bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Induction of Virulence and Antibiotic Resistance Transfer in
Enterococcus faecalis
Mediated by the Sex Pheromone-Sensing System of pCF10
Authors: Hirt Helmut , Schlievert Patrick M. , Dunny Gary M.
Published: July 2002

Enterococcus faecalis
has become one of the most notable nosocomial pathogens in the last decade. Aggregation substance (AS) on the sex pheromone plasmids of
E. faecalis
has been implicated as a virulence factor in several model systems. We investigated the AS-encoding plasmid pCF10 for its ability to increase virulence in a rabbit endocarditis model. Cells containing pCF10 increased the virulence in the model significantly, as assessed by an increase in aortic valve vegetation size. The results confirmed in vivo induction of the normally tightly controlled AS. In addition to the expression of AS when
E. faecalis
cells were in contact with plasma, plasmid transfer of the tetracycline resistance-carrying plasmid was also activated in vitro and in vivo. In vivo, plasmid transfer reached remarkable frequencies of 8 × 10
−2
to 9 × 10
−2
. These values are comparable to the highest frequencies ever observed in vitro. Cells harboring pCF10 had a significant survival advantage over plasmid-free cells indicated by pCF10 present in two-thirds of the recipient population. Plasma induction was dependent on the presence of the plasmid-encoded PrgZ protein, indicating the requirement of the pheromone-sensing system in the induction process. The data suggested that the mechanism of in vivo induction may involve interference of plasma with the normal function of the pheromone peptide and its inhibitor.
Babesia bovisMerozoite Surface Antigen 1 and Rhoptry-Associated Protein 1 Are Expressed in Sporozoites, and Specific Antibodies Inhibit Sporozoite Attachment to Erythrocytes
Authors: Mosqueda Juan , McElwain Terry F. , Stiller David , Palmer Guy H.
Published: July 2002

We examinedBabesia bovissporozoites for the expression of two molecules, merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), that are postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites. Importantly, monospecific MSA-1 and RAP-1 antisera each inhibited sporozoite invasion of erythrocytes in vitro. This is the first identification of antigens expressed inBabesiasp. sporozoites and establishes that, at least in part, sporozoites and merozoites share common targets of antibody mediated inhibition of erythrocyte invasion.
Growth of
Neisseria gonorrhoeae
in the Female Mouse Genital Tract Does Not Require the Gonococcal Transferrin or Hemoglobin Receptors and May Be Enhanced by Commensal Lactobacilli
Authors: Jerse Ann E. , Crow Emily T. , Bordner Amy N. , Rahman Ishrat , Cornelissen Cynthia Nau , Moench Thomas R. , Mehrazar Karim
Published: July 2002

Neisseria gonorrhoeae
is capable of utilizing a variety of iron sources in vitro, including human transferrin, human lactoferrin, hemoglobin, hemoglobin-haptoglobin complexes, heme, and heterologous siderophores. Transferrin has been implicated as a critical iron store for
N. gonorrhoeae
in the human male urethra. The demonstration that gonococci can infect the lower genital tracts of estradiol-treated BALB/c mice in the absence of human transferrin, however, suggests that other usable iron sources are present in the murine genital tract. Here we demonstrate that gonococcal transferrin and hemoglobin receptor mutants are not attenuated in mice, thereby ruling out transferrin and hemoglobin as essential for murine infection. An increased frequency of phase variants with the hemoglobin receptor “on” (Hg
+
) occurred in ca. 50% of infected mice; this increase was temporally associated with an influx of neutrophils and detectable levels of hemoglobin in the vagina, suggesting that the presence of hemoglobin in inflammatory exudates selects for Hg
+
phase variants during infection. We also demonstrate that commensal lactobacilli support the growth of
N. gonorrhoeae
in vitro unless an iron chelator is added to the medium. We hypothesize that commensal lactobacilli may enhance growth of gonococci in vivo by promoting the solubilization of iron on mucosal surfaces through the production of metabolic intermediates. Finally, transferrin-binding lipoprotein (TbpB) was detected on gonococci in vaginal smears, suggesting that although gonococci replicate within the genital tracts of mice, they may be sufficiently iron-stressed to express iron-repressible proteins. In summary, these studies support the potential role of nontransferrin, nonhemoglobin iron sources during gonococcal infection of the female genital tract.
Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of the
Escherichia coli rhaBAD
Operon
Authors: Holcroft Carolyn C. , Egan Susan M.
Published: July 2002

The
Escherichia coli rhaBAD
operon encodes the enzymes for catabolism of the sugar
l
-rhamnose. Full
rhaBAD
activation requires the AraC family activator RhaS (bound to a site that overlaps the −35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at −92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on
rhaBAD
activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from
rhaBAD
promoter fusions. We also expressed a derivative of the α subunit of RNA polymerase deleted for the entire C-terminal domain (α-Δ235) and assayed expression from
rhaBAD
promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the α subunit (α-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on α-CTD. Overall, our results suggest that CRP interaction with α-CTD may not be required for
rhaBAD
activation; however, α-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.
Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by
Pseudomonas abietaniphila
BKME-9
Authors: Martin Vincent J. J. , Mohn William W.
Published: July 2002

We have cloned and sequenced the
dit
gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by
Pseudomonas abietaniphila
BKME-9. The
dit
gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes
ditA1A2A3
encode the α and β subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (
ditA1
::Tn
5
) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of
xylE
transcriptional fusion strains showed induction of
ditA1
and
ditA3
by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, the
dit
cluster includes
ditC
, encoding an extradiol ring cleavage dioxygenase, and
ditR
, encoding an IclR-type transcriptional regulator. Although
ditR
is not strictly required for the growth of strain BKME-9 on abietanes, a
ditR
::Km
r
mutation in a
ditA3
::
xylE
reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of
ditA3
. An ORF with sequence similarity to genes encoding permeases (
ditE
) is linked with genes involved in abietane degradation.
The Redox-Sensitive Transcriptional Activator OxyR Regulates the Peroxide Response Regulon in the Obligate Anaerobe
Bacteroides fragilis
Authors: Rocha Edson R. , Owens Gary , Smith C. Jeffrey
Published: July 2002

The peroxide response-inducible genes
ahpCF
,
dps
, and
katB
in the obligate anaerobe
Bacteroides fragilis
are controlled by the redox-sensitive transcriptional activator OxyR. This is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria.
oxyR
and
dps
were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions.
B. fragilis
OxyR and Dps proteins showed high identity to homologues from a closely related anaerobe,
Porphyromonas gingivalis
. Northern blot analysis revealed that
oxyR
was expressed as a monocistronic 1-kb mRNA and that
dps
mRNA was approximately 500 bases in length.
dps
mRNA was induced over 500-fold by oxidative stress in the parent strain and was constitutively induced in the peroxide-resistant mutant IB263. The constitutive peroxide response in strain IB263 was shown to have resulted from a missense mutation at codon 202 (GAT to GGT) of the
oxyR
gene [
oxyR
(Con)] with a predicted D202G substitution in the OxyR protein. Transcriptional fusion analysis revealed that deletion of
oxyR
abolished the induction of
ahpC
and
katB
following treatment with hydrogen peroxide or oxygen exposure. However,
dps
expression was induced approximately fourfold by oxygen exposure in Δ
oxyR
strains but not by hydrogen peroxide. This indicates that
dps
expression is also under the control of an oxygen-dependent OxyR-independent mechanism. Complementation of Δ
oxyR
mutant strains with wild-type
oxyR
and
oxyR
(Con) restored the inducible peroxide response and the constitutive response of the
ahpCF
,
katB
, and
dps
genes, respectively. However, overexpression of OxyR abolished the catalase activity but not
katB
expression, suggesting that higher levels of intracellular OxyR may be involved in other physiological processes. Analysis of
oxyR
expression in the parents and in Δ
oxyR
and overexpressing
oxyR
strains by Northern blotting and
oxyR
′::
xylB
fusions revealed that
B. fragilis
OxyR does not control its own expression.
The ς
70
Transcription Factor TyrR Has Zinc-Stimulated Phosphatase Activity That Is Inhibited by ATP and Tyrosine
Authors: Zhao Shimin , Zhu Qin , Somerville Ronald L.
Published: July 2002

The TyrR protein of
Escherichia coli
(513 amino acid residues) is the chief transcriptional regulator of a group of genes that are essential for aromatic amino acid biosynthesis and transport. The TyrR protein can function either as a repressor or as an activator. The central region of the TyrR protein (residues 207 to 425) is similar to corresponding polypeptide segments of the NtrC protein superfamily. Like the NtrC protein, TyrR has intrinsic ATPase activity. Here, we report that TyrR possesses phosphatase activity. This activity is subject to inhibition by
l
-tyrosine and its analogues and by ATP and ATP analogues. Zinc ion (2 mM) stimulated the phosphatase activity of the TyrR protein by a factor of 57. The phosphatase-active site of TyrR was localized to a 31-kDa domain (residues 191 to 467) of the protein. However, mutational alteration of distant amino acid residues at both the N terminus and the C terminus of TyrR altered the phosphatase activity.
Haemophilus influenzae
TyrR (318 amino acid residues), a protein with a high degree of sequence similarity to the C terminus of the
E. coli
TyrR protein, exhibited a phosphatase activity similar to that of
E. coli
TyrR.
Characterization and Role of
tbuX
in Utilization of Toluene by
Ralstonia pickettii
PKO1
Authors: Kahng Hyung-Yeel , Byrne Armando M. , Olsen Ronald H. , Kukor Jerome J.
Published: July 2002

The
tbu
regulon of
Ralstonia pickettii
PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the
tbu
pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of
tbuT
, which is located immediately downstream of
tbuA1UBVA2C
, and the associated promoter (
PtbuA1
) is unique in that it results in a cascade type of up-regulation of
tbuT
in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of
tbuT
, 63 bp downstream of the
tbuT
stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated
tbuX
. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in
Escherichia coli
. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in
R. pickettii
PKO1. In addition, the expression of
tbuX
appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.
FabF Is Required for Piezoregulation of
cis
-Vaccenic Acid Levels and Piezophilic Growth of the Deep-Sea Bacterium
Photobacterium profundum
Strain SS9
Authors: Allen Eric E. , Bartlett Douglas H.
Published: July 2002

To more fully explore the role of unsaturated fatty acids in high-pressure, low-temperature growth, the
fabF
gene from the psychrotolerant, piezophilic deep-sea bacterium
Photobacterium profundum
strain SS9 was characterized and its role and regulation were examined. An SS9 strain harboring a disruption in the
fabF
gene (strain EA40) displayed growth impairment at elevated hydrostatic pressure concomitant with diminished
cis
-vaccenic acid (18:1) production. However, growth ability at elevated pressure could be restored to wild-type levels by the addition of exogenous 18:1 to the growth medium. Transcript analysis did not indicate that the SS9
fabF
gene is transcriptionally regulated, suggesting that the elevated 18:1 levels produced in response to pressure increase result from posttranscriptional changes. Unlike many pressure-adapted bacterial species such as SS9, the mesophile
Escherichia coli
did not regulate its fatty acid composition in an adaptive manner in response to changes in hydrostatic pressure. Moreover, an
E. coli fabF
strain was as susceptible to elevated pressure as wild-type cells. It is proposed that the SS9
fabF
product, β-ketoacyl–acyl carrier protein synthase II has evolved novel pressure-responsive characteristics which facilitate SS9 growth at high pressure.
Cloning, Expression, and Characterization of
cis
-Polyprenyl Diphosphate Synthase from the Thermoacidophilic Archaeon
Sulfolobus acidocaldarius
Authors: Hemmi Hisashi , Yamashita Satoshi , Shimoyama Takefumi , Nakayama Toru , Nishino Tokuzo
Published: July 2002

cis
-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms. However, only little is known about this enzyme of archaea. In this report, we isolated the gene of
cis
-polyprenyl diphosphate synthase from a thermoacidophilic archaeon,
Sulfolobus acidocaldarius
, and characterized the recombinant enzyme.
Functional and Mutational Analysis of P19, a DNA Transfer Protein with Muramidase Activity
Authors: Bayer Michaela , Iberer Robert , Bischof Karin , Rassi Edith , Stabentheiner Edith , Zellnig Günther , Koraimann Günther
Published: July 2002

Protein P19 encoded by the conjugative resistance plasmid R1 has been identified as being one member of a large family of muramidases encoded by bacteriophages and by type III and type IV secretion systems. We carried out a mutational analysis to investigate the function of protein P19 and used in vivo complementation assays to test those of several P19 mutants. The results indicated that conserved residues present in the presumed catalytic center of P19 are absolutely essential for its function in conjugation of plasmid R1 and infection by the RNA phage R17. Overexpression of protein P19 in an early growth phase resulted in a massive lysis of
Escherichia coli
cells in liquid culture, as indicated by a rapid and distinct decrease in cell culture densities after induction. Change of the proposed catalytic glutamate at position 44 to glutamine completely abolished this effect. P19-induced cell lysis was directly shown by transmission and scanning electron microscopy. Typically, P19-overexpressing cells showed bulges protruding from the cell surfaces. Our interpretation is that these protrusions arose from a localized and spatially confined disruption of the bacterial cell wall. To our knowledge such an effect has not previously been documented for any member of the lytic transglycosylase family. From the data presented here, we conclude that protein P19 possesses the proposed localized peptidoglycan-hydrolyzing activity. This activity would be a prerequisite for efficient penetration of the cell envelope by the DNA translocation complex encoded by the conjugative plasmid.
Characterization of VPI Pathogenicity Island and CTXφ Prophage in Environmental Strains of
Vibrio cholerae
Authors: Mukhopadhyay Asish K. , Chakraborty Soumen , Takeda Yoshifumi , Nair G. Balakrish , Berg Douglas E.
Published: July 2002

Environmental isolates of
Vibrio cholerae
of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into
V. cholerae
evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of
tcpF
(which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical
tcpF
; (ii) a new allele of
toxT
(virulence regulatory gene), 36% divergent (at the protein level) in its 5′ half and nearly identical in its 3′ half to canonical
toxT
; (iii) a new
tcpA
(pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of
toxT
. Also found were transpositions of an IS
903
-related element and function-unknown genes to sites in VPI. Cholera toxin (
ctx
) genes were found in isolates of two RAPD types, in each case embedded in CTXφ-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic
V. cholerae
strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species.
Residue Aspartate-147 from the Third Transmembrane Region of Na
+
/H
+
Antiporter NhaB of
Vibrio alginolyticus
Plays a Role in Its Activity
Authors: Nakamura Tatsunosuke , Fujisaki Yumiko , Enomoto Hiromi , Nakayama Yuji , Takabe Teruhiro , Yamaguchi Naoto , Uozumi Nobuyuki
Published: July 2002

NhaB is a bacterial Na
+
/H
+
antiporter with unique topology. The pH dependence of NhaB from
Vibrio alginolyticus
differs from that of the
Escherichia coli
NhaB homolog. Replacement of Asp-147 with Glu made high H
+
concentrations a requirement for the NhaB activity. Replacement of Asp-147 with neutral amino acids inactivated NhaB.
Differential Effects of ReplacingEscherichiacoliRibosomal Protein L27 with Its Homologue fromAquifexaeolicus
Authors: Maguire Bruce A. , Manuilov Anton V. , Zimmermann Robert A.
Published: July 2002

TherpmAgene, which encodes 50S ribosomal subunit protein L27, was cloned from the extreme thermophileAquifexaeolicus, and the protein was overexpressed and purified. Comparison of theA.aeolicusprotein with its homologue fromEscherichiacoliby circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas theE.coliprotein is unstructured under the same conditions. A mutant ofE.colithat lacks L27 was found earlier to be impaired in the assembly and function of the 50S subunit; both defects could be corrected by expression ofE.coliL27 from an extrachromosomal copy of therpmAgene. WhenA.aeolicusL27 was expressed in the same mutant, an increase in the growth rate occurred and the “foreign” L27 protein was incorporated intoE.coliribosomes. However, the presence ofA.aeolicusL27 did not promote 50S subunit assembly. Thus, while theA.aeolicusprotein can apparently replace itsE.colihomologue functionally in completed ribosomes, it does not assist in the assembly ofE.coliribosomes that otherwise lack L27. Possible explanations for this paradoxical behavior are discussed.
Characterization ofPneumocystis carinii PHR1, a pH-Regulated Gene Important for Cell Wall Integrity
Authors: Kottom Theodore J. , Thomas Charles F. , Limper Andrew H.
Published: July 2002

Pneumocystis cariniiremains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region codingGSC-1, a gene mediating β-glucan cell wall synthesis inP. carinii. The predicted amino acid sequence of this new gene, termedP. carinii PHR1,exhibited 38% homology toSaccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology toCandida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression ofP. carinii PHR1was examined. As inC. albicans,P. carinii PHR1expression was repressed under acidic conditions but induced at neutral and more alkaline pH.PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties withP. cariniiculture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression ofP. cariniigenes in related fungi to address functional correlates ofP. carinii-encoded proteins. Therefore, the potential role ofP. carinii PHR1in cell wall integrity was examined by assessing its ability to rescue anS. cerevisiae gas1mutant with absent endogenous Phr1p-like activity. Interestingly,P. carinii PHR1DNA successfully restored proliferation ofS. cerevisiae gas1mutants under lethal conditions of cell wall stress. These results indicate thatP. carinii PHR1encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.
Characterization of
Pseudomonas aeruginosa
Chitinase, a Gradually Secreted Protein
Authors: Folders Jindra , Algra Jon , Roelofs Marc S. , van Loon Leendert C. , Tommassen Jan , Bitter Wilbert
Published: July 2002

The gram-negative bacterium
Pseudomonas aeruginosa
secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene,
chiC
, coding for an extracellular chitinolytic enzyme, was identified. The
chiC
gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of
Serratia marcescens
, ChiA of
Vibrio harveyi,
and ChiD of
Bacillus circulans
and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and
p
-nitrophenyl-β-
d
-
N
,
N
′,
N
"-triacetylchitotriose, but not on
p
-nitrophenyl-β-
d
-
N
-acetylglucosamine, indicating that it is an endochitinase. Expression of the
chiC
gene appears to be regulated by the quorum-sensing system of
P. aeruginosa
, since this gene was not expressed in a
lasIR vsmI
mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.
Overexpression of the Response Regulator
evgA
of the Two-Component Signal Transduction System Modulates Multidrug Resistance Conferred by Multidrug Resistance Transporters
Authors: Nishino Kunihiko , Yamaguchi Akihito
Published: July 2002

Overexpression of
evgA
, a response regulator of a two-component system, increased multidrug efflux in
Escherichia coli
. Since overexpression of the
emrKY
operon, which is controlled by
evgAS
, could account only for deoxycholate resistance, the
evgAS
locus apparently controls expression of at least one other multidrug efflux operon.
The −10 Region Is a Key Promoter Specificity Determinant for the
Bacillus subtilis
Extracytoplasmic-Function ς Factors ς
X
and ς
W
Authors: Qiu Jian , Helmann John D.
Published: July 2002

Transcriptional selectivity derives, in large part, from the sequence-specific DNA-binding properties of the ς subunit of RNA polymerase. There are 17 ς factors in
Bacillus subtilis
which, in general, recognize distinct sets of promoters. However, some ς factors have overlapping promoter selectivity. We hypothesize that the overlap between the regulons activated by the ς
X
and ς
W
factors can be explained by overlapping specificity for the −10 region: ς
X
recognizes −10 elements with the sequence CGAC and ς
W
recognizes CGTA, while both can potentially recognize CGTC. To test this model, we mutated the ς
X
-specific autoregulatory site (P
X
), containing the −10 element CGAC, to either CGTC or GCTA. Conversely, the ς
W
autoregulatory site (P
W
) was altered from CGTA to CGTC or CGAC. Transcriptional analyses, both in vitro and in vivo, indicate that changes to the −10 element are sufficient to switch a promoter from the ς
X
to the ς
W
regulon or, conversely, from the ς
W
to the ς
X
regulon, but context effects clearly play an important role in determining promoter strength. It seems likely that these subtle differences in promoter selectivity derive from amino acid differences in conserved region 2 of ς, which contacts the −10 element. However, we were unable to alter promoter selectivity by replacements of two candidate recognition residues in ς
W
.
Requirement for NusG for Transcription Antitermination In Vivo by the λ N Protein
Authors: Zhou Ying , Filter Joshua J. , Court Donald L. , Gottesman Max E. , Friedman David I.
Published: July 2002

Transcription antitermination by the bacteriophage λ N protein is stimulated in vitro by the
Escherichia coli
NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription antitermination in intact cells.
TonB Interacts with Nonreceptor Proteins in the Outer Membrane ofEscherichia coli
Authors: Higgs Penelope I. , Letain Tracy E. , Merriam Kelley K. , Burke Neal S. , Park HaJeung , Kang ChulHee , Postle Kathleen
Published: July 2002

TheEscherichia coliTonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B12across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.
Selenium Is Mobilized In Vivo from Free Selenocysteine and Is Incorporated Specifically into Formate Dehydrogenase H and tRNA Nucleosides
Authors: Lacourciere Gerard M.
Published: July 2002

Selenophosphate synthetase (SPS), the
selD
gene product from
Escherichia coli
, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. It was recently demonstrated that selenium delivered from selenocysteine by an
E. coli
NifS-like protein could replace free selenide in the in vitro SPS assay for selenophosphate formation (G. M. Lacourciere, H. Mihara, T. Kurihara, N. Esaki, and T. C. Stadtman, J. Biol. Chem. 275:23769-23773, 2000). During growth of
E. coli
in the presence of 0.1 μM
75
SeO
3
2−
and increasing amounts of
l
-selenocysteine, a concomitant decrease in
75
Se incorporation into formate dehydrogenase H and nucleosides of bulk tRNA was observed. This is consistent with the mobilization of selenium from
l
-selenocysteine in vivo and its use in selenophosphate formation. The ability of
E. coli
to utilize selenocysteine as a selenium source for selenophosphate biosynthesis in vivo supports the participation of the NifS-like proteins in selenium metabolism.
Characterization of Newly Emerging Newcastle Disease Virus Isolates from the People's Republic of China and Taiwan
Authors: Yu Li , Wang Zhiliang , Jiang Yihai , Chang Leo , Kwang Jimmy
Published: July 2002

Seven Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken and pigeon flocks in China and Taiwan between 1996 and 2000 were genotypically and pathotypically characterized. By phylogenetic analysis of the fusion protein genes, isolates Ch-A7/96, Ch/98-3, Ch/99, Ch/2000, and TW/2000 were placed into two novel subgenotypes, VIIc and VIId. Isolate Ch/98-1 was grouped into subgenotype VIb, while Ch-W6/96 was proven to be a mixture of isolates Ch-A7/96 and Ch/98-1. These isolates were pathotyped as viscerotropic velogenic for Ch/98-3, Ch/99, Ch/2000, and TW/2000; neurotropic velogenic for Ch-A7/96; and mesogenic for Ch/98-1. Three separate, comparative, genetic analyses of the F genes, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, were performed with our isolates and selected NDV strains from GenBank. Results showed that the close genetic similarity provided evidence for the epidemiological linkage between the outbreaks in China and Taiwan and that the 1990s outbreaks in Asia, the Middle East, Africa, and Europe constituted the fourth panzootic of ND. In combination with epidemiological analysis, an evolutionary model of the NDV strains, representative of the direction of transmission within the NDV strains, was proposed, and epidemiology of NDV transmission was evaluated with emphasis on molecular aspects. Finally, a cross-protective experiment indicated that at least one strain (Ch-A7/96) among our NDV isolates was an antigenic variant, responsible for recent outbreaks of ND in vaccinated chicken flocks.
Use of Random Amplified Polymorphic DNA PCR To Examine Epidemiology of
Stenotrophomonas maltophilia
and
Achromobacter
(
Alcaligenes
)
xylosoxidans
from Patients with Cystic Fibrosis
Authors: Krzewinski Jay W. , Nguyen Chau D. , Foster Jessica M. , Burns Jane L.
Published: July 2002

Stenotrophomonas maltophilia
and
Achromobacter
(
Alcaligenes
)
xylosoxidans
have been increasingly recognized as a cause of respiratory tract colonization in cystic fibrosis (CF). Although both organisms have been associated with progressive deterioration of pulmonary function, demonstration of causality is lacking. To examine the molecular epidemiology of
S. maltophilia
and
A. xylosoxidans
in CF, isolates from patients monitored for up to 2 years were fingerprinted using a PCR-based randomly amplified polymorphic DNA (RAPD-PCR) method. Sixty-one of 69 CF centers screened had 183
S. maltophilia
culture-positive patients, and 46 centers had 92
A. xylosoxidans
-positive patients. At least one isolate from each patient was genotyped, and patients with ≥10 positive cultures (12 S
. maltophilia
cultures, 15
A. xylosoxidans
cultures) had serial isolates genotyped. In addition, centers with multiple culture-positive patients were examined for evidence of shared clones. There were no instances of shared genotypes among different CF centers. Some patients demonstrated isolates with a single genotype throughout the observation period, and others had intervening or sequential genotypes. At the six centers with multiple
S. maltophilia
culture-positive patients and the seven centers with multiple
A. xylosoxidans
-positive patients, there were three and five instances of shared genotypes, respectively. The majority of shared isolates were from pairs who were siblings or otherwise epidemiologically linked. These findings suggest RAPD-PCR typing can distinguish unique CF isolates of
S. maltophilia
and
A. xylosoxidans
, person-to-person transmission may occur, there are not a small number of clones infecting CF airways, and patients with long-term colonization may either have a persistent organism or may acquire additional organisms over time.
Evaluation of the Hexaplex Assay for Detection of Respiratory Viruses in Children
Authors: Kehl Sue C. , Henrickson Kelly J. , Hua Weimin , Fan Jiang
Published: July 2002

The Hexaplex assay (Prodesse, Inc., Milwaukee, Wis.) is a multiplex reverse transcriptase (RT)-PCR assay for the detection of parainfluenza virus types 1, 2, and 3, respiratory syncytial virus (RSV) types A and B, and influenza virus types A and B. We evaluated the Hexaplex assay in comparison with conventional viral cell cultures and rapid enzyme immunoassays (EIAs) for RSV (Directigen; Becton Dickinson Inc., Cockeysville, Md.) and influenza A virus (Abbott Test Pack; Abbott Laboratories, Abbott Park, Ill.) for the detection of respiratory viruses from pediatric respiratory specimens obtained from children seen at Children's Hospital of Wisconsin from December 1997 through May 1998. A total of 363 respiratory specimens were evaluated. The tissue culture prevalence of parainfluenza virus during this period of time was low (1.1%). The sensitivity, specificity, and positive and negative predictive value of Hexaplex compared to tissue culture for the detection of parainfluenza virus were 100, 95.8, 19.0, and 100%, respectively. Only one specimen was determined to contain influenza B virus by Hexaplex; it was tissue culture negative. A specimen was considered to contain RSV or influenza A virus when it was either culture positive or culture negative but Hexaplex and EIA positive. Prior to the analysis of discrepant results, the sensitivity, specificity, and positive and negative predictive value for the detection of RSV were 91.2, 100, 100, and 98.0%, respectively, for tissue culture; 84.5, 100, 100, and 96.6% for EIA; and 98.5, 91.5, 72.8, and 99.6% for Hexaplex, respectively. The sensitivity, specificity, and positive and negative predictive value for the detection of influenza A virus prior to the analysis of discrepant results were 100, 100, 100, and 100%, respectively, for culture, 78.0, 100, 100, and 89.4% for EIA, respectively, and 95.1, 94.1, 67.2, and 99.3% for Hexaplex, respectively. Culture- and/or EIA-negative, Hexaplex-positive specimens were analyzed by a second RT-PCR assay which used primers specific for a different genomic region than that used in the Hexaplex assay. After analysis of these discrepant results, the sensitivity, specificity, and positive and negative predictive value for the detection of RSV were 74.3, 100, 100, and 93.5%, respectively, for tissue culture; 70.3, 100, 100, and 92.5% for EIA; and 98.6, 97.4, 91.2, and 99.6% for Hexaplex. The sensitivity, specificity, and positive and negative predictive value for the detection of influenza A virus were 83.3, 100, 100, and 97.4%, respectively, for tissue culture; 69.4, 100, 100, and 83.3% for EIA; and 95.8, 98.7, 92.0, and 99.3% for Hexaplex. Hexaplex is a rapid, sensitive, and specific method for the detection of the seven most common respiratory viruses in children.
Real-Time PCR Assay Targeting IS
481
of
Bordetella pertussis
and Molecular Basis for Detecting
Bordetella holmesii
Authors: Reischl Udo , Lehn Norbert , Sanden Gary N. , Loeffelholz Mike J.
Published: July 2002

Detection of
Bordetella holmesii
by a real-time PCR assay targeting IS
481
of
Bordetella pertussis
is reported. Sequencing of IS
481
-specific PCR products from
B. pertussis
and
B. holmesii
isolates revealed sequence homology. Restriction fragment length polymorphism demonstrated a low copy number of IS
481
-like sequences in
B. holmesii
. These results, and culture of
B. holmesii
from patients with cough, suggest that the specificity and predictive value of IS
481
-based PCR assays for pertussis may be compromised.
Efficacy of a Swab Transport System in Maintaining Viability of
Neisseria gonorrhoeae
and
Streptococcus pneumoniae
Authors: Farhat Shaker E. , Thibault Mary , Devlin Roslyn
Published: July 2002

The efficacy of swab transport systems in maintaining viability of
Neisseria gonorrhoeae
and
Streptococcus pneumoniae
is crucial both for establishing definitive diagnosis and for monitoring emerging resistance. We tested the efficacy of a newly modified Amies charcoal swab transport system, the StarSwab SP131X (Starplex Scientific, Inc., Etobicoke, Ontario, Canada), by using a combined total of 31 clinical and American Type Culture Collection stock reference strains of
N. gonorrhoeae
and
S. pneumoniae
in 46 suspensions of concentrations ranging from 10
5
to 10
8
CFU/ml. Triplicate swabs per strain held at room temperature for 0, 24, and 48 h were plated without prior vortexing, and their growths were graded. All 31 strains were viable at 0 and 24 h. Gonococcal viability at 48 h varied considerably, even among strains with comparable inoculum sizes, suggesting that viability might be strain dependent and confirming the different structural and growth profiles of gonococcal strains.
S. pneumoniae
strains showed consistent viability, with all strains recovered at all holding periods. This study demonstrates that the StarSwab SP131X is capable of maintaining the viability of
N. gonorrhoeae
and
S. pneumoniae
for at least 24 and 48 h, respectively, and reinforces the need for adequate sampling and for timely processing of specimens to maintain optimum performance.
Evaluation of the Affirm Ambient Temperature Transport System for the Detection and Identification of
Trichomonas vaginalis
,
Gardnerella vaginalis
, and
Candida
Species from Vaginal Fluid Specimens
Authors: Brown Haywood L. , Fuller DeAnna D. , Davis Thomas E. , Schwebke Jane R. , Hillier Sharon L.
Published: July 2002

The objective of this study was to measure the performance of the Affirm Ambient Temperature Transport System (ATTS) over time and to estimate the length of time the system can preserve a vaginal specimen containing the three common organisms causing vaginitis:
Trichomonas vaginalis
,
Candida
species, and
Gardnerella vaginalis
(one of the causative agents of bacterial vaginosis). Women with symptoms of vaginitis presenting to one of three clinical centers were evaluated over a 4- to 8-week period. Four simultaneously obtained swabs were collected and tested by the Affirm VPIII assay at time zero with and without a preservative reagent, at 24 h with reagent, and at either 48 or 72 h with reagent. For each of the three organisms,
Trichomonas
,
Gardnerella
, and
Candida
, positivity at each time point was evaluated and compared to that at reference time zero with and without the ATTS. A total of 940 specimens were obtained from the three clinical sites. Eight hundred three were positive for one or more of the three organisms.
Gardnerella
had the highest overall positive rate (62%), followed by
Candida
with 18% and
Trichomonas
at 9%. The percent sensitivity versus control for
Trichomonas
ranged from 100% at time zero with and without reagent to 91% by 72 h.
Gardnerella
and
Candida
sensitivity remained at 100% for each time period. The Affirm VPIII ATTS system performed within 10% of the control swab (no transport reagent) at all four time points (0, 24, 48, and 72 h) for
Trichomonas
,
Gardnerella
, and
Candida
.
In Vitro Activities of Terbinafine in Combination with Fluconazole, Itraconazole, Voriconazole, and Posaconazole against Clinical Isolates of
Candida glabrata
with Decreased Susceptibility to Azoles
Authors: Perea Sofia , Gonzalez Gloria , Fothergill Annette W. , Sutton Deanna A. , Rinaldi Michael G.
Published: July 2002

A checkerboard microdilution method, performed according to the recommendations of the National Committee for Clinical Laboratory Standards, was used to study the in vitro interaction of terbinafine (TRB) with fluconazole (FLU), itraconazole (ITRA), voriconazole (VRC), and posaconazole (PSZ) in 24 isolates of
Candida glabrata
with decreased susceptibility to azoles isolated from the oral cavities of human immunodeficiency virus patients. Synergy, defined as a fractional inhibitory concentration index of ≤0.5, was observed in 17% of TRB-FLU interactions, 21% of TRB-ITRA interactions, 33% of TRB-VRC interactions, and 12% of TRB-PSZ interactions. Where synergy was not achieved, there was still a decrease in the MIC of one or both drugs when used in combination. Antagonism was not observed in any drug combination. Clinical studies are warranted to elucidate the potential utility of these combination therapies.
Quantitative Detection of Respiratory
Chlamydia pneumoniae
Infection by Real-Time PCR
Authors: Kuoppa Yvonne , Boman Jens , Scott Lena , Kumlin Urban , Eriksson Iréne , Allard Annika
Published: July 2002

Real-time PCR was evaluated as a quantitative diagnostic method for
Chlamydia pneumoniae
infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing
C. pneumoniae
infection.
Modification of the Cytoplasmic Domain of Influenza Virus Hemagglutinin Affects Enlargement of the Fusion Pore
Authors: Kozerski Christine , Ponimaskin Evgeni , Schroth-Diez Britta , Schmidt Michael F. G. , Herrmann Andreas
Published: July 2002

The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.
Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells
Authors: Xiang Jinhua , Wünschmann Sabina , Schmidt Warren , Shao Jianqiang , Stapleton Jack T.
Published: July 2002

GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro.