bluuebunny/crossref_metadata_2025 huggingface dataset:
Tunable optical properties in atomic layer deposition grown ZnO thin films
Authors: Pal Dipayan , Mathur Aakash , Singh Ajaib , Singhal Jaya , Sengupta Amartya , Dutta Surjendu , Zollner Stefan , Chattopadhyay Sudeshna
Published: November 2016

ZnO thin films with very low surface roughness and bulklike electron density were grown on Si and SiO2 by atomic layer deposition. The real and imaginary parts of the complex dielectric function of ZnO on Si show monotonically decreasing values with decreasing film thickness at and below a threshold of about 20 nm. On the other hand, x-ray reflectivity results show that the electron density of our ZnO films is close to that of bulk ZnO and does not vary considerably with film thickness. While the reduction of the dielectric function cannot be explained by the electron density of our ZnO films, the Tanguy–Elliott amplitude prefactor governing the strength of optical interband transitions can explain our results consistently through the lowering of the electron–hole overlap factor at the ZnO/Si interface. In the case of ZnO/Si, a staggered type-II (spatially indirect) quantum well, holes are scattered into the Si substrate, causing a lowering of the electron–hole overlap factor and thus the reduction of excitonic absorption, consequently a decrease in the real and the imaginary parts of the dielectric function. This hypothesis was confirmed with ZnO films grown on SiO2, where a thin type-I quantum well, consisting of a narrower-bandgap semiconductor grown on a wider-bandgap (insulator) substrate, in which both the electron and the hole are confined in the ZnO thin film, leads to an increase in the electron–hole overlap matrix element with decreasing film thickness due to confinement, resulting in enhancement of the excitonic absorption in thinner ZnO films on SiO2.
Vapor deposition of copper(I) bromide films via a two-step conversion process
Authors: Heasley Rachel , Chang Christina M. , Davis Luke M. , Liu Kathy , Gordon Roy G.
Published: November 2016

Thin films of Cu2S grown by pulsed-chemical vapor deposition of bis(N,N′-di-sec-butylacetamidinato)dicopper(I) and hydrogen sulfide were converted to CuBr upon exposure to anhydrous hydrogen bromide. X-ray diffraction shows that the as-deposited films have a polycrystalline Cu2S structure. After exposure to HBr gas, the surface of the films is transformed to a γ-CuBr polycrystalline structure. Scanning electron microscopy and X-ray photoelectron spectroscopy reveal complete conversion of up to 100 nm of film. However, when the conversion to CuBr approaches the interface between as-deposited Cu2S and the SiO2 substrate, the morphology of the film changes from continuous and nanocrystalline to sparse and microcrystalline.
Phosphorylation of Janus kinase 1 (JAK1) by AMP-activated protein kinase (AMPK) links energy sensing to anti-inflammatory signaling
Authors: Rutherford Claire , Speirs Claire , Williams Jamie J. L. , Ewart Marie-Ann , Mancini Sarah J. , Hawley Simon A. , Delles Christian , Viollet Benoit , Costa-Pereira Ana P. , Baillie George S. , Salt Ian P. , Palmer Timothy M.
Published: November 2016

The energy-sensing pathway kinase AMPK inhibits proinflammatory JAK1 signaling.
IP
3
, still on the move but now in the slow lane
Authors: Leybaert Luc
Published: November 2016

Studies using mammalian cells challenge the conventional view of rapid diffusion of IP
3
(Dickinson
et al
., in 08 November 2016 issue).
The leucine-responsive regulatory protein of Escherichia coli negatively regulates transcription of ompC and micF and positively regulates translation of ompF
Authors: Ferrario M , Ernsting B R , Borst D W , Wiese D E , Blumenthal R M , Matthews R G
Published: November 2016

The two major porins of Escherichia coli K-12 strains, OmpC and OmpF, are inversely regulated with respect to one another. The expression of OmpC and OmpF has been shown to be influenced by the leucine-responsive regulatory protein (Lrp): two-dimensional gel electrophoresis of proteins from strains with and strains without a functional Lrp protein revealed that OmpC expression is increased in an lrp strain, while OmpF expression is decreased. In agreement with these findings, we now present evidence that transcriptional (operon) fusions of lacZ+ to ompC and micF are negatively regulated by Lrp. Lrp binds specifically to the intergenic region between micF and ompC, as indicated by mobility shift assays and by DNase I footprinting. The expression of an ompF'-lacZ+ gene (translational) fusion is increased 3.7-fold in an lrp+ background compared with an lrp background, but expression of an ompF-lacZ+ operon fusion is not. Studies of in vivo expression of the outer membrane porins during growth on glucose minimal medium showed that the OmpF/OmpC ratio is higher in lrp+ strains than it is in isogenic lrp strains. The effect of Lrp was not seen in a strain containing a deletion of micF. Our studies suggest that the positive effect of Lrp on OmpF expression stems from a negative effect of Lrp on the expression of micF, an antisense RNA that inhibits ompF translation.
Purification and characterization of a novel extracellular Streptomyces lividans 66 enzyme inactivating fusidic acid
Authors: von der Haar B , Schrempf H
Published: November 2016

The wild-type strain Streptomyces lividans 66 is resistant against the steroid-like antibiotic fusidic acid. Comparative studies of the wild-type strain and a fusidic acid-sensitive mutant allowed the identification of an extracellular enzyme which inactivates fusidic acid. With the help of a combination of ultrafiltration and chromatographies with Phenyl-Sepharose and an anion exchanger, the enzyme was highly purified. Its apparent molecular mass is 48 kDa, its optimal activity ranges between 45 and 55 degrees C, and its optimal pH is 6.0 to 9.0. It is stimulated by neither monovalent nor divalent ions. The enzyme acts as a specific esterase which removes the acetyl group at C-16 from fusidic acid. The resulting intermediate is unstable, and spontaneous lactonization between C-21 and C-16 occurs rapidly.
The hsp70 gene family of Neurospora crassa: cloning, sequence analysis, expression, and genetic mapping of the major stress-inducible member
Authors: Kapoor M , Curle C A , Runham C
Published: November 2016

The gene encoding the major heat shock-inducible member of the HSP70 family of Neurospora crassa was cloned and characterized. The 5' nontranscribed region shows the presence of consensus sequence motifs resembling the classical heat shock elements found in many heat shock-responsive eukaryotic promoters, as well as metal-responsive-element sequences. The coding region of the gene contains four introns with boundaries and internal consensus motifs typical of genes of filamentous fungi. None of the other stress-inducible hsp70 genes of fungal origin have, so far, been reported to contain introns. The sequence adjoining the transcriptional initiation zone shows the presence of prominent CT-rich stretches, characteristic of highly expressed fungal genes. The deduced amino acid sequence corresponds to a 646-residue polypeptide, with a calculated molecular mass of 70,561 Da and an average pI of 6.01, exhibiting strong sequence homology with many other eukaryotic HSP70s, with typical HSP70 family signatures 1 and 2 and a bipartite nuclear targeting sequence. Experiments with primer extension revealed the presence of one minor and two major transcriptional start sites. This gene, designated hsps-1, was mapped to a locus on the left arm of linkage group II, in close proximity to the AR-30 translocation breakpoint.
The TyrR protein of Escherichia coli is a class I transcription activator
Authors: Lawley B , Fujita N , Ishihama A , Pittard A J
Published: November 2016

The purified TyrR protein and phenylalanine were sufficient to activate in vitro transcription from the tyrP promoter by wild-type RNA polymerase. Such TyrR-mediated activation did not occur when the mutant alpha 235 RNA polymerase was used, indicating that TyrR is a class I transcription activator.
Isolation, sequencing, and mutagenesis of the gene encoding NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) from Paracoccus denitrificans, in which GD-FALDH is essential for methylotrophic growth
Authors: Ras J , Van Ophem P W , Reijnders W N , Van Spanning R J , Duine J A , Stouthamer A H , Harms N
Published: November 2016

NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) of Paracoccus denitrificans has been purified as a tetramer with a relative molecular mass of 150 kDa. The gene encoding GD-FALDH (flhA) has been isolated, sequenced, and mutated by insertion of a kanamycin resistance gene. The mutant strain is not able to grow on methanol, methylamine, or choline, while heterotrophic growth is not influenced by the mutation. This finding indicates that GD-FALDH of P. denitrificans is essential for the oxidation of formaldehyde produced during methylotrophic growth.
Bordetella pertussis fur gene restores iron repressibility of siderophore and protein expression to deregulated Bordetella bronchiseptica mutants
Authors: Brickman T J , Armstrong S K
Published: November 2016

We report the isolation and preliminary phenotypic characterization of manganese-resistant Bordetella bronchiseptica mutants with respect to deregulation of siderophore and iron-regulated protein expression. The fur gene of Bordetella pertussis was cloned by genetic complementation of this deregulated phenotype and confirmed as fur by nucleotide sequence analysis.
Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis
Authors: Martinussen J , Glaser P , Andersen P S , Saxild H H
Published: November 2016

Uracil phosphoribosyltransferase (UPRTase) catalyzes the key reaction in the salvage of uracil in many microorganisms. Surprisingly, two genes encoding UPRTase activity were cloned from Bacillus subtilis by complementation of an Escherichia coli mutant. The genes were sequenced, and the putative amino acid sequences were deduced. One gene showed a high level of homology to UPRTases from other organisms, whereas the other gene with a low level of homology to other UPRTases turned out to be the pyrR gene--the repressor of the pyr operon. The role of these genes in uracil metabolism was established by an analysis of the phenotypes of upp and pyrR mutants.
Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4
Authors: Gibson D T , Resnick S M , Lee K , Brand J M , Torok D S , Wackett L P , Schocken M J , Haigler B E
Published: November 2016

The stereospecific oxidation of indan and indene was examined with mutant and recombinant strains expressing naphthalene dioxygenase of Pseudomonas sp. strain 9816-4. Pseudomonas sp. strain 9816/11 and Escherichia coli JM109(DE3)[pDTG141] oxidized indan to (+)-(1S)-indanol, (+)-cis-(1R,2S)-indandiol, (+)-(1S)-indenol, and 1-indanone. The same strains oxidized indene to (+)-cis-(1R,2S)-indandiol and (+)-(1S)-indenol. Purified naphthalene dioxygenase oxidized indan to the same four products formed by strains 9816/11 and JM109(DE3)[pDTG141]. In addition, indene was identified as an intermediate in indan oxidation. The major products formed from indene by purified naphthalene dioxygenase were (+)-(1S)-indenol and (+)-(1R,2S)-indandiol. The results show that naphthalene dioxygenase catalyzes the enantiospecific monooxygenation of indan to (+)-(1S)-indanol and the desaturation of indan to indene, which then serves as a substrate for the formation of (+)-(1R,2S)-indandiol and (+)-(1S)-indenol. The relationship of the desaturase, monooxygenase, and dioxygenase activities of naphthalene dioxygenase is discussed with reference to reactions catalyzed by toluene dioxygenase, plant desaturases, cytochrome P-450, methane monooxygenase, and other bacterial monooxygenases.
Magnesium transport in Salmonella typhimurium: mgtA encodes a P-type ATPase and is regulated by Mg2+ in a manner similar to that of the mgtB P-type ATPase
Authors: Tao T , Snavely M D , Farr S G , Maguire M E
Published: November 2016

Salmonella typhimurium has three distinct Mg2+ transport systems: CorA, MgtA, and MgtB, each encoded by its respective gene. corA and mgtB have been previously sequenced and characterized. This report details the sequence and properties of mgtA. Like mgtB, mgtA encodes a P-type ATPase. The mgtA gene encodes a slightly smaller protein than does mgtB, with a predicted molecular mass of about 95 kDa, running at 91 kDa on protein gels, which compares with values of 101 and 102 kDa, respectively, for the MgtB protein. The deduced amino acid sequence of MgtA is only 50% identical to that of MgtB, with a further 25% conservative amino acid substitutions, surprisingly low for such otherwise functionally similar proteins from the same organism. Codon usage for each gene is normal for S. typhimurium, however, indicating that neither gene is the result of a recent acquisition from another organism. A single open reading frame at mgtA encodes MgtA, in contrast to mgtB, which is shown to be an operon encoding (5' to 3') the 22.5-kDa MgtC and the MgtB proteins. Genetic constructs were used to show that deletion of MgtC does not alter the expression or transport properties of MgtB, making the role of the companion MgtC protein unclear. (The S. typhimurium homolog of treR, which encodes a putative repressor for trehalose uptake, is encoded by a gene adjacent to mgtA, and its sequence is also reported. Finally, exteremely strong Mg(2+) regulation of the mgtA and mgtB promoters but not of the corA or treR promoters was demonstrated by cloning the appropriate DNA sequences with luxAB and measuring enhancement of light production as a function of extracellular Mg(2+) concentration. Lowering the extracellular Mg(2+) concentration from 10 mM to 1 or 10 microM elicited a transcriptional response of several thousandfold from both the mgtA and mgtB promoters.
Isolation of a novel paraquat-inducible (pqi) gene regulated by the soxRS locus in Escherichia coli
Authors: Koh Y S , Roe J H
Published: November 2016

We have isolated promoters inducible by paraquat, a superoxide radical-generating agent, from Escherichia coli, using promoter-probing plasmid pJAC4 (Y.S. Koh and J.H. Roe, Korean J. Microbiol. 31:267-273, 1993). One promoter clone pqi-5 (pqi denotes paraquat-inducible gene) was mapped at 21.8 min on the E. coli chromosome by using the Kohara phage library. We constructed an operon fusion of the lacZ gene with the pqi-5 promoter to monitor the expression of the gene in the single-copy state. LacZ expression was induced about 7- to 13-fold by 77 to 780 microM paraquat. Other known superoxide generators such as menadione, phenazine methosulfate, and plumbagin also induced the expression of beta-galactosidase in this fusion strain. On the other hand, no significant induction was observed with treatment with hydrogen peroxide, ethanol, and heat shock. Induction of beta-galactosidase was significantly reduced by introducing a delta sox-8::cat or soxS3::Tn10 mutation into the fusion strain, indicating that pqi-5 is a member of the soxRS regulon. A DNA fragment containing the pqi-5 promoter was cloned and sequenced from the Kohara phage E2E5. We identified two pqi-5 open reading frames (ORFs); ORF-A encodes a predicted protein of 342 amino acids, and ORF-B is truncated at the cloning site. The transcription start site from the pqi-5 promoter was determined by primer extension and S1 nuclease protection analyses. Northern (RNA) and S1 analyses indicated that there are two kinds of pqi-5 transcript; one covers ORF-A only and the other covers ORF-A and possibly also ORF-B.
Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor
Authors: Cunliffe H E , Merriman T R , Lamont I L
Published: November 2016

Cells of Pseudomonas aeruginosa secrete a fluorescent yellow-green siderophore, pyoverdine, when grown under iron-deficient conditions. We describe here the cloning and characterization of a gene, pvdS, which is required for this process. The pvdS gene is required for expression from promoters of at least two pyoverdine synthesis genes and can cause expression from these promoters in Escherichia coli, where they are otherwise inactive. Sequencing of pvdS revealed that it is a member of a subfamily of RNA polymerase sigma factors which direct the synthesis of extracellular products by bacteria. The pvdS gene is expressed only in iron-starved bacteria, and in E. coli cells at least, expression is regulated by the Fur repressor protein. We propose that in iron-rich cells of P. aeruginosa, Fur binds to the pvdS promoter and prevents expression of the gene; under conditions of iron starvation, repression is relieved and PvdS is made, reprogramming the cells for pyoverdine synthesis.
Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli
Authors: Hopper S , Böck A
Published: November 2016

The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli. The protein shares homology with the sigma 54-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N terminus lacking the aspartate residue which is the site of phosphorylation. Purified FHLA displays intrinsic ATPase activity which is stimulated weakly by formate and DNA. The presence of both formate and DNA carrying the upstream regulatory sequence to which FHLA binds leads to a large increase in the rate of ATP hydrolysis. Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, also stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA. Half-maximal saturation of FHLA with formate took place at around 5 mM, and half-maximal saturation with target DNA took place at around 50 nM. The stimulation of ATPase activity by formate was conferred by a decrease in the apparent Km for ATP, whereas the effect of the DNA binding site also affected the Kcat of the reaction. The other nucleoside triphosphates, GTP, UTP, and CTP, competed with ATP cleavage by FHLA, suggesting at least their binding to FHLA. The specific ATPase activity of FHLA was dependent on the concentration of FHLA in the assay, especially in the presence of DNA and formate. Direct liganding of the effector, therefore, leads to the same consequence as phosphorylation for the NTRC-type regulators, namely, stimulation of ATPase activity.
The dnaA gene of Rhizobium meliloti lies within an unusual gene arrangement
Authors: Margolin W , Bramhill D , Long S R
Published: November 2016

Rhizobium meliloti exists either as a free-living soil organism or as a differentiated endosymbiont bacteroid form within the nodules of its host plant, alfalfa (Medicago sativa), where it fixes atmospheric N2. Differentiation is accompanied by major changes in DNA replication and cell division. In addition, R. meliloti harbors three unique large circular chromosome-like elements whose replication coordination may be complex. As part of a study of DNA replication control in R. meliloti, we isolated a dnaA homolog. The deduced open reading frame predicts a protein of 57 kDa that is 36% identical to the DnaA protein of Escherichia coli, and the predicted protein was confirmed by immunoblot analysis. In a comparison with the other known DnaA proteins, this protein showed the highest similarity to that of Caulobacter crescentus and was divergent in some domains that are highly conserved in other unrelated species. The dnaA genes of a diverse group of bacteria are adjacent to a common set of genes. Surprisingly, analysis of the DNA sequence flanking dnaA revealed none of these genes, except for an rpsT homolog, also found upstream of dnaA in C. crescentus. Instead, upstream of rpsT lie homologs of fpg, encoding a DNA glycosylase, and fadB1, encoding an enoyl-coenzyme A hydratase with a strikingly high (53 to 55%) level of predicted amino acid identity to two mammalian mitochondrial homologs. Downstream of dnaA, there are two open reading frames that are probably expressed but are not highly similar to any genes in the databases. These results show that R. meliloti dnaA is located within a novel gene arrangement.
The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16
Authors: Jensen G B , Wilcks A , Petersen S S , Damgaard J , Baum J A , Andrup L
Published: November 2016

The aggregation phenotypes Agr+ and Agr- of Bacillus thuringiensis subsp. israelensis are correlated with a conjugation-like plasmid transfer and characterized by the formation of aggregates when the bacteria are socialized during exponential growth. We present evidence for the association of the Agr+ phenotype with the presence of the large (135-MDa) self-transmissible plasmid pXO16.
DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication
Authors: Sweasy J B , Chen M , Loeb L A
Published: November 2016

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.
Nucleotide sequences and expression of genes from Streptomyces purpurascens that cause the production of new anthracyclines in Streptomyces galilaeus
Authors: Niemi J , Mäntsälä P
Published: November 2016

Six open reading frames, rdmA to rdmF, in a 6,077-bp segment of Streptomyces purpurascens DNA which caused the production of hybrid anthracyclines were identified. The minimal fragment that produced anthracyclines modified at the 10th position contained rdmB to rdmD; rdmE is the gene for aklavinone-11-hydroxylase. RdmC is similar to a putative open reading frame in the daunorubicin biosynthetic cluster of Streptomyces peucetius and is likely to participate in the removal of the side chain at the 10th position.
Characterization of the 23S and 5S rRNA genes of Coxiella burnetii and identification of an intervening sequence within the 23S rRNA gene
Authors: Afseth G , Mo Y Y , Mallavia L P
Published: November 2016

Characterization of the rRNA operon from the obligate intracellular bacterium Coxiella burnetii has determined the order of the rRNA genes to be 16S-23S-5S. A 444-bp intervening sequence (IVS) was identified to interrupt the 23S rRNA gene beginning at position 1176. The IVS is predicted to form a stem-loop structure formed by flanking inverted repeats, and the absence of intact 23S rRNA molecules suggests that the loop is removed. An open reading frame in the IVS has been identified that shows 70% similarity at the amino acid level to IVS open reading frames characterized from four species of Leptospira.
Production and reutilization of an extracellular phosphatidylinositol catabolite, glycerophosphoinositol, by Saccharomyces cerevisiae
Authors: Patton J L , Pessoa-Brandao L , Henry S A
Published: November 2016

Phosphatidylinositol catabolism in Saccharomyces cerevisiae is known to result in the formation of extracellular glycerophosphoinositol (GroPIns). We now report that S. cerevisiae not only produces but also reutilizes extracellular GroPIns and that these processes are regulated in response to inositol availability. A wild-type strain uniformly prelabeled with [3H] inositol displayed dramatically higher extracellular GroPIns levels when cultured in medium containing inositol than when cultured in medium lacking inositol. This difference in extracellular accumulation of GroPIns in response to inositol availability was shown to be a result of both regulated production and regulated reutilization. In a strain in which a negative regulator of phospholipid and inositol biosynthesis had been deleted (an opi1 mutant), this pattern of extracellular GroPIns accumulation in response to inositol availability was altered. An inositol permease mutant (itr1 itr2), which is unable to transport free inositol, was able to incorporate label from exogenous glycerophospho [3H]inositol, indicating that the inositol label did not enter the cell solely via the transporters encoded by itr1 and itr2. Kinetic studies of a wild-type strain and an itr1 itr2 mutant strain revealed that at least two mechanisms exist for the utilization of exogenous GroPIns: an inositol transporter-dependent mechanism and an inositol transporter-independent mechanism. The inositol transporter-independent pathway of exogenous GroPIns utilization displayed saturation kinetics and was energy dependent. Labeling studies employing [14C]glycerophospho[3H] inositol indicated that, while GroPIns enters the cell intact, the inositol moiety but not the glycerol moiety is incorporated into lipids.
Characterization of cotJ, a sigma E-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores
Authors: Henriques A O , Beall B W , Roland K , Moran C P
Published: November 2016

The outermost protective structure found in endospores of Bacillus subtilis is a thick protein shell known as the coat, which makes a key contribution to the resistance properties of the mature spore and also plays a role in its interaction with compounds able to trigger germination. The coat is organized as a lamellar inner layer and an electron-dense outer layer and has a complex polypeptide composition. Here we report the cloning and characterization of an operon, cotJ, located at about 62 degrees on the B. subtilis genetic map, whose inactivation results in the production of spores with an altered pattern of coat polypeptides. The cotJ operon was identified by screening a random library of lacZ transcriptional fusions for a conditional (inducer-dependent) Lac+ phenotype in cells of a strain in which the structural gene (spoIIGB) for the early-acting, mother-cell-specific transcriptional factor sigma E was placed under the control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible Pspac promoter. Sequence analysis of cloned DNA from the cotJ region complemented by genetic experiments revealed a tricistronic operon preceded by a strong sigma E-like promoter. Expression of an SP beta-borne cotJ-lacZ fusion commences at around h 2 of sporulation, as does expression of other sigma E-dependent genes, and shows an absolute requirement for sigma E. Studies with double-reporter strains bearing a cotJ-gusA fusion and lacZ fusions to other cot genes confirmed that expression of cotJ is initiated during sporulation prior to activation of genes known to encode coat structural proteins (with the sole exception of cotE). An in vitro-constructed insertion-deletion mutation in cotJ resulted in the formation of spores with no detectable morphological or resistance deficiency. However, examination of the profile of electrophoretically separated spore coat proteins from the null mutant revealed a pattern that was essentially identical to that of a wild-type strain in the range of 12 to 65 kDa, except for polypeptides of 17 and 24 kDa, the putative products of the second (cotJB) and third (cotJC) cistrons of the operon, that were missing or reduced in amount in the coat of the mutant. Polypeptides of the same apparent sizes are detected in spores of a cotE null mutant, on which basis we infer that the products of the cotJ operon are required for the normal formation of the inner layers of the coat or are themselves structural components of the coat. Because the onset of cotJ transcription is temporally coincident with the appearance of active sigma E, we speculate that the cotJ-encoded products may be involved in an early state of coat assembly.
Characterization of the basic replicon of pCM1, a narrow-host-range plasmid from the moderate halophile Chromohalobacter marismortui
Authors: Mellado E , Asturias J A , Nieto J J , Timmis K N , Ventosa A
Published: November 2016

The moderately halophilic bacterium Chromohalobacter marismortui contains a 17.5-kb narrow-host-range plasmid, pCM1, which shows interesting properties for the development of cloning vectors for the genetic manipulation of this important group of extremophiles. Plasmid pCM1 can stably replicate and is maintained in most gram-negative moderate halophiles tested. The replication origin has been identified and sequenced, and the minimal pCM1 replicon has been localized to a 1,600-bp region which includes two functionally discrete regions, the oriV region and the repA gene. oriV, located on a 700-bp fragment, contains four iterons 20 bp in length adjacent to a DnaA box that is dispensable but required for efficient replication of pCM1, and it requires trans-acting functions. The repA gene, which encodes a replication protein of 289 residues, is similar to the replication proteins of other gram-negative bacteria.
DNA restriction-modification systems mediate plasmid maintenance
Authors: Kulakauskas S , Lubys A , Ehrlich S D
Published: November 2016

Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.
The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides
Authors: Juillard V , Laan H , Kunji E R , Jeronimus-Stratingh C M , Bruins A P , Konings W N
Published: November 2016

The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.
Regulated underexpression and overexpression of the FliN protein of Escherichia coli and evidence for an interaction between FliN and FliM in the flagellar motor
Authors: Tang H , Billings S , Wang X , Sharp L , Blair D F
Published: November 2016

The FliN protein of Escherichia coli is essential for the assembly and function of flagella. Here, we report the effects of regulated underexpression and overexpression of FliN in a fliN null strain. Cells that lack the FliN protein do not make flagella. When FliN is underexpressed, cells produce relatively few flagella and those made are defective, rotating at subnormal, rapidly varying speeds. These results are similar to what was seen previously when the flagellar protein FliM was underexpressed and unlike what was seen when the motility proteins MotA and MotB were underexpressed. Overexpression of FliN impairs motility and flagellation, as has been reported previously for FliM, but when FliN and FliM are co-overexpressed, motility is much less impaired. This and additional evidence presented indicate that FliM and FliN are associated in the flagellar motor, in a structure distinct from the MotA/MotB torque generators. A recent study showed that FliN might be involved in the export of flagellar components during assembly (A. P. Vogler, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). We show here that approximately 50 amino acid residues from the amino terminus of FliN are dispensable for function and that the remaining, essential part of FliN has sequence similarity to a part of Spa33, a protein that functions in transmembrane export in Shigella flexneri. Thus, FliN might function primarily in flagellar export, rather than in torque generation, as has sometimes been supposed.
A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins
Authors: Denoya C D , Fedechko R W , Hafner E W , McArthur H A , Morgenstern M R , Skinner D D , Stutzman-Engwall K , Wax R G , Wernau W C
Published: November 2016

A second cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1 alpha), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1 beta) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1 alpha subunit of several prokaryotic and eukaryotic BCDH complexes. An S. avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5' end of bkdF is also described. The mutant exhibited a typical Bkd- phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources. Since BCDH provides an alpha-branched-chain fatty acid starter unit, either S(+)-alpha-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S. avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(+)-alpha-methylbutyrate and isobutyrate. Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins. These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S. avermitilis.
A gene at 333 degrees on the Bacillus subtilis chromosome encodes the newly identified sigma B-dependent general stress protein GspA
Authors: Antelmann H , Bernhardt J , Schmid R , Hecker M
Published: November 2016

In Bacillus subtilis, general stress proteins (Gsps) are induced in response to different stresses (heat, salt, or ethanol) or after nutrient starvation. The majority of the genes for the Gsps are organized in a very large stationary-phase or stress regulon which is controlled by alternative sigma factor sigma B. The most striking spots on Coomassie-stained two-dimensional gels belong to GsiB and GspA, which are synthesized at extremely high levels in response to different stresses. Therefore, we determined the N-terminal protein sequence of GspA, which exhibited total identity to a hypothetical 33.5-kDa protein of B. subtilis encoded by open reading frame 2 (ipa-12d) in the sacY-tyrS1 intergenic region. The GspA-encoding gene gspA and the upstream and downstream regions were cloned with the aid of the PCR technique. By primer extension experiments, one sigma B-dependent promoter immediately upstream of the coding region was identified. A putative factor-independent terminator closely followed the coding region. By Northern (RNA) blot analysis, a 0.95-kb transcript was detected which indicates a monocistronic transcriptional unit. The gspA mRNA was strongly induced by different stimuli like heat or salt stress and starvation for glucose. Analysis of RNA isolated from a sigma B deletion mutant revealed that the transcription of gspA is sigma B dependent. Insertional inactivation of the B. subtilis chromosomal gspA gene confirmed that the gspA gene is not essential for either vegetative growth or growth under the influence of different stresses. In gspA mutant cells, the level of flagellin was increased severalfold over that in wild-type cells.
ccrA1: a mutation in Streptomyces coelicolor that affects the control of catabolite repression
Authors: Ingram C , Delic I , Westpheling J
Published: November 2016

The regulation of carbon utilization is of central importance in the gene expression pathways for both morphological development and antibiotic production in Streptomyces species. We report the identification and characterization of a mutation in Streptomyces coelicolor, ccrA1, that affects the expression of several catabolite-controlled promoters. ccrA1 mutants are altered in expression of galP1, the glucose-sensitive, galactose-dependent promoter of the galactose utilization operon; in expression of the glycerol utilization operon, which is glucose sensitive and glycerol dependent; and in expression of chi63, the glucose-sensitive chitin-dependent promoter of a gene involved in chitin utilization. ccrA1 has no effect on the expression of galP2, a promoter that directs constitutive transcription of the galE and galK genes. ccrA1 maps to a region of the S. coelicolor genome which distinguishes it from other mutations known to be involved in catabolite control. We suggest that ccrA1 identifies a gene whose product may be involved in the general regulation of carbon catabolite repression in this complex bacterium.
The putative fabJ gene of Escherichia coli fatty acid synthesis is the fabF gene
Authors: Magnuson K , Carey M R , Cronan J E
Published: November 2016

Siggaard-Andersen and coworkers (M. Siggaard-Andersen, M. Wissenbach, J. Chuck, I. Svendsen, J. G. Olsen, and P. von Wettstein-Knowles, Proc. Natl. Acad. Sci. USA 91:11027-11031, 1994) recently reported the DNA sequence of a gene encoding a beta-ketoacyl-acyl carrier protein synthase from Escherichia coli. These workers assigned this gene the designation fabJ and reported that the gene encoded a new beta-ketoacyl-acyl carrier protein synthase. We report that the fabJ gene is the previously reported fabF gene that encodes the known beta-ketoacyl-acyl carrier protein synthase II.
Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP
Authors: Kaiser M , Sawers G
Published: November 2016

The pfl operon is expressed at high levels anaerobically. Growth of Escherichia coli in the presence of nitrate or nitrite led to a 45% decrease in expression when cells were cultivated in rich medium. Nitrate repression, however, was significantly enhanced (sevenfold) when the cells were cultured in minimal medium. Regulation of pfl expression by nitrate was dependent on the NarL, NarP, NarQ, and NarX proteins but independent of FNR, ArcA, and integration host factor, which are additional regulators of pfl expression. Strains unable to synthesize any one of the NarL, NarP, NarQ, or NarX proteins, but retaining the capacity to synthesize the remaining three, exhibited essentially normal nitrate regulation. In contrast, narL narP and narX narQ double null mutants were devoid of nitrate regulation when cultured in rich medium but they retained some nitrate repression (1.3-fold) when grown in minimal medium. By using lacZ fusions, it was possible to localize the DNA sequences required to mediate nitrate repression to the pfl promoter-regulatory region. DNase I footprinting studies identified five potential binding sites for the wild-type NarL protein in the pfl promoter-regulatory region. Specific footprints were obtained only when NarL was phosphorylated with acetyl phosphate before the binding reaction was performed. Each of the protected regions contained at least one heptamer sequence which has been deduced from mutagenesis studies to be essential for NarL binding (K. Tyson, A. Bell, J. Cole, and S. Busby, Mol. Microbiol. 7:151-157, 1993).
Pressure effects on the composition and thermal behavior of lipids from the deep-sea thermophile Methanococcus jannaschii
Authors: Kaneshiro S M , Clark D S
Published: November 2016

The deep-sea archaeon Methanococcus jannaschii was grown at 86 degrees C and under 8, 250, and 500 atm (1 atm = 101.29 kPa) of hyperbaric pressure in a high-pressure, high-temperature bioreactor. The core lipid composition of cultures grown at 250 or 500 atm, as analyzed by supercritical fluid chromatography, exhibited an increased proportion of macrocyclic archaeol and corresponding reductions in aracheol and caldarchaeol compared with the 8-atm cultures. Thermal analysis of a model core-lipid system (23% archaeol, 37% macrocyclic archaeol, and 40% caldarchaeol) using differential scanning calorimetry revealed no well-defined phase transition in the temperature range of 20 to 120 degrees C. Complementary studies of spin-labeled samples under 10 and 500 atm in a special high-pressure, high-temperature electron paramagnetic resonance spectroscopy cell supported the differential scanning calorimetry phase transition data and established that pressure has a lipid-ordering effect over the full range of M. jannaschii's growth temperatures. Specifically, pressure shifted the temperature dependence of lipid fluidity by ca. 10 degrees C/500 atm.
A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes
Authors: Schupp T , Toupet C , Cluzel B , Neff S , Hill S , Beck J J , Ligon J M
Published: November 2016

A 40-kb region of DNA from Sorangium cellulosum So ce26, which contains polyketide synthase (PKS) genes for synthesis of the antifungal macrolide antibiotic soraphen A, was cloned. These genes were detected by homology to Streptomyces violaceoruber genes encoding components of granaticin PKS, thus extending this powerful technique for the identification of bacterial PKS genes, which has so far been applied only to actinomycetes, to the gram-negative myxobacteria. Functional analysis by gene disruption has indicated that about 32 kb of contiguous DNA of the cloned region contains genes involved in soraphen A biosynthesis. The nucleotide sequence of a 6.4-kb DNA fragment, derived from the region with homology to granaticin PKS genes, was determined. Analysis of this sequence has revealed the presence of a single large open reading frame beginning and ending outside the 6.4-kb fragment. The deduced amino acid sequence indicates the presence of a domain with a high level of similarity to beta-ketoacyl synthases that are involved in polyketide synthesis. Other domains with high levels of similarity to regions of known polyketide biosynthetic functions were identified, including those for acyl transferase, acyl carrier protein, ketoreductase, and dehydratase. We present data which indicate that soraphen A biosynthesis is catalyzed by large, multifunctional enzymes analogous to other bacterial PKSs of type I.
Quantitative differences in adhesiveness of type 1 fimbriated Escherichia coli due to structural differences in fimH genes
Authors: Sokurenko E V , Courtney H S , Maslow J , Siitonen A , Hasty D L
Published: November 2016

Type 1 fimbriae are heteropolymeric surface organelles responsible for the D-mannose-sensitive (MS) adhesion of Escherichia coli. We recently reported that variation of receptor specificity of type 1 fimbriae can result solely from minor alterations in the structure of the gene for the FimH adhesin subunit. To further study the relationship between allelic variation of the fimH gene and adhesive properties of type 1 fimbriae, the fimH genes from five additional strains were cloned and used to complement the FimH deletion in E. coli KB18. When the parental and recombinant strains were tested for adhesion to immobilized mannan, a wide quantitative range in the ability of bacteria to adhere was noted. The differences in adhesion do not appear to be due to differences in the levels of fimbriation or relative levels of incorporation of FimH, because these parameters were similar in low-adhesion and high-adhesion strains. The nucleotide sequence for each of the fimH genes was determined. Analysis of deduced FimH sequences allowed identification of two sequence homology groups, based on the presence of Asn-70 and Ser-78 or Ser-70 and Asn-78 residues. The consensus sequences for each group conferred very low adhesion activity, and this low-adhesion phenotype predominated among a group of 43 fecal isolates. Strains isolated from a different host niche, the urinary tract, expressed type 1 fimbriae that conferred an increased level of adhesion. The results presented here strongly suggest that the quantitative variations in MS adhesion are due primarily to structural differences in the FimH adhesin. The observed differences in MS adhesion among populations of E. coli isolated from different host niches call attention to the possibility that phenotypic variants of FimH may play a functional role in populations dynamics.
A single amino acid substitution in sigma E affects its ability to bind core RNA polymerase
Authors: Shuler M F , Tatti K M , Wade K H , Moran C P
Published: November 2016

We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis. sigma E is required for the production of endospores in B. subtilis but not for vegetative growth. Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores. Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase. The substitution at position 68 did not affect the stability of the protein in B. subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor. The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.