bluuebunny/crossref_metadata_2025 huggingface dataset:
Results From Kenya’s 2016 Report Card on Physical Activity for Children and Youth
Authors: Onywera Vincent O. , Muthuri Stella K. , Hayker Sylvester , Wachira Lucy-Joy M. , Kyallo Florence , Mang’eni Robert O. , Bukhala Peter , Mireri Caleb
Published: November 2016

Background:

Kenya’s 2016 report card aimed to highlight the health and well-being of Kenyan children and youth using the best available evidence on the physical activity of Kenyan children and youth. The report pointed at areas where Kenya was succeeding and areas where more action is required.

Methods:

Inclusive analyses of available data sources on the core indicators related to physical activity and body weights of Kenyan children and youth (5 to 17 years) were conducted. These were assigned grades based on a set of specific criteria.

Results:

Results show that Active Play, Active Transportation, Overweight and Obesity, and Sedentary Behavior were favorable with a grade of B. Overall Physical Activity, Organized Sport Participation, and School (infrastructure, policies, and programs) each received a grade of C, while Family and Peers, Government and Nongovernment organizations, as well as the Community and the Built Environment were assigned grade D.

Conclusions:

Over 72% of Kenyan children and youth use active transportation to and from school and in their daily lives. Although majority of the children and youth have normal body weight, there is need to ensure that they meet and maintain the physical activity levels recommended by the World Health Organization. More needs to be done especially in relation to the governmental and nongovernmental organizations, organized sports participation, as well as involvement of family and peers in promoting healthy active lifestyles among Kenyan children and youth. More representative data for all indicators are required in Kenya.
Results From Qatar’s 2016 Active Healthy Kids Report Card on Physical Activity for Children and Youth
Authors: Al-Kuwari Mohamed G. , Ibrahim Izzeldin A. , Hammadi Eiman M. Al , Reilly John J.
Published: November 2016

Background:

The first Qatar Active Healthy Kids (QAHK) Report Card was developed in 2015–2016. It is a synthesis of the available evidence on physical activity in children and youth in the state of Qatar—an assessment of the state of the nation. The report card is important for future physical activity advocacy, policy, and program development.

Methods:

The QAHK Report Card was inspired by the Active Healthy Kids Scotland 2013 Report Card. The methodology used in Scotland’s report card was adapted for Qatar. A Working Group identified indicators for physical activity and related health behaviors, and evaluated the available data on these indicators. The card grades were determined by the percentage of children meeting guidelines or recommendations.

Results:

The 2016 QAHK Report Card consisted of 9 indicators: 6 Physical Activity and Health Behaviors and Outcomes, and 3 Settings and Influences on these health behaviors and outcomes. The indicator National Policy, Strategy, and Investment was assigned the highest grade (B). Four indicators were assigned D grades: Sedentary Behavior, Dietary Habits, Organized Sports Participation, and Family and Peer Influence. Physical Activity and Obesity were both graded F. Two indicators could not be graded due to insufficient data and/or absence of a recommendation: Active and Outdoor Play, and Community and School Influence.

Conclusions:

The QAHK Report Card identified weaknesses and gaps in the evidence on physical activity and health in children and youth in Qatar. The quality of evidence was poor for some indicators, with some data collection methods of limited validity and reliability, or only available for a limited age range, so the grades are best estimates of the current situation in Qatar. Future surveys and research using objective physical activity measures will support the development of a second QAHK Report Card by 2018.
Results From Denmark’s 2016 Report Card on Physical Activity for Children and Youth
Authors: Larsen Lisbeth Runge , Troelsen Jens , Kirkegaard Kasper Lund , Riiskjær Søren , Krølner Rikke , Østergaard Lars , Kristensen Peter Lund , Møller Niels Christian , Christensen Bjørn Friis Neerfeldt , Jensen Jens-Ole , Østergård Charlotte , Skovgaard Thomas
Published: November 2016

Background:

The first Danish Report Card on Physical Activity (PA) for Children and Youth describes Denmark’s efforts in promoting and facilitating PA and PA opportunities for children and youth.

Methods:

The report card relies primarily on a synthesis of the best available research and policy strategies identified by the Report Card Research Committee consisting of a wide presentation of researchers and experts within PA health behaviors and policy development. The work was coordinated by Research and Innovation Centre for Human Movement and Learning situated at the University of Southern Denmark and the University College Lillebaelt. Nine PA indicators were graded using the Active Healthy Kids Canada Report Card development process.

Results:

Grades from A (highest) to F (lowest) varied in Denmark as follows: 1) Overall Physical Activity (D+), 2) Organized Sport Participation (A), 3) Active Play (INC; incomplete), 4) Active Transportation (B), 5) Sedentary Behaviors (INC), 6) Family and Peers (INC), 7) School (B), 8) Community and the Built Environment (B+), and 9) Government strategies and investments (A-).

Conclusions:

A large proportion of children in Denmark do not meet the recommendations for PA despite the favorable investments and intensions from the government to create good facilities and promote PA.
A ribosomal strategy for magnesium deficiency
Authors: Mushegian Alexandra A.
Published: November 2016

Under low-magnesium stress, bacteria suppress ribosome synthesis.
Location of the dipteran specificity region in a lepidopteran-dipteran crystal protein from Bacillus thuringiensis
Authors: Widner W R , Whiteley H R
Published: November 2016

Two highly related crystal protein genes from Bacillus thuringiensis subsp. kurstaki HD-1, designated cryIIA and cryIIB (previously named cryB1 and cryB2, respectively), were used to study host range specificity. Their respective gene products are 87% identical but exhibit different toxicity spectra; CryIIA is toxic to both mosquito and tobacco hornworm larvae, whereas CryIIB is toxic only to the latter. Hybrids of the cryIIA and cryIIB genes were generated, and their resultant gene products were assayed for toxicity. A short segment of CryIIA corresponding to residues 307 through 382 was shown to be sufficient for altering host range specificity-i.e., when this region replaced the corresponding segment of CryIIB, the resulting hybrid protein acquired toxicity against mosquitoes. The CryIIA and CryIIB polypeptides differ by only 18 amino acids in this region, indicating that very few amino acid changes can have a substantial effect on the toxicity spectra of these proteins.
SecY, a multispanning integral membrane protein, contains a potential leader peptidase cleavage site
Authors: Akiyama Y , Inada T , Nakamura Y , Ito K
Published: November 2016

SecY is an Escherichia coli integral membrane protein required for efficient translocation of other proteins across the cytoplasmic membrane; it is embedded in this membrane by the 10 transmembrane segments. Among several SecY-alkaline phosphatase (PhoA) fusion proteins that we constructed previously, SecY-PhoA fusion 3-3, in which PhoA is fused to the third periplasmic region of SecY just after the fifth transmembrane segment, was found to be subject to rapid proteolytic processing in vivo. Both the SecY and PhoA products of this cleavage have been identified immunologically. In contrast, cleavage of SecY-PhoA 3-3 was barely observed in a lep mutant with a temperature-sensitive leader peptidase. The full-length fusion protein accumulated in this mutant was cleaved in vitro by the purified leader peptidase. A sequence Ala-202-Ile-Ala located near the proposed interface between transmembrane segment 5 and periplasmic domain 3 of SecY was found to be responsible for the recognition and cleavage by the leader peptidase, since a mutated fusion protein with Phe-Ile-Phe at this position was no longer cleaved even in the wild-type cells. These results indicate that SecY contains a potential leader peptidase cleavage site that undergoes cleavage if the PhoA sequence is attached carboxy terminally. Thus, transmembrane segment 5 of SecY can fulfill both of the two important functions of the signal peptide, translocation and cleavage, although the latter function is cryptic in the normal SecY protein.
Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili
Authors: Nunn D , Bergman S , Lory S
Published: November 2016

The polar pili of Pseudomonas aeruginosa are composed of monomers of the pilin structural subunits. The biogenesis of pili involves the synthesis of pilin precursor, cleavage of a six-amino-acid leader peptide, membrane translocation, and assembly of monomers into a filamentous structure extending from the bacterial surface. This report describes three novel genes necessary for the formation of pili. DNA sequences adjacent to pilA, the pilin structural gene, were cloned and mutagenized with transposon Tn5. Each of the insertions were introduced into the chromosome of P. aeruginosa PAK by gene replacement. The effect of the Tn5 insertions in the bacterial chromosome on pilus assembly was assessed by electron microscopy and sensitivity of mutants to a pilus-specific bacteriophage. The resultant mutants were also tested for synthesis and membrane localization of the pilin antigen in order to define the genes required for maturation, export, and assembly of pilin. A 4.0-kilobase-pair region of DNA adjacent to the pilin structural gene was found to be essential for formation of pili. This region was sequenced and found to contain three open reading frames coding for 62-, 38- to 45-, and 28- to 32-kilodalton proteins (pilB, pilC, and pilD, respectively). Three proteins of similar molecular weight were expressed in Escherichia coli from the 4.0-kilobase-pair fragment flanking pilA with use of a T7 promoter-polymerase expression system. The results of the analyses of the three genes and the implications for pilin assembly and maturation are discussed.
tRNA genes in mycobacteria: organization and molecular cloning
Authors: Bhargava S , Tyagi A K , Tyagi J S
Published: November 2016

DNAs from nine mycobacteria cleaved with restriction endonucleases were hybridized with cDNA probes synthesized to tRNAs from Mycobacterium tuberculosis and Mycobacterium smegmatis. The tRNA genes are conserved, but their gross genomic organization has diverged in six of the nine species examined. Organisms of the M. tuberculosis H37Ra and H37Rv-M. bovis BCG complex appeared to have identical tRNA gene organization and were indistinguishable from each other. M. tuberculosis and M. smegmatis tRNA-derived cDNA probes hybridized differentially to tRNA-coding DNA segments in five of the species examined, suggesting the existence of qualitatively different tRNA pools in these slow- and fast-growing species. Mycobacterial DNAs hybridized with cDNA synthesized to 23S plus 16S rRNAs from Escherichia coli, and the data suggested that the tRNA genes map close to the rRNA genes. A gene bank of M. tuberculosis H37Rv DNA was constructed, and a recombinant plasmid, pSB2, coding for tRNA(s) and rRNA(s) was partially characterized. Plasmid pSB2 recognized a SalI restriction fragment length polymorphism (RFLP) in M. tuberculosis H37Rv and H37Ra; however, the RFLP is not linked to the tRNA-coding region. To the best of our knowledge, this is the first report of an RFLP which distinguishes the pathogenic strain M. tuberculosis H37Rv from its avirulent derivative H37Ra.
Bacteriophage T4 deoxynucleotide kinase: gene cloning and enzyme purification
Authors: Brush G S , Bhatnagar S K , Bessman M J
Published: November 2016

Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein.
Activation of a new proline transport system in Salmonella typhimurium
Authors: Ekena K , Liao M K , Maloy S
Published: November 2016

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.
Mutations in a new Streptomyces coelicolor locus which globally block antibiotic biosynthesis but not sporulation
Authors: Adamidis T , Riggle P , Champness W
Published: November 2016

Streptomyces coelicolor produces four known antibiotics. To define genetic elements that regulate antibiotic synthesis, we screened for mutations that visibly blocked synthesis of the two pigmented antibiotics and found that the mutant strains which we recovered were of two classes--double mutants and mutants in which all four antibiotics were blocked. The mutations in these multiply blocked strains define a new locus of S. coelicolor which we have named absA. The genetic location of absA, at 10 o'clock, is distinct from the locations of the antibiotic gene clusters and from other known mutations that affect antibiotic synthesis. The phenotype of the absA mutants suggests that all S. coelicolor antibiotic synthesis genes are subject to a common global regulation that is at least in part distinct from sporulation and that absA is a genetic component of the regulatory mechanism.
A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase
Authors: Williams M V , Pollack J D
Published: November 2016

The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.
The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation
Authors: Miyake K , Kuzuyama T , Horinouchi S , Beppu T
Published: November 2016

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.
RecA protein of Escherichia coli has a third essential role in SOS mutator activity
Authors: Sweasy J B , Witkin E M , Sinha N , Roegner-Maniscalco V
Published: November 2016

The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity.
The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC
Authors: Kornacki J A , Burlage R S , Figurski D H
Published: November 2016

Broad-host-range plasmid RK2 encodes several kil operons (kilA, kilB, kilC, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kil-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mapping revealed the precise location of korC in a region near transposon Tn1. We determined the nucleotide sequence of this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC alleles from a bacteriophage T7 RNA polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA and KorC proteins act as corepressors in the control of the kilC and kilE operons. Finally, we show that korC is expressed from the bla promoters within the upstream transposon Tn1, suggesting that insertion of Tn1 interrupted a plasmid operon that may have originally included korC and kilC.
Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12
Authors: Alexander-Caudle C , Latinwo L M , Jackson J H
Published: November 2016

Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C. The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions. Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case. These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.
Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other gram-positive organisms
Authors: Josson K , Soetaert P , Michiels F , Joos H , Mahillon J
Published: November 2016

A Lactobacillus hilgardii plasmid, pLAB1000, was studied to understand the organization of autonomous replicons from lactobacilli. Two cassettes could be identified. First, the replication region consisted of a sequence coding for a replication protein (Rep) and its corresponding target site, similar to those from plasmids pUB110, pC194 (Staphylococcus aureus), pFTB14, pBAA1 (Bacillus sp.), and pLP1 (Lactobacillus sp.). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate Rep production. The results also suggested that pLAB1000 replicates via a single-stranded DNA intermediate, and a putative lagging-strand initiation site was found that had similarities to those of alpha 3, St-1, and G4 isometric bacteriophages. The second cassette of pLAB1000 consisted of a sequence coding for a putative mobilization protein (Mob) and its corresponding RSA site. This cassette was similar to those found in pT181, pUB110, pE194 (S. aureus), and pG12 (Bacillus sp.), and it was found to be conserved among different Lactobacillus plasmid replicons. The origin and evolution of these functional cassettes are also discussed.
Use of nonmotile mutants to identify a set of membrane proteins related to gliding motility in Cytophaga johnsonae
Authors: Pate J L , De Jong D M
Published: November 2016

Nonmotile mutants of the gliding bacterium Cytophaga johnsonae were examined to identify proteins that might be involved in gliding motility. Wild-type and mutant cell proteins were solubilized and fractionated by using Triton X-114, and the proteins that partitioned into the aqueous phase or the detergent phase were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for proteins that differed between wild-type and mutant cells. Seventeen proteins, ranging in size from 16 to 150 kilodaltons, were implicated by this technique as having some relationship to gliding and were designated Gld-1 through Gld-17. All Gld proteins behaved as integral membrane proteins, partitioning into the detergent phase. All 56 mutants examined exhibited changes in 1 or more of the Gld proteins, with the number of proteins altered in any mutant varying from 1 to 11. Several lines of evidence suggested that proteins Gld-12 through Gld-15 are glycoproteins. Analysis of banding patterns of detergent-fraction proteins of motile revertants supported the idea that the Gld proteins have a role in gliding motility.
The lcrE gene is part of an operon in the lcr region of Yersinia enterocolitica O:3
Authors: Viitanen A M , Toivanen P , Skurnik M
Published: November 2016

The low-calcium response (lcr) region of the virulence plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica has been associated with calcium-dependent growth of bacteria. Mutations in the previously identified lcrE locus within the lcr region lack the repressor control of production of the lcr specific proteins, Yersinia outer membrane proteins (Yops) and V and W antigens. We sequenced a 3.3-kilobase-pair BamHI-ClaI fragment of the lcrE locus of pYVO3, the virulence plasmid of Y. enterocolitica O:3. The sequence of lcrE locus revealed six tightly packed open reading frames (ORFs), one of which was identified as the structural gene, lcrE, of the 32.9-kilodalton outer membrane protein LcrE (formerly known as Yop4b or YopN). Detection of large (greater than 2.3-kilobase-pair) transcripts strongly supports the conclusion that the lcrE gene and ORF1 to -5 function as an operon. Transcription of the lcrE-containing operon and the adjacent lcrB locus was found to be divergent, and the corresponding transcripts overlapped about 1,200 nucleotides. This extremely long overlap of the 5' ends of the transcripts produced from face-to-face promoters is a new finding; the longest overlap thus far found has been a few hundred nucleotides. Temperature was found to play the major role in regulation of transcription of the lcrE-containing operon of pYVO3, whereas Ca2+ concentration seemed to affect it only moderately.
Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography
Authors: Levinson B L , Kasyan K J , Chiu S S , Currier T C , González J M
Published: November 2016

An improved high-performance liquid chromatography separation was developed to detect and quantify beta-exotoxin production in Bacillus thuringiensis culture supernatants. Exotoxin production was assigned to a plasmid in five strains, from three subspecies (B. thuringiensis subsp. thuringiensis serotype 1, B. thuringiensis subsp. tolworthi serotype 9, and B. thuringiensis subsp. darmstadiensis serotype 10). A new exotoxin, called type II beta-exotoxin in this report, was discovered in B. thuringiensis subsp. morrisoni serotype 8ab, purified, and partially characterized. This material is more specific than type I beta-exotoxin and is very active against the Colorado potato beetle, Leptinotarsa decemlineata.
Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions
Authors: Metcalf W W , Steed P M , Wanner B L
Published: November 2016

Twenty-four independent phosphate starvation-inducible (psi) transcriptional fusions made with Mu d1(lacZbla) were analyzed by sequencing the psi::lacZ(Mu d1) chromosomal junctions by using DNAs amplified with the polymerase chain reaction or mini-Mu cloning. Our DNA sequence analysis showed that the MuR DNA in Mu d1 has an unexpected structure that is comprised of 104 bases of MuR DNA in the form of a large inverted repeat, which we denoted Mu d1-R. Also, Mu d1s in the phoA and phn (psiD) loci of the phosphate regulon showed regional specificities for the insertion sites despite the randomness of Mu d1 insertions into the genome as a whole. Gene products or open reading frames were identified for seven unknown psi::lacZ(Mu d1) transcriptional fusions by searching DNA data bases with the sequences adjacent and upstream of the Mu d1s. One psiC::lacZ(Mu d1) lies in the ugpB gene of the ugpBAEC operon, which encodes a periplasmic sn-glycerol-3-phosphate-binding protein; two psiQ::lacZ(Mu d1)s lie in the gltB gene, and one psiQ::lacZ(Mu d1) lies in the gltD gene of the gltBDF operon, encoding the large and small subunits of glutamate synthase, respectively; and the psi-51::lacZ(Mu d1) lies in the glpB gene of the glpABC operon, which codes for the anaerobically regulated glycerol-3-phosphate dehydrogenase. psiE and psiF::lacZ(Mu d1)s lie in uncharacterized open reading frames near the xylE and phoA genes, respectively. Six other psi::lacZ(Mu d1)s lie in yet unreported Escherichia coli sequences.
Characterization of a gene involved in histidine biosynthesis in Halobacterium (Haloferax) volcanii: isolation and rapid mapping by transformation of an auxotroph with cosmid DNA
Authors: Conover R K , Doolittle W F
Published: November 2016

Techniques for the transformation of halophilic archaebacteria have been developed recently and hold much promise for the characterization of these organisms at the molecular level. In order to understand genome organization and gene regulation in halobacteria, we have begun the characterization of genes involved in amino acid biosynthesis in Halobacterium (Haloferax) volcanii. These studies are facilitated by the many auxotrophic mutants of H. volcanii that have been isolated. In this project we demonstrate that cosmid DNA prepared from Escherichia coli can be used to transform an H. volcanii histidine auxotroph to prototrophy. A set of cosmid clones covering most of the genome of H. volcanii was used to isolate the gene which is defective in H. volcanii WR256. Subcloning identified a 1.6-kilobase region responsible for transformation. DNA sequence analysis of this region revealed an open reading frame encoding a putative protein 361 amino acids in length. A search of the DNA and protein data bases revealed that this open reading frame encodes histidinol-phosphate aminotransferase (EC 2.6.1.9), the sequence of which is also known for E. coli, Bacillus subtilis, and Saccharomyces cerevisiae.
Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor
Authors: Chang B Y , Doi R H
Published: November 2016

By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.
Characterization of repetitive sequences controlling phase variation of Haemophilus influenzae lipopolysaccharide
Authors: Weiser J N , Maskell D J , Butler P D , Lindberg A A , Moxon E R
Published: November 2016

Phase variation of lipopolysaccharide epitopes of an Haemophilus influenzae serotype b strain (strain RM.7004) occurs through a mechanism which depends on multiple tandem repeats of the DNA sequence 5'-CAAT-3' situated within the chromosomal locus lic1. We report here that the same tetranucleotide repeats are also found in two other genomic loci (lic2 and lic3) of RM.7004. Similar to lic1, there are multiple tandem repeats of 5'-CAAT-3' present at the 5' ends of long open reading frames in lic2 and lic3. Variation in the number of repeats of CAAT, by shifting the upstream initiation codons in or out of phase with the remainder of the open reading frame, could switch on or off the translation of downstream genes. Similar to previously reported findings for lic1, site-directed mutations in the open reading frame downstream (3') from the repeats of CAAT in lic2 abolished phase variation and identified DNA sequences required for the expression of additional oligosaccharide epitopes. When we used an oligonucleotide comprising five repeats of CAAT or DNA sequences specific for lic1, lic2, and lic3 as probes, a survey of other encapsulated H. influenzae strains (serotypes a through f) and nontypable H. influenzae strains (including biotype aegyptius) showed that the chromosome of H. influenzae can have from two to five regions which contain multiple tandem repeats of CAAT in addition to other sequences which hybridize to lic1 and lic2.
The Rhizobium meliloti trpE(G) gene is regulated by attenuation, and its product, anthranilate synthase, is regulated by feedback inhibition
Authors: Bae Y M , Crawford I P
Published: November 2016

In Rhizobium meliloti, the genes involved in biosynthesis of the amino acid tryptophan are found at three separate chromosomal locations. Of the three gene clusters, trpE(G), trpDC, and trpFBA, only the trpE(G) gene is regulated by the end product of the pathway, tryptophan. We found that trpE(G) mRNA contains a leader transcript that terminates at a stem-loop structure in a putative transcription attenuator. The level of this leader transcript was constant regardless of the amount of tryptophan in the growth medium. However, the level of full-length trpE(G) mRNA decreased as the amount of tryptophan increased. The beta-galactosidase activity of an R. meliloti strain carrying a trpL'-'lacZ fusion remained constant at different tryptophan concentrations, but the beta-galactosidase activity of the same strain carrying a trpE(G)'-'lacZ fusion decreased as the tryptophan concentration increased. These data indicate that transcription of the R. meliloti trpE(G) gene is regulated only by attenuation. We also found that the product of the trpE(G) gene, anthranilate synthase, is feedback inhibited by tryptophan.
Surface proteins of the gliding bacterium Cytophaga sp. strain U67 and its mutants defective in adhesion and motility
Authors: Burchard R P , Bloodgood R A
Published: November 2016

Surface proteins of the gliding bacterium Cytophaga sp. strain U67 that make contact with glass substrata were radioiodinated, using a substratum-immobilized catalyst (Iodo-Gen). At least 15 polypeptides were iodinated, fewer than the number labeled by surface biotinylation of whole cells; these polypeptides define the set of possible candidates for the surface protein(s) that mediates gliding-associated substratum adhesion. The labeling of three adhesion-defective mutants exhibited two characteristic patterns of surface iodination which involved addition, loss, or alteration of several polypeptides of high molecular weight. An adhesion-competent revertant of mutant Adh3 and one of Adh2 exhibited the wild-type labeling pattern. Two other Adh2 revertants resembled their adhesion-defective parent. The labeling pattern of surface polypeptides of a nongliding but adhesive cell strain was similar to that of the wild type.
Mutations that affect control of the methylesterase activity of CheB, a component of the chemotaxis adaptation system in Escherichia coli
Authors: Stewart R C , Roth A F , Dahlquist F W
Published: November 2016

Sensory adaptation by the chemotaxis system of Escherichia coli requires adjustments of the extent of methyl esterification of the chemotaxis receptor proteins. One mechanism utilized by E. coli to make such adjustments is to control the activity of CheB, the enzyme responsible for removing receptor methyl ester groups. Previous work has established the existence of a multicomponent signal transduction pathway that enables the chemotaxis receptor proteins to control the methylesterase activity in response to chemotactic stimuli. We isolated and characterized CheB mutants that do not respond normally to this control mechanism. In intact cells these CheB variants could not be activated in response to negative chemotaxis stimuli. Further characterization indicated that these CheB variants could not be phosphorylated by the chemotaxis protein kinase CheA. Disruption of the mechanism responsible for regulating methylesterase activity was also observed in cells carrying chromosomal deletions of either cheA or cheW as well as in cells expressing mutant versions of CheA that lacked kinase activity. These results provide further support for recent proposals that activation of the methylesterase activity of CheB involves phosphorylation of CheB by CheA. Furthermore, our findings suggest that CheW plays an essential role in enabling the chemotaxis receptor proteins to control the methylesterase activity, possibly by controlling the CheA-CheB phosphotransfer reaction.
Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola
Authors: Choquet C G , Kushner D J
Published: November 2016

In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichia coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNA(FMet) (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA(FMet) showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4+ (as glutamate). Higher salt concentrations, especially those with Cl- as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl- ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl- ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4Cl, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl- ions inhibited the attachment of initiation factors to tRNA(FMet) but had little effect on binding of initiation factors to R17 RNA.
High-frequency, site-specific recombination between lactococcal and pAM beta 1 plasmid DNAs
Authors: Hayes F , Daly C , Fitzgerald G F
Published: November 2016

In vivo recombination events involving the 75-kilobase lactose proteinase plasmid pCI301 of Lactococcus lactis subsp. lactis UC317 and the conjugative enterococcal plasmid pAM beta 1 were analyzed. A fragment, identified as containing the pCI301 recombination site, mediated greatly elevated levels of mobilization and recombination with pAM beta 1 when cloned in a nonmobilizable L. lactis-Escherichia coli shuttle vector. This latter recombination event was site and orientation specific on both plasmids. Recombination on pAM beta 1 was within the region associated with plasmid replication, but no effect on pAM beta 1 replication functions was detected. Resolution of recombinant plasmids generated derivatives indistinguishable from the parental plasmids.
Detection of the surface-exposed 18-kilodalton binding protein in Chlamydia trachomatis by immunogold staining
Authors: Gray G J , Kaul R , Sherburne R , Wenman W M
Published: November 2016

Dot-blot analysis of Chlamydia trachomatis elementary bodies (EBs) with monospecific polyclonal antibodies demonstrated that the 18-kilodalton binding protein is surface exposed. Immunoelectron microscopy with whole serovar L2 EBs and ultrathin sections confirmed this finding. In addition, only the extracellular EBs and not the intracellular reticulate bodies were labeled with immunogold.
The response of a Bacillus subtilis temperature-sensitive sigA mutant to heat stress
Authors: Chang B Y , Chen K Y , Wen Y D , Liao C T
Published: November 2016

The mutant sigA allele of Bacillus subtilis DB1005 was confirmed to be temperature sensitive (ts) and transferable among strains of B. subtilis by chromosomal transformation and gene conversion. This ts sigA allele had a pleiotropic effect on gene expression of DB1005. The induction of certain heat shock proteins in DB1005 was markedly less significant than that observed in the wild-type strain (DB2) under heat stress. In contrast, some proteins required for coping with oxidative stress and glucose starvation were induced abruptly in DB1005 but not in DB2. Heat induction of the groEL gene in vivo at both transcription and translation levels was much lower in DB1005 than in DB2. Besides, the putative sigma A-type promoter from the groESL operon of B. subtilis was able to be transcribed by the reconstituted sigma A RNA polymerase in vitro at both 37 and 49 degrees C. These results strongly suggest that the expression of the groEL gene of B. subtilis under heat stress is regulated at least in part by sigma A at the level of transcription. Our results also showed that DB1005 did not respond too differently from the wild type to ethanol stress, except after a relatively long exposure.
Growth and bioenergetics of alkaliphilic Bacillus firmus OF4 in continuous culture at high pH
Authors: Sturr M G , Guffanti A A , Krulwich T A
Published: November 2016

The effect of external pH on growth of alkaliphilic Bacillus firmus OF4 was studied in steady-state, pH-controlled cultures at various pH values. Generation times of 54 and 38 min were observed at external pH values of 7.5 and 10.6, respectively. At more alkaline pH values, generation times increased, reaching 690 min at pH 11.4; this was approximately the upper limit of pH for growth with doubling times below 12 h. Decreasing growth rates above pH 11 correlated with an apparent decrease in the ability to tightly regulate cytoplasmic pH and with the appearance of chains of cells. Whereas the cytoplasmic pH was maintained at pH 8.3 or below up to external pH values of 10.8, there was an increase up to pH 8.9 and 9.6 as the growth pH was increased to 11.2 and 11.4, respectively. Both the transmembrane electrical potential and the phosphorylation potential (delta Gp) generally increased over the total pH range, except for a modest fall-off in the delta Gp at pH 11.4. The capacity for pH homeostasis rather than that for oxidative phosphorylation first appeared to become limiting for growth at the high edge of the pH range. No cytoplasmic or membrane-associated organelles were observed at any growth pH, confirming earlier conclusions that structural sequestration of oxidative phosphorylation was not used to resolve the discordance between the total electrochemical proton gradient (delta p) and the delta Gp as the external pH is raised. Were a strictly bulk chemiosmotic coupling mechanism to account for oxidative phosphorylation over the entire range, the deltaGp/deltap ration (which would equal the H+/ATP ratio) would rise from about 3 at pH 7.5 to 13 at pH 11.2, dropping to 7 at pH 11.4 only because of the rise in cytoplasmic pH relative to other parameters. Moreover, the molar growth yields on malate were higher at pH 10.5 than at pH 7.5, indicating greater rather than lesser efficiency in the use of substrate at the more alkaline pH.
Cloning and sequencing of a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase gene involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis
Authors: Nagata Y , Ohtomo R , Miyauchi K , Fukuda M , Yano K , Takagi M
Published: November 2016

In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL), which is then metabolized to 2,5-dichlorohydroquinone. Here, we isolated from the genomic library of UT26 two genes which expressed 2,5-DDOL dehydrogenase activity when they were transformed into P. putida and Escherichia coli. Both gene products had an apparent molecular size of 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first gene, named linC, located separately from the two genes (linA and linB) which we had already cloned as genes involved in the gamma-HCH degradation. The other, named linX, located about 1 kb upstream of the linA gene encoding gamma-HCH dehydrochlorinase. A gamma-HCH degradation-negative mutant, named UT72, which lacked the whole linC gene but had the intact linX gene was isolated. The linC gene given in a plasmid could complement UT72. These results strongly suggest that the linC gene but not the linX gene is essential for the assimilation of gamma-HCH in UT26. Deduced amino acid sequences of LinC and LinX show homology to those of members of the short-chain alcohol dehydrogenase family.