bluuebunny/crossref_metadata_2025 huggingface dataset:
Virulence Plasmid ofRhodococcus equiContains Inducible Gene Family Encoding Secreted Proteins
Authors: Byrne Barbara A. , Prescott John F. , Palmer Guy H. , Takai Shinji , Nicholson Vivian M. , Alperin Debra C. , Hines Stephen A.
Published: July 2002

Rhodococcus equicauses severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains ofR. equibear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the sameR. equiisolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid,vapC, -D, and -E, that are tandemly arranged downstream ofvapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other.vapC, -D, and -Eare found only inR. equistrains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed whenR. equiis cultured at 37°C but not at 30°C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins.
Familial Correlation of Immunoglobulin G Subclass Responses to
Plasmodium falciparum
Antigens in Burkina Faso
Authors: Aucan Christophe , Traoré Yves , Fumoux Francis , Rihet Pascal
Published: July 2002

Host genes are thought to determine the immune response to malaria infection and the outcome. Cytophilic antibodies have been associated with protection, whereas noncytophilic antibodies against the same epitopes may block the protective activity of the protective ones. To assess the contribution of genetic factors to immunoglobulin G (IgG) subclass responses against conserved epitopes and
Plasmodium falciparum
blood-stage extracts, we analyzed the isotypic distribution of the IgG responses in 366 individuals living in two differently exposed areas in Burkina Faso. We used one-way analysis of variance and pairwise estimators to calculate sib-sib and parent-offspring correlation coefficients, respectively. Familial patterns of inheritance of IgG subclass responses to defined antigens and
P. falciparum
extracts appear to be similar in the two areas. We observed a sibling correlation for the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against ring-infected-erythrocyte surface antigen, merozoite surface protein 1 (MSP-1), MSP-2, and
P. falciparum
extract. Moreover, a parent-offspring correlation was found for several IgG subclass responses, including the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against conserved MSP-2 epitopes. Our results indicated that the IgG subclass responses against
P. falciparum
blood-stage antigens are partly influenced by host genetic factors. The localization and identification of these genes may have implications for immunoepidemiology and vaccine development.
Effects of Tumor Necrosis Factor Alpha on Host Immune Response in Chronic Persistent Tuberculosis: Possible Role for Limiting Pathology
Authors: Mohan Vellore P. , Scanga Charles A. , Yu Keming , Scott Holly M. , Tanaka Kathryn E. , Tsang Enders , Tsai Ming Chih , Flynn JoAnne L. , Chan John
Published: July 2002

Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused byMycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-α), we examined the effects of TNF-α on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-α in the containment of persistent tuberculosis. TNF-α neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-α-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-α-neutralized mice. In summary, results of this study suggest that TNF-α plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.
Identification of Polymorphic Outer Membrane Proteins ofChlamydia psittaci6BC
Authors: Tanzer Regina J. , Longbottom David , Hatch Thomas P.
Published: July 2002

The genomes ofChlamydiaspp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC ofChlamydia psittaciby polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovineC. psittacistrain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.
Granulomatous Skin Lesions in Moray Eels Caused by a NovelMycobacteriumSpecies Related toMycobacterium triplex
Authors: Herbst Lawrence H. , Costa Sylvia F. , Weiss Louis M. , Johnson Linda K. , Bartell John , Davis Raymond , Walsh Michael , Levi Michael
Published: July 2002

An outbreak of granulomatous dermatitis was investigated in a captive population of moray eels. The affected eels had florid skin nodules concentrated around the head and trunk. Histopathological examination revealed extensive granulomatous inflammation within the dermis and subcutaneous fascial plane between the fat and axial musculature. Acid-fast rods were detected within the smallest lesions, which were presumably the ones that had developed earliest. Eventually, after several months of incubation at room temperature, a very slowly growing acid-fast organism was isolated. Sequencing of the 16S rRNA gene identified it as aMycobacteriumspecies closely related (0.59% divergence) toM. triplex, an SAV mycobacterium. Intradermal inoculation of healthy green moray eels with this organism reliably reproduced the lesion. Experimentally induced granulomatous dermatitis appeared within 2 weeks of inoculation and slowly but progressively expanded during the 2 months of the experiment. Live organisms were recovered from these lesions at all time points, fulfilling Koch's postulates for this bacterium. In a retrospective study of tissues collected between 1993 and 1999 from five spontaneous disease cases, acid-fast rods were consistently found within lesions, and a nested PCR for the rRNA gene also demonstrated the presence of mycobacteria within affected tissues.
SarT, a Repressor of α-Hemolysin in
Staphylococcus aureus
Authors: Schmidt Katherine A. , Manna Adhar C. , Gill Steven , Cheung Ambrose L.
Published: July 2002

In searching the
Staphylococcus aureus
genome, we found several homologs to SarA. One of these genes,
sarT
, codes for a basic protein with 118 residues and a predicted molecular size of 16,096 Da. Northern blot analysis revealed that the expression of
sarT
was repressed by
sarA
and
agr
. An insertion
sarT
mutant generated in
S. aureus
RN6390 and 8325-4 backgrounds revealed minimal effect on the expression of
sarR
and
sarA
. The RNAIII level was notably increased in the
sarT
mutant, particularly in postexponential-phase cells, while the augmentative effect on RNAII was less. SarT repressed the expression of α-hemolysin, as determined by Northern blotting, Western blotting, and a rabbit erythrocyte hemolytic assay. This repression was relieved upon complementation. Similar to
agr
and
sarA
mutants, which predictably displayed a reduction in
hla
expression, the
agr sarT
mutant exhibited a lower level of
hla
transcription than the
sarT
mutant. In contrast,
hla
transcription was enhanced in the
sarA sarT
mutant compared with the single
sarA
mutant. Collectively, these results indicated that the
sarA
locus, contrary to the regulatory action of
agr
, induced α-hemolysin production by repressing
sarT
, a repressor of
hla
transcription.
Ongoing Horizontal and Vertical Transmission of Virulence Genes and
papA
Alleles among
Escherichia coli
Blood Isolates from Patients with Diverse-Source Bacteremia
Authors: Johnson James R. , O'Bryan Timothy T. , Kuskowski Michael , Maslow Joel N.
Published: July 2002

The phylogenetic distributions of multiple putative virulence factors (VFs) and
papA
(P fimbrial structural subunit) alleles among 182
Escherichia coli
blood isolates from patients with diverse-source bacteremia were defined. Phylogenetic correspondence among these strains, the
E. coli
Reference (ECOR) collection, and other collections of extraintestinal pathogenic
E. coli
(ExPEC) was assessed. Although among the 182 bacteremia isolates phylogenetic group B2 predominated, exhibited the greatest concentration of individual VFs, and contained the largest number of familiar virulent clones, other phylogenetic groups exhibited greater concentrations of certain VFs than did group B2 and included several additional virulent clones. Certain of the newly detected VF genes, e.g.,
fyuA
(yersiniabactin; 76%) and
focG
(F1C fimbriae; 25%), were as prevalent or more prevalent than their more familiar traditional counterparts, e.g.,
iut
(aerobactin; 57%) and
sfaS
(S fimbriae; 14%), thus possibly offering additional useful targets for preventive interventions. Considerable diversity of VF profiles was observed at every level within the phylogenetic tree, including even within individual lineages. This suggested that many different pathways can lead to extraintestinal virulence in
E. coli
and that the evolution of ExPEC, which involves extensive horizontal transmission of VFs and continuous remodeling of pathogenicity-associated islands, is a highly active, ongoing process.
CD8+-T-Cell Immunity againstToxoplasma gondiiCan Be Induced but Not Maintained in Mice Lacking Conventional CD4+T Cells
Authors: Casciotti Lori , Ely Kenneth H. , Williams Martha E. , Khan Imtiaz A.
Published: July 2002

T-cell immunity is critical for survival of hosts infected withToxoplasma gondii.Among the cells in the T-cell population, CD8+T cells are considered the major effector cells against this parasite. It is believed that CD4+T cells may be crucial for induction of the CD8+-T-cell response againstT. gondii. In the present study, CD4−/−mice were used to evaluate the role of conventional CD4+T cells in the immune response againstT. gondiiinfection. CD4−/−mice infected withT. gondiiexhibited lower gamma interferon (IFN-γ) messages in the majority of their tissues. As a result, mortality due to a hyperinflammatory response was prevented in these animals. Interestingly,T. gondiiinfection induced a normal antigen-specific CD8+-T-cell immune response in CD4−/−mice. No difference in generation of precursor cytotoxic T lymphocytes (pCTL) or in IFN-γ production by the CD8+-T-cell populations from the knockout and wild-type animals was observed. However, the mutant mice were not able to sustain CD8+-T-cell immunity. At 180 days after infection, the CD8+-T-cell response in the knockout mice was depressed, as determined by pCTL and IFN-γ assays. Loss of CD8+-T-cell immunity at this time was confirmed by adoptive transfer experiments. Purified CD8+T cells from CD4−/−donors that had been immunized 180 days earlier failed to protect the recipient mice against a lethal infection. Our study demonstrated that although CD8+-T-cell immunity can be induced in the absence of conventional CD4+T cells, it cannot be maintained without such cells.
Adherence ofBurkholderia pseudomalleiCells to Cultured Human Epithelial Cell Lines Is Regulated by Growth Temperature
Authors: Brown Nat F. , Boddey Justin A. , Flegg Cameron P. , Beacham Ifor R.
Published: July 2002

We have investigated the adherence ofBurkholderia pseudomallei, cultured under a number of different conditions, to six human epithelial cell lines. While several complex medium compositions had relatively little effect on adherence, growth at 30°C was found to significantly increase adherence to all cell lines relative to that of cultures grown at 37°C (P< 0.001).
Identification of a SecondArcanobacterium pyogenesNeuraminidase and Involvement of Neuraminidase Activity in Host Cell Adhesion
Authors: Jost B. Helen , Songer J. Glenn , Billington Stephen J.
Published: July 2002

Arcanobacterium pyogenes, a common inhabitant of the upper respiratory and urogenital tracts of economically important animals, such as cattle and swine, is also an opportunistic pathogen associated with suppurative infections in these animals.A. pyogenesexpresses neuraminidase activity encoded by thenanHgene, and previously, construction of ananHmutant ofA. pyogenesBBR1 indicated that a second neuraminidase is present in this strain. A 5,112-bp gene,nanP, was cloned and sequenced, and this gene conferred neuraminidase activity on anEscherichia colihost strain. The predicted 186.8-kDa NanP protein exhibited similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. As expected, insertional inactivation of thenanPgene inA. pyogenesBBR1 resulted in a mutant with reduced neuraminidase activity. However, insertional inactivation of thenanPgene in thenanHmutant strain resulted in a complete lack of neuraminidase activity. Like NanH, NanP was localized to theA. pyogenescell wall. However, unlike thenanHgene, which was present in 100% of the strains examined,nanPwas present in only 64.2% of the isolates (n= 53).A. pyogenesadheres to HeLa cells, and ananPmutant displayed a wild-type adhesion phenotype with these cells. In contrast, the ability of ananHnanPdouble mutant to bind to HeLa cells was reduced by 53%. The wild-type adhesion phenotype was restored by providingnanPintrans. These data indicate that the neuraminidases ofA. pyogenesplay a role in adhesion of this organism to host epithelial cells.
Molecular Analysis of the Contribution of the Capsular Polysaccharide and the Lipopolysaccharide O Side Chain to the Virulence of
Klebsiella pneumoniae
in a Murine Model of Pneumonia
Authors: Cortés Guadalupe , Borrell Nuria , de Astorza Beatriz , Gómez Cristina , Sauleda Jaume , Albertí Sebastián
Published: July 2002

Klebsiella pneumoniae
is a common cause of gram-negative bacterial nosocomial pneumonia. Two surface polysaccharides, lipopolysaccharide (LPS) O side chain and capsular polysaccharide (CPS), are critical for the microorganism in causing sepsis, but little is known about their role in pneumonia. To investigate their contribution in the pathogenesis of
K. pneumoniae
pneumonia, we characterized the host response to bacterial challenge with a highly virulent clinical isolate or with isogenic insertion-duplication mutants deficient in CPS or LPS O side chain in a murine model of pneumonia. Animals challenged intratracheally with the wild-type or LPS O side chain-deficient strain developed pneumonia and became bacteremic before death. Extensive lung lesions as well as pleuritis, vasculitis, and edema were observed by histopathological examination, and polymorphonuclear infiltration was also demonstrated. In contrast, none of the animals challenged with the unencapsulated strain developed pneumonia or bacteremia. Examination of tissue from this group did not identify lung lesions, and none of the infected animals died. Analysis of the early host defense mechanisms that contributed to the clearance of the unencapsulated mutant showed that the levels of C3 deposited on the unencapsulated mutant surface were threefold higher than those for the wild-type and LPS O side chain-deficient strains. Furthermore, phagocytosis of the unencapsulated mutant by human alveolar macrophages (AM) was more efficient than that of the wild-type and LPS O side chain-deficient strains. We conclude that CPS, but not LPS O side chain, is essential for
Klebsiella
pneumonia because it modulates the deposition of C3 and protects the microorganisms against human AM phagocytosis.
Mycobacterium tuberculosis
Genes Induced during Infection of Human Macrophages
Authors: Dubnau Eugenie , Fontán Patricia , Manganelli Riccardo , Soares-Appel Sonia , Smith Issar
Published: July 2002

We identified
Mycobacterium tuberculosis
genes preferentially expressed during infection of human macrophages using a promoter trap adapted for this pathogen.
inhA
encodes an enoyl-acyl carrier protein reductase that is required for mycolic acid biosynthesis (A. Quemard et al., Biochemistry
34:
8235-8241, 1995) and is a major target for isoniazid (INH) in mycobacterial species (A. Banerjee et al., Science
263:
227-230, 1994). Since overexpression of
inhA
confers INH resistance in
Mycobacterium smegmatis
(Banerjee et al., Science
263:
227-230, 1994), we designed a promoter trap based on this gene. A library of clones, containing small fragments of
M. tuberculosis
DNA cloned upstream of
inhA
in a plasmid vector, was electroporated into
M. tuberculosis
, and the resulting culture was used to infect the human monocytic THP-1 cell line. Selection was made for clones surviving INH treatment during infection but retaining INH sensitivity on plates. The DNA upstream of
inhA
was sequenced in each clone to identify the promoter driving
inhA
expression. Thirteen genes identified by this method were analyzed by quantitative reverse transcription-PCR (R. Manganelli et al., Mol. Microbiol.
31:
715-724, 1999), and eight of them were found to be differentially expressed from cultures grown in macrophages compared with broth-grown cultures. Several of these genes are presumed to be involved in fatty acid metabolism; one potentially codes for a unique DNA binding protein, one codes for a possible potassium channel protein, and the others code for proteins of unknown function. Genes which are induced during infection are likely to be significant for survival and growth of the pathogen; our results lend support to the view that fatty acid metabolism is essential for the virulence of
M. tuberculosis.
The
lbgAB
Gene Cluster of
Haemophilus ducreyi
Encodes a β-1,4-Galactosyltransferase and an α-1,6-
dd
-Heptosyltransferase Involved in Lipooligosaccharide Biosynthesis
Authors: Tullius Michael V. , Phillips Nancy J. , Scheffler N. Karoline , Samuels Nicole M. , Munson Robert S. , Hansen Eric J. , Stevens-Riley Marla , Campagnari Anthony A. , Gibson Bradford W.
Published: July 2002

All
Haemophilus ducreyi
strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep
3
-phosphorylated 3-deoxy-
d
-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes,
lbgA
and
lbgB
, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the
lbgB
(
losB
) gene was found to be required for transfer of the second sugar,
d
-
glycero
-
d
-
manno
-heptose (
dd
-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene,
lbgA
, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked β1→4, to the
dd
-Hep. LOS from an
lbgA
mutant and an
lbgAB
double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (
lbgAB
) or the disaccharide
dd
-Hepα1→6Glcβ1 (
lbgA
) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of
H. ducreyi
infection.
LsaA, an Antigen Involved in Cell Attachment and Invasion, Is Expressed byLawsonia intracellularisduring Infection In Vitro and In Vivo
Authors: McCluskey Jackie , Hannigan Joanne , Harris Jennifer D. , Wren Brendan , Smith David G. E.
Published: July 2002

Lawsonia intracellularishas been identified recently as the etiological agent of proliferative enteropathies, which are characterized by intestinal epithelial hyperplasia and associated moderate immune responses. This disease complex has been reported in a broad range of animals, prevalently in pigs, andL. intracellularishas been linked with ulcerative colitis in humans.L. intracellularisis an obligate intracellular bacterium, and the pathogenic mechanisms used to cause disease are unknown. Using in vitro-grown organisms as a source of genomic DNA, we identified aLawsoniagene which encodes a surface antigen, LsaA (forLawsoniasurface antigen), associated with attachment to and entry into cells. The deduced amino acid sequence of this protein showed some similarity to members of a novel protein family identified in a number of other bacterial pathogens but for which roles are not fully defined. Transcription of this gene was detected by reverse transcription-PCR inL. intracellularisgrown in vitro in IEC18 cells and in bacteria present in ileal tissue from infected animals. Immunohistochemistry with specific monoclonal antibody and immunoblotting with sera from infected animals demonstrated that LsaA protein is synthesized byL. intracellularisduring infection. Expression of this gene during infection in vitro and in vivo suggests that this surface antigen is involved during infection, and phenotypic analysis indicated a role duringL. intracellularisattachment to and entry into intestinal epithelial cells
Construction and Phenotypic Characterization of an Auxotrophic Mutant of
Mycobacterium tuberculosis
Defective in
l
-Arginine Biosynthesis
Authors: Gordhan Bhavna G. , Smith Debbie A. , Alderton Heidi , McAdam Ruth A. , Bancroft Gregory J. , Mizrahi Valerie
Published: July 2002

A mutant of
Mycobacterium tuberculosis
defective in the metabolism of
l
-arginine was constructed by allelic exchange mutagenesis. The
argF
mutant strain required exogenous
l
-arginine for growth in vitro, and in the presence of 0.96 mM
l
-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by
l
-citrulline, suggesting that the Δ
argF
::
hyg
mutation exerted a polar effect on the downstream
argG
gene but not on
argH
. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that
l
-arginine availability is restricted in vivo.
Uptake ofAspergillus fumigatusConidia by Phagocytic and Nonphagocytic Cells In Vitro: Quantitation Using Strains Expressing Green Fluorescent Protein
Authors: Wasylnka Julie A. , Moore Margo M.
Published: July 2002

Several pathogenic fungal organisms enter eukaryotic cells and manipulate the host cell environment to favor their own growth and survival.Aspergillus fumigatusis a saprophytic fungus that causes invasive lung disease in the immunocompromised host. To determine whetherA. fumigatuscould enter eukaryotic cells, we studied the uptake of two different GFP-expressingA. fumigatusstrains into A549 lung epithelial cells, human umbilical vein endothelial (HUVE) cells, and J774 murine macrophages in vitro. A549 cells internalized 30% of the bound conidia whereas HUVE and J774 cells internalized 50 and 90%, respectively. Conidia within A549 cells remained viable for 6 h; however, 60 to 80% of conidia within J774 cells were killed after only 4 h. Live and heat-killed conidia were internalized to the same extent by A549 cells. After 6 h, almost none of the conidia inside A549 cells had germinated, whereas extracellular conidia had developed germ tubes. Internalization of conidia by A549 cells was a temperature-dependent process and required rearrangement of the underlying host cell cytoskeleton; uptake was inhibited by 75% with 0.5 μM cytochalasin D and by 65% with 5 μM colchicine. Fluorescent labeling of infected A549 cells with rhodamine phalloidin provided visible evidence of cytoskeletal alteration as many of the intracellular conidia were contained in actin-coated phagosomes. These data provide evidence that significant numbers ofA. fumigatusconidia can be internalized by nonprofessional phagocytes in vitro and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host.
Activation of the
Pseudomonas aeruginosa
Type III Secretion System Requires an Intact Pyruvate Dehydrogenase
aceAB
Operon
Authors: Dacheux Denis , Epaulard Olivier , de Groot Arjan , Guery Benoit , Leberre Rozen , Attree Ina , Polack Benoit , Toussaint Bertrand
Published: July 2002

Pseudomonas aeruginosa
clinical cystic fibrosis isolate CHA was mutagenized with Tn
5
Tc to identify new genes involved in type III secretion system (TTSS)-dependent cytotoxicity toward human polymorphonuclear neutrophils. Among 25 mutants affected in TTSS function, 14 contained the insertion at different positions in the
aceAB
operon encoding the PDH-E1 and -E2 subunits of pyruvate dehydrogenase. In PDH mutants, no transcriptional activation of TTSS genes in response to calcium depletion occurred. Expression in
trans
of ExsA restored TTSS function and cytotoxicity.
An Essential Two-Component Signal Transduction System in
Mycobacterium tuberculosis
Authors: Zahrt Thomas C. , Deretic Vojo
Published: July 2002

The bacterial two-component signal transduction systems regulate adaptation processes and are likely to play a role in
Mycobacterium tuberculosis
physiology and pathogenesis. The previous initial characterization of an
M. tuberculosis
response regulator from one of these systems,
mtrA-mtrB
, suggested its transcriptional activation during infection of phagocytic cells. In this work, we further characterized the
mtrA
response regulator from
M. tuberculosis
H37Rv. Inactivation of
mtrA
on the chromosome of
M. tuberculosis
H37Rv was possible only in the presence of plasmid-borne functional
mtrA
, suggesting that this response regulator is essential for
M. tuberculosis
viability. In keeping with these findings, expression of
mtrA
in
M. tuberculosis
H37Rv was detectable during in vitro growth, as determined by S1 nuclease protection and primer extension analyses of mRNA levels and mapping of transcript 5′ ends. The
mtrA
gene was expressed differently in virulent
M. tuberculosis
and the vaccine strain
M. tuberculosis
var.
bovis
BCG during infection of macrophages, as determined by monitoring of
mtrA-gfp
fusion activity. In
M. bovis
BCG,
mtrA
was induced upon entry into macrophages. In
M. tuberculosis
H37Rv, its expression was constitutive and unchanged upon infection of murine or human monocyte-derived macrophages. In conclusion, these results identify
mtrA
as an essential response regulator gene in
M. tuberculosis
which is differentially expressed in virulent and avirulent strains during growth in macrophages.
Metabolic Defects Caused by Mutations in the
isc
Gene Cluster in
Salmonella enterica
Serovar Typhimurium: Implications for Thiamine Synthesis
Authors: Skovran Elizabeth , Downs Diana M.
Published: July 2002

The metabolic consequences of two insertions,
iscR1
::MudJ and
iscA2
::MudJ, in the
isc
gene cluster of
Salmonella enterica
serovar Typhimurium were studied. Each of these insertions had polar effects and caused a nutritional requirement for the thiazole moiety of thiamine. Data showed that IscS was required for the synthesis of nicotinic acid and the thiazole moiety of thiamine and that one or more additional
isc
gene products were required for a distinct step in the thiazole biosynthetic pathway. Strains with
isc
lesions had reduced succinate dehydrogenase and aconitase activities. Furthermore,
isc
mutants accumulated increased levels of pyruvate in the growth medium in response to exogenously added iron (FeCl
3
), and this response required a functional ferric uptake regulator, Fur.
Genome Structure of the GenusAzospirillum
Authors: Martin-Didonet Claudia C. G. , Chubatsu Leda S. , Souza Emanuel M. , Kleina Margareth , Rego Fabiane G. M. , Rigo Liu U. , Yates M. Geoffrey , Pedrosa Fabio O.
Published: July 2002

Azospirillumspecies are plant-associated diazotrophs of the alpha subclass ofProteobacteria. The genomes of five of the sixAzospirillumspecies were analyzed by pulsed-field gel electrophoresis. All strains possessed several megareplicons, some probably linear, and 16S ribosomal DNA hybridization indicated multiple chromosomes in genomes ranging in size from 4.8 to 9.7 Mbp. ThenifHDKoperon was identified in the largest replicon.
Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds inAgrobacterium tumefaciens: Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene
Authors: Parke Donna
Published: July 2002

A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain ofAgrobacterium tumefaciensA348. The nucleotide sequence of the A348pcaHGBgenes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and β-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into thepcaBgene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described forAcinetobactersp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker inpcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained withAcinetobactersp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting thepcaHGgenes ofA. tumefaciensfor spontaneous mutation. Sixteen strains carrying putative second-site mutations inpcaHor -Gwere subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (−1) frameshifts, one (+1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG fromPseudomonas putidaandAcinetobactersp. as well as of residues in other parts of the enzyme.
Escherichia coliTehB RequiresS-Adenosylmethionine as a Cofactor To Mediate Tellurite Resistance
Authors: Liu Mingfu , Turner Raymond J. , Winstone Tara L. , Saetre Andrea , Dyllick-Brenzinger Melanie , Jickling Glen , Tari Leslie W. , Weiner Joel H. , Taylor Diane E.
Published: July 2002

TheEscherichia colichromosomal determinant for tellurite resistance consists of two genes (tehAandtehB) which, when expressed on a multicopy plasmid, confer resistance to K2TeO3at 128 μg/ml, compared to the MIC of 2 μg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found inS-adenosyl-l-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a ∼20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.
A Set of Genes Encoding a Second Toluene Efflux System in
Pseudomonas putida
DOT-T1E Is Linked to the
tod
Genes for Toluene Metabolism
Authors: Mosqueda Gilberto , Ramos Juan-Luis
Published: July 2002

Sequence analysis in
Pseudomonas putida
DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the
ttgDEF
genes, which are adjacent to the
tod
genes. The
ttgDEF
genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in
P. putida
, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn
5
insertion in the
ttgABC
gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the
ttgD
gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single
ttgD
mutant, DOT-T1E-1, and a double mutant with knock outs in the
ttgD
and
ttgA
genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the single
ttgD
mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type and
ttgD
mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in
P. putida
DOT-T1E.
Evidence for Na+Influx via the NtpJ Protein of the KtrII K+Uptake System inEnterococcus hirae
Authors: Kawano Miyuki , Abuki Ryoko , Igarashi Kazuei , Kakinuma Yoshimi
Published: July 2002

ThentpJgene, a cistron located at the tail end of the vacuolar-type Na+-ATPase (ntp) operon ofEnterococcus hirae, encodes a transporter of the KtrII K+uptake system. We found that K+accumulation in thentpJ-disrupted mutant JEM2 was markedly enhanced by addition of valinomycin at pH 10. Studies of the membrane potential (ΔΨ; inside negative) by 3,3′-dihexyloxacarbocyanine iodide fluorescence revealed that the ΔΨ was hyperpolarized at pH 10 in JEM2; the ΔΨ values of the parent strain ATCC 9790 and JEM2, estimated by determining the equilibrium distribution of K+or Rb+in the presence of valinomycin, were −118 and −160 mV, respectively. ΔΨ generation at pH 10 was accomplished by an electrogenic Na+efflux via the Na+-ATPase, whose levels in the two strains were quite similar. Na+uptake driven by an artificially imposed ΔΨ (inside negative) was missing in JEM2, suggesting that NtpJ mediates Na+movement in addition to K+movement. Finally, the growth of JEM2 arrested in K+-limited high-Na+medium at pH 10 was restored by addition of valinomycin. These results suggest that NtpJ mediates electrogenic transport of K+as well as Na+, that it likely mediates K+and Na+cotransport, and that Na+movement via NtpJ is the major Na+reentry pathway at high pH values.
Improvement in K+-Limited Growth Rate Associated with Expression of the N-Terminal Fragment of One Subunit (KdpA) of the Multisubunit Kdp Transporter inEscherichia coli
Authors: Sardesai Abhijit A. , Gowrishankar J.
Published: July 2002

Mutations in any one of three genes,kdpA, -B,or -C,inEscherichia coliabolish the activity of Kdp, a multisubunit K+-ATPase that belongs to the P-type ATPase family of cation transporters. We found in this study that expression in vivo of a 135-amino-acid-long N-terminal fragment (KdpA′), less than one-quarter the length of native KdpA, was able to mediate an improvement in K+-limited growth rates in two different contexts, even in the absence of both KdpC and the ATPase subunit KdpB. The first context was when KdpA′ was overexpressed in cells from a heterologous inducible promoter, and the second was when KdpA′ was provided with a C-terminally altered extension (following a spontaneous genetic rearrangement). Our results suggest that KdpA′ provides an incipient pathway for K+translocation which can serve to transport K+into the cells in response to the cytoplasmic membrane potential.
Characterization of theUstilago maydis sid2Gene, Encoding a Multidomain Peptide Synthetase in the Ferrichrome Biosynthetic Gene Cluster
Authors: Yuan Walter M. , Gentil Guillaume D. , Budde Allen D. , Leong Sally A.
Published: July 2002

Ustilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation ofl-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase.sid1encodesl-ornithineN5-oxygenase, which catalyzes hydroxylation ofl-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis inU. maydis. In this report we characterizesid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed thatsid2is located 3.7 kb upstream ofsid1and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulatedsid2transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption ofurbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis inUstilago. Multiple GATA motifs are present in the intergenic region betweensid1andsid2, suggesting bidirectional transcription regulation byurbs1of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation ofsid1, altered the differential regulation ofsid2by iron.
Juxtaposition of an Active Promoter to
vsp
Genes via Site-Specific DNA Inversions Generates Antigenic Variation in
Mycoplasma bovis
Authors: Lysnyansky Innesa , Ron Yael , Yogev David
Published: July 2002

Mycoplasma bovis
, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent
vsp
genes, which occur as single chromosomal copies, was recently found in the chromosome of
M. bovis
. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON→OFF→ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each
vsp
gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all
vsp
genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of
vsp
upstream cassettes involving three
vsp
genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A
2
, is an active promoter and that juxtaposition of this regulatory element to a silent
vsp
gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.
Pseudomonas aeruginosa
Exhibits Directed Twitching Motility Up Phosphatidylethanolamine Gradients
Authors: Kearns Daniel B. , Robinson Jayne , Shimkets Lawrence J.
Published: July 2002

Pseudomonas aeruginosa
translocates over solid surfaces by a type IV pilus-dependent form of multicellular motility known as twitching. We wondered whether cells utilize endogenous factors to organize twitching, and we purified from wild-type cells a lipid that caused directed movement. Wild-type
P. aeruginosa
, but not a
pilJ
pilus-deficient mutant, showed biased movement up gradients of phosphatidylethanolamine (PE) established in agar. Activity was related to the fatty acid composition of the lipid, as two synthetic PE species, dilauroyl and dioleoyl PE, were capable of directing
P. aeruginosa
motility while many other species were inactive.
P. aeruginosa
PE did not contain either laurate or oleate, implying that the native attractant species contains different fatty acids. Uniform concentrations of PE increased cell velocity, suggesting that chemokinesis may be at least partly responsible for directed movement. We speculate that PE-directed twitching motility may be involved in biofilm formation and pathogenesis.
Functional Elements of the Strong
psbAI
Promoter of
Synechococcus elongatus
PCC 7942
Authors: Nair Usha , Thomas Colleen , Golden Susan S.
Published: July 2002

The
psbAI
gene of the cyanobacterium
Synechococcus elongatus
PCC 7942 is one of three
psbA
genes that encode a critical photosystem II reaction center protein, D1. Regulation of the gene family in response to changes in the light environment is complex, occurs at transcriptional and posttranscriptional levels, and results in an interchange of two different forms of D1 in the membrane. Expression of
psbAI
is downregulated under high-intensity light (high light) in contrast to induction of the other two family members. We show that, in addition to a known accelerated degradation of the
psbAI
message, promoter activity decreases upon exposure to high light. Unlike the other
psbA
genes, additional sequences upstream of the
psbAI
−35 element are required for expression. Mutagenizing the atypical
psbAI
−10 element from TCTCCT to TATAAT increased the magnitude of expression from both
psbAI
::
lacZ
and
psbAI
::
luxAB
fusions but did not affect downregulation under high light. Inactivation of group 2 sigma factor genes
rpoD2
and
sigC
, in both wild-type and −10-element mutagenized backgrounds, resulted in elevated
psbAI
::
luxAB
expression but did not alter the response to high light. The results are consistent with redundancy of promoter recognition among cyanobacterial group 2 sigma factors. Electrophoretic mobility shift assays showed that the DNA sequence corresponding to the untranslated leader of the
psbAI
message binds one or more proteins from an
S. elongatus
extract. The corresponding region of
psbAII
efficiently competed for this binding activity, suggesting a shared regulatory factor among these disparately regulated genes.
Involvement of a CbbR Homolog in Low CO
2
-Induced Activation of the Bicarbonate Transporter Operon in Cyanobacteria
Authors: Omata Tatsuo , Gohta Satoshi , Takahashi Yukari , Harano Yoshimi , Maeda Shin-ichi
Published: July 2002

The
cmpABCD
operon of
Synechococcus
sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is transcribed only under CO
2
-limited conditions. In
Synechocystis
sp. strain PCC 6803, the
slr0040, slr0041, slr0043
, and
slr0044
genes, forming an operon with a putative porin gene (
slr0042
), were identified as the
cmpA, cmpB, cmpC
, and
cmpD
genes, respectively, on the basis of their strong similarities to the corresponding
Synechococcus cmp
genes and their induction under low CO
2
conditions. Immediately upstream of and transcribed divergently from the
Synechocystis cmp
operon is a gene (
sll0030
) encoding a homolog of CbbR, a LysR family transcriptional regulator of the CO
2
fixation operons of chemoautotrophic and purple photosynthetic bacteria. Inactivation of
sll0030
, but not of another closely related
cbbR
homolog (
sll1594
), abolished low CO
2
induction of
cmp
operon expression. Gel retardation assays showed specific binding of the Sll0030 protein to the
sll0030-cmpA
intergenic region, suggesting that the protein activates transcription of the
cmp
operon by interacting with its regulatory region. A
cbbR
homolog similar to
sll0030
and
sll1594
was cloned from
Synechococcus
sp. strain PCC 7942 and shown to be involved in the low CO
2
-induced activation of the
cmp
operon. We hence designated the
Synechocystis sll0030
gene and the
Synechococcus cbbR
homolog
cmpR
. In the mutants of the
cbbR
homologs, upregulation of ribulose-1,5-bisphosphate carboxylase/oxygenase operon expression by CO
2
limitation was either unaffected (strain PCC 6803) or enhanced (strain PCC 7942), suggesting existence of other low CO
2
-responsive transcriptional regulator(s) in cyanobacteria.