bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression Characteristics of the Transfer-Related
kilB
Gene Product of
Streptomyces
Plasmid pIJ101: Implications for the Plasmid Spread Function
Authors: Pettis Gregg S. , Ward Naomi , Schully Kevin L.
Published: July 2002

Intermycelial transfer of
Streptomyces
plasmid pIJ101 occurs prior to cellular differentiation and is mediated by plasmid functions that are also required for production of zones of growth-inhibited recipient cells (i.e., pocks) that develop around individual donors during mating on agar medium. Several other pIJ101 functions, including that of the
kilB
gene, whose unregulated expression on pIJ101 is lethal, are required for normal pock size and so have been postulated to mediate intramycelial spread of the plasmid throughout recipient cells. Using antibodies raised against a KilB fusion protein expressed in
Escherichia coli
, native KilB protein was detected throughout development of pIJ101-containing
Streptomyces lividans
cells, with the concentration of KilB increasing dramatically and reaching a maximum during the final stages (i.e., sporulation and secondary metabolism) of cellular differentiation. Insertion of the
kilB
gene of pIJ101 into the
S. lividans
chromosome in cells lacking the pIJ101 KorB protein, which normally represses
kilB
gene transcription, resulted in elevated but still temporally increasing amounts of KilB. The increased expression or accumulation of the KilB spread protein throughout cellular differentiation of
S. lividans
, which leads to maximum KilB concentrations during developmental stages that occur far later than when intermycelial transfer of pIJ101 is mediated, supports the existence of a subsequent intramycelial component to the pIJ101 spread function. The results also suggest that intramycelial spread of pIJ101 molecules within the recipient extends beyond intercompartmental movements within the substrate mycelia and includes undetermined steps within the spore-yielding aerial hyphae as well.
Identification of Specific Residues in Colicin E1 Involved in Immunity Protein Recognition
Authors: Lindeberg Magdalen , Cramer William A.
Published: July 2002

The basis of specificity between pore-forming colicins and immunity proteins was explored by interchanging residues between colicins E1 (ColE1) and 10 (Col10) and testing for altered recognition by their respective immunity proteins, Imm and Cti. A total of 34 divergent residues in the pore-forming domain of ColE1 between residues 419 and 501, a region previously shown to contain the specificity determinants for Imm, were mutagenized to the corresponding Col10 sequences. The residue changes most effective in converting ColE1 to the Col10 phenotype are residue 448 at the N terminus of helix VI and residues 470, 472, and 474 at the C terminus of helix VII. Mutagenesis of helix VI residues 416 to 419 in Col10 to the corresponding ColE1 sequence resulted in increased recognition by Imm and loss of recognition by Cti.
Transcriptional Activation of the
Bacillus subtilis ackA
Promoter Requires Sequences Upstream of the CcpA Binding Site
Authors: Moir-Blais Tessa R. , Grundy Frank J. , Henkin Tina M.
Published: July 2002

Carbon catabolite protein A (CcpA) is a global regulator of carbon metabolism in gram-positive bacteria, repressing transcription of genes for the utilization of secondary carbon sources in the presence of a readily metabolized carbon source and activating transcription of genes, such as
ackA
and
pta
, that are required for carbon excretion. The promoter region of the
Bacillus subtilis ackA
gene contains two catabolite responsive elements (
cre
sites), of which only the site closest to the promoter (
cre2
) binds CcpA to activate transcription. A region immediately upstream of the
cre2
site is also important for transcriptional activation. The required elements in this region were further defined by mutagenesis. CcpA binds to the
ackA
promoter region in gel shift assays even in the presence of mutations in the upstream element that block transcriptional activation, indicating that this region has a function other than promoting binding of CcpA.
Roles ofhilCandhilDin Regulation ofhilAExpression inSalmonella entericaSerovar Typhimurium
Authors: Lucas Robin L. , Lee Catherine A.
Published: July 2002

Sequences between −332 and −39 upstream of thehilApromoter are required for repression ofhilA. An unidentified repressor is thought to bind these upstream repressing sequences (URS) to inhibithilAexpression. Two AraC-like transcriptional regulators encoded onSalmonellapathogenicity island 1 (SPI1), HilC and HilD, bind to the URS to counteract the repression ofhilA. The URS is required for regulation ofhilAby osmolarity, oxygen, PhoP/PhoQ, and SirA/BarA. Here, we show that FadD, FliZ, PhoB, and EnvZ/OmpR also require the URS to regulatehilA. These environmental and regulatory factors may affecthilAexpression by altering the expression or activity of HilC, HilD, or the unknown repressor. To begin investigating these possibilities, we tested the effects of environmental and regulatory factors onhilCandhilDexpression. We also examinedhilAregulation whenhilCorhilDwas disrupted or expressed to a high level. AlthoughhilCis regulated by all environmental conditions and regulatory factors that modulatehilAexpression,hilCis not required for the regulation ofhilAby any conditions or factors except EnvZ/OmpR. In contrast,hilDis absolutely required forhilAexpression, but environmental conditions and regulatory factors have little or no effect onhilDexpression. We speculate that EnvZ/OmpR regulateshilAby altering the expression and/or activity ofhilC, while all other regulatory conditions and mutations regulatehilAby modulatinghilDposttranscriptionally. We also discuss models in which the regulation ofhilAexpression is mediated by modulation of the expression or activity of one or more repressors.
Trypsin Mediates Growth Phase-Dependent Transcriptional Regulation of Genes Involved in Biosynthesis of Ruminococcin A, a Lantibiotic Produced by a
Ruminococcus gnavus
Strain from a Human Intestinal Microbiota
Authors: Gomez Ana , Ladiré Monique , Marcille Françoise , Fons Michel
Published: July 2002

Ruminococcin A (RumA) is a trypsin-dependent lantibiotic produced by
Ruminococcus gnavus
E1, a gram-positive strict anaerobic strain isolated from a human intestinal microbiota. A 12.8-kb region from
R. gnavus
E1 chromosome, containing the biosynthetic gene cluster of RumA, has been cloned and sequenced. It consisted of 13 open reading frames, organized in three operons with predicted functions in lantibiotic biosynthesis, signal transduction regulation, and immunity. One unusual feature of the locus is the presence of three almost identical structural genes, all of them encoding the RumA precursor. In order to determine the role of trypsin in RumA production, the transcription of the
rum
genes has been investigated under inducing and noninducing conditions. Trypsin activity is needed for the growth phase-dependent transcriptional activation of RumA operons. Our results suggest that bacteriocin production by
R. gnavus
E1 is controlled through a complex signaling mechanism involving the proteolytic processing of a putative extracellular inducer-peptide by trypsin, a specific environmental cue of the digestive ecosystem.
Escherichia coliDNA Polymerase III Can Replicate Efficiently past a T-Tcis-synCyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded bydnaQAre Inactivated
Authors: Borden Angela , O'Grady Paul I. , Vandewiele Dominique , Fernández de Henestrosa Antonio R. , Lawrence Christopher W. , Woodgate Roger
Published: July 2002

Although very little replication past a T-Tcis-syncyclobutane dimer normally takes place inEscherichia coliin the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (ΔumuDC) strains if the 3′ to 5′ exonuclease proofreading activity of the Pol III ε subunit was also disabled bymutD5. This observation might be explained in at least two ways. In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases. Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function. We have investigated this issue by examining dimer bypass frequencies in ΔumuDC mutD5strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations. Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V. Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted bymutD5, indicating the surprising conclusion that bypass was probably performed by themutD5Pol III enzyme itself. This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally.
Nitric Oxide Metabolism in
Neisseria meningitidis
Authors: Anjum Muna F. , Stevanin Tânia M. , Read Robert C. , Moir James W. B.
Published: July 2002

Neisseria meningitidis
, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of
N. meningitidis
includes the genes
aniA
and
norB
, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that
N. meningitidis
can support growth microaerobically by the denitrification of nitrite via NO and that
norB
is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the
cycP
gene product cytochrome
c
′ are able to counteract toxicity due to exogenously added NO. Expression of these genes by
N. meningitidis
during colonization and disease may confer protection against exogenous or endogenous nitrosative stress.
Characterization of the Class 3 Integron and the Site-Specific Recombination System It Determines
Authors: Collis Christina M. , Kim Mi-Jurng , Partridge Sally R. , Stokes H. W. , Hall Ruth M.
Published: July 2002

Integrons capture gene cassettes by using a site-specific recombination mechanism. As only one class of integron and integron-determined site-specific recombination system has been studied in detail, the properties of a second class, the only known class 3 integron, were examined. The configuration of the three potentially definitive features of integrons, theintI3gene, the adjacentattI3recombination site, and the Pcpromoter that directs transcription of the cassettes, was similar to that found in the corresponding region (5′ conserved segment) of class 1 integrons. The integron features are flanked by a copy of the terminal inverted repeat, IRi, from class 1 integrons on one side and a resolvase-encodingtniRgene on the other, suggesting that they are part of a transposable element related to Tn402but with the integron module in the opposite orientation. The IntI3 integrase was active and able to recognize and recombine both known types of IntI-specific recombination sites, theattI3site in the integron, and different cassette-associated 59-be (59-base element) sites. Both integration of circularized cassettes into theattI3site and excision of integrated cassettes were also catalyzed by IntI3. TheattI3site was localized to a short region adjacent to theintI3gene. Recombination between a 59-be and secondary sites was also catalyzed by IntI3, but at frequencies significantly lower than observed with IntI1, the class 1 integron integrase.
The InhA2 Metalloprotease of
Bacillus thuringiensis
Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route
Authors: Fedhila Sinda , Nel Patricia , Lereclus Didier
Published: July 2002

The entomopathogenic bacterium
Bacillus thuringiensis
is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts. We identified a second copy of the
inhA
gene, named
inhA2
, in
B. thuringiensis
strain 407 Cry
−
. The
inhA2
gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional
inhA2
′-
lacZ
fusion to show that
inhA2
expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence of a reverse Spo0A box in the promoter region of
inhA2
suggests that Spo0A directly regulates the transcription of
inhA2
. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized
Bombyx mori
larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against
Galleria mellonella
larvae infected via the oral pathway. The spores of Δ
inhA2
mutant strain were ineffective in providing synergism whereas those of the Δ
inhA
mutant strain were not. These results indicate that the
B. thuringiensis
InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route.
Phage TP901-1 Site-Specific Integrase Functions in Human Cells
Authors: Stoll Stephanie M. , Ginsburg Daniel S. , Calos Michele P.
Published: July 2002

We demonstrate that the site-specific integrase encoded by phage TP901-1 of
Lactococcus lactis
subsp.
cremoris
has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in
Escherichia coli
and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in
E. coli
and determined that for complete reaction in
E. coli,
the minimal sizes of
attB
and
attP
are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.
Molecular Cloning and Expression of Mn
2+
-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium,
Pseudomonas
sp. Strain TK4
Authors: Sueyoshi Noriyuki , Kita Katsuhiro , Okino Nozomu , Sakaguchi Keishi , Nakamura Takashi , Ito Makoto
Published: July 2002

We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium,
Pseudomonas
sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn
2+
. The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from
Bacillus cereus
,
Staphylococcus aureus
,
Listeria ivanovii
, and
Leptospira interrogans
, as well as a hypothetical protein encoded in
Chromobacterium violaceum
, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.
VmrA, a Member of a Novel Class of Na
+
-Coupled Multidrug Efflux Pumps from
Vibrio parahaemolyticus
Authors: Chen Jing , Morita Yuji , Huda M. Nazmul , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa
Published: July 2002

Gene
vmrA
, cloned from
Vibrio parahaemolyticus
, made
Escherichia coli
resistant to 4prime;,6-diamino-2-phenylindol, tetraphenylphosphonium chloride, acriflavine, and ethidium bromide. VmrA belongs to the DinF branch of MATE family efflux transporters. VmrA catalyzed acriflavine efflux and showed Na
+
/drug transporter activity because the addition of tetraphenylphosphonium to Na
+
-loaded cells caused Na
+
efflux.
FimZ Is a Molecular Link between Sticking and Swimming in
Salmonella enterica
Serovar Typhimurium
Authors: Clegg Steven , Hughes Kelly T.
Published: July 2002

Salmonella enterica
serovar Typhimurium produces two types of filamentous appendages on its surface. Fimbriae mediate adherence to tissues and cells via receptor-specific interactions, and flagella are the organelles of motility. These appendages play a role in colonization and dissemination, respectively, from infected surfaces and may be important components of bacterial survival. Increased expression of FimZ in serovar Typhimurium resulted in bacteria which were hyperfimbriated but were nonmotile in soft agar. This lack of motility was associated with down regulation of the
flhDC
master flagellar operon. Therefore, FimZ represents a molecular connection between flagella and fimbrial formation in serovar Typhimurium, indicating that the synthesis of flagella and fimbriae are oppositely controlled.
Endotoxin of
Neisseria meningitidis
Composed Only of Intact Lipid A: Inactivation of the Meningococcal 3-Deoxy-
d
-Manno-Octulosonic Acid Transferase
Authors: Tzeng Yih-Ling , Datta Anup , Kolli V. Kumar , Carlson Russell W. , Stephens David S.
Published: July 2002

Lipopolysaccharide, lipooligosaccharide (LOS), or endotoxin is important in bacterial survival and the pathogenesis of gram-negative bacteria. A necessary step in endotoxin biosynthesis is 3-deoxy-
d
-manno-octulosonic acid (Kdo) glycosylation of lipid A, catalyzed by the Kdo transferase KdtA (WaaA). In enteric gram-negative bacteria, this step is essential for survival. A nonpolar
kdtA
::
aphA-3
mutation was created in
Neisseria meningitidis
via allelic exchange, and the mutant was viable. Detailed structural analysis demonstrated that the endotoxin of the
kdtA
::
aphA-3
mutant was composed of fully acylated lipid A with variable phosphorylation but without Kdo glycosylation. In contrast to what happens in other gram-negative bacteria, tetra-acylated lipid IV
A
did not accumulate. The LOS structure of the
kdtA
::
aphA-3
mutant was restored to the wild-type structure by complementation with
kdtA
from
N. meningitidis
or
Escherichia coli
. The expression of a fully acylated, unglycosylated lipid A indicates that lipid A biosynthesis in
N. meningitidis
can proceed without the addition of Kdo and that KdtA is not essential for survival of the meningococcus.
Characterization of a
Mycobacterium intracellulare
Variant Strain by Molecular Techniques
Authors: Menendez M. C. , Palenque E. , Navarro M. C. , Nuñez M. C. , Rebollo M. J. , Garcia M. J.
Published: July 2002

This paper describes a
Mycobacterium intracellulare
variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the
Mycobacterium avium
complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as
mig
(macrophage-induced gene). The results obtained identify the isolates as a variant of
M. intracellulare
, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members.
New LightCycler PCR for Rapid and Sensitive Quantification of Parvovirus B19 DNA Guides Therapeutic Decision-Making in Relapsing Infections
Authors: Harder Timm C. , Hufnagel Markus , Zahn Katrin , Beutel Karin , Schmitt Heinz-Josef , Ullmann Uwe , Rautenberg Peter
Published: July 2002

Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically ≥5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus.
Mycobacterium interjectum
as Causative Agent of Cervical Lymphadenitis
Authors: De Baere Thierry , Moerman Mieke , Rigouts Leen , Dhooge Catharina , Vermeersch Hubert , Verschraegen Gerda , Vaneechoutte Mario
Published: July 2002

A mycobacterial strain isolated from a lymph node of a 3-year-old female with cervical lymphadenitis was identified as
Mycobacterium interjectum
by means of sequencing of the 16S rRNA gene. Analysis of this case and previously published cases demonstrates the importance of
M. interjectum
as a causative agent of cervical lymphadenitis in young children.
Isolation of Amantadine-Resistant Influenza A Viruses (H3N2) from Patients following Administration of Amantadine in Japan
Authors: Iwahashi Jun , Tsuji Katsuro , Ishibashi Tetsuya , Kajiwara Junboku , Imamura Yoshihiro , Mori Ryoichi , Hara Koyu , Kashiwagi Takahito , Ohtsu Yasushi , Hamada Nobuyuki , Maeda Hisao , Toyoda Michiko , Toyoda Tetsuya
Published: July 2002

In Japan, the use of amantadine for treatment of influenza A virus infection was not accepted until November 1998, although it was widely used for treatment of Parkinsonism. Since then, we have monitored the emergence of amantadine-resistant viruses and isolated two viruses from patients on long-term treatment with amantadine.
Does Blood of Healthy Subjects Contain Bacterial Ribosomal DNA?
Authors: Nikkari Simo , McLaughlin Ian J. , Bi Wanli , Dodge Deborah E. , Relman David A.
Published: July 2002

Real-time PCR methods with primers and a probe targeting conserved regions of the bacterial 16S ribosomal DNA (rDNA) revealed a larger amount of rDNA in blood specimens from healthy individuals than in matched reagent controls. However, the origins and identities of these blood-associated bacterial rDNA sequences remain obscure.
Evaluation of a Reformulated CHROMagar Candida
Authors: Jabra-Rizk Mary Ann , Brenner Troy M. , Romagnoli Mark , Baqui A. A. M. A. , Merz William G. , Falkler William A. , Meiller Timothy F.
Published: July 2002

CHROMagar Candida is a differential culture medium for the isolation and presumptive identification of clinically important yeasts. Recently the medium was reformulated by Becton Dickinson. This study was designed to evaluate the performance of the new formula of CHROMagar against the original CHROMagar Candida for recovery, growth, and colony color with stock cultures and with direct plating of clinical specimens. A total of 90 stock yeast isolates representing nine yeast species, including
Candida dubliniensis
, as well as 522 clinical specimens were included in this study. No major differences were noted in growth rate or colony size between the two media for most of the species. However, all 10
Candida albicans
isolates evaluated consistently gave a lighter shade of green on the new CHROMagar formulation. In contrast, all 26
C. dubliniensis
isolates gave the same typical dark green color on both media. A total of 173 of the 522 clinical specimens were positive for yeast, with eight yeast species recovered. The recovery rates for each species were equivalent on both media, with no consistent species-associated differences in colony size or color. Although both media were comparable in performance, the lighter green colonies of
C. albicans
isolates on the new CHROMagar made it easier to differentiate between
C. albicans
and
C. dubliniensis
isolates. In conclusion, the newly formulated Becton Dickinson CHROMagar Candida medium is as equally suited as a differential medium for the presumptive identification of yeast species and for the detection of multiple yeast species in clinical specimens as the original CHROMagar Candida medium.
Serosubtypes and PorA Types of
Neisseria meningitidis
Serogroup B Isolated in Brazil during 1997-1998: Overview and Implications for Vaccine Development
Authors: Sacchi Claudio T. , Lemos Ana Paula S. , Popovic Tanja , Cassio de Morais Jose , Whitney Anne M. , Melles Carmo Elias A. , Brondi Luciana M. G. , Monteiro Lucia M. C. , Paiva Maria V. , Solari Claude A. , Mayer Leonard W.
Published: July 2002

Meningococcal disease caused by
N. meningitidis
serogroup B (MenB) has been endemic in Brazil since 1997. In this study, we determined the prevalence of serosubtypes of MenB isolated in 10 Brazilian states and the Federal District during 1997 and 1998 and investigated the extent of PorA VR sequence variation among the most prevalent serosubtypes to evaluate the possible use of an outer membrane vesicle (OMV)-, PorA-based vaccine to prevent meningococcal disease in Brazil. During this period, a total of 8,932 cases of meningococcal disease were reported. Only 42% (
n
= 3,751) of the reported cases were laboratory confirmed, and about 60% (
n
= 2,255) of those were identified as MenB. Among 1,297 MenB strains selected for this study, the most prevalent serosubtypes were P1.19,15 (66%), P1.7,1 (11%), and P1.7,16 (4%). PorA VR typing showed that 91% of the P1.19,15 strains analyzed had VR1 and VR2 sequences identical to those of the prototype strain. No sequence variation was detected among the 40 strains representing all isolated MenB P1.7,16 strains in the three southern states, where this serosubtype accounts for 75% of the serosubtypes identified. Similarly, all P1.7,1 strains were identified by PorA typing as P1.7-1,1. Although further improvements in the reporting of cases and collection of strains in Brazil are needed, our data suggest that a trivalent OMV-based vaccine prepared with PorA types P1.19,15, P1.7-1,1, and P1.7,16 may be appropriate to control serogroup B meningococcal disease in most of the Brazilian states.
Identification and Characterization of Variable-Number Tandem Repeats in the
Yersinia pestis
Genome
Authors: Klevytska Alexandra M. , Price Lance B. , Schupp James M. , Worsham Patricia L. , Wong Jane , Keim Paul
Published: July 2002

Yersinia pestis
, the infamous plague-causing pathogen, appears to have emerged in relatively recent history. Evidence of this fact comes from several studies that document a lack of nucleotide diversity in the
Y. pestis
genome. In contrast, we report that variable-number tandem repeat (VNTR) sequences are common in the
Y. pestis
genome and occur frequently in gene coding regions. Larger tandem repeat arrays, most useful for phylogenetic analysis, are present at an average of 2.18 arrays per 10 kbp and are distributed evenly throughout the genome and the two virulence plasmids, pCD1 and pMT1. We examined allelic diversity at 42 chromosomal VNTR loci in 24 selected isolates (12 globally distributed and 12 from Siskiyou County, Calif.). Vast differences in diversity were observed among the 42 VNTR loci, ranging from 2 to 11 alleles. We found that the maximum copy number of repeats in an array was highly correlated with diversity (
R
= 0.86). VNTR-based phylogenetic analysis of the 24 strains successfully grouped isolates from biovar orientalis and most of the antiqua and mediaevalis strains. Hence, multiple-locus VNTR analysis (MLVA) appears capable of both distinguishing closely related strains and successfully classifying more distant relationships. Harnessing the power of MLVA to establish standardized databases will enable researchers to better understand plague ecology and evolution around the world.
Diagnosis of Placental Malaria
Authors: Mockenhaupt Frank P. , Ulmen Ulrike , von Gaertner Christiane , Bedu-Addo George , Bienzle Ulrich
Published: July 2002

In a group of 596 delivering Ghanaian women, the sensitivities of peripheral blood thick film microscopy, ICT Malaria P.f/P.v test, and PCR in detecting microscopically confirmed placental
Plasmodium falciparum
infection were 42, 80, and 97%, respectively. In addition to the gross underestimation of placental malaria by peripheral blood film microscopy, submicroscopic infections were found to be a risk factor for maternal anemia.
Geographic Distribution of Mating Type Alleles of
Cryptococcus neoformans
in Four Areas of the United States
Authors: Yan Zhun , Li Xiaogang , Xu Jianping
Published: July 2002

To better understand the epidemiology and population structure of
Cryptococcus neoformans
, we determined mating types for 358
C. neoformans
strains isolated through the active surveillance program from 1992 to 1994 in four geographic areas in the United States: San Francisco, California; Georgia; Texas; and Alabama. Two assays were used to determine mating types: (i) crossing with standard laboratory tester strains JEC20 and JEC21 on V8 agar medium; and (ii) PCR with the mating type α allele-specific primer of the
STE12
gene and with serotype (A and D)- and mating type (a and α)-specific primers of the
STE20
gene. Using these two methods, we found that this sample consisted of the following: (i) 324 serotype A, mating type (
MAT
) α (Aα) strains; (ii) 12 serotype D, α (Dα) strains; (iii) 14 serotype AD strains with mating type alleles Aa and Dα (AaDα); (iv) 2 serotype AD strains with mating type alleles Aα and Da (AαDa); (v) 3 serotype B, α (Bα) strains; and (vi) 3 serotype AD strains but with only one mating type allele. No strain with
MAT
a was found within serotype A, B, or D in this collection. Interestingly, 14 of the 19 serotype AD strains contained the Aa allele at the
STE20
locus; 13 of these 14 were from San Francisco. Our results suggest that the environment in San Francisco might contain Aa strains capable of mating with Dα strains. In addition, our result demonstrate that the sample from San Francisco had a significantly higher proportion of self-fertile strains than those from the other three areas.
Prospective Evaluation of the New Chromogenic Medium Candida ID, in Comparison with Candiselect, for Isolation of Molds and Isolation and Presumptive Identification of Yeast Species
Authors: Letscher-Bru Valerie , Meyer Marie-Helene , Galoisy Anne-Cecile , Waller Jocelyn , Candolfi Ermanno
Published: July 2002

We conducted a prospective evaluation of Candida ID chromogenic medium (bioMérieux, Marcy l'Etoile, France) with 786 clinical specimens in comparison with Candiselect medium (Bio-Rad, Marnes la Coquette, France). Candida ID chromogenic medium identified 97.7% of
Candida albicans
strains; enabled presumptive identification of
C. tropicalis
,
C. lusitaniae
,
C. guillermondii
, and
C. kefyr
and better detection of yeast combinations (11.4% more often); and was more sensitive for the isolation of filamentous fungi (17.7% more often). However, Candida ID chromogenic medium appeared to be less selective vis-à-vis bacteria, with bacterial colonies sometimes pigmented blue.
Comparison of Chromogenic and Fluorogenic Membrane Filtration Methods for Detection of Four
Candida
Species
Authors: Bauters T. G. , Nelis H. J.
Published: July 2002

CHROMagar Candida medium was inoculated with a variety of clinical samples (
n
= 282) by using membrane filtration instead of swabbing or streaking. This modified CHROMagar procedure increased the ability to detect yeast by 16.7%. Compared to an enzymatic two-step membrane filtration method with fluorogenic substrates, it yielded similar recovery but was slower.
Global Molecular Epidemiology of the O15:K52:H1 Extraintestinal Pathogenic
Escherichia coli
Clonal Group: Evidence of Distribution beyond Europe
Authors: Johnson James R. , Stell Adam L. , O'Bryan Timothy T. , Kuskowski Michael , Nowicki Bogdan , Johnson Candice , Maslow Joel N. , Kaul Anil , Kavle Justine , Prats Guillem
Published: July 2002

Escherichia coli
O15:K52:H1 is a significant extraintestinal pathogen in Europe (G. Prats et al., J. Clin. Microbiol. 38:201-209, 2000). To search for evidence of this clonal group outside of Europe, 75 non-European
E. coli
isolates of serogroup O15 were compared with five members of the O15:K52:H1 clonal group from Barcelona, Spain, according to genomic background, virulence genotypes, and antimicrobial resistance profiles. Amplification phylotyping showed that 16 (21%) of the 75 non-European O15 isolates corresponded with the O15:K52:H1 clonal group. The 16 non-European O15:K52:H1 clonal group members represented diverse geographic locales. They were isolated almost exclusively from humans with extraintestinal infections and accounted for 50% of all O15 isolates from five human clinical collections studied. Most non-European clonal group members exhibited a consensus virulence factor profile that included the F16 or F7-2
papA
alleles (P fimbrial structural subunit),
papG
allele II (P fimbrial adhesin),
iha
(putative adhesin siderophore), and
iutA
(aerobactin receptor). This resembles the virulence profiles of (i) European representatives of the O15:K52:H1 clonal group and (ii) phylogenetically related “clonal group A,” a recently recognized significant contributor to trimethoprim-sulfamethoxazole resistance in the United States (A. R. Manges et al., N. Engl. J. Med. 345:1007-1013, 2001). Antimicrobial resistance profiles were variable, and resistance was inconsistently transferred by conjugation. These findings indicate that the O15:K52:H1 clonal group is broadly distributed beyond Europe, exhibits previously unrecognized phenotypic and genotypic diversity, and contributes significantly to extraintestinal infections in humans.
Frequent Transmission of Enterococcal Strains between Mechanically Ventilated Patients Treated at an Intensive Care Unit
Authors: Lund Bodil , Agvald-Öhman Christina , Hultberg Anna , Edlund Charlotta
Published: July 2002

The objectives of this investigation were to study the respiratory tract colonization and transmission of enterococci between 20 patients treated with mechanical ventilation at an intensive care unit (ICU), to compare genotyping with phenotyping, and to determine the antibiotic susceptibilities of the isolated enterococci. Samples were collected from the oropharynx, stomach, subglottic space, and trachea within 24 h of intubation, every third day until day 18, and thereafter every fifth day until day 33. Enterococcal isolates (
n
= 170) were analyzed by pulsed-field gel electrophoresis and with the PhenePlate (PhP) system. The antimicrobial susceptibilities to five agents were determined. Seventeen of the 20 subjects were colonized with enterococci in the respiratory tract; 12 were colonized in the lower respiratory tract. Genotype analyses suggested that 13 patients were involved in a transmission event, including all patients intubated more than 12 days. In conclusion, colonization of resistant enterococci in the respiratory tract of intubated patients treated at an ICU was common. Transmission of enterococci between patients occurred frequently. Prolonged intubation period seems to be a risk factor for enterococcal cross-transmission.
Comparison of Human Immunodeficiency Virus (HIV)-Specific Infection-Enhancing and -Inhibiting Antibodies in AIDS Patients
Authors: Subbramanian Ramu A. , Xu Jingwu , Toma Emil , Morisset Richard , Cohen Eric A. , Menezes, José , Ahmad Ali
Published: July 2002

The humoral immune response of the human host against the human immunodeficiency virus (HIV) type 1 (HIV-1) envelope glycoproteins comprises virus-neutralizing antibodies (NAs), antibody-dependent cellular cytotoxicity-mediating (ADCC) antibodies, and infection-enhancing antibodies (IEAs). Because of their potential significance for the outcome of infection with this virus, we have studied the relative prevalence of NAs, ADCC antibodies, and IEAs in the sera of patients infected with HIV. Our results demonstrate that while ≥60% of serum samples are positive for NAs or ADCC antibodies, 72% of these serum samples mediate the enhancement of infection in the presence of complement. In patients with low CD4 counts, NA and ADCC antibody levels tend to decrease, while IEA levels increase. A significant positive correlation was found only between the presence of ADCC antibodies and the presence of antibodies that neutralized HIV-1 in the presence of complement. These results show that the anti-HIV-1 humoral immune response consists of a mixture of antibodies that may inhibit or enhance HIV infection and whose ratios may vary in different stages of the infection.
Mouse Hepatitis Virus Replicase Proteins Associate with Two Distinct Populations of Intracellular Membranes
Authors: Sims Amy C. , Ostermann Joachim , Denison Mark R.
Published: July 2002

The coronavirus replicase gene (gene 1) is translated into two co-amino-terminal polyproteins that are proteolytically processed to yield more than 15 mature proteins. Several gene 1 proteins have been shown to localize at sites of viral RNA synthesis in the infected cell cytoplasm, notably on late endosomes at early times of infection. However, both immunofluorescence and electron microscopic studies have also detected gene 1 proteins at sites distinct from the putative sites of viral RNA synthesis or virus assembly. In this study, mouse hepatitis virus (MHV)-infected cells were fractionated and analyzed to determine if gene 1 proteins segregated to more than one membrane population. Following differential centrifugation of lysates of MHV-infected DBT cells, gene 1 proteins as well as the structural N and M proteins were detected almost exclusively in a high-speed small membrane pellet. Following fractionation of the small membrane pellet on an iodixanol density gradient, the gene 1 proteins p28 and helicase cofractionated with dense membranes (1.12 to 1.13 g/ml) that also contained peak concentrations of N. In contrast, p65 and p1a-22 were detected in a distinct population of less dense membranes (1.05 to 1.09 g/ml). Viral RNA was detected in membrane fractions containing helicase, p28, and N but not in the fractions containing p65 and p1a-22. LAMP-1, a marker for late endosomes and lysosomes, was detected in both membrane populations. These results demonstrate that multiple gene 1 proteins segregate into two biochemically distinct but tightly associated membrane populations and that only one of these populations appears to be a site for viral RNA synthesis. The results further suggest that p28 is a component of the viral replication complex whereas the gene 1 proteins p1a-22 and p65 may serve roles during infection that are distinct from viral RNA transcription or replication.
Epstein-Barr Virus EB2 Protein Exports Unspliced RNA via a Crm-1-Independent Pathway
Authors: Farjot Géraldine , Buisson Monique , Duc Dodon Madeleine , Gazzolo Louis , Sergeant Alain , Mikaelian Ivan
Published: July 2002

Human herpesviruses encode posttranscriptional activators that are believed to up-regulate viral replication by facilitating early and late gene expression. We have reported previously that the Epstein-Barr virus protein EB2 (also called M or SM) promotes nuclear export of RNAs that are poor substrates for spliceosome assembly, an effect that closely resembles the human immunodeficiency virus type 1 Rev-dependent nuclear export of unspliced viral RNA. Here we present experimental data showing that EB2 efficiently promotes the nuclear export of unspliced RNA expressed from a Rev reporter construct. Site-directed mutagenesis as well as domain swapping experiments indicate that a leucine-rich region found in the EB2 protein, which matches the consensus sequence for the leucine-rich nuclear export signal, is not a nuclear export signal per se. Accordingly, leptomycin B (LMB), a specific Crm-1 inhibitor, impairs Rev- but not EB2-dependent nuclear export of unspliced RNA. Moreover, EB2 nucleocytoplasmic shuttling visualized by a heterokaryon assay is, unlike Rev shuttling, not affected by LMB. We also show that overexpression of an N-terminal deletion mutant of Nup214/can, a major nucleoporin of the nuclear pore complex involved in several aspects of nuclear transport, blocks both Rev- and EB2-dependent nuclear export of RNA. These results strongly suggest that EB2 nuclear export of unspliced RNA is mediated by a Crm-1-independent pathway.
Characterization of a Region of the Measles Virus Hemagglutinin Sufficient for Its Dimerization
Authors: Plemper Richard K. , Hammond Anthea L. , Cattaneo Roberto
Published: July 2002

Attachment of measles virus (MV) to its cellular receptor is mediated by the viral envelope glycoprotein hemagglutinin (H). H exists at the viral surface as a disulfide-linked dimer which may associate into a tetramer. We aimed to define regions of H essential for its homo-oligomerization. To delineate these more precisely, we have generated a series of H ectodomain truncation mutants and studied their abilities to form both homotypic complexes and heterotypic complexes with full-length H. We define a “minimal unit” which is sufficient for MV H dimerization as that encompassing residues 1 to 151. This unit forms both homodimers and heterodimers with full-length H protein, although neither is transported to the cell surface even in the presence of other MV proteins. We show that cysteine residues at positions 139 and 154 are both critical in mediating covalent dimerization, not only of the truncated H mutants but also of full-length MV H protein. Even those cysteine mutants unable to form covalent intermolecular interactions are biologically active, mediating the formation of syncytia, albeit at a reduced rate. We demonstrate that this impaired capacity to mediate cell-to-cell fusion is based mainly on a reduced transport rate of the mutant molecules to the cell surface, indicating a role for covalent intermolecular interactions in efficient transport of MV H dimers to the cell surface.
The Human Cytomegalovirus 86-Kilodalton Major Immediate-Early Protein Interacts Physically and Functionally with Histone Acetyltransferase P/CAF
Authors: Bryant L. A. , Mixon P. , Davidson M. , Bannister A. J. , Kouzarides T. , Sinclair J. H.
Published: July 2002

The major immediate-early proteins of human cytomegalovirus (HCMV) play a pivotal role in controlling viral and cellular gene expression during productive infection. As well as negatively autoregulating its own promoter, the HCMV 86-kDa major immediate early protein (IE86) activates viral early gene expression and is known to be a promiscuous transcriptional regulator of cellular genes. IE86 appears to act as a multimodal transcription factor. It is able to bind directly to target promoters to activate transcription but is also able to bridge between upstream binding factors such as CREB/ATF and the basal transcription complex as well as interacting directly with general transcription factors such as TATA-binding protein and TFIIB. We now show that IE86 is also able to interact directly with histone acetyltransferases during infection. At least one of these factors is the histone acetyltransferase CBP-associated factor (P/CAF). Furthermore, we show that this interaction results in synergistic transactivation by IE86 of IE86-responsive promoters. Recruitment of such chromatin-remodeling factors to target promoters by IE86 may help explain the ability of this viral protein to act as a promiscuous transactivator of cellular genes.
The Structure of Cucumber Mosaic Virus and Comparison to Cowpea Chlorotic Mottle Virus
Authors: Smith Thomas J. , Chase Elaine , Schmidt Timothy , Perry Keith L.
Published: July 2002

The structure of cucumber mosaic virus (CMV; strain Fny) has been determined to a 3.2-Å resolution using X-ray crystallography. Despite the fact that CMV has only 19% capsid protein sequence identity (34% similarity) to cowpea chlorotic mottle virus (CCMV), the core structures of these two members of the
Bromoviridae
family are highly homologous. As suggested by a previous low-resolution structural study, the 305-Å diameter (maximum) of CMV is ∼12 Å larger than that of CCMV. In CCMV, the structures of the A, B, and C subunits are nearly identical except in their N termini. In contrast, the structures of two loops in subunit A of CMV differ from those in B and C. These loops are 6 and 7 residues longer than the analogous regions in CCMV. Unlike that of CCMV, the capsid of CMV does not undergo swelling at pH 7.0 and is stable at pH 9.0. This may be partly due to the fact that the N termini of the B and C subunits form a unique bundle of six amphipathic helices oriented down into the virion core at the threefold axes. In addition, while CCMV has a cluster of aspartic acid residues at the quasi-threefold axis that are proposed to bind metal in a pH-dependent manner, this cluster is replaced by complementing acids and bases in CMV. Finally, this structure clearly demonstrates that the residues important for aphid transmission lie at the outermost portion of the βH-βI loop and yields details of the portions of the virus that are hypothesized to mediate binding to aphid mouthparts.
Early Steps of Polyomavirus Entry into Cells
Authors: Gilbert Joanna M. , Benjamin Thomas L.
Published: July 2002

The mechanism by which murine polyomavirus penetrates cells and arrives at the nucleus, the site of viral replication, is not well understood. Simian virus 40 and JC virus, two closely related members of the polyomavirus subfamily, use caveola- and clathrin-mediated uptake pathways for entry, respectively. The data presented here indicate that compounds that block endocytosis of both caveola- and clathrin-derived vesicles have no effect on polyomavirus infectivity. Polyomavirus does not appear to colocalize with either clathrin light chain or caveolin-1 by immunofluorescence microscopy. Additionally, expression of a dominant-negative form of dynamin I has no effect on polyomavirus uptake and infectivity. Therefore, polyomavirus uptake occurs through a class of uncoated vesicles in a clathrin-, caveolin-1-, and dynamin I-independent manner.