bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene
Authors: DeShazer D , Brett P J , Carlyon R , Woods D E
Published: November 2016

Burkholderia pseudomallei is a human and animal pathogen in tropical regions, especially Southeast Asia and northern Australia. Currently little is known about the genetics and molecular biology of this organism. In this report, we describe the mutagenesis of B. pseudomallei with the transposon Tn5-OT182. B. pseudomallei 1026b transposon mutants were obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome. We used Tn5-OT182 to identify the flagellin structural gene, fliC. We screened 3,500 transposon mutants and identified 28 motility mutants. Tn5-OT182 integrated into 19 unique genetic loci encoding proteins with homology to Escherichia coli and Salmonella typhimurium flagellar and chemotaxis proteins. Two mutants, MM35 and MM36, contained Tn5-OT182 integrations in fliC. We cloned and sequenced fliC and used it to complement MM35 and MM36 in trans. The fliC transcriptional start site and a sigmaF-like promoter were identified by primer extension analysis. We observed a significant difference in the expression of two distinct fliC-lacZ transcriptional fusions during bacterial growth, suggesting the presence of a latent intragenic transcriptional terminator in fliC. There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these animal models of B. pseudomallei infection. A phylogenetic analysis based on the flagellins from a variety of bacterial species supported the recent transfer of B. pseudomallei from the genus Pseudomonas to Burkholderia.
Role of the RepA1 protein in RepFIC plasmid replication
Authors: Maas R , Wang C
Published: November 2016

Using a sensitive primer extension technique, we have carried out studies to localize the start site of replication of the replicon RepFIC. In the course of these studies, we have found evidence that supports the hypothesis that transcription is an integral component of the initiation of replication. On the basis of our findings, we suggest that the transcript is processed to act as a primer, and therefore we propose that the transcript has a dual role as primer of replication and mRNA for the RepA1 protein. We present a model, based on our evidence, for the initiation of replication of the replicon RepFIC. This model provides as well an alternative explanation for what has been called the cis action of RepA1, and we show that RepA1 may act in trans as well as in cis.
Activation of the catBCA promoter: probing the interaction of CatR and RNA polymerase through in vitro transcription
Authors: Chugani S A , Parsek M R , Hershberger C D , Murakami K , Ishihama A , Chakrabarty A M
Published: November 2016

The soil bacterium Pseudomonas putida is capable of degrading many aromatic compounds, including benzoate, through catechol as an intermediate. The catabolism of catechol is mediated by the catBCA operon, whose induction requires the pathway intermediate cis,cis-muconate as an inducer and the regulatory protein, CatR. CatR also regulates the plasmid-borne pheBA operon of P. putida PaW85, which is involved in phenol catabolism. We have used an in vitro transcription system to study the roles of CatR, cis,cis-muconate, Escherichia coli RNA polymerase, and promoter sequences in expression of the cat and phe operons. The assay confirmed the requirement of both CatR and cis,cis-muconate for transcript formation. We also examined the in vitro transcription of three site-directed mutants of the catBCA promoter; the results obtained compared favorably with previous in vivo data. The requirement of the alpha subunit of RNA polymerase for expression of the catBCA and the pheBA transcripts was also examined. The C-terminal region of the alpha subunit of RNA polymerase has been implicated in direct protein-protein contact with transcriptional regulatory proteins and/or direct contact with the DNA. We show that the carboxyl terminus of the alpha subunit is required for the expression of the catBCA and the pheBA operons because RNA polymerases with truncated alpha subunits were deficient in activation. Further experiments demonstrated the arginine at position 265 and the asparagine at position 268 of the alpha subunit as possible amino acids involved in activation. On the basis of these and previous results, we propose a model to explain the interaction of the different regulatory components leading to CatR-dependent activation of the catBCA operon.
Multiple loci of Pseudomonas syringae pv. syringae are involved in pathogenicity on bean: restoration of one lesion-deficient mutant requires two tRNA genes
Authors: Rich J J , Willis D K
Published: November 2016

A mutational analysis of lesion-forming ability was undertaken in Pseudomonas syringae pv. syringae B728a, causal agent of bacterial brown spot disease of bean. Following a screen of 6,401 Tn5-containing derivatives of B728a on bean pods, 26 strains that did not form disease lesions were identified. Nine of the mutant strains were defective in the ability to elicit the hypersensitive reaction (HR) and were shown to contain Tn5 insertions within the P. syringae pv. syringae hrp region. Ten HR+ mutants were defective in the production of the toxin syringomycin, and a region of the chromosome implicated in the biosynthesis of syringomycin was deleted in a subset of these mutants. The remaining seven lesion-defective mutants retained the ability to produce protease and syringomycin. Marker exchange mutagenesis confirmed that the Tn5 insertion was causal to the mutant phenotype in several lesion-defective, HR+ strains. KW239, a lesion- and syringomycin-deficient mutant, was characterized at the molecular level. Sequence analysis of the chromosomal region flanking the Tn5 within KW239 revealed strong similarities to a number of known Escherichia coli gene products and DNA sequences: the nusA operon, including the complete initiator tRNA(Met) gene, metY; a tRNA(Leu) gene; the tpiA gene product; and the MrsA protein. Removal of sequences containing the two potential tRNA genes prevented restoration of mutant KW239 in trans. The Tn5 insertions within the lesion-deficient strains examined, including KW239, were not closely linked to each other or to the lemA or gacA genes previously identified as involved in lesion formation by P. syringae pv. syringae.
Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus sigma70 promoter
Authors: Ge Y , Old I G , Saint Girons I , Charon N W
Published: November 2016

A large motility operon, referred to as the flgB operon, was identified, characterized, and mapped at 310 to 320 kb on the linear chromosome of the spirochete Borrelia burgdorferi. This is the first report that a sigma70-like promoter rather than a sigma28-like promoter is involved in the transcription of a major motility operon in bacteria. From these results in conjunction with results from a previous study (Y. Ge and N. W. Charon, Gene, in press), we have identified 26 genes in this operon that are relevant to motility and flagellar synthesis. With few exceptions, the gene order and deduced gene products were most similar to those of other spirochetes and Bacillus subtilis. Primer extension analysis indicated that transcription initiated from a conserved sigma70-like promoter immediately upstream of flgB; this promoter mapped within the heat-shock-induced protease gene hslU. Reverse transcriptase PCR analysis indicated that a single transcript of 21 kb initiated at this promoter and extended through flgE and (with our previous results) onto the putative motility gene flbE. The flgB promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. As expected, a mutant of S. typhimurium with an inactivated flagellum-specific sigma28 factor did not affect the function of this promoter. Western blot analysis indicated that B. burgdorferi recombinant FliG and FliI were antigenically similar to those of E. coli and other spirochetes. Although complementation of E. coli or S. typhimurium fliG or fliI mutants with the B. burgdorferi genes was unsuccessful, B. burgdorferi recombinant FliI completely inhibited flagellar synthesis and motility of wild-type E. coli and S. typhimurium. These results show that spirochete motility genes can influence flagellar synthesis in other species of bacteria. Finally, Western blot analysis with sera from infected humans and animals indicated a weak or nondetectable response to recombinant FliG and FliI. These results indicate that these antigens are not favorable candidate reagents to be used in the diagnosis of Lyme disease.
Variable efficiency of a Ti plasmid-encoded VirA protein in different agrobacterial hosts
Authors: Bélanger C , Loubens I , Nester E W , Dion P
Published: November 2016

The transconjugant CB100, harboring the Ti plasmid from the Agrobacterium tumefaciens biovar 2 strain D10B/87 in the chromosomal background of the biovar 1 strain C58, was defective in vir gene induction. This defect was corrected in the presence of virA from pTiA6. Based on this complementation result and an analysis of the induction requirements of the transconjugant CB100 and its parent strains, it was hypothesized that the defective vir gene induction in CB100 was related to a dysfunctional interaction between the pTi-encoded D10B/87 VirA and the chromosome-encoded C58 ChvE. To verify this hypothesis, D10B/87 and C58 virA were compared, and conclusions from this first set of analyses were then corroborated by comparing D10B/87 and C58 chvE. Whereas only a few nucleotide differences were identified in the promoters and 5' ends of the coding regions of D10B/87 and C58 virA, analysis of hybrid virA genes showed that these differences collectively accounted for the poor vir gene induction of strain CB100. In contrast with the sequence similarity of the VirA proteins, extensive divergence was seen between the chromosome-encoded D10B/87 and C58 ChvE. Although D10B/87 chvE introduced in trans had little effect on vir gene induction of CB100, it enhanced the induction response of a strain CB100 derivative in which the chromosomal C58 chvE had been inactivated by marker exchange. These results suggest that chromosomal backgrounds provided by different strains of A. tumefaciens are not equivalent for VirA function. Following conjugative transfer of certain Ti plasmids to a new agrobacterial host, evolution of the newly introduced virA, or coevolution of chvE and virA, may lead to optimization of ChvE-VirA interaction and vir gene induction levels.
Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid
Authors: Köhler G A , White T C , Agabian N
Published: November 2016

An IMP dehydrogenase gene was isolated from Candida albicans on a approximately 2.9-kb XbaI genomic DNA fragment. The putative Candida IMP dehydrogenase gene (IMH3) encodes a protein of 521 amino acids with extensive sequence similarity to the IMP dehydrogenases of Saccharomyces cerevisiae and various other organisms. Like the S. cerevisiae IMH3 sequence characterized in the genome sequencing project, the open reading frame of the C. albicans IMH3 gene is interrupted by a small intron (248 bp) with typical exon-intron boundaries and a consensus S. cerevisiae branchpoint sequence. IMP dehydrogenase mRNAs are detected in both the yeast and hyphal forms of C. albicans as judged by Northern hybridization. Growth of wild-type (sensitive) C. albicans cells is inhibited at 1 microg of mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenases, per ml, whereas transformants hosting a plasmid with the IMH3 gene are resistant to MPA levels of up to at least 40 microg/ml. The resistance of cells to MPA is gene dosage dependent and suggests that IMH3 can be used as a dominant selection marker in C. albicans.
Inversion of Moraxella lacunata type 4 pilin gene sequences by a Neisseria gonorrhoeae site-specific recombinase
Authors: Rozsa F W , Meyer T F , Fussenegger M
Published: November 2016

A plasmid library of Neisseria gonorrhoeae sequences was screened for the ability to mediate recombinations on a sequence containing the Moraxella lacunata type 4 pilin gene invertible region in Escherichia coli. A plasmid containing the N. gonorrhoeae sequence encoding the putative recombinase (gcr) was identified and sequenced. Plasmids containing gcr were able to mediate site-specific recombinations despite a weak amino acid homology to Piv, the native M. lacunata pilin gene invertase. The gcr gene is present only in pathogenic strains of Neisseria tested; however, in our assays gene knockouts of gcr did not alter the variation of surface features that play a role in the pathogenesis of N. gonorrhoeae.
Cyclic AMP receptor protein functions as a repressor of the osmotically inducible promoter proP P1 in Escherichia coli
Authors: Xu J , Johnson R C
Published: November 2016

Transcription of the proP gene, encoding a transporter of the osmoprotectants proline and glycine betaine, is controlled from two promoters, P1 and P2, that respond primarily to osmotic and stationary-phase signals, respectively. The P1 promoter is normally expressed at a very low level under low or normal medium osmolarity. We demonstrate that the binding of the cyclic AMP (cAMP) receptor protein (CRP) to a site centered at -34.5 within the promoter is responsible for the low promoter activity under these conditions. A brief period of reduced CRP binding in early log phase corresponds to a transient burst of P1 transcription upon resumption of growth in Luria-Bertani broth. A CRP binding-site mutation or the absence of a functional crp gene leads to high constitutive expression of P1. We show that the binding of CRP-cAMP inhibits transcription by purified RNA polymerase in vitro at P1, but this repression is relieved at moderately high potassium glutamate concentrations. Likewise, open-complex formation at P1 in vivo is inhibited by the presence of CRP under low-osmolarity conditions. Because P1 expression can be further induced by osmotic upshifts in a delta crp strain or in the presence of the CRP binding-site mutation, additional controls exist to osmotically regulate P1 expression.
Protein-tyrosine phosphorylation in the Archaea
Authors: Smith S C , Kennelly P J , Potts M
Published: November 2016

Sulfolobus sulfataricus ATCC 35091, Haloferax volcanii, and Methanosarcina thermophila TM-1, representing the Euryarchaeota and Crenarchaeota subdomains of the Archaea, contain proteins which are phosphorylated on tyrosine. These data raise fundamental questions as to the origin and evolution of tyrosine phosphorylation, a protein modification that is of pivotal importance in the regulation of the physiology of eukaryotic cells.
A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp
Authors: Runyen-Janecky L J , Sample A K , Maleniak T C , West S E
Published: November 2016

The Pseudomonas aeruginosa homolog of the Escherichia coli global transcriptional regulator CRP (or CAP) was recently identified and designated Vfr (S. E. H. West, A. K. Sample, and L. J. Runyen-Janecky, J. Bacteriol. 176:7532-7542, 1994). Nucleotide sequence analysis of the region 5' to vfr identified a 423-bp open reading frame (ORF), which was designated orfX. The deduced amino acid sequence of ORFX was 53% identical and 87% similar to a divergent ORF of unknown function located 5' to the E. coli crp gene. When orfX was expressed from a phage T7 promoter in E. coli, a protein with an apparent molecular mass of approximately 18 kDa was produced. We constructed a chromosomal deletion of the region containing the 5' end of orfX (orfX'), vfr, and the 3' end of trpC (trpC') in P. aeruginosa strains PAO1 and PA103. The cloned vfr gene restored Vfr-dependent production of exotoxin A and protease in the PA103 orfX'-vfr-trpC' deletion mutant, suggesting that ORFX is not required for Vfr production or activity. To determine whether transcription of orfX and vfr are controlled by the same mechanisms that control transcription of the region of the divergent ORF (dorf) and of crp, we compared the vfr-orfX and crp-dorf intergenic regions. Using S1 nuclease analysis, we determined that the distance between the orfX and vfr transcriptional start sites was 105 bp. Thus, the P. aeruginosa orfX and vfr promoters are arranged in a back-to-back orientation rather than the face-to-face orientation of the dorf and crp promoters. A CRP recognition site is associated with each promoter in the crp-dorf intergenic region; binding of the CRP-cyclic AMP complex to the stronger dorf CRP recognition site activates transcription from the dorf promoter and represses transcription from the crp promoter. The vfr-orfX intergenic region does not contain an obvious CRP recognition site. In addition, vfr was not required for transcription of orfX. Unlike the dorf and crp mRNAs, the 5' ends of the orfX and vfr mRNAs were not complementary. Thus, the orfX mRNA cannot hybridize to the 5' end of the vfr mRNA to inhibit vfr transcription, a mechanism that has been postulated to control crp transcription in E. coli.
Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation
Authors: Pagès S , Gal L , Bélaïch A , Gaudin C , Tardif C , Bélaïch J P
Published: November 2016

The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated. The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined. At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.28, 0.38, and 0.55 micromol of miniCipC1 per g, respectively. The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M. These results are discussed with regard to the properties of the various substrates. The synergistic action of miniCipC1 and two forms of endoglucanase CelA (with and without the dockerin domain [CelA2 and CelA3, respectively]) in cellulose degradation was also studied. Although only CelA2 interacted with miniCipC1 (K(d), 7 x 10(-9) M), nonhydrolytic miniCipC1 enhanced the activities of endoglucanases CelA2 and CelA3 with all of the insoluble substrates tested. This finding shows that miniCipC1 plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area. In addition, the data obtained with a hybrid protein, CelA3-CBD(CipC), which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation.
Unbalanced membrane phospholipid compositions affect transcriptional expression of certain regulatory genes in Escherichia coli
Authors: Inoue K , Matsuzaki H , Matsumoto K , Shibuya I
Published: November 2016

The amount of porin protein OmpF in the outer membrane of Escherichia coli was reduced to one-third by the pgsA3 mutation that diminishes the amount of phosphatidylglycerol and cardiolipin in the membrane, whereas a cls (cardiolipin synthase) null mutation had no effect. Osmoregulation of OmpF was functional in the pgsA3 mutant. As assessed by the beta-galactosidase activities of lacZ fusions, the ompF expression was not reduced at the transcriptional level but was reduced about threefold at the posttranscriptional level by pgsA3. This reduction was mostly restored by a micF null mutation, and the micF RNA that inhibits the ompF mRNA translation was present 1.3 to 1.4 times more in the pgsA3 mutant, as assayed by RNase protection and Northern blot analyses. Elevation of the level of micF RNA was not restricted to acidic-phospholipid deficiency: OmpF was hardly detected and micF RNA was present 2.7 to 2.8 times more in a pssA null mutant that lacked phosphatidylethanolamine. Other common phenotypes of pgsA3 and pssA null mutants, reduced rates of cell growth and phospholipid synthesis, were not the cause of micF activation. Salicylate, which activates micF expression and inhibits cell motility, did not repress the flagellar master operon. These results imply that an unbalanced phospholipid composition, rather than a decrease or increase in the amount of specific phospholipid species, induces a phospholipid-specific stress signal to which certain regulatory genes respond positively or negatively according to their intrinsic mechanisms.
The Bacillus subtilis sigma(X) protein is an extracytoplasmic function sigma factor contributing to survival at high temperature
Authors: Huang X , Decatur A , Sorokin A , Helmann J D
Published: November 2016

The sigX gene, identified as part of the international effort to sequence the Bacillus subtilis genome, has been proposed to encode an alternative sigma factor of the extracytoplasmic function (ECF) subfamily. The sigX gene is cotranscribed with a downstream gene, ypuN, during logarithmic and early stationary phases of growth. We now report that strains lacking sigma(X) are impaired in the ability to survive at high temperature whereas a ypuN mutant has increased thermotolerance. We overproduced and purified sigma(X) from Escherichia coli and demonstrate that in vitro, both sigma(A) and sigma(X) holoenzymes recognize promoter elements within the sigX-ypuN control region. However, they have distinct salt optima such that sigma(A)-dependent transcription predominates at low salt while sigma(X)-dependent transcription predominates at high salt. A 54-bp region upstream of sigX suffices as a sigma(X)-dependent promoter in vivo, demonstrating that sigX is at least partially under positive autoregulatory control. Mutation of ypuN increases expression from the sigma(X)-dependent promoter in vivo, suggesting that ypuN may encode a negative regulator of sigma(X) activity.
Primary structure and functional analysis of the soluble transducer protein HtrXI in the archaeon Halobacterium salinarium
Authors: Brooun A , Zhang W , Alam M
Published: November 2016

Signal transduction in the archaeon Halobacterium salinarium is mediated by a family of 13 soluble and membrane-bound transducers. Here, we report the primary structure and functional analysis of one of the smallest halobacterial putative transducers, HtrXI. Hydropathy plot analysis of the primary structure predicts no membrane-spanning segments in HtrXI. The fractionation of the H. salinarium proteins confirmed that HtrXI is a soluble protein. Capillary assay with an HtrXI deletion mutant and a complemented strain revealed that this soluble transducer is involved in Asp and Glu taxis. In vivo analysis of the methylesterase activity of the htrXI-1 deletion mutant suggests that HtrXI plays an important role in the adaptation of the chemotactic responses to His, Asp, and Glu, which are attractants for halobacteria. Stimulation by Asp and Glu causes demethylation of HtrXI and of another putative transducer, HtrVII. But addition of His to halobacterial cells increases HtrXI methylation together with that of other putative transducers. In the absence of HtrXI, stimulation by either Glu or His does not decrease or increase the methylation of any putative transducers. Therefire, the HtrXI transducer appears to have a complex role in chemotaxis signal transduction.
An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB
Authors: Togashi F , Yamaguchi S , Kihara M , Aizawa S I , Macnab R M
Published: November 2016

Pseudorevertants (second-site suppressor mutants) were isolated from a set of parental mutants of Salmonella with defects in the flagellar switch genes fliG and fliM. Most of the suppressing mutations lay in flagellar region IIIb of the chromosome. One fliG mutant, SJW2811, gave rise to a large number of suppressor mutations in the motility genes motA and motB, which are in flagellar region II. SJW2811, which has a three-amino-acid deletion (delta Pro-Ala-Ala) at positions 169 to 171 of FliG, had an extreme clockwise motor bias that produced inverse smooth swimming (i.e., swimming by means of clockwise rotation of a hydrodynamically induced right-handed helical bundle), and formed Mot(-)-like colonies on semisolid medium. Unlike previously reported inverse-swimming mutants, it did not show a chemotactic response to serine, and it remained inverse even in a delta che background; thus, its switch is locked in the clockwise state. The location of the mutation further underscores the conclusion from a previous study of spontaneous missense mutants (V. M. Irikura, M. Kihara, S. Yamaguchi, H. Sockett, and R. M. Macnab, J. Bacteriol. 175:802-810, 1993) that a relatively localized region in the central part of the FliG sequence is critically important for switching. All of the second-site mutations in motA and motB caused some impairment of motility, both in the pseudorevertants and in a wild-type fliG background. The mechanism of suppression of the fliG mutation by the mot mutations is complex, involving destabilization of the right-handed flagellar bundle as a result of reduced motor speed. The mutations in the MotA and MotB sequences were clustered to a considerable degree as follows: in transmembrane helices 3 and 4 of MotA and the sole transmembrane helix of MotB, at helix-membrane interfaces, in the cytoplasmic domains of MotA, and in the vicinity of the peptidoglycan binding region of the periplasmic domain of MotB. The potential importance of Lys28 and Asp33 of the MotB sequence for proton delivery to the site of torque generation is discussed.
NodV and NodW, a second flavonoid recognition system regulating nod gene expression in Bradyrhizobium japonicum
Authors: Loh J , Garcia M , Stacey G
Published: November 2016

In Bradyrhizobium japonicum, members of two global regulatory families, a LysR-type regulator, NodD1, and a two-component regulatory system, NodVW, positively regulate nod gene expression in response to plant-produced isoflavone signals. By analogy to other two-component systems, NodV and NodW are thought to activate transcription via a series of phosphorylation steps. These include the phosphorylation of NodV in response to the plant signal and the subsequent activation of NodW via the transfer of the phosphoryl group to an aspartate residue in the receiver domain of NodW. In this study, we demonstrated that NodW can be phosphorylated in vitro by both acetyl phosphate and its cognate kinase, NodV. In addition, in vivo experiments indicate that phosphorylation is induced by genistein, a known isoflavone nod gene inducer in B. japonicum. By using site-directed mutagenesis, a NodWD70N mutant in which the aspartate residue at the proposed phosphorylation site was converted to an asparagine residue was generated. This mutant was not phosphorylated in either in vitro or in vivo assays. Comparisons of the biological activity of both the wild-type and mutant proteins indicate that phosphorylation of NodW is essential for the ability of NodW to activate nod gene expression.
Characterization of the Bacillus subtilis thiC operon involved in thiamine biosynthesis
Authors: Zhang Y , Taylor S V , Chiu H J , Begley T P
Published: November 2016

The characterization of a three-gene operon (the thiC operon) at 331 min, which is involved in thiamine biosynthesis in Bacillus subtilis, is described. The first gene in the operon is homologous to transcription activators in the lysR family. The second and third genes (thiK and thiC) have been subcloned and overexpressed in Escherichia coli. ThiK (30 kDa) catalyzes the phosphorylation of 4-methyl-5-(beta-hydroxyethyl)thiazole. ThiC (27 kDa) catalyzes the substitution of the pyrophosphate of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate to yield thiamine phosphate. Transcription of the thiC operon is not regulated by thiamine or 2-methyl-4-amino-5-hydroxymethylpyrimidine and is only slightly repressed by 4-methyl-5-(beta-hydroxyethyl)thiazole.
The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli
Authors: Okada K , Minehira M , Zhu X , Suzuki K , Nakagawa T , Matsuda H , Kawamukai M
Published: November 2016

The Escherichia coli ispB gene encoding octaprenyl diphosphate synthase is responsible for the synthesis of the side chain of isoprenoid quinones. We tried to construct an E. coli ispB-disrupted mutant but could not isolate the chromosomal ispB disrupted mutant unless the ispB gene or its homolog was supplied on a plasmid. The chromosomal ispB disruptants that harbored plasmids carrying the ispB homologs from Haemophilus influenzae and Synechocystis sp. strain PCC6803 produced mainly ubiquinone 7 and ubiquinone 9, respectively. Our results indicate that the function of the ispB gene is essential for normal growth and that this function can be substituted for by homologs of the ispB gene from other organisms that produce distinct forms of ubiquinone.
Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria
Authors: Piard J C , Hautefort I , Fischetti V A , Ehrlich S D , Fons M , Gruss A
Published: November 2016

The M6 protein from Streptococcus pyogenes is the best-characterized member of a family of cell envelope-associated proteins. Based on the observation that the C-terminal sorting signals of these proteins can drive cell wall anchoring of heterologous unanchored proteins, we have cloned and expressed the emm6 structural gene for the M6 protein in various lactic acid bacteria (LAB). The emm6 gene was successfully expressed from lactococcal promoters in several Lactococcus lactis strains, an animal-colonizing Lactobacillus fermentum strain, Lactobacillus sake, and Streptococcus salivarius subsp. thermophilus. The M6 protein was efficiently anchored to the cell wall in all strains tested. In lactobacilli, essentially all detectable M6 protein was cell wall associated. These results suggest the feasibility of using the C-terminal anchor moiety of M6 for protein surface display in LAB.
Ganglioneuroma of the pancreas in a 4-year-old girl
Authors: Ikoma Naruhiko , Santamaria-Barria Juan A , Wray Curtis , Tsao KuoJen
Published: November 2016

Ganglioneuroma (GN) is the uncommon, benign representative of the peripheral neuroblastic tumours (PNTs), which arise from primitive sympathetic ganglion cells. PNTs comprise one of the most common groups of neoplastic diseases in infants and children, but its occurrence in the pancreas is rare. We report a 4-year-old girl with GN of the pancreas requiring pancreaticoduodenectomy as a definitive therapy and with a great outcome, and we review the published literature.
Rare urological manifestation of Henoch-Schönlein purpura: testicular torsion
Authors: Oomens Pim , Derix Malou , Fossion Laurent
Published: November 2016

The Henoch-Schönlein purpura (HSP) is a systemic vasculitis that mostly occurs in children. Besides the most common clinical manifestations such as palpable purpura, arthralgia, abdominal pain and renal disease, it can have urological manifestations. We report a rare case of a 2-year-old boy with the HSP who developed a testicular torsion under corticosteroid treatment.
Recurrence and return to play after shoulder instability events in young and adolescent athletes: a systematic review and meta-analysis
Authors: Zaremski Jason L , Galloza Juan , Sepulveda Fernando , Vasilopoulos Terrie , Micheo William , Herman Daniel C
Published: November 2016

Background

Shoulder instability in athletics is a common occurrence. However, there is controversy as to whether non-operative versus operative management is the most effective treatment in youth athletes. We systematically reviewed recurrence and return to play (RTP) after shoulder instability events in youth athletes based on surgical versus non-surgical intervention.

Design

The systematic review concerned studies published before August 2016. Statistical analysis was performed to compare rates of recurrence for each extracted risk factor. Pooled ORs were analysed using random-effects meta-analysis.

Results

17 studies comprising 654 total shoulder instability events met the criteria for inclusion (438 men and 158 women; 507 cases traumatic and 20 atraumatic). Patients were grouped by whether they received non-operative or operative treatment. The primary non-operative group was more likely to have recurrence compared to the primary operative group (OR=13.41; 99% CI 3.60 to 49.93, p<0.001). Patients <14 years old in the primary non-operative group were less likely to recur compared to those aged ≥14 years (OR=0.16; 99% CI 0.06 to 0.43, p<0.001). The rate of recurrence in patients aged <14 years was high (44.44%). For RTP, there is evidence that RTP rates were higher for primary operative patients (95.3%) versus primary non-operative (41.3%, Z=6.12, p<0.001) and secondary operative patients (77.6%, Z=2.66, p=0.008).

Conclusions

This meta-analysis summarises a mix of 17 acceptable quality level II and III prospective and retrospective cohort studies. Given the superior rates of recurrence and RTP, primary operative treatment for shoulder instability should be considered in youth athletes aged ≥14 years. Additionally, the recurrence rate in athletes aged <14 years is significant.
Patterns of health-related behaviours among adolescents: a cross-sectional study based on the National Survey of School Health Brazil 2012
Authors: Azeredo Catarina Machado , Levy Renata Bertazzi , Peres Maria Fernanda Tourinho , Menezes Paulo Rossi , Araya Ricardo
Published: November 2016

Objectives

The aim of this study was to analyse the clustering of multiple health-related behaviours among adolescents and describe which socio-demographic characteristics are associated with these patterns.

Design

Cross-sectional study.

Setting

Brazilian schools assessed by the National Survey of School Health (PeNSE, 2012).

Participants

104 109 Brazilian ninth-grade students from public and private schools (response rate=82.7%).

Methods

Exploratory and confirmatory factor analyses were performed to identify behaviour clustering and linear regression models were used to identify socio-demographic characteristics associated with each one of these behaviour patterns.

Results

We identified a good fit model with three behaviour patterns. The first was labelled ‘problem-behaviour’ and included aggressive behaviour, alcohol consumption, smoking, drug use and unsafe sex; the second was labelled ‘health-compromising diet and sedentary behaviours’ and included unhealthy food indicators and sedentary behaviour; and the third was labelled ‘health-promoting diet and physical activity’ and included healthy food indicators and physical activity. No differences in behaviour patterns were found between genders. The problem-behaviour pattern was associated with male gender, older age, more developed region (socially and economically) and public schools (compared with private). The ‘health-compromising diet and sedentary behaviours’ pattern was associated with female gender, older age, mothers with higher education level and more developed region. The ‘health-promoting diet and physical activity’ pattern was associated with male gender and mothers with higher education level.

Conclusions

Three health-related behaviour patterns were found among Brazilian adolescents. Interventions to decrease those negative patterns should take into account how these behaviours cluster together and the individuals most at risk.
Protective factors for child development at age 2 in the presence of poor maternal mental health: results from the All Our Babies (AOB) pregnancy cohort
Authors: McDonald Sheila W , Kehler Heather L , Tough Suzanne C
Published: November 2016

Objective

To identify the combination of factors most protective of developmental delay at age 2 among children exposed to poor maternal mental health.

Design

Observational cohort study.

Setting

Pregnant women were recruited from primary healthcare offices, the public health laboratory service and community posters in Calgary, Alberta, Canada.

Participants

1596 mother–child dyads who participated in the All Our Babies study and who completed a follow-up questionnaire when their child was 2 years old. Among participants who completed the 2-year questionnaire and had complete mental health data (n=1146), 305 women (27%) were classified as high maternal mental health risk.

Primary measures

Child development at age 2 was described and a resilience analysis was performed among a subgroup of families at maternal mental health risk. The primary outcome was child development problems. Protective factors were identified among families at risk, defined as maternal mental health risk, a composite measure created from participants’ responses to mental health life course questions and standardised mental health measures.

Results

At age 2, 18% of children were classified as having development problems, 15% with behavioural problems and 13% with delayed social–emotional competencies. Among children living in a family with maternal mental health risk, protective factors against development problems included higher social support, higher optimism, more relationship happiness, less difficulty balancing work and family responsibilities, limiting the child's screen time to <1 hour per day and the child being able to fall asleep in <30 min and sleeping through the night by age 2.

Conclusions

Among families where the mother has poor mental health, public health and early intervention strategies that support interpersonal relationships, social support, optimism, work–life balance, limiting children's screen time and establishing good sleep habits in the child's first 2 years show promise to positively influence early child development.
Wordless intervention for people with epilepsy and learning disabilities (WIELD): a randomised controlled feasibility trial
Authors: Mengoni Silvana E , Gates Bob , Parkes Georgina , Wellsted David , Barton Garry , Ring Howard , Khoo Mary Ellen , Monji-Patel Deela , Friedli Karin , Zia Asif , Irvine Lisa , Durand Marie-Anne
Published: November 2016

Objective

To investigate the feasibility of a full-scale randomised controlled trial of a picture booklet to improve quality of life for people with epilepsy and learning disabilities.

Trial design

A randomised controlled feasibility trial. Randomisation was not blinded and was conducted using a centralised secure database and a blocked 1:1 allocation ratio.

Setting

Epilepsy clinics in 1 English National Health Service (NHS) Trust.

Participants

Patients with learning disabilities and epilepsy who had: a seizure within the past 12 months, meaningful communication and a carer with sufficient proficiency in English.

Intervention

Participants in the intervention group used a picture booklet with a trained researcher, and a carer present. These participants kept the booklet, and were asked to use it at least twice more over 20 weeks. The control group received treatment as usual, and were provided with a booklet at the end of the study.

Outcome measures

7 feasibility criteria were used relating to recruitment, data collection, attrition, potential effect on epilepsy-related quality of life (Epilepsy and Learning Disabilities Quality of Life Scale, ELDQOL) at 4-week, 12-week and 20-week follow-ups, feasibility of methodology, acceptability of the intervention and potential to calculate cost-effectiveness.

Outcome

The recruitment rate of eligible patients was 34% and the target of 40 participants was reached. There was minimal missing data and attrition. An intention-to-treat analysis was performed; data from the outcome measures suggest a benefit from the intervention on the ELDQOL behaviour and mood subscales at 4 and 20 weeks follow-up. The booklet and study methods were positively received, and no adverse events were reported. There was a positive indication of the potential for a cost-effectiveness analysis.

Conclusions

All feasibility criteria were fully or partially met, therefore confirming feasibility of a definitive trial.

Trial registration number

ISRCTN80067039.
Cohort profile: the Finnish Genetics of Pre-eclampsia Consortium (FINNPEC)
Authors: Jääskeläinen Tiina , Heinonen Seppo , Kajantie Eero , Kere Juha , Kivinen Katja , Pouta Anneli , Laivuori Hannele
Published: November 2016

Purpose

The Finnish Genetics of Pre-eclampsia Consortium (FINNPEC) Study was established to set up a nationwide clinical and DNA database on women with and without pre-eclampsia (PE), including their partners and infants, in order to identify genetic risk factors for PE.

Participants

FINNPEC is a cross-sectional case–control cohort collected from 5 university hospitals in Finland during 2008–2011. A total of 1450 patients with PE and 1065 pregnant control women without PE (aged 18–47 years) were recruited. Altogether, there were 1377 full triads (625 PE and 752 control triads).

Findings to date

The established cohort holds both clinical and genetic information of mother–infant–father triads representing a valuable resource for studying the pathogenesis of the disease. Furthermore, maternal biological samples (first and third trimester serum and placenta) will provide additional information for PE research. Until now, research has encompassed studies on candidate genes, Sanger and next-generation sequencing, and various studies on the placenta. FINNPEC has also participated in the InterPregGen study, which is the largest investigation on maternal and fetal genetic factors underlying PE until now.

Future plans

Ongoing studies focus on elucidating the role of immunogenetic and metabolic factors in PE. Data on morbidity and mortality will be collected from mothers and fathers through links to the nationwide health registers.
Computed tomography interobserver agreement in the assessment of aneurysmal subarachnoid hemorrhage and predictors for clinical outcome
Authors: Woo Peter Y M , Tse Teresa P K , Chan Robert S K , Leung Lianne N Y , Liu Stephanie K K , Leung Andrew Y T , Wong Hoi-Tung , Chan Kwong-Yau
Published: November 2016

Background

The severity of aneurysmal subarachnoid hemorrhage (SAH) is often assessed by the clinical state of the patient on presentation, but radiological evaluation of the extent of hemorrhage has rarely been examined in the literature. Several CT scan based grading systems exist yet only a few studies have investigated interobserver agreement. We evaluated five radiological grading systems and assessed their clinical value for early prognostication.

Methodology

This was a retrospective study of patients diagnosed with aneurysmal SAH with a CT scan performed within 72 hours of symptom onset. Four independent observers, blinded to patient outcome, evaluated each scan using the five grading systems. A separate assessor determined 6 month outcome from clinical records. The primary outcome was interobserver agreement for each grading system using the Fleiss κ statistic. The secondary endpoint was the 6 month modified Rankin Scale score, with poor outcome defined as a score of 4–6.

Results

165 patients with a mean age of 59 years were assessed. Interobserver agreement for the Fisher, modified Fisher, Claassen, Barrow Neurological Institute, and Hijdra grading systems were as follows: k=0.53 (moderate), k=0.42 (moderate), k=0.38 (mild), k=0.20 (poor), and k=0.66 (good), respectively. The only independent clinical risk factor for poor outcome was a World Federation of Neurological Surgeons (WFNS) grade of 4 or 5 (adjusted OR 6.55; p<0.05). After adjusting for confounders, Fisher grade 4 (adjusted OR 17.84), modified Fisher grade 4 (adjusted OR 5.65), and Hijdra grade 3 (adjusted OR 3.34) were associated with poor outcome. Receiver operator characteristic analysis revealed that the Hijdra grading system (area under the curve=0.76) was more predictive of outcome compared with the Fisher and modified Fisher systems. A Hijdra cut-off score of 22 was associated with poor outcome (adjusted OR 5.92).

Conclusions

The Hijdra grading system had the best interobserver agreement and was a better independent early predictor for 6 month clinical outcome than the other systems. A Hijdra score ≥22 was associated with poor outcome.
Outcomes of ischaemic mitral regurgitation in anterior versus inferior ST elevation myocardial infarction
Authors: Mentias Amgad , Raza Mohammad Q , Barakat Amr F , Hill Elizabeth , Youssef Dalia , Krishnaswamy Amar , Desai Milind Y , Griffin Brian , Ellis Stephen , Menon Venu , Tuzcu E Murat , Kapadia Samir R
Published: November 2016

Background

Ischaemic mitral regurgitation (IMR) is a detrimental complication of ST elevation myocardial infarction (STEMI).

Objective

We sought to determine patient characteristics and outcomes of patients with IMR with focus on anterior or inferior location of STEMI.

Methods

All patients presenting with STEMI complicated by IMR to our centre who underwent primary percutaneous coronary intervention within the first 12 hours of presentation from 1995 to 2014 were included. IMR was graded from 1+ to 4+ within 3 days of index myocardial infarction by echocardiography, divided into 2 groups based on infarct location and outcomes were compared.

Results

Overall, 805 patients were included. There were 302 (17.8%) patients with mitral regurgitation (MR) out of the 1700 patients with anterior STEMI while 503 (21.8%) had MR out of the 2305 patients with inferior STEMI. There was no significant difference between both groups in comorbidities, clinical presentation or door-to-balloon time (DBT; median 104 vs 106 min, p=0.5). 30-day and 1-year mortality were higher in anterior STEMI compared with inferior STEMI (14.9% vs 6.8% and 26.4% vs 14.3%, respectively, p<0.001 both), as well as 5-year mortality (39.7% vs 24.8%, p<0.01). When analysis was performed for each grade of IMR, anterior was associated with worse outcomes in every grade. On multivariate cox survival analysis, after adjustment for age, gender, comorbidities, grade of IMR, ejection fraction and DBT, anterior STEMI was still associated with worse outcomes (HR 1.62 (95% CI 1.23 to 2.12), p<0.001).

Conclusions

Although IMR occurs more frequently with inferior infarction, outcomes are worse following anterior infarction.
Brexit: what does it mean for the veterinary profession?
Authors: Simmons A.
Published: November 2016

Alick Simmons, chair of the BVA's Brexit working group, discusses the implications of Brexit for the veterinary profession and the opportunities it might provide
My working week: Robert Huey
Authors: Huey Robert
Published: November 2016

Robert Huey is the Chief Veterinary Officer for Northern Ireland. Here, he describes a week in October.