bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of Glucose byBifidobacterium animalissubsp.lactisOccurs via Facilitated Diffusion
Authors: Briczinski E. P. , Phillips A. T. , Roberts R. F.
Published: September 2008

Two strains ofBifidobacterium animalissubsp.lactiswere indistinguishable by several nucleic acid-based techniques; however, the type strain DSMZ 10140 was glucose utilization positive, while RB 4825, an industrially employed strain, was unable to grow rapidly on glucose as the principal carbon source. This difference was attributed to the presence of a low-affinity facilitated-diffusion glucose transporter identified in DSMZ 10140 but lacking in RB 4825. Uptake ofd-[U-14C]glucose in DSMZ 10140 was stimulated by monovalent cations (ammonium, sodium, potassium, and lithium) and inhibited by divalent cations (calcium and magnesium). When competitor carbohydrates were included in the uptake assays, stereospecific inhibition was exhibited, with greater competition by methyl-β-glucoside than methyl-α-glucoside. Significant inhibition (>30%) was observed with phloretin, an inhibitor of facilitated diffusion of glucose, whereas there was no inhibition by sodium fluoride, iodoacetate, sodium arsenate, sodium azide, 2,4-dinitrophenol, monensin, or valinomycin, which typically reduce energy-driven transport. Based on kinetic analyses, the mean values forKtandVmaxwere 14.8 ± 3.4 mMd-glucose and 0.13 ± 0.03 μmol glucose/min/mg cell protein, respectively. Glucose uptake by several glucose-utilizing commercial strains ofB. animalissubsp.lactiswas also inhibited by phloretin, indicating the presence of facilitated diffusion glucose transporters in those strains. Since DSMZ 10140 has been previously reported to lack a functional glucose phosphoenolpyruvate phosphotransferase system, the glucose transporter identified here is responsible for much of the organism's glucose uptake.
Host Ranges of
Listeria
-Specific Bacteriophages from the Turkey Processing Plant Environment in the United States
Authors: Kim Jae-Won , Siletzky Robin M. , Kathariou Sophia
Published: September 2008

Even though at least 400
Listeria
phages have been isolated from various sources, limited information is available on phages from the food processing plant environment. Phages in the processing plant environment may play critical roles in determining the
Listeria
population that becomes established in the plant. In this study, we pursued the isolation of
Listeria
-specific phages from environmental samples from four turkey processing plants in the United States. These environmental samples were also utilized to isolate
Listeria
spp. Twelve phages were isolated and classified into three groups in terms of their host range. Of these, nine (group 1) showed a wide host range, including multiple serotypes of
Listeria monocytogenes
, as well as other
Listeria
spp. (
L. innocua
,
L. welshimeri
,
L. seeligeri
, and
L. ivanovii
). The remaining phages mostly infected
L. monocytogenes
serotype 4b as well as
L. innocua
,
L. ivanovii
, and/or
L. welshimeri
. All but one of the strains of the serotype 4b complex (4b, 4d, 4e) from the processing plant environment could be readily infected by the wide-host-range phages isolated from the environment of the processing plants. However, many strains of other serotypes (1/2a [or 3a] and 1/2b [or 3b]), which represented the majority of
L. monocytogenes
strains isolated from the environmental samples, were resistant to infection by these phages. Experiments with two phage-resistant strains showed reduced phage adsorption onto the host cells. These findings suggest that phage resistance may be an important component of the ecology of
L. monocytogenes
in the turkey processing plants.
Diverse Phage-Encoded Toxins in a Protective Insect Endosymbiont
Authors: Degnan Patrick H. , Moran Nancy A.
Published: September 2008

The lysogenic bacteriophage APSE infects “CandidatusHamiltonella defensa,” a facultative endosymbiont of aphids and other sap-feeding insects. This endosymbiont has established a beneficial association with aphids, increasing survivorship following attack by parasitoid wasps. Although APSE and “Ca. Hamiltonella defensa” are effectively maternally transmitted between aphid generations, they can also be horizontally transferred among insect hosts, which results in genetically distinct “Ca. Hamiltonella defensa” strains infecting the same aphid species and sporadic distributions of both APSE and “Ca. Hamiltonella defensa” among hosts. Aphids infected only with “Ca. Hamiltonella defensa” have significantly less protection than those infected with both “Ca. Hamiltonella defensa” and APSE. This protection has been proposed to be connected to eukaryote-targeted toxins previously discovered in the genomes of two characterized APSE strains. In this study, we have sequenced partial genomes from seven additional APSE strains to address the evolution and extent of toxin variation in this phage. The APSE lysis region has been a hot spot for nonhomologous recombination of novel virulence cassettes. We identified four new toxins from three protein families, Shiga-like toxin, cytolethal distending toxin, and YD-repeat toxins. These recombination events have also resulted in reassortment of the downstream lysozyme and holin genes. Analysis of the conserved APSE genes flanking the variable toxin cassettes reveals a close phylogenetic association with phage sequences from two other facultative endosymbionts of insects. Thus, phage may act as a conduit for ongoing gene exchange among heritable endosymbionts.
Mechanistic Dissection of the Enzyme Complexes Involved in Biosynthesis of Lacticin 3147 and Nisin
Authors: Kuipers Anneke , Meijer-Wierenga Jenny , Rink Rick , Kluskens Leon D. , Moll Gert N.
Published: September 2008

The thioether rings in the lantibiotics lacticin 3147 and nisin are posttranslationally introduced by dehydration of serines and threonines, followed by coupling of these dehydrated residues to cysteines. The prepeptides of the two-component lantibiotic lacticin 3147, LtnA1 and LtnA2, are dehydrated and cyclized by two corresponding bifunctional enzymes, LtnM1 and LtnM2, and are subsequently processed and exported via one bifunctional enzyme, LtnT. In the nisin synthetase complex, the enzymes NisB, NisC, NisT, and NisP dehydrate, cyclize, export, and process prenisin, respectively. Here, we demonstrate that the combination of LtnM2 and LtnT can modify, process, and transport peptides entirely different from LtnA2 and that LtnT can process and transport unmodified LtnA2 and unrelated peptides. Furthermore, we demonstrate a higher extent of NisB-mediated dehydration in the absence of thioether rings. Thioether rings apparently inhibited dehydration, which implies alternating actions of NisB and NisC. Furthermore, certain (but not all) NisC-cyclized peptides were exported with higher efficiency as a result of their conformation. Taken together, these data provide further insight into the applicability of
Lactococcus lactis
strains containing lantibiotic enzymes for the design and production of modified peptides.
Codon Optimization Increases Steady-State mRNA Levels in
Aspergillus oryzae
Heterologous Gene Expression
Authors: Tokuoka Masafumi , Tanaka Mizuki , Ono Kazuhisa , Takagi Shinobu , Shintani Takahiro , Gomi Katsuya
Published: September 2008

We investigated the effect of codon optimization on the expression levels of heterologous proteins in
Aspergillus oryzae
, using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in
A. oryzae
by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with
A. oryzae
glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3′ rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization in
A. oryzae
results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.
Kinetics of Reduction of Fe(III) Complexes by Outer Membrane Cytochromes MtrC and OmcA ofShewanella oneidensisMR-1
Authors: Wang Zheming , Liu Chongxuan , Wang Xuelin , Marshall Matthew J. , Zachara John M. , Rosso Kevin M. , Dupuis Michel , Fredrickson James K. , Heald Steve , Shi Liang
Published: September 2008

Because of their cell surface locations, the outer membranec-type cytochromes MtrC and OmcA ofShewanella oneidensisMR-1 have been suggested to be the terminal reductases for a range of redox-reactive metals that form poorly soluble solids or that do not readily cross the outer membrane. In this work, we determined the kinetics of reduction of a series of Fe(III) complexes with citrate, nitrilotriacetic acid (NTA), and EDTA by MtrC and OmcA using a stopped-flow technique in combination with theoretical computation methods. Stopped-flow kinetic data showed that the reaction proceeded in two stages, a fast stage that was completed in less than 1 s, followed by a second, relatively slower stage. For a given complex, electron transfer by MtrC was faster than that by OmcA. For a given cytochrome, the reaction was completed in the order Fe-EDTA > Fe-NTA > Fe-citrate. The kinetic data could be modeled by two parallel second-order bimolecular redox reactions with second-order rate constants ranging from 0.872 μM−1s−1for the reaction between MtrC and the Fe-EDTA complex to 0.012 μM−1s−1for the reaction between OmcA and Fe-citrate. The biphasic reaction kinetics was attributed to redox potential differences among the heme groups or redox site heterogeneity within the cytochromes. The results of redox potential and reorganization energy calculations showed that the reaction rate was influenced mostly by the relatively large reorganization energy. The results demonstrate that ligand complexation plays an important role in microbial dissimilatory reduction and mineral transformation of iron, as well as other redox-sensitive metal species in nature.
Formate-Dependent H
2
Production by the Mesophilic Methanogen
Methanococcus maripaludis
Authors: Lupa Boguslaw , Hendrickson Erik L. , Leigh John A. , Whitman William B.
Published: September 2008

Methanococcus maripaludis
, an H
2
- and formate-utilizing methanogen, produced H
2
at high rates from formate. The rates and kinetics of H
2
production depended upon the growth conditions, and H
2
availability during growth was a major factor. Specific activities of resting cells grown with formate or H
2
were 0.4 to 1.4 U·mg
−1
(dry weight). H
2
production in formate-grown cells followed Michaelis-Menten kinetics, and the concentration of formate required for half-maximal activity (

K
f

) was 3.6 mM. In contrast, in H
2
-grown cells this process followed sigmoidal kinetics, and the

K
f

was 9 mM. A key enzyme for formate-dependent H
2
production was formate dehydrogenase, Fdh. H
2
production and growth were severely reduced in a mutant containing a deletion of the gene encoding the Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H
2
production by a mutant containing a deletion of the coenzyme F
420
-reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H
2
production comprised Fdh1 and Fru. Because a Δ
fru
-Δ
frc
mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F
420
-dependent methylene-H
4
MTP dehydrogenase (Mtd) or the H
2
-forming methylene-H
4
MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H
2
production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H
2
production was not a direct intermediate of methanogenesis. In conclusion, high rates of formate-dependent H
2
production demonstrated the potential of
M. maripaludis
for the microbial production of H
2
from formate.
Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the
Sinorhizobium meliloti
ORFeome
Authors: Humann Jodi L. , Schroeder Brenda K. , Mortimer Michael W. , House Brent L. , Yurgel Svetlana N. , Maloney Scott C. , Ward Kristel L. , Fallquist Heather M. , Ziemkiewicz Hope T. , Kahn Michael L.
Published: September 2008

The
Sinorhizobium meliloti
ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (
gusA
) and green fluorescent protein (
gfp
) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the
S. meliloti
SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.
Hydrogen Peroxide Induces Hyphal Differentiation in
Candida albicans
Authors: Nasution Olviyani , Srinivasa Kavitha , Kim Minsun , Kim Yeo-Jung , Kim Wankee , Jeong Woojin , Choi Wonja
Published: September 2008

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H
2
O
2
in
Candida albicans.
This finding is confirmed by the changing intracellular concentration of H
2
O
2
. In order to induce the same level of differentiation, low concentrations of exogenous H
2
O
2
are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.
African Trypanosomes Contain 5-Methylcytosine in Nuclear DNA
Authors: Militello Kevin T. , Wang Ping , Jayakar Sangeeta K. , Pietrasik Rebecca L. , Dupont Christopher D. , Dodd Kristi , King Anthony M. , Valenti Paul R.
Published: September 2008

It is currently unclear if there are modified DNA bases in
Trypanosoma brucei
other than J-base. We identify herein a cytosine-5 DNA methyltransferase gene and report the presence and location of 5-methylcytosine in genomic DNA. Our data demonstrate that African trypanosomes contain a functional cytosine DNA methylation pathway.
Role of the Histone-Like Nucleoid Structuring Protein in the Regulation of
rpoS
and RpoS-Dependent Genes in
Vibrio cholerae
Authors: Silva Anisia J. , Sultan Syed Zafar , Liang Weili , Benitez Jorge A.
Published: September 2008

Production of the Zn-metalloprotease hemagglutinin (HA)/protease by
Vibrio cholerae
has been reported to enhance enterotoxicity in rabbit ileal loops and the reactogenicity of live cholera vaccine candidates. Expression of HA/protease requires the alternate sigma factor σ
S
(RpoS), encoded by
rpoS
. The histone-like nucleoid structuring protein (H-NS) has been shown to repress
rpoS
expression in
Escherichia coli
. In
V. cholerae
strains of the classical biotype, H-NS has been reported to silence virulence gene expression. In this study we examined the role of H-NS in the expression of HA/protease and motility in an El Tor biotype strain by constructing a Δ
hns
mutant. The Δ
hns
mutant exhibited multiple phenotypes, such as production of cholera toxin in nonpermissive LB medium, reduced resistance to high osmolarity, enhanced resistance to low pH and hydrogen peroxide, and reduced motility. Depletion of H-NS by overexpression of a dominant-negative allele or by deletion of
hns
resulted in diminished expression of HA/protease. Epistasis analysis of HA/protease expression in Δ
hns
, Δ
rpoS
, and Δ
hns
Δ
rpoS
mutants, analysis of RpoS reporter fusions, quantitative reverse transcription-PCR measurements, and ectopic expression of RpoS in Δ
rpoS
and Δ
rpoS
Δ
hns
mutants showed that H-NS posttranscriptionally enhances RpoS expression. The Δ
hns
mutant exhibited a lower degree of motility and lower levels of expression of
flaA
,
flaC
,
cheR
-
2
, and
motX
mRNAs than the wild type. Comparison of the mRNA abundances of these genes in wild-type, Δ
hns
, Δ
rpoS
, and Δ
hns
Δ
rpoS
strains revealed that deletion of
rpoS
had a more severe negative effect on their expression. Interestingly, deletion of
hns
in the
rpoS
background resulted in higher expression levels of
flaA
,
flaC
, and
motX
, suggesting that H-NS represses the expression of these genes in the absence of σ
S
. Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.
A Possible Extended Family of Regulators of Sigma Factor Activity inStreptomyces coelicolor
Authors: Kim Eun Sook , Song Ju Yeon , Kim Dae Wi , Chater Keith F. , Lee Kye Joon
Published: September 2008

SCO4677 is one of a large number of similar genes inStreptomyces coelicolorthat encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB ofBacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific σF, suggesting that it is an antagonistic regulator of σF. Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-σFduring a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA ofB. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.
The ABC-Type Multidrug Resistance Transporter LmrCD Is Responsible for an Extrusion-Based Mechanism of Bile Acid Resistance in
Lactococcus lactis
Authors: Zaidi Arsalan Haseeb , Bakkes Patrick J. , Lubelski Jacek , Agustiandari Herfita , Kuipers Oscar P. , Driessen Arnold J. M.
Published: September 2008

Upon prolonged exposure to cholate and other toxic compounds,
Lactococcus lactis
develops a multidrug resistance phenotype that has been attributed to an elevated expression of the heterodimeric ABC-type multidrug transporter LmrCD. To investigate the molecular basis of bile acid resistance in
L. lactis
and to evaluate the contribution of efflux-based mechanisms in this process, the drug-sensitive
L. lactis
NZ9000 Δ
lmrCD
strain was challenged with cholate. A resistant strain was obtained that, compared to the parental strain, showed (i) significantly improved resistance toward several bile acids but not to drugs, (ii) morphological changes, and (iii) an altered susceptibility to antimicrobial peptides. Transcriptome and transport analyses suggest that the acquired resistance is unrelated to elevated transport activity but, instead, results from a multitude of stress responses, changes to the cell envelope, and metabolic changes. In contrast, wild-type cells induce the expression of
lmrCD
upon exposure to cholate, whereupon the cholate is actively extruded from the cells. Together, these data suggest a central role for an efflux-based mechanism in bile acid resistance and implicate LmrCD as the main system responsible in
L. lactis
.
Identification and Characterization of Cyclic Diguanylate Signaling Systems Controlling Rugosity in
Vibrio cholerae
Authors: Beyhan Sinem , Odell Lindsay S. , Yildiz Fitnat H.
Published: September 2008

Vibrio cholerae
, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the
V. cholerae
genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes,
cdgG
and
cdgH
, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in
V. cholerae
. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in
V. cholerae
.
Lipoteichoic Acid Is a Major Component of the
Bacillus subtilis
Periplasm
Authors: Matias Valério R. F. , Beveridge Terry J.
Published: September 2008

Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive bacteria, this method reveals the presence of a periplasmic space between the plasma membrane and an often differentiated cell wall matrix. Since virtually nothing is known about the composition of its constituent matter (i.e., the periplasm), it is still unclear what structures (or mechanism) sustain a gram-positive periplasmic space. Here we have used cryo-EM of frozen-hydrated sections in combination with various labels to probe the model gram-positive organism
Bacillus subtilis
for major periplasmic components. Incubation of cells with positively charged gold nanoparticles showed almost similar levels of gold binding to the periplasm and the cell wall. On cells whose cell walls were enzymatically hydrolyzed (i.e., on protoplasts), a surface diffuse layer extending ∼30 nm from the membrane was revealed. The thickness and density of this layer were not significantly altered after treatment with a nonspecific protease, whereas it was labeled with anti-lipoteichoic acid (LTA) antibodies conjugated to nanogold. Further, the LTA layer spans most of the thickness of the periplasmic space, which strongly suggests that LTA is a major component of the
B. subtilis
periplasm.
Outer and Inner Membrane Proteins Compose an Arginine-Agmatine Exchange System inChlamydophila pneumoniae
Authors: Smith Conor B. , Graham David E.
Published: September 2008

Most chlamydial strains have a pyruvoyl-dependent decarboxylase protein that convertsl-arginine to agmatine. However, chlamydiae do not produce arginine, so they must import it from their host.Chlamydophila pneumoniaehas a gene cluster encoding a putative outer membrane porin (CPn1033 oraaxA), an arginine decarboxylase (CPn1032 oraaxB), and a putative cytoplasmic membrane transporter (CPn1031 oraaxC). TheaaxCgene was expressed inEscherichia coliproducing an integral cytoplasmic membrane protein that catalyzed the exchange ofl-arginine for agmatine. Expression of theaaxAgene produced an outer membrane protein that enhanced the arginine uptake and decarboxylation activity of cells coexpressingaaxBandaaxC. This chlamydial arginine/agmatine exchange system complemented anE. colimutant missing the native arginine-dependent acid resistance system. These cells survived extreme acid shock in the presence ofl-arginine. Biochemical and evolutionary analysis showed theaaxABCgenes evolved convergently with the enteric arginine degradation system, and they could have a different physiological role in chlamydial cells. The chlamydial system uniquely includes an outer membrane porin, and it is most active at a higher pH from 3 to 5. The chlamydial AaxC transporter was resistant to cadaverine,l-lysine andl-ornithine, which inhibit theE. coliAdiC antiporter.
Hypernegative Supercoiling Inhibits Growth by Causing RNA Degradation
Authors: Baaklini Imad , Usongo Valentine , Nolent Flora , Sanscartier Patrick , Hraiky Chadi , Drlica Karl , Drolet Marc
Published: September 2008

Transcription-induced hypernegative supercoiling is a hallmark ofEscherichia colitopoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA on ribosomes was reduced forfisand shorter forcrp, polysomes were much less abundant relative to monosomes, and protein synthesis rate dropped, as did the ratio of large to small proteins. Altered processing and degradation oflacAandfismRNA was also observed. These data are consistent with truncation of mRNA during growth arrest. These effects were not affected by a mutation in the gene encoding RNase E, indicating that this endonuclease is not involved in the abnormal mRNA processing. They were also unaffected by spectinomycin, an inhibitor of protein synthesis, which argued against induction of RNase activity. In vitro transcription revealed that R-loop formation is more extensive on hypernegatively supercoiled templates. These results allow us, for the first time, to present a model by which hypernegative supercoiling inhibits growth. In this model, the introduction of hypernegative supercoiling by gyrase facilitates degradation of nascent RNA; overproduction of RNase HI limits the accumulation of hypernegative supercoiling, thereby preventing extensive RNA degradation.
The Complete Genome Sequence of
Thermococcus onnurineus
NA1 Reveals a Mixed Heterotrophic and Carboxydotrophic Metabolism
Authors: Lee Hyun Sook , Kang Sung Gyun , Bae Seung Seob , Lim Jae Kyu , Cho Yona , Kim Yun Jae , Jeon Jeong Ho , Cha Sun-Shin , Kwon Kae Kyoung , Kim Hyung-Tae , Park Cheol-Joo , Lee Hee-Wook , Kim Seung Il , Chun Jongsik , Colwell Rita R. , Kim Sang-Jin , Lee Jung-Hyun
Published: September 2008

Members of the genus
Thermococcus
, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of
Thermococcus onnurineus
NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis,
T. onnurineus
NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO
2
, thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for
T. onnurineus
NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in
Thermococcus
.
RstA-Promoted Expression of the Ferrous Iron Transporter FeoB under Iron-Replete Conditions Enhances Fur Activity in
Salmonella enterica
Authors: Jeon Jihye , Kim Hyunkeun , Yun Jiae , Ryu Sangryeol , Groisman Eduardo A. , Shin Dongwoo
Published: September 2008

The Fur protein is a primary regulator that monitors and controls cytoplasmic iron levels. We now report the identification of a regulatory pathway mediated by the
Salmonella
response regulator RstA that promotes Fur activity. Genome-wide expression experiments revealed that under iron-replete conditions, expression of the RstA protein from a plasmid lowered transcription levels of various genes involved in iron acquisition. The RstA protein controlled iron-responsive genes through the Fur-Fe(II) protein because deletion of the
fur
gene or iron depletion abrogated RstA-mediated repression of these genes. The RstA protein maintained wild-type levels of the Fur protein but exceptionally activated transcription of the
feoAB
operon encoding the ferrous iron transporter FeoB by binding directly to the
feoA
promoter. This FeoB induction resulted in increased ferrous iron uptake, which associates with the Fur protein because lack of RstA-dependent transcriptional activation of the
feoA
promoter and
feoB
-deletion abolished repression of the Fur target genes by the RstA protein. Under iron-replete conditions, RstA expression retarded
Salmonella
growth but enabled the Fur protein to repress the target genes beyond the levels which were simply accomplished by iron.
DNA Microarray Comparisons of Plant Factor- and Nitrogen Deprivation-Induced Hormogonia Reveal Decision-Making Transcriptional Regulation Patterns in
Nostoc punctiforme
Authors: Campbell Elsie L. , Christman Harry , Meeks John C.
Published: September 2008

Hormogonia are nongrowing filaments, motile by means of a gliding mechanism, that are produced by certain cyanobacteria. Their differentiation is induced by positive and negative factors for growth, such as deprivation of combined nitrogen (nitrogen stress induction [NSI]). In
Nostoc punctiforme
, they are also induced by the exudate (hormogonium-inducing factor [HIF]) of a symbiotic plant partner. Time course (0.5 to 24 h) transcription profiles were determined by DNA microarray assays for hormogonia of
N. punctiforme
following induction by HIF and NSI. Clustering analysis revealed both common and distinct transcriptional patterns for the two methods of induction. By 24 h, a common set of 1,328 genes was identified. This 24-h common set of genes arose by the transition of 474 genes from an 819-member common set of genes at 1 h after induction; 405 and 51 genes unique to the HIF and NSI groups at 1 h, respectively; and 398 genes differentially transcribed at later time points. The NSI hormogonia showed a transcriptional checkpoint at 12 h following induction in which up- and downregulated genes were transiently down- or upregulated, respectively. The transient changes in these 1,043 genes appeared to reflect a switch back to a vegetative growth state. Such a checkpoint was not seen in HIF hormogonia. Genes uniquely upregulated in HIF hormogonia included those encoding proteins hypothesized to synthesize a metabolite repressor of hormogonium differentiation. Approximately 34 to 42% of the 6,893 printed genes were differentially transcribed during hormogonium differentiation; about half of those genes were upregulated, and 1,034 genes responded within 0.5 h after induction. These collective results indicate extensive and rapid global changes in the transcription of specific genes during the differentiation of these specialized filaments.
Divergence among Genes Encoding the Elongation Factor Tu of
Yersinia
Species
Authors: Isabel Sandra , Leblanc Éric , Boissinot Maurice , Boudreau Dominique K. , Grondin Myrian , Picard François J. , Martel Eric A. , Parham Nicholas J. , Chain Patrick S. G. , Bader Douglas E. , Mulvey Michael R. , Bryden Louis , Roy Paul H. , Ouellette Marc , Bergeron Michel G.
Published: September 2008

Elongation factor Tu (EF-Tu), encoded by
tuf
genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated
tuf
genes (
tufA
and
tufB
), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of
tuf
genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus
Yersinia
(representing 12
Yersinia
species). The levels of intragenomic divergence between
tufA
and
tufB
sequences ranged from 8.3 to 16.2% for the genus
Yersinia
, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored
tuf
gene evolution in
Yersinia
and other
Enterobacteriaceae
by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated
tufA
and
tufB
sequences revealed a monophyletic genus
Yersinia
in the family
Enterobacteriaceae
. Moreover,
Yersinia
strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between
Yersinia tufA
and
tufB
sequences, a feature unique among sequenced
Enterobacteriaceae
and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of
Yersinia
species.
Characterization of Genes Regulated Directly by the VirR/VirS System in
Clostridium perfringens
Authors: Okumura Kayo , Ohtani Kaori , Hayashi Hideo , Shimizu Tohru
Published: September 2008

Analysis of the complete sequence of the genome of
Clostridium perfringens
strain 13 resulted in identification of five genes, including
pfoA
(encoding theta toxin) and
vrr
(encoding VirR/VirS-regulated RNA), with consensus VirR-binding sequences upstream of the open reading frame (ORF), suggesting that expression of these genes may be regulated directly by the two-component VirR/VirS system. To test this possibility, we examined VirR/VirS system-mediated transcriptional regulation of three genes,
virT
,
ccp
(encoding alpha-clostripain), and
virU
, with the novel VirR-binding sequences. Northern analysis revealed that the steady-state levels (increases or decreases in the amounts of RNA expressed) of
virT
,
ccp
, and
virU
mRNAs were lower in a
virR
mutant strain than in the wild-type strain, as were the levels of the
pfoA
and
vrr
transcripts. The consensus VirR-binding sites were located similarly relative to the transcription start sites in the
virT
,
ccp
, and
virU
promoters. Mutation and overexpression analyses with
virT
and
virU
revealed that the
virT
gene product has a negative effect on expression of
pfoA
and
ccp
, whereas the
virU
gene product positively affects expression of
pfoA
,
virT
,
ccp
, and
vrr
. Nonsense and frameshift mutations in the
virT
or
virU
putative ORF did not affect the regulatory functions, suggesting that
virT
and
virU
may encode RNA regulators rather than proteins. These results suggest that a complex regulatory network, perhaps involving several regulatory RNA molecules, governs the expression of the VirR/VirS regulon in
C. perfringens
.
Molecular Types and Genetic Profiles of
Staphylococcus aureus
Strains Isolated from Bovine Intramammary Infections and Extramammary Sites
Authors: Haveri M. , Hovinen M. , Roslöf A. , Pyörälä S.
Published: September 2008

Staphylococcus aureus
isolates collected from sites of intramammary infection during a 10-month period and from extramammary sites (dairy cow teat skin, teat canals, and skin lesions; milking liners; and hands and nostrils of milking personnel) at two separately managed Finnish dairy herd establishments were analyzed to study the sources and reservoirs of bovine
S. aureus
intramammary infection. Selected isolates were subjected to pulsed-field gel electrophoresis (PFGE) typing and PCR analysis for genes encoding hemolysins (
hla
to
hlg
), leukocidins (
lukED
and
lukM
), superantigens (
sea
,
sec
,
sed
,
seg
to
seo
,
seu
, and
tst
), adhesins (
fnbA
and
fnbB
), and penicillin and methicillin resistance (
blaZ
and
mecA
).
S. aureus
was found throughout the herds in 94% of the cows. Nine PFGE types were found, with the herds each having their own predominant type and sharing one type. The degree of diversity of PFGE types in herd II, which integrated foreign heifers, was higher than that in herd I. For both herds, the majority of the PFGE-typed isolates both from milk and from extramammary sites represented the predominant PFGE types. In isolates from herd I, the most prevalent genes were
hla
-
hlg
,
lukED
, and
fnbA
; in those from herd II, they were
hla
,
hld
,
hlg
,
lukED
, and
fnbA
. The other genes were pulsotype linked within the herds. The predominant PFGE types carried both
fnbA
and
fnbB
; only
fnbA
was detected in the other PFGE types. No connection between specific virulence genes and the origins of isolates was found. The results suggest that for the two herds, most
S. aureus
isolates from extramammary sites were indistinguishable from the isolates infecting the mammary gland and that those sites can thus act as origins and reservoirs of intramammary infections. However, contamination in the opposite direction cannot be excluded.
Use of Fungal Blood Cultures in an Academic Medical Center
Authors: Kosmin Aaron R. , Fekete Thomas
Published: September 2008

We studied the use of fungal blood cultures in our hospital. They added little compared to routine culture results, but clinicians ordered them for sicker patients, when facing diagnostic uncertainty, or after prior candidemia. We need a practical guideline for when to order fungal blood cultures.
Kinetics of Epstein-Barr Virus DNA Load in Different Blood Compartments of Pediatric Recipients of T-Cell-Depleted HLA-Haploidentical Stem Cell Transplantation
Authors: Baldanti Fausto , Gatti Marta , Furione Milena , Paolucci Stefania , Tinelli Carmine , Comoli Patrizia , Merli Pietro , Locatelli Franco
Published: September 2008

Epstein-Barr virus (EBV) DNA levels in whole-blood samples of 54 pediatric patients receiving T-cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT) in 2003 to 2007 were retrospectively compared with EBV DNA loads in peripheral blood mononuclear cells (PBMC). Determination of EBV DNA in whole blood missed 1 of 19 patients (5.2%), who tested positive for EBV DNA in PBMC. The analytical sensitivity of EBV DNA detection in whole-blood samples relative to that in PBMC was 94.7%. Regression analysis showed a significant correlation between DNA levels in PBMC and whole blood (
r
= 0.81;
P
< 0.001). Relative to that in PBMC, the appearance of EBV DNA in whole blood was delayed in 9/18 patients (median, 49 days; range, 6 to 226 days), while peak levels and clearance were reached simultaneously. Following peak levels, EBV DNA showed a slower decline in whole blood than in PBMC. In conclusion, (i) EBV DNA levels in PBMC were significantly correlated with those in whole blood; (ii) a differential kinetics of EBV DNA in the two blood compartments was observed; and (iii) monitoring of EBV DNA levels in whole blood appears to be a valuable alternative to PBMC in the follow-up of pediatric recipients of haploidentical T-cell-depleted HSCT.
Bioinformatic and Physical Characterizations of Genome-Scale Ordered RNA Structure in Mammalian RNA Viruses
Authors: Davis Matthew , Sagan Selena M. , Pezacki John P. , Evans David J. , Simmonds Peter
Published: September 2008

By the analysis of thermodynamic RNA secondary structure predictions, we previously obtained evidence for evolutionarily conserved large-scale ordering of RNA virus genomes (P. Simmonds, A. Tuplin, and D. J. Evans, RNA 10:1337-1351, 2004). Genome-scale ordered RNA structure (GORS) was widely distributed in many animal and plant viruses, much greater in extent than RNA structures required for viral translation or replication, but in mammalian viruses was associated with host persistence. To substantiate the existence of large-scale RNA structure differences between viruses, a large set of alignments of mammalian RNA viruses and rRNA sequences as controls were examined by thermodynamic methods (to calculate minimum free energy differences) and by algorithmically independent RNAz and Pfold methods. These methods produced generally concordant results and identified substantial differences in the degrees of evolutionarily conserved, sequence order-dependent RNA secondary structure between virus genera and groups. A probe hybridization accessibility assay was used to investigate the physical nature of GORS. Transcripts of hepatitis C virus (HCV), hepatitis G virus/GB virus-C (HGV/GBV-C), and murine norovirus, which are predicted to be structured, were largely inaccessible to hybridization in solution, in contrast to the almost universal binding of probes to a range of unstructured virus transcripts irrespective of G+C content. Using atomic force microscopy, HCV and HGV/GBV-C RNA was visualized as tightly compacted prolate spheroids, while under the same experimental conditions the predicted unstructured poliovirus and rubella virus RNA were pleomorphic and had extensively single-stranded RNA on deposition. Bioinformatic and physical characterization methods both identified fundamental differences in the configurations of viral genomic RNA that may modify their interactions with host cell defenses and their ability to persist.
Utilization of DC-SIGN for Entry of Feline Coronaviruses into Host Cells
Authors: Regan Andrew D. , Whittaker Gary R.
Published: September 2008

The entry and dissemination of viruses in several families can be mediated by C-type lectins such as DC-SIGN. We showed that entry of the serotype II feline coronavirus strains feline infectious peritonitis virus (FIPV) WSU 79-1146 and DF2 into nonpermissive mouse 3T3 cells can be rescued by the expression of human DC-SIGN (hDC-SIGN) and that infection of a permissive feline cell line (Crandall-Reese feline kidney) was markedly enhanced by the overexpression of hDC-SIGN. Treatment with mannan considerably reduced infection of feline monocyte-derived cells expressing DC-SIGN, indicating a role for FIPV infection in vivo.
Host Ethnicity and Virus Genotype Shape the Hepatitis B Virus-Specific T-Cell Repertoire
Authors: Tan Anthony Tanoto , Loggi Elisabetta , Boni Carolina , Chia Adeline , Gehring Adam J. , Sastry Konduru S. R. , Goh Vera , Fisicaro Paola , Andreone Pietro , Brander Christian , Lim Seng Gee , Ferrari Carlo , Bihl Florian , Bertoletti Antonio
Published: September 2008

Repertoire composition, quantity, and qualitative functional ability are the parameters that define virus-specific T-cell responses and are linked with their potential to control infection. We took advantage of the segregation of different hepatitis B virus (HBV) genotypes in geographically and genetically distinct host populations to directly analyze the impact that host and virus variables exert on these virus-specific T-cell parameters. T-cell responses against the entire HBV proteome were analyzed in a total of 109 HBV-infected subjects of distinct ethnicities (47 of Chinese origin and 62 of Caucasian origin). We demonstrate that HBV-specific T-cell quantity is determined by the virological and clinical profiles of the patients, which outweigh any influence of race or viral diversity. In contrast, HBV-specific T-cell repertoires are divergent in the two ethnic groups, with T-cell epitopes frequently found in Caucasian patients seldom detected in Chinese patients. In conclusion, we provide a direct biological evaluation of the impact that host and virus variables exert on virus-specific T-cell responses. The discordance between HBV-specific CD8 T-cell repertoires present in Caucasian and Chinese subjects shows the ability of HLA micropolymorphisms to diversify T-cell responses and has implications for the rational development of therapeutic and prophylactic vaccines for worldwide use.
Role of the Hepatitis C Virus Core+1 Open Reading Frame and Corecis-Acting RNA Elements in Viral RNA Translation and Replication
Authors: Vassilaki Niki , Friebe Peter , Meuleman Philipe , Kallis Stephanie , Kaul Artur , Paranhos-Baccalà Glaucia , Leroux-Roels Geert , Mavromara Penelope , Bartenschlager Ralf
Published: September 2008

Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.
First Cohort of Young-of-the-year Dungeness Crab, Cancer magister, Reduces Abundance of Subsequent Cohorts in Intertidal Shell Habitat
Authors: Fernandez Miriam , Armstrong David , Iribarne Oscar
Published: September 2008

Arrival and settlement of successive cohorts of Dungeness crab, Cancer magister, megalopae were observed in Grays Harbor, Washington, in 1991. The first cohort of megalopae entered the estuary between May 15 and 20 and settled in ("occupied") previously constructed artificial, intertidal oyster shell habitats at densities ranging from 155 to 196 first instar juvenile (J1) crabs∙m−2. Subsequently, a second set of shell habitats was constructed that did not contain crabs of the first cohort ("unoccupied"). Between June 15 and 18, when crabs of the first cohort were a mix of second and third instars, a second cohort of megalopae settled in both occupied and unoccupied shell habitat at respective J1 crab densities of 9–37 and 168–298 crabs∙m−2. The possible roles of cannibalism, competition, and conspecific avoidance are proposed as alternative hypotheses to explain significantly lower density of the second cohort in shell habitats previously occupied by larger conspecifics of the first cohort. Based on laboratory experiments, we suggest that cannibalism is a plausible explanation of our field observations.