bluuebunny/crossref_metadata_2025 huggingface dataset:
Description of a 23.9-Kilobase Chromosomal Deletion Containing a Region Encoding
fsr
Genes Which Mainly Determines the Gelatinase-Negative Phenotype of Clinical Isolates of
Enterococcus faecalis
in Urine
Authors: Nakayama Jiro , Kariyama Reiko , Kumon Hiromi
Published: July 2002

Expression of virulence-related extracellular proteases, gelatinase, and serine protease of
Enterococcus faecalis
is regulated by a quorum-sensing system encoded by the
fsr
gene cluster. In this study, a 23.9-kb chromosomal deletion containing the
fsr
gene cluster region was found to be present in the majority (79%) of gelatinase-negative clinical isolates of
E. faecalis
from urine.
Bacterial Adhesion: Seen Any Good Biofilms Lately?
Authors: Dunne W. Michael
Published: July 2002

The process of surface adhesion and biofilm development is a survival strategy employed by virtually all bacteria and refined over millions of years. This process is designed to anchor microorganisms in a nutritionally advantageous environment and to permit their escape to greener pastures when essential growth factors have been exhausted. Bacterial attachment to a surface can be divided into several distinct phases, including primary and reversible adhesion, secondary and irreversible adhesion, and biofilm formation. Each of these phases is ultimately controlled by the expression of one or more gene products. Ultrastructurally, the mature bacterial biofilm resembles an underwater coral reef containing pyramidal or mushroom-shaped microcolonies of organisms embedded within an extracellular glycocalyx, with channels and cavities to allow the exchange of nutrients and waste. The biofilm protects its inhabitants from predators, dehydration, biocides, and other environmental extremes while regulating population growth and diversity through primitive cell signals. From a physiological standpoint, surface-bound bacteria behave quite differently from their planktonic counterparts. Recognizing that bacteria naturally occur as surface-bound and often polymicrobic communities, the practice of performing antimicrobial susceptibility tests using pure cultures and in a planktonic growth mode should be questioned. That this model does not reflect conditions found in nature might help explain the difficulties encountered in the management and treatment of biomedical implant infections.
Effect of α-Oligosaccharide Phenotype of
Neisseria gonorrhoeae
Strain MS11 on Invasion of Chang Conjunctival, HEC-1-B Endometrial, and ME-180 Cervical Cells
Authors: Minor Susie Y. , Banerjee Asesh , Gotschlich Emil C.
Published: July 2002

The genes encoding the glycosyltransferases responsible for the addition of the five sugars in the α-oligosaccharide (α-OS) moiety of lipooligosaccharide (LOS) have been identified. Disruption of these glycosyltransferase genes singly or in combination results in corresponding truncations in LOS. In the present work we show that sequential deletion of the terminal four sugar residues of gonococcal α-OS had no discernible effect on the invasion of human conjunctival, endometrial, and cervical cell lines. However, deletion of the proximal glucose, which resulted in the complete deletion of α-OS, significantly impaired invasion of the gonococci into all three cell lines. The effect of deleting α-OS on invasion was independent of and additive to the known invasion-promoting factor OpaA. These data suggest that the proximal glucose residue of the α-OS chain of LOS is required for efficient invasion of gonococci into host mucosa.
Phylogeny of
Vibrio cholerae
Based on
recA
Sequence
Authors: Stine O. Colin , Sozhamannan Shanmuga , Gou Qing , Zheng Siqen , Morris J. Glenn , Johnson Judith A.
Published: July 2002

We sequenced a 705-bp fragment of the
recA
gene from 113
Vibrio cholerae
strains and closely related species. One hundred eighty-seven nucleotides were phylogenetically informative, 55 were phylogenetically uninformative, and 463 were invariant. Not unexpectedly,
Vibrio parahaemolyticus
and
Vibrio vulnificus
strains formed out-groups; we also identified isolates which resembled
V. cholerae
biochemically but which did not cluster with
V. cholerae
. In many instances,
V. cholerae
serogroup designations did not correlate with phylogeny, as reflected by
recA
sequence divergence. This observation is consistent with the idea that there is horizontal transfer of O-antigen biosynthesis genes among
V. cholerae
strains.
Impact of Siderophore Production on
Pseudomonas aeruginosa
Infections in Immunosuppressed Mice
Authors: Takase Hiroyuki , Nitanai Hironobu , Hoshino Kazuki , Otani Tsuyoshi
Published: July 2002

Pseudomonas aeruginosa
produces siderophores, pyoverdin and pyochelin, for high-affinity iron uptake. To investigate their contribution to
P. aeruginosa
infections, we constructed allelic exchange mutants from strain PAO1 which were deficient in producing one or both of the siderophores. When inoculated into the calf muscles of immunosuppressed mice, pyochelin-deficient and pyoverdin-deficient mutants grew and killed the animals as efficiently as PAO1. In contrast, the pyochelin- and pyoverdin-deficient (double) mutant did not show lethal virulence, although it did infect the muscles. On the other hand, when inoculated intranasally, all mutants grew in the lungs and killed immunosuppressed mice. Compared with PAO1, however, the pyoverdin-deficient mutant and the double mutant grew poorly in the lungs, and the latter was significantly attenuated for virulence. Irrespective of the inoculation route, the pyoverdin-deficient and doubly deficient mutants detected in the blood were significantly less numerous than PAO1. Additionally, in vitro examination demonstrated that the growth of the double mutant was extremely reduced under a free-iron-restricted condition with apotransferrin but that the growth reduction was completely canceled by supplementation with hemoglobin as a heme source. These results suggest that both pyoverdin and pyochelin are required for efficient bacterial growth and full expression of virulence in
P. aeruginosa
infection, although pyoverdin may be comparatively more important for bacterial growth and dissemination. However, the siderophores were not always required for infection. It is possible that non-siderophore-mediated iron acquisition, such as via heme uptake, might also play an important role in
P. aeruginosa
infections.
The
galE
Gene of
Campylobacter jejuni
Is Involved in Lipopolysaccharide Synthesis and Virulence
Authors: Fry Benjamin N. , Feng Shi , Chen Yuen-Yuen , Newell Diane G. , Coloe Peter J. , Korolik Victoria
Published: July 2002

Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS from
Campylobacter
spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (
wla
) which is involved in the synthesis of the
Campylobacter jejuni
81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with
galE
genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A
Salmonella galE
mutant was transformed with the
galE
gene from
C. jejuni
. The LPS analysis of wild-type,
galE
, and complemented
galE Salmonella
strains showed that the
C. jejuni galE
gene could restore the smooth wild-type
Salmonella
LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the
galE
gene from
C. jejuni
encoded this epimerase. We constructed a
C. jejuni galE
mutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the
galE
gene in the synthesis of
C. jejuni
LPS. The
galE
mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of
C. jejuni
is an important virulence factor.
Immune Response to an 18-Kilodalton Outer Membrane Antigen Identifies Lipoprotein 20 as a
Helicobacter pylori
Vaccine Candidate
Authors: Keenan Jacqueline , Oliaro Jane , Domigan Neil , Potter Howard , Aitken Geoff , Allardyce Randall , Roake Justin
Published: July 2002

Experiments were performed using the standardized murine model of
Helicobacter pylori
infection to determine the immunogenicity of
H. pylori
outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in
H. pylori
-infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.
Cooperation between Reactive Oxygen and Nitrogen Intermediates in Killing ofRhodococcus equiby Activated Macrophages
Authors: Darrah Patricia A. , Hondalus Mary K. , Chen Quiping , Ischiropoulos Harry , Mosser David M.
Published: July 2002

Rhodococcus equiis a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense againstR. equiby using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-γ), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO−), which killsR. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS−/−) or the oxidative burst (gp91phox−/−) are more susceptible to lethalR. equiinfection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O2−) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays,R. equiremains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO−efficiently kills virulentR. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO−in situ. Inhibition of nitric oxide production by activated macrophages by usingNG-monomethyl-l-arginine blocks their production of ONOO−and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing ofR. equiby IFN-γ-activated macrophages.
Shigella flexneriIpaH7.8Facilitates Escape of Virulent Bacteria from the Endocytic Vacuoles of Mouse and Human Macrophages
Authors: Fernandez-Prada Carmen M. , Hoover David L. , Tall Ben D. , Hartman Antoinette B. , Kopelowitz June , Venkatesan Malabi M.
Published: July 2002

The behavior ofShigella flexneri ipaHmutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, anipaH7.8deletion mutant ofS. flexneri2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting thatipaH7.8deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressingipaH7.8restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions inipaH4.5oripaH9.8, however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulentShigella. These results suggest that a role of theipaH7.8gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.
Characterization of a Streptococcal Endopeptidase with Homology to Human Endothelin-Converting Enzyme
Authors: Oetjen Joyce , Fives-Taylor Paula , Froeliger Eunice
Published: July 2002

A gene encoding an endopeptidase from
Streptococcus parasanguis
FW213 has been cloned and shown to have high sequence homology to genes encoding mammalian metalloendopeptidases. The gene, designated
S. parasanguis pepO
, was cloned into the pET28a expression vector, resulting in a fusion of vector sequences encoding a hexahistidine tag at the carboxyl terminus. The recombinant PepO (rPepO) was expressed in
Escherichia coli
and purified using an Ni
2+
affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and immunoblot analysis revealed that other oral streptococci contain regions of DNA with homology to
pepO
and produce a protein with antigenic properties similar to that of FW213 PepO. Enzymatic activity assays indicated that only
S. parasanguis
species possess the ability to cleave metenkephalin, the natural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that
S. parasanguis
PepO is a member of the M13 category of metalloendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE-1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that
S. parasanguis
PepO is more similar to ECE-1 than to NEP.
Gonococcal Genes Encoding Transferrin-Binding Proteins A and B Are Arranged in a Bicistronic Operon but Are Subject to Differential Expression
Authors: Ronpirin Chalinee , Jerse Ann E. , Cornelissen Cynthia Nau
Published: July 2002

Neisseria gonorrhoeaeis capable of utilizing host iron-binding proteins, such as transferrin, lactoferrin, and hemoglobin, as the sole source of iron. The receptor involved in transferrin iron acquisition is composed of two distinct transferrin-binding proteins, TbpA and TbpB. The genes that encode these proteins are linked on the chromosome in the ordertbpB-tbpAbut are separated by an inverted repeat of unknown function. In this study, we sought to understand the transcriptional organization and regulation of thetbpgenes, using a combination oflacZtranscriptional fusion analysis and reverse transcriptase PCR (RT-PCR). First, we demonstrated thattbpBandtbpAare cotranscribed and coregulated from the common upstream promoter that precedestbpB. Using β-galactosidase activity as a surrogate fortbp-specific transcription, we found thattbpB-specific transcripts were more prevalent thantbpA-specific transcripts after 2 h of growth under iron stress conditions. We confirmed the results obtained by fusion analysis by using RT-PCR applied to native RNA isolated from wild-type gonococci. Three different varieties of RT-PCR were employed: relative, competitive, and real time quantitative. The results of all analyses indicated thattbpB-specific transcripts were approximately twofold more prevalent thantbpA-specific transcripts at steady state. In iron-stressed cultures, the ratio oftbpB- totbpA-specific message was approximately 2; however, in iron-replete cultures, this ratio dropped to 1. Using these techniques, we also quantitated the effects of iron, external pH, and presence of ligand ontbpmRNA levels.
Mouse Strain-Dependent Chemokine Regulation of the Genital Tract T Helper Cell Type 1 Immune Response
Authors: Darville Toni , Andrews Charles W. , Sikes James D. , Fraley Patrick L. , Braswell Leah , Rank Roger G.
Published: July 2002

Vaginal infection with the mouse pneumonitis agent ofChlamydia trachomatis(MoPn) produces shorter courses of infection in C57BL/6 and BALB/c mice than in C3H/HeN mice, while C57BL/6 mice are more resistant to oviduct pathology. A robust Th1 response is extremely important in host defense against chlamydia. In this study we examined gamma interferon (IFN-γ), interleukin 10 (IL-10), and the T-cell-regulatory chemokines macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1) to determine if differences in these responses were associated with the differential courses of infection seen in these three strains of mice. Increased and prolonged IFN-γ responses and lower IL-10 responses were observed in the C57BL/6 strain compared to BALB/c and C3H. Examination of genital tract chemokines revealed a marked predominance of MIP-1α over MCP-1 only in the C57 strain. Thus, a pattern of high MIP-1α and low MCP-1 levels during the first week of infection is associated with an increased Th1 response and a shorter, more benign chlamydial infection. Inhibition of the MCP-1 response in C3H mice increased their later T-cell production of IFN-γ but decreased their early IFN-γ response and had no effect on the course or outcome of infection. Inhibition of MCP-1 is not beneficial in chlamydial infection because of its pleiotropic effects.
Molecular Characterization of a NovelStaphylococcus aureusSerine Protease Operon
Authors: Reed Samantha B. , Wesson Carla A. , Liou Linda E. , Trumble William R. , Schlievert Patrick M. , Bohach Gregory A. , Bayles Kenneth W.
Published: July 2002

The present study identified and characterized a unique operon (spl) encoding six serine protease-like proteins. In addition, native Spl proteins were isolated and characterized. Typical of most exoproteins, thesplgene products contain putative 35- or 36-amino-acid signal peptides. The Spl proteins share 44 to 95% amino acid sequence identity with each other and 33 to 36% sequence identity with V8 protease. They also contain amino acids found in catalytic triads of enzymes in the trypsin-like serine protease family, and SplB and SplC were shown to degrade casein. Thesploperon is transcribed on a 5.5-kb transcript, but several nonrandom degradation products of this transcript were also identified. Similar to otherS. aureusexoprotein genes, thesploperon is maximally expressed during the transition into stationary phase and is positively controlled by the Agr virulence factor regulator. The Sar regulatory system did not affectsploperon expression. PCR analysis revealed the presence of thesploperon in 64% of theS. aureusisolates tested, although onesploperon-negative isolate was shown to contain at least two of thesplgenes. Finally, intraperitoneal injection of ansploperon deletion mutant revealed no major differences in virulence compared to the parental strain.
Role of Alginate O Acetylation in Resistance of MucoidPseudomonas aeruginosato Opsonic Phagocytosis
Authors: Pier Gerald B. , Coleman Fadie , Grout Martha , Franklin Michael , Ohman Dennis E.
Published: July 2002

Establishment and maintenance of chronic lung infections with mucoidPseudomonas aeruginosain patients with cystic fibrosis (CF) require that the bacteria avoid host defenses. Elaboration of the extracellular, O-acetylated mucoid exopolysaccharide, or alginate, is a major microbial factor in resistance to immune effectors. Here we show that O acetylation of alginate maximizes the resistance of mucoidP. aeruginosato antibody-independent opsonic killing and is the molecular basis for the resistance of mucoidP. aeruginosato normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients. O acetylation of alginate appears to be critical forP. aeruginosaresistance to host immune effectors in CF patients.
Evidence that the
Campylobacter fetus sap
Locus Is an Ancient Genomic Constituent with Origins before Mammals and Reptiles Diverged
Authors: Tu Zheng-Chao , Dewhirst Floyd E. , Blaser Martin J.
Published: July 2002

Campylobacter fetus
bacteria, isolated from both mammals and reptiles, may be either subsp.
fetus
or subsp.
venerealis
and either serotype A or serotype B. Surface layer proteins, expressed and secreted by genes in the
sap
locus, play an important role in
C. fetus
virulence. To assess whether the
sap
locus represents a pathogenicity island and to gain further insights into
C. fetus
evolution, we examined several
C. fetus
genes in 18 isolates. All of the isolates had 5 to 9
sapA
or
sapB
homologs. One strain (85-387) possessed both
sapA
and
sapB
homologs, suggesting a recombinational event in the
sap
locus between
sapA
and
sapB
strains. When we amplified and analyzed nucleotide sequences from portions of housekeeping gene
recA
(501 bp) and
sapD
(450 bp), a part of the 6-kb
sap
invertible element, the phylogenies of the genes were highly parallel. Among the 15 isolates from mammals, serotype A and serotype B strains generally had consistent positions. The fact that the serotype A
C. fetus
subsp.
fetus
and subsp.
venerealis
strains were on the same branch suggests that their differentiation occurred after the type A-type B split. Isolates from mammals and reptiles formed two distinct tight phylogenetic clusters that were well separated. Sequence analysis of 16S rRNA showed that the reptile strains form a distinct phylotype between mammalian
C. fetus
and
Campylobacter hyointestinalis
. The phylogenies and sequence results showing that
sapD
and
recA
have similar G + C contents and substitution rates suggest that the
sap
locus is not a pathogenicity island but rather is an ancient constituent of the
C. fetus
genome, integral to its biology.
Cloning, Expression, and Catalytic Activity of
Helicobacter hepaticus
Urease
Authors: Beckwith Catherine S. , McGee David J. , Mobley Harry L. T. , Riley Lela K.
Published: July 2002

Helicobacter hepaticus
causes disease in the liver and lower intestinal tract of mice. It is strongly urease positive, although it does not live in an acidic environment. The
H. hepaticus
urease gene cluster was expressed in
Escherichia coli
with and without coexpression of the
Helicobacter pylori
nickel transporter NixA. As for
H. pylori
, it was difficult to obtain enzymatic activity from recombinant
H. hepaticus
urease; special conditions including NiCl
2
supplementation were required. The
H. hepaticus
urease cluster contains a homolog of each gene in the
H. pylori
urease cluster, including the urea transporter gene
ureI
. Downstream genes were homologs of the
nik
nickel transport operon of
E. coli
. Nongastric
H. hepaticus
produces urease similar to that of
H. pylori
.
Enhanced Delivery of Exogenous Peptides into the Class I Antigen Processing and Presentation Pathway
Authors: de Haan Lolke , Hearn Arron R. , Rivett A. Jennifer , Hirst Timothy R.
Published: July 2002

Current immunization strategies, using peptide or protein antigens, generally fail to elicit cytotoxic-T-lymphocyte responses, since these antigens are unable to access intracellular compartments where loading of major histocompatibility complex class I (MHC-I) molecules occurs. In an attempt to circumvent this, we investigated whether the GM1 receptor-binding B subunit ofEscherichia coliheat-labile toxin (EtxB) could be used to deliver class I epitopes. When a class I epitope was conjugated to EtxB, it was delivered into the MHC-I presentation pathway in a GM1-binding-dependent fashion and resulted in the appearance of MHC-I-epitope complexes at the cell surface. Importantly, we show that the efficiency of EtxB-mediated epitope delivery could be strikingly enhanced by incorporating, adjacent to the class I epitope, a 10-amino-acid segment from the C terminus of the DNA polymerase (Pol) of herpes simplex virus. The replacement of this 10-amino-acid segment by a heterologous sequence or the introduction of specific amino acid substitutions within this segment either abolished or markedly reduced the efficiency of class I epitope delivery. If the epitope was extended at its C terminus, EtxB-mediated delivery into the class I presentation pathway was found to be completely dependent on proteasome activity. Thus, by combining the GM1-targeting function of EtxB with the 10-amino-acid Pol segment, highly efficient delivery of exogenous epitopes into the endogenous pathway of class I antigen processing and presentation can be achieved.
Tol1, a Fission Yeast Phosphomonoesterase, Is an In Vivo Target of Lithium, and Its Deletion Leads to Sulfite Auxotrophy
Authors: Miyamoto Rumi , Sugiura Reiko , Kamitani Shinya , Yada Tomoko , Lu Yabin , Sio Susie O. , Asakura Masahiro , Matsuhisa Akio , Shuntoh Hisato , Kuno Takayoshi
Published: July 2002

Lithium is the drug of choice for the treatment of bipolar affective disorder. The identification of an in vivo target of lithium in fission yeast as a model organism may help in the understanding of lithium therapy. For this purpose, we have isolated genes whose overexpression improved cell growth under high LiCl concentrations. Overexpression of
tol1
+
, one of the isolated genes, increased the tolerance of wild-type yeast cells for LiCl but not for NaCl.
tol1
+
encodes a member of the lithium-sensitive phosphomonoesterase protein family, and it exerts dual enzymatic activities, 3′(2′),5′-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase.
tol1
+
gene-disrupted cells required high concentrations of sulfite in the medium for growth. Consistently, sulfite repressed the sulfate assimilation pathway in fission yeast. However,
tol1
+
gene-disrupted cells could not fully recover from their growth defect and abnormal morphology even when the medium was supplemented with sulfite, suggesting the possible implication of inositol polyphosphate 1-phosphatase activity for cell growth and morphology. Given the remarkable functional conservation of the lithium-sensitive dual-specificity phosphomonoesterase between fission yeast and higher-eukaryotic cells during evolution, it may represent a likely in vivo target of lithium action across many species.
Cloning of the Gene Coding for
Staphylococcus hyicus
Exfoliative Toxin B and Its Expression in
Escherichia coli
Authors: Watanabe Takao , Sato Hisaaki , Hatakeyama Yu , Matsuzawa Takeshi , Kawai Masanori , Aizawa Chikara , Danbara Hirofumi , Maehara Nobutoshi
Published: July 2002

The
Staphylococcus hyicus
exfoliative toxin B (SHETB) gene was cloned into pUC118 and expressed in
Escherichia coli
. The nucleotide sequence of the SHETB gene consists of a coding region of 804 bp specifying a polypeptide of 268 amino acid residues, which included a putative 20-residue signal sequence.
A New Thermoactive Pullulanase from
Desulfurococcus mucosus
: Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in
Bacillus subtilis
Authors: Duffner Fiona , Bertoldo Costanzo , Andersen Jens T. , Wagner Karen , Antranikian Garabed
Published: July 2002

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon
Desulfurococcus mucosus
(
apuA
) was cloned in
Escherichia coli
and sequenced.
apuA
from
D. mucosus
showed 45.4% pairwise amino acid identity with the pullulanase from
Thermococcus aggregans
and contained the four regions conserved among all amylolytic enzymes.
apuA
encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The
apuA
gene was then expressed in
Bacillus subtilis
and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a
Bacillus
strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed α-1,6 glycosidic linkages of pullulan, producing maltotriose, and also α-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from
Archaea
are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from
D. mucosus
should be considered an archaeal pullulanase type II with a wider substrate specificity.
ADP-Ribosylation of Variants of
Azotobacter vinelandii
Dinitrogenase Reductase by
Rhodospirillum rubrum
Dinitrogenase Reductase ADP-Ribosyltransferase
Authors: Grunwald Sandra K. , Ryle Matthew J. , Lanzilotta William N. , Ludden Paul W.
Published: July 2002

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from
Azotobacter vinelandii
is known. In this study, mutant forms of dinitrogenase reductase from
A. vinelandii
that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from
Rhodospirillum rubrum
. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe
4
S
4
cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073–2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.
Characterization of a Putative Pathogenicity Island from Bovine
Staphylococcus aureus
Encoding Multiple Superantigens
Authors: Fitzgerald J. Ross , Monday Steven R. , Foster Timothy J. , Bohach Gregory A. , Hartigan Patrick J. , Meaney William J. , Smyth Cyril J.
Published: July 2002

Previous studies have demonstrated that a proportion of
Staphylococcus aureus
isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (
tst
), the SEC bovine variant (
sec
-bovine), and a gene (
sel
) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3′ end of the GMP synthase gene (
gmps
) in the
S. aureus
chromosome. SaPIbov contains a central region of sequence identity with the previously characterized
tst
pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527–543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of
S. aureus
strain RF122 were constructed by allele replacement. In vitro bovine Vβ-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of
tst
and
sec
-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.
Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene fromRalstonia eutrophaH16 and Characterization of the Gene Product
Authors: Saegusa Haruhisa , Shiraki Mari , Kanai Chie , Saito Terumi
Published: July 2002

An intracellular poly[d(−)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned fromRalstonia eutrophaH16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract ofEscherichia colicontaining the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomericd(−)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed inR. eutrophacells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant ofR. eutrophawhosephaZgene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the clonedphaZgene encodes an intracellular PHB depolymerase inR. eutropha.
Novel Type of Glucose-6-Phosphate Isomerase in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Hansen Thomas , Oehlmann Margitta , Schönheit Peter
Published: July 2002

Glucose-6-phosphate isomerase (phosphoglucose isomerase [PGI]) (EC
5.3.1.9
) from the hyperthermophilic archaeon
Pyrococcus furiosus
was purified 500-fold to homogeneity. The enzyme had an apparent molecular mass of 43 kDa and was composed of a single type of subunit of 23 kDa indicating a homodimeric (α
2
) structure. Kinetic constants of the enzyme were determined at the optimal pH 7 and at 80°C. Rate dependence on both substrates followed Michaelis-Menten kinetics. The apparent

K
m

values for glucose-6-phosphate and fructose-6-phosphate were 8.7 and 1.0 mM, respectively, and the corresponding apparent
V
max
values were 800 and 130 U/mg. The enzyme had a temperature optimum of 96°C and showed a significant thermostability up to 100°C, which is in accordance with its physiological function under hyperthermophilic conditions. Based on the N-terminal amino acid sequence of the subunit, a single open reading frame (ORF; Pf_209264) was identified in the genome of
P. furiosus
. The ORF was characterized by functional overexpression in
Escherichia coli
as a gene,
pgi
, encoding glucose-6-phosphate isomerase. The recombinant PGI was purified and showed molecular and kinetic properties almost identical to those of the native PGI purified from
P. furiosus
. The deduced amino acid sequence of
P. furiosus
PGI did not reveal significant similarity to the conserved PGI superfamily of eubacteria and eucarya. This is the first description of an archaeal PGI, which represents a novel type of PGI.
Functional Analysis ofrelAandrshA, TworelA/spoTHomologues ofStreptomyces coelicolorA3(2)
Authors: Sun Jongho , Hesketh Andrew , Bibb Mervyn
Published: July 2002

Deletion of the (p)ppGpp synthetase gene,relA, ofStreptomyces coelicolorA3(2) results in loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) and delayed morphological differentiation when the mutant is grown under conditions of nitrogen limitation. To analyze the role of (p)ppGpp as an intracellular signaling molecule for the initiation of antibiotic production, several C-terminally deleted derivatives ofS. coelicolor relAthat could potentially function in the absence of ribosome activation were placed under the control of the thiostrepton-inducibletipApromoter. While 0.82- and 1.28-kb N-terminal segments failed to restore (p)ppGpp and antibiotic production upon induction in arelAnull mutant, 1.46- and 2.07-kb segments did. Under conditions of phosphate limitation, deletion ofrelAhad little or no effect on Act or Red synthesis, potentially reflecting an alternative mechanism for ppGpp synthesis. A secondS. coelicolorRelA homologue (RshA, with 42% identity toS. coelicolorRelA) was identified in the genome sequence. However, deletion ofrshAhad no effect on the ability of therelAmutant to make Act and Red when grown under conditions of phosphate limitation. While high-level induction oftipAp::rshAin therelAmutant resulted in growth inhibition, low-level induction restored antibiotic production and sporulation. In neither case, nor in therelAmutant that was grown under phosphate limitation and producing Act and Red, could (p)ppGpp synthesis be detected. Thus, a ppGpp-independent mechanism exists to activate antibiotic production under conditions of phosphate limitation that can be mimicked by overexpression ofrshA.
Activity of the
Kluyveromyces lactis
Pdr5 Multidrug Transporter Is Modulated by the Sit4 Protein Phosphatase
Authors: Chen Xin Jie
Published: July 2002

A possible role for posttranslational modifications in regulating the activity of ATP-binding cassette (ABC) transporters has not been well established. In this study, the drug efflux ABC transporter gene
KlPDR5
was isolated from the budding yeast
Kluyveromyces lactis
, and it was found that the encoded KlPdr5 drug pump is posttranslationally regulated by the type 2A-related Ser/Thr protein phosphatase, Sit4p. The KlPdr5 transporter is a protein of 1,525 amino acids sharing 63.8% sequence identity with its
Saccharomyces cerevisiae
counterpart, ScPdr5p. Overexpression of the
KlPDR5
gene confers resistance to oligomycin, antimycin, econazole, and ketoconazole, whereas cells with a disrupted allele of
KlPDR5
are hypersensitive to the drugs and have a decreased capacity to carry out efflux of the anionic fluorescent dye rhodamine 123. It was found that a chromosomal disruption of
KlPDR5
abolishes the drug-resistant phenotype associated with
sit4
mutations and that a synergistic hyperresistance to the drugs can be created by overexpressing
KlPDR5
in
sit4
mutants. These data strongly indicate that the multidrug-resistant phenotype of
sit4
mutants is mediated by negatively modulating the activity of KlPdr5p. As the transcriptional level of
KlPDR5
and the steady-state level of KlPdr5p are not significantly affected by mutations in
SIT4
, the regulation by Sit4p appears to be a posttranslational process.
Three Efflux Pumps Are Required To Provide Efficient Tolerance to Toluene in
Pseudomonas putida
DOT-T1E
Authors: Rojas Antonia , Duque Estrella , Mosqueda Gilberto , Golden Geir , Hurtado Ana , Ramos Juan L. , Segura Ana
Published: July 2002

In
Pseudomonas putida
DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The
ttgGHI
genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single
ttgH
mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene,
m
-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.
OhrR Is a Repressor of
ohrA
, a Key Organic Hydroperoxide Resistance Determinant in
Bacillus subtilis
Authors: Fuangthong Mayuree , Atichartpongkul Sopapan , Mongkolsuk Skorn , Helmann John D.
Published: July 2002

Bacillus subtilis
displays a complex adaptive response to the presence of reactive oxygen species. To date, most proteins that protect against reactive oxygen species are members of the peroxide-inducible PerR and ς
B
regulons. We investigated the function of two
B. subtilis
homologs of the
Xanthomonas campestris
organic hydroperoxide resistance (
ohr
) gene. Mutational analyses indicate that both
ohrA
and
ohrB
contribute to organic peroxide resistance in
B. subtilis
, with the OhrA protein playing the more important role in growing cells. Expression of
ohrA
, but not
ohrB
, is strongly and specifically induced by organic peroxides. Regulation of
ohrA
requires the convergently transcribed gene,
ohrR
, which encodes a member of the MarR family of transcriptional repressors. In an
ohrR
mutant,
ohrA
expression is constitutive, whereas expression of the neighboring
ohrB
gene is unaffected. Selection for mutant strains that are derepressed for
ohrA
transcription identifies a perfect inverted repeat sequence that is required for OhrR-mediated regulation and likely defines an OhrR binding site. Thus,
B. subtilis
contains at least three regulons (ς
B
, PerR, and OhrR) that contribute to peroxide stress responses.
Reduced Function of a Phenylacetate-Oxidizing Cytochrome P450 Caused Strong Genetic Improvement in Early Phylogeny of Penicillin-Producing Strains
Authors: Rodrı́guez-Sáiz M. , Barredo J. L. , Moreno M. A. , Fernández-Cañón J. M. , Peñalva M. A. , Dı́ez B.
Published: July 2002

The single-copy
pahA
gene from
Penicillium chrysogenum
encodes a phenylacetate 2-hydroxylase that catalyzes the first step of phenylacetate catabolism, an oxidative route that decreases the precursor availability for penicillin G biosynthesis. PahA protein is homologous to cytochrome P450 monooxygenases involved in the detoxification of xenobiotic compounds, with 84% identity to the
Aspergillus nidulans
homologue PhacA. Expression level of
pahA
displays an inverse correlation with the penicillin productivity of the strain and is subject to induction by phenylacetic acid. Gene expression studies have revealed a reduced oxidative activity of the protein encoded by
pahA
genes from penicillin-overproducing strains of
P. chrysogenum
compared to the activity conferred by
phacA
of
A. nidulans
. Sequencing and expression of wild-type
pahA
from
P. chrysogenum
NRRL 1951 revealed that an L181F mutation was responsible for the reduced function in present industrial strains. The mutation has been tracked down to Wisconsin 49–133, a mutant obtained at the Department of Botany of the University of Wisconsin in 1949, at the beginning of the development of the Wisconsin family of strains.
Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of
Escherichia coli
Authors: Yang Xiaoming , Ishiguro Edward E.
Published: July 2002

Amino acid-deprived
rplK
(previously known as
relC
) mutants of
Escherichia coli
cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The
rplK
gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative of
rplK
encoding L11 lacking the N-terminal 36 amino acids (designated ′L11) was constructed. Bacteria overexpressing ′L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed ′L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.
The First Step in Polyethylene Glycol Degradation by Sphingomonads Proceeds via a Flavoprotein Alcohol Dehydrogenase Containing Flavin Adenine Dinucleotide
Authors: Sugimoto Manabu , Tanabe Miwa , Hataya Misako , Enokibara Shogo , Duine Johannis A. , Kawai Fusako
Published: July 2002

Several
Sphingomonas
spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from
Sphingomonas terrae
revealed that the enzyme is membrane bound. The gene encoding this enzyme (
pegA
) was cloned, sequenced, and expressed in
Escherichia coli
. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from
Pseudomonas oleovorans
(46%); choline dehydrogenase from
E. coli
(40%);
l
-sorbose dehydrogenase from
Gluconobacter oxydans
(38%); and 4-nitrobenzyl alcohol dehydrogenase from a
Pseudomonas
species (35%).