bluuebunny/crossref_metadata_2025 huggingface dataset:
Organization of the PlasmidcpeLocus inClostridium perfringensType A Isolates
Authors: Miyamoto Kazuaki , Chakrabarti Ganes , Morino Yosiharu , McClane Bruce A.
Published: July 2002

Clostridium perfringenstype A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), whileC. perfringenstype A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmidcpegene. In the present study, the plasmidcpelocus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of thecpelocus in other type A isolates. Those analyses determined that the region upstream of the plasmidcpegene is highly conserved among type A isolates carrying acpeplasmid. The organization of the type A plasmidcpelocus was also found to be unique, as it contains IS1469sequences located similarly to those in the chromosomalcpelocus but lacks the IS1470sequences found upstream of IS1469in the chromosomalcpelocus. Instead of those upstream IS1470sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469in the plasmidcpelocus of all type A isolates tested. Similardcmsequences were also detected in severalcpe-negativeC. perfringensisolates carrying plasmids but not in type A isolates carrying a chromosomalcpegene. Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmidcpegene in most type A isolates tested. Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470sequences found downstream of the chromosomalcpegene. Collectively, these and previous results suggest that thecpeplasmid of many type A isolates originated from integration of acpe-containing genetic element near thedcmsequences of aC. perfringensplasmid. The similarity of the plasmidcpelocus in many type A isolates is consistent with horizontal transfer of a commoncpeplasmid amongC. perfringenstype A strains.
Identification of an Aggregative Adhesion Fimbria (AAF) Type III-Encoding Operon in Enteroaggregative
Escherichia coli
as a Sensitive Probe for Detecting the AAF-Encoding Operon Family
Authors: Bernier Christine , Gounon Pierre , Le Bouguénec Chantal
Published: July 2002

Enteroaggregative
Escherichia coli
(EAEC) is recognized as an emerging cause of diarrhea in children and adults worldwide, and recent studies have implicated EAEC in persistent diarrhea in patients infected with human immunodeficiency virus (HIV). In this study, we identified aggregative adhesion fimbria type III (AAF-III) in isolate 55989, a typical EAEC strain. Analysis of the sequence of the plasmid-borne
agg-3
gene cluster encoding AAF-III showed this cluster to be closely related to the
agg
and
aaf
operons and to the
afa
operons carried by diffusely adherent pathogenic
E. coli
. We investigated the adhesion properties of a collection of 25 EAEC strains isolated from HIV-infected patients presenting with persistent diarrhea. We found that a minority of strains (36%) carried sequences similar to those of the
agg
and
aaf
operons, which encode AAF-I and AAF-II, respectively. We developed PCR assays specific for the
agg-3
operon. In our collection, the frequency of AAF-III strains was similar (12%) to that of AAF-I strains (16%) but higher than that of AAF-II isolates (0%). Differences between EAEC strains in terms of the virulence factors present render detection of these strains difficult with the available DNA probes. Based on comparison of the
agg
,
aaf
, and
agg-3
operons, we defined an AAF probe internal to the adhesion gene clusters and demonstrated that it was efficient for the identification of EAEC strains. We investigated 32 EAEC isolates, of which only 34.4% were detected with the classical CVD432 probe (detecting pAA virulence plasmids) whereas 65.6% were detected with the AAF probe.
Clostridium septicumAlpha-Toxin Is Active against the Parasitic ProtozoanToxoplasma gondiiand Targets Members of the SAG Family of Glycosylphosphatidylinositol-Anchored Surface Proteins
Authors: Wichroski Michael J. , Melton Jody A. , Donahue Carolyn G. , Tweten Rodney K. , Ward Gary E.
Published: July 2002

As is the case with many other protozoan parasites, glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface ofToxoplasma gondiitachyzoites. The mechanisms by whichT. gondiiGPI-anchored proteins are synthesized and transported through the unusual triple-membrane structure of the parasite pellicle to the plasma membrane remain largely unknown. As a first step in developing tools to study these processes, we show here thatClostridium septicumalpha-toxin, a pore-forming toxin that targets GPI-anchored protein receptors on the surface of mammalian cells, is active againstT. gondiitachyzoites (50% effective concentration, 0.2 nM). Ultrastructural studies reveal that a tight physical connection between the plasma membrane and the underlying membranes of the inner membrane complex is locally disrupted by toxin treatment, resulting in a massive outward extension of the plasma membrane and ultimately lysis of the parasite. Toxin treatment also causes swelling of the parasite endoplasmic reticulum, providing the first direct evidence that alpha-toxin is a vacuolating toxin. Alpha-toxin binds to several parasite GPI-anchored proteins, including surface antigen 3 (SAG3) and SAG1. Interestingly, differences in the toxin-binding profiles between the virulent RH and avirulent P strain were observed. Alpha-toxin may prove to be a powerful experimental tool for molecular genetic analysis of GPI anchor biosynthesis and GPI-anchored protein trafficking inT. gondiiand other susceptible protozoa.
Tissue Tropism of Enteropathogenic
Escherichia coli
Strains Belonging to the O55 Serogroup
Authors: Fitzhenry R. J. , Reece S. , Trabulsi L. R. , Heuschkel R. , Murch S. , Thomson M. , Frankel G. , Phillips A. D.
Published: July 2002

Four enteropathogenic
Escherichia coli
(EPEC) strains belonging to the O55 serogroup (G21 and G30 [both O55:H6], G35 [O55:H−], and G58 [O55:H7]) were tested for their tissue tropism by using human intestinal in vitro organ culture. Strains showed restricted adhesion with attaching-and-effacing activity to follicle-associated epithelium of Peyer's patches, with no apparent adhesion to duodenum or colon. G35 and G58 express intimin γ and show a similar tropism to intimin γ-expressing enterohemorrhagic
E. coli
(EHEC) O157:H7. However, strains G21 and G30 were unusual because they expressed intimin α and had a restricted tissue tropism of intimin γ phenotype. The amino acid sequence of the carboxy-terminal 280 amino acids of intimin from G21 was determined. Comparison with the prototype intimin α from strain E2348/69 (O127:H6) showed a single amino acid difference (corresponding to Val907 and Ala907 in the whole intimins). This mutation was reproduced by site-directed mutagenesis in an intimin α plasmid template, pCVD438, with the hypothesis that it may induce a change in tropism. However, when the mutated plasmid was placed in both EPEC and EHEC backgrounds, duodenal adhesion in a manner similar to strain E2348/69 was evident upon in vitro organ culture. Thus, additional factor(s) unrelated to intimin exist in the O55:H6 genome that influence human intestinal tissue tropism.
Characterization of Pit, a
Streptococcus pneumoniae
Iron Uptake ABC Transporter
Authors: Brown Jeremy S. , Gilliland Sarah M. , Ruiz-Albert Javier , Holden David W.
Published: July 2002

Bacteria frequently have multiple mechanisms for acquiring iron, an essential micronutrient, from the environment. We have identified a four-gene
Streptococcus pneumoniae
operon, named
pit
, encoding proteins with similarity to components of a putative
Brachyspira hyodysenteriae
iron uptake ABC transporter, Bit. An
S. pneumoniae
strain containing a defined mutation in
pit
has impaired growth in medium containing the iron chelator ethylenediamine di-
o
-hydroxyphenylacetic acid, reduced sensitivity to the iron-dependent antibiotic streptonigrin, and impaired virulence in a mouse model of
S. pneumoniae
systemic infection. Furthermore, addition of a mutation in
pit
to a strain containing mutations in the two previously described
S. pneumoniae
iron uptake ABC transporters,
piu
and
pia
, resulted in a strain with impaired growth in two types of iron-deficient medium, a high degree of resistance to streptonigrin, and a reduced rate of iron uptake. Comparison of the susceptibilities to streptonigrin of the individual
pit
,
piu
, and
pia
mutant strains and comparison of the growth in iron-deficient medium and virulence of single and double mutant strains suggest that
pia
is the dominant iron transporter during in vitro and in vivo growth.
Defective Hyphal Induction of a
Candida albicans
Phosphatidylinositol 3-Phosphate 5-Kinase Null Mutant on Solid Media Does Not Lead to Decreased Virulence
Authors: Augsten Martin , Hübner Claudia , Nguyen Monika , Künkel Waldemar , Härtl Albert , Eck Raimund
Published: July 2002

A phosphatidylinositol 3-phosphate [PI(3)P] 5-kinase gene (
CaFAB1
) of the most important human pathogenic yeast,
Candida albicans
, was cloned and sequenced. An open reading frame was detected which encodes a 2,369-amino-acid protein with a calculated molecular mass of 268 kDa and a relative isoelectric point of 6.76. This protein exhibits 38% overall amino acid sequence identity with
Saccharomyces cerevisiae
Fab1p. We localized the
CaFAB1
gene on chromosome R. To determine the influence of the PI(3)P 5-kinase CaFab1p on processes involved in
C. albicans
morphogenesis and pathogenicity, we sequentially disrupted both copies of the gene. Homozygous deletion of
C. albicans CaFAB1
resulted in a mutant strain which exhibited defects in morphogenesis. A
Cafab1
null mutant had enlarged vacuoles, an acidification defect, and increased generation times and was unable to form hyphae on different solid media. The sensitivities to hyperosmotic and high-temperature stresses, adherence, and virulence compared to those of wild-type strain SC5314 were not affected.
Structure-Function Analysis of Decay-Accelerating Factor: Identification of Residues Important for Binding of theEscherichia coliDr Adhesin and Complement Regulation
Authors: Hasan Rafia J. , Pawelczyk Edyta , Urvil Petri T. , Venkatarajan Mathura S. , Goluszko Pawel , Kur Jozef , Selvarangan Rangaraj , Nowicki Stella , Braun Werner A. , Nowicki Bogdan J.
Published: July 2002

Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearingEscherichia coli.The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a−); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids—predominantly those in close proximity to Ser165 to alanine—and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are ≈20 Å apart.
Identification and Characterization of a Novel Heme-Associated Cell Surface Protein Made by
Streptococcus pyogenes
Authors: Lei Benfang , Smoot Laura M. , Menning Heather M. , Voyich Jovanka M. , Kala Subbarao V. , Deleo Frank R. , Reid Sean D. , Musser James M.
Published: July 2002

Analysis of the genome sequence of a serotype M1 group A
Streptococcus
(GAS) strain identified a gene encoding a previously undescribed putative cell surface protein. The gene was cloned from a serotype M1 strain, and the recombinant protein was overexpressed in
Escherichia coli
and purified to homogeneity. The purified protein was associated with heme in a 1:1 stoichiometry. This streptococcal heme-associated protein, designated Shp, was produced in vitro by GAS, located on the bacterial cell surface, and accessible to specific antibody raised against the purified recombinant protein. Mice inoculated subcutaneously with GAS and humans with invasive infections and pharyngitis caused by GAS seroconverted to Shp, indicating that Shp was produced in vivo. The blood of mice actively immunized with Shp had significantly higher bactericidal activity than the blood of unimmunized mice. The
shp
gene was cotranscribed with eight contiguous genes, including homologues of an ABC transporter involved in iron uptake in gram-negative bacteria. Our results indicate that Shp is a novel cell surface heme-associated protein.
Sat, the Secreted Autotransporter Toxin of Uropathogenic
Escherichia coli
, Is a Vacuolating Cytotoxin for Bladder and Kidney Epithelial Cells
Authors: Guyer Debra M. , Radulovic Suzana , Jones Faye-Ellen , Mobley Harry L. T.
Published: July 2002

The secreted autotransporter toxin (Sat) of uropathogenic
Escherichia coli
exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a
sat
mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type
E. coli
CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type
E. coli
CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic
E. coli
CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection.
Helicobacter pylori-Induced Activation of Human Endothelial Cells
Authors: Innocenti M. , Thoreson A.-C. , Ferrero R. L. , Strömberg E. , Bölin I. , Eriksson L. , Svennerholm A.-M. , Quiding-Järbrink M.
Published: July 2002

Helicobacter pyloriinfection causes active chronic inflammation with a continuous recruitment of neutrophils to the inflamed gastric mucosa. To evaluate the role of endothelial cells in this process, we have examined adhesion molecule expression and chemokine and cytokine production from human umbilical vein endothelial cells stimulated with well-characterizedH. pyloristrains as well as purified proteins. Our results indicate that endothelial cells actively contribute to neutrophil recruitment, since stimulation withH. pyloribacteria induced upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin as well as the chemokines interleukin 8 (IL-8) and growth-related oncogene alpha (GRO-α) and the cytokine IL-6. However, there were large variations in the ability of the differentH. pyloristrains to stimulate endothelial cells. These interstrain variations were seen irrespective of whether the strains had been isolated from patients with duodenal ulcer disease or asymptomatic carriers and were not solely related to the expression of known virulence factors, such as the cytotoxin-associated gene pathogenicity island, vacuolating toxin A, and Lewis blood group antigens. In addition, one or several unidentified proteins which act via NF-κB activation seem to induce endothelial cell activation. In conclusion, human endothelial cells produce neutrophil-recruiting factors and show increased adhesion molecule expression after stimulation with certainH. pyloristrains. These effects probably contribute to the continuous recruitment of neutrophils toH. pylori-infected gastric mucosa and may also contribute to tissue damage and ulcer formation.
Fusobacterium necrophorumLeukotoxin Induces Activation and Apoptosis of Bovine Leukocytes
Authors: Narayanan Sanjeevkumar , Stewart George C. , Chengappa M. M. , Willard Lloyd , Shuman Wilma , Wilkerson Melinda , Nagaraja T. G.
Published: July 2002

Fusobacterium necrophorum, a gram-negative, rod-shaped, anaerobic bacterium, is a primary or secondary etiological agent in a variety of necrotic, purulent infections in humans and animals. Its major virulence factor is leukotoxin, a high-molecular-weight secreted protein, primarily toxic to ruminant leukocytes. In this study, bovine peripheral blood leukocytes were exposed to various concentrations of immunoaffinity-purified leukotoxin and the cytotoxicity was analyzed by flow cytometry and scanning and transmission electron microscopy. At very low toxin concentrations, polymorphonuclear leukocytes (PMNs) showed activation, as indicated by translocation of primary and secondary granules to the periphery of the cytoplasm. Furthermore, these cells showed changes characteristic of apoptosis, including decreased cell size, organelle condensation, cytoplasmic membrane blebbing (zeiosis), and chromatin condensation and margination, and decrease in cellular DNA content. At moderately high concentrations of leukotoxin, bovine mononuclear cells were also induced to undergo programmed cell death. At very high concentrations, leukotoxin caused necrotic cell death of bovine peripheral leukocytes. The ability ofF. necrophorumleukotoxin to modulate the host immune system by its toxicity, including cellular activation of PMNs and apoptosis-mediated killing of phagocytes and immune effector cells, represents a potentially important mechanism of its pathogenesis.
Expression of Inducible Nitric Oxide Synthase in Skin Lesions of Patients with American Cutaneous Leishmaniasis
Authors: Qadoumi Muna , Becker Inge , Donhauser Norbert , Röllinghoff Martin , Bogdan Christian
Published: July 2002

Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is indispensable for the resolution of
Leishmania major
or
Leishmania donovani
infections in mice. In contrast, little is known about the expression and function of iNOS in human leishmaniasis. Here, we show by immunohistological analysis of skin biopsies from Mexican patients with local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the expression of iNOS was most prominent in LCL lesions with small numbers of parasites whereas lesions with a high parasite burden (LCL or DCL) contained considerably fewer iNOS-positive cells. This is the first study to suggest an antileishmanial function of iNOS in human
Leishmania
infections in vivo.
Role of the High-Affinity Zinc Uptake
znuABC
System in
Salmonella enterica
Serovar Typhimurium Virulence
Authors: Campoy Susana , Jara Mónica , Busquets Núria , Pérez de Rozas Ana M. , Badiola Ignacio , Barbé Jordi
Published: July 2002

The
Salmonella enterica
serovar Typhimurium
znuABC
genes encoding a high-affinity zinc uptake system and its regulatory
zur
gene have been cloned.
Salmonella
serovar Typhimurium
zur
and
znuC
knockout mutants have been constructed by marker exchange. The 50% lethal dose of the
znuC
mutant increased when either orally or intraperitoneally inoculated in BALB/c mice, while virulence of the
zur
mutant decreased only when mice were intraperitoneally challenged.
Inactivation of Cytochrome
o
Ubiquinol Oxidase Relieves Catabolic Repression of the
Pseudomonas putida
GPo1 Alkane Degradation Pathway
Authors: Dinamarca M. Alejandro , Ruiz-Manzano Ana , Rojo Fernando
Published: July 2002

Expression of the alkane degradation pathway encoded by the OCT plasmid of
Pseudomonas putida
GPo1 is regulated by two control systems. One relies on the transcriptional regulator AlkS, which activates expression of the pathway in the presence of alkanes. The other, which is a dominant global regulation control, represses the expression of the pathway genes when a preferred carbon source is present in the growth medium in addition to alkanes. This catabolite repression control occurs through a poorly characterized mechanism that ultimately regulates transcription from the two AlkS-activated promoters of the pathway. To identify the factors involved, a screening method was developed to isolate mutants without this control. Several isolates were obtained, all of which contained mutations that mapped to genes encoding cytochrome
o
ubiquinol oxidase, the main terminal oxidase of the electron transport chain under highly aerobic conditions. Elimination of this terminal oxidase led to a decrease in the catabolic repression observed both in rich Luria-Bertani medium and in a defined medium containing lactate or succinate as the carbon source. This suggests that catabolic repression could monitor the physiological or metabolic status by using information from the electron transport chain or from the redox state of the cell. Since inactivation of the
crc
gene also reduces catabolic repression in rich medium (although not that observed in a defined medium), a strain was generated lacking both the Crc function and the cytochrome
o
terminal oxidase. The two mutations had an additive effect in relieving catabolic repression in rich medium. This suggests that
crc
and
cyo
belong to different regulation pathways, both contributing to catabolic repression.
Family Shuffling of a Targeted
bphA
Region To Engineer Biphenyl Dioxygenase
Authors: Barriault Diane , Plante Marie-Michèle , Sylvestre Michel
Published: July 2002

In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs). Instead of shuffling the whole gene, we have targeted a fragment of
bphA
that is critical for enzyme specificity. We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls. Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of
bphA
genes from
Burkholderia
sp. strain LB400,
Comamonas testosteroni
B-356, and
Rhodococcus globerulus
P6. Unlike all parents, these variants exhibited high activity toward 2,2′-, 3,3′-, and 4,4′-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl. The data showed that the replacement of a short segment (
335
TFNNIRI
341
) of LB400 BphA by the corresponding segment (
333
GINTIRT
339
) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates.
The RNA Polymerase α Subunit from
Sinorhizobium meliloti
Can Assemble with RNA Polymerase Subunits from
Escherichia coli
and Function in Basal and Activated Transcription both In Vivo and In Vitro
Authors: Peck Melicent C. , Gaal Tamas , Fisher Robert F. , Gourse Richard L. , Long Sharon R.
Published: July 2002

Sinorhizobium meliloti
, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in
S. meliloti
, we cloned and characterized the gene for the α subunit of RNA polymerase (RNAP).
S. meliloti rpoA
encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding
rpoA
identified six open reading frames that are found in the conserved gene order
secY
(SecY)-
adk
(Adk)-
rpsM
(S13)-
rpsK
(S11)-
rpoA
(α)-
rplQ
(L17) found in the α-proteobacteria. In vivo, S
. meliloti rpoA
expressed in
Escherichia coli
complemented a temperature sensitive mutation in
E. coli rpoA
, demonstrating that
S. meliloti
α supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of
E. coli.
In vitro, we reconstituted RNAP holoenzyme from
S. meliloti
α and
E. coli
β, β′, and σ subunits. Similar to
E. coli
RNAP, the hybrid RNAP supported transcription from an
E. coli
core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from
S. meliloti
. Our results demonstrate that
S. meliloti
α functions are conserved in heterologous host
E. coli
even though the two α subunits are only 51% identical. The ability to utilize
E. coli
as a heterologous system in which to study the regulation of
S. meliloti
genes could provide an important tool for our understanding and manipulation of these processes.
Activity of
Sinorhizobium meliloti
NodAB and NodH Enzymes on Thiochitooligosaccharides
Authors: Southwick Audrey M. , Wang Lai-Xi , Long Sharon R. , Lee Yuan C.
Published: July 2002

Rhizobium
bacteria synthesize signal molecules called Nod factors that elicit responses in the legume root during nodulation. Nod factors, modified N-acylated β-(1,4)-
N
-acetylglucosamine, are synthesized by the nodulation (
nod
) gene products. We tested the ability of three
Sinorhizobium meliloti nod
gene products to modify Nod factor analogs with thio linkages instead of O-glycosidic bonds in the oligosaccharide backbone.
RsmA and the Quorum-Sensing Signal,N-[3-Oxohexanoyl]-l-Homoserine Lactone, Control the Levels ofrsmBRNA inErwinia carotovorasubsp.carotovoraby Affecting Its Stability
Authors: Chatterjee Asita , Cui Yaya , Chatterjee Arun K.
Published: July 2002

RsmA (for regulator of secondary metabolism), RsmC, andrsmBRNA, the components of a posttranscriptional regulatory system, control extracellular protein production and pathogenicity inErwinia carotovorasubsp. carotovora.RsmA, an RNA binding protein, acts as a negative regulator by promoting message decay.rsmBRNA, on the other hand, acts as a positive regulator by neutralizing the effect of RsmA. RsmC modulates the levels of RsmA andrsmBRNA by positively regulatingrsmAand negatively controllingrsmB.The level ofrsmBRNA is substantially higher in RsmA+bacteria than in RsmA−mutants. We show thatrsmBRNA is more stable in the presence of RsmA than in its absence. RsmA does not stimulate the expression of anrsmB-lacZtranscriptional fusion; in fact, the β-galactosidase level is somewhat higher in RsmA−bacteria than in RsmA+bacteria. We also investigated the basis for increased levels ofrsmAandrsmBRNAs in the absence of the quorum-sensing signal,N-[3-oxohexanoyl]-l-homoserine lactone (OHL). The absence of OHL activates transcription ofrsmAbut not ofrsmB.Instead, increased stability ofrsmBRNA in the presence of RsmA accounts for the elevated levels of thersmBRNA in OHL−bacteria. Mutant studies disclosed that while RsmA, OHL, and RsmC control the levels ofrsmBRNA, high levels ofrsmBRNA occur in the absence of RsmC or OHL only in RsmA+bacteria, indicating a critical role for RsmA in modulating the levels ofrsmBRNA. The findings reported here firmly establish that the quorum-sensing signal is channeled inE. carotovorasubsp.carotovoravia thersmA-rsmBposttranscriptional regulatory system.
Evidence for Temporal Regulation of the Two
Pseudomonas cellulosa
Xylanases Belonging to Glycoside Hydrolase Family 11
Authors: Emami Kaveh , Nagy Tibor , Fontes Carlos M. G. A. , Ferreira Luis M. A. , Gilbert Harry J.
Published: July 2002

Pseudomonas cellulosa
is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of
xyn10A
,
xyn10B
,
xyn10C
,
xyn10D
, and
xyn11A
, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene,
xyn11B
, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound.
xyn10C
,
xyn10D
,
xyn11A
, and
xyn11B
were all abundantly expressed when the bacterium was cultured on xylan or β-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of
xyn11A
and
xyn11B
was apparent on xylan-containing media. Transcription of
xyn11A
occurred earlier than transcription of
xyn11B
, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of
P. cellulosa
in which
xyn11A
and
xyn11B
were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type
P. cellulosa
. Based on the data presented here, we propose a model for xylan degradation by
P. cellulosa
in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.
The 2-Aminoethylphosphonate-Specific Transaminase of the 2-Aminoethylphosphonate Degradation Pathway
Authors: Kim Alexander D. , Baker Angela S. , Dunaway-Mariano Debra , Metcalf W. W. , Wanner B. L. , Martin Brian M.
Published: July 2002

The 2-aminoethylphosphonate transaminase (AEPT; the
phnW
gene product) of the
Salmonella enterica
serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and
l
-alanine (
l
-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log

V
m

and log

V
m

/

K
m

versus pH) revealed a pH optimum of 8.5. At pH 8.5,
K
eq
is equal to 0.5 and the
k
cat
values of the forward and reverse reactions are 7 and 9 s
−1
, respectively. The

K
m

for AEP is 1.11 ± 0.03 mM; for pyruvate it is 0.15 ± 0.02 mM, for P-Ald it is 0.09 ± 0.01 mM, and for
l
-Ala it is 1.4 ± 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.
Characterization of
Salmonella
Serovars by PCR-Single-Strand Conformation Polymorphism Analysis
Authors: Nair Satheesh , Lin Thong Kwai , Pang Tikki , Altwegg Martin
Published: July 2002

PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb
groEL
gene from 41 strains of 10 different
Salmonella
serovars. Three
Hae
III RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the
groEL
genes of various
Salmonella
serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different
Salmonella
serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11
Salmonella enterica
serovar Enteritidis strains, and two SSCP profiles were generated for the 7
S. enterica
serovar Infantis and five
S. enterica
serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of
Salmonella
serovars due to its rapidity, simplicity, and typeability.
Cross-Reactivity among Several Recombinant Calicivirus Virus-Like Particles (VLPs) with Monoclonal Antibodies Obtained from Mice Immunized Orally with One Type of VLP
Authors: Kitamoto Noritoshi , Tanaka Tomoyuki , Natori Katsurou , Takeda Naokazu , Nakata Shuji , Jiang Xi , Estes Mary K.
Published: July 2002

Human caliciviruses (HuCVs) are classified into the Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV) as genera within the family
Caliciviridae.
The NLV genus is further classified into genogroups I and II, based on sequence similarities. To study the antigenic determinants on the HuCV capsid protein and develop new diagnostic tools for field samples, we established and characterized monoclonal antibodies (MAbs) against baculovirus-expressed recombinant HuCV virus-like particles (VLPs). Hybrid clones producing MAbs were obtained from cultures of PAI myeloma cells fused with spleen or mesenteric lymph node cells from mice immunized orally with either a single type of recombinant Norwalk virus (rNV), Kashiwa 47 virus (rKAV), Snow Mountain agent (rSMA), or Sapporo virus (rSV) VLP or with mixtures of two types of VLPs from different genogroups. Twenty MAbs, obtained as mouse ascites, were characterized and classified into six groups according to their enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) cross-reactivity patterns to VLPs. Five groups of MAbs reacted by both WB and ELISA and were classified as follows: common cross-reactive MAbs for four genogroup I and six genogroup II VLPs (group A), genogroup I-specific MAbs (group B), genogroup II-specific MAbs (group C), and strain-specific MAbs (groups D and E). One MAb group (group F) reacted only by ELISA. The group A MAbs, which showed broad cross-reactivity with VLPs of both NLV genogroups, were obtained from mice immunized orally with a single type of VLP (either rNV or rKAV). Two MAbs, which were obtained from mice immunized with rSV, reacted with rSV but not with any NLV VLP. These are the first MAbs to be reported for any SLV. These strain-, genogroup-, and genus-reactive MAbs will be useful tools for further study of the antigenic and structural topography of the HuCV virion and for diagnostic assays for HuCVs.
Nosocomial Spread of a
Staphylococcus capitis
Strain with Heteroresistance to Vancomycin in a Neonatal Intensive Care Unit
Authors: Van Der Zwet Wil C. , Debets-Ossenkopp Yvette J. , Reinders Erik , Kapi Maria , Savelkoul Paul H. M. , Van Elburg Ruurd M. , Hiramatsu Keiichi , Vandenbroucke-Grauls Christina M. J. E.
Published: July 2002

A premature infant in a neonatal intensive care unit (NICU) developed a bloodstream infection caused by coagulase-negative staphylococci (CoNS) sensitive to vancomycin. The infection persisted for 3 weeks, despite therapy with vancomycin and replacement of all intravenous catheters. The neonate died due to necrotizing enterocolitis which developed during the ongoing sepsis. We screened this strain and 216 other strains of CoNS from cultures of blood obtained from neonates between 1997 and 2000 for heteroresistance to vancomycin. Forty-eight isolates, including the strain that caused ongoing sepsis, proved heteroresistant. All isolates were identified as
Staphylococcus capitis
and were identical, just as their resistant stable subcolonies were, when they were genetically fingerprinted by amplified-fragment length polymorphism analysis. The heteroresistant phenotype of this endemic strain was confirmed by population analysis. We conclude that heteroresistance to vancomycin occurs in
S. capitis
and might be the cause of therapeutic failures in NICUs. Moreover, heteroresistant strains can become endemic in such units.
Use of a Fragment of Glycoprotein G-2 Produced in the Baculovirus Expression System for Detecting Herpes Simplex Virus Type 2-Specific Antibodies
Authors: Ikoma Minako , Liljeqvist Jan-Åke , Groen Jan , Glazenburg Koen L. , The T. Hauw , Welling-Wester Sytske
Published: July 2002

Fragments of glycoprotein G (gG-2
281-594His
), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1
t26-189His
), and glycoprotein D of HSV-1 (gD-1
1-313
), were expressed in the baculovirus expression system to develop an assay for the detection of HSV-1 and HSV-2 type-specific antibodies. The expression of the gG-1
t26-189His
and gG-2
281-594His
fragments was analyzed by Western blotting using monoclonal antibodies LP10 and AP1, respectively. The molecular masses of the major products of gG-1
t26-189His
and the fragment of gG-2
281-594His
were 36 to 39 kDa and 64 to 72 kDa, respectively. Human sera positive for HSV-1 reacted with gG-1
t26-189His
, sera positive for HSV-2 reacted with the gG-2
281-594His
fragment, and sera positive for both types reacted with gG-1
t26-189His
and gG-2
281-594His
in Western blotting. The human sera recognized polypeptides of gG-2
281-594His
with molecular masses of 57 to 67 and 120 to 150 kDa and additional faint bands of 21, 29, and 45 kDa. The recombinant gG-1
t26-189His
and the recombinant gG-2
281-594His
fragment were used as type-specific antigens for the detection of HSV-1- and HSV-2-specific antibody responses in human sera, respectively. As type-common antigens, an extract of HSV-1-infected Vero cells and recombinant gD-1
1-313
were used. An enzyme-linked immunosorbent assay to detect type-specific antibodies was developed, and the sensitivity and specificity were evaluated by comparison with commercial tests by using sera obtained from different sources. The sensitivity and specificity were 91.5 and 95.5%, respectively, compared to the Gull assay. The gG-2
281-594His
fragment can be obtained in relatively large quantities at low cost.
Improvement of AMPLICOR Human Immunodeficiency Virus Type 1 Viral Load Test (Version 1.5) by Addition of a Coprecipitant during the RNA Isolation Step
Authors: Xu Min , Chan Yung , Fischer Steven H. , Remaley Alan T.
Published: July 2002

The effect of the addition of a coprecipitant during the RNA isolation step on the analytical performance of the COBAS AMPLICOR human immunodeficiency virus type 1 (HIV-1) Monitor (version 1.5; Roche) viral load test was tested. Thirty-six specimens including patient samples, positive control samples, and negative control samples were processed in the presence and absence of the Pellet Paint coprecipitant. Specimens processed without the coprecipitant had lower RNA yields, as evidenced by a lower signal for the quantitation standard (QS). In addition, the results for all samples processed with the coprecipitant were acceptable on the basis of the optical density (OD) reading for the QS, whereas the result for one specimen processed without the coprecipitant was unacceptable on the basis of the OD reading for the QS, which required the assay to be repeated. Furthermore, the use of the coprecipitant improved the overall precision of the assay.
Antibiotic Resistance in
Staphylococcus
Isolates Obtained from Fecal Samples of Healthy Children
Authors: Domínguez Elena , Zarazaga Myriam , Torres Carmen
Published: July 2002

Thirty-nine
Staphylococcus
isolates with different mechanisms of resistance were recovered from the feces of 50 healthy children and tested for their susceptibilities to 17 antibiotics. The percentages of resistance of the staphylococci to some antibiotics were as follows: penicillin, 87%; erythromycin, 64%; tobramycin, 36%; tetracycline, 20.5%; kanamycin, 15%; and gentamicin, 13%. The
mecA
gene was detected in nine coagulase-negative staphylococci.
Multidrug and Broad-Spectrum Cephalosporin Resistance among
Salmonella enterica
Serotype Enteritidis Clinical Isolates in Southern Italy
Authors: Villa Laura , Mammina Caterina , Miriagou Vivi , Tzouvelekis Leonidas S. , Tassios Panayotis T. , Nastasi Antonino , Carattoli Alessandra
Published: July 2002

From 1992 to 1997, only six sporadic isolates of
Salmonella enterica
serotype Enteritidis from patients with cases of gastroenteritis in southern Italy exhibited resistance to broad-spectrum cephalosporins. Five isolates produced SHV-12, and one isolate encoded a class C β-lactamase. The
bla
SHV-12
gene was located in at least two different self-transferable plasmids, one of which also carried a novel class 1 integron.
Characterization of Human Immunodeficiency Virus Type 1 (HIV-1) Gag- and Gag Peptide-Specific CD4+T-Cell Clones from an HIV-1-Seronegative Donor following In Vitro Immunization
Authors: Venturini Sara , Mosier Donald E. , Burton Dennis R. , Poignard Pascal
Published: July 2002

Substantial evidence argues that human immunodeficiency virus type 1 (HIV-1)-specific CD4+T cells play an important role in the control of HIV-1 replication in infected individuals. Moreover, it is increasingly clear that an HIV vaccine should elicit potent cytotoxic lymphocyte and antibody responses that will likely require an efficient CD4+T-cell response. Therefore, understanding and characterizing HIV-specific CD4+T-cell responses is an important aim. Here we describe the generation of HIV-1 Gag- and Gag peptide-specific CD4+T-cell clones from an HIV-1-seronegative donor by in vitro immunization with HIV-1 Gag peptides. The Gag peptides were able to induce a strong CD4+T-cell immune response in peripheral blood mononuclear cells from the HIV-1-seronegative donor. Six Gag peptide-specific CD4+T-cell clones were isolated and their epitopes were mapped. The region of p24 between amino acids 201 and 300 of Gag was defined as the immunodominant region of Gag. A new T helper epitope in the p6 protein of Gag was identified. Two clones were shown to recognize Gag peptides and processed Gag protein, while the other four clones reacted only to Gag peptides under the experimental conditions used. Functional analysis of the clones indicated that both Th1 and Th2 types of CD4+T cells were obtained. One clone showed direct antigen-specific cytotoxic activity. These clones represent a valuable tool for understanding the cellular immune response to HIV-1, and the study provides new insights into the HIV-1-specific CD4+T-cell response and the induction of an anti-Gag and -Gag peptide cellular primary immune response in vitro.
Herpes Simplex Virus Type 1 Origins of DNA Replication Play No Role in the Regulation of Flanking Promoters
Authors: Summers Bretton C. , Leib David A.
Published: July 2002

Herpes simplex virus (HSV) exhibits altered gene regulation in neuronal compared to nonneuronal tissues. It has been hypothesized that initiation of DNA synthesis at the viral origins of replication (oriS and oriL) is a critical step in the upregulation of transcriptional activity of flanking divergent promoters, thereby increasing productive gene expression in neurons. Notably, oriS is flanked by the immediate-early (IE) ICP4 and ICP22/47 promoters, and oriL is flanked by the early (E) UL29 and UL30 promoters. To test this hypothesis further, a series of constructs were generated in which these promoters were placed upstream of luciferase genes. In addition, DNA replication origins were deleted in the context of these promoter constructs. All cassettes were recombined into the viral genome of HSV type 1 strain KOS at a site distal to its native origins. Recombinant reporter expression was monitored in vitro and in vivo to determine the role of viral origins of DNA replication in the regulation of their flanking promoters. Reporter gene expression was unaffected by the presence or absence of oriS or oriL, with the exception of a twofold increase in ICP22/47 promoter activity in the absence of oriS. DNA synthesis inhibitors resulted in a decrease of both IE- and E-promoter activity in primary cells but not continuous cell cultures. Reporter activity was readily assayed in vivo during acute infection and reactivation from latency and was also sensitive to DNA synthesis inhibitors. In all assays, reporter gene expression was unaffected by the presence or absence of either oriS or oriL. These data support the requirement of DNA synthesis for full viral gene expression in vivo but suggest that the origin elements play no role in the regulation of their flanking promoters.
Processing Map and Essential Cleavage Sites of the Nonstructural Polyprotein Encoded by ORF1 of the Feline Calicivirus Genome
Authors: Sosnovtsev Stanislav V. , Garfield Mark , Green Kim Y.
Published: July 2002

Feline calicivirus (FCV) nonstructural proteins are translated as part of a large polyprotein that undergoes autocatalytic processing by the virus-encoded 3C-like proteinase. In this study, we mapped three new cleavage sites (E
46
/A
47
, E
331
/D
332
, and E
685
/N
686
) recognized by the virus proteinase in the N-terminal part of the open reading frame 1 (ORF1) polyprotein to complete the processing map. Taken together with two sites we identified previously (E
960
/A
961
and E
1071
/S
1072
), the FCV ORF1 polyprotein contains five cleavage sites that define the borders of six proteins with calculated molecular masses of 5.6, 32, 38.9, 30.1, 12.7, and 75.7 kDa, which we designated p5.6, p32, p39 (NTPase), p30, p13 (VPg), and p76 (Pro-Pol), respectively. Mutagenesis of the E to A in each of these cleavage sites in an infectious FCV cDNA clone was lethal for the virus, indicating that these cleavages are essential in a productive virus infection. Mutagenesis of two cleavage sites (E
1345
/T
1346
and E
1419
/G
1420
) within the 75.7-kDa Pro-Pol protein previously mapped in bacterial expression studies was not lethal.
Reactivation of Latent Cytomegalovirus Infection in Mouse Brain Cells Detected after Transfer to Brain Slice Cultures
Authors: Tsutsui Yoshihiro , Kawasaki Hideya , Kosugi Isao
Published: July 2002

Cytomegalovirus (CMV) is the most significant infectious cause of brain disorders in humans involving the developing brain. It is hypothesized that the brain disorders occur after recurrent reactivation of the latent infection in some kinds of cells in the brains. In order to test this hypothesis, we examined the reactivation of latent murine CMV (MCMV) infection in the mouse brain by transfer to brain slice culture. We infected neonatal and young adult mice intracerebrally with recombinant MCMV in which the
lacZ
gene was inserted into a late gene. The brains were removed 6 months after infection and used to prepare brain slices that were then cultured for up to 4 weeks. Reactivation of latent infection in the brains was detected by β-galactosidase (β-Gal) staining to assess β-galactosidase expression. Viral replication was also confirmed by the plaque assay. Reactivation was observed in about 75% of the mice infected during the neonatal period 6 months after infection. Unexpectedly, reactivation was also observed in 75% of mice infected as young adults, although the infection ratio in the brain slices was significantly lower than that in neonatally infected mice. β-Gal-positive cells were observed in marginal regions of the brains or immature neural cells in the ventricular walls. Immunohistochemical staining showed that the β-Gal-positive reactivated cells were neural stem or progenitor cells. These results suggest that brain disorders may occur long after infection by reactivation of latent infection in the immature neural cells in the brain.