bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of nitric oxide from the two equivalent guanidino nitrogens of L-arginine by Lactobacillus fermentum
Authors: Morita H , Yoshikawa H , Sakata R , Nagata Y , Tanaka H
Published: November 2016

Ten strains of Lactobacillus fermentum that differed in origin converted metmyoglobin to nitrosylmyoglobin [a pentacoordinate nitric oxide (NO) complex of Fe(II) myoglobin] in MRS broth at pH 4.3. Of the 10 strains, L. fermentum IFO 3956 possessed the strongest capacity to convert metmyoglobin to nitrosylmyoglobin. This strain synthesizes NO enzymatically from the two equivalent guanidino nitrogens of L-arginine. To our knowledge, this demonstrates for the first time the production of NO synthesized from the guanidino nitrogens of L-arginine by lactic acid bacteria. IFO 3956 may possess a bacterial NO synthase.
Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1
Authors: Tomita H , Fujimoto S , Tanimoto K , Ike Y
Published: November 2016

The pheromone-responsive conjugative plasmid pPD1 (59 kb) of Enterococcus faecalis encodes the bacteriocin 21 (bac21) determinant. Cloning, transposon insertion mutagenesis and sequence analysis of the bac21 determinant showed that an 8.5-kb fragment lying between kb 27.1 and 35.6 of the pPD1 map is required for complete expression of the bacteriocin. The 8.5-kb fragment contained nine open reading frames (ORFs), bacA to bac1, which were oriented in the same (upstream-to-downstream) direction. Transposon insertions into the bacA to bacE ORFs, which are located in the proximal half of bac21, resulted in defective bacteriocin expression. Insertions into the bacF to bac1 ORFs, which are located in the distal half of bac21, resulted in reduced bacteriocin expression. Deletion mutant analysis of the cloned 8.5-kb fragment revealed that the deletion of segments between kb 31.6 and 35.6 of the pPD1 map, which contained the distal region of the determinant encoding bacF to bac1, resulted in reduced bacteriocin expression. The smallest fragment (4.5 kb) retaining some degree of bacteriocin expression contained the bacA to bacE sequences located in the proximal half of the determinant. The cloned fragment encoding the 4.5-kb proximal region and a Tn916 insertion mutant into pPD1 bacB trans-complemented intracellularly to give complete expression of the bacteriocin. bacA encoded a 105-residue sequence with a molecular mass of 11.1 kDa. The deduced BacA protein showed 100% homology to the broad-spectrum antibiotic peptide AS-48, which is encoded on the E. faecalis conjugative plasmid pMB2 (58 kb). bacH encoded a 195-residue sequence with a molecular mass of 21.9 kDa. The deduced amino acid sequence showed significant homology to the C-terminal region of HlyB (31.1% identical residues), a protein located in the Escherichia coli alpha-hemolysin operon that is a representative bacterial ATP-binding cassette export protein.
Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73
Authors: Luo Y , Glisson J R , Jackwood M W , Hancock R E , Bains M , Cheng I H , Wang C
Published: November 2016

The major outer membrane protein (OmpH) of Pasteurella multocida X-73 was purified by selective extraction with detergents, followed by size exclusion chromatography. The planar lipid bilayer assay showed that OmpH has pore-forming function. The average single channel conductance in 1.0 M KCl was 0.62 nS. The gene (ompH) encoding OmpH has been isolated and sequenced by construction of a genomic library and PCR techniques. The coding region of this gene is 1,059 bp long. The predicted primary protein is composed of 353 amino acids, with a 20-amino-acid signal peptide. The mature protein is composed of 333 amino acids with a molecular mass of 36.665 kDa. The ompH gene encoding mature protein has been expressed in Escherichia coli by using a regulatable expression system. The ompH gene was distributed among 15 P. multocida serotypes and strain CU. Protection studies showed that OmpH was able to induce homologous protection in chickens. These findings demonstrate that OmpH is a protective outer membrane porin of strain X-73 and is conserved among P. multocida somatic serotypes.
Secretion of nuclease across the outer membrane of Serratia marcescens and its energy requirements
Authors: Suh Y , Benedik M J
Published: November 2016

Extracellular secretion of Serratia marcescens nuclease occurs as a two-step process via a periplasmic intermediate. Unlike other extracellular proteins secreted by gram-negative bacteria by the general secretory pathway, nuclease accumulates in the periplasm in its active form for an unusually long time before its export into the growth medium. The energy requirements for extracellular secretion of nuclease from the periplasm were investigated. Our results suggest that the second step of secretion across the outer membrane is dependent upon the external pH; acidic pH effectively but reversibly blocks extracellular secretion. However, electrochemical proton gradient, and possibly ATP hydrolysis, are not required for this step. We suggest that nuclease uses a novel mechanism for the second step of secretion in S. marcescens.
Characterization of in-frame proteins encoded by cvaA, an essential gene in the colicin V secretion system: CvaA* stabilizes CvaA to enhance secretion
Authors: Hwang J , Manuvakhova M , Tai P C
Published: November 2016

Colicin V (ColV), an antibacterial peptide toxin, uses a dedicated signal sequence-independent export system for its extracellular secretion in Escherichia coli. The products of at least three genes (a chromosomal tolC gene and two plasmid-born cvaA and cvaB genes) are involved in this process. To characterize the gene products, the cvaA gene was subcloned and expressed under the control of T7 RNA polymerase promoter. Two in-frame proteins, CvaA and CvaA*, were expressed and identified. DNA sequences predicted that both proteins have two potential translational initiation sites. N-terminal peptide sequencing showed that the translation of CvaA starts from a TTG, 11 amino acids upstream of the previously proposed ATG initiation site. CvaA* is translated from an upstream ATG. Expression of both CvaA and CvaA* was induced by the iron chelator 2,2'-dipyridyl, indicating that cvaA is negatively regulated at least partially by Fur. CvaA*-depleted cells were found to secrete less ColV, based on reduced activity in the supernatant, than did wild type, which was recovered by the addition of a plasmid producing CvaA*. Interestingly, CvaA*-depleted and wild-type cells had similar levels of intracellular ColV activity. Translational fusions showed that the syntheses of ColV and CvaA are not affected by CvaA* depletion. However, CvaA in CvaA*-depleted cells was less stable than that in wild-type cells, indicating that CvaA* may directly or indirectly affect the stability of CvaA. We conclude that CvaA* is not essential for ColV secretion but that it enhances the ColV secretion by stabilizing the CvaA protein.
Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis
Authors: Martin G , Haehnel W , Böger P
Published: November 2016

In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.
Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of atropine by Pseudomonas sp. strain AT3
Authors: Long M T , Bartholomew B A , Smith M J , Trudgill P W , Hopper D J
Published: November 2016

Pseudomonas sp. strain AT3 grew with dl-tropic acid, the aromatic component of the alkaloid atropine, as the sole source of carbon and energy. Tropic acid-grown cells rapidly oxidized the growth substrate, phenylacetaldehyde, and phenylacetic acid. Crude cell extracts, prepared from dl-tropic acid-grown cells, contained two NAD+-linked dehydrogenases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenylacetaldehyde. Phenylacetaldehyde dehydrogenase was relatively unstable. The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatography. It had a pH optimum of 9.5 and was equally active with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation. The formation of phenylacetaldehyde from tropic acid requires, in addition to dehydrogenation, a decarboxylation step. By analogy with NAD+-specific isocitrate and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde. Other workers have established that isocitrate and malate dehydrogenases catalyze the decarboxylation of enzyme-bound or added 3-oxoacid intermediates, a reaction that requires Mn2+ or Mg2+ ions. Studies with tropic acid dehydrogenase were hampered by lack of availability of phenylmalonic semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we have not, by a number of approaches, found any evidence for the transient accumulation of phenylmalonic semialdehyde.
Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp
Authors: Fenno J C , Wong G W , Hannam P M , Müller K H , Leung W K , McBride B C
Published: November 2016

The major surface protein (Msp) of Treponema denticola has been implicated as a mediator of the interaction between the spirochete and the gingival epithelium in periodontal diseases. Previous studies showed that the Msp of T. denticola ATCC 35405 had porin activity, depolarized epithelial cell membranes, bound to extracellular matrix components of epithelial cells, and formed a regular hexagonal surface array in the treponemal outer membrane. The gene encoding Msp in ATCC 35405 was recently cloned, sequenced, and expressed in Escherichia coli (J. C. Fenno, K.-H. Müller, and B. C. McBride, J. Bacteriol. 178:2489-2496, 1996). In the present study, we identified genes encoding Msp-like proteins in several oral spirochetes. A prominent heat-modifiable Msp-like protein having an apparent molecular mass of between 43 and 64 kDa was present in all oral spirochete strains tested. Antibodies raised against the ATCC 35405 Msp reacted strongly with the Msp proteins of T. denticola ATCC 35404 and T. vincentii, reacted very weakly with the Msp protein of T. denticola ATCC 33520, and did not react with T. denticola OTK, T. socranskii, and T. pectinovorum. The msp loci of the T. denticola strains and T. vincentii were identified in analyses using PCR with oligonucleotide primers derived from the DNA sequence flanking msp in ATCC 35405. Southern blot analysis showed at least three groups of related msp DNA sequences. Comparison of DNA sequences of the 5' and 3' ends of the msp genes showed high sequence homology in the flanking regions and signal peptide coding regions, while the homologies between regions encoding the mature peptide were as low as 50%. The entire msp DNA sequences of T. denticola ATCC 33520 and OTK were determined, and the deduced Msp amino acid sequences were compared to the sequence of the previously reported Msp of ATCC 35405. The results show that the msp locus is conserved in oral treponemes but that there are significant differences between the mature Msp peptides of different strains. Further studies of the antigenic domains, functional domains, and physical structures of Msp proteins, based on these results, will enhance understanding of the role of Msp in the cytopathology associated with oral spirochetes.
A conserved glutamate residue, Glu-257, is important for substrate binding and transport by the Escherichia coli mannitol permease
Authors: Saraceni-Richards C A , Jacobson G R
Published: November 2016

The mannitol permease, or D-mannitol-specific enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, both transports and phosphorylates its substrate. Previous analyses of the amino acid sequences of PTS permeases specific for various carbohydrates in different species of bacteria revealed several regions of similarity. The most highly conserved region includes a GIXE motif, in which the glutamate residue is completely conserved among the permeases that contain this motif. The corresponding residue in the E. coli mannitol permease is Glu-257, which is located in a large putative cytoplasmic loop of the transmembrane domain of the protein. We used site-directed mutagenesis to investigate the role of Glu-257. The properties of proteins with mutations at position 257 suggest that a carboxylate side chain at this position is essential for mannitol binding. E257A and E257Q mutant proteins did not bind mannitol detectably, while the E257D mutant could still bind this substrate. Kinetic studies with the E257D mutant protein also showed that a glutamate residue at position 257 of this permease is specifically required for efficient mannitol transport. While the E257D permease phosphorylated mannitol with kinetic parameters similar to those of the wild-type protein, the Vmax for mannitol uptake by this mutant protein is less than 5% that of the wild type. These results suggest that Glu-257 of the mannitol permease and the corresponding glutamate residues of other PTS permeases play important roles both in binding the substrate and in transporting it through the membrane.
Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2
Authors: Dombek P , Ream W
Published: November 2016

The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2.
DNA-binding determinants of sigma 54 as deduced from libraries of mutations
Authors: Guo Y , Gralla J D
Published: November 2016

PCR mutagenesis was used to obtain libraries of mutations in the region between amino acids 300 and 400 in the DNA-binding domain of Escherichia coli sigma 54. Two hundred changes that did not alter function were identified. These were compared with a somewhat smaller number of changes that did alter function. Several important regions were identified. Single point mutations in two of these, near amino acids 363 and 383, destroyed the ability of sigma to bind DNA, as assayed by band shift analysis. A third segment from amino acids 327 to 347 is also a candidate for contributing to DNA binding. Comparison with data in the literature leads to testable proposals for the complex mode of DNA binding that is associated with sigma 54.
Translation of the mRNA for the sporulation gene spoIIID of Bacillus subtilis is dependent upon translation of a small upstream open reading frame
Authors: Decatur A , McMurry M T , Kunkel B N , Losick R
Published: November 2016

We report the existence of a small open reading frame (usd) that is located between the promoter and coding sequence for the sporulation gene spoIIID in Bacillus subtilis. The mRNA from the usd-spoIIID operon contains an inverted repeat sequence that is predicted to form a stem-loop structure that would sequester the ribosome binding site for spoIIID. A mutation eliminating the ribosome binding site for the upstream open reading frame caused an oligosporogenous phenotype and interfered with the translation, but not the transcription, of the downstream gene spoIIID. We propose that efficient synthesis of SpoIIID requires that the putative stem-loop structure be disrupted by translation through the upstream open reading frame.
Plasmid virulence gene expression induced by short-chain fatty acids in Salmonella dublin: identification of rpoS-dependent and rpo-S-independent mechanisms
Authors: El-Gedaily A , Paesold G , Chen C Y , Guiney D G , Krause M
Published: November 2016

The Salmonella plasmid virulence spvABCD genes are growth phase regulated and require RpoS for maximal expression in stationary phase. We identified a growth phase-independent expression of spv which is mediated by short-chain fatty acids. During this fatty acid-mediated expression of spv, RpoS is required for induction only during exponential phase. In stationary phase, an rpoS-independent mechanism is responsible for expression of spv.
The inactivated plasmid inititator protein RepC/RepC* may have a regulatory role
Authors: Rasooly A , Rasooly R S
Published: November 2016

During replication of the plasmid pT181, the initiator protein RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. Here we show that during in vitro replication, RepC* is radioactively labeled, suggesting that the source of the RepC* oligodeoxynucleotide is the newly synthesized pT181 DNA. The RepC/RepC* heterodimer retains its ability to bind the pT181 double-strand origin and, therefore, it may act as a competitive inhibitor of the RepC homodimer during replication.
Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon
Authors: Peng H L , Yang Y H , Deng W L , Chang H Y
Published: November 2016

By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli. The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon. This gene, designated acoK, is capable of encoding a protein with an overall 58.4% amino acid identity with MalT, the transcriptional activator of the E. coli maltose regulon. A conserved sequence for nucleotide binding at the N-terminal region, as well as a helix-turn-helix motif belonging to the LuxR family of transcriptional regulators at the C terminus, was also identified. Primer extension analysis identified two transcription initiation sites, S1 and S2, located 319 and 267 bp, respectively, upstream of the putative start codon of acoK. Several copies of NtrC recognition sequence [CAC-(N11 to N18)-GTG] were found in the promoter regions of both the acoK gene and the acoABCD operon. Acetoin-dependent expression of the acoABCD operon could be restored in the E. coli acoK mutants by supplying a plasmid carrying an intact acoK, suggesting a transactivating function of the gene product. The AcoK protein overproduced in E. coli was approximately 100 kDa, which is in good agreement with the molecular mass deduced from the nucleotide sequence. A specific DNA binding property and an ATPase activity of the purified AcoK were also demonstrated.
Characterization of UDP amino sugars as major phosphocompounds in the hyperthermophilic archaeon Pyrococcus furiosus
Authors: Ramakrishnan V , Teng Q , Adams M W
Published: November 2016

The archaeon Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally at 100 degrees C by the fermentation of carbohydrates. It is known to contain high concentrations of novel intracellular solutes such as beta-mannosylglycerate and di-myo-inositol 1,1'-phosphate (DIP) (L. O. Martins and H. Santos, Appl. Environ. Microbiol. 61:3299-3303, 1995). Here, 31P nuclear magnetic resonance (NMR) spectroscopy was used to show that this organism also accumulates another type of phospho compound, as revealed by a major multiplet signal in the pyrophosphate region. The compounds were purified from cell extracts of P. furiosus by anion-exchange and gel filtration chromatographic procedures and were structurally analyzed by 1H, 13C, and 31P NMR spectroscopy. They were identified as two uridylated amino sugars, UDP N-acetylglucosamine and UDP N-acetylgalactosamine. Unambiguous characterizations and complete assignments of 1H and 13C resonances from such sugars have not been previously reported. In vitro 31P NMR spectroscopic analyses showed that, in contrast to DIP, which is maintained at a constant intracellular concentration (approximately 32 mM) throughout the growth phase of P. furiosus, the UDP amino sugars accumulated (to approximately 14 mM) only during the late log phase. The possible biochemical roles of these compounds in P. furiosus are discussed.
A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes
Authors: Ripio M T , Domínguez-Bernal G , Lara M , Suárez M , Vazquez-Boland J A
Published: November 2016

Virulence genes in Listeria monocytogenes are coordinately expressed under the control of the transcriptional activator PrfA, encoded by prfA, a member of the cyclic AMP (cAMP) receptor protein (CRP)/FNR family of bacterial regulators. Strain P14-A is a spontaneous mutant of L. monocytogenes serovar 4b which produces elevated levels of virulence factors (M. T. Ripio, G. Domínguez-Bernal, M. Suárez, K. Brehm, P. Berche, and J. A. Vázquez-Boland, Res. Microbiol. 147:371-384, 1996). Here we report that P14-A and other variant strains with the same phenotype carry a point mutation in codon 145 of prfA, leading to a Gly-->Ser substitution. trans-complementation experiments with PrfA-deficient mutants demonstrated that the Gly145Ser prfA allele causes overexpression of virulence factors in L. monocytogenes, to the levels found in the virulence factor-overexpressing variants. In strain P14-A with a chromosomal Glyl45Ser prfA background, transcription of prfA and of PrfA-dependent virulence genes remained constitutively high under culture conditions in which virulence factor expression is downregulated in wild-type L. monocytogenes. The Glyl45Ser substitution is located in a PrfA stretch (residues 141 to 151) showing high sequence similarity to the D alpha-helix of CRP. Interestingly, well-characterized crp* mutations, which make CRP functionally active in the absence of cAMP, map in this region (i.e., Gly141Ser and Ala144Thr substitutions). By analogy with the CRP model, the phenotype conferred to L. monocytogenes by the Gly145Ser substitution in PrfA could be due to the mutant regulatory protein being locked in a transcriptionally active, cofactor-independent conformational state. Our observations allow the construction of a model for PrfA-dependent virulence gene regulation in which the levels of virulence factor expression depend primarily on the conformational state of the PrfA protein, which alternates between active and inactive forms according to its interaction with an environmentally regulated signal molecule or cofactor.
Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae
Authors: Murley Y M , Edlind T D , Pozsgay J M , LiPuma J J
Published: November 2016

The bacteriocin haemocin is produced by most type b strains of Haemophilus influenzae, including strains of diverse genetic lineage, and is toxic to virtually all nontypeable H. influenzae strains. An H. influenzae transformant bearing a plasmid with a 1.5-kbp chromosomal fragment capable of conferring haemocin immunity on a haemocin-susceptible H. influenzae mutant was selected by using partially purified haemocin. Deletional and site-directed mutagenesis localized the haemocin immunity gene to the 3' open reading frame (ORF) within this chromosomal fragment. Subcloning of this ORF demonstrated that it was sufficient to confer haemocin immunity on wild-type haemocin-susceptible H. influenzae strains as well as haemocin-susceptible strains of Escherichia coli. This ORF, designated hmcl, encodes a 105-amino-acid protein with an estimated molecular mass of 12.6 kDa. Primer extension analysis revealed a putative transcriptional start site 34 bp upstream of the start codon, and the presence of a promoter immediately upstream of hmcI was confirmed by cloning the gene into a promoterless chloramphenicol acetyltransferase vector. To characterize the hmcI gene product, a His-HmcI fusion protein was constructed.
In vivo evidence that acyl coenzyme A regulates DNA binding by the Escherichia coli FadR global transcription factor
Authors: Cronan J E
Published: November 2016

In vitro experiments point to fatty acyl coenzymes A (acyl-CoAs) rather than unesterified fatty acids as the small-molecule ligands regulating DNA binding by the FadR protein of Escherichia coli. To provide an in vivo test of this specificity, unesterified fatty acids were generated within the cellular cytosol. These fatty acids were found to be efficient modulators of FadR action only when the acids could be converted to acyl-CoAs.
Isolation and characterization of a molecular chaperone, gp57A, of bacteriophage T4
Authors: Matsui T , Griniuviené B , Goldberg E , Tsugita A , Tanaka N , Arisaka F
Published: November 2016

A molecular chaperone of bacteriophage T4, gp57A, which facilitates the formation of the long and short tail fibers, was isolated and characterized by peptide analysis, sedimentation equilibrium, and circular dichroism (CD). Sequence analysis confirmed the predicted sequence of 79 amino acids from the nucleotide sequence of the gene with the N-terminal methionine removed. The result led to the conclusion that the apparent smaller molecular weight of 6,000 from Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the expected molecular weight of 8,710 was due to its abnormal electrophoretic behavior instead of cleavage or processing of the gene product. Estimation of the secondary structure from far-UV CD indicated a 94% alpha-helix content, which was in accord with the prediction from the primary structure. A sedimentation equilibrium study, on the other hand, revealed that gp57A assumes a tetrameric subunit structure.
UV-B-induced synthesis of photoprotective pigments and extracellular polysaccharides in the terrestrial cyanobacterium Nostoc commune
Authors: Ehling-Schulz M , Bilger W , Scherer S
Published: November 2016

Liquid cultures of the terrestrial cyanobacterium Nostoc commune derived from field material were treated with artificial UV-B and UV-A irradiation. We studied the induction of various pigments which are though to provide protection against damaging UV-B irradiation. First, UV-B irradiation induced an increase in carotenoids, especially echinenone and myxoxanthophyll, but did not influence production of chlorophyll a. Second, an increase of an extracellular, water-soluble UV-A/B-absorbing mycosporine occurred, which was associated with extracellular glycan synthesis. Finally, synthesis of scytonemin, a lipid-soluble, extracellular pigment known to function as a UV-A sunscreen, was observed. After long-time exposure, the UV-B effect on carotenoid and scytonemin synthesis ceased whereas the mycosporine content remained constantly high. The UV-B sunscreen mycosporine is exclusively induced by UV-B (< 315 nm). The UV-A sunscreen scytonemin is induced only slightly by UV-B (< 315 nm), very strongly by near UV-A (350 to 400 nm), and not at all by far UV-A (320 to 350 nm). These results may indicate that the syntheses of these UV sunscreens are triggered by different UV photoreceptors.
Transposition without transposase: a spontaneous mutation in bacteria
Authors: Rappleye C A , Roth J R
Published: November 2016

Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.
Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors
Authors: Tardy F , Nasser W , Robert-Baudouy J , Hugouvieux-Cotte-Pattat N
Published: November 2016

In Erwinia chrysanthemi 3937, pectate lyase activity mainly results from the cumulative action of five major isoenzymes, PelA to PelE. Comparison of their amino acid sequences revealed two families, PelB-C and PelA-D-E. Molecular cloning permitted expression of the different pel genes in Escherichia coli and the isolation of each Pel independently from the other isoenzymes. We used similar experimental conditions to overproduce and purify the five Pels in a one-step chromatography method. We analyzed some of the basic enzymatic properties of these five isoenzymes. PelA has a low specific activity compared to the other four enzymes. PelB and PelC have a high affinity for their substrate: about 10-fold higher than the enzymes of the PelA-D-E group. The optimum pH is more alkaline for PelB and PelC (about 9.2) than for PelA, PelD, and PelE (from 8 to 8.8). Below pH 7, activity was negligible for PelB and PelC, while PelA, PelD, and PelE retained 25 to 30% of their activities. The temperature optima were determined to be 50 degrees C for PelD and PelE, 55 degrees C for PelA, and 60 degrees C for PelB and PelC. Enzymes of the PelB-C group are more stable than those of the PelA-D-E group. Use of substrates presenting various degrees of methylation revealed that PelA, PelD, and PelE are active only for very low levels of methylation, while PelB and PelC are more active on partially methylated pectins (up to 22% for PelC and up to 45% for PelB). Pectate lyases have an absolute requirement for Ca2+ ions. For the five isoenzymes, maximal activity was obtained at a Ca2+ concentration of 0.1 mM. None of the tested cations (Ba2+, Co2+, Cu2+, Mg2+, Mn2+, Sr2+, Zn2+) can substitute for Ca2+. At a high concentration (1 mM), most of the divalent cations inhibited pectate lyase activity. In addition, we demonstrated that two compounds present in plant tissues, epicatechin and salicylic acid, inhibit the pectate lyases at a concentration of 0.2 mM.
Molecular cloning and characterization of the genes encoding the L1 and L2 components of hemolysin BL from Bacillus cereus
Authors: Ryan P A , Macmillan J D , Zilinskas B A
Published: November 2016

Hemolysin BL, which is composed of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal food poisoning syndrome caused by strains of Bacillus cereus. To further characterize the toxin, we sought to clone and sequence the genes encoding the L1 and L2 proteins. A genomic library was screened with polyclonal antibody to the L1 and L2 proteins to identify recombinant clones containing the genes. Five clones reacted with the antibody to L2, but none reacted with the antibody to L1. Southern hybridization analysis with oligonucleotide probes designed from the N-terminal amino acid sequences of the L1 and L2 proteins, in conjunction with immunoblot and nucleotide sequence analysis, revealed that the recombinant plasmid from one of the clones contained two genes, hblC and hblD, which encode L2 and L1, respectively. The two genes are arranged in tandem and are separated by only 37 bases. The gene which encodes the B component of hemolysin BL (hblA) is located immediately downstream from the gene encoding the L1 protein. Northern blot analysis of B. cereus RNA showed a 5.5-kb transcript which hybridized with DNA fragments internal to, or including a portion of, the coding sequences of the B, L1, and L2 genes, suggesting that the clustered genes which encode the components of hemolysin BL are cotranscribed and constitute an operon.
Characterization of the stringent and relaxed responses of Streptococcus equisimilis
Authors: Mechold U , Malke H
Published: November 2016

The 739-codon rel(Seq) gene of Streptococcus equisimilis H46A is bifunctional, encoding a strong guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) and a weaker ribosome-independent ATP:GTP 3'-pyrophosphoryltransferase [(p)ppGpp synthetase]. To analyze the function of this gene, (p)ppGpp accumulation patterns as well as protein and RNA synthesis were compared during amino acid deprivation and glucose exhaustion between the wild type and an insertion mutant carrying a rel(Seq) gene disrupted at codon 216. We found that under normal conditions, both strains contained basal levels of (p)ppGpp. Amino acid deprivation imposed by pseudomonic acid or isoleucine hydroxamate triggered a rel(Seq)-dependent stringent response characterized by rapid (p)ppGpp accumulation at the expense of GTP and abrupt cessation of net RNA accumulation in the wild type but not in the mutant. Tetracycline added to block (p)ppGpp synthesis caused the accumulated (p)ppGpp to degrade rapidly, with a concomitant increase of the GTP pool (decay constant of ppGpp, approximately 0.7 min(-1)). Simultaneous addition of pseudomonic acid and tetracycline to mimic a relaxed response caused wild-type RNA synthesis to proceed at rates approximating those seen under either condition in the mutant. Glucose exhaustion provoked the (p)ppGpp accumulation response in both the wild type and the rel(Seq) insertion mutant, consistent with the block of net RNA accumulation in both strains. Although the source of (p)ppGpp synthesis during glucose exhaustion remains to be determined, these findings reinforce the idea entertained previously that rel(Seq) fulfils functions that reside separately in the paralogous reL4 and spoT genes of Escherichia coli. Analysis of (p)ppGpp accumulation patterns was complicated by finding an unknown phosphorylated compound that comigrated with ppGpp under two standard thin-layer chromatography conditions. Unlike ppGpp, this compound did not adsorb to charcoal and did not accumulate appreciably during isoleucine deprivation. Like ppGpp, the unknown compound did accumulate during energy source starvation.
Transposon mutagenesis reinforces the correlation between Mycoplasma pneumoniae cytoskeletal protein HMW2 and cytadherence
Authors: Krause D C , Proft T , Hedreyda C T , Hilbert H , Plagens H , Herrmann R
Published: November 2016

A new genetic locus associated with Mycoplasma pneumoniae cytadherence was previously identified by transposon mutagenesis with Tn4001. This locus maps approximately 160 kbp from the genes encoding cytadherence-associated proteins HMW1 and HMW3, and yet insertions therein result in loss of these proteins and a hemadsorption-negative (HA-) phenotype, prompting the designation cytadherence-regulatory locus (crl). In the current study, passage of transformants in the absence of antibiotic selection resulted in loss of the transposon, a wild-type protein profile, and a HA+ phenotype, underscoring the correlation between crl and M. pneumoniae cytadherence. Nucleotide sequence analysis of crl revealed open reading frames (ORFs) orfp65, orfp216, orfp41, and orfp24, arranged in tandem and flanked by a promoter-like and a terminator-like sequence, suggesting a single transcriptional unit, the P65 operon. The 5' end of orfp65 mRNA was mapped by primer extension, and a likely promoter was identified just upstream. The product of each ORF was identified by using antisera prepared against fusion proteins. The previously characterized surface protein P65 is encoded by orfp65, while the 190,000 Mr cytadherence-associated protein HMW2 is a product of orfp216. Proteins with sizes of 47,000 and 41,000 Mr and unknown function were identified for orfp41 and orfp24, respectively. Structural analyses of HMW2 predict a periodicity highly characteristic of a coiled-coil conformation and five leucine zipper motifs, indicating that HMW2 probably forms dimers in vivo, which is consistent with a structural role in cytadherence. Each transposon insertion mapped to orfp216 but affected the levels of all products of the P65 operon. HMW2 is thought to form a disulfide-linked dimer, formerly designated HMW5, and examination of an hmw2 deletion mutant confirms that HMW5 is a product of the hmw2 gene.
Transcription of glutamine synthetase genes (glnA and glnN) from the cyanobacterium Synechocystis sp. strain PCC 6803 is differently regulated in response to nitrogen availability
Authors: Reyes J C , Muro-Pastor M I , Florencio F J
Published: November 2016

In the cyanobacterium Synechocystis sp. strain PCC 6803 we have previously reported the presence of two different proteins with glutamine synthetase activity: GSI, encoded by the glnA gene, and GSIII, encoded by the glnN gene. In this work we show that expression of both the glnA and glnN genes is subjected to transcriptional regulation in response to changes in nitrogen availability. Northern blot experiments and transcriptional fusions demonstrated that the glnA gene is highly transcribed in nitrate- or ammonium-grown cells and exhibits two- to fourfold-higher expression in nitrogen-starved cells. In contrast, the glnN gene is highly expressed only under nitrogen deficiency. Half-lives of both mRNAs, calculated after addition of rifampin or ammonium to nitrogen-starved cells, were not significantly different (2.5 or 3.4 min, respectively, for glnA mRNA; 1.9 or 1.4 min, respectively, for glnN mRNA), suggesting that changes in transcript stability are not involved in the regulation of the expression of both genes. Deletions of the glnA and glnN upstream regions were used to delimit the promoter and the regulatory sequences of both genes. Primer extension analysis showed that structure of the glnA gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified, Escherichia coli-expressed Synechocystis NtcA protein binds to the promoter of the glnA gene. Primer extension also revealed the existence of a sequence related to the NtcA binding site upstream from the glnN promoter. However, E. coli-expressed NtcA failed to bind to this site. These findings suggest that an additional modification of NtcA or an additional factor is required for the regulation of glnN gene expression.
Coactivation in vitro of the sigma54-dependent promoter Pu of the TOL plasmid of Pseudomonas putida by HU and the mammalian HMG-1 protein
Authors: Pérez-Martín J , De Lorenzo V
Published: November 2016

The mechanism by which the prokaryotic histone-like protein HU replaces the integration host factor (IHF) in the coactivation of the sigma54-dependent promoter Pu of Pseudomonas putida has been investigated. By using a preactivated form of the cognate activator protein XylR, we show that the functional replacement of IHF with HU previously suggested in vivo can be faithfully reproduced in vitro with purified components. Furthermore, the coactivation effect of IHF on Pu could be mimicked not only by HU but also by the mammalian nonhistone chromatin protein HMG-1 and could be bypassed by intrinsically curved DNA. These results suggest that either of two different mechanisms (generation of a site-specific static DNA bend or a general flexibilization of the promoter region) gives rise to the same structural effect of stimulating transcription from Pu through changes in promoter architecture.
An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity
Authors: Zanin Mark , Duan Susu , Wong Sook-San , Kumar Gyanendra , Baviskar Pradyumna , Collin Emily , Russell Charles , Barman Subrata , Hause Benjamin , Webby Richard
Published: November 2016

Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1
low
-1) and A/swine/Virginia/1814-2/2012 (pH1N1
low
-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1
low
-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1
low
-1 and pH1N1
low
-2. pH1N1
low
-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1
low
-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

IMPORTANCE
Neuraminidase (NA) is a sialidase that is one of the major surface glycoproteins of influenza A viruses and the target for the influenza drugs oseltamivir and zanamivir. NA is important as it releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. Mutations in the globular head domain that decrease enzymatic activity but confer resistance to NA inhibitors have been characterized; however, the importance of specific mutations in the stalk domain is unknown. We identified 66Y (N1 numbering), a highly conserved amino acid that was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.
Replacement of Porcine CD163 Scavenger Receptor Cysteine-Rich Domain 5 with a CD163-Like Homolog Confers Resistance of Pigs to Genotype 1 but Not Genotype 2 Porcine Reproductive and Respiratory Syndrome Virus
Authors: Wells Kevin D. , Bardot Rachel , Whitworth Kristin M. , Trible Benjamin R. , Fang Ying , Mileham Alan , Kerrigan Maureen A. , Samuel Melissa S. , Prather Randall S. , Rowland Raymond R. R.
Published: November 2016

CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection ofCD163-modified pigs with representative type 1 and type 2 viruses confirmed thein vitroresults. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163.

IMPORTANCEGenetic modification of theCD163gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification ofCD163with the goal of making pigs refractory to infection while retaining important CD163 functions.