bluuebunny/crossref_metadata_2025 huggingface dataset:
Is our Youth Cycling to Health? Results From the Netherlands’ 2016 Report Card on Physical Activity for Children and Youth
Authors: Burghard Marcella , Knitel Karlijn , van Oost Iris , Tremblay Mark S. , Takken Tim , _ _
Published: November 2016

Background:

The Active Healthy Kids the Netherlands (AHKN) Report Card consolidates and translates research and assesses how the Netherlands is being responsible in providing physical activity (PA) opportunities for children and youth (<18 years). The primary aim of this article is to summarize the results of the 2016 AHKN Report Card.

Methods:

Nine indicators were graded using the Active Healthy Kids Global Alliance report card development process, which includes a synthesis of the best available research, surveillance, policy and practice findings, and expert consensus.

Results:

Grades assigned were: Overall Physical Activity Levels, D; Organized Sport Participation, B; Active Play, B; Active Transportation, A; Sedentary Behaviors, C; Family and Peers, B; School, C; Community and the Built Environment, A; Government Strategies and Investments, INC.

Conclusions:

Sedentary behavior and overall PA levels are not meeting current guidelines. However, the Dutch youth behaviors in sports, active transportation, and active play are satisfactory. Several modifiable factors of influence might be enhanced to improve these indicators or at least prevent regression. Although Dutch children accumulate a lot of daily PA through cycling, it is not enough to meet the current national PA guidelines of 60 minutes of moderate-to-vigorous PA per day.
Results From Hong Kong’s 2016 Report Card on Physical Activity for Children and Youth
Authors: Huang Wendy Yajun , Wong Stephen Heung-Sang , Wong Martin Chi-Sang , Sit Cindy Hui-Ping , Sum Raymond Kim-Wai , He Gang
Published: November 2016

Background:

Hong Kong’s 2016 Report Card on Physical Activity (PA) for Children and Youth is the first evidence-based synthesis of various indicators related to individual behaviors that contribute to overall PA levels, settings and sources of influence, and strategies and investments in Hong Kong.

Methods:

Following a standardized protocol, currently best available data for Hong Kong youth were collated and evaluated by an expert consensus panel on 9 indicators (5 activity behaviors and 4 influences on these behaviors).

Results:

Less than half of the children and youth met the recommended PA level. As a result, a D grade was given for Overall PA levels. Organized Sport Participation and Active Transportation received grades of C- and B, respectively. Sedentary Behaviors and School scored a C grade. Community and the Built Environment scored a grade of B. Family Influence received as low a score as Overall PA (D). Active Play and Government were not graded due to incomplete data.

Conclusions:

PA levels are low and sedentary behaviors are high for children and youth in Hong Kong. Promising policies exist in schools and features of community and the built environment are favorable. Increasing family support should be emphasized for future PA promotion.
Results From the United Arab Emirates’ 2016 Report Card on Physical Activity for Children and Youth
Authors: Zaabi Mouza Al , Shah Syed Mahboob , Sheek-Hussein Mohamud , Abdulle Abdishakur , Junaibi Abdulla Al , Loney Tom
Published: November 2016

Background:

The Active Healthy Kids 2016 United Arab Emirates (UAE) Report Card provides a systematic evaluation of how the UAE is performing in supporting and engaging physical activity (PA) in children and adolescents.

Methods:

The Active Healthy Kids Global Alliance framework and standardized set of procedures were used to perform the systematic assessment of PA in UAE youth and children. Indicator grades were based on the proportion of children and youth achieving a defined benchmark: A = 81% to 100%; B = 61% to 80%; C = 41% to 60%; D = 21% to 40%; F = 0% to 20%; INC = incomplete data.

Results:

Overall Physical Activity Level and Active Transportation both received a grade of D-/F-. Sedentary Behavior and Family and Peers both received a C- minus grade and School was graded D. Minus grades indicate PA disparities related to age, gender, nationality, socioeconomic status, and geographic location. Government Strategies and Investments received a B+ grade. Sport Participation, Active Play, and Community and the Built Environment were graded INC due to a lack of nationally representative data for all 7 emirates.

Conclusions:

The majority of UAE children are not achieving the daily recommended level of PA. The UAE leadership has invested significant resources into improving PA through school- and community-based PA interventions; however, inter- and intraemirate population-based strategies remain fragmented.
Results From Chile’s 2016 Report Card on Physical Activity for Children and Youth
Authors: Aguilar-Farias Nicolas , Cortinez-O’Ryan Andrea , Sadarangani Kabir P. , Von Oetinger Astrid , Leppe Jaime , Valladares Macarena , Balboa-Castillo Teresa , Cobos Carolina , Lemus Nicolas , Walbaum Magdalena , Cristi-Montero Carlos
Published: November 2016

Background:

The 2016 Chilean Report Card on Physical Activity for Children and Youth is a review of the evidence across indicators of behaviors, settings, and sources of influence associated with physical activity (PA) of Chilean children and youth.

Methods:

A Research Work Group reviewed available evidence from publications, surveys, government documents and datasets to assign a grade for 11 indicators for PA behavior based on the percentage of compliance for defined benchmarks. Grades were defined as follows: A, 81% to 100% of children accomplishing a given benchmark; B, 61% to 80%; C, 41% to 60%; D, 21% to 40%; F, 0% to 20%; INC, incomplete data available to assign score.

Results:

Grades assigned were for i) ‘Behaviors that contribute to overall PA levels’: Overall PA, F; Organized Sport Participation, D; Active Play, INC; and Active Transportation, C-; ii) ‘Factors associated with cardiometabolic risk’: Sedentary Behavior, D; Overweight and Obesity, F; Fitness, F; and iii) ‘Factors that influence PA’: Family and Peers, D; School, D; Community and Built Environment, C; Government Strategies and Investments, C.

Conclusions:

Chile faces a major challenge as most PA indicators scored low. There were clear research and information gaps that need to be filled with the implementation of consistent and regular data collection methods.
Results From Brazil’s 2016 Report Card on Physical Activity for Children and Youth
Authors: Nardo Nelson , Silva Diego Augusto Santos , de Moraes Ferrari Gerson Luis , Petroski Edio Luiz , Pacheco Ricardo Lucas , Martins Priscila Custódio , Oliveira Luis Carlos , Araújo Timóteo Leandro , Mendes Anselmo Alexandre , Lazarin Samara Pereira Brito , dos Santos Tamires Leal Cordeiro , Matsudo Victor
Published: November 2016

Background:

Very few studies have comprehensively analyzed the physical activity of children and adolescents in Brazil. The purpose of this article is to show the methodology and summarize findings from the first Brazilian Report Card on Physical Activity for Children and Youth.

Methods:

Three Brazilian research institutions coordinated the activities to develop the Brazilian 2016 Report Card. The data available were collected independently and then synthesized by the Research Work Group using the grade system developed for the First Global Matrix released in 2014, which included 9 indicators of physical activity. Where possible, grades were assigned based on the percentage of children and youth meeting each indicator: A is 81% to 100%; B is 61% to 80%; C is 41% to 60%; D is 21% to 40%; F is 0% to 20%; INC is incomplete data.

Results:

Among the 9 indicators, only 5 had sufficient data for grading. Overall Physical Activity received a C- grade, Active Transportation received a C+ grade, Sedentary Behavior received a D+ grade, and Government Strategies and Investments received a D grade.

Conclusions:

The low grades observed highlight the need for continued efforts aimed at improving physical activity in Brazilian children.
Results From South Korea’s 2016 Report Card on Physical Activity for Children and Youth
Authors: Song Yoonkyung , Yang Hyuk In , Lee Eun-Young , Yu Mi-Seong , Kang Min Jae , Kang Hyun Joo , Song Wook , Kim YeonSoo , Park Hyon , Lee Han Joo , Suh Sang-hoon , Spence John C. , Jeon Justin Y.
Published: November 2016

Background:

South Korea’s 2016 Report Card on Physical Activity for Children and Youth is the first assessment of physical activity according to the indicators set by Active Healthy Kids Global Alliance.

Methods:

National surveys were used as preferred sources of data. This was then supported by peer-reviewed papers and government reports identified by a systematic search of the literature written in English or Korean. A Research Working Group then graded indicators based on the collected evidence.

Results:

Each indicator was graded as follows: Overall Physical Activity, D-; Organized Sport and Physical Activity Participation, C-; Active Transport, C+; Sedentary Behavior, F; School, D; Government and Investment, C; Active Play, Physical Literacy, Family and Peers, and Community and Built Environment were graded INC (incomplete) due to lack of available evidence.

Conclusions:

Though the final grades of key indicators for South Korean children and youth are not satisfactory, increasing interests and investments have been demonstrated at a national level. More evidence is required for comprehensive assessment on all indicators to better inform policy and practice. This should be accompanied by the use of consistent criteria to contribute to global efforts for active healthy kids.
Global Matrix 2.0: Report Card Grades on the Physical Activity of Children and Youth Comparing 38 Countries
Authors: Tremblay Mark S. , Barnes Joel D. , González Silvia A. , Katzmarzyk Peter T. , Onywera Vincent O. , Reilly John J. , Tomkinson Grant R. , _ _
Published: November 2016

The Active Healthy Kids Global Alliance organized the concurrent preparation of Report Cards on the physical activity of children and youth in 38 countries from 6 continents (representing 60% of the world’s population). Nine common indicators were used (Overall Physical Activity, Organized Sport Participation, Active Play, Active Transportation, Sedentary Behavior, Family and Peers, School, Community and the Built Environment, and Government Strategies and Investments), and all Report Cards were generated through a harmonized development process and a standardized grading framework (from A = excellent, to F = failing). The 38 Report Cards were presented at the International Congress on Physical Activity and Public Health in Bangkok, Thailand on November 16, 2016. The consolidated findings are summarized in the form of a Global Matrix demonstrating substantial variation in grades both within and across countries. Countries that lead in certain indicators often lag in others. Average grades for both Overall Physical Activity and Sedentary Behavior around the world are D (low/poor). In contrast, the average grade for indicators related to supports for physical activity was C. Lower-income countries generally had better grades on Overall Physical Activity, Active Transportation, and Sedentary Behaviors compared with higher-income countries, yet worse grades for supports from Family and Peers, Community and the Built Environment, and Government Strategies and Investments. Average grades for all indicators combined were highest (best) in Denmark, Slovenia, and the Netherlands. Many surveillance and research gaps were apparent, especially for the Active Play and Family and Peers indicators. International cooperation and cross-fertilization is encouraged to address existing challenges, understand underlying determinants, conceive innovative solutions, and mitigate the global childhood inactivity crisis. The paradox of higher physical activity and lower sedentary behavior in countries reporting poorer infrastructure, and lower physical activity and higher sedentary behavior in countries reporting better infrastructure, suggests that autonomy to play, travel, or chore requirements and/or fewer attractive sedentary pursuits, rather than infrastructure and structured activities, may facilitate higher levels of physical activity.
RasGRP1 promotes amphetamine-induced motor behavior through a Rhes interaction network (“Rhesactome”) in the striatum
Authors: Shahani Neelam , Swarnkar Supriya , Giovinazzo Vincenzo , Morgenweck Jenny , Bohn Laura M. , Scharager-Tapia Catherina , Pascal Bruce , Martinez-Acedo Pablo , Khare Kshitij , Subramaniam Srinivasa
Published: November 2016

Protein interaction networks of the GTPase Rhes regulate motor control in the striatum.
A unique type of GSK-3 inhibitor brings new opportunities to the clinic
Authors: Licht-Murava Avital , Paz Rom , Vaks Lilach , Avrahami Limor , Plotkin Batya , Eisenstein Miriam , Eldar-Finkelman Hagit
Published: November 2016

A substrate peptide that the kinase GSK-3 converts into its own inhibitor improves symptoms and cognitive function in an Alzheimer’s disease model.
Focus Issue: New horizons for treating neurological disease
Authors: Ferrarelli Leslie K.
Published: November 2016

Kinases and protein interaction networks offer new strategies and experimental targets to treat neurological disease.
Auxin signaling without Aux/IAAs
Authors: Gough Nancy R.
Published: November 2016

Auxin regulates the interaction of the transcription factor ETTIN (also known as ARF3) with other developmentally important transcription factors.
Inhibiting the insulin receptor with galectin3
Authors: Wong Wei
Published: November 2016

Obesity-associated insulin resistance may be caused by a lectin released by macrophages that blocks insulin receptor signaling.
Structure and transcription of the genes encoding the B1015 light-harvesting complex beta and alpha subunits and the photosynthetic reaction center L, M, and cytochrome c subunits from Rhodopseudomonas viridis
Authors: Wiessner C , Dunger I , Michel H
Published: November 2016

The genes encoding the beta and alpha subunits of the B1015 light-harvesting complex (LHC) and the L, M, and cytochrome c subunits of the photosynthetic reaction center from Rhodopseudomonas viridis are organized in an operon, in analogy to other nonsulfur purple bacteria, named the puf operon. In photoheterotrophically grown cells, two abundant puf operon mRNA species of 3,581 and 621 bases were present. The large transcript encoded the LHC beta, LHC alpha, and reaction center L, M, and cytochrome c polypeptides, whereas the small transcript only coded for the LHC beta and alpha polypeptides. Both transcripts share a common 5' end which is located 115 bases upstream from the initiation codon of the LHC beta gene. Two additional low-level transcripts of 3,718 and 758 bases with 5' ends 254 +/- 3 bases upstream from the LHC beta gene were detected. Analysis of the DNA sequence preceding the different 5' ends revealed DNA elements of striking homology. The 3' ends of the small transcripts were mapped within the alpha-L intercistronic DNA region downstream from a sequence capable of forming a very stable stem-loop when transcribed into RNA. The 3' termini of the large transcripts are located immediately downstream from the region coding the cytochrome c subunit in two areas resembling rho-independent transcription terminators. No open reading frames corresponding to pufQ and pufX from Rhodobacter capsulatus and Rhodobacter sphaeroides were present in the flanking DNA regions of the puf operon. In contrast, an open reading frame ending 191 base pairs upstream from the LHC beta gene showed 50% homology at the amino acid level to the available sequence of the bchA gene from R. capsulatus. The genes coding for the B1015 LHC subunits had C-terminal extensions of 13 (beta) and 10 (alpha) amino acids which were not present in the proteins isolated from intracytoplasmic membranes.
Copper-zinc superoxide dismutase of Caulobacter crescentus: cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme
Authors: Steinman H M , Ely B
Published: November 2016

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extracellular oxidative stress against which the periplasmic CuZnSOD may defend more effectively than the cytoplasmic FeSOD. Amino acid sequence alignments of C. crescentus CuZnSOD with eucaryotic CuZnSODs and with CuZnSOD of Photobacterium leiognathi (the only other bacterium from which CuZnSOD has been isolated and sequenced) suggest similar supersecondary structures for bacterial and eucaryotic CuZnSODs but reveal four novel substitutions in C. crescentus CuZnSOD: a phenylalanine critical to intrasubunit hydrophobic bonding replaced by alanine, a histidine ligand of zinc replaced by aspartate, and substitutions of two other previously invariant residues that stabilize zinc or both copper and zinc. These amino acid substitutions in C. crescentus CuZnSOD may have implications for its catalysis and stability.
Heat mutagenesis in bacteriophage T4: another walk down the transversion pathway
Authors: Kricker M C , Drake J W
Published: November 2016

Extracellular nonreplicating bacteriophage T4 particles accumulate mutations as functions of temperature, time, pH, and ionic environment via two mechanisms: 5-hydroxymethylcytidine deamination produces G.C----A.T transitions while a guanosine modification produces transversions. Neither frameshift mutations nor mutations at A.T base pairs are appreciably induced. We now show that heat induces G.C----T.A transversions which we suggest may arise via a G*.A mispair, in which G* is a modified guanosine that has experienced a glycosylic bond migration. The rate of this reaction at 37 degrees C is sufficient to present a genetic hazard, particularly to large genomes; thus, the lesion is probably efficiently repaired in cellular genomes.
tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon
Authors: Gollnick P , Yanofsky C
Published: November 2016

Tryptophanase (tna) operon expression in Escherichia coli is induced by tryptophan. This response is mediated by features of a 319-base-pair leader region preceding the major structural genes of the operon. Translation of the coding region (tnaC) for a 24-amino-acid leader peptide is essential for induction. We have used site-directed mutagenesis to investigate the role of the single Trp codon, at position 12 in tnaC, in regulation of the operon. Codon 12 was changed to either a UAG or UGA stop codon or to a CGG arginine codon. Induction by tryptophan was eliminated by any of these changes. Studies with suppressor tRNAs indicated that tRNA(Trp) translation of codon 12 in tnaC is essential for induction of the operon. Reduction of tna expression by a miaA mutation supports a role for translation by tRNA(Trp) in regulation of the operon. Frameshift mutations and suppression that allows translation of tnaC to proceed beyond the normal stop codon result in constitutive tna operon expression. Deletion of a potential site for Rho factor utilization just beyond tnaC also results in partial constitutive expression. These studies suggest possible models for tryptophan induction of tna operon expression involving tRNA(Trp)-mediated frame shifting or readthrough at the tnaC stop codon.
Streptomyces genes involved in biosynthesis of the peptide antibiotic valinomycin
Authors: Perkins J B , Guterman S K , Howitt C L , Williams V E , Pero J
Published: November 2016

We have identified genes from Streptomyces levoris A-9 involved in the biosynthesis of the peptide antibiotic valinomycin. Two segments of chromosomal DNA were recovered from genomic libraries, constructed by using the low-copy-number plasmid pIJ922, by complementation of valinomycin-deficient (vlm) mutants of S. levoris A-9. One set of plasmids restored valinomycin production to only one mutant, that carrying vlm-1, whereas a second set of plasmids restored productivity to seven vlm mutants, those carrying vlm-2 through vlm-8. Additional complementation studies using subcloned restriction enzyme fragments showed that the vlm-1+ gene was contained within a 2.5-kilobase (kb) DNA region, whereas alleles vlm-2+ through vlm-8+ were contained in a 12-kb region, representing at least three genes. Physical mapping experiments based on the isolation of cosmid clones showed that the two vlm loci were 50 to 70 kb apart. Southern hybridization experiments demonstrated that the vlm-2+ gene cluster was highly conserved among other valinomycin-producing Streptomyces strains, whereas the vlm-1+ gene was ubiquitous among Streptomyces species tested. Increasing the copy number of the vlm-2+ gene cluster in S. levoris A-9 by the introduction of low-copy-number recombinant plasmids resulted in a concomitant increase in the level of valinomycin production.
Identification and characterization of hetA, a gene that acts early in the process of morphological differentiation of heterocysts
Authors: Holland D , Wolk C P
Published: November 2016

Envelope polysaccharide is a major early diagnostic of differentiating heterocysts. The mutation in mutant EF116 of Anabaena sp. strain PCC 7120 reduces the cohesiveness of this polysaccharide. A 3.5-kilobase fragment of DNA cloned from the wild type of this Anabaena sp. was previously shown to complement this mutation. We present the nucleotide sequence of a 2,555-base-pair portion of this fragment containing an open reading frame (ORF) of 601 amino acids. Complementation analysis using deletion derivatives of the 3.5-kilobase fragment showed that the gene mutated in EF116, which we designate hetA, lies within this ORF. Transcription of hetA was induced as a result of deprivation for nitrate and yielded a monocistronic mRNA that was present at greatest abundance 7 h after nitrogen stepdown. At that time, proheterocysts could not be distinguished by light microscopy; transcription of nifHD, structural genes of nitrogenase, peaked much later. Situated 3' to hetA are 4 identical repeats of the sequence 5'-TTCAAAA-3' and 12 repeats (10 identical) of the sequence 5'-CCCCAAT-3'. The 12 repeats, present within and near the 5' end of a second ORF, are almost identical to repeats that have been reported to be present in the region between the petC and petA genes of a related cyanobacterium.
Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences
Authors: Cai Y P , Wolk C P
Published: November 2016

Use of the sacB gene (J. L. Ried and A. Collmer, Gene 57:239-246, 1987) provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, we mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.
Escherichia coli rna gene encoding RNase I: cloning, overexpression, subcellular distribution of the enzyme, and use of an rna deletion to identify additional RNases
Authors: Zhu L Q , Gangopadhyay T , Padmanabha K P , Deutscher M P
Published: November 2016

The cloning and overexpression of the Escherichia coli rna gene encoding RNase I are described. Only a single copy of the rna gene is present on the E. coli chromosome. Although cells with as much as a 100-fold increase in RNase I activity were constructed, little effect on cell growth was observed. Overexpressed RNase I was found in the periplasmic space to the same degree (approximately 85%) as wild-type enzyme, suggesting no limitation in RNase I transport. The rna clone was used to identify a deletion strain totally lacking the rna gene. The normal growth of this strain showed that RNase I is not essential for cell viability. Extracts from the RNase I deletion strain still retained a low level of RNase activity in the presence of EDTA, conclusively demonstrating the existence of additional EDTA-active RNases in E. coli. The possibility of a RNase I inhibitor is also discussed.
In vivo rearrangement of foreign DNA by Fusarium oxysporum produces linear self-replicating plasmids
Authors: Powell W A , Kistler H C
Published: November 2016

Particular combinations of fungal strains and transformation vectors allow for fungal rearrangement of normally integrative plasmids, resulting in the creation of linear self-replicating plasmids in Fusarium oxysporum. The rearrangement results in the addition of fungal DNA, including telomere consensus sequences, to plasmid termini. The mechanism by which this rearrangement occurs is unclear, but it has similarities to extrachromosomal gene amplification. A DNA fragment which allows for linear autonomous replication upon reintroduction to the fungus was subcloned and sequenced. This DNA sequence contains the repeated telomeric sequence TTAGGG flanked by a region of twofold symmetry consisting primarily of pUC12 DNA. Isolation and identification of this sequence is the first step toward development of vectors that function as artificial chromosomes in filamentous fungi. This sequence was shown to promote autonomous replication and enhance transformation in several strains of F. oxysporum, Nectria haematococca, and Cryphonectria parasitica.
Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei
Authors: Southam G , Kalmokoff M L , Jarrell K F , Koval S F , Beveridge T J
Published: November 2016

In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella. However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell. Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm. Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing. Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol). The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000. However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1. This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins. On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated. This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels. Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.
Mobilization of closely related plasmids pUB110 and pBC16 by Bacillus plasmid pXO503 requires trans-acting open reading frame beta
Authors: Selinger L B , McGregor N F , Khachatourians G G , Hynes M F
Published: November 2016

Genetic analysis of the closely related nonconjugative plasmids pUB110 and pBC16 has demonstrated that the open reading frame beta (ORF-beta) region in pUB110 and the corresponding homologous region in pBC16 are essential for mobilization of these plasmids by pLS20 or its derivatives. Deletions in this region or insertions that interrupted ORF-beta severely impaired or eliminated the mobilization of pUB110::pUC18 and pBC16::pUC18 hybrids. In contrast, a hybrid in which pUC18 was inserted into pBC16 at a point outside ORF-beta transferred at a frequency comparable to that of intact pUB110 or pBC16 (10(-4) transcipients per donor cell). The defect of most transfer-deficient (Mob-) hybrid plasmids could be complemented by an intact sister plasmid (i.e., pBC16 for pUB110::pUC18 Mob- hybrids). The inability to complement certain constructs suggested that the origin of transfer might be located in an area 5' to ORF-beta. Furthermore, cloning the region 5' to ORF-beta onto a nonmobilizable pC194::pUC18 construct resulted in a hybrid plasmid, pUCCoriTBC16, that could be mobilized with complementation. These results indicate that mobilization of pUB110 and pBC16 by conjugative helper plasmids requires ORF-beta in trans and at least one other region, including the RSA sequence, which presumably functions as an origin of transfer, in cis.
Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2)
Authors: Buttner M J , Chater K F , Bibb M J
Published: November 2016

The rpoD gene of Myxococcus xanthus was used as a probe to isolate three Streptomyces coelicolor genes, hrdB, hrdC, and hrdD, which appear to encode RNA polymerase sigma factors extremely similar to the sigma 70 polypeptide of Escherichia coli. Gene disruption experiments suggested that hrdB is essential in S. coelicolor A3(2) but showed that hrdC and hrdD mutants are viable and are apparently unaffected in differentiation, gross morphology, and antibiotic production. S1 nuclease mapping showed that hrdB and hrdD, but not hrdC, were transcribed in liquid culture. The most upstream of two hrdD promoters is internal to an open reading frame (ORF X) on the opposite strand. The predicted product of this gene is homologous to the phosphinothricin acetyltransferases of Streptomyces hygroscopicus and Streptomyces viridochromogenes. The possible significance of the overlapping and divergent transcription of hrdD and ORF X is discussed. A general method for in vivo gene replacement was developed that allowed a positive selection for the desired mutants even in the absence of a mutant phenotype; it was used to isolate a stable hrdC mutant.
A new Escherichia coli heat shock gene, htrC, whose product is essential for viability only at high temperatures
Authors: Raina S , Georgopoulos C
Published: November 2016

We identified and characterized a new Escherichia coli gene, htrC. Inactivation of the htrC gene results in the inability to form colonies at 42 degrees C. An identical bacterial phenotype is found whether the htrC gene is inactivated either by Tn5 insertions or by a deletion spanning the entire gene. The htrC gene has been localized at 90 min, immediately downstream of the rpoC gene, and has been previously sequenced. It codes for a basic polypeptide with an Mr of 21,130. The htrC gene is under heat shock regulation, since it is transcribed actively only in bacteria possessing functional sigma 32. Inactivation of htrC results in (i) bacterial filamentation at intermediate temperatures, (ii) cell lysis at temperatures above 42 degrees C, (iii) overproduction of sigma 32-dependent heat shock proteins at all temperatures, (iv) overproduction of a few additional polypeptides, (v) underproduction of many polypeptides, and (vi) an overall defect in cellular proteolysis as judged by the reduced rate of puromycyl polypeptide degradation. In addition, the presence of an htrC mutation eliminates the UV sensitivity normally exhibited by lon mutant bacteria.
Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms
Authors: Gollop N , Damri B , Chipman D M , Barak Z
Published: November 2016

Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically important reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as follows: VAHB/VAL = R [2-ketobutyrate]/[pyruvate] for 14 enzymes from 10 procaryotic and eucaryotic organisms. Each organism considered has at least one AHAS of R greater than 20, and some appear to contain but a single biosynthetic AHAS. The implications of this for the design of the pathway are discussed. The selective pressure for high specificity for 2-ketobutyrate versus pyruvate implies that the 2-ketobutyrate concentration is much lower than the pyruvate concentration in all these organisms. It seems important for 2-ketobutyrate levels to be relatively low to avoid a variety of metabolic interferences. These results also reinforce the conclusion that biosynthetic AHAS isozymes of low R (1 to 2) are a special adaptation for heterotrophic growth on certain poor carbon sources. Two catabolic "pH 6 AL-synthesizing enzymes" are shown to be highly specific for AL formation only (R less than 0.1).
Trehalose transport and metabolism in Escherichia coli
Authors: Boos W , Ehmann U , Forkl H , Klein W , Rimmele M , Postma P
Published: November 2016

Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITre/IIIGlc)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EIITre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate- and IITre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.
Further electron microscopic studies on the expression of Escherichia coli group II capsules
Authors: Kröncke K D , Golecki J R , Jann K
Published: November 2016

The de novo expression of Escherichia coli K1, K5, and K12 capsules was analyzed with immunoelectron microscopy in temperature upshift experiments, with upshift from 18 degrees C (capsule restrictive) to 37 degrees C (capsule permissive). Newly produced capsular polysaccharides appeared at the cell surface atop membrane adhesion sites (Bayer's junctions). After plasmolysis of the bacteria at an early expression stage, the capsular polysaccharides were labeled at discrete sites in the periplasm by the immunogold technique. After temperature upshift in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or chloramphenicol, the polysaccharides were labeled in the cytoplasm.
Amino-terminal deletions define a glutamine amide transfer domain in glutamine phosphoribosylpyrophosphate amidotransferase and other PurF-type amidotransferases
Authors: Mei B G , Zalkin H
Published: November 2016

A series of deletions was constructed in cloned Escherichia coli purF encoding glutamine phosphoribosylpyrophosphate amidotransferase. These deletions extended into the NH2 terminus of the protein and removed amino acids that are required for glutamine-dependent enzyme activity. Enzyme function, ascribed to the NH3-dependent activity, was retained in deletions that removed up to 237 amino acids. This result supports a model in which PurF-type amidotransferases contain an NH2-terminal glutamine amide transfer domain of approximately 194 to 200 amino acids fused to an aminator domain with NH3-dependent function.
Plasmid pSC101 harbors a recombination site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif
Authors: Cornet F , Mortier I , Patte J , Louarn J M
Published: November 2016

Plasmid pSC101 harbors a 28-bp sequence which is homologous to dif, the target site of the XerC/XerD-dependent recombination system in Escherichia coli. Using a technique which allows very sensitive detection of plasmid loss, we show that recombination at this site, termed psi for pSC101 stabilized inheritance, causes a moderate increase in pSC101 stability. The role of the psi sequence in site-specific recombination has been explored in two other contexts. It was cloned in a derivative of plasmid p15A and inserted into the chromosome in place of dif. In the first situation, psi activity requires accessory sequences and results in multimer resolution; in the second situation, it suppresses the effects of the dif deletion and can promote intermolecular exchanges. Thus, psi is a site whose recombinational activity depends on the context, the first in the cer/dif family known to exhibit such flexibility.
DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli
Authors: Mackay W J , Han S , Samson L D
Published: November 2016

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.
Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements
Authors: Rådström P , Sköld O , Swedberg G , Flensburg J , Roy P H , Sundström L
Published: November 2016

Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086. Several characteristics suggest that this element is a transposon, which we call Tn5090. Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins. The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein. The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly. Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism. The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.
Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB-dependent proteins
Authors: Henderson D P , Payne S M
Published: November 2016

The regulation of hutA, the Vibrio cholerae gene encoding a 77-kDa iron-regulated outer membrane protein required for heme iron utilization, was characterized, and the DNA sequence of the gene was determined. A hutA::Tn5 lac fusion generated previously (D. P. Henderson and S. M. Payne, Mol. Microbiol. 7:461-469, 1993) was transformed into Fur- and Fur+ strains of Escherichia coli and V. cholerae. The results of beta-galactosidase assays on the transformed strains demonstrated that transcription of hutA is regulated by the Fur repressor protein in E. coli and at least partially regulated by Fur in V. cholerae. Analysis of the DNA sequence of hutA indicated that a sequence homologous to the E. coli consensus Fur box was present in the promoter region of hutA. The amino acid sequence of HutA is homologous to those of several TonB-dependent outer member proteins. However, when the V. cholerae heme utilization system, which requires one or more genes encoded by the recombinant plasmid pHUT10 in addition to hutA carried on a second vector, was transferred to a wild-type strain and an isogenic tonB mutant of E. coli, the tonB mutant could utilize heme iron as efficiently as the wild-type strain. These data indicate that the V. cholerae heme utilization system reconstituted in E. coli does not require a functional TonB protein. The tonB mutant transformed with the heme utilization plasmids could not utilize the siderophore ferrichrome as an iron source, indicating that none of the genes encoded on the heme utilization plasmids complements the tonB defect in E. coli. It is possible that a gene(s) encoded by the recombinant heme utilization plasmids encodes a protein serving a TonB-like function in V. cholerae. A region in the carboxy terminus of HutA is homologous to the horse hemoglobin gamma chain, and the amino acids involved in forming the heme pocket in the gamma chain are conserved in HutA. These data suggest that this region of HutA is involved in heme binding.
Roles of LysP and CadC in mediating the lysine requirement for acid induction of the Escherichia coli cad operon
Authors: Neely M N , Dell C L , Olson E R
Published: November 2016

Expression of the Escherichia coli cadBA operon, encoding functions required for the conversion of lysine to cadaverine and for cadaverine excretion, requires at least two extracellular signals: low pH and a high concentration of lysine. To better understand the nature of the lysine-dependent signal, mutants were isolated which expressed a cadA-lacZ transcription fusion in the absence of lysine while retaining pH regulation. The responsible mutation in one of these isolates (EP310) was in cadC, a gene encoding a function necessary for transcriptional activation of cadBA. This mutation (cadC310) is in a part of the gene encoding the periplasmic domain of CadC and results in an Arg-to-Cys change at position 265, indicating that this part of the protein is involved in responding to the presence of lysine. Three other mutants had mutations mapping in or near lysP (cadR), a gene encoding a lysine transport protein that has previously been shown to regulate cadA expression. One of these mutations is an insertion in the lysP coding region. Thus, in the absence of exogenous lysine, LysP is a negative regulator of cadBA expression. Negative regulation by LysP was further demonstrated by showing that lysP expression from a high-copy-number plasmid rendered cadA-lacZ uninducible. Expression of cadA-lacZ in a strain carrying the cadC310 allele, however, was not affected by the plasmid-expressed lysP. Cadaverine was shown to inhibit expression of the cadA-lacZ fusion in cadC+ cells but not in a cadC310 background.