bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential inactivation of alcohol dehydrogenase isoenzymes in Zymomonas mobilis by oxygen
Authors: Tamarit J , Cabiscol E , Aguilar J , Ros J
Published: November 2016

Zymomonas mobilis is endowed with two isoenzymes of fermentative alcohol dehydrogenase, a zinc-containing enzyme (ADH I) and an iron-containing enzyme (ADH II). The activity of ADH I remains fully conserved, while ADH II activity decays when anaerobic cultures are shifted to aerobiosis. This differential response depends on the metal present on each isoenzyme, since pure preparations of ADH I are resistant to oxidative inactivation and preparations of zinc-containing ADH II, obtained by incubation of pure ADH II with ZnCl2, showed no modification of the target for oxidative damage (His277-containing peptide). It was consistently found that the activity of the zinc-containing ADH II, once submitted to oxidative treatment, was fully restored when iron was reintroduced into the enzyme structure. These results indicate that zinc bound to these proteins plays an important role in the protection of their active centers against oxidative damage and may have relevant biochemical and physiological consequences in this species.
Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7
Authors: Suzuki T , Inoki Y , Yamagishi A , Iwasaki T , Wakagi T , Oshima T
Published: November 2016

The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.
Biochemical and serological evidence for an RNase E-like activity in halophilic Archaea
Authors: Franzetti B , Sohlberg B , Zaccai G , von Gabain A
Published: November 2016

Endoribonuclease RNase E appears to control the rate-limiting step that mediates the degradation of many mRNA species in bacteria. In this work, an RNase E-like activity in Archaea is described. An endoribonucleolytic activity from the extreme halophile Haloarcula marismortui showed the same RNA substrate specificity as the Escherichia coli RNase E and cross-reacted with a monoclonal antibody raised against E. coli RNase E. The archaeal RNase E activity was partially purified from the extreme halophilic cells and shown, contrary to the E. coli enzyme, to require a high salt concentration for cleavage specificity and stability. These data indicate that a halophilic RNA processing enzyme can specifically recognize and cleave mRNA from E. coli in an extremely salty environment (3 M KCI). Having recently been shown in mammalian cells (A. Wennborg, B. Sohlberg, D. Angerer, G. Klein, and A. von Gabain, Proc. Natl. Acad. Sci. USA 92:7322-7326, 1995), RNase E-like activity has now been identified in all three evolutionary domains: Archaea, Bacteria, and Eukarya. This strongly suggests that mRNA decay mechanisms are highly conserved despite quite different environmental conditions.
VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1
Authors: Baron C , Llosa M , Zhou S , Zambryski P C
Published: November 2016

During genetic transformation of plant cells by Agrobacterium tumefaciens, 11 VirB proteins and VirD4 are proposed to form a transmembrane bridge to transfer a DNA-protein complex (T-complex) into the plant cytoplasm. In this study, the localization of the first product of the virB operon, VirB1, was studied in detail. While full-length VirB1 localized mostly to the inner membrane, an immunoreactive VirB1 product was found as soluble processed form, designated VirB1*. Equal amounts of VirB1* could be detected in concentrated culture supernatants versus associated with the cell. VirB1* was purified from the supernatant of vir-induced cells by ammonium sulfate precipitation and Q-Sepharose chromatography. Sequence analysis of the N terminus of VirB1* localized the processing site after amino acid 172 of VirB1. Cell-associated VirB1* was partly removed by vortexing, suggesting a loose association with the cell or active secretion. However, cross-linking and coimmunoprecipitation showed a close association of cell-bound VirB1* with the VirB9-VirB7 heterodimer, a membrane-associated component of the T-complex transfer machinery. Homologies of the N-terminal part of VirB1 to bacterial transglycosylases suggest that it may assist T-complex transfer by local lysis of the bacterial cell wall, whereas the exposed localization of the C-terminal processing product VirB1* predicts direct interaction with the plant. Thus, VirB1 may be a bifunctional protein where both parts have different functions in T-complex transfer from Agrobacterium to plant cells.
Characterization of the fimA gene encoding bundle-forming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria
Authors: Ojanen-Reuhs T , Kalkkinen N , Westerlund-Wikström B , van Doorn J , Haahtela K , Nurmiaho-Lassila E L , Wengelnik K , Bonas U , Korhonen T K
Published: November 2016

The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5' end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::omega mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.
Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae
Authors: Hsu T , Artiushin S , Minion F C
Published: November 2016

Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells. Previous studies have implicated a 97-kDa outer membrane-associated protein, P97, that appeared to mediate this interaction. In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed. Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas. The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones. DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain. The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M. hyopneumoniae. Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M. hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo.
Identification of the epitope for a highly cross-reactive monoclonal antibody on the major sigma factor of bacterial RNA polymerase
Authors: Breyer M J , Thompson N E , Burgess R R
Published: November 2016

A highly cross-reactive monoclonal antibody (MAb), 2G10, was found to react in a conserved region of Escherichia coli RNA polymerase sigma70. The epitope was localized to amino acids 470 to 486, which included part of conserved region 3.1. The epitope for MAb 3D3, a MAb which maps close to the 2G10 epitope, was also determined.
Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes
Authors: Schmitz A , Josenhans C , Suerbaum S
Published: November 2016

Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa. While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis. We have cloned and characterized an H. pylori homolog (named flbA) of the lcrD/flbF family of genes. Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence-associated proteins via type III secretion systems. The H. pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequence similarity with other known members of the LcrD/FlbF protein family. An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis. The mutant strain expressed neither the FlaA nor the FlaB flagellin protein. The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent. The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level. The flbA mutants were aflagellate and completely nonmotile. Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product. The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein. Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H. pylori. The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression was not affected by the flbA gene disruption and which might represent further H. pylori homologs of the LcrD/FlbF protein family.
Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number
Authors: Nesvera J , Pátek M , Hochmannová J , Abrhámová Z , Becvárová V , Jelínkova M , Vohradský J
Published: November 2016

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.
Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa
Authors: Liu S , Yahr T L , Frank D W , Barbieri J T
Published: November 2016

Genetic studies have shown that the 53-kDa (Exo53) and 49-kDa (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa are encoded by separate genes, termed exoT and exoS, respectively. Although ExoS and Exo53 possess 76% primary amino acid homology, Exo53 has been shown to express ADP-ribosyltransferase activity at about 0.2% of the specific activity of ExoS. The mechanism for the lower ADP-ribosyltransferase activity of Exo53 relative to ExoS was analyzed by using a recombinant deletion protein which contained the catalytic domain of Exo53, comprising its 223 carboxyl-terminal residues (termed N223-53). N223-53 was expressed in Escherichia coli as a stable, soluble fusion protein which was purified to >80% homogeneity. Under linear velocity conditions, N223-53 catalyzed the FAS (for factor activating exoenzyme S)-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) at 0.4% and of the Ras protein at 1.0% of the rates of catalysis by N222-49. N222-49 is a protein comprising the 222 carboxyl-terminal residues of ExoS, which represent its catalytic domain. N223-53 possessed binding affinities for NAD and SBTI similar to those of N222-49 (less than fivefold differences in Kms) but showed a lower velocity rate for the ADP-ribosylation of SBTI. This indicated that the primary defect for ADP-ribosylation by Exo53 resided within its catalytic capacity. Analysis of hybrid proteins, composed of reciprocal halves of N223-53 and N222-49, localized the catalytic defect to residues between positions 235 and 349 of N223-53. E385 was also identified as a potential active site residue of Exo53.
Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter
Authors: Boucher P E , Murakami K , Ishihama A , Stibitz S
Published: November 2016

The expression of virulence factor genes in Bordetella pertussis is mediated by the BvgA-BvgS two-component signal transduction system. The response regulator, BvgA, acts directly as a transcriptional activator at the loci encoding pertussis toxin (ptx) and filamentous hemagglutinin (fha). Previous studies have demonstrated that these two loci are differentially regulated by BvgA. As an initial step in gaining insight into the mechanism underlying this differential regulation, we initiated DNA binding and in vitro transcription analyses to examine the activities of BvgA and RNA polymerase (RNAP) purified from both B. pertussis and Escherichia coli at the fha promoter. We discovered that unphosphorylated BvgA binds to a single region (-100 to -70, relative to the start of transcription), whereas phosphorylated BvgA binds both this region and another, farther downstream, that extends to the -35 nucleotide. In the absence of BvgA, RNAP binds a region farther upstream than expected (-104 to -35). However, occupation of both sites by BvgA phosphate repositions RNAP to the site used in vivo. The binding of BvgA phosphate to the downstream site correlates with in vitro transcriptional activity at the fha promoter. As the DNA binding and transcription activities of the E. coli-derived RNAP are similar to those observed for the B. pertussis enzyme, we employed several mutant E. coli proteins in in vitro transcription analyses. We observed that polymerases carrying either a deletion of the C-terminal domain of the alpha subunit or substitution of alanine at either of two critical residues within this domain were severely impaired in the ability to mediate BvgA-activated transcription at fha.
Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae
Authors: Sheldon P J , Johnson D A , August P R , Liu H W , Sherman D H
Published: November 2016

In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an Mr of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.
Most of the propeptide is dispensable for stability and autoprocessing of the zymogen of the germination protease of spores of Bacillus species
Authors: Pedersen L B , Nessi C , Setlow P
Published: November 2016

Loss of 3, 7, or 10 of the amino-terminal 15 residues removed upon autoactivation of the zymogen of the germination protease (GPR), which initiates protein degradation during germination of spores of Bacillus species, did not result in significant changes in (i) the lack of enzymatic activity of the zymogen, (ii) the rate of zymogen autoactivation, or (iii) the unreactivity of the zymogen's single SH group. Removal of 13 amino-terminal residues resulted in a partially active enzyme whose SH group was as reactive as the fully active enzyme. These findings suggest that at least a part of the propeptide blocks access to the enzyme's active site. However, the free propeptide did not inhibit the enzyme.
Characterization of the pH signal transduction pathway gene palA of Aspergillus nidulans and identification of possible homologs
Authors: Negrete-Urtasun S , Denison S H , Arst H N
Published: November 2016

We have cloned the palA gene of Aspergillus nidulans, one of six genes participating in ambient pH signal transduction in a regulatory circuit mediating pH regulation of gene expression. The derived 798-residue PalA protein is 29.4% identical over its entire length to a hypothetical protein from the nematode Caenorhabditis elegans and also has possible yeast homologs.
Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4
Authors: Auvray F , Coddeville M , Ritzenthaler P , Dupont L
Published: November 2016

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.
Evidence for the role of redox carriers in photosynthesis gene expression and carotenoid biosynthesis in Rhodobacter sphaeroides 2.4.1
Authors: O'Gara J P , Kaplan S
Published: November 2016

Previous work from this laboratory revealed that alterations in the structure of the ccoNOQP operon of Rhodobacter sphaeroides 2.4.1 could lead to induction of the photosynthetic apparatus under aerobic growth conditions. Immediately downstream of the ccoNOQP operon is the rdxB gene, the first gene of the rdxBHIS cluster. The rdxB gene product is predicted to encode a membrane protein which can bind two [4Fe-4S] clusters. The ccoP gene product is a diheme cytochrome which is a component of the cbb3-type cytochrome oxidase. Under aerobic growth conditions, strains possessing ccoP and rdxB mutations both singly and in combination produced light-harvesting complexes, suggesting that normal functioning of these proteins is required to maintain repression of photosynthesis gene expression in the presence of oxygen. Analysis of the expression of puc::lacZ fusions under aerobic conditions revealed an approximately 12-fold increase in puc operon expression in the RDXB1 and CCOP1 mutant strains compared with that for wild-type 2.4.1. Similarly, puf::lacZ activity was observed to be elevated fourfold above wild-type levels. Further indication of the importance of the RdxB and CcoP proteins was derived from studies of mutant and wild-type cells grown under anoxygenic photosynthetic and nitrogen-fixing conditions. These mutant strains were observed to accumulate spheroidenone to approximately 50% or more of the total carotenoid. In wild-type cultures, spheroidenone normally accumulates to approximately 10 to 20% of the total carotenoid under the same growth conditions. This effect was most pronounced when both the rdxB and the ccoP mutations were present together in cells cultured under nitrogen-fixing photosynthetic growth conditions in which spheroidenone represented approximately 90% of the total carotenoid. We propose that mutations in the rdxB or ccoP gene may lead to changes in a membrane-generated redox signal or the accumulation of a critical redox intermediate in the mutant strains which results in increased photosynthesis gene expression under aerobic conditions by alteration of the activity of a transcriptional regulator(s) of photosynthesis gene expression. Mutations in these genes also appear to posttranscriptionally influence the terminal step of carotenoid biogenesis. Potential regulators interacting with an aberrant redox signal in the mutants and the possible nature of such a redox signal are discussed.
Reduction and removal of heptavalent technetium from solution by Escherichia coli
Authors: Lloyd J R , Cole J A , Macaskie L E
Published: November 2016

Anaerobic, but not aerobic, cultures of Escherichia coli accumulated Tc(VII) and reduced it to a black insoluble precipitate. Tc was the predominant element detected when the precipitate was analyzed by proton-induced X-ray emission. Electron microscopy in combination with energy-dispersive X-ray analysis showed that the site of Tc deposition was intracellular. It is proposed that Tc precipitation was a result of enzymatically mediated reduction of Tc(VII) to an insoluble oxide. Formate was an effective electron donor for Tc(VII) reduction which could be replaced by pyruvate, glucose, or glycerol but not by acetate, lactate, succinate, or ethanol. Mutants defective in the synthesis of the transcription factor FNR, in molybdenum cofactor (molybdopterin guanine dinucleotide [MGD]) synthesis, or in formate dehydrogenase H synthesis were all defective in Tc(VII) reduction, implicating a role for the formate hydrogenlyase complex in Tc(VII) reduction. The following observations confirmed that the hydrogenase III (Hyc) component of formate hydrogenlyase in both essential and sufficient for Tc(VII) reduction: (i) dihydrogen could replace formate as an effective electron donor for Tc(VII) reduction by wild-type bacteria and mutants defective in MGD synthesis; (ii) the inability of fnr mutants to reduce Tc(VII) can be suppressed phenotypically by growth with 250 microM Ni2+ and formate; (iii) Tc(VII) reduction is defective in a hyc mutant; (iv) the ability to reduce Tc(VII) was repressed during anaerobic growth in the presence of nitrate, but this repression was counteracted by the addition of formate to the growth medium; (v) H2, but not formate, was an effective electron donor for a Sel- mutant which is unable to incorporate selenocysteine into any of the three known formate dehydrogenases of E. coli. This appears to be the first report of Hyc functioning as an H2-oxidizing hydrogenase or as a dissimilatory metal ion reductase in enteric bacteria.
Role of mRNA termination in regulation of ermK
Authors: Choi S S , Kim S K , Oh T G , Choi E C
Published: November 2016

To study the role of mRNA termination in the regulation of ermK, we introduced mismatches into terminators by in vitro mutagenesis. In wild-type ermK, only truncated transcription products were detected in the absence of induction. In contrast, only the full-length transcript was synthesized in the terminator 1 and terminator 2 double mutants, even in the absence of erythromycin. These results indicate that the expression of ermK is primarily regulated by transcriptional attenuation rather than translational attenuation. We also tested the possible contribution of translational attenuation control to the regulation of ermK by constructing a triple mutant (terminator 1 plus terminator 2 plus the methylase Shine-Dalgarno region). A higher level of beta-galactosidase synthesis was seen in the triple mutant. Therefore, unlike with previously described attenuators, it can be concluded that both transcriptional and translational attenuation contribute to the regulation of ermK, although transcriptional attenuation plays a larger role.
Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations
Authors: Cai Z H , Hwang Y , Cue D , Catalano C , Feiss M
Published: November 2016

The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme. Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA. After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs. cosB, a site adjacent to cosN, is a terminase binding site. cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase. cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage. The role of complex I is to bind the prohead. Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects. The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB. Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1. Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented. It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA. Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases.
DNA binding by the Xis protein of the conjugative transposon Tn916
Authors: Rudy C K , Scott J R , Churchward G
Published: November 2016

We purified the Xis protein of the conjugative transposon Tn916 and showed by nuclease protection experiments that Xis bound specifically to sites close to each end of Tn916. These specific binding sites are close to, and in the same relative orientation to, binding sites for the N-terminal domain of Tn916 integrase protein. These results suggest that Xis is involved in the formation of nucleoprotein structures at the ends of Tn916 that help to correctly align the ends so that excision can occur.
Molecular characterization of genes of Pseudomonas sp. strain HR199 involved in bioconversion of vanillin to protocatechuate
Authors: Priefert H , Rabenhorst J , Steinbüchel A
Published: November 2016

The gene loci vdh, vanA, and vanB, which are involved in the bioconversion of vanillin to protocatechuate by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a novel vanillin dehydrogenase (vdh) and the two subunits of a vanillate demethylase (vanA and vanB), respectively. These genes were localized on an EcoRI fragment (E230), which was cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The vdh gene was identified on a subfragment (HE35) of E230, and the vanA and vanB genes were localized on a different subfragment (H110) of E230. The nucleotide sequences of fragment HE35 and part of fragment H110 were determined, revealing open reading frames of 1062, 951, and 1446 bp, representing vanA, vanB, and vdh, respectively. The vdh gene was organized in one operon together with a fourth open reading frame (ORF2), of 735 bp, which was located upstream of vdh. The deduced amino acid sequences of vanA and vanB exhibited 78.8 and 62.1% amino acid identity, respectively, to the corresponding gene products from Pseudomonas sp. strain ATCC 19151 (F. Brunel and J. Davison, J. Bacteriol. 170:4924-4930, 1988). The deduced amino acid sequence of the vdh gene exhibited up to 35.3% amino acid identity to aldehyde dehydrogenases from different sources. The deduced amino acid sequence of ORF2 exhibited up to 28.4% amino acid identity to those of enoyl coenzyme A hydratases. Escherichia coli strains harboring fragment E230 cloned in pBluescript SK- converted vanillin to protocatechuate via vanillate, indicating the functional expression of vdh, vanA, and vanB in E. coli. High expression of vdh in E. coli was achieved with HE35 cloned in pBluescript SK-. The resulting recombinant strains converted vanillin to vanillate at a rate of up to 0.3 micromol per min per ml of culture. Transfer of vanA, vanB, and vdh to Alcaligenes eutrophus and to different Pseudomonas strains, which were unable to utilize vanillin or vanillate as carbon sources, respectively, conferred the ability to grow on these substrates to these bacteria.
Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4
Authors: Domanski T L , de Jonge B L , Bayles K W
Published: November 2016

The high level of cross-linking found in Staphylococcus aureus peptidoglycan is dependent on the low-molecular-weight penicillin-binding protein PBP4. Recently, the PBP4 gene, pbpD, was cloned and shown to be adjacent to and divergently transcribed relative to the putative ABC-type transporter gene, abcA. Disruption of abcA (in strain KB400) was previously shown to result in heightened resistance to several antibiotics known to interact with PBP4, suggesting that the regulation of pbpD is affected by abcA. In this report, this hypothesis was confirmed by use of a Northern (RNA) blot analysis which revealed increased accumulation of pbpD-specific transcripts in KB400 compared to that in the wild-type strain, 8325-4. By using reverse-phase high-performance liquid chromatography to examine the structure of the peptidoglycan, it was demonstrated that the increased expression of pbpD resulted in an increased level of peptidoglycan cross-linking in the staphylococcal cell wall. Promoter fusion studies demonstrated that the abcA mutation caused approximately 7-fold and 100-fold increases in pbpD and abcA promoter activities, respectively. Primer extension experiments revealed that these genes have long, untranslated leader sequences that result in a transcriptional overlap of 80 bp. Interestingly, deletion of a 26-bp region containing an inverted repeat sequence resulted in the loss of expression from both the abcA and the pbpD promoters. These data provide evidence that abcA and pbpD are under the control of a common regulatory mechanism that may involve the transport function of the abcA gene product.
A mutation at proline-115 in the A-factor receptor protein of Streptomyces griseus abolishes DNA-binding ability but not ligand-binding ability
Authors: Onaka H , Sugiyama M , Horinouchi S
Published: November 2016

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein (ArpA) are required for streptomycin production and aerial mycelium formation in Streptomyces griseus. A mutant strain HO1 that produced streptomycin and formed aerial mycelium and spores was derived from an A-factor-deficient mutant, S. griseus HH1. The phenotypes of mutant HO1 were found to result from a single amino acid replacement of ArpA; the proline residue at position 115 in the wild-type ArpA was replaced by serine, yielding mutant ArpA (P115S). The mutant ArpA (P115S) was still able to form a homodimer and possessed A-factor-binding ability but lost the ability to bind DNA. The properties of P115S suggest that ArpA consists of two independently functional domains, one for A-factor binding and one for DNA binding, and that proline-115 plays an important role in DNA binding. This is in agreement with the idea that A-factor binding to the COOH-terminal domain of ArpA causes a subtle conformational change of the distal NH2-terminal DNA-binding domain, resulting in dissociation of ArpA from DNA.
Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens
Authors: French C E , Boonstra B , Bufton K A , Bruce N C
Published: November 2016

The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.
Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System
Authors: Banada Padmapriya P. , Deshpande Srinidhi , Chakravorty Soumitesh , Russo Riccardo , Occi James , Meister Gabriel , Jones Kelly J. , Gelhaus Carl H. , Valderas Michelle W. , Jones Martin , Connell Nancy , Alland David
Published: November 2016

Francisella tularensis
is a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitive
F. tularensis
assay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system.
F. tularensis
DNA in buffer or CFU of
F. tularensis
was spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected with
F. tularensis
Schu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/ml
F. tularensis
in both human and macaque blood. In infected macaques, the assay detected
F. tularensis
on days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detect
F. tularensis
in bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection.
Multiplex Urinary Antigen Detection for 13 Streptococcus pneumoniae Serotypes Improves Diagnosis of Pneumococcal Pneumonia in South African HIV-Infected Adults
Authors: Albrich Werner C. , Pride Michael W. , Madhi Shabir A. , Callahan Jan , Adrian Peter V. , French Roger , van Niekerk Nadia , Sebastian Shite , Souza Victor , Telles Jean-Noel , Paranhos-Baccalà Glaucia , Jansen Kathrin U. , Klugman Keith P.
Published: November 2016

A serotype-specific urinary antigen detection (UAD) assay for 13 serotypes included in the pneumococcal conjugate vaccine (PCV13) was recently reported as a useful diagnostic tool for pneumococcal pneumonia. We aimed to assess the diagnostic accuracy of the UAD in HIV-infected South African adults. Urine specimens from a well-defined cohort of HIV-infected South African adults with pneumonia were evaluated retrospectively in the UAD assay. Pneumonia was considered pneumococcal if either sputum Gram stain, sputum culture, blood culture, or the immunochromatographic (ICT) BinaxNow
S. pneumoniae
test (composite diagnostic) was positive. Among 235 enrolled pneumonia patients, the UAD assay was more frequently positive (104 [44.3%]) than the composite diagnostic (71 [30.2%];
P
< 0.001) and increased the pneumococcal etiology from 30.2% by an additional 22.6% to 52.8%. The UAD assay detected more pneumococcal etiologies (45.0%) than the serotype-independent ICT (23.4%,
P
< 0.001). UAD identified 6/7 patients with PCV13 serotype bacteremia without misclassification of bacteremia episodes due to non-PCV13 serotypes. UAD was positive for 5.1% of asymptomatic HIV-infected persons, with higher rates among those with nasopharyngeal carriage. Concordance between serotypes identified by UAD and by Quellung reaction and PCR serotyping was 70/86 (81.4%). UAD identified the dominant serotype in multiple serotype carriage. This study confirms the utility of the UAD assay for HIV-infected adults comparing favorably with other diagnostic tests. A highly valent UAD may become a new standard for detection of pneumococcal pneumonia in adults. Prior to PCV introduction, at least 53% of pneumonia cases were due to pneumococci in HIV-infected South African adults.
Evaluation of Veterinary-Specific Interpretive Criteria for Susceptibility Testing of Streptococcus equi Subspecies with Trimethoprim-Sulfamethoxazole and Trimethoprim-Sulfadiazine
Authors: Sadaka Carmen , Kanellos Theo , Guardabassi Luca , Boucher Joseph , Watts Jeffrey L.
Published: November 2016

Antimicrobial susceptibility test results for trimethoprim-sulfadiazine withStreptococcus equisubspecies are interpreted based on human data for trimethoprim-sulfamethoxazole. The veterinary-specific data generated in this study support a single breakpoint for testing trimethoprim-sulfamethoxazole and/or trimethoprim-sulfadiazine withS. equi. This study indicates trimethoprim-sulfamethoxazole as an acceptable surrogate for trimethoprim-sulfadiazine withS. equi.
Relative Prevalence and Antimicrobial Susceptibility of Clinical Isolates of Elizabethkingia Species Based on 16S rRNA Gene Sequencing
Authors: Han Mi-Soon , Kim Hyunsoo , Lee Yangsoon , Kim Myungsook , Ku Nam Su , Choi Jun Yong , Yong Dongeun , Jeong Seok Hoon , Lee Kyungwon , Chong Yunsop
Published: November 2016

Some of the previously reported clinical isolates of
Elizabethkingia meningoseptica
may be later named species of
Elizabethkingia
. We determined the accuracy of species identification (with two matrix-assisted laser desorption ionization–time of flight mass spectrometry [MALDI-TOF MS] systems and the Vitek 2 GN card), relative prevalence of three
Elizabethkingia
spp. in clinical specimens, and antimicrobial susceptibility of the species identified by 16S rRNA gene sequencing. Specimens for culture were collected from patients in a university hospital in Seoul, South Korea, between 2009 and 2015. All 3
Elizabethkingia
spp. were detected in patients; among the 86 isolates identified by 16S rRNA gene sequencing, 17 (19.8%) were
E. meningoseptica
, 18 (20.9%) were
Elizabethkingia
miricola
, and 51 (59.3%) were
Elizabethkingia
anophelis
. Only the MALDI-TOF Vitek MS system with an amended database correctly identified all of the isolates. The majority (76.7%) of the isolates were from the lower respiratory tract, and 8 (9.3%) were from blood. Over 90% of
E. meningoseptica
and
E. anophelis
isolates were susceptible to piperacillin-tazobactam and rifampin. In contrast, all
E. miricola
isolates were susceptible to fluoroquinolones except ciprofloxacin. Further studies are urgently needed to determine the optimal antimicrobial agents for the treatment of infections due to each individual
Elizabethkingia
species.
Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages
Authors: Callaway Heather M. , Feng Kurtis H. , Lee Donald W. , Allison Andrew B. , Pinard Melissa , McKenna Robert , Agbandje-McKenna Mavis , Hafenstein Susan , Parrish Colin R.
Published: November 2016

Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids.

IMPORTANCE
Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.
A Highly Conserved Residue in HIV-1 Nef Alpha Helix 2 Modulates Protein Expression
Authors: Johnson Aaron L. , Dirk Brennan S. , Coutu Mathieu , Haeryfar S. M. Mansour , Arts Eric J. , Finzi Andrés , Dikeakos Jimmy D.
Published: November 2016

The HIV-1 Nef protein has been established as a key pathogenic determinant of HIV/AIDS, but there is little knowledge of how the extensive genetic diversity of HIV-1 affects Nef function. Upon compiling a set of subtype-specific reference strains, we identified a subtype C reference strain, C.BR92025, that contained natural polymorphisms at otherwise highly conserved residues 13, 84, and 92. Interestingly, strain C.BR92025 Nef displayed impaired Nef function and had decreased protein expression. We have demonstrated that strain C.BR92025 Nef has a higher rate of protein turnover than highly expressed Nef proteins and that this higher rate of protein turnover is due to an alanine-to-valine substitution at Nef residue 84. These findings highlight residue A84 as a major determinant of HIV-1 Nef expression.
Symptomatic adrenal suppression among children in Canada
Authors: Goldbloom Ellen B , Mokashi Arati , Cummings Elizabeth A , Abish Sharon , Benseler Susanne M , Huynh Hien Q , Watson Wade , Ahmet Alexandra
Published: November 2016

Background

Adrenal suppression (AS) is an under-recognised side effect of glucocorticoid (GC) use. AS may go undetected until a physiological stress precipitates an adrenal crisis. The incidence of AS has not been established. We sought to estimate the minimum national incidence and presenting features of paediatricsymptomaticAS.

Methods

Through the established methodology of the Canadian Paediatric Surveillance Program, over 2500 paediatricians were surveyed monthly for 2 years (April 2010–March 2012) to report new cases of symptomatic AS.

Results

Forty-six cases of symptomatic AS were confirmed. The estimated annual incidence is 0.35/100 000 children aged 0–18 years (95% CI 0.26 to 0.47). The most common presentations were growth failure (35%), non-specific symptoms (28%) or both (13%). Adrenal crisis occurred in six cases (13%). Thirty-seven children (80%) had received inhaled corticosteroid (ICS) alone or in combination with other GC forms. Many children received high but commonly prescribed doses of ICS.

Conclusions

AS is responsible for significant morbidity in children, including susceptibility to adrenal crisis. The minimal estimated incidence reported is for the entire paediatric population and would be much higher in the at-risk group (ie, children treated with GCs). Close monitoring of growth and possible symptoms of AS, which may be non-specific, are important in children on all forms of GC therapy including ICS. To reduce the risk of AS, physicians must be aware of the risk of AS, revisit GC doses frequently and use the lowest effective dose.