bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmids of Distinct IncK Lineages Show Compatible Phenotypes
Authors: Rozwandowicz Marta , Brouwer Michael S. M. , Zomer Aldert L. , Bossers Alex , Harders Frank , Mevius Dik J. , Wagenaar Jaap A. , Hordijk Joost
Published: January 2017

IncK plasmids are some of the main carriers of
bla
CTX-M-14
and
bla
CMY-2
genes and show high similarity to other plasmids belonging to the I complex, including IncB/O plasmids. Here, we studied the phylogenetic relationship of 37 newly sequenced IncK and IncB/O plasmids. We show that IncK plasmids can be divided into two compatible lineages named IncK1 and IncK2.
Human Three-Dimensional Endometrial Epithelial Cell Model To Study Host Interactions with Vaginal Bacteria and Neisseria gonorrhoeae
Authors: Łaniewski Paweł , Gomez Adriana , Hire Geoffrey , So Magdalene , Herbst-Kralovetz Melissa M.
Published: January 2017

Colonization of the endometrium by pathogenic bacteria ascending from the lower female reproductive tract (FRT) is associated with many gynecologic and obstetric health complications. To study these host-microbe interactions
in vitro
, we developed a human three-dimensional (3-D) endometrial epithelial cell (EEC) model using the HEC-1A cell line and the rotating wall vessel (RWV) bioreactor technology. Our model, composed of 3-D EEC aggregates, recapitulates several functional/structural characteristics of human endometrial epithelial tissue, including cell differentiation, the presence of junctional complexes/desmosomes and microvilli, and the production of membrane-associated mucins and Toll-like receptors (TLRs). TLR function was evaluated by exposing the EEC aggregates to viral and bacterial products. Treatment with poly(I·C) and flagellin but not with synthetic lipoprotein (fibroblast-stimulating lipoprotein 1 [FSL-1]) or lipopolysaccharide (LPS) significantly induced proinflammatory mediators in a dose-dependent manner. To simulate ascending infection, we infected EEC aggregates with commensal and pathogenic bacteria:
Lactobacillus crispatus
,
Gardnerella vaginalis
, and
Neisseria gonorrhoeae
. All vaginal microbiota and
N. gonorrhoeae
efficiently colonized the 3-D surface, localizing to crevices of the EEC model and interacting with multiple adjacent cells simultaneously. However, only infection with pathogenic
N. gonorrhoeae
and not infection with the other bacteria tested significantly induced proinflammatory mediators and significant ultrastructural changes to the host cells. The latter observation is consistent with clinical findings and illustrated the functional specificity of our system. Additionally, we highlighted the utility of the 3-D EEC model for the study of the pathogenesis of
N. gonorrhoeae
using a well-characterized Δ
pilT
mutant. Overall, this study demonstrates that the human 3-D EEC model is a robust tool for studying host-microbe interactions and bacterial pathogenesis in the upper FRT.
The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria
Authors: Stahl D A , Urbance J W
Published: January 2017

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationships among selected species of mycobacteria and related organisms. The phylogeny inferred reflects the traditional classification, with major branches of the phylogenetic tree in general correspondence to the four Runyon groups and with numerical classification analyses. All the mycobacterial species compared, with the exception of M. chitae, are closely related (average similarity values greater than 95%). The slow growers form a coherent line of descent, distinct from the rapid growers, within which the overt pathogens are clustered. The distant relationship between M. chitae and the remaining mycobacteria suggests that this organism is incorrectly classified with the mycobacteria. M. paratuberculosis 18 was indistinguishable from M. avium-M. intracellulare-M. scrofulaceum serovar 1 by this analysis.
recB recC-dependent processing of heteroduplex DNA stimulates recombination of an adjacent gene in Escherichia coli
Authors: Kraczkiewicz-Dowjat A , Fishel R
Published: January 2017

The effect of DNA mismatched repair on the genetic recombination of a gene adjacent to the mismatch site (MS) was tested by using four mismatch configurations. An MS was constructed in a well-characterized plasmid recombination substrate, and recombination with a resident compatible plasmid was measured after transformation of the mismatched plasmid into Escherichia coli. The mismatched plasmids were constructed such that one of the DNA strands was methylated by the DNA adenine methylase (Dam), while the other strand was unmethylated. The processing of a hemimethylated single-base-pair mismatch had no effect on the recombination of the adjacent gene, suggesting that the most efficient (Dam-instructed) mismatch repair process does not secondarily promote genetic recombination. However, mismatches that could form an ordered secondary structure resembling a cruciform increased the recombination of this adjacent gene at least 20-fold. An identical mismatch that could not form an ordered secondary structure had no effect in this system. The increased frequency of recombination observed was found to require the recB or recC gene product or both. Furthermore, the recombination appeared unidirectional, in that the cruciform-containing plasmid did not produce stable transformants. Our results support a model in which the cruciform-containing plasmid can participate in recombination with the resident plasmid but is unable to produce stable transformant progeny. A proposed role for the RecBCD enzyme (ExoV) in this process is discussed.
Conservation of a dual-start motif in P22 lysis gene regulation
Authors: Nam K , Bläsi U , Zagotta M T , Young R
Published: January 2017

Gene 13 of bacteriophage P22 is functionally equivalent to lambda lysis gene S. Gene S codes for two products, the polypeptides S105 and S107, produced from translational initiation events at the third and first codon, respectively. We have shown that the two polypeptides have opposing functions in lysis: S105 is the lethal lysis effector, and S107 acts as an inhibitor of lysis (U. Bläsi, K. Nam, D. Hartz, L. Gold, and R. Young, EMBO J. 11:3501-3510, 1989). Gene 13 has a 108-codon reading frame and its product begins with a similar motif: Met-1-Lys-2-Lys-3-Met-4. Here, we present in vivo and in vitro evidence for the expression of a 13(108) and a 13(105) product and show that the lambda lysis control mechanisms is evolutionarily conserved in phage P22. In this case 13(108), like S107 in lambda, functions as the inhibitor of the lysis effector 13(105). Although the DNA sequences upstream of the S and 13 gene starts showed less homology, the same structural characteristics, i.e., stem-loop structures immediately upstream and about 10 codons downstream of the start region, were present in both reading frames. Using in vitro mutagenesis and toeprinting, we show that the upstream stem-loop structures of genes 13 and S, containing the Shine-Dalgarno sequence for initiations at Met-1, are interchangeable. Moreover, our data indicate that the stability of the secondary structures present in the translational initiation regions of genes S and 13 is set to create a particular ratio of initiation events at Met-1 and Met-3 or Met-4. The ratio of effector to inhibitor was much higher in P22 than in lambda. We propose that this reflects less transcriptional readthrough at the late terminator t(R) and suggests that the dual-start motif in genes 13 and S may be important for establishment of maintenance of the lysogenic state.
Aspartokinase III, a new isozyme in Bacillus subtilis 168
Authors: Graves L M , Switzer R L
Published: January 2017

A previously undetected Bacillus subtilis aspartokinase isozyme, which we have called aspartokinase III, has been characterized. The new isozyme was most readily detected in extracts of cells grown with lysine, which repressed aspartokinase II and induced aspartokinase III, or in extracts of strain VS11, a mutant lacking aspartokinase II. Antibodies against aspartokinase II did not cross-react with aspartokinase III. Aspartokinases II and III coeluted on gel filtration chromatography at Mr 120,000, which accounts for the previous inability to detect it. Aspartokinase III was induced by lysine and repressed by threonine. It was synergistically inhibited by lysine and threonine. Aspartokinase III activity, like aspartokinase II activity, declined rapidly in B. subtilis cells that were starved for glucose. In contrast, the specific activity of aspartokinase I, the diaminopimelic acid-inhibitable isozyme, was constant under all growth conditions examined.
The Myxococcus xanthus FprA protein causes increased flavin biosynthesis in Escherichia coli
Authors: Shimkets L J
Published: January 2017

The fprA gene is immediately adjacent to the csgA gene (formerly known as spoC) of Myxococcus xanthus. Whereas the csgA gene has an essential role in cell interactions during the developmental cycle, the function of the fprA gene is unknown. Gene disruption was used to determine what affect a null mutation in this gene has on the phenotype of the cell. A csgA-fprA deletion and an fprA frameshift mutation were constructed in vitro in a cloned copy of this locus and then inserted into the M. xanthus chromosome to create a merodiploid with the wild-type and mutant alleles in tandem. The merodiploid was then allowed to segregate one of the two alleles along with the vector sequences in an effort to replace the wild-type allele with the mutant allele. All of the segregants had the wild-type allele, suggesting that a functional fprA gene is essential for vegetative growth. The fprA gene was placed under control of the lacZ transcriptional and translational signals and overexpressed in Escherichia coli, and the new host was examined for any phenotypic changes. A 27-kilodalton protein was observed in sodium dodecyl sulfate-polyacrylamide gels of total-cell protein as predicted from the DNA sequence of this gene. Overexpression of FprA caused the accumulation of a yellow pigment with spectral and redox properties similar to that of the flavins. The pigment cochromatographed with flavin mononucleotide by Silica Gel G thin-layer chromatography. Approximately two-thirds of the total cellular flavin was associated with soluble protein. The major soluble flavin-associated protein was purified on DEAE-Bio-Gel A and Phenyl-Sepharose CL-4B and by polyacrylamide gel electrophoresis. The amino acid composition of the purified protein was similar to that predicted from the DNA sequence of the FprA fusion protein. Apparently, overproduction of FprA (for flavin-associated protein A) in E. coli resulted in a large increase in flavin biosynthesis. Together, these results suggest that the fprA gene encodes a protein that is associated with flavin mononucleotide and has an essential function in M. xanthus.
Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes
Authors: Dietrichs D , Andreesen J R
Published: January 2017

Three different dihydrolipoamide dehydrogenases were purified to homogenity from the anaerobic glycine-utilizing bacteria Clostridium cylindrosporum, Clostridium sporogenes, and Peptostreptococcus glycinophilus, and their basic properties were determined. The enzyme isolated from P. glycinophilus showed the properties typical of dihydrolipoamide dehydrogenases: it was a dimer with a subunit molecular mass of 53,000 and contained 1 mol of flavin adenine dinucleotide and 2 redox-active sulfhydryl groups per subunit. Only NADH was active as a coenzyme for reduction of lipoamide. Spectra of the oxidized enzyme exhibited maxima at 230, 270, 353, and 453 nm, with shoulders at 370, 425, and 485 nm. The dihydrolipoamide dehydrogenases of C. cylindrosporum and C. sporogenes were very similar in their structural properties to the enzyme of P. glycinophilus except for their coenzyme specificity. The enzyme of C. cylindrosporum used NAD(H) as well as NADP(H), whereas the enzyme of C. sporogenes reacted only with NADP(H), and no reaction could be detected with NAD(H). Antibodies raised against the dihydrolipoamide dehydrogenase of C. cylindrosporum reacted with extracts of Clostridium acidiurici, Clostridium purinolyticum, and Eubacterium angustum, whereas antibodies raised against the enzymes of P. glycinophilus and C. sporogenes showed no cross-reaction with extracts from 42 organisms tested.
Chorismic acid, a key metabolite in modification of tRNA
Authors: Hagervall T G , Jönsson Y H , Edmonds C G , McCloskey J A , Björk G R
Published: January 2017

Chorismic acid is the common precursor for the biosynthesis of the three aromatic amino acids as well as for four vitamins. Mutants of Escherichia coli defective in any of the genes involved in the synthesis of chorismic acid are also unable to synthesize uridine 5-oxyacetic acid (cmo5U) and its methyl ester (mcmo5U). Both modified nucleosides are normally present in the wobble position of some tRNA species. Mutants defective in any of the specific pathways leading to phenylalanine, tyrosine, tryptophan, folate, enterochelin, ubiquinone, and menaquinone have normal levels of cmo5U and mcmo5U in their tRNA. The presence of shikimic acid in the growth medium restores the ability of an aroD mutant to synthesize cmo5U, while O-succinylbenzoate, which is an early intermediate in the synthesis of menaquinone, does not. Thus, chorismic acid is a key metabolite in the synthesis of these two modified nucleosides in tRNA. The absence of chorismic acid blocks the formation of cmo5U and mcmo5U at the first step, which might be the formation of 5-hydroxyuridine. This results in an unmodified U in the wobble position of tRNA(1Val) and in most of the tRNAs normally containing cmo5U and mcmo5U. Since cmo5U and mcmo5U are synthesized under anaerobic conditions, the formation of these nucleosides does not require molecular oxygen. One of the carbon atoms of the side chain, --O--CH2--COOH, originates from the methyl group of methionine. The other carbon atom does not originate directly from the C-1 pool, from the carboxyl group methionine, or from bicarbonate. This metabolic link between intermediary metabolism and translation also exists for another member of the family Enterobacteriaceae, Salmonella typhimurium, as well as for the distantly related gram-positive organism Bacillus subtilis.
Positioning of replicated chromosomes in Escherichia coli
Authors: Hiraga S , Ogura T , Niki H , Ichinose C , Mori H
Published: January 2017

The positioning of replicated chromosomes at one-fourth and three-fourths of the cell length was inhibited when protein synthesis was inhibited by chloramphenicol or rifampin or by starvation for amino acids. Under these conditions, the progress of chromosome replication continued and replicated chromosomes were located close to each other as one nucleoid mass at midcell. Cells which already had two separate daughter chromosomes located at the cell quarters divided into two daughter cells under these conditions. When protein synthesis resumed, daughter chromosomes moved from midcell to the cell quarters, respectively, before any detectable increase in cell length was observed. The chromosome positioning occurred even under inhibition of the initiation of chromosome replication and under inactivation of DNA gyrase. The chromosome positioning presumably requires new synthesis of a particular protein(s) or translation itself.
Sequence analysis of the inversion region containing the pilin genes of Moraxella bovis
Authors: Fulks K A , Marrs C F , Stevens S P , Green M R
Published: January 2017

Moraxella bovis EPP63 is able to produce two antigenically distinct pili called Q and I pili (previously called beta and alpha pili). Hybridization studies have shown that the transition between the types is due to inversion of a 2.1-kilobase segment of chromosomal DNA. We present the sequence of a 4.1-kilobase region of cloned DNA spanning the entire inversion region in orientation 1 (Q pilin expressed). Comparison of this sequence with the sequence of the polymerase chain reaction-amplified genomic DNA from orientation 2 (I pilin expressed) allows the site-specific region of recombination to be localized to a 26-base-pair region in which sequence similarity to the left inverted repeat of the Salmonella typhimurium hin system was previously noted. In addition, 50% sequence similarity was seen in a 60-base-pair segment of our sequence to the recombinational enhancer of bacteriophage P1, an inversion system related to the hin system of S. typhimurium. Finally, two open reading frames representing potential genes were identified.
Cu,Zn superoxide dismutase and copper deprivation and toxicity in Saccharomyces cerevisiae
Authors: Greco M A , Hrab D I , Magner W , Kosman D J
Published: January 2017

A wild-type strain of the yeast Saccharomyces cerevisiae grown at a medium [Cu] of less than or equal to 50 nM contained less Cu,Zn superoxide dismutase (SOD) mRNA (60%), protein (50%), and activity (50%) in comparison with control cultures grown in normal synthetic dextrose medium ([Cu] approximately 150 nM). A compensating increase in the activity of MnSOD was observed, as well as a smaller increase in MnSOD mRNA. These medium [Cu]-dependent differences were observed in cultures under N2 as well. Addition of Cu2+ (100 microM) to Cu-depleted cultures resulted in a rapid (30 min) increase in Cu,ZnSOD mRNA (2.5-fold), protein (3.5-fold), and activity (4-fold). Ethidium bromide (200 micrograms/ml of culture) inhibited by 50% the increase in Cu,ZnSOD mRNA, while cycloheximide (100 micrograms/ml of culture) inhibited completely the increase in protein and activity. Addition of Cu2+ to greater than or equal to 100 microM caused no further increase in these parameters but did result in a loss of total cellular RNA and translatable RNA, a decline in the population of specific mRNAs, a decrease in total soluble protein and the activity of specific enzymes, and an inhibition of incorporation of [3H]uracil and [3H]leucine into trichloroacetic acid-insoluble material. Cu,ZnSOD mRNA, protein, and activity appeared relatively more resistant to these effects of Cu toxicity than did the other cellular constituents examined. When evaluated in cultures under N2, the cellular response to [Cu] of greater than or equal to 100 microM was limited to the inhibition of radiolabel incorporation into trichloroacetic acid-insoluble material. All other effects were absent in the absence of O2. The data indicated that medium (cellular) Cu alters the steady-state level of Cu, ZnSOD. This regulation may be at the level of transcription. In addition, Cu,ZnSOD exhibits the characteristics of Cu-stress protein in that it and its mRNA are enhanced relative to other cellular species under conditions of Cu excess. This observation and the O2-dependence of some of the manifestations of Cu excess suggest that one mechanism of Cu toxicity involves the superoxide radical anion O2-.
Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2)
Authors: Narva K E , Feitelson J S
Published: January 2017

Previous genetic evidence suggested that the redD gene product might be involved in the regulation of undecylprodigiosin (Red) biosynthesis in Streptomyces coelicolor. The redD+ gene was subcloned on a 2.2-kilobase-pair restriction fragment from the S. coelicolor redCD region by complementation of S. coelicolor JF1 (redD42). The DNA sequence of the 2.2-kilobase-pair redD-complementing region was determined, and the redD coding sequence was identified by computer analysis and deletion subcloning. Transcription at the redD locus was analyzed by using in vivo promoter probing, high resolution S1 mapping, and in vitro runoff transcription. A face-to-face arrangement of promoters was deduced, in which the proposed redD promoter was opposed by a cluster of four other promoters for another unidentified open reading frame. In time course experiments, redD transcription preceded that at two biosynthetic loci, redE and redBF; transcription at the latter two loci was reduced in redD42 mutants. The putative redD polypeptide lacked any strong sequence similarities to other known proteins.
Repellent response functions of the Trg and Tap chemoreceptors of Escherichia coli
Authors: Yamamoto K , Macnab R M , Imae Y
Published: January 2017

The chemoreceptors responsible for the repellent response of Escherichia coli to phenol were investigated. In the absence of all four known methyl-accepting chemoreceptors (Tar, Tsr, Trg, and Tap), cells showed no response to phenol. However, when Trg, which mediates the attractant response to ribose and galactose, was introduced via a plasmid, the cells acquired a repellent response to phenol. About 1 mM phenol induced a clear repellent response; this response was suppressed by 1 mM ribose. Thus, Trg mediates the repellent response to phenol. Mutant Trg proteins with altered sensing for ribose and galactose showed a normal response to phenol, indicating that the interaction site for phenol differs from that for the ribose- and galactose-binding proteins. Tap, which mediates the attractant response to dipeptides, mediated a weaker repellent response to phenol. Tsr, which mediates the attractant response to serine, mediated an even weaker response to phenol. Trg and Tap were also found to function as intracellular pH sensors. Upon a pH decrease, Trg mediated an attractant response, whereas Tap mediated a repellent response. These results indicate that all the receptors in E. coli have dual functions, mediating both attractant and repellent responses.
Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence
Authors: Seifert H S , Ajioka R S , Paruchuri D , Heffron F , So M
Published: January 2017

The method of shuttle mutagenesis has been extended to Neisseria gonorrhoeae. We have constructed a defective mini-Tn3 derivative that encodes chloramphenicol resistance in both N. gonorrhoeae and Escherichia coli and selected for mutations in the chloramphenicol resistance gene that express higher levels of antibiotic resistance in N. gonorrhoeae. Isogenic N. gonorrhoeae strains that differ only in pilin expression were constructed and used to test the effect of pilin null mutations on DNA transformation competence.
Energy-dependent uptake of 4-chlorobenzoate in the coryneform bacterium NTB-1
Authors: Groenewegen P E , Driessen A J , Konings W N , de Bont J A
Published: January 2017

The uptake of 4-chlorobenzoate (4-CBA) in intact cells of the coryneform bacterium NTB-1 was investigated. Uptake and metabolism of 4-CBA were observed in cells grown in 4-CBA but not in glucose-grown cells. Under aerobic conditions, uptake of 4-CBA occurred with a high apparent affinity (apparent Kt, 1.7 microM) and a maximal velocity (Vmax) of 5.1 nmol min-1 mg of protein-1. At pH values below 7, the rate of 4-CBA uptake was greatly reduced by nigericin, an ionophore which dissipates the pH gradient across the membrane (delta pH). At higher pH values, inhibition was observed only with valinomycin, an ionophore which collapses the electrical potential across the membrane (delta psi). Under anaerobic conditions, no uptake of 4-CBA was observed unless an alternative electron acceptor was present. With nitrate as the terminal electron acceptor, 4-CBA was rapidly accumulated by the cells to a steady-state level, at which uptake of 4-CBA was balanced by excretion of 4-hydroxybenzoate. The mechanism of energy coupling to 4-CBA transport under anaerobic conditions was further examined by the imposition of an artificial delta psi, delta pH, or both. Uptake of 4-CBA was shown to be coupled to the proton motive force, suggesting a proton symport mechanism. Competition studies with various substrate analogs revealed a very narrow specificity of the 4-CBA uptake system. This is the first report of carrier-mediated transport of halogenated aromatic compounds in bacteria.
Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product
Authors: Sweet G , Gandor C , Voegele R , Wittekindt N , Beuerle J , Truniger V , Lin E C , Boos W
Published: January 2017

The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.
Structure of the rhsA locus from Escherichia coli K-12 and comparison of rhsA with other members of the rhs multigene family
Authors: Feulner G , Gray J A , Kirschman J A , Lehner A F , Sadosky A B , Vlazny D A , Zhang J , Zhao S , Hill C W
Published: January 2017

The complete nucleotide sequence of the rhsA locus and selected portions of other members of the rhs multigene family of Escherichia coli K-12 have been determined. A definition of the limits of the rhsA and rhsC loci was established by comparing sequences from E. coli K-12 with sequences from an independent E. coli isolate whose DNA contains no homology to the rhs core. This comparison showed that rhsA comprises 8,249 base pairs (bp) in strain K-12 and that the Rhs0 strain, instead, contains an unrelated 32-bp sequence. Similarly, the K-12 rhsC locus is 9.6 kilobases in length and a 10-bp sequence resides at its location in the Rhs0 strain. The rhsA core, the highly conserved portion shared by all rhs loci, comprises a single open reading frame (ORF) 3,714 bp in length. The nucleotide sequence of the core ORF predicts an extremely hydrophilic 141-kilodalton peptide containing 28 repeats of a motif whose consensus is GxxxRYxYDxxGRL(I or T). One of the most novel aspects of the rhs family is the extension of the core ORF into the divergent adjacent region. Core extensions of rhsA, rhsB, rhsC, and rhsD add 139, 173, 159, and 177 codons to the carboxy termini of the respective core ORFs. For rhsA, the extended core protein would have a molecular mass of 156 kilodaltons. Core extensions of rhsB and rhsD are related, exhibiting 50.3% conservation of the predicted amino acid sequence. However, comparison of the core extensions of rhsA and rhsC at both the nucleotide and the predicted amino acid level reveals that each is highly divergent from the other three rhs loci. The highly divergent portion of the core extension is joined to the highly conserved core by a nine-codon segment of intermediate conservation. The rhsA and rhsC loci both contain partial repetitions of the core downstream from their primary cores. The question of whether the rhs loci should be considered accessory genetic elements is discussed but not resolved.
Purification and properties of ferredoxinNAP, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816
Authors: Haigler B E , Gibson D T
Published: January 2017

One of the three components of the naphthalene dioxygenase occurring in induced cells of Pseudomonas sp. strain NCIB 9816 has been purified to homogeneity. The protein contained 2 g-atoms each of iron and acid-labile sulfur and had an apparent molecular weight of 13,600. The evidence indicates that it is a ferredoxin-type protein that functions as an intermediate electron transfer protein in naphthalene dioxygenase activity.
Cloning and nucleotide sequence of the Enterobacter aerogenes signal peptidase II (lsp) gene
Authors: Isaki L , Kawakami M , Beers R , Hom R , Wu H C
Published: January 2017

In Escherichia coli, prolipoprotein signal peptidase is encoded by the lsp gene, which is organized into an operon consisting of ileS, lsp, and three open reading frames, designated genes x, orf-149, and orf-316. The Enterobacter aerogenes lsp gene was cloned and expressed in E. coli. The nucleotide sequence of the Enterobacter aerogenes lsp gene and a part of its flanking sequences were determined. A high degree of homology was found between the E. coli ileS-lsp operon and the corresponding genes in Enterobacter aerogenes. Furthermore, the same five genes which constitute an operon in E. coli were found in Enterobacter aerogenes in the same order.
Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lactis subsp. lactis
Authors: van der Lelie D , Wösten H A , Bron S , Oskam L , Venema G
Published: January 2017

pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcus faecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAM beta 1 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 did not require the presence of a conjugative plasmid in the donor strain but, rather, appeared to be dependent on putative chromosomal functions in strain IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.
Intracellular serine protease 1 of Bacillus subtilis is formed in vivo as an unprocessed, active protease in stationary cells
Authors: Sheehan S M , Switzer R L
Published: January 2017

Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium. No evidence for an inactive precursor form of the protease was found.
Construction of a stable shuttle vector for high-frequency transformation in Pseudomonas syringae pv. syringae
Authors: Mukhopadhyay P , Mukhopadhyay M , Mills D
Published: January 2017

A cryptic 80.3-kilobase plasmid, pOSU900, in Pseudomonas syringae pv. syringae strain J900 could be cured by treatment with mitomycin without affecting the pathogenicity of J900 on the host, Phaseolus vulgaris L. The replication region of pOSU900 was identified, subcloned, and modified for construction of a high-copy cloning vector. This vector could be transformed into Pseudomonas strains with high efficiency (ca. 10(6) transformants per microgram of DNA) and was very stable during growth of the host bacteria in planta.
Defects in fruiting body development caused by Tn5 lac insertions in Myxococcus xanthus
Authors: Kroos L , Kuspa A , Kaiser D
Published: January 2017

Mutations caused by insertions of Tn5 lac that block development are rare. At least six of the eight mutations examined appeared to be regulatory. Three of these were found to disrupt social motility, suggesting a particular importance for this function. One other occurred in a known cell-cell interaction gene, bsgA, and the remaining two were located in genes operative early in the developmental program.
Evidence for multiple OmpR-binding sites in the upstream activation sequence of the ompC promoter in Escherichia coli: a single OmpR-binding site is capable of activating the promoter
Authors: Maeda S , Mizuno T
Published: January 2017

We present experimental evidence for the existence of multiple activator-binding sites in the upstream sequence of the ompC promoter, the expression of which is activated by the positive regulator OmpR in response to the osmolarity of the medium. We also found that a single OmpR-binding site can activate the ompC promoter, providing that the binding site is close and placed stereospecifically with respect to the canonical-35 and -10 regions.
Dramatic increase in negative superhelicity of plasmid DNA in the forespore compartment of sporulating cells of Bacillus subtilis
Authors: Nicholson W L , Setlow P
Published: January 2017

Plasmid pUB110, isolated from vegetative cells of Bacillus subtilis, has an average of 34 negative supertwists (tau av = -34). This value falls to -30 early in sporulation, and the plasmid in the mother cell compartment maintains a tau av of -30. However, the plasmid within the developing forespore becomes much more negatively supercoiled, reaching a tau av of -47 in the dormant spore. This increased negative supercoiling in the forespore plasmid takes place in parallel with the synthesis of small, acid-soluble spore proteins, alpha and beta; and the plasmid from spores lacking small, acid-soluble proteins alpha and beta has a tau av of -40. The large increase in negative supercoiling of spore plasmid was also observed with Bacillus megaterium and in B. subtilis containing a plasmid with an origin different from that of pUB110. During spore germination plasmid pUB110 rapidly relaxed back to the tau av value characteristic of vegetative cells. It is possible that the observed changes in forespore plasmid topology are involved in modulating gene expression, DNA photochemistry, or both of these parameters in this compartment.
Topology of penicillin-binding protein 1b of Escherichia coli and topography of four antigenic determinants studied by immunocolabeling electron microscopy
Authors: den Blaauwen T , Nanninga N
Published: January 2017

A method has been developed to study the orientation of proteins in the cytoplasmic membrane of Escherichia coli. Vesicles from sonicated cells were incubated in droplets on electron microscope support grids in sequence with a monoclonal antibody (MAb) against a protein with an unknown orientation (PBP 1b) followed by a MAb against a periplasmic component (peptidoglycan). The different MAbs were made visible with 5- and 10-nm gold-conjugated secondary antibodies, respectively. PBP 1b appeared to colabel with peptidoglycan. The labeling of PBP 1b in membrane vesicles with MAbs against four different epitopes was further used to estimate the number of PBP 1b molecules per cell. Approximately 1,400 PBP 1b molecules per cell grown in broth were labeled. The spatial distribution of the epitopes of the MAbs was studied by immunocolabeling of pairs of MAbs and by competitive antibody-binding inhibition. It could be tentatively concluded that the four epitopes form a cluster of antigenic determinants which occupy less than half of the surface of PBP 1b.
Molecular Epidemiology of Clostridium difficile Infection in Hospitalized Patients in Eastern China
Authors: Jin Dazhi , Luo Yun , Huang Chen , Cai Jian , Ye Julian , Zheng Yi , Wang Liqian , Zhao Peng , Liu Anbing , Fang Weijia , Wang Xianjun , Xia Shichang , Jiang Jianmin , Tang Yi-Wei
Published: January 2017

Few studies on risk factors for and transmission of
Clostridium difficile
infection (CDI) in China have been reported. A cross-sectional study was conducted for 3 years in eastern China. Consecutive stool specimens from hospitalized patients with diarrhea were cultured for
C. difficile. C. difficile
isolates from these patients then were analyzed for toxin genes, genotypes, and antimicrobial resistance. A severity score for the CDI in each patient was determined by a blinded review of the medical record, and these scores ranged from 1 to 6. A total of 397 out of 3,953 patients (10.0%) with diarrhea were found to have CDI. Severity of CDI was mild to moderate, and the average (± standard deviation) severity score was 2.61 ± 1.01.
C. difficile
was isolated from stool specimens in 432 (10.9%) of all the patients who had diarrhea.
C. difficile
genotypes were determined by multilocus sequence analysis and PCR ribotyping; sequence type 37 (ST37)/ribotype 017 (RT017) (
n
= 68, 16.5%) was the dominant genotype. Eleven patients (16.2%) with this genotype had a CDI severity score of 5. Overall, three RTs and four STs were predominant; these genotypes were associated with significantly different antimicrobial resistance patterns in comparison to all genotypes (χ
2
= 79.56 to 97.76;
P
< 0.001). Independent risk factors associated with CDI included age greater than 55 years (odds ratio [95% confidence interval], 26.80 [18.76 to 38.29]), previous hospitalization (12.42 [8.85 to 17.43]), previous antimicrobial treatment within 8 weeks (150.56 [73.11 to 310.06]), hospital stay more than 3 days before sampling (2.34 [1.71 to 3.22]), undergoing chemotherapy (3.31 [2.22 to 4.92]), and undergoing abdominal surgery (4.82 [3.54 to 6.55]). CDI is clearly a problem in eastern China and has a prevalence of 10.0% in hospitalized patients. Among risk factors for CDI, the advanced age threshold was younger for Chinese patients than that reported for patients in developed countries.
Multilocus Phylogeny and Antifungal Susceptibility of Aspergillus Section Circumdati from Clinical Samples and Description of A. pseudosclerotiorum sp. nov
Authors: Siqueira J. P. Z. , Sutton D. A. , Gené J. , García D. , Wiederhold N. , Peterson S. W. , Guarro J.
Published: January 2017

A multilocus phylogenetic study was carried out to assess species identity of a set of 34 clinical isolates from
Aspergillus
section
Circumdati
from the United States and to determine their
in vitro
antifungal susceptibility against eight antifungal drugs. The genetic markers used were the internal transcribed spacer (ITS) region, and fragments of the beta-tubulin (
BenA
), calmodulin (
CaM
), and RNA polymerase II second largest subunit (
RPB2
) genes. The drugs tested were amphotericin B, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, micafungin, and terbinafine. The most common species sampled was
A. westerdijkiae
(29.4%), followed by a novel species, which was described here as
A. pseudosclerotiorum
(23.5%). Other species identified were
A. sclerotiorum
(17.6%),
A. ochraceus
(8.8%),
A. subramanianii
(8.8%), and
A. insulicola
and
A. ochraceopetaliformis
, with two isolates (5.9%) of each. The drugs that showed the most potent activity were caspofungin, micafungin, and terbinafine, while amphotericin B showed the least activity.
Singleton Sequence Type 382, an Emerging Clonal Group of Listeria monocytogenes Associated with Three Multistate Outbreaks Linked to Contaminated Stone Fruit, Caramel Apples, and Leafy Green Salad
Authors: Chen Yi , Luo Yan , Pettengill James , Timme Ruth , Melka David , Doyle Matthew , Jackson Alikeh , Parish Mickey , Hammack Thomas S. , Allard Marc W. , Brown Eric W. , Strain Errol A.
Published: January 2017

Three multistate outbreaks between 2014 and 2016, involving case patients in and outside the United States, were linked to stone fruit, caramel apples, and packaged leafy green salad contaminated with
Listeria monocytogenes
singleton sequence type 382 (ST382), a serotype IVb-v1 clone with limited genomic divergence. Isolates from these outbreaks and other ST382 isolates not associated with these outbreaks were analyzed by whole-genome sequencing (WGS) analysis. The primary differences among ST382 strains were single nucleotide polymorphisms (SNPs). WGS analysis differentiated ST382 from a clonal complex 1 outbreak strain co-contaminating the caramel apples. WGS clustered food, environmental, and clinical isolates within each outbreak, and also differentiated among the three outbreak strains and epidemiologically unrelated ST382 isolates, which were indistinguishable by pulsed-field gel electrophoresis. ST382 appeared to be an emerging clone that began to diverge from its ancestor approximately 32 years before 2016. We estimated that there was 1.29 nucleotide substitution per genome (2.94 Mbp) per year for this clone.
Nosocomial Outbreak of Drug-Resistant Streptococcus pneumoniae Serotype 9V in an Adult Respiratory Medicine Ward
Authors: Jauneikaite Elita , Khan-Orakzai Zareena , Kapatai Georgia , Bloch Susannah , Singleton Julie , Atkin Sara , Shah Victoria , Hatcher James , Samarasinghe Dunisha , Sheppard Carmen , Fry Norman K. , Satta Giovanni , Sriskandan Shiranee
Published: January 2017

Streptococcus pneumoniae
infections arising in hospitalized patients are often assumed to be sporadic and linked to community acquisition. Here, whole-genome sequencing was used to demonstrate nosocomial acquisition of antimicrobial-resistant sequence type 156 (ST156) serotype 9V
S. pneumoniae
in 3 respiratory patients that resulted in two bacteremias and one lower respiratory tract infection. Two of the cases arose in patients who had recently been discharged from the hospital and were readmitted from the community. Nosocomial spread was suspected solely because of the highly unusual resistance pattern and case presentations within 24 h of one another. The outbreak highlights the potential for rapid transmission and the short incubation period in the respiratory ward setting.
The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share an Essential Role for Viral Replication
Authors: Gallo Antonio , Lampe Melanie , Günther Thomas , Brune Wolfram
Published: January 2017

KS-Bcl-2 is a Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral Bcl-2 (vBcl-2) homolog which has apoptosis- and autophagy-inhibiting activity when expressed in transfected cells. However, little is known about its function during viral infection. As KS-Bcl-2 is expressed during the lytic replication cycle, we used constitutively lytic and inducibly lytic KSHV mutants to investigate the role of KS-Bcl-2 during the lytic cycle. We show that KSHV cannot complete the lytic replication cycle and produce infectious progeny in the absence of KS-Bcl-2, indicating that the protein is essential for KSHV replication. Replacement of the KS-Bcl-2 coding sequence, ORF16, by sequences encoding a potent cellular apoptosis and autophagy inhibitor, Bcl-XL, or the cytomegalovirus mitochondrial inhibitor of apoptosis, vMIA, did not rescue KSHV replication, suggesting that KS-Bcl-2 has a function that goes beyond apoptosis and autophagy inhibition. Strikingly, the vBcl-2 proteins of the related γ
2
-herpesviruses murine herpesvirus 68 and herpesvirus saimiri did not rescue the replication of a KS-Bcl-2 deletion mutant, but rhesus rhadinovirus (RRV) vBcl-2 did. Deletion of ORF16 from the RRV genome abrogated viral replication, but its replacement by KSHV ORF16 rescued RRV replication, indicating that the essential vBcl-2 function is conserved between these two primate rhadinoviruses. We further show that the KSHV and RRV Bcl-2 homologs localize to the mitochondria and nuclei of infected cells. Deletion of 17 amino acids from the N terminus of KS-Bcl-2 abrogates nuclear localization and KSHV replication, suggesting that KS-Bcl-2 might execute its essential function in the nuclei of infected cells.

IMPORTANCE
Several viruses express proteins homologous to cellular Bcl-2. Viral Bcl-2 proteins have functions similar to those of cellular Bcl-2: they can inhibit apoptosis, a form of programmed cell death, and autophagy, a self-degradative process for the disposal of dysfunctional or unwanted components. This study shows that the vBcl-2 proteins of KSHV and RRV differ from other vBcl-2 proteins in that they are essential for viral replication. The essential function is separate from the apoptosis- and autophagy-inhibiting activity but correlates with an unusual localization within the cell nucleus, suggesting that these proteins exert a novel function in the nucleus.
Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Upregulates Glucose Transporter 1 Transcription via the mTORC1/NF-κB Signaling Pathways
Authors: Zhang Jun , Jia Lin , Lin Weitao , Yip Yim Ling , Lo Kwok Wai , Lau Victoria Ming Yi , Zhu Dandan , Tsang Chi Man , Zhou Yuan , Deng Wen , Lung Hong Lok , Lung Maria Li , Cheung Lai Man , Tsao Sai Wah
Published: January 2017

Accumulating evidence indicates that oncogenic viral protein plays a crucial role in activating aerobic glycolysis during tumorigenesis, but the underlying mechanisms are largely undefined. Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a transmembrane protein with potent cell signaling properties and has tumorigenic transformation property. Activation of NF-κB is a major signaling pathway mediating many downstream transformation properties of LMP1. Here we report that activation of mTORC1 by LMP1 is a key modulator for activation of NF-κB signaling to mediate aerobic glycolysis. NF-κB activation is involved in the LMP1-induced upregulation of glucose transporter 1 (Glut-1) transcription and growth of nasopharyngeal carcinoma (NPC) cells. Blocking the activity of mTORC1 signaling effectively suppressed LMP1-induced NF-κB activation and Glut-1 transcription. Interfering NF-κB signaling had no effect on mTORC1 activity but effectively altered Glut-1 transcription. Luciferase promoter assay of Glut-1 also confirmed that the Glut-1 gene is a direct target gene of NF-κB signaling. Furthermore, we demonstrated that C-terminal activating region 2 (CTAR2) of LMP1 is the key domain involved in mTORC1 activation, mainly through IKKβ-mediated phosphorylation of TSC2 at Ser
939
. Depletion of Glut-1 effectively led to suppression of aerobic glycolysis, inhibition of cell proliferation, colony formation, and attenuation of tumorigenic growth property of LMP1-expressing nasopharyngeal epithelial (NPE) cells. These findings suggest that targeting the signaling axis of mTORC1/NF-κB/Glut-1 represents a novel therapeutic target against NPC.

IMPORTANCE
Aerobic glycolysis is one of the hallmarks of cancer, including NPC. Recent studies suggest a role for LMP1 in mediating aerobic glycolysis. LMP1 expression is common in NPC. The delineation of essential signaling pathways induced by LMP1 in aerobic glycolysis contributes to the understanding of NPC pathogenesis. This study provides evidence that LMP1 upregulates Glut-1 transcription to control aerobic glycolysis and tumorigenic growth of NPC cells through mTORC1/NF-κB signaling. Our results reveal novel therapeutic targets against the mTORC1/NF-κB/Glut-1 signaling axis in the treatment of EBV-infected NPC.
Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production in Streptococcus pneumoniae D39
Authors: Lisher John P. , Tsui Ho-Ching Tiffany , Ramos-Montañez Smirla , Hentchel Kristy L. , Martin Julia E. , Trinidad Jonathan C. , Winkler Malcolm E. , Giedroc David P.
Published: January 2017

Adaptation to endogenous oxidative stress is an integral aspect of
Streptococcus pneumoniae
colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H
2
O
2
and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in
S. pneumoniae
.
Urinary C peptide creatinine ratio in pregnant women with normal glucose tolerance and type 1 diabetes: evidence for insulin secretion
Authors: Markoska Ankica , Valaiyapathi Rajalakshmi , Thorn Chloe , Dornhorst Anne
Published: January 2017

Hypothesis

In pregnancy, urinary C peptide creatinine ratio (UCPCR) reflects endogenous insulin secretion in women with normal glucose tolerance and type 1 diabetes.

Research design and methods

UCPCR and serum C peptide were measured in 90 glucose-tolerant women at 0 and 120 min during a 75 g oral glucose tolerance test (OGTT) at 28 weeks of gestation. UCPCR was measured in 2 samples obtained over 10 weeks apart in 7 pregnant women with longstanding type 1 diabetes.

Results

UCPCROGTTand serum C peptideOGTTof glucose-tolerant women were significantly correlated at 0 and 120 min (rs0.675, 0.541 respectively, p<0.0001). All 7 pregnant women with type 1 diabetes had detectable first sample UCPCR (median (range) 49 (6–1038) pmol/mmol) that rose in 6 women by 477 (29–1491) pmol/mmol.

Conclusions

Detectable UCPCR in pregnant women with normal glucose tolerance and type 1 diabetes is likely to reflect endogenous insulin secretion and hence β-cell activity.