bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational Analysis of the Flagellar Protein FliG: Sites of Interaction with FliM and Implications for Organization of the Switch Complex
Authors: Brown Perry N. , Terrazas Moises , Paul Koushik , Blair David F.
Published: December 2006

The switch complex at the base of the bacterial flagellum is essential for flagellar assembly, rotation, and switching. In
Escherichia coli
and
Salmonella
, the complex contains about 26 copies of FliG, 34 copies of FliM, and more then 100 copies of FliN, together forming the basal body C ring. FliG is involved most directly in motor rotation and is located in the upper (membrane-proximal) part of the C ring. A crystal structure of the middle and C-terminal parts of FliG shows two globular domains connected by an α-helix and a short extended segment. The middle domain of FliG has a conserved surface patch formed by the residues EHPQ
125-128
and R
160
(the EHPQR motif), and the C-terminal domain has a conserved surface hydrophobic patch. To examine the functional importance of these and other surface features of FliG, we made mutations in residues distributed over the protein surface and measured the effects on flagellar assembly and function. Mutations preventing flagellar assembly occurred mainly in the vicinity of the EHPQR motif and the hydrophobic patch. Mutations causing aberrant clockwise or counterclockwise motor bias occurred in these same regions and in the waist between the upper and lower parts of the C-terminal domain. Pull-down assays with glutathione
S
-transferase-FliM showed that FliG interacts with FliM through both the EHPQR motif and the hydrophobic patch. We propose a model for the organization of FliG and FliM subunits that accounts for the FliG-FliM interactions identified here and for the different copy numbers of FliG and FliM in the flagellum.
Global Gene Expression and Phenotypic Analysis of a
Vibrio cholerae rpoH
Deletion Mutant
Authors: Slamti Leyla , Livny Jonathan , Waldor Matthew K.
Published: December 2006

Vibrio cholerae
, the cause of cholera, can grow in a variety of environments outside of human hosts. During infection, this pathogen must adapt to significant environmental alterations, including the elevated temperature of the human gastrointestinal tract. σ
32
, an alternative sigma factor encoded by
rpoH
, activates transcription of genes involved in the heat shock response in several bacterial species. Here, we assessed the role of σ
32
in
V. cholerae
physiology. In aggregate, our findings suggest that σ
32
promotes
V. cholerae
growth at temperatures ranging at least from 15°C to 42°C. Growth of the
rpoH
mutant was severely attenuated within the suckling mouse intestine, suggesting that σ
32
-regulated genes are critical for
V. cholerae
adaptation to conditions within the gastrointestinal tract. We defined the
V. cholerae
RpoH regulon by comparing the whole-genome transcription profiles of the wild-type and
rpoH
mutant strains after a temperature up-shift. Most of the
V. cholerae
genes expressed in an RpoH-dependent manner after heat shock encode proteins that influence protein fate, such as proteases and chaperones, or are of unknown function. Bioinformatic analyses of the microarray data were used to define a putative σ
32
consensus binding sequence and subsequently to identify genes that are likely to be directly regulated by RpoH in the whole
V. cholerae
genome.
Complete and Integrated Pyrene Degradation Pathway inMycobacterium vanbaaleniiPYR-1 Based on Systems Biology
Authors: Kim Seong-Jae , Kweon Ohgew , Jones Richard C. , Freeman James P. , Edmondson Ricky D. , Cerniglia Carl E.
Published: December 2006

Mycobacterium vanbaaleniiPYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway forM. vanbaaleniiPYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with theM. vanbaaleniiPYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates througho-phthalate and the β-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism inM. vanbaaleniiPYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.
Phosphotransfer Reactions of the CbbRRS Three-Protein Two- Component System from
Rhodopseudomonas palustris
CGA010 Appear To Be Controlled by an Internal Molecular Switch on the Sensor Kinase
Authors: Romagnoli Simona , Tabita F. Robert
Published: December 2006

The CbbRRS system is an atypical three-protein two-component system that modulates the expression of the
cbb
I
CO
2
fixation operon of
Rhodopseudomonas palustris
, possibly in response to a redox signal. It consists of a membrane-bound hybrid sensor kinase, CbbSR, with a transmitter and receiver domain, and two response regulator proteins, CbbRR1 and CbbRR2. No detectable helix-turn-helix DNA binding domain is associated with either response regulator, but an HPt domain and a second receiver domain are predicted at the C-terminal region of CbbRR1 and CbbRR2, respectively. The abundance of conserved residues predicted to participate in a His-Asp phosphorelay raised the question of their de facto involvement. In this study, the role of the multiple receiver domains was elucidated in vitro by generating site-directed mutants of the putative conserved residues. Distinct phosphorylation patterns were obtained with two truncated versions of the hybrid sensor kinase, CbbSR
T189
and CbbSR
R96
(CbbSR beginning at residues T189 and R96, respectively). These constructs also exhibited substantially different affinities for ATP and phosphorylation stability, which was found to be dependent on a conserved Asp residue (Asp-696) within the kinase receiver domain. Asp-696 also played an important role in defining the specificity of phosphorylation for response regulators CbbRR1 or CbbRR2, and this residue appeared to act in conjunction with residues within the region from Arg-96 to Thr-189 at the N terminus of the sensor kinase. The net effect of concerted interactions at these distinct regions of CbbSR created an internal molecular switch that appears to coordinate a unique branched phosphorelay system.
Naturally Occurring Adenines within mRNA Coding Sequences Affect Ribosome Binding and Expression in
Escherichia coli
Authors: Brock Jay E. , Paz Robert L. , Cottle Patrick , Janssen Gary R.
Published: December 2006

Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many
Escherichia coli
genes, and addition of downstream adenine-rich sequences increases expression from several genes in
E. coli
. Here we describe site-directed mutagenesis of the
E. coli aroL
,
pncB
, and
cysJ
coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from
aroL-
,
pncB-
, and
cysJ-lacZ
fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from
aroL
- and
cysJ
-
lacZ
fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of
E. coli
translation.
Coordinate Expression of the Acetyl Coenzyme A Carboxylase Genes,
accB
and
accC
, Is Necessary for Normal Regulation of Biotin Synthesis in
Escherichia coli
Authors: Abdel-Hamid Ahmed M. , Cronan John E.
Published: December 2006

Transcription of the biotin (
bio
) biosynthetic operon of
Escherichia coli
is negatively regulated by the BirA protein, an atypical repressor protein in that it is also an enzyme. The BirA-catalyzed reaction involves the covalent attachment of biotin to AccB, a subunit of acetyl coenzyme (acetyl-CoA) carboxylase. The two functions of BirA allow regulation of the
bio
operon to respond to the intracellular concentrations of both biotin and unbiotinylated AccB. We report here that
bio
operon expression is down-regulated by overproduction of AccC, another acetyl-CoA carboxylase subunit known to form a complex with AccB. This down-regulation is eliminated when AccB and AccC are coordinately overexpressed, but only when the AccB partner is competent to bind AccC. Under AccC overexpression conditions AccB is underbiotinylated. These findings can be explained by a model in which excess AccC sequesters AccB in a complex that is a poor substrate for biotinylation. The observed disruption of biotin synthesis and attachment provides an excellent rationale for the observation that in the vast majority of sequenced bacterial genomes AccB and AccC are encoded in a two-gene operon.
Heme Concentration Dependence and Metalloporphyrin Inhibition of the System I and II CytochromecAssembly Pathways
Authors: Richard-Fogal Cynthia L. , Frawley Elaine R. , Feissner Robert E. , Kranz Robert G.
Published: December 2006

Studies have indicated that specific heme delivery to apocytochromecis a critical feature of the cytochromecbiogenesis pathways called system I and II. To determine directly the heme requirements of each system, including whether other metal porphyrins can be incorporated into cytochromesc, we engineeredEscherichia coliso that the natural system I (ccmABCDEFGH) was deleted and exogenous porphyrins were the sole source of porphyrins (ΔhemA). The engineeredE. colistrains that produced recombinant system I (fromE. coli) or system II (fromHelicobacter) facilitated studies of the heme concentration dependence of each system. Using this exogenous porphyrin approach, it was shown that in system I the levels of heme used are at least fivefold lower than the levels used in system II, providing an important advantage for system I. Neither system could assemble holocytochromescwith other metal porphyrins, suggesting that the attachment mechanism is specific for Fe protoporphyrin. Surprisingly, Zn and Sn protoporphyrins are potent inhibitors of the pathways, and exogenous heme competes with this inhibition. We propose that the targets are the heme binding proteins in the pathways (CcmC, CcmE, and CcmF for system I and CcsA for system II).
New Functional Identity for the DNA Uptake Sequence in Transformation and Its Presence in Transcriptional Terminators
Authors: Ambur O. Herman , Frye Stephan A. , Tønjum Tone
Published: December 2006

The frequently occurring DNA uptake sequence (DUS), recognized as a 10-bp repeat, is required for efficient genetic transformation in the human pathogens
Neisseria meningitidis
and
Neisseria gonorrhoeae
. Genome scanning for DUS occurrences in three different species of
Neisseria
demonstrated that 76% of the nearly 2,000 neisserial DUS were found to have two semiconserved base pairs extending from the 5′ end of DUS to constitute a 12-mer repeat. Plasmids containing sequential variants of the neisserial DUS were tested for their ability to transform
N. meningitidis
and
N. gonorrhoeae
, and the 12-mer was found to outperform the 10-mer DUS in transformation efficiency. Assessment of meningococcal uptake of DNA confirmed the enhanced performance of the 12-mer compared to the 10-mer DUS. An inverted repeat DUS was not more efficient in transformation than DNA species containing a single or direct repeat DUS. Genome-wide analysis revealed that half of the nearly 1,500 12-mer DUS are arranged as inverted repeats predicted to be involved in rho-independent transcriptional termination or attenuation. The distribution of the uptake signal sequence required for transformation in the
Pasteurellaceae
was also biased towards transcriptional terminators, although to a lesser extent. In addition to assessing the intergenic location of DUS, we propose that the 10-mer identity of DUS should be extended and recognized as a 12-mer DUS. The dual role of DUS in transformation and as a structural component on RNA affecting transcription makes this a relevant model system for assessing significant roles of repeat sequences in biology.
Complete and SOS-Mediated Response of
Staphylococcus aureus
to the Antibiotic Ciprofloxacin
Authors: Cirz Ryan T. , Jones Marcus B. , Gingles Neill A. , Minogue Timothy D. , Jarrahi Behnam , Peterson Scott N. , Romesberg Floyd E.
Published: December 2006

Staphylococcus aureus
infections can be difficult to treat due to both multidrug resistance and the organism's remarkable ability to persist in the host. Persistence and the evolution of resistance may be related to several complex regulatory networks, such as the SOS response, which modifies transcription in response to environmental stress. To understand how
S. aureus
persists during antibiotic therapy and eventually emerges resistant, we characterized its global transcriptional response to ciprofloxacin. We found that ciprofloxacin induces prophage mobilization as well as significant alterations in metabolism, most notably the up-regulation of the tricarboxylic acid cycle. In addition, we found that ciprofloxacin induces the SOS response, which we show, by comparison of a wild-type strain and a non-SOS-inducible
lexA
mutant strain, includes the derepression of 16 genes. While the SOS response of
S. aureus
is much more limited than those of
Escherichia coli
and
Bacillus subtilis
, it is similar to that of
Pseudomonas aeruginosa
and includes RecA, LexA, several hypothetical proteins, and a likely error-prone Y family polymerase whose homologs in other bacteria are required for induced mutation. We also examined induced mutation and found that either the inability to derepress the SOS response or the lack of the LexA-regulated polymerase renders
S. aureus
unable to evolve antibiotic resistance in vitro in response to UV damage. The data suggest that up-regulation of the tricarboxylic acid cycle and induced mutation facilitate
S. aureus
persistence and evolution of resistance during antibiotic therapy.
Substrate Specificity of the
Escherichia coli
Outer Membrane Protease OmpP
Authors: Hwang Bum-Yeol , Varadarajan Navin , Li Haixin , Rodriguez Sarah , Iverson Brent L. , Georgiou George
Published: December 2006

Escherichia coli
OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (
k
cat
/

K
m

= 3.0 × 10
6
M
−1
s
−1
). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1′ sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3′. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1′, site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F′ episome results in increased resistance to the antimicrobial peptide protamine both in
ompT
mutants and in wild-type
E. coli
cells.
Escherichia coli
HdeB Is an Acid Stress Chaperone
Authors: Kern Renée , Malki Abderrahim , Abdallah Jad , Tagourti Jihen , Richarme Gilbert
Published: December 2006

We cloned, expressed, and purified the
hdeB
gene product, which belongs to the
hdeAB
acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The
hdeA
and
hdeB
mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that
Escherichia coli
possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.
Expression of the Iron-ActivatednspAandsecYGenes inNeisseria meningitidisGroup B by Fur-Dependent and -Independent Mechanisms
Authors: Shaik Yazdani B. , Grogan Susan , Davey Michael , Sebastian Shite , Goswami Sulip , Szmigielski Borys , Genco Caroline A.
Published: December 2006

Our whole-genome microarray studies ofNeisseria meningitidisMC58 previously identified a set of 153 genes whose transcription was activated during growth in iron. In this study, Fur-mediated regulation of the iron-activatednspAgene was confirmed, whereas iron-activated regulation of thesecYgene was demonstrated to be Fur independent. Analysis of the Fur binding sequences in thenspAgene and an additional iron-activated and Fur-regulated gene identified a hexameric (G/T)ATAAT unit in the operator regions of these genes similar to that observed in Fur- and iron-repressed genes. These studies indicate that the expression of the iron-activatednspAandsecYgenes inN. meningitidisoccur by Fur-dependent and -independent mechanisms, respectively.
Diversity of the Abundant pKLC102/PAGI-2 Family of Genomic Islands inPseudomonas aeruginosa
Authors: Klockgether Jens , Würdemann Dieco , Reva Oleg , Wiehlmann Lutz , Tümmler Burkhard
Published: December 2006

The known genomic islands ofPseudomonas aeruginosaclone C strains are integrated into tRNALys(pKLC102) or tRNAGly(PAGI-2 and PAGI-3) genes and differ from their core genomes by distinctive tetranucleotide usage patterns. pKLC102 and the related island PAPI-1 fromP. aeruginosaPA14 were spontaneously mobilized from their host chromosomes at frequencies of 10% and 0.3%, making pKLC102 the most mobile genomic island known with a copy number of 30 episomal circular pKLC102 molecules per cell. The incidence of islands of the pKLC102/PAGI-2 type was investigated in 71 unrelatedP. aeruginosastrains from diverse habitats and geographic origins. pKLC102- and PAGI-2-like islands were identified in 50 and 31 strains, respectively, and 15 and 10 subtypes were differentiated by hybridization on pKLC102 and PAGI-2 macroarrays. The diversity of PAGI-2-type islands was mainly caused by one large block of strain-specific genes, whereas the diversity of pKLC102-type islands was primarily generated by subtype-specific combination of gene cassettes. Chromosomal loss of PAGI-2 could be documented in sequentialP. aeruginosaisolates from individuals with cystic fibrosis. PAGI-2 was present in most testedCupriavidus metalliduransandCupriavidus campinensisisolates from polluted environments, demonstrating the spread of PAGI-2 across habitats and species barriers. The pKLC102/PAGI-2 family is prevalent in numerous beta- and gammaproteobacteria and is characterized by high asymmetry of the cDNA strands. This evolutionarily ancient family of genomic islands retained its oligonucleotide signature during horizontal spread within and among taxa.
Comparison between Nasal Swabs and Nasopharyngeal Aspirates for, and Effect of Time in Transit on, Isolation of
Streptococcus pneumoniae
,
Staphylococcus aureus
,
Haemophilus influenzae
, and
Moraxella catarrhalis
Authors: Carville Kylie S. , Bowman Jacinta M. , Lehmann Deborah , Riley Thomas V.
Published: December 2006

We assessed the impact of the use of nasal swabs or nasopharyngeal aspirates and the time from specimen collection to storage at −70°C on bacterial isolation.
Haemophilus influenzae
was isolated significantly less often from swabs than from nasopharyngeal aspirates. Samples in transit for >3 days were half as likely to grow
Streptococcus pneumoniae
and
H. influenzae
as those in transit for ≤3 days. There was no statistically significant difference for either
Moraxella catarrhalis
or
Staphylococcus aureus
.
Assessment of an Optimized Mycobacterial Interspersed Repetitive- Unit-Variable-Number Tandem-Repeat Typing System Combined with Spoligotyping for Population-Based Molecular Epidemiology Studies of Tuberculosis
Authors: Oelemann Mara Cardoso , Diel Roland , Vatin Vincent , Haas Walter , Rüsch-Gerdes Sabine , Locht Camille , Niemann Stefan , Supply Philip
Published: December 2006

An optimized set of 24 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci, including a discriminatory subset of 15 loci, has recently been defined for the typing ofMycobacterium tuberculosis. Here, we evaluated the performances of this MIRU-VNTR typing system in combination with spoligotyping for the detection of transmission chains in a population-based study comprising 91% of culture-confirmed tuberculosis patients reported in 2003 in Hamburg, Germany. Of the 154 isolates investigated, more than 90% had high IS6110copy numbers (≥6). IS6110restriction fragment length polymorphism (RFLP) typing resulted in 13 clusters, 5 of which had a confirmed epidemiological link. All five, as well as six of the eightIS6110clusters with no identified epidemiological link, were perfectly matched by MIRU-VNTR typing with the 24 loci. Two IS6110clusters were split by differences into 6 to 12 MIRU-VNTR loci, clearly supporting the absence of a link, as judged by contact tracing data. In contrast, only one MIRU-VNTR cluster, grouping what were probably epidemiologically unlinked isolates, was split by IS6110RFLP. However, these isolates were also distinguished by spoligotyping. Both the optimized 24-locus and 15-locus sets thus showed a comparable to slightly better predictive value, especially when combined with spoligotyping, than the current gold standard IS6110RFLP for the study of tuberculosis transmission in Hamburg. Because the epidemiological characteristics of this setting are similar to those of many developed countries, these results support the wide applicability of this real-time genotyping approach for population-based studies ofM. tuberculosistransmission.
tcdC
Genotypes Associated with Severe TcdC Truncation in an Epidemic Clone and Other Strains of
Clostridium difficile
Authors: Curry Scott R. , Marsh Jane W. , Muto Carlene A. , O'Leary Mary M. , Pasculle A. William , Harrison Lee H.
Published: December 2006

Severe
Clostridium difficile
associated disease is associated with outbreaks of the recently described BI/NAP1 epidemic clone. This clone is characterized by an 18-bp deletion in the
tcdC
gene and increased production of toxins A and B in vitro. TcdC is a putative negative regulator of toxin A&B production. We characterized
tcdC
genotypes from a collection of
C. difficile
isolates from a hospital that experienced an outbreak caused by the BI/NAP1 epidemic clone. Sequence analysis of
tcdC
was performed on DNA samples isolated from 199 toxigenic
C. difficile
isolates (31% BI/NAP1) from 2001 and 2005. Sequences obtained from 36 (18.6%) isolates predicted wild-type TcdC (232 amino acid residues), whereas 12 (6.1%) isolates had
tcdC
genotypes with previously described 18- or 39-bp deletions. The remaining isolates comprised 15 unique genotypes. Of these, 5 genotypes contain 18- or 36-bp deletions. Of these five genotypes, one is characterized by a single nucleotide deletion at position 117 resulting in a frameshift that introduces a stop codon at position 196, truncating the predicted TcdC to 65 amino acid residues. All 62 of the isolates in this collection comprising the epidemic clone are characterized by this genotype. This result suggests that severe truncation of TcdC is responsible for the increased toxin production observed in strains belonging to the BI/NAP1 clone and that the 18-bp deletion is probably irrelevant to TcdC function. Further investigations are required to determine the effect of this and other
tcd
C genotypes on toxin production and clinical disease.
Staphylococcal Cassette Chromosome
mec
and Panton-Valentine Leukocidin Characterization of Methicillin-Resistant
Staphylococcus aureus
Clones
Authors: Moroney Shannon M. , Heller Loree C. , Arbuckle Jesse , Talavera Monica , Widen Ray H.
Published: December 2006

Staphylococcal cassette chromosome
mec
(SCC
mec
) types and Panton-Valentine leukocidin (PVL) gene carriage were compared among suspected community-associated methicillin-resistant
Staphylococcus aureus
MRSA (CA-MRSA) and health care-associated MRSA (HA-MRSA) isolates. CA-MRSA isolates carried the SCC
mec
type IV complex, and most were PVL positive. The HA-MRSA isolates carried the SCC
mec
type II complex and did not harbor the PVL genes.
Genotypic Analysis of Invasive
Streptococcus pneumoniae
from Mali, Africa, by Semiautomated Repetitive-Element PCR and Pulsed-Field Gel Electrophoresis
Authors: Harrington S. M. , Stock F. , Kominski A. L. , Campbell J. D. , Hormazabal J. C. , Livio S. , Rao L. , Kotloff K. L. , Sow S. O. , Murray P. R.
Published: December 2006

As part of a large, ongoing study of invasive infections in pediatric patients in Bamako, Mali, 106 cases of invasive pneumococcal disease were identified from June 2002 to July 2003 (J. D. Campbell et al., Pediatr. Infect. Dis. J. 23:642-649, 2004). Of the 12 serotypes present, the majority of isolates were not contained in PCV7 (the 7-valent pneumococcal conjugate vaccine), including 1 isolate that was serotype 1, 12 isolates that were serotype 2, 58 isolates that were serotype 5, 7 isolates that were serotype 7F, and 1 isolate that was serotype 12F. To determine whether clonal dissemination of the predominant serotypes had taken place, genotyping was performed on 100
S. pneumoniae
isolates by using two methods: pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, and the Bacterial Barcodes repetitive-element PCR (rep-PCR) method. Criteria for delineating rep-PCR genotypes were established such that isolates of different serotypes were generally not grouped together. The two methods were equally discriminatory within a given pneumococcal serotype. PFGE separated the isolates into 15 genotypes and 7 subtypes; rep-PCR separated isolates into 15 genotypes and 6 subtypes. Using either method, isolates within serotypes 2, 5, and 7 formed three large, separate clusters containing 1 genotype each. Both methods further distinguished related subtypes within serotypes 2 and 5. Interestingly, one of the PFGE subtypes of serotype 5 is indistinguishable from the Columbia
5
-19 clone circulating in Latin America since 1994. The data support that serotypes 2 and 5 were likely to be the result of dissemination of particular clones, some of which are responsible for invasive disease over a broad population range.
Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site
Authors: Deng Qiang , Zhai Jian-wei , Michel Marie-Louise , Zhang Jun , Qin Jun , Kong Yu-ying , Zhang Xin-xin , Budkowska Agata , Tiollais Pierre , Wang Yuan , Xie You-hua
Published: December 2006

A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W
1
T
2
X
3
W
4
W
5
) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.
Conserved Determinants for Membrane Association of Nonstructural Protein 5A fromHepatitis C Virus and Related Viruses
Authors: Brass Volker , Pal Zsuzsanna , Sapay Nicolas , Deléage Gilbert , Blum Hubert E. , Penin François , Moradpour Darius
Published: December 2006

Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic
alpha helix mediates in-plane membrane association of HCV NS5A and at
the same time is likely involved in specific protein-protein
interactions required for the assembly of a functional replication
complex. The aim of this study was to identify the determinants for
membrane association of NS5A from the related GB viruses and
pestiviruses. Although primary amino acid sequences differed
considerably, putative membrane anchor domains with amphipathic
features were predicted in the N-terminal domains of NS5A proteins from
these viruses. Confocal laser scanning microscopy, as well as membrane
flotation analyses, demonstrated that NS5As from GB virus B (GBV-B),
GBV-C, and bovine viral diarrhea virus, the prototype pestivirus,
display membrane association characteristics very similar to those of
HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A
proteins were sufficient for membrane association. Circular dichroism
analyses confirmed the capacity of these segments to fold into alpha
helices upon association with lipid-like molecules. Despite structural
conservation, only very limited exchanges with sequences from related
viruses were tolerated in the context of functional HCV RNA
replication, suggesting virus-specific interactions of these segments.
In conclusion, membrane association of NS5A by an N-terminal
amphipathic alpha helix is a feature shared by HCV and related members
of the family
Flaviviridae
. This observation points to
conserved roles of the N-terminal amphipathic alpha helices of NS5A in
replication complex
formation.
Cytomegalovirus Primary Envelopment Occurs at Large Infoldings of the Inner Nuclear Membrane
Authors: Buser Christopher , Walther Paul , Mertens Thomas , Michel Detlef
Published: December 2006

We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis.
Oncolytic Vesicular Stomatitis Virus Induces Apoptosis via Signaling through PKR, Fas, and Daxx
Authors: Gaddy Daniel F. , Lyles Douglas S.
Published: December 2006

Matrix (M) protein mutants of vesicular stomatitis virus (VSV) are promising oncolytic agents for cancer therapy. Previous research has implicated Fas and PKR in apoptosis induced by other viruses. Here, we show that dominant-negative mutants of Fas and PKR inhibit M protein mutant virus-induced apoptosis. Most previous research has focused on the adapter protein FADD as a necessary transducer of Fas-mediated apoptosis. However, the expression of dominant-negative FADD had little effect on the induction of apoptosis by M protein mutant VSV. Instead, virus-induced apoptosis was inhibited by the expression of a dominant-negative mutant of the adapter protein Daxx. These data indicate that Daxx is more important than FADD for apoptosis induced by M protein mutant VSV. These results show that PKR- and Fas-mediated signaling play important roles in cell death during M protein mutant VSV infection and that Daxx has novel functions in the host response to virus infection by mediating virus-induced apoptosis.
Nuclear Import of Bovine Papillomavirus Type 1 E1 Protein Is Mediated by Multiple Alpha Importins and Is Negatively Regulated by Phosphorylation near a Nuclear Localization Signal
Authors: Bian Xue-Lin , Rosas-Acosta Germán , Wu Yu-Chieh , Wilson Van G.
Published: December 2006

Papillomavirus DNA replication occurs in the nucleus of infected cells and requires the viral E1 protein, which enters the nuclei of host epithelial cells and carries out enzymatic functions required for the initiation of viral DNA replication. In this study, we investigated the pathway and regulation of the nuclear import of the E1 protein from bovine papillomavirus type 1 (BPV1). Using an in vitro binding assay, we determined that the E1 protein interacted with importins α3, α4, and α5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified E1 protein was effectively imported into the nucleus of digitonin-permeabilized HeLa cells after incubation with importin α3, α4, or α5 and other necessary import factors. We also observed that in vitro binding of E1 protein to all three α importins was significantly decreased by the introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated E1 protein failed to enter the nucleus of digitonin-permeabilized HeLa cells in vitro. Likewise, the pseudophosphorylation mutant showed aberrant intracellular localization in vivo and accumulated primarily on the nuclear envelope in transfected HeLa cells, while the corresponding alanine replacement mutant displayed the same cellular location pattern as wild-type E1 protein. Collectively, our data demonstrate that BPV1 E1 protein can be transported into the nucleus by more than one importin α and suggest that E1 phosphorylation by host cell kinases plays a regulatory role in modulating E1 nucleocytoplasmic localization. This phosphoregulation of nuclear E1 protein uptake may contribute to the coordination of viral replication with keratinocyte proliferation and differentiation.
The Antiviral Efficacy of Simian Immunodeficiency Virus-Specific CD8
+
T Cells Is Unrelated to Epitope Specificity and Is Abrogated by Viral Escape
Authors: Loffredo John T. , Burwitz Benjamin J. , Rakasz Eva G. , Spencer Sean P. , Stephany Jason J. , Giraldo Vela Juan Pablo , Martin Sarah R. , Reed Jason , Piaskowski Shari M. , Furlott Jessica , Weisgrau Kim L. , Rodrigues Denise S. , Soma Taeko , Napoé Gnankang , Friedrich Thomas C. , Wilson Nancy A. , Kallas Esper G. , Watkins David I.
Published: December 2006

CD8
+
T lymphocytes appear to play a role in controlling human immunodeficiency virus (HIV) replication, yet routine immunological assays do not measure the antiviral efficacy of these cells. Furthermore, it has been suggested that CD8
+
T cells that recognize epitopes derived from proteins expressed early in the viral replication cycle can be highly efficient. We used a functional in vitro assay to assess the abilities of different epitope-specific CD8
+
T-cell lines to control simian immunodeficiency virus (SIV) replication. We compared the antiviral efficacies of 26 epitope-specific CD8
+
T-cell lines directed against seven SIV epitopes in Tat, Nef, Gag, Env, and Vif that were restricted by either Mamu-A*01 or Mamu-A*02. Suppression of SIV replication varied depending on the epitope specificities of the CD8
+
T cells and was unrelated to whether the targeted epitope was derived from an early or late viral protein. Tat
28-35
SL8- and Gag
181-189
CM9-specific CD8
+
T-cell lines were consistently superior at suppressing viral replication compared to the other five SIV-specific CD8
+
T-cell lines. We also investigated the impact of viral escape on antiviral efficacy by determining if Tat
28-35
SL8- and Gag
181-189
CM9-specific CD8
+
T-cell lines could suppress the replication of an escaped virus. Viral escape abrogated the abilities of Tat
28-35
SL8- and Gag
181-189
CM9-specific CD8
+
T cells to control viral replication. However, gamma interferon (IFN-γ) enzyme-linked immunospot and IFN-γ/tumor necrosis factor alpha intracellular-cytokine-staining assays detected cross-reactive immune responses against the Gag escape variant. Understanding antiviral efficacy and epitope variability, therefore, will be important in selecting candidate epitopes for an HIV vaccine.
Reduced Apoptosis in Human Intestinal Cells Cured of Persistent Poliovirus Infection
Authors: Labadie Karine , Saulnier Aure , Martin-Latil Sandra , Colbère-Garapin Florence
Published: December 2006

Cells cured of persistent virus infection can be used to investigate cellular pathways of resistance to viral cytopathic effects. Persistent poliovirus (PV) infections were established in human intestinal Caco-2 cells, and spontaneously cured cell cultures were obtained. Two cell clones, cl6 and b13, cured of type 3 PV mutant infection and their parental Caco-2 cells were compared for susceptibility to PV infection, PV receptor CD155 expression, capacity to differentiate into polarized enterocytes, and PV-, staurosporine-, and actinomycin D-induced apoptosis. Our results strongly suggest that cells that are partially resistant to apoptosis can be selected during persistent virus infection.
Phylogenetic Analysis of Feline Immunodeficiency Virus in Feral and Companion Domestic Cats of New Zealand
Authors: Hayward Jessica J. , Taylor John , Rodrigo Allen G.
Published: December 2006

Nested PCR was used to amplify envelope V3-V6 gene fragments of feline immunodeficiency virus (FIV) from New Zealand cats. Phylogenetic analyses established that subtypes A and C predominate among New Zealand cats, with clear evidence of intersubtype recombination. In addition, 17 sequences were identified that were distinct from all known FIV clades, and we tentatively suggest these belong to a novel subtype.
Complete Genome Analysis of 33 Ecologically and Biologically Diverse Rift Valley Fever Virus Strains Reveals Widespread Virus Movement and Low Genetic Diversity due to Recent Common Ancestry
Authors: Bird Brian H. , Khristova Marina L. , Rollin Pierre E. , Ksiazek Thomas G. , Nichol Stuart T.
Published: December 2006

Rift Valley fever (RVF) virus is a mosquito-borne RNA virus responsible for large explosive outbreaks of acute febrile disease in humans and livestock in Africa with significant mortality and economic impact. The successful high-throughput generation of the complete genome sequence was achieved for 33 diverse RVF virus strains collected from throughout Africa and Saudi Arabia from 1944 to 2000, including strains differing in pathogenicity in disease models. While several distinct virus genetic lineages were determined, which approximately correlate with geographic origin, multiple exceptions indicative of long-distance virus movement have been found. Virus strains isolated within an epidemic (e.g., Mauritania, 1987, or Egypt, 1977 to 1978) exhibit little diversity, while those in enzootic settings (e.g., 1970s Zimbabwe) can be highly diverse. In addition, the large Saudi Arabian RVF outbreak in 2000 appears to have involved virus introduction from East Africa, based on the close ancestral relationship of a 1998 East African virus. Virus genetic diversity was low (∼5%) and primarily involved accumulation of mutations at an average of 2.9 × 10
−4
substitutions/site/year, although some evidence of RNA segment reassortment was found. Bayesian analysis of current RVF virus genetic diversity places the most recent common ancestor of these viruses in the late 1800s, the colonial period in Africa, a time of dramatic changes in agricultural practices and introduction of nonindigenous livestock breeds. In addition to insights into the evolution and ecology of RVF virus, these genomic data also provide a foundation for the design of molecular detection assays and prototype vaccines useful in combating this important disease.
Suppression of the Sendai Virus M Protein through a Novel Short Interfering RNA Approach Inhibits Viral Particle Production but Does Not Affect Viral RNA Synthesis
Authors: Mottet-Osman Geneviève , Iseni Frédéric , Pelet Thierry , Wiznerowicz Maciej , Garcin Dominique , Roux Laurent
Published: December 2006

Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3′ untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50- to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.
Caspase 3-Dependent Cell Death of Neurons Contributes to the Pathogenesis of West Nile Virus Encephalitis
Authors: Samuel Melanie A. , Morrey John D. , Diamond Michael S.
Published: December 2006

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that has become a significant global cause of viral encephalitis. To examine the mechanisms of WNV-induced neuronal death and the importance of apoptosis in pathogenesis, we evaluated the role of a key apoptotic regulator, caspase 3. WNV infection induced caspase 3 activation and apoptosis in the brains of wild-type mice. Notably, congenic caspase 3
−/−
mice were more resistant to lethal WNV infection, although there were no significant differences in the tissue viral burdens or the kinetics of viral spread. Instead, decreased neuronal death was observed in the cerebral cortices, brain stems, and cerebella of caspase 3
−/−
mice. Analogously, primary central nervous system (CNS)-derived neurons demonstrated caspase 3 activation and apoptosis after WNV infection, and treatment with caspase inhibitors or a genetic deficiency in caspase 3 significantly decreased virus-induced death. These studies establish that caspase 3-dependent apoptosis contributes to the pathogenesis of lethal WNV encephalitis and suggest possible novel therapeutic targets to restrict CNS injury.
Evolution and Molecular Epidemiology of H9N2 Influenza A Viruses from Quail in Southern China, 2000 to 2005
Authors: Xu K. M. , Li K. S. , Smith G. J. D. , Li J. W. , Tai H. , Zhang J. X. , Webster R. G. , Peiris J. S. M. , Chen H. , Guan Y.
Published: December 2006

H9N2 influenza viruses have become established and maintain long-term endemicity in terrestrial poultry in Asian countries. Occasionally these viruses transmit to other mammals, including humans. Increasing epidemiological and laboratory findings suggest that quail may be an important host, as they are susceptible to different subtypes of influenza viruses. To better understand the role of quail in influenza virus ecology and evolution, H9N2 viruses isolated from quail during 2000 to 2005 were antigenically and genetically characterized. Our results showed that H9N2 viruses are prevalent year-round in southern China and replicate mainly asymptomatically in the respiratory tract of quail. Genetic analysis revealed that both the G1-like and Ck/Bei-like H9N2 lineages were cocirculating in quail since 2000. Phylogenetic analyses demonstrated that most of the isolates tested were double- or multiple-reassortant variants, with four G1-like and 16 Ck/Bei-like genotypes recognized. A novel genotype of G1-like virus became predominant in quail since 2003, while multiple Ck/Bei-like genotypes were introduced into quail, wherein they incorporated G1-like gene segments, but none of them became established in this host. Those Ck/Bei-like reassortants generated in quail have then been introduced into other poultry. These complex interactions form a two-way transmission system between quail and other types of poultry. The present study provides evidence that H9N2 and H5N1 subtype viruses have also exchanged gene segments to generate currently circulating reassortants of both subtypes that have pandemic potential. Continuing influenza virus surveillance in poultry is critical to understanding the genesis and emergence of potentially pandemic strains in this region.