanthus (W. Shi, T. Köhler, and D. R. Zusman, Mol. Microbiol. 9:601-611, 1993). However it was unclear from that study how chemotaxis might be achieved at the cellular level. In this study, we showed that M. xanthus cells not only modulate the reversal frequency of cell movement in response to repellent stimuli but also exhibit sensory adaptation in response to the continuous presence of nonsaturating repellent stimuli. The sensory adaptation behavior requires FrzF (a putative methyltransferase) and is correlated with the methylation-demethylation of FrzCD, a methyl-accepting chemotaxis protein. These results indicate that negative chemotaxis in M. xanthus is achieved by chemokinesis plus sensory adaptation in a manner analogous to that of the free-swimming enteric bacteria.
Role of endoglucanases in Erwinia chrysanthemi 3937 virulence on Saintpaulia ionantha
Authors: Boccara M , Aymeric J L , Camus C
Published: November 2016

The role of endoglucanases (endoglucanases Z and Y) in Erwinia chrysanthemi pathogenicity on Saintpaulia ionantha was assessed by mutagenizing cloned cel genes (celZ and celY) and recombining them with the chromosomal alleles. Strains with an omega interposon in celZ, a deletion in celY, or a double cel mutant were as virulent as the wild-type strain. However, in the strain with a deletion in celY, a delay in the appearance of symptoms was observed, and then maceration progressed as in plants infected with the wild-type strain, suggesting that E. chrysanthemi endoglucanases play a minor role in soft rot disease development.