bluuebunny/crossref_metadata_2025 huggingface dataset:
A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly
Authors: von Eiff C , Heilmann C , Proctor R A , Woltz C , Peters G , Götz F
Published: November 2016

Although small-colony variants (SCVs) of Staphylococcus aureus have been recognized for many years, this phenotype has only recently been related to persistent and recurrent infections. Clinical S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain elements menaquinone and cytochromes, respectively. While this observation as well as other biochemical characteristics of SCVs suggests a link between electron-transport-defective strains and persistent infections, the strains examined thus far have been genetically undefined SCVs. Therefore, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in S. aureus by inserting an ermB cassette into hemB. We isolated a hemB mutant, due to homologous recombination, by growth at a nonpermissive temperature and selection for erythromycin resistance. This mutant showed typical characteristics of clinical SCVs, such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Additionally, the mutant was able to persist within cultured endothelial cells due to decreased alpha-toxin production. Northern and Western blot analyses showed that expression of alpha-toxin and that of protein A were markedly reduced, at both the mRNA and the protein level. The SCV phenotype of the hemB mutant was reversed by growth with hemin or by complementation with intact hemB. Hence, a defect in the electron transport system allows S. aureus SCVs to resist aminoglycosides and persist intracellularly.
A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator
Authors: Hay N A , Tipper D J , Gygi D , Hughes C
Published: November 2016

Proteus swarming is the rapid cyclical population migration across surfaces by elongated cells that hyperexpress flagellar and virulence genes. The mini-Tn5 transposon mutant mns2 was isolated as a tight nonswarming mutant that did not elongate or upregulate flagellar and hemolysin genes. Individual cell motility was retained but was reduced. The transposon had inserted in the gene encoding the global transcriptional regulator Lrp (leucine-responsive regulatory protein), expression of which was upregulated in differentiating swarm cells. Swarming was restored to the lrp mutant by artificial overexpression of the flhDC flagellar regulatory master operon. Lrp may be a key component in generating or relaying signals that are required for flagellation and swarming, possibly acting through the flhDC operon.
Lipase secretion by bacterial hybrid ATP-binding cassette exporters: molecular recognition of the LipBCD, PrtDEF, and HasDEF exporters
Authors: Akatsuka H , Binet R , Kawai E , Wandersman C , Omori K
Published: November 2016

Serratia marcescens secretes several proteins, such as the lipase LipA, the metalloprotease PrtA, and the heme-binding protein HasA, which is required for heme acquisition, through two N-terminal signal peptide-independent systems that are classified as bacterial ATP-binding cassette (ABC) exporters. One is the ABC exporter for HasA, consisting of the ABC protein HasD, the membrane fusion protein (MFP) HasE, and the outer membrane protein (OMP) HasF. The second, composed of LipB (an ABC protein), LipC (an MFP), and LipD (an OMP), promotes secretion of LipA and PrtA in Escherichia coli recombinant clones. PrtA, which shows homology to the Erwinia chrysanthemi metalloproteases, is efficiently secreted by E. coli cells carrying the E. chrysanthemi ABC exporter PrtD (ABC protein)-PrtE (MFP)-PrtF (OMP). The existence of distinct systems in this bacterium and of various substrates for these systems allowed the study of protein secretion by heterologous Has, Lip, and Prt systems and by Has-Lip and Lip-Prt hybrid exporters in the genuine host as well as in E. coli. For that purpose, lipB-, lipC-, and lipD-deficient mutants were isolated from S. marcescens 8000 and their secretion of LipA and PrtA was analyzed. This demonstrated that a unique exporter, the Lip apparatus, in S. marcescens secretes both LipA and PrtA. Hybrid exporters were tested for secretion of HasA and LipA. The LipB-HasE-HasF exporter allowed secretion of LipA but not HasA, showing that the ABC protein LipB is responsible for the substrate specificity. LipA, HasA, and E. chrysanthemi PrtC were secreted via heterologous exporters and via some hybrid exporters. Analysis of secretion via hybrid exporters showed that specific interactions occur between MFPs and OMPs in these systems. These genetic experiments demonstrated that specific interactions between the ABC protein and the MFP are required for the formation of active exporters.
The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin
Authors: Aduse-Opoku J , Slaney J M , Rangarajan M , Muir J , Young K A , Curtis M A
Published: November 2016

The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association. The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a regulatory relationship between tla and other members of this gene family.
Cloning and expression in Escherichia coli of genetic determinants for production of and immunity to microcin E492 from Klebsiella pneumoniae
Authors: Wilkens M , Villanueva J E , Cofré J , Chnaiderman J , Lagos R
Published: November 2016

Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor.
A new Thermus-Escherichia coli shuttle integration vector system
Authors: Tamakoshi M , Uchida M , Tanabe K , Fukuyama S , Yamagishi A , Oshima T
Published: November 2016

We established a Thermus thermophilus strain in which the pyrE gene (coding for orotate phosphoribosyltransferase of the pyrimidine biosynthetic pathway) was totally deleted. We also constructed an integration vector, which consisted of the Escherichia coli plasmid vector pBluescript and a 2.1-kb segment of the T. thermophilus leu operon sequence, for the integration of a foreign gene into a chromosome of the thermophile. pyrE and leuB genes were used as probes to test the integration vector. The integration vector pINV, bearing the pyrE gene, transformed the delta pyrE strain at a frequency of 6 x 10(-5) through a single crossover event. The leuB gene could also be used as another marker of the integration vector system. The vector could be integrated at the expected site. By digesting the chromosomal DNA of the T. thermophilus transformants with a unique restriction enzyme, the vector could be recovered into E. coli after the recircularization in vitro. The kanamycin nucleotidyltransferase gene could be successfully expressed in the thermophile by using pINV.
Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae
Authors: Fukui T , Doi Y
Published: November 2016

A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.
Osmoprotectant-dependent expression of plcH, encoding the hemolytic phospholipase C, is subject to novel catabolite repression control in Pseudomonas aeruginosa PAO1
Authors: Sage A E , Vasil M L
Published: November 2016

Expression of the hemolytic phospholipase C (PlcH) of Pseudomonas aeruginosa is induced under phosphate starvation conditions or in the presence of the osmoprotectants choline and glycine betaine. Because choline and glycine betaine may serve as carbon and energy sources in addition to conferring osmoprotection to P. aeruginosa, it seemed possible that induction of plcH is subject to catabolite repression control (CRC) by tricarboxylic cycle intermediates such as succinate. Total phospholipase (PLC) activity in osmoprotectant-induced cultures of P. aeruginosa PAO1 supplemented with 20 mM succinate was three- to fourfold lower than the levels in cultures supplemented with the non-catabolite-repressive substrate lactate. Analyses of osmoprotectant-dependent plcH expression in a derivative of strain PAO1 containing a plcH::lacZ operon fusion showed that (i) succinate prevented induction of plcH expression by osmoprotectants; and (ii) addition of succinate reduced or shut down further expression of plcH in osmoprotectant-induced bacteria, while cultures supplemented with lactate had little or no change in plcH expression. RNase protection analysis confirmed that repression of plcH occurs at the transcriptional level. However, a P. aeruginosa mutant decoupled in CRC exhibited a phenotype similar to that of the wild-type strain (PAO1) with respect to succinate-dependent repression of plcH expression. Osmoprotectant-induced total PLC activities, levels of expression of plcH measured with the same plcH::lacZ fusion, and levels of plcH transcription in a CRC-deficient strain reflected those seen in strain PAO1. This indicates that CRC of plcH functions by a distinct mechanism which differs from that regulating the glucose or mannitol catabolic pathway. A strain carrying a mutation in vfr, which encodes the Escherichia coli Crp homolog in P. aeruginosa, still exhibited a wild-type phenotype with respect to osmoprotectant-dependent expression and CRC of plcH. These data indicate that there is a novel CRC system that regulates the expression of plcH in P. aeruginosa.
Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase
Authors: Hanzelka B L , Stevens A M , Parsek M R , Crone T J , Greenberg E P
Published: November 2016

Synthesis of the Vibrio fischeri autoinducer, a signal involved in the cell density-dependent activation of bioluminescence, is directed by the luxI gene product. The LuxI protein catalyzes the synthesis of N-acyl-homoserine lactones from S-adenosylmethionine and acylated-acyl carrier protein. We have gained an appreciation of the LuxI regions and amino acid residues involved in autoinducer synthesis by isolating and analyzing mutations generated by random and site-specific mutagenesis of luxI. By random mutagenesis we isolated 13 different single amino acid substitutions in the LuxI polypeptide. Eleven of these substitutions resulted in no detectable autoinducer synthase activity, while the remaining two amino acid substitutions resulted in reduced but detectable activity. The substitutions that resulted in no detectable autoinducer synthase activity mapped to two small regions of LuxI. In Escherichia coli, wild-type luxI showed dominance over all of the mutations. Because autoinducer synthesis has been proposed to involve formation of a covalent bond between an acyl group and an active-site cysteine, we constructed site-directed mutations that altered each of the three cysteine residues in LuxI. All of the cysteine mutants retained substantial activity as an autoinducer synthase in E. coli. Based on the analysis of random mutations we propose a model in which there are two critical regions of LuxI, at least one of which is an intimate part of an active site, and based on the analysis of site-directed mutations we conclude that an active-site cysteine is not essential for autoinducer synthase activity.
Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics
Authors: Zhao G , Yeh W K , Carnahan R H , Flokowitsch J , Meier T I , Alborn W E , Becker G W , Jaskunas S R
Published: November 2016

To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.
Colicin U, a novel colicin produced by Shigella boydii
Authors: Smajs D , Pilsl H , Braun V
Published: November 2016

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.
A Bacillus subtilis gene encoding a protein similar to nucleotide sugar transferases influences cell shape and viability
Authors: Price K D , Roels S , Losick R
Published: November 2016

Bacillus subtilis gene ypfP, which is located at 196 degrees on the genetic map, shows similarity to both the monogalactosyldiacylglycerol synthase gene of Cucumis sativus, which encodes a galactosyltransferase, and the murG genes of B. subtilis, Escherichia coli, Haemophilus influenzae, and Synechocystis sp. strain PCC6803, which encode N-acetylglucosaminyltransferases involved in peptidoglycan biosynthesis. Cells containing a null mutation of ypfP are shorter and rounder than wild-type cells during growth in Luria-Bertani medium and glucose minimal medium. In addition, the mutant cells preferentially undergo lysis when grown on solid Luria-Bertani medium.
Cell cycle characteristics of thermophilic archaea
Authors: Bernander R , Poplawski A
Published: November 2016

We have performed a cell cycle analysis of organisms from the Archaea domain. Exponentially growing cells of the thermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius were analyzed by flow cytometry, and several unusual cell cycle characteristics were found. The cells initiated chromosome replication shortly after cell division such that the proportion of cells with a single chromosome equivalent was low in the population. The postreplication period was found to be long; i.e., there was a considerable time interval from termination of chromosome replication until cell division. A further unusual feature was that cells in stationary phase contained two genome equivalents, showing that they entered the resting stage during the postreplication period. Also, a reduction in cellular light scatter was observed during entry into stationary phase, which appeared to reflect changes not only in cell size but also in morphology and/or composition. Finally, the in vivo organization of the chromosome DNA appeared to be different from that of eubacteria, as revealed by variation in the relative binding efficiency of different DNA stains.
Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons
Authors: Hoflack L , Seurinck J , Mahillon J
Published: November 2016

The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).
Control of poly-beta-hydroxybutyrate synthase mediated by acetyl phosphate in cyanobacteria
Authors: Miyake M , Kataoka K , Shirai M , Asada Y
Published: November 2016

Poly-beta-hydroxybutyrate (PHB) synthesis in a cyanobacterium, Synechococcus sp. strain MA19, is controlled at the enzyme level and is dependent on the C/N balance in the culture medium. The control involves at least two enzymes. The first enzyme is PHB synthase. Little PHB synthase activity was detected in crude extracts from cells grown under nitrogen-sufficient conditions (MA19(+N)). The activity was detected exclusively in membrane fractions from nitrogen-deprived cells (MA19(-N)) under light but not dark conditions. The shift in the enzyme activity was insensitive to chloramphenicol, which suggests posttranslational activation. Acetyl phosphate activated PHB synthase in membrane fractions from MA19(+N). In vitro, the activation level of PHB synthase changed, depending on the concentration of acetyl phosphate. The second enzyme was phosphotransacetylase (EC 2.3.1.8), which catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate. The activity was detected in crude extracts from MA19(-N) but not in those from MA19(+N). The results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C/N balance and intracellular acetyl-CoA concentration. Acetyl phosphate probably acts as a signal of C/N balance affecting PHB metabolism in MA19.
Characterization of a transposon Tn916-generated mutant of Haemophilus ducreyi 35000 defective in lipooligosaccharide biosynthesis
Authors: Gibson B W , Campagnari A A , Melaugh W , Phillips N J , Apicella M A , Grass S , Wang J , Palmer K L , Munson R S
Published: November 2016

To define the role of the surface lipooligosaccharide (LOS) of Haemophilus ducreyi in the pathogenesis of chancroid, Tn916 mutants of H. ducreyi 35000 defective in expression of the murine monoclonal antibody (MAb) 3F11 epitope on H. ducreyi LOS were identified by immunologic screening. One mutant, designated 1381, has an LOS which lacks the MAb 3F11 epitope and migrates with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene disrupted by the Tn916 element in strain 1381 was identified by cloning the sequences flanking the Tn916 element. The sequences were then used to probe a lambda DASHII genomic library. In strain 1381, Tn916 interrupts a gene which encodes an open reading frame (ORF) with an Mr of 40,246. This ORF has homology to the product of the rfaK gene of Escherichia coli. The major LOS glycoform produced by strain 1381 was analyzed by using a combination of mass spectrometry, linkage and composition analysis, and 1H nuclear magnetic resonance spectroscopy. The major LOS species was found to terminate in a single glucose attached to the heptose (L-glycero-D-manno-heptose, or Hep) trisaccharide core. In the wild-type strain 35000, glucose serves as the acceptor for the addition of the D-glycero-D-manno-heptose (or DDHep), which extends to form the mature branch of the H. ducreyi LOS. This mature oligosaccharide is in turn partially capped by the addition of sialic acid (NeuAc), i.e., NeuAc2 alpha-->3Gal beta1-->4GlcNAc beta1-->3Gal beta1-->4DDHep alpha1-->6Glc beta1 (W. Melaugh et al., Biochemistry 33:13070-13078, 1994). Since this LOS terminates prior to the addition of the branch DD-heptose, this gene is likely to encode the D-glycero-D-manno-heptosyltransferase. Strain 1381 exhibits a significant reduction in adherence to and invasion of primary human keratinocytes. This defect was complemented by the cloned heptosyltransferase gene, indicating that the terminal portion of the LOS oligosaccharide plays an important role in adherence to human keratinocytes.
Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli
Authors: Guzman L M , Weiss D S , Beckwith J
Published: November 2016

FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle.
Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium
Authors: Asai Y , Kojima S , Kato H , Nishioka N , Kawagishi I , Homma M
Published: November 2016

The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel. MotX was proposed to be a component of a sodium channel. Here we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions. These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene, suggesting that newly synthesized PomA proteins, which are components of a force-generating unit, were successively integrated into the defective motor complexes. These findings imply that Na+-driven flagellar motors may have similar structure and function as proton-driven motors, but with some interesting differences as well, and it is possible to compare and study the coupling mechanisms of the sodium and proton ion flux for the force generation.
D-erythro-neopterin biosynthesis in the methanogenic archaea Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH
Authors: Howell D M , White R H
Published: November 2016

The steps in the biosynthetic transformation of GTP to 7,8-dihydro-D-erythro-neopterin (H2neopterin), the precursor to the modified folates found in the methanogenic archaea, has been elucidated for the first time in two members of the domain Archaea. In Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH, it has been demonstrated that H2neopterin 2':3'-cyclic phosphate is an intermediate in this conversion. In addition, the formation of the pterin ring of the H2neopterin 2':3'-cyclic phosphate is catalyzed not by a single enzyme, as is known to occur with GTP cyclohydrolase I in the Eucarya and Bacteria, but rather by two or more enzymes. A 2,4,5-triamino-4(3H)-pyrimidinone-containing molecule, most likely 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, has been identified as an intermediate in the formation of the H2neopterin 2':3'-cyclic phosphate. Synthetic H2neopterin 2':3'-cyclic phosphate was found to be readily hydrolyzed by cell extracts of M. thermophila via the H2neopterin 3'-phosphate to H2neopterin, a known precursor to the pterin portion of methanopterin.
The IncP plasmid-encoded cell envelope-associated DNA transfer complex increases cell permeability
Authors: Daugelavicius R , Bamford J K , Grahn A M , Lanka E , Bamford D H
Published: November 2016

IncP-type plasmids are broad-host-range conjugative plasmids. DNA translocation requires DNA transfer-replication functions and additional factors required for mating pair formation (Mpf). The Mpf system is located in the cell membranes and is responsible for DNA transport from the donor to the recipient. The Mpf complex acts as a receptor for IncP-specific phages such as PRD1. In this investigation, we quantify the Mpf complexes on the cell surface by a phage receptor saturation technique. Electrochemical measurements are used to show that the Mpf complex increases cell envelope permeability to lipophilic compounds and ATP. In addition it reduces the ability of the cells to accumulate K+. However, the Mpf complex does not dissipate the membrane voltage. The Mpf complex is rapidly disassembled when intracellular ATP concentration is decreased, as measured by a PRD1 adsorption assay.
Autogenous control of PspF, a constitutively active enhancer-binding protein of Escherichia coli
Authors: Jovanovic G , Dworkin J , Model P
Published: November 2016

Escherichia coli sigma54-dependent phage shock protein operon (pspA to -E) transcription is under the control of PspF, a constitutively active activator. Sigma70-dependent transcription of pspF is under autogenous control by wild-type PspF but not by a DNA-binding mutant, PspF deltaHTH. Negative autoregulation of PspF is continual and not affected by stimuli, like f1 pIV, that induce the pspA to -E operon. PspF production is independent of PspA (the negative regulator of the pspA to -E operon) and of PspB and -C (positive regulators).
Role of the ESCRT Complexes in Telomere Biology
Authors: Dieckmann Anna K. , Babin Vera , Harari Yaniv , Eils Roland , König Rainer , Luke Brian , Kupiec Martin
Published: November 2016

Eukaryotic chromosomal ends are protected by telomeres from fusion, degradation, and unwanted double-strand break repair events. Therefore, telomeres preserve genome stability and integrity. Telomere length can be maintained by telomerase, which is expressed in most human primary tumors but is not expressed in the majority of somatic cells. Thus, telomerase may be a highly relevant anticancer drug target. Genome-wide studies in the yeast
Saccharomyces cerevisiae
identified a set of genes associated with telomere length maintenance (
TLM
genes). Among the
tlm
mutants with short telomeres, we found a strong enrichment for those affecting vacuolar and endosomal traffic (particularly the
e
ndosomal
s
orting
c
omplex
r
equired for
t
ransport [ESCRT] pathway). Here, we present our results from investigating the surprising link between telomere shortening and the ESCRT machinery. Our data show that the whole ESCRT system is required to safeguard proper telomere length maintenance. We propose a model of impaired end resection resulting in too little telomeric overhang, such that Cdc13 binding is prevented, precluding either telomerase recruitment or telomeric overhang protection.

IMPORTANCE
Telomeres are the ends of eukaryotic chromosomes. They are necessary for the proper replication of the genome and protect the chromosomes from degradation. In a large-scale systematic screen for mutants that affect telomere length in yeast, we found that mutations in any of the genes encoding the ESCRT complexes, required for the formation of transport vesicles within the cell, cause telomere shortening. We carried out an analysis of the mechanisms disrupted in these mutants and found that they are defective for the ability to elongate short telomeres, probably due to faulty end processing. We discuss the significance of these findings and how they could be relevant to anticancer therapies.
One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the
Shigella
VirF Regulator
Authors: Di Martino Maria Letizia , Romilly Cédric , Wagner E. Gerhart H. , Colonna Bianca , Prosseda Gianni
Published: November 2016

VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of
Shigella
spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the
virF
mRNA is translated into two proteins, the major form, VirF
30
(30 kDa), and the shorter VirF
21
(21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF
30
and VirF
21
and showed that the two different forms of VirF arise from differential translation. Interestingly,
in vitro
and
in vivo
translation experiments showed that VirF
21
is also translated from a leaderless mRNA (llmRNA) whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF
21
. The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF
30
is responsible for activation of the virulence system, VirF
21
negatively autoregulates
virF
expression itself. Since VirF
21
modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in
virF
mRNA transcription and preferential translation of llmRNA.

IMPORTANCE
Shigella
spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the
Shigella virF
gene encodes two proteins of different sizes, VirF
30
and VirF
21
, that are functionally distinct. The major form, VirF
30
, activates the genes necessary for virulence, whereas the minor VirF
21
, which shares the C-terminal two-thirds of VirF
30
, negatively autoregulates
virF
expression itself. VirF
21
is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the
Shigella
invasive process and may help development of new therapies for shigellosis.
Rigorous Science: a How-To Guide
Authors: Casadevall Arturo , Fang Ferric C.
Published: November 2016

Proposals to improve the reproducibility of biomedical research have emphasized scientific rigor. Although the word “rigor” is widely used, there has been little specific discussion as to what it means and how it can be achieved. We suggest that scientific rigor combines elements of mathematics, logic, philosophy, and ethics. We propose a framework for rigor that includes redundant experimental design, sound statistical analysis, recognition of error, avoidance of logical fallacies, and intellectual honesty. These elements lead to five actionable recommendations for research education.
Expanding the Host Range of Hepatitis C Virus through Viral Adaptation
Authors: von Schaewen Markus , Dorner Marcus , Hueging Kathrin , Foquet Lander , Gerges Sherif , Hrebikova Gabriela , Heller Brigitte , Bitzegeio Julia , Doerrbecker Juliane , Horwitz Joshua A. , Gerold Gisa , Suerbaum Sebastian , Rice Charles M. , Meuleman Philip , Pietschmann Thomas , Ploss Alexander
Published: November 2016

Hepatitis C virus (HCV) species tropism is incompletely understood. We have previously shown that at the level of entry, human CD81 and occludin (OCLN) comprise the minimal set of human factors needed for viral uptake into murine cells. As an alternative approach to genetic humanization, species barriers can be overcome by adapting HCV to use the murine orthologues of these entry factors. We previously generated a murine tropic HCV (mtHCV or Jc1/mCD81) strain harboring three mutations within the viral envelope proteins that allowed productive entry into mouse cell lines. In this study, we aimed to characterize the ability of mtHCV to enter and infect mouse hepatocytes
in vivo
and
in vitro
. Using a highly sensitive, Cre-activatable reporter, we demonstrate that mtHCV can enter mouse hepatocytes
in vivo
in the absence of any human cofactors. Viral entry still relied on expression of mouse CD81 and SCARB1 and was more efficient when mouse CD81 and OCLN were overexpressed. HCV entry could be significantly reduced in the presence of anti-HCV E2 specific antibodies, suggesting that uptake of mtHCV is dependent on viral glycoproteins. Despite mtHCV’s ability to enter murine hepatocytes
in vivo
, we did not observe persistent infection, even in animals with severely blunted type I and III interferon signaling and impaired adaptive immune responses. Altogether, these results establish proof of concept that the barriers limiting HCV species tropism can be overcome by viral adaptation. However, additional viral adaptations will likely be needed to increase the robustness of a murine model system for hepatitis C.

IMPORTANCE
At least 150 million individuals are chronically infected with HCV and are at risk of developing serious liver disease. Despite the advent of effective antiviral therapy, the frequency of chronic carriers has only marginally decreased. A major roadblock in developing a vaccine that would prevent transmission is the scarcity of animal models that are susceptible to HCV infection. It is poorly understood why HCV infects only humans and chimpanzees. To develop an animal model for hepatitis C, previous efforts focused on modifying the host environment of mice, for example, to render them more susceptible to HCV infection. Here, we attempted a complementary approach in which a laboratory-derived HCV variant was tested for its ability to infect mice. We demonstrate that this engineered HCV strain can enter mouse liver cells but does not replicate efficiently. Thus, additional adaptations are likely needed to construct a robust animal model for HCV.
Reconstructing the end of the Appalachian orogeny
Authors: Hopper Emily , Fischer Karen M. , Wagner Lara S. , Hawman Robert B.
Published: November 2016

In contrast to crustal deformation observed in the actively forming Himalayas, where shallowly dipping crustal detachments extend over hundreds of kilometers, prior work on the Paleozoic southern Appalachian orogeny inferred that the final continental collision occurred on a steeply dipping crustal suture, permitting collision models that are dominated by strike-slip motion. Here, we use scattered seismic phases to instead reveal the Appalachian (Alleghanian) crustal suture as a low-angle (<∼15°) southward-dipping interface that soles into a flat-lying mid-crustal detachment. The observed suture geometry implies more than 300 km of head-on shortening across a plate boundary structure similar to the Himalayan mid-crustal detachment, indicating that this mode of deformation has been fundamental in continental collisions over hundreds of millions of years.
Ultrapotassic rocks and xenoliths from South Tibet: Contrasting styles of interaction between lithospheric mantle and asthenosphere during continental collision
Authors: Xu Bo , Griffin William L. , Xiong Qing , Hou Zeng-Qian , O’Reilly Suzanne Y. , Guo Zhen , Pearson Norman J. , Gréau Yoann , Yang Zhi-Ming , Zheng Yuan-Chuan
Published: November 2016

Widespread Miocene (24–8 Ma) ultrapotassic rocks and their entrained xenoliths provide information on the composition, structure, and thermal state of the sub-continental lithospheric mantle in southern Tibet during the India-Asia continental collision. The ultrapotassic rocks along the Lhasa block delineate two distinct lithospheric domains with different histories of depletion and enrichment. The eastern ultrapotassic rocks (89°E–92°E) reveal a depleted, young, and fertile lithospheric mantle (87Sr/86Srt = 0.704–0.707 [t is eruption time]; Hf depleted-mantle model age [TDM] = 377–653 Ma). The western ultrapotassic rocks (79°E–89°E) and their peridotite xenoliths (81°E) reflect a refractory harzburgitic mantle refertilized by ancient metasomatism (lavas: 87Sr/86Srt = 0.714–0.734; peridotites: 87Sr/86Srt = 0.709–0.716). These data integrated with seismic tomography suggest that upwelling asthenosphere was diverted away from the deep continental root beneath the western Lhasa block, but rose to shallower depths beneath a thinner lithosphere in the eastern part. Heating of the lithospheric mantle by the rising asthenosphere ultimately generated the ultrapotassic rocks with regionally distinct geochemical signatures reflecting the different nature of the lithospheric mantle.
Isotopic signatures of mercury contamination in latest Permian oceans
Authors: Grasby Stephen E. , Shen Wenjie , Yin Runsheng , Gleason James D. , Blum Joel D. , Lepak Ryan F. , Hurley James P. , Beauchamp Benoit
Published: November 2016

Sedimentary records from the northwest margin of Pangea and the Tethys show anomalously high Hg levels at the latest Permian extinction boundary. Background δ202Hg values are consistent with normal marine conditions but exhibit negative shifts coincident with increased Hg concentrations. Hg isotope mass-independent fractionation (Δ199Hg) trends are consistent with volcanic input in deep-water marine environments. In contrast, nearshore environments have Δ199Hg signatures consistent with enhanced soil and/or biomass input. We hypothesize that the deep-water signature represents an overall global increase in volcanic Hg input and that this isotope signature is overwhelmed in nearshore locations due to Hg from terrestrial sources. High-productivity nearshore regions may have experienced stressed marine ecosystems due to enhanced Hg loading.
Association between metabolic syndrome and liver histology among NAFLD patients without diabetes
Authors: Kanwar Pushpjeet , Nelson James E , Yates Katherine , Kleiner David E , Unalp-Arida Aynur , Kowdley Kris V
Published: November 2016

Background

Metabolic syndrome (MetS) and obesity are associated with non-alcoholic fatty liver disease (NAFLD). The aim of this observational study was to examine the relationship of MetS and a diagnosis of non-alcoholic steatohepatitis (NASH) in patients without diabetes in the NASH Clinical Research Network (CRN).

Methods

Clinical, demographic, histological, laboratory and anthropometric data were collected on 356 adult patients without diabetes with NAFLD. Obesity was defined as body mass index ≥30.0. MetS was determined using the National Cholesterol Education Program-Adult Treatment Panel III (NCEP ATPIII) 2001 criteria to include 3 or more of the following: increased waist circumference, elevated triglycerides, reduced high-density lipoprotein cholesterol, hypertension and elevated fasting blood glucose.

Results

Most patients were obese (71%) and had MetS (67%). Obesity was more prevalent among patients with MetS (80% vs 52%; p≤0.001). Markers of insulin resistance such as homoeostasis model assessment of insulin resistance (6.5 vs 4.9, p≤0.001) were higher among those with MetS compared with those without MetS. Histologically, patients without MetS had higher hepatocellular (p=0.04) and reticuloendothelial system iron (p=0.04). Patients with MetS were more likely to have severe hepatic steatosis (p=0.04) and chronic portal inflammation (p=0.01). On multiple logistic regression analysis, patients with definite NASH were almost 2.5 times more likely to have MetS than those without definite NASH (OR=2.41, p=0.01).

Conclusions

MetS is common in patients without diabetes with NAFLD and is associated with greater insulin resistance, hepatic steatosis and portal inflammation. While patients without MetS have greater iron overload, patients with MetS may have an increased propensity to have NASH. Therefore, presence of MetS in patients without diabetes with NAFLD may serve as a potential criterion for liver biopsy.

Trial registration number

NCT00063622; Pre-results.
Preference-based disease-specific health-related quality of life instrument for glaucoma: a mixed methods study protocol
Authors: Muratov Sergei , Podbielski Dominik W , Jack Susan M , Ahmed Iqbal Ike K , Mitchell Levine A H , Baltaziak Monika , Xie Feng
Published: November 2016

Introduction

A primary objective of healthcare services is to improve patients' health and health-related quality of life (HRQoL). Glaucoma, which affects a substantial proportion of the world population, has a significant detrimental impact on HRQoL. Although there are a number of glaucoma-specific questionnaires to measure HRQoL, none is preference-based which prevent them from being used in health economic evaluation. The proposed study is aimed to develop a preference-based instrument that is capable of capturing important effects specific to glaucoma and treatments on HRQoL and is scored based on the patients' preferences.

Methods

A sequential, exploratory mixed methods design will be used to guide the development and evaluation of the HRQoL instrument. The study consists of several stages to be implemented sequentially: item identification, item selection, validation and valuation. The instrument items will be identified and selected through a literature review and the conduct of a qualitative study. Validation will be conducted to establish psychometric properties of the instrument followed by a valuation exercise to derive utility scores for the health states described.

Ethics and dissemination

This study has been approved by the Trillium Health Partners Research Ethics Board (ID number 753). All personal information will be de-identified with the identification code kept in a secured location including the rest of the study data. Only qualified and study-related personnel will be allowed to access the data. The results of the study will be distributed widely through peer-reviewed journals, conferences and internal meetings.