bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Culture of
Borrelia garinii
Results in Loss of Flagella and Decreased Invasiveness
Authors: Sellek Ricela E. , Escudero Raquel , Gil Horacio , Rodríguez Isabel , Chaparro Escolástica , Pérez-Pastrana Esperanza , Vivo Amparo , Anda Pedro
Published: October 2002

A virulent, low-passage culture of a tick-derived strain of
Borrelia garinii
was subjected to serial in vitro passages, from which inoculations were made into C3H/HeN mice. A full display of pathogenicity was observed through passage 4, as measured by cultures of ear punch biopsy samples and internal organs and determination of tibiotarsal joint swelling. Decreased dissemination through skin and infection of internal organs were observed beginning at passage 6. These losses correlated with both the selection of clones harboring 21% less flagella than the parent strain, as seen by electron microscopy, and loss of the motility of the higher passages, as demonstrated by a swarm assay. However, during the chronic phase (3 months after infection), spirochetes were cultured from the bladder and kidney of a mouse inoculated with passage 12. The kidney isolate had the same number of flagella and motility as the original low-passage isolate. Although we can't exclude the possibility that other subtle variations may be arising given the uncloned nature of the isolate, we have found a strong association between loss of flagella and decreased invasiveness. Arthritogenicity progressively decreased with passages, so that only 12.5% of chronically infected mice inoculated with passage 29 still presented with joint swelling, concurrent with a decrease in the staining intensity in a Southern blot with a
vlsE
-based probe. These results suggest a multifactorial model in which the number of flagella drives the invasiveness of this agent, while plasmid-associated factors are responsible for triggering arthritogenicity.
The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of
Streptococcus pyogenes
Authors: Ricci Susanna , Janulczyk Robert , Björck Lars
Published: October 2002

Ferric uptake regulator (Fur) and Fur-like proteins form an important family of transcriptional regulators in many bacterial species. In this work we have characterized a Fur-like protein, the peroxide regulator PerR, in an M1 serotype of
Streptococcus pyogenes
. To determine the role of PerR in
S. pyogenes
, we inactivated the gene by allelic replacement. PerR-deficient bacteria showed 48% reduction of
55
Fe incorporation from the culture medium. Transcriptional analysis revealed that
mtsA
, encoding a metal-binding protein of an ABC transporter in
S. pyogenes
, was transcribed at lower levels than were wild-type cells. Although total iron accumulation was reduced, the growth of the mutant strain was not significantly hampered. The mutant showed hyperresistance to hydrogen peroxide, and this response was induced in wild-type cells by growth in aerobiosis, suggesting that PerR acts as an oxidative stress-responsive repressor. PerR may also participate in the response to superoxide stress, as the
perR
mutant was more sensitive to the superoxide anion and had a reduced transcription of
sodA
, which encodes the sole superoxide dismutase of
S. pyogenes
. Complementation of the mutation with a functional
perR
gene restored
55
Fe incorporation, response to peroxide stress, and transcription of both
mtsA
and
sodA
to levels comparable to those of wild-type bacteria. Finally, the
perR
mutant was attenuated in virulence in a murine air sac model of infection (
P
< 0.05). These results demonstrate that PerR is involved in the regulation of iron homeostasis and oxidative stress responses and that it contributes to the virulence of
S. pyogenes
.
Structure-Function Relationships for Human Antibodies to Pneumococcal Capsular Polysaccharide from Transgenic Mice with Human Immunoglobulin Loci
Authors: Chang Q. , Zhong Z. , Lees A. , Pekna M. , Pirofski L.
Published: October 2002

To investigate the influence of antibody structure and specificity on antibody efficacy againstStreptococcus pneumoniae, human monospecific antibodies (MAbs) to serotype 3 pneumococcal capsular polysaccharide (PPS-3) were generated from transgenic mice reconstituted with human immunoglobulin loci (XenoMouse mice) vaccinated with a PPS-3-tetanus toxoid conjugate and their molecular genetic structures, epitope specificities, and protective efficacies in normal and complement-deficient mice were determined. Nucleic acid sequence analysis of three MAbs (A7, 1A2, and 7C5) revealed that they use two different VH3 genes (A7 and 1A2 both use V3-15) and three different Vκgene segments. The MAbs were found to have similar affinities for PPS-3 but different epitope specificities and CDR3 regions. Both A7 and 7C5 had a lysine at the VH-D junction, whereas 1A2 had a threonine. Challenge experiments with serotype 3S. pneumoniaein BALB/c mice revealed that both 10- and 1-μg doses of A7 and 7C5 were protective, while only a 10-μg dose of 1A2 was protective. Both A7 and 7C5 were also protective in mice lacking either an intact alternative (FB−/−) or classical (C4−/−) complement pathway, but 1A2 was not protective in either strain. Our data suggest that PPS-3 consists of epitopes that can elicit both highly protective and less protective antibodies and that the superior efficacies of certain antibodies may be a function of their structures and/or specificities. Further investigation of relationships between structure, specificity, and efficacy for defined MAbs to PPS may identify antibody features that might be useful surrogates for antibody (and vaccine) efficacy.
Virulent Combinations of Adhesin and Toxin Genes in Natural Populations of
Staphylococcus aureus
Authors: Peacock Sharon J. , Moore Catrin E. , Justice Anita , Kantzanou Maria , Story Lisa , Mackie Kathryn , O'Neill Gael , Day Nicholas P. J.
Published: October 2002

Most cases of severe
Staphylococcus aureus
disease cannot be explained by the action of a single virulence determinant, and it is likely that a number of factors act in combination during the infective process. This study examined the relationship between disease in humans and a large number of putative virulence determinants, both individually and in combination.
S. aureus
isolates (
n
= 334) from healthy blood donors and from patients with invasive disease were compared for variation in the presence of 33 putative virulence determinants. After adjusting for the effect of clonality, seven determinants (
fnbA
,
cna
,
sdrE
,
sej
,
eta
,
hlg
, and
ica
) were significantly more common in invasive isolates. All seven factors contributed independently to virulence. No single factor predominated as the major predictor of virulence, their effects appearing to be cumulative. No combinations of the seven genes were either more or less likely to cause disease than others with the same number of virulence-associated genes. There was evidence of considerable horizontal transfer of genes on a background of clonality. Our findings also suggested that allelic variants of a polymorphic locus can make different contributions to the disease process, further study of which is likely to expand our understanding of staphylococcal disease pathogenesis.
Bovine CD18 Is Necessary and Sufficient To Mediate
Mannheimia
(
Pasteurella
)
haemolytica
Leukotoxin-Induced Cytolysis
Authors: Deshpande M. S. , Ambagala T. C. , Ambagala A. P. N. , Kehrli M. E. , Srikumaran S.
Published: October 2002

Leukotoxin (Lkt) secreted by
Mannheimia
(
Pasteurella
)
haemolytica
is an RTX toxin which is specific for ruminant leukocytes. Lkt binds to β
2
integrins on the surface of bovine leukocytes. β
2
integrins have a common β subunit, CD18, that associates with three distinct α chains, CD11a, CD11b, and CD11c, to give rise to three different β
2
integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (CR4), respectively. Our earlier studies revealed that Lkt binds to all three β
2
integrins, suggesting that the common β subunit, CD18, may be the receptor for Lkt. In order to unequivocally elucidate the role of bovine CD18 as a receptor for Lkt, a murine cell line nonsusceptible to Lkt (P815) was transfected with cDNA for bovine CD18. One of the transfectants, 2B2, stably expressed bovine CD18 on the cell surface. The 2B2 transfectant was effectively lysed by Lkt in a concentration-dependent manner, whereas the P815 parent cells were not. Immunoprecipitation of cell surface proteins of 2B2 with monoclonal antibodies specific for bovine CD18 or murine CD11a suggested that bovine CD18 was expressed on the cell surface of 2B2 as a heterodimer with murine CD11a. Expression of bovine CD18 and the Lkt-induced cytotoxicity of 2B2 cells were compared with those of bovine polymorphonuclear neutrophils and lymphocytes. There was a strong correlation between cell surface expression of bovine CD18 and percent cytotoxicity induced by Lkt. These results indicate that bovine CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells.
Cruzipain Induces Both Mucosal and Systemic Protection againstTrypanosoma cruziin Mice
Authors: Schnapp Anita R. , Eickhoff Chris S. , Sizemore Donata , Curtiss Roy , Hoft Daniel F.
Published: October 2002

Cruzipain, the major cysteinyl proteinase ofTrypanosoma cruzi, is expressed by all developmental forms and strains of the parasite and stimulates potent humoral and cellular immune responses during infection in both humans and mice. This information suggested that cruzipain could be used to develop an effectiveT. cruzivaccine. To study whether cruzipain-specific T cells could inhibitT. cruziintracellular replication, we generated cruzipain-reactive CD4+Th1 cell lines. These T cells produced large amounts of gamma interferon when cocultured with infected macrophages, resulting in NO production and decreased intracellular parasite replication. To study the protective effects in vivo of cruzipain-specific Th1 responses against systemicT. cruzichallenges, we immunized mice with recombinant cruzipain plus interleukin 12 (IL-12) and a neutralizing anti-IL-4 MAb. These immunized mice developed potent cruzipain-specific memory Th1 cell responses and were significantly protected against normally lethal systemicT. cruzichallenges. Although cruzipain-specific Th1 responses were associated withT. cruziprotective immunity in vitro and in vivo, adoptive transfer of cruzipain-specific Th1 cells alone did not protect BALB/c histocompatible mice, indicating that additional immune mechanisms are important for cruzipain-specific immunity. To study whether cruzipain could induce mucosal immune responses relevant for vaccine development, we prepared recombinant attenuatedSalmonella entericaserovar Typhimurium vaccines expressing cruzipain. BALB/c mice immunized with salmonella expressing cruzipain were significantly protected againstT. cruzimucosal infection. Overall, these data indicate that cruzipain is an importantT. cruzivaccine candidate and that protectiveT. cruzivaccines will need to induce more than CD4+Th1 cells alone.
AChlamydia trachomatis-Specific Th2 Clone Does Not Provide Protection against a Genital Infection and Displays Reduced Trafficking to the Infected Genital Mucosa
Authors: Hawkins Raymond A. , Rank Roger G. , Kelly Kathleen A.
Published: October 2002

A T helper type 1 (Th1) response is essential for resolving genital infections with the mouse pneumonitis biovar ofChlamydia trachomatis(MoPn). However, T-cell-dependent anti-chlamydial antibody is produced and may also contribute to protective immunity. We produced a MoPn-specific CD4 Th2 clone (Th2-MoPn) to study the role of a Th2 response during infection. We found that Th2-MoPn was unable to eradicate chlamydiae from the genital tract (GT) when it was transferred into MoPn-infected nude mice. Mice that received Th2-MoPn produced greater titers of MoPn-specific serum immunoglobulin G (IgG) antibody than mice that received a MoPn-specific Th1 clone (Th1-MoPn) (log10titers, 1.89 ± 0.84 and 0.58 ± 0.76 [mean ± standard deviation], respectively [P< 0.01]). Also, the IgG isotypes were different for the two groups; whereas IgG1 was associated with Th2-MoPn, IgG2a was associated with Th1-MoPn. Also, infected nude mice that received Th2-MoPn produced higher levels of IgA in vaginal secretions. Although clone Th2-MoPn was detected in the GT, it was less efficient at migrating (112 ± 35.6 labeled Th2 clone cells/105GT cells) than Th1-MoPn (505 ± 51.6 Th1 clone cells/105GT cells) (P< 0.001, as determined by attest). This may have been due to reduced expression of α4β7 and P-selectin ligand 1 on Th2-MoPn. However, Th2-MoPn cells were retained in the GT during chronic infection and comprised 10 to 15% of the total GT cells 80 days after transfer. The data show that the MoPn-specific Th2 cells are important for serum and vaginal antibody production and may accumulate in the GT during chronic infection.
Efa1 Influences Colonization of the Bovine Intestine by Shiga Toxin-ProducingEscherichia coliSerotypes O5 and O111
Authors: Stevens Mark P. , van Diemen Pauline M. , Frankel Gad , Phillips Alan D. , Wallis Timothy S.
Published: October 2002

Shiga toxin-producingEscherichia coli(STEC) comprises a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in animals and humans. Non-O157 STEC serotypes contain a gene (efa1) that mediates attachment to cultured epithelial cells. An almost-identical gene in enteropathogenicE. coli(lifA) encodes lymphostatin, which inhibits the proliferation of mitogen-activated lymphocytes and the synthesis of proinflammatory cytokines. We have investigated the role of theefa1gene in colonization of 4- and 11-day-old conventional calves by STEC serotypes O5 and O111. Our findings show that Efa1 is required for efficient colonization of the bovine intestinal tract by STEC, sinceefa1deletion and insertion mutants were shed in the feces in significantly lower numbers. In addition,efa1mutations dramatically reduced the number of bacteria associated with the intestinal epithelium. Expression and secretion of locus for enterocyte effacement-encoded type III secreted proteins that are required for adhesion and AE-lesion formation were impaired by mutation ofefa1in STEC but not by mutation oflifAin enteropathogenicE. coli. However, STECefa1mutants retain the ability to nucleate filamentous actin under sites of bacterial attachment to cultured eukaryotic cells. Efa1 is only the second STEC factor shown to influence carriage of the bacteria in the bovine intestine. Our data may have implications for strategies to reduce the prevalence of STEC in cattle.
Analysis of Pathogen-Host Cell Interactions in Purpura Fulminans: Expression of Capsule, Type IV Pili, and PorA by
Neisseria meningitidis
In Vivo
Authors: Harrison O. B. , Robertson B. D. , Faust S. N. , Jepson M. A. , Goldin R. D. , Levin M. , Heyderman R. S.
Published: October 2002

The pattern of meningococcal surface structure expression in different microenvironments following bloodstream invasion in vivo is not known. We used immunohistochemistry to determine the expression of capsule, type IV pili, and PorA by meningococci residing in the skin lesions of children with purpura fulminans. All the skin biopsy samples showed evidence of thrombosis and, frequently, a perivascular inflammatory cell infiltrate consisting of neutrophils (elastase positive) and monocytes/macrophages (CD68 positive). Modified Gram staining revealed 20 to over 100 gram-negative diplococci in each 4-μm-thick section, usually grouped into microcolonies. Immunoperoxidase staining demonstrated that the invading meningococci expressed PorA, capsule, and type IV pilin. Expression of these antigens was not restricted to any particular environment and was found in association with meningococci located in leukocytes, small blood vessels, and the dermal interstitium. Confocal laser scanning microscopy demonstrated coexpression of pilin and capsule by numerous microcolonies. However, there was some discordance in capsule and pilin expression within the microcolonies, suggesting phase variation. The strategy employed in this study will be helpful in investigating invasive bacterial diseases where antigenic and phase variation has a significant impact on virulence and on vaccine design.
Hydrogen Peroxide-Mediated Killing ofCaenorhabditis elegansbyStreptococcus pyogenes
Authors: Jansen W. T. M. , Bolm M. , Balling R. , Chhatwal G. S. , Schnabel R.
Published: October 2002

Caenorhabditis elegansis currently introduced as a new, facile, and cheap model organism to study the pathogenesis of gram-negative bacteria such asPseudomonas aeruginosaandSalmonella entericaserovar Typhimurium. The mechanisms of killing involve either diffusible exotoxins or infection-like processes. Recently, it was shown that also some gram-positive bacteria killC. elegans, although the precise mechanisms of killing remained open. We examinedC. elegansas a pathogenesis model for the gram-positive bacteriumStreptococcus pyogenes, a major human pathogen capable of causing a wide spectrum of diseases. We demonstrate thatS. pyogeneskillsC. elegans, both on solid and in liquid medium. UnlikeP. aeruginosaandS. entericaserovar Typhimurium, the killing byS. pyogenesis solely mediated by hydrogen peroxide. Killing required live streptococci; the killing capacity depends on the amount of hydrogen peroxide produced, and killing can be inhibited by catalase. Major exotoxins ofS. pyogenesare not involved in the killing process as confirmed by using specific toxin inhibitors and knockout mutants. Moreover, no accumulation ofS. pyogenesinC. elegansis observed, which excludes the involvement of infection-like processes. Preliminary results show thatS. pneumoniaecan also killC. elegansby hydrogen peroxide production. Hydrogen peroxide-mediated killing might represent a common mechanism by which gram-positive, catalase-negative pathogens killC. elegans.
Molecular Genetic Analyses of Mating Pheromones Reveal Intervariety Mating or Hybridization in
Cryptococcus neoformans
Authors: Chaturvedi Vishnu , Fan Jinjiang , Stein Birgit , Behr Melissa J. , Samsonoff William A. , Wickes Brian L. , Chaturvedi Sudha
Published: October 2002

The sexual mating of the pathogenic yeast
Cryptococcus neoformans
is important for pathogenesis studies because the fungal virulence is linked to the
α
mating type (
MATα
). We characterized
C. neoformans
mating pheromones (
MFα 1
and
MFa1
) from 122 strains to understand intervariety hybridization or mating and intervariety virulence.
MFα 1
in three
C. neoformans
varieties showed (a) specific nucleotide polymorphisms, (b) different copy numbers and chromosomal localizations, and (c) unique deduced amino acids in two geographic populations of
C. neoformans
var.
gattii. MFα 1
of different varieties cross-hybridized in Southern hybridizations. Their phylogenetic analyses showed purifying selection (neutral evolution). These observations suggested that
MATα
strains from any of the three
C. neoformans
varieties could mate or hybridize in nature with
MAT
a
strains of
C. neoformans
var.
neoformans
. A few serotype A/D diploid strains provided evidence for mating or hybridization, while a majority of A/D strains tested positive for haploid
MFα 1
identical to that of
C. neoformans
var.
grubii
.
MFα 1
sequence and copy numbers in diploids were identical to those of
C. neoformans
var.
grubii
, while their
MFa1
sequences were identical to those of
C. neoformans
var.
neoformans
; thus, these strains were hybrids. The mice survival curves and histological lesions revealed A/D diploids to be highly pathogenic, with pathogenicity levels similar to that of the
C. neoformans
var.
grubii
type strain and unlike the low pathogenicity levels of
C. neoformans
var.
neoformans
strains. In contrast to
MFα 1
in three varieties,
MFa1
amplicons and hybridization signals could be obtained only from two
C. neoformans
var.
neoformans
reference strains and eight A/D diploids. This suggested that a yet undiscovered
MFa
pheromone(s) in
C. neoformans
var.
gattii
and
C. neoformans
var.
grubii
is unrelated to, highly divergent from, or rarer than that in
C. neoformans
var.
neoformans
. These observations could form the basis for future studies on the role of intervariety mating in
C. neoformans
biology and virulence.
Identification and Disruption of the Gene Encoding the Third Member of the Low-Molecular-Mass Rhoptry Complex in
Plasmodium falciparum
Authors: Baldi Deborah L. , Good Robert , Duraisingh Manoj T. , Crabb Brendan S. , Cowman Alan F.
Published: October 2002

The low-molecular-mass rhoptry complex of
Plasmodium falciparum
consists of three proteins, rhoptry-associated protein 1 (RAP1), RAP2, and RAP3. The genes encoding RAP1 and RAP2 are known; however, the
RAP3
gene has not been identified. In this study we identify the
RAP3
gene from the
P. falciparum
genome database and show that this protein is part of the low-molecular-mass rhoptry complex. Disruption of
RAP3
demonstrated that it is not essential for merozoite invasion, probably because RAP2 can complement the loss of RAP3. RAP3 has homology with RAP2, and the genes are encoded on chromosome 5 in a head-to-tail fashion. Analysis of the genome databases has identified homologous genes in all
Plasmodium
spp., suggesting that this protein plays a role in merozoite invasion. The region surrounding the
RAP3
homologue in the
Plasmodium yoelii
genome is syntenic with the same region in
P. falciparum
; however, there is a single gene. Phylogenetic comparison of the RAP2/3 protein family from
Plasmodium
spp. suggests that the RAP2/3 duplication occurred after divergence of these parasite species.
The Siderophore Iron Transporter ofCandida albicans(Sit1p/Arn1p) Mediates Uptake of Ferrichrome-Type Siderophores and Is Required for Epithelial Invasion
Authors: Heymann Petra , Gerads Michaela , Schaller Martin , Dromer Francoise , Winkelmann Günther , Ernst Joachim F.
Published: October 2002

The human fungal pathogenCandida albicanscontains a close homologue of yeast siderophore transporters, designated Sit1p/Arn1p. We have characterized the function ofSIT1inC. albicansby constructingsit1deletion strains and testing their virulence and ability to utilize a range of siderophores and other iron complexes.sit1mutant strains are defective in the uptake of ferrichrome-type siderophores including ferricrocin, ferrichrysin, ferrirubin, coprogen, and triacetylfusarinine C. A mutation ofFTR1did not impair the use of these siderophores but did affect the uptake of ferrioxamines E and B, as well as of ferric citrate, indicating that their utilization was independent of Sit1p. Hemin was a source of iron for bothsit1andftr1mutants, suggesting a pathway of hemin uptake distinct from that of siderophores and iron salts. Heterologous expression ofSIT1in the yeastSaccharomyces cerevisiaeconfirmed the function of Sit1p as a transporter for ferrichrome-type siderophores. Thesit1mutant was defective in infection of a reconstituted human epithelium as a model for human oral mucosa, while theSIT1strain was invasive. In contrast, bothsit1andSIT1strains were equally virulent in the mouse model of systemic infection. These results suggest that siderophore uptake by Sit1p/Arn1p is required in a specific process ofC. albicansinfection, namely epithelial invasion and penetration, while in the blood or within organs other sources of iron, including heme, may be used.
Mixed Periodontal Th1-Th2 Cytokine Profile in
Actinobacillus actinomycetemcomitans
-Specific Osteoprotegerin Ligand (or RANK-L)- Mediated Alveolar Bone Destruction In Vivo
Authors: Teng Yen-Tung A.
Published: October 2002

The Th1/Th2 cytokines involved in human periodontitis remain unclear; therefore, we established a humanized mouse model to investigate this issue in
Actinobacillus actinomycetemcomitans
-mediated periodontal infection. Quantitative-PCR analysis clearly demonstrates a predominantly mixed Th1 and Th2 expression profile associated with pathogen-specific cell-mediated immunity via osteoprotegerin ligand (or RANK-L)-mediated alveolar bone destruction in vivo.
β-1,2-Mannosylation of
Candida albicans
Mannoproteins and Glycolipids Differs with Growth Temperature and Serotype
Authors: Trinel P. A. , Jouault T. , Cutler J. E. , Poulain D.
Published: October 2002

Increasing the growth temperature from 28 to 37°C reduced the expression of β-1,2-oligomannoside epitopes on mannoproteins of
Candida albicans
serotypes A and B. In contrast, β-1,2-mannosylation of phospholipomannan (PLM) remained constant despite a slight decrease in the relative molecular weight (
M
r
) of this compound. At all growth temperatures investigated, serotype A PLM displayed an
M
r
and an antigenicity different from those of serotype B PLM when they were tested with a panel of monoclonal antibodies.
Functional Domain of Bovine Milk Lactoferrin Which Inhibits the Adherence of
Streptococcus mutans
Cells to a Salivary Film
Authors: Oho Takahiko , Mitoma Morihide , Koga Toshihiko
Published: October 2002

The bovine lactoferrin molecule and relatively long lactoferrin fragments containing residues 473 to 538 strongly inhibited adherence of
Streptococcus mutans
to saliva-coated hydroxyapatite beads. Each cysteine residue in Lf411 (residues 473 to 538) was replaced by a serine residue, and the mutants Lf411-C481S and Lf411-C532S strongly inhibited
S. mutans
adherence. These results suggest that the functional domain of lactoferrin that binds to a salivary film lies in residues 473 to 538 and that the region might be concealed by disulfide bond formation between Cys481 and Cys532 in the Lf411 fragment.
ADirofilaria immitisPolyprotein Up-Regulates Nitric Oxide Production
Authors: Tezuka Hiroyuki , Imai Shinjiro , Tsukidate Setsuko , Fujita Koichiro
Published: October 2002

We investigated the effect of recombinantDirofilaria immitispolyprotein (rDiAg) on nitric oxide (NO) production by peritoneal macrophages. rDiAg induced NO production by macrophages from wild-type and lipopolysaccharide-hyporesponsive C3H/HeJ, but not CD40−/−, mice. These results suggest that CD40 is involved in rDiAg-driven NO production by murine macrophages.
Differential Expression of the p44 Gene Family in the Agent of Human Granulocytic Ehrlichiosis
Authors: IJdo Jacob W. , Wu Caiyun , Telford Sam R. , Fikrig Erol
Published: October 2002

Using reverse transcription-PCR targeting of the p44 genes of the agent of human granulocytic ehrlichiosis (HGE) with primers flanking the hypervariable region, we show differential expression in a murine model of HGE infection and during tick transmission. The p44 genes were differentially expressed in salivary glands of infected nymphal ticks removed during transmission feeding but not in nonfeeding infected ticks. Similarly, the p44 genes were differentially expressed in infected C3H mice, in SCID mice, and in cultured HGE bacteria. Thus, differential p44 expression exists in vivo and in vitro and could provide a basis for antigenic variation.
Roles for the Two 1-Butanol Dehydrogenases ofPseudomonas butanovorain Butane and 1-Butanol Metabolism
Authors: Vangnai Alisa S. , Sayavedra-Soto Luis A. , Arp Daniel J.
Published: October 2002

Pseudomonas butanovoragrown on butane or 1-butanol expresses two 1-butanol dehydrogenases, a quinoprotein (BOH) and a quinohemoprotein (BDH). BOH exhibited high affinity towards 1-butanol (Km= 1.7 ± 0.2 μM). BOH also oxidized butyraldehyde and 2-butanol (Km= 369 ± 85 μM andKm= 662 ± 98 μM, respectively). The mRNA induction profiles of BOH and BDH at three different levels of 1-butanol, a nontoxic level (0.1 mM), a growth-supporting level (2 mM), and a toxic level (40 mM), were similar. When cells were grown in citrate-containing medium in the presence of different levels of 1-butanol, wild-typeP. butanovoracould tolerate higher levels of 1-butanol than theP. butanovora boh::tetstrain and theP. butanovora bdh::kanstrain. A model is proposed in which the electrons from 1-butanol oxidation follow a branched electron transport chain. BOH may be coupled to ubiquinone, with the electrons being transported to a cyanide-sensitive terminal oxidase. In contrast, electrons from BDH may be transferred to a terminal oxidase that is less sensitive to cyanide. The former pathway may function primarily in energy generation, while the latter may be more important in the detoxification of 1-butanol.
ZntB Is a Novel Zn
2+
Transporter in
Salmonella enterica
Serovar Typhimurium
Authors: Worlock Ashley J. , Smith Ronald L.
Published: October 2002

A Zn
2+
transport system encoded by the
zntB
locus of
Salmonella enterica
serovar Typhimurium has been identified. The protein encoded by this locus is homologous to the CorA family of Mg
2+
transport proteins and is widely distributed among the eubacteria. Mutations at
zntB
confer an increased sensitivity to the cytotoxic effects of Zn
2+
and Cd
2+
, a phenotype that suggests that the encoded protein mediates the efflux of both cations. A direct analysis of transport activity identified a capacity for Zn
2+
efflux. These data identify ZntB as a zinc efflux pathway in the enteric bacteria and assign a new function to the CorA family of cation transporters.
Tools for Characterization of
Escherichia coli
Genes of Unknown Function
Authors: Merlin Christophe , McAteer Sean , Masters Millicent
Published: October 2002

Despite the power of sequencing and of emerging high-throughput technologies to collect data rapidly, the definitive functional characterization of unknown genes still requires biochemical and genetic analysis in case-by-case studies. This often involves the deletion of target genes and phenotypic characterization of the deletants. We describe here modifications of an existing deletion method which facilitates the deletion process and enables convenient analysis of the expression properties of the target gene by replacing it with an FRT-
lacZ
-
aph
-

P
lac

-FRT cassette. The
lacZ
gene specifically reports the activity of the deleted gene and therefore allows the determination of the conditions under which it is actively expressed. The
aph
gene, encoding resistance to kanamycin, provides a selectable means of transducing a deleted locus between strains so that the deletion can be combined with other relevant mutations. The
lac
promoter helps to overcome possible polar effects on downstream genes within an operon. Because the cassette is flanked by two directly repeated FRT sites, the cassette can be excised by the Flp recombinase provided in
trans
. Removing the cassette leaves an in-frame deletion with a short scar which should not interfere with downstream expression. Replacements of
yacF
,
yacG
,
yacH
,
yacK
(
cueO
),
yacL
,
ruvA
,
ruvB
,
yabB
, and
yabC
made with the cassette were used to verify its properties.
Genome Sequence of
Yersinia pestis
KIM
Authors: Deng Wen , Burland Valerie , Plunkett Guy , Boutin Adam , Mayhew George F. , Liss Paul , Perna Nicole T. , Rose Debra J. , Mau Bob , Zhou Shiguo , Schwartz David C. , Fetherston Jaqueline D. , Lindler Luther E. , Brubaker Robert R. , Plano Gregory V. , Straley Susan C. , McDonough Kathleen A. , Nilles Matthew L. , Matson Jyl S. , Blattner Frederick R. , Perry Robert D.
Published: October 2002

We present the complete genome sequence of
Yersinia pestis
KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of
Escherichia coli
K-12. The KIM genome sequence was compared with that of
Y. pestis
CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and
E. coli
K-12 genome proteins were also compared, exposing surprising amounts of locally colinear “backbone,” or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of
E. coli
pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.
Mutational Analysis of the TolA C-Terminal Domain ofEscherichia coliand Genetic Evidence for an Interaction between TolA and TolB
Authors: Dubuisson Jean François , Vianney Anne , Lazzaroni Jean Claude
Published: October 2002

The Tol proteins are involved in the outer membrane stability of gram-negative bacteria. The C-terminal domain of TolA was mutagenized to identify residues important for its functions. The isolation of suppressor mutants oftolAmutations in thetolBgene confirmed an interaction between TolAIII and the N-terminal domain of TolB.
Recombination-Promoting Activity of the Bacteriophage λ Rap Protein in
Escherichia coli
K-12
Authors: Poteete Anthony R. , Fenton Anita C. , Wang Hsinju R.
Published: October 2002

The
rap
gene of bacteriophage λ was placed in the chromosome of an
Escherichia coli
K-12 strain in which the
recBCD
gene cluster had previously been replaced by the λ
red
genes and in which the
recG
gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the
recG
Δ
red
+
rap
+
strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the
cat
gene, with flanking
lac
sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type
lacZ
with
lacZ
::
cat
was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the λ
rap
gene could functionally substitute for the
E. coli ruvC
gene in Red-mediated recombination.
Genomic Variability of O Islands Encoding Tellurite Resistance in Enterohemorrhagic
Escherichia coli
O157:H7 Isolates
Authors: Taylor Diane E. , Rooker Michelle , Keelan Monika , Ng Lai-King , Martin Irene , Perna Nicole T. , Burland N. T. Valerie , Blattner Fredrick R.
Published: October 2002

Strains of
Escherichia coli
causing enterohemorrhagic colitis belonging to the O157:H7 lineage are reported to be highly related. Fifteen strains of
E. coli
O157:H7 and 1 strain of
E. coli
O46:H
−
(nonflagellated) were examined for the presence of potassium tellurite resistance (Te
r
). Te
r
genes comprising
terABCDEF
were shown previously to be part of a pathogenicity island also containing integrase, phage, and urease genes. PCR analysis, both conventional and light cycler based, demonstrated that about one-half of the Te
r
E. coli
O157:H7 strains (6 of 15), including the Sakai strain, which has been sequenced, carried a single copy of the Te
r
genes. Five of the strains, including EDL933, which has also been sequenced, contained two copies. Three other O157:H7 strains and the O46:H
−
strain did not contain the Te
r
genes. In strains containing two copies, the Te
r
genes were associated with the
serW
and
serX
tRNA genes. Five O157:H7 strains resembled the O157 Sakai strain whose sequence contained one copy, close to
serX
, whereas in one isolate the single copy was associated with
serW
. There was no correlation between Te
r
and the ability to produce Shiga toxin ST1 or ST2. The Te
r
MIC for most strains, containing either one or two copies, was 1,024 μg/ml, although for a few the MIC was intermediate, 64 to 128 μg/ml, which could be increased to 512 μg/ml by pregrowth of strains in subinhibitory concentrations of potassium tellurite. Reverse transcriptase PCR analysis confirmed that in most strains Te
r
was constitutive but that in the rest it was inducible and involved induction of
terB
and
terC
genes. Only the
terB
, -
C
, -
D
, and -
E
genes are required for Te
r
. The considerable degree of homology between the
ter
genes on IncH12 plasmid R478, which originated in
Serratia marcescens
, and pTE53, from an
E. coli
clinical isolate, suggests that the pathogenicity island was acquired from a plasmid. This work demonstrates diversity among
E. coli
O157:H7 isolates, at least as far as the presence of Te
r
genes is concerned.
Roles of RecJ, RecO, and RecR in RecET-Mediated Illegitimate Recombination in
Escherichia coli
Authors: Shiraishi Kouya , Hanada Katsuhiro , Iwakura Yoichiro , Ikeda Hideo
Published: October 2002

We analyzed effects of overexpression of RecE and RecT on illegitimate recombination during prophage induction in
Escherichia coli
and found that frequencies of spontaneous and UV-induced illegitimate recombination are enhanced by coexpression of RecE and RecT in the wild type, but the enhanced recombination was reduced by
recJ, recO
, or
recR
mutation. The results indicated that RecET-mediated illegitimate recombination depends on the functions of RecJ, RecO, and RecR, suggesting that the RecE and RecJ exonucleases play different roles in this recombination pathway and that the RecO and RecR proteins also play important roles in the recombination. On the other hand, the frequency of the RecET-mediated illegitimate recombination was enhanced by a
recQ
mutation, implying that the RecQ protein plays a role in suppression of RecET-mediated illegitimate recombination. It was also found that RecET-mediated illegitimate recombination is independent of the RecA function with UV irradiation, but it is enhanced by the
recA
mutation without UV irradiation. Based on these results, we propose a model for the roles of RecJOR on RecET-mediated illegitimate recombination.
Tet(L) and Tet(K) Tetracycline-Divalent Metal/H
+
Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences
Authors: Jin Jie , Guffanti Arthur A. , Bechhofer David H. , Krulwich Terry A.
Published: October 2002

The Tet(L) protein encoded in the
Bacillus subtilis
chromosome and the closely related Tet(K) protein from
Staphylococcus aureus
plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me
2+
) complex that bears a net single positive charge, Na
+
, and K
+
. Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H
+
as the coupling ion. In this study, competitive cross-inhibition between K
+
and other cytoplasmic efflux substrates was demonstrated. Tet(L) and Tet(K) had also been shown to use K
+
as an alternate coupling ion in support of Na
+
or K
+
efflux. Here they were shown to couple TC-Me
2+
efflux to K
+
uptake as well, exhibiting greater use of K
+
as a coupling ion as the external pH increased. The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K
+
, both as a cytoplasmic efflux substrate and as an external coupling ion. Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative “antiporter motif,” motif C, of Tet proteins would be involved in these characteristic preferences. Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K
+
preference both as coupling ion and efflux substrate. A reciprocal S157A mutant of Tet(K) exhibited reduced K
+
preference. Competitive inhibition among substrates and the parallel effects of the single mutation upon K
+
preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion. However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates.
Genome-Wide Analysis of the Stationary-Phase Sigma Factor (Sigma-H) Regulon ofBacillus subtilis
Authors: Britton Robert A. , Eichenberger Patrick , Gonzalez-Pastor Jose Eduardo , Fawcett Paul , Monson Rita , Losick Richard , Grossman Alan D.
Published: October 2002

Sigma-H is an alternative RNA polymerase sigma factor that directs the transcription of many genes that function at the transition from exponential growth to stationary phase inBacillus subtilis. Twenty-three promoters, which drive transcription of 33 genes, are known to be recognized by sigma-H-containing RNA polymerase. To identify additional genes under the control of sigma-H on a genome-wide basis, we carried out transcriptional profiling experiments using a DNA microarray containing >99% of the annotatedB.subtilisopen reading frames. In addition, we used a bioinformatics-based approach aimed at the identification of promoters recognized by RNA polymerase containing sigma-H. This combination of approaches was successful in confirming most of the previously described sigma-H-controlled genes. In addition, we identified 26 putative promoters that drive expression of 54 genes not previously known to be under the direct control of sigma-H. Based on the known or inferred function of most of these genes, we conclude that, in addition to its previously known roles in sporulation and competence, sigma-H controls genes involved in many physiological processes associated with the transition to stationary phase, including cytochrome biogenesis, generation of potential nutrient sources, transport, and cell wall metabolism.
Surface Loop Motion in FepA
Authors: Scott Daniel C. , Newton Salete M. C. , Klebba Phillip E.
Published: October 2002

Using a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops ofEscherichia coliFepA during its transport of the siderophore ferric enterobactin. Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein. The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2. Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332. These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 Å, from a ligand-free open state to a ligand-bound closed state. The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanidem-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide.
The Membrane-Bound α-Glucuronidase from
Pseudomonas cellulosa
Hydrolyzes 4-
O-
Methyl-
d
-Glucuronoxylooligosaccharides but Not 4-
O-
Methyl-
d
-Glucuronoxylan
Authors: Nagy Tibor , Emami Kaveh , Fontes Carlos M. G. A. , Ferreira Luis M. A. , Humphry David R. , Gilbert Harry J.
Published: October 2002

The microbial degradation of xylan is a key biological process. Hardwood 4-
O-
methyl-
d
-glucuronoxylans are extensively decorated with 4-
O
-methyl-
d
-glucuronic acid, which is cleaved from the polysaccharides by α-glucuronidases. In this report we describe the primary structures of the α-glucuronidase from
Cellvibrio mixtus
(
C. mixtus
GlcA67A) and the α-glucuronidase from
Pseudomonas cellulosa
(
P. cellulosa
GlcA67A) and characterize
P. cellulosa
GlcA67A. The primary structures of
C. mixtus
GlcA67A and
P. cellulosa
GlcA67A, which are 76% identical, exhibit similarities with α-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad α-glucuronidase released 4-
O
-methyl-
d
-glucuronic acid from 4-
O
-methyl-
d
-glucuronoxylooligosaccharides but not from 4-
O-
methyl-
d
-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-
O-
methyl-
d
-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.
Filamentous Bacteriophages of Vibrios Are Integrated into the
dif
-Like Site of the Host Chromosome
Authors: Iida Tetsuya , Makino Kozo , Nasu Hatsumi , Yokoyama Katsushi , Tagomori Kenichi , Hattori Akiko , Okuno Toshihiro , Shinagawa Hideo , Honda Takeshi
Published: October 2002

The
dif
site is located in the replication terminus region of bacterial chromosomes, having a function of resolving dimeric chromosomes formed during replication. We demonstrate that filamentous bacteriophages of vibrios, such as f237 (
Vibrio parahaemolyticus
) and CTXφ (
V. cholerae
), are integrated into the
dif
-like site of host chromosome.
Conserved Amplification of Chemotactic Responses through Chemoreceptor Interactions
Authors: Lamanna Allison C. , Gestwicki Jason E. , Strong Laura E. , Borchardt Sara L. , Owen Robert M. , Kiessling Laura L.
Published: October 2002

Many bacteria concentrate their chemoreceptors at the cell poles. Chemoreceptor location is important in
Escherichia coli
, since chemosensory responses are sensitive to receptor proximity. It is not known, however, whether chemotaxis in other bacteria is similarly regulated. To investigate the importance of receptor-receptor interactions in other bacterial species, we synthesized saccharide-bearing multivalent ligands that are designed to cluster relevant chemoreceptors. As has been shown with
E. coli
, we demonstrate that the behaviors of
Bacillus subtilis
,
Spirochaete aurantia
, and
Vibrio furnissii
are sensitive to the valence of the chemoattractant. Moreover, in
B. subtilis
, chemotactic responses to serine were increased by pretreatment with saccharide-bearing multivalent ligands. This result indicates that, as in
E. coli
, signaling information is transferred among chemoreceptors in
B. subtilis
. These results suggest that interreceptor communication may be a general mechanism for modulating chemotactic responses in bacteria.
Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination
Authors: Arkov Alexey L. , Hedenstierna Klas O. F. , Murgola Emanuel J.
Published: October 2002

Nucleotide 1093 in domain II of
Escherichia coli
23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.