bluuebunny/crossref_metadata_2025 huggingface dataset:
Salmonella
Pathogenicity Island 2-Encoded Proteins SseC and SseD Are Essential for Virulence and Are Substrates of the Type III Secretion System
Authors: Klein Joanna R. , Jones Bradley D.
Published: July 2002

Survival of
Salmonella enterica
serovar Typhimurium within host phagocytic cells is a critical step in establishing systemic infection in mice. Genes within
Salmonella
pathogenicity island 2 (SPI-2) encode a type III secretion system that is required for establishment of systemic infection. Several proteins encoded by SPI-2 have homology to type III secreted proteins from enteropathogenic
Escherichia coli
and
Yersinia
and, based on that homology, are predicted to be secreted through the SPI-2 type III secretion system. We have investigated the roles of two of these proteins, SseC and SseD. We demonstrate here that the SseD protein is required for systemic
Salmonella
infection of the mouse, and we confirmed the virulence requirement for the SseC protein. Experiments were performed, using cellular fractionation and immunoblotting, to identify the subcellular location of the SseC and SseD proteins. Both proteins were found to localize predominantly to the bacterial cell membrane. In addition, our work revealed that SseC and SseD are exposed to the extracellular environment and are loosely associated with the bacterial membrane. Furthermore, localization of SseC and SseD to the bacterial membrane was found to require a functional SPI-2 type III secretion system. Collectively, these results indicate that the SseC and SseD proteins are secreted by the SPI-2 type III secretion system to the bacterial membrane in order to perform their virulence functions.
Conserved DegP Protease in Gram-Positive Bacteria Is Essential for Thermal and Oxidative Tolerance and Full Virulence in
Streptococcus pyogenes
Authors: Jones C. Hal , Bolken Tove' C. , Jones Kevin F. , Zeller Gloria O. , Hruby Dennis E.
Published: July 2002

The DegP protease, a multifunctional chaperone and protease, has been shown to be essential for virulence in gram-negative pathogens such as
Salmonella enterica
serovar Typhimurium,
Brucella abortus, Yersinia enterocolitica
, and
Pseudomonas aeruginosa
. The function of DegP in pathogenesis appears to be the degradation of damaged proteins that accumulate as a result of the initial host response to infection, which includes the release of reactive oxygen intermediates. Additionally, the DegP protease plays a major role in monitoring and maintaining the
Escherichia coli
periplasm and influences
E. coli
pilus biogenesis. We report here the identification of highly homologous enzymes in
Streptococcus pyogenes, Streptococcus gordonii, Streptococcus mutans, Staphylococcus aureus
, and
Enterococcus faecalis
. Moreover, the phenotype of an insertionally inactivated
degP
allele in
S. pyogenes
is similar to that reported for
E. coli
, with temperature sensitivity for growth and enhanced sensitivity to reactive oxygen intermediates. Virulence studies in a mouse model of streptococcal infection indicate that a functional DegP protease is required for full virulence. These results suggest DegP as an attractive broad-spectrum target for future anti-infective drug development.
Absence of All Components of the Flagellar Export and Synthesis Machinery Differentially Alters Virulence ofSalmonella entericaSerovar Typhimurium in Models of Typhoid Fever, Survival in Macrophages, Tissue Culture Invasiveness, and Calf Enterocolitis
Authors: Schmitt Clare K. , Ikeda Jack S. , Darnell Stephen C. , Watson Patricia R. , Bispham Jennifer , Wallis Timothy S. , Weinstein Debra L. , Metcalf Eleanor S. , O'Brien Alison D.
Published: July 2002

In this study, we constructed anflhD(the master flagellar regulator gene) mutant ofSalmonella entericaserovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that theflhDmutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, theflhDmutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on anfljB fliCmutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.
Conformationally Correct Expression of Membrane-AnchoredToxoplasma gondiiSAG1 in the Primitive ProtozoanGiardia duodenalis
Authors: Marti Matthias , Li Yajie , Köhler Peter , Hehl Adrian B.
Published: July 2002

To explore the possibility of expressing membrane-anchored exodomains of heterologous surface antigens inGiardia, a chimeric construct containing theToxoplasma gondii SAG1gene was made. TheGiardiasystem is shown here to provide a means of generating correctly folded chimeric surface proteins in a native and unmodified form.
Urease of Enterohemorrhagic
Escherichia coli
: Evidence for Regulation by Fur and a
trans
-Acting Factor
Authors: Heimer Susan R. , Welch Rod A. , Perna Nicole T. , Pósfai György , Evans Peter S. , Kaper James B. , Blattner Fred R. , Mobley Harry L. T.
Published: July 2002

Recent genomic analyses of
Escherichia coli
O157:H7 strain EDL933 revealed two loci encoding urease gene homologues (
ureDABCEFG
), which are absent in nonpathogenic
E. coli
strain K-12. This report demonstrates that the cloned EDL933
ure
gene cluster is capable of synthesizing urease in an
E. coli
DH5α background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified
trans
-acting factor in enterohemorrhagic
E. coli
(EHEC) is responsible for this regulation of
ure
expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of
ureD
and
ureA
. Enzymatic assays confirmed a difference in urease expression of cloned EHEC
ure
clusters in
E. coli
MC3100Δ
fur
. Likewise, interruption of
fur
in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of
Salmonella enterica
serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.
Identification and Characterization of the ESAT-6 Homologue ofMycobacterium lepraeand T-Cell Cross-Reactivity withMycobacterium tuberculosis
Authors: Geluk Annemieke , van Meijgaarden Krista E. , Franken Kees L. M. C. , Subronto Yanri W. , Wieles Brigitte , Arend Sandra M. , Sampaio Elizabeth P. , de Boer Tjitske , Faber William R. , Naafs Ben , Ottenhoff Tom H. M.
Published: July 2002

In this paper we describe identification and characterization ofMycobacterium lepraeESAT-6 (L-ESAT-6), the homologue ofM. tuberculosisESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to theM. tuberculosiscomplex but is absent from virtually all other mycobacterial species, and it is a promising antigen for immunodiagnosis of tuberculosis (TB). Therefore, we analyzed whether L-ESAT-6 is a similarly powerful tool for the study of leprosy by examining T-cell responses against L-ESAT-6 in leprosy patients, TB patients, and exposed or nonexposed healthy controls from areas where leprosy and TB are endemic and areas where they are not endemic. L-ESAT-6 was recognized by T cells from leprosy patients, TB patients, individuals who had contact with TB patients, and healthy individuals from an area where TB and leprosy are endemic but not by T cells from individuals who were not exposed toM. tuberculosisandM. leprae. Moreover, leprosy patients who were not responsive toM. lepraefailed to respond to L-ESAT-6. A very similar pattern was obtained with T-ESAT-6. These results show that L-ESAT-6 is a potentM. lepraeantigen that stimulates T-cell-dependent gamma interferon production in a large proportion of individuals exposed toM. leprae. Moreover, our results suggest that there is significant cross-reactivity between T-ESAT-6 and L-ESAT-6, which has implications for the use of ESAT-6 as tool for diagnosis of leprosy and TB in areas where both diseases are endemic.
Mechanisms of Monophosphoryl Lipid A Augmentation of Host Responses to Recombinant HagB fromPorphyromonas gingivalis
Authors: Yang Qiu-Bo , Martin Michael , Michalek Suzanne M. , Katz Jannet
Published: July 2002

Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection toP. gingivalisinfection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 μg) alone or with MPL (25 μg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P< 0.05) and serum IgG (P< 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P< 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was ≈1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.
Essential Role of Ferritin Pfr in
Helicobacter pylori
Iron Metabolism and Gastric Colonization
Authors: Waidner Barbara , Greiner Stefan , Odenbreit Stefan , Kavermann Holger , Velayudhan Jyoti , Stähler Frank , Guhl Johannes , Bissé Emmanuel , van Vliet Arnoud H. M. , Andrews Simon C. , Kusters Johannes G. , Kelly David J. , Haas Rainer , Kist Manfred , Bereswill Stefan
Published: July 2002

The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the
Helicobacter pylori
ferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled
H. pylori
to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the
pfr
gene restricted growth of
H. pylori
under these conditions. The lowered total iron content in the
pfr
mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an
H. pylori feoB
mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of
pfr
transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant
H. pylori
Fur to the
pfr
promoter region. The functions of
H. pylori
Pfr in iron metabolism are essential for survival in the gastric mucosa, as the
pfr
mutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the
pfr
phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for
H. pylori
to survive in its hostile natural environment.
Organization, Structure, and Variability of the rRNA Operon of the Whipple's Disease Bacterium (
Tropheryma whippelii
)
Authors: Maiwald Matthias , von Herbay Axel , Lepp Paul W. , Relman David A.
Published: July 2002

Whipple's disease is a systemic disorder associated with a cultivation-resistant, poorly characterized actinomycete,
Tropheryma whippelii
. We determined a nearly complete rRNA operon sequence of
T. whippelii
from specimens from 3 patients with Whipple's disease, as well as partial operon sequences from 43 patients. Variability was observed in the 16S-23S rRNA spacer sequences, leading to the description of five distinct sequence types. One specimen contained two spacer sequence types, raising the possibility of a double infection. Secondary structure models for the primary rRNA transcript and mature rRNAs revealed rare or unique features.
Sequential Inactivation of
rdxA
(HP0954) and
frxA
(HP0642) Nitroreductase Genes Causes Moderate and High-Level Metronidazole Resistance in
Helicobacter pylori
Authors: Jeong Jin-Yong , Mukhopadhyay Asish K. , Dailidiene Daiva , Wang Yipeng , Velapatiño Billie , Gilman Robert H. , Parkinson Alan J. , Nair G. Balakrish , Wong Benjamin C. Y. , Lam Shiu Kum , Mistry Rajesh , Segal Isidore , Yuan Yuan , Gao Hua , Alarcon Teresa , Brea MaNuel Lopez , Ito Yoshiyuki , Kersulyte Dangeruta , Lee Hae-Kyung , Gong Yan , Goodwin Avery , Hoffman Paul S. , Berg Douglas E.
Published: July 2002

Helicobacter pylori
is a human-pathogenic bacterial species that is subdivided geographically, with different genotypes predominating in different parts of the world. Here we test and extend an earlier conclusion that metronidazole (Mtz) resistance is due to mutation in
rdxA
(HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We found that (i)
rdxA
genes PCR amplified from 50 representative Mtz
r
strains from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtz
s
H. pylori
to Mtz
r
; (ii) Mtz
r
mutant derivatives of a cultured Mtz
s
strain resulted from mutation in
rdxA
; and (iii) transformation of Mtz
s
strains with
rdxA
-null alleles usually resulted in moderate level Mtz resistance (16 μg/ml). However, resistance to higher Mtz levels was common among clinical isolates, a result that implicates at least one additional gene. Expression in
Escherichia coli
of
frxA
(HP0642; flavin oxidoreductase), an
rdxA
paralog, made this normally resistant species Mtz
s
, and
frxA
inactivation enhanced Mtz resistance in
rdxA
-deficient cells but had little effect on the Mtz susceptibility of
rdxA
+
cells. Strains carrying
frxA
-null and
rdxA
-null alleles could mutate to even higher resistance, a result implicating one or more additional genes in residual Mtz susceptibility and hyperresistance. We conclude that most Mtz resistance in
H. pylori
depends on
rdxA
inactivation, that mutations in
frxA
can enhance resistance, and that genes that confer Mtz resistance without
rdxA
inactivation are rare or nonexistent in
H. pylori
populations.
Functional Organization and Insertion Specificity of IS607, a Chimeric Element ofHelicobacter pylori
Authors: Kersulyte Dangeruta , Mukhopadhyay Asish K. , Shirai Mutsinori , Nakazawa Teruko , Berg Douglas E.
Published: July 2002

A search by subtractive hybridization for sequences present in only certain strains ofHelicobacter pyloriled to the discovery of a 2-kb transposable element to be called IS607, which further PCR and hybridization tests indicated was present in about one-fifth ofH. pyloristrains worldwide. IS607contained two open reading frames (ORFs) of possibly different phylogenetic origin. One ORF (orfB) exhibited protein-level homology to one of two putative transposase genes found in several other chimeric elements including IS605(also ofH. pylori) and IS1535(ofMycobacterium tuberculosis). The second IS607gene (orfA) was unrelated to the second gene of IS605and might possibly be chimeric itself: it exhibited protein-level homology tomerRbacterial regulatory genes in the first ∼50 codons and homology to the second gene of IS1535(annotated as “resolvase,” apparently due to a weak short recombinase motif) in the remaining three-fourths of its length. IS607was found to transpose inEscherichia coli, and analyses of sequences of IS607-target DNA junctions inH. pyloriandE. coliindicated that it inserted either next to or between adjacent GG nucleotides, and generated either a 2-bp or a 0-bp target sequence duplication, respectively. Mutational tests showed that its transposition inE. colirequiredorfAbut notorfB, suggesting that OrfA protein may represent a new, previously unrecognized, family of bacterial transposases.
Novel Role for an HPt Domain in Stabilizing the Phosphorylated State of a Response Regulator Domain
Authors: Janiak-Spens Fabiola , Sparling David P. , West Ann H.
Published: July 2002

Two-component regulatory systems that utilize a multistep phosphorelay mechanism often involve a histidine-containing phosphotransfer (HPt) domain. These HPt domains serve an essential role as histidine-phosphorylated protein intermediates during phosphoryl transfer from one response regulator domain to another. In
Saccharomyces cerevisiae
, the YPD1 protein facilitates phosphoryl transfer from a hybrid sensor kinase, SLN1, to two distinct response regulator proteins, SSK1 and SKN7. Because the phosphorylation state largely determines the functional state of response regulator proteins, we have carried out a comparative study of the phosphorylated lifetimes of the three response regulator domains associated with SLN1, SSK1, and SKN7 (R1, R2, and R3, respectively). The isolated regulatory domains exhibited phosphorylated lifetimes within the range previously observed for other response regulator domains (i.e., several minutes to several hours). However, in the presence of YPD1, we found that the half-life of phosphorylated SSK1-R2 was dramatically extended (almost 200-fold longer than in the absence of YPD1). This stabilization effect was specific for SSK1-R2 and was not observed for SLN1-R1 or SKN7-R3. Our findings suggest a mechanism by which SSK1 is maintained in its phosphorylated state under normal physiological conditions and demonstrate an unprecedented regulatory role for an HPt domain in a phosphorelay signaling system.
Cloning and Functional Characterization of an NAD
+
-Dependent DNA Ligase from
Staphylococcus aureus
Authors: Kaczmarek Frank S. , Zaniewski Richard P. , Gootz Thomas D. , Danley Dennis E. , Mansour Mahmoud N. , Griffor Matt , Kamath Ajith V. , Cronan Melissa , Mueller John , Sun Dongxu , Martin Patrick K. , Benton Bret , McDowell Laura , Biek Donald , Schmid Molly B.
Published: July 2002

A
Staphylococcus aureus
mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the
S. aureus lig
A gene. Orthologues of the putative
S. aureus
NAD
+
-dependent DNA ligase could be identified in the genomes of
Bacillus stearothermophilus
and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the
ligA
genes from wild type and temperature-sensitive
S. aureus
strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The
S. aureus ligA
gene was cloned and overexpressed in
Escherichia coli
, and the enzyme was purified to near homogeneity. NAD
+
-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of
32
P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified
S. aureus
DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in
E. coli
and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the
S. aureus
LigA protein expressed in
E. coli
did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD
+
-dependent DNA ligase from
B. stearothermophilus
, two independent functional domains exist in
S. aureus
DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen
S. aureus
indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.
Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon,
Haloarcula marismortui
Authors: Ichiki Hirotaka , Tanaka Yoko , Mochizuki Kiyotaka , Yoshimatsu Katsuhiko , Sakurai Takeshi , Fujiwara Taketomo
Published: July 2002

Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon,
Haloarcula marismortui
. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (g
II
= 2.232; A
II
= 4.4 mT) and type 2 Cu centers (g
II
= 2.304; A
II
= 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A
nirK
gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 μM NO
2
−
· min
−1
· mg
−1
.
uvrA
Is an Acid-Inducible Gene Involved in the Adaptive Response to Low pH in
Streptococcus mutans
Authors: Hanna Michael N. , Ferguson Ronald J. , Li Yung-Hua , Cvitkovitch Dennis G.
Published: July 2002

The pH-inducible acid tolerance response (ATR) is believed to play a major role in acid adaptation and virulence of
Streptococcus mutans
. To study this phenomenon in
S
.
mutans
JH1005, differential display PCR was used to identify and clone 13 cDNA products that had increased expression in response to pH 5.0 compared to that of pH 7.5-grown cells. One of these products, confirmed to be pH inducible by RNA dot blot and reverse transcription-PCR analyses, had 67% identity to a
uvrA
-UV repair excinuclease gene in
Bacillus subtilis
. Further sequence analysis of the
uvrA
homologue using the
S
.
mutans
genome database revealed that the complete gene was encoded in an open reading frame (ORF) of 2,829 bp (944 amino acids; 104.67 kDa). Immediately 3′ of
uvrA
was an ORF encoding a putative aminopeptidase gene (
pepP
).
uvrA
knockouts were constructed in
S
.
mutans
strains JH1005, NG8, and UA159 using allelic-exchange mutagenesis, replacing the entire gene with an erythromycin resistance cassette. As with
uvrA
mutants in other bacteria, the
S
.
mutans uvrA
mutants were extremely sensitive to UV irradiation. The
uvrA
mutant of
S
.
mutans
JH1005 was also more sensitive than the wild type to growth at pH 5.0, showing a 15% reduction in growth rate and a 14% reduction in final resting culture density. Acid-adapted
S
.
mutans
JH1005
uvrA
mutants were shown to be more resistant to UV irradiation than was the parent but were unable to survive exposure to a killing pH of 3.0. Moreover, agarose gel electrophoretic analysis of chromosomal DNA isolated from
uvrA
-deficient cells exposed to low pH demonstrated more DNA damage than that for the wild-type strain. Here we suggest that
uvrA
and the nucleotide excision repair pathway are involved in the repair of acid-induced DNA damage and are associated with successful adaptation of
S
.
mutans
to low pH.
T7 Single Strand DNA Binding Protein but Not T7 Helicase Is Required for DNA Double Strand Break Repair
Authors: Yu Man , Masker Warren
Published: July 2002

An in vitro system based on
Escherichia coli
infected with bacteriophage T7 was used to test for involvement of host and phage recombination proteins in the repair of double strand breaks in the T7 genome. Double strand breaks were placed in a unique
Xho
I site located approximately 17% from the left end of the T7 genome. In one assay, repair of these breaks was followed by packaging DNA recovered from repair reactions and determining the yield of infective phage. In a second assay, the product of the reactions was visualized after electrophoresis to estimate the extent to which the double strand breaks had been closed. Earlier work demonstrated that in this system double strand break repair takes place via incorporation of a patch of DNA into a gap formed at the break site. In the present study, it was found that extracts prepared from uninfected
E. coli
were unable to repair broken T7 genomes in this in vitro system, thus implying that phage rather than host enzymes are the primary participants in the predominant repair mechanism. Extracts prepared from an
E. coli recA
mutant were as capable of double strand break repair as extracts from a wild-type host, arguing that the
E. coli
recombinase is not essential to the recombinational events required for double strand break repair. In T7 strand exchange during recombination is mediated by the combined action of the helicase encoded by gene 4 and the annealing function of the gene 2.5 single strand binding protein. Although a deficiency in the gene 2.5 protein blocked double strand break repair, a gene 4 deficiency had no effect. This argues that a strand transfer step is not required during recombinational repair of double strand breaks in T7 but that the ability of the gene 2.5 protein to facilitate annealing of complementary single strands of DNA is critical to repair of double strand breaks in T7.
Link between the Membrane-Bound Pyridine Nucleotide Transhydrogenase and Glutathione-Dependent Processes in
Rhodobacter sphaeroides
Authors: Hickman Jason W. , Barber Robert D. , Skaar Eric P. , Donohue Timothy J.
Published: July 2002

The presence of a glutathione-dependent pathway for formaldehyde oxidation in the facultative phototroph
Rhodobacter sphaeroides
has allowed the identification of gene products that contribute to formaldehyde metabolism. Mutants lacking the glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) are sensitive to metabolic sources of formaldehyde, like methanol. This growth phenotype is correlated with a defect in formaldehyde oxidation. Additional methanol-sensitive mutants were isolated that contained Tn
5
insertions in
pntA
, which encodes the α subunit of the membrane-bound pyridine nucleotide transhydrogenase. Mutants lacking transhydrogenase activity have phenotypic and physiological characteristics that are different from those that lack GSH-FDH activity. For example, cells lacking transhydrogenase activity can utilize methanol as a sole carbon source in the absence of oxygen and do not display a formaldehyde oxidation defect, as determined by whole-cell
13
C-nuclear magnetic resonance. Since transhydrogenase can be a major source of NADPH, loss of this enzyme could result in a requirement for another source for this compound. Evidence supporting this hypothesis includes increased specific activities of other NADPH-producing enzymes and the finding that glucose utilization by the Entner-Doudoroff pathway restores aerobic methanol resistance to cells lacking transhydrogenase activity. Mutants lacking transhydrogenase activity also have higher levels of glutathione disulfide under aerobic conditions, so it is consistent that this strain has increased sensitivity to oxidative stress agents like diamide, which are known to alter the oxidation reduction state of the glutathione pool. A model will be presented to explain the role of transhydrogenase under aerobic conditions when cells need glutathione both for GSH-FDH activity and to repair oxidatively damaged proteins.
Phylogeny and Functional Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Autotrophic Ammonia-Oxidizing Bacterium
Nitrosospira
sp.Isolate 40KI
Authors: Utåker Janne B. , Andersen Kjell , Aakra Ågot , Moen Birgitte , Nes Ingolf F.
Published: July 2002

The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO
2
by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (
cbb
) genes and proteins from the AOB. The
cbbLS
genes from
Nitrosospira
sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the β and γ subgroups of the class
Proteobacteria
are also presented. All except one of the β-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the
Ralstonia eutropha
enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB,
Nitrosococcus halophilus
Nc4 and
Nitrosomonas europaea
Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in
N. europaea
was surprising, as all of the other β-subgroup AOB had red-like RubisCOs. The green-like enzyme of
N. europaea
Nm50 was probably acquired by horizontal gene transfer. Functional expression of
Nitrosospira
sp. isolate 40KI RubisCO in the chemoautotrophic host
R. eutropha
was demonstrated. Use of an expression vector harboring the
R. eutropha cbb
control region allowed regulated expression of
Nitrosospira
sp. isolate 40KI RubisCO in an
R. eutropha cbb
deletion strain. The
Nitrosospira
RubisCO supported autotrophic growth of
R. eutropha
with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB
cbb
genes.
Speed versus Efficiency in Microbial Growth and the Role of Parallel Pathways
Authors: Helling Robert B.
Published: July 2002

Many microorganisms have sets of parallel pathways for ATP production in respiration and for ATP utilization in glutamate synthesis. The alternatives differ in efficiency of ATP production and utilization. The choice among these parallel pathways has been hypothesized to control the speed and efficiency of growth. Thus, the organism should be able to alleviate (or exaggerate) deficiency in one pathway by deleting another. I show here that inEscherichia colithe effect of lack of the glutamate-synthesizing enzyme glutamate dehydrogenase on glucose-limited growth is altered predictably byndh,cyo, andcydmutations affecting parallel pathways leading to ATP synthesis in respiration.
Mobility of a Restriction-Modification System Revealed by Its Genetic Contexts in Three Hosts
Authors: Naderer Marc , Brust Jessica R. , Knowle Dieter , Blumenthal Robert M.
Published: July 2002

The flow of genes among prokaryotes plays a fundamental role in shaping bacterial evolution, and restriction-modification systems can modulate this flow. However, relatively little is known about the distribution and movement of restriction-modification systems themselves. We have isolated and characterized the genes for restriction-modification systems from two species of
Salmonella
,
S. enterica serovar
Paratyphi A and
S. enterica
serovar Bareilly. Both systems are closely related to the
Pvu
II restriction-modification system and share its target specificity. In the case of
S. enterica
serovar Paratyphi A, the restriction endonuclease is inactive, apparently due to a mutation in the subunit interface region. Unlike the chromosomally located
Salmonella
systems, the
Pvu
II system is plasmid borne. We have completed the sequence characterization of the
Pvu
II plasmid pPvu1, originally from
Proteus vulgaris
, making this the first completely sequenced plasmid from the genus
Proteus
. Despite the pronounced similarity of the three restriction-modification systems, the flanking sequences in
Proteus
and
Salmonella
are completely different. The
Spt
AI and
Sba
I genes lie between an equivalent pair of bacteriophage P4-related open reading frames, one of which is a putative integrase gene, while the
Pvu
II genes are adjacent to a
mob
operon and a XerCD recombination (
cer
) site.
Variation in the Spacer Regions Separating tRNA Genes in
Renibacterium salmoninarum
Distinguishes Recent Clinical Isolates from the Same Location
Authors: Alexander Sarah M. , Grayson T. Hilton , Chambers Edel M. , Cooper Lynne F. , Barker Gavin A. , Gilpin Martyn L.
Published: July 2002

A means for distinguishing between clinical isolates of
Renibacterium salmoninarum
that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of
R. salmoninarum
tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of
R. salmoninarum
from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS
994
. Our method enabled us to make divisions between closely related clinical isolates of
R. salmoninarum
that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS
994
profiles.
Limited Genetic Diversity of
Brucella
spp
Authors: Gándara Benjamı́n , Merino Ahidé López , Rogel Marco Antonio , Martı́nez-Romero Esperanza
Published: July 2002

Multilocus enzyme electrophoresis (MLEE) of 99
Brucella
isolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of a monospecific genus. In MLEE-derived dendrograms,
Brucella abortus
and a marine
Brucella
sp. grouped into a single electrophoretic type related to
Brucella neotomae
and
Brucella ovis. Brucella suis
and
Brucella canis
formed another cluster linked to
Brucella melitensis
and related to
Rhizobium tropici
. The
Brucella
strains tested that were representatives of the six electrophoretic types had the same rRNA gene restriction fragment length polymorphism patterns and identical ribotypes. All 99 isolates had similar chromosome profiles as revealed by the Eckhardt procedure.
Rapid Detection of Smear-Negative
Mycobacterium tuberculosis
by PCR and Sequencing for Rifampin Resistance with DNA Extracted Directly from Slides
Authors: Patnaik Madhumita , Liegmann Karsten , Peter James B.
Published: July 2002

Conventional methods for identification of
Mycobacterium tuberculosis
from culture can take 6 weeks. To facilitate the rapid detection of
M. tuberculosis
and to assess the risks of drug resistance, we developed a technique of eluting DNA directly from sputum slides and performing PCR for the detection of
M. tuberculosis
DNA, followed by sequencing the
rpoB
gene to detect rifampin resistance. This entire process requires only 48 h. Forty-seven sputum specimens submitted for microscopy for detection of acid-fast bacilli (AFB) and for mycobacterial culture and susceptibility testing were assessed after elution from the slides and extraction.
M. tuberculosis
-specific DNA was amplified in a nested PCR with previously described primers (primers rpo95-rpo293 and rpo105-rpo273), followed by analysis on a 4% agarose gel for a 168-bp product. Automated sequencing was performed, and the sequences were aligned against a database for detection of anomalies in the
rpoB
gene (codons 511 to 533) which indicate rifampin resistance. Of the 47 sputum specimens tested, 51% (24 of 47) were culture positive (time to positive culture, 2 to 6 weeks). Smears for AFB were positive for 58% (14 of 24) of the specimens and were negative for 42% (10 of 24) of the specimens. All 24 culture-positive sputum specimens (14 microscopy-positive and 10 microscopy-negative sputum specimens) were positive by PCR with eluates from the smears. Forty-nine percent (23 of 47) of the sputum specimens were negative for
M. tuberculosis
by smear, culture, and PCR. Of the isolates from the culture-positive samples, five were rifampin resistant by sequencing; all five were also rifampin resistant by in vitro susceptibility testing. Of these rifampin-resistant
M. tuberculosis
isolates, two were microscopy negative for AFB. Patients who are negative for AFB and culture positive for
M. tuberculosis
can now be identified within a day, allowing institution of therapy and reducing isolation time and medical costs.
Qualitative Plasma PCR Assay (AMPLICOR CMV Test) versus pp65 Antigenemia Assay for Monitoring Cytomegalovirus Viremia and Guiding Preemptive Ganciclovir Therapy in Allogeneic Stem Cell Transplantation
Authors: Solano Carlos , Muñoz Isabel , Gutiérrez Antonio , Farga Amparo , Prósper Felipe , Garcı́a-Conde Javier , Navarro David , Gimeno Concepción
Published: July 2002

The performances of a commercially available qualitative plasma PCR assay (AMPLICOR CMV test; Roche Diagnostics) and the pp65 antigenemia assay (AG) were evaluated for the monitoring of cytomegalovirus (CMV) viremia in 43 allogeneic stem cell transplant recipients. In addition, the suitabilities of both assays for triggering the initiation of preemptive ganciclovir therapy were assessed. A total of 37 CMV viremic episodes were detected in 28 patients. Positivity of plasma PCR testing in one or more consecutive specimens was the only marker of CMV viremia in 18 of the 37 episodes (PCR positive and AG negative,
n
= 50 specimens). Five episodes were diagnosed on the basis of a single positive AG result (AG positive and PCR negative,
n
= 5 specimens); both assays were eventually positive (PCR positive and AG positive,
n
= 27 specimens) for 14 viremic episodes; for these episodes, conversion of the PCR assay result to a positive result occurred an average of 1 week before conversion of the AG result. Overall, the concordance between the two methods was 90%, and the sensitivities of the plasma PCR assay and AG for the detection of CMV viremic episodes were 86.5 and 51.3%, respectively. Two patients who tested positive by both assays simultaneously progressed to CMV end-stage organ disease, despite the initiation of preemptive ganciclovir therapy. Conversion of the AG result to a negative result upon administration of preemptive ganciclovir therapy occurred a median of 7.5 days earlier than conversion of the plasma PCR assay result. Nineteen of the 28 patients with CMV viremia received AG-guided preemptive ganciclovir therapy; had the positivity of the plasma PCR assay triggered the initiation of preemptive therapy, 9 additional patients would have been unnecessarily treated since none of them developed CMV end-stage organ disease. Although the AMPLICOR CMV assay is more sensitive than AG, the latter appears to be more suitable both for guiding the initiation of preemptive therapy and for monitoring a patient's response to antiviral therapy.
Unusual Occurrence of M Type 77, Antibiotic-Resistant Group A Streptococci in Southern Sweden
Authors: Jasir A. , Tanna A. , Efstratiou A. , Schalén C.
Published: July 2002

For many years group A streptococci of T type 28 (T28) have been common in southern Sweden; however, since 1995 resistance to both macrolide-lincosamide-streptogramin B (MLS) antibiotics and tetracycline was observed among T28 isolates, which prompted the present studies on clonal relatedness of antibiotic-resistant T28 strains. By extended T typing, 95 of 100 examined tetracycline-resistant strains showed the combination T9-T13-T28; of these, 94 belonged to M type 77 (M77) and one belonged to M73. Three strains were T28-M28 and two were T28-M nontypeable. The serological M77 was confirmed by PCR capture enzyme-linked immunosorbent assay,
emm
amplicon restriction profiling, and
emm
sequence typing. Fifty strains were examined for superantigen genes:
speA
was detected in three blood isolates only, whereas all isolates harbored
speB
, and only two of the strains were negative for
speC
. Eighty-nine of the 100 strains were also macrolide resistant, of which 59 were inducibly MLS resistant (IR) and 21 were constitutively MLS resistant (CR), 6 were noninducibly resistant (NI), and 3 had novel subphenotypes recently reported by our group. Resistance genes were determined by PCR and hybridization methods. Eighty-four of the 100 strains harbored
tetM. ermB
was detected in all CR and IR strains, and
mefA
was found in all NI strains; both
ermB
and
mefA
were identified in two strains with novel subphenotypes. Pulsed-field gel electrophoresis showed that these antibiotic-resistant M77 strains belonged to at least five different clones.
Chronic Melioidosis in a Patient with Cystic Fibrosis
Authors: Schülin Tanja , Steinmetz Ivo
Published: July 2002

Burkholderia pseudomallei
, the causative agent of melioidosis, is endemic in Southeast Asia and northern Australia, where it can be found in soil and surface water. We report a case of chronic pulmonary melioidosis in a patient with cystic fibrosis who had traveled to an area where
B. pseudomallei
is endemic.
Persistence of Two Invasive
Streptococcus pneumoniae
Clones of Serotypes 1 and 5 in Comparison to That of Multiple Clones of Serotypes 6B and 23F among Children in Southern Israel
Authors: Porat Nurith , Trefler Ronit , Dagan Ron
Published: July 2002

We conducted a study to examine the clonal distribution of invasive serotype 1 and 5 isolates as representatives of serotypes that are rarely carried by healthy individuals compared to that of invasive serotype 6B and 23F isolates as representatives of serotypes often carried by young children for prolonged periods. All invasive serotype 1, 5, 6B, and 23F isolates recovered from blood cultures during January 1995 to May 1999 were analyzed; these included 66 serotype 1, 30 serotype 5, 11 serotype 6B, and 15 serotype 23F isolates. One hundred thirty-three nasopharyngeal (NP) isolates of the indicated four serotypes from healthy children were also studied. The strains were characterized using serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis profiling. We found that both invasive and NP serotype 1 and 5 isolates were susceptible to penicillin and that each serotype showed only one clonal type. In contrast, serotype 6B and 23F strains showed different phenotypic characteristics as well as multiple clonal types; 10 clones were identified among 6B isolates, and 11 clones were identified among 23F isolates.
Variable Oncogene Promoter Activity of Human Papillomavirus Type 16 Cervical Cancer Isolates from Australia
Authors: Watts Kylie J. , Thompson Carol H. , Cossart Yvonne E. , Rose Barbara R.
Published: July 2002

The functional significance of sequence variation within the upstream regulatory region (URR) of six human papillomavirus type 16 (HPV16) cervical cancer isolates from Australia was investigated. Specific changes in transcription factor binding sites leading to increased promoter activity may explain the transforming ability of some episomal HPV16 isolates.
Performance of the Chromogenic Medium CHROMagar Staph Aureus and the Staphychrom Coagulase Test in the Detection and Identification of
Staphylococcus aureus
in Clinical Specimens
Authors: Carricajo Anne , Treny Axel , Fonsale Nathalie , Bes Michele , Reverdy Marie Elisabeth , Gille Yves , Aubert Gerald , Freydiere Anne Marie
Published: July 2002

CHROMagar Staph aureus (CSAM) (CHROMagar Microbiology, Paris, France) is a new chromogenic medium designed to enable detection of colonies of
Staphylococcus aureus
by their pink color. A total of 775 specimens were cultured in parallel on CHROMagar Staph aureus and conventional media. Among the 267
S. aureus
strains recovered on at least one medium, 263 were isolated on CSAM medium (sensitivity, 98.5%), and 245 (sensitivity, 91.8%) were isolated on conventional media. The specificity of presumptive identification of
S. aureus
on the basis of pink colony color on CSAM medium was 97% (493 of 508). This specificity increased to 100% when coagulase detection with the Staphychrom coagulase test was added and to 98.8% when
S. aureus
surface components were detected by agglutination in the Pastorex Staph Plus test. Susceptibility testing of 67
S. aureus
strains, performed in parallel on pink CSAM colonies and on colonies grown on blood agar, gave similar results. Thus, rapid and accurate recognition and identification of
S. aureus
isolates were achieved with CSAM as the primary isolation medium, followed by the staphylocoagulase Staphychrom test. Antimicrobial susceptibility testing (disk-diffusion method or ATB STAPH System) can be performed directly on pink CSAM colonies.