bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantification of
phnAc
and
nahAc
in Contaminated New Zealand Soils by Competitive PCR
Authors: Laurie Andrew D. , Lloyd-Jones Gareth
Published: July 2002

Unculturable polycyclic aromatic hydrocarbon (PAH)-degrading bacteria are a significant reservoir of the microbial potential to catabolize low-molecular-weight PAHs. The population of these bacteria is larger than the population of
nah
-like bacteria that are the dominant organisms in culture-based studies. We used the recently described
phn
genes of
Burkholderia
sp. strain RP007, which feature only rarely in culture-based studies, as an alternative genotype for naphthalene and phenanthrene degradation and compared this genotype with the genotypically distinct but ubiquitous
nah
-like class in different soils. Competitive PCR quantification of
phnAc
and
nahAc
, which encode the iron sulfur protein large (α) subunits of PAH dioxygenases in
nah
-like and
phn
catabolic operons, revealed that the
phn
genotype can have a greater ecological significance than the
nah
-like genotype.
Production of Volatile Derivatives of Metal(loid)s by Microflora Involved in Anaerobic Digestion of Sewage Sludge
Authors: Michalke K. , Wickenheiser E. B. , Mehring M. , Hirner A. V. , Hensel R.
Published: July 2002

Gases released from anaerobic wastewater treatment facilities contain considerable amounts of volatile methyl and hydride derivatives of metals and metalloids, such as arsine (AsH
3
), monomethylarsine, dimethylarsine, trimethylarsine, trimethylbismuth (TMBi), elemental mercury (Hg
0
), trimethylstibine, dimethyltellurium, and tetramethyltin. Most of these compounds could be shown to be produced by pure cultures of microorganisms which are representatives of the anaerobic sewage sludge microflora, i.e., methanogenic archaea (
Methanobacterium formicicum
,
Methanosarcina barkeri
,
Methanobacterium thermoautotrophicum
), sulfate-reducing bacteria (
Desulfovibrio vulgaris
,
D. gigas
), and a peptolytic bacterium (
Clostridium collagenovorans
). Additionally, dimethylselenium and dimethyldiselenium could be detected in the headspace of most of the pure cultures. This is the first report of the production of TMBi, stibine, monomethylstibine, and dimethylstibine by a pure culture of
M. formicicum
.
Intracellular Accumulation of Polyphosphate by the Yeast
Candida humicola
G-1 in Response to Acid pH
Authors: McGrath John W. , Quinn John P.
Published: July 2002

Cells of a newly isolated environmental strain of
Candida humicola
accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by
C. humicola
G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.
Factors Influencing Numbers of
Mycobacterium avium
,
Mycobacterium intracellulare
, and Other Mycobacteria in Drinking Water Distribution Systems
Authors: Falkinham Joseph O. , Norton Cheryl D. , LeChevallier Mark W.
Published: July 2002

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly
Mycobacterium avium
and
Mycobacterium intracellulare
, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter
−1
. The number of
M. avium
in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (
r
2
= 0.65,
P
= 0.03). Although
M. intracellulare
was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm
2
). Evidently, the ecological niches of
M. avium
and
M. intracellulare
are distinct.
Degradation of Phenanthrene and Anthracene by Cell Suspensions ofMycobacteriumsp. Strain PYR-1
Authors: Moody Joanna D. , Freeman James P. , Doerge Daniel R. , Cerniglia Carl E.
Published: July 2002

Cultures ofMycobacteriumsp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry,1H and13C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified ascis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. WhenMycobacteriumsp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified ascis- andtrans-9,10-dihydroxy-9,10-dihydrophenanthrene andcis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2′-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.
Natural Communities of Novel Archaea and Bacteria Growing in Cold Sulfurous Springs with a String-of-Pearls-Like Morphology
Authors: Rudolph Christian , Wanner Gerhard , Huber Robert
Published: July 2002

We report the identification of novel archaea living in close association with bacteria in the cold (approximately 10°C) sulfurous marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. These microorganisms form a characteristic, macroscopically visible structure, morphologically comparable to a string of pearls. Tiny, whitish globules (the pearls; diameter, about 0.5 to 3.0 mm) are connected to each other by thin, white-colored threads. Fluorescent in situ hybridization (FISH) studies have revealed that the outer part of the pearls is mainly composed of bacteria, with a filamentous bacterium predominating. Internally, archaeal cocci are the predominant microorganisms, with up to 10
7
cells estimated to be present in a single pearl. The archaea appear to be embedded in a polymer of unknown chemical composition. According to FISH and 16S rRNA gene sequence analysis, the archaea are affiliated with the euryarchaeal kingdom. The new euryarchaeal sequence represents a deep phylogenetic branch within the 16S rRNA tree and does not show extensive similarity to any cultivated archaea or to 16S rRNA gene sequences from environmental samples.
Purification and Characterization of Pyranose Oxidase from the White Rot Fungus
Trametes multicolor
Authors: Leitner Christian , Volc Jindrich , Haltrich Dietmar
Published: July 2002

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus
Trametes multicolor
by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are
d
-glucose,
d-
xylose, and
l-
sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (
k
cat
/

K
m

), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H
2
O
2
. An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.
Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of
pmoA
,
mmoX
,
mxaF
, and 16S rRNA and Ribosomal DNA, Including
pmoA
-
Based Terminal Restriction Fragment Length Polymorphism Profiling
Authors: Horz Hans-Peter , Yimga Merlin Tchawa , Liesack Werner
Published: July 2002

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of
pmoA
, which encodes the α subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by
pmoA
-based denaturing gradient gel electrophoresis. The comparison of
pmoA
-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera
Methylomonas
,
Methylobacter
,
Methylococcus
, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the
Methylocystis
-
Methylosinus
group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel
pmoA
lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of
pmoA
provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (
mmoX
) and methanol dehydrogenase (
mxaF
). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of
mmoX
,
mxaF
, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of
pmoA
.
Isolation and Characterization of
Novosphingobium
sp. Strain MT1, a Dominant Polychlorophenol-Degrading Strain in a Groundwater Bioremediation System
Authors: Tiirola Marja A. , Männistö Minna K. , Puhakka Jaakko A. , Kulomaa Markku S.
Published: July 2002

A high-rate fluidized-bed bioreactor has been treating polychlorophenol-contaminated groundwater in southern Finland at 5 to 8°C for over 6 years. We examined the microbial diversity of the bioreactor using three 16S ribosomal DNA (rDNA)-based methods: denaturing gradient gel electrophoresis, length heterogeneity-PCR analysis, and restriction fragment length polymorphism analysis. The molecular study revealed that the process was dependent on a stable bacterial community with low species diversity. The dominant organism,
Novosphingobium
sp. strain MT1, was isolated and characterized.
Novosphingobium
sp. strain MT1 degraded the main contaminants of the groundwater, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, at 8°C. The strain carried a homolog of the
pcpB
gene, coding for the pentachlorophenol-4-monooxygenase in
Sphingobium chlorophenolicum
. Spontaneous deletion of the
pcpB
gene homolog resulted in the loss of degradation ability. Phenotypic dimorphism (planktonic and sessile phenotypes), low growth rate (0.14 to 0.15 h
−1
), and low-copy-number 16S rDNA genes (single copy) were characteristic of strain MT1 and other MT1-like organisms isolated from the bioreactor.
Cloning and Characterization of the Gene Encoding the Glutamate Dehydrogenase of
Streptococcus suis
Serotype 2
Authors: Okwumabua Ogi , Persaud Julia S. , Reddy P. G.
Published: July 2002

Given the lack of effective vaccines to control
Streptococcus suis
infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, a polyclonal antibody was raised against the whole-cell protein of
S. suis
type 2 and used to screen an
S. suis
gene library in an effort to identify protective antigen(s) and antigens of diagnostic importance. A clone that produced a 45-kDa
S. suis
-specific protein was identified by Western blotting. Restriction analysis showed that the gene encoding the 45-kDa protein was present on a 1.6-kb pair
Dra
I region on the cloned chromosomal fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 448 amino acid residues with a calculated molecular mass of 48.8 kDa, in close agreement with the size observed on Western blots. A GenBank database search revealed that the derived amino acid sequence is homologous to the sequence of glutamate dehydrogenase (GDH) protein isolated from various sources, including conserved motifs and functional domains typical of the family 1-type hexameric GDH proteins, thus placing it in that family. Because of these similarities, the protein was designated the GDH of
S. suis
. Hybridization studies showed that the gene is conserved among the
S. suis
type 2 strains tested. Antiserum raised against the purified recombinant protein was reactive with a protein of the same molecular size as the recombinant protein in
S. suis
strains, suggesting expression of the gene in all of the isolates and antigenic conservation of the protein. The recombinant protein was reactive with serum from pigs experimentally infected with a virulent strain of
S. suis
type 2, suggesting that the protein might serve as an antigen of diagnostic importance to detect
S. suis
infection. Activity staining showed that the
S. suis
GDH activity is NAD(P)H dependent but, unlike the NAD(P)H-dependent GDH from various other sources, that of
S. suis
utilizes
l
-glutamate rather than α-ketoglutarate as the substrate. Highly virulent strains of
S. suis
type 2 could be distinguished from moderately virulent and avirulent strains on the basis of their GDH protein profile following activity staining on a nondenaturing gel. We examined the cellular location of the protein using a whole-cell enzyme-linked immunosorbent assay and an immunogold-labeling technique. Results showed that the
S. suis
GDH protein is exposed at the surface of intact cells.
Immune Responses in Hookworm Infections
Authors: Loukas Alex , Prociv Paul
Published: July 2002

Hookworms infect perhaps one-fifth of the entire human population, yet little is known about their interaction with our immune system. The two major species are Necator americanus, which is adapted to tropical conditions, and Ancylostoma duodenale, which predominates in more temperate zones. While having many common features, they also differ in several key aspects of their biology. Host immune responses are triggered by larval invasion of the skin, larval migration through the circulation and lungs, and worm establishment in the intestine, where adult worms feed on blood and mucosa while injecting various molecules that facilitate feeding and modulate host protective responses. Despite repeated exposure, protective immunity does not seem to develop in humans, so that infections occur in all age groups (depending on exposure patterns) and tend to be prolonged. Responses to both larval and adult worms have a characteristic T-helper type 2 profile, with activated mast cells in the gut mucosa, elevated levels of circulating immunoglobulin E, and eosinoophilia in the peripheral blood and local tissues, features also characteristic of type I hypersensitivity reactions. The longevity of adult hookworms is determined probably more by parasite genetics than by host immunity. However, many of the proteins released by the parasites seem to have immunomodulatory activity, presumably for self-protection. Advances in molecular biotechnology enable the identification and characterization of increasing numbers of these parasite molecules and should enhance our detailed understanding of the protective and pathogenetic mechanisms in hookworm infections.
Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat
Authors: Bradford Patricia A.
Published: July 2002

β-Lactamases continue to be the leading cause of resistance to β-lactam antibiotics among gram-negative bacteria. In recent years there has been an increased incidence and prevalence of extended-spectrum β-lactamases (ESBLs), enzymes that hydrolyze and cause resistance to oxyimino-cephalosporins and aztreonam. The majority of ESBLs are derived from the widespread broad-spectrum β-lactamases TEM-1 and SHV-1. There are also new families of ESBLs, including the CTX-M and OXA-type enzymes as well as novel, unrelated β-lactamases. Several different methods for the detection of ESBLs in clinical isolates have been suggested. While each of the tests has merit, none of the tests is able to detect all of the ESBLs encountered. ESBLs have become widespread throughout the world and are now found in a significant percentage of Escherichia coli and Klebsiella pneumoniae strains in certain countries. They have also been found in other Enterobacteriaceae strains and Pseudomonas aeruginosa. Strains expressing these β-lactamases will present a host of therapeutic challenges as we head into the 21st century.
Recent Advances in Biology and Immunobiology ofEimeriaSpecies and in Diagnosis and Control of Infection with These Coccidian Parasites of Poultry
Authors: Allen P. C. , Fetterer R. H.
Published: July 2002

Avian coccidiosis, an intestinal disease caused by protozoan parasites of the genus Eimeria, occurs worldwide. It is considered to be one of the most economically important diseases of domestic poultry. For many years, prophylactic use of anticoccidial feed additives has been the primary means of controlling coccidiosis in the broiler industry and has played a major role in the growth of this industry, which now can produce about 7.6 billion chickens annually. However, development of anticoccidial resistance has threatened the economic stability of the broiler industry. Although there has been little effort by the pharmaceutical industry to develop new anticoccidials, the mounting problem of drug resistance of Eimeria species has prompted major research efforts to seek alternative means of control through increased knowledge of parasite biology, host response, and nutritional modulation. As a consequence, important advancements have been made, particularly in defining parasite antigens that have potential use in vaccines, defining the Eimeria genome, understanding the immunology of coccidial infections, and the practical applications of live vaccines. This review describes the progress in these areas, most of which has occurred within the past 10 to 15 years.
Gene Expression and Production of Tumor Necrosis Factor Alpha, Interleukin-1β (IL-1β), IL-8, Macrophage Inflammatory Protein 1α (MIP-1α), MIP-1β, and Gamma Interferon-Inducible Protein 10 by Human Neutrophils Stimulated with Group B Meningococcal Outer Membrane Vesicles
Authors: Lapinet José A. , Scapini Patrizia , Calzetti Federica , Pérez Oliver , Cassatella Marco A.
Published: July 2002

Accumulation of polymorphonuclear neutrophils (PMN) into the subarachnoidal space is one of the hallmarks ofNeisseria meningitidisinfection. In this study, we evaluated the ability of outer membrane vesicles (OMV) fromN. meningitidisB to stimulate cytokine production by neutrophils. We found that PMN stimulated in vitro by OMV produce proinflammatory cytokines and chemokines including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β. A considerable induction of gamma interferon (IFN-γ)-inducible protein 10 (IP-10) mRNA transcripts, as well as extracellular IP-10 release, was also observed when neutrophils were stimulated by OMV in combination with IFN-γ. Furthermore, PMN stimulated by OMV in the presence of IFN-γ demonstrated an enhanced capacity to release TNF-α, IL-1β, IL-8, and MIP-1β compared to stimulation with OMV alone. In line with its downregulatory effects on neutrophil-derived proinflammatory cytokines, IL-10 potently inhibited TNF-α, IL-1β, IL-8, and MIP-1β production triggered by OMV. Finally, a neutralizing anti-TNF-α monoclonal antibody (MAb) did not influence the release of IL-8 and MIP-1β induced by OMV, therefore excluding a role for endogenous TNF-α in mediating the induction of chemokine release by OMV. In contrast, the ability of lipopolysaccharide fromN. meningitidisB to induce the production of IL-8 and MIP-1β was significantly inhibited by anti-TNF-α MAb. Our results establish that, in response to OMV, neutrophils produce a proinflammatory profile of cytokines and chemokines which may not only play a role in the pathogenesis of meningitis but may also contribute to the development of protective immunity to serogroup B meningococci.
Isolation of
Neisseria gonorrhoeae
Mutants That Show Enhanced Trafficking across Polarized T84 Epithelial Monolayers
Authors: Hopper Sylvia , Wilbur J. Scott , Vasquez Brandi L. , Larson Jason , Clary Susan , Mehr Ian J. , Seifert H. S. , So Magdalene
Published: July 2002

Initiation of a gonococcal infection involves attachment of
Neisseria gonorrhoeae
to the plasma membrane of an epithelial cell in the mucosal epithelium and its internalization, transepithelial trafficking, and exocytosis from the basal membrane. Piliation and expression of certain Opa proteins and the immunoglobulin A1 protease influence the transcytosis process. We are interested in identifying other genetic determinants of
N. gonorrhoeae
that play a role in transcellular trafficking. Using polarized T84 monolayers as a model epithelial barrier, we have assayed an
N. gonorrhoeae
FA1090 minitransposon (mTn) mutant bank for isolates that traverse the monolayer more quickly than the isogenic wild-type (WT) strain. From an initial screen, we isolated four mutants, defining three genetic loci, that traverse monolayers significantly more quickly than their WT parent strain. These mutants adhere to and invade cells normally and do not affect the integrity of the monolayer barrier. Backcrosses of the mutations into the WT FA1090 strain yielded mutants with a similar fast-trafficking phenotype. In two mutants, the mTns had inserted 370 bp apart into the same locus, which we have named
fit
, for fast intracellular trafficker. Backcrosses of one of these mutants into the MS11A genetic background also yielded a fast-trafficking mutant. The
fit
locus contains two overlapping open reading frames,
fitA
and
fitB
, whose deduced amino acid sequences have predicted molecular weights of 8.6 and 15.3, respectively. Neither protein contains a signal sequence. FitA has a potential helix-turn-helix motif, while the deduced sequence of FitB offers no clues to its function.
fitA
or
fitB
homologues are present in the genomes of
Pseudomonas syringae
and
Rhizobium meliloti
, but not
Neisseria meningitidis
. Replication of the MS11A
fitA
mutant in A431 and T84 cells is significantly accelerated compared to that of the isogenic WT strain. In contrast, growth of this mutant in liquid media is normal. Taken together, these results strongly suggest that traversal of
N. gonorrhoeae
across an epithelial barrier is linked to intracellular bacterial growth.
Humanized In Vivo Model for Streptococcal Impetigo
Authors: Scaramuzzino Dominick A. , McNiff Jennifer M. , Bessen Debra E.
Published: July 2002

An in vivo model for group A streptococcal (GAS) impetigo was developed, whereby human neonatal foreskin engrafted onto SCID mice was superficially damaged and bacteria were topically applied. Severe infection, indicated by a purulent exudate, could be induced with as few as 1,000 CFU of a virulent strain. Early findings (48 h) showed a loss of stratum corneum and adherence of short chains of gram-positive cocci to the external surface of granular keratinocytes. This was followed by an increasing infiltration of polymorphonuclear leukocytes (neutrophils) of mouse origin, until a thick layer of pus covered an intact epidermis, with massive clumps of cocci accumulated at the outer rim of the pus layer. By 7 days postinoculation, the epidermis was heavily eroded; in some instances, the dermis contained pockets (ulcers) filled with cocci, similar to that observed for ecthyma. Importantly, virulent GAS underwent reproduction, resulting in a net increase in CFU of 20- to 14,000-fold. The majority ofemmpattern D strains had a higher gross pathology score thanemmpattern A, B, or C (A–C) strains, consistent with epidemiological findings that pattern D strains have a strong tendency to cause impetigo, whereas pattern A–C strains are more likely to cause pharyngitis.
Production of Toxic Shock Syndrome Toxin 1 by
Staphylococcus aureus
Requires Both Oxygen and Carbon Dioxide
Authors: Ross Robin A. , Onderdonk Andrew B.
Published: July 2002

The effect of O
2
and CO
2
on expression of toxic shock syndrome toxin 1 (TSST-1) by
Staphylococcus aureus
was investigated under controlled growth conditions with continuous-culture techniques. To stimulate TSST-1 production, air and anaerobic gas were premixed before delivery to the culture vessel. At a growth rate—or mass doubling time (
t
d
)—of 3 h, production of specific TSST-1 (expressed as micrograms per milligram of cell dry weight) was 5.9-fold greater at an O
2
concentration of 4% than under anaerobic conditions. Increasing the O
2
concentration to 11% did not result in a significant increase (
P
> 0.05) in the rate of toxin production over that during growth in 4% O
2
but did result in a significant increase (4.9-fold;
P
< 0.001) in the rate of toxin production over that during anaerobic growth. At a
t
d
of 9 h, addition of 3.5% O
2
resulted in a 7.6-fold increase in specific TSST-1 production. When room air was sparged through a culture growing at a
t
d
of 9 h, TSST-1 production increased significantly (by 3.4-fold) over that during anaerobic growth. When a growth environment of 4% O
2
–remainder N
2
was studied, no increase in TSST-1 production was observed; this was also the case with 8% O
2
at gas-flow rates of 0.1, 0.2, and 0.4 liters/min. In all experiments, production of biomass (expressed as milligrams of cell dry weight per milliliter) increased, indicating that O
2
was metabolized by
S. aureus
. Addition of CO
2
to the gas mix (4% O
2
, 10% CO
2
, 86% N
2
) resulted in a 5.1- to 6.8-fold increase in TSST-1 production over that during anaerobic growth and a 3.6-fold increase over that during growth in an environment of 4% O
2
–remainder N
2
. The
agr
mutant strain tested produced 6.1-fold more specific TSST-1 in a growth environment of 4% O
2
–10% CO
2
–86% N
2
than during anaerobic growth. These data suggest that in this system, O
2
alone does not trigger production of TSST-1; rather, both CO
2
and O
2
are required.
ς
B
Activity in
Staphylococcus aureus
Is Controlled by RsbU and an Additional Factor(s) during Bacterial Growth
Authors: Palma Marco , Cheung Ambrose L.
Published: July 2002

Two genes of the
sigB
operon,
rsbU
and
rsbV
, were deleted in an
rsbU
+
strain (FDA486) to evaluate the contribution of these two genes to ς
B
activity in
Staphylococcus aureus.
The ς
B
protein level and the transcription of two ς
B
-dependent promoters (
sigB
and
sarA
P3 transcripts) were analyzed in the constructed mutants. A deletion of the first gene (
rsbU
) within the
sigB
operon led only to a partial reduction in ς
β
activity. A deletion of the second gene (
rsbV
) resulted in a more dramatic reduction in the ς
B
protein level and its activity than did the deletion of
rsbU
, thus indicating that RsbV can be activated independent of RsbU. In the parental strain, the ς
B
-dependent transcript initiated upstream of
rsbV
was 28-fold higher than the ς
A
-dependent transcript originating from the
rsbU
promoter. The level of the ς
B
-dependent transcript decreased up to 50% in the
rsbU
mutant and up to 90% in the
rsbV
mutant compared with the transcript in the wild type. The yellow pigment of
S
.
aureus
colonies, a ς
B
-dependent phenotype, was partially reduced in the
rsbU
and
rsbV
mutants, whereas alpha-hemolysin was increased. Additionally, the
sarA
P3 promoter activity of the parental strain was induced to a higher level in response to pH 5.5 than was that of the
rsbU
or
rsbV
mutant, indicating that RsbU is the major activator of the ς
B
response to acid stress. Using a tetracycline-inducible system to modulate the expression of RsbW, we progressively repressed pigment production, presumably by reducing the free ς
B
level. Collectively, our data indicated that RsbU and RsbV in
S
.
aureus
contributed to different levels of ς
B
protein expression and varying ς
B
activities. Although RsbV can activate ς
B
independent of RsbU, RsbU remains the major activator of ς
B
during acid stress.
Cu,Zn Superoxide Dismutase ofMycobacterium tuberculosisContributes to Survival in Activated Macrophages That Are Generating an Oxidative Burst
Authors: Piddington Debra L. , Fang Ferric C. , Laessig Tracey , Cooper Andrea M. , Orme Ian M. , Buchmeier Nancy A.
Published: July 2002

Macrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Resistance to killing by macrophages is critical to the virulence ofMycobacterium tuberculosis. M. tuberculosishas two genes encoding superoxide dismutase proteins,sodAandsodC. SodC is a Cu,Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity ofM. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protectM. tuberculosisfrom reactive oxygen intermediates at the bacterial surface. To examine the role of the Cu,Zn superoxide dismutase in protectingM. tuberculosisfrom the toxic effects of exogenously generated reactive oxygen species, we constructed a null mutation in thesodCgene. In this report, we show that theM. tuberculosis sodCmutant is readily killed by superoxide generated externally, while the isogenic parentalM. tuberculosisis unaffected under these conditions. Furthermore, thesodCmutant has enhanced susceptibility to killing by gamma interferon (IFN-γ)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-γ or by macrophages from respiratory burst-deficient mice. These observations establish that the Cu,Zn superoxide dismutase contributes to the resistance ofM. tuberculosisagainst oxidative burst products generated by activated macrophages.
Comparative Immune Response to PE and PE_PGRS Antigens of
Mycobacterium tuberculosis
Authors: Delogu Giovanni , Brennan Michael J.
Published: July 2002

Sequencing of the entire genome of
Mycobacterium tuberculosis
identified a novel multigene family composed of two closely related subfamilies designated PE and PE_PGRS. The major difference between these two families is the presence of a domain containing numerous Gly-Ala repeats extending to the C terminus of the PE_PGRS genes. We have used a representative PE_PGRS gene from
M. tuberculosis, Rv1818c
(1818
PE_PGRS
), and its amino-terminal PE region (1818
PE
), to investigate the immunological response to these proteins during experimental tuberculosis and following immunization with DNA constructs. During infection of mice with
M. tuberculosis
, a significant humoral immune response was observed against recombinant 1818
PE_PGRS
but not toward the 1818
PE
protein. Similarly, immunization with a 1818
PE_PGRS
DNA construct induced antibodies directed against 1818
PE_PGRS
but not against 1818
PE
proteins, and no humoral response was induced by 1818
PE
DNA. These results suggest that certain PE_PGRS genes are expressed during infection of the host with
M. tuberculosis
and that an antibody response is directed solely against the Gly-Ala-rich PGRS domain. Conversely, splenocytes from 1818
PE
-vaccinated mice but not mice immunized with 1818
PE_PGRS
secreted gamma interferon following in vitro restimulation and demonstrated protection in the mouse tuberculosis challenge model. These results suggest that the PE vaccine can elicit an effective cellular immune response and that immune recognition of the PE antigen is influenced by the Gly-Ala-rich PGRS domain.
NhaA, an Na
+
/H
+
Antiporter Involved in Environmental Survival of
Vibrio cholerae
Authors: Vimont Sophie , Berche Patrick
Published: July 2002

Vibrio cholerae
, the agent of cholera, is a normal inhabitant of aquatic environments, in which it survives under a wide range of conditions of pH and salinity. In this work, we identified the
nhaA
gene in a wild-type epidemic strain of
V. cholerae
O1.
nhaA
encodes a protein of 382 amino acids that is very similar to the proteins NhaA of
Vibrio parahaemolyticus
,
Vibrio alginolyticus
(∼87% identity), and
Escherichia coli
(56% identity).
V. cholerae
NhaA complements an
E. coli nhaA
mutant, enabling it to grow in 700 mM NaCl, pH 7.5, indicating functional homology to
E. coli
NhaA. However, unlike
E. coli
, the growth of a
nhaA
-inactivated mutant of
V. cholerae
was not restricted at various pH and NaCl concentrations, although it was inhibited in the presence of 120 mM LiCl at pH 8.5. Nevertheless, using a
nhaA′-lacZ
transcriptional fusion, we observed induction of
nhaA
transcription by Na
+
, Li
+
, and K
+
. These results strongly suggest that NhaA is an Na
+
/H
+
antiporter contributing to the Na
+
/H
+
homeostasis of
V. cholerae. nhaA
-related sequences were detected in all strains of
V. cholerae
from the various serogroups. This gene is presumably involved in the survival and persistence of free-living bacteria in their natural habitat.
Population Studies of Methicillin-Resistant and -Sensitive
Staphylococcus aureus
Strains Reveal a Lack of Variability in the
agrD
Gene, Encoding a Staphylococcal Autoinducer Peptide
Authors: van Leeuwen Willem , van Nieuwenhuizen Wendy , Gijzen Christel , Verbrugh Henri , van Belkum Alex
Published: July 2002

The virulence of
Staphylococcus aureus
is controlled by the accessory gene regulator (
agr
) system, including an extracellular inducer encoded by
agrD
. Variable
agr
PCR restriction fragment length polymorphism (RFLP) patterns of unique
S. aureus
strains (
n
= 192) were determined for a region comprising
agrD
and parts of the neighboring
agrC
and
agrB
genes. Twelve unique RFLP patterns were identified among
S. aureus
strains in general; these patterns were further specified by sequencing. All sequences could be catalogued in the three current
agr
groups. A major proportion of the
S. aureus
strains belong to
agr
group 1, whereas only 6% of the methicillin-susceptible
S. aureus
strains and 5% of the methicillin-resistant
S. aureus
strains belong to
agr
groups 2 and 3, respectively. The homology between groups varied from 75 to 80%, and within groups it varied from 96 to 100%. Different levels of sequence variability were observed in the different
agr
genes.
agr
-related bacterial interference among colonizing
S. aureus
strains in the noses of persistent and intermittent human carriers was studied.
S. aureus
strains belonging to different agr groups were encountered in the same individual. This may suggest that the activity of the
agrD
gene product does not define colonization dynamics, which is further substantiated by the rarity of
agr
group 2 and 3 strains.
A Phosphopantetheinyl Transferase Homolog Is Essential for
Photorhabdus luminescens
To Support Growth and Reproduction of the Entomopathogenic Nematode
Heterorhabditis bacteriophora
Authors: Ciche Todd A. , Bintrim Scott B. , Horswill Alexander R. , Ensign Jerald C.
Published: July 2002

The bacterium
Photorhabdus luminescens
is a symbiont of the entomopathogenic nematode
Heterorhabditis bacteriophora.
The nematode requires the bacterium for infection of insect larvae and as a substrate for growth and reproduction. The nematodes do not grow and reproduce in insect hosts or on artificial media in the absence of viable
P. luminescens
cells. In an effort to identify bacterial factors that are required for nematode growth and reproduction, transposon-induced mutants of
P. luminescens
were screened for the loss of the ability to support growth and reproduction of
H. bacteriophora
nematodes. One mutant, NGR209, consistently failed to support nematode growth and reproduction. This mutant was also defective in the production of siderophore and antibiotic activities. The transposon was inserted into an open reading frame homologous to
Escherichia coli
EntD, a 4′-phosphopantetheinyl (Ppant) transferase, which is required for the biosynthesis of the catechol siderophore enterobactin. Ppant transferases catalyze the transfer of the Ppant moiety from coenzyme A to a holo-acyl, -aryl, or -peptidyl carrier protein(s) required for the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Possible roles of a Ppant transferase in the ability of
P. luminescens
to support nematode growth and reproduction are discussed.
Genetic Requirements for Induction of Germination of Spores of
Bacillus subtilis
by Ca
2+
-Dipicolinate
Authors: Paidhungat Madan , Ragkousi Katerina , Setlow Peter
Published: July 2002

Dormant
Bacillus subtilis
spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca
2+
and dipicolinic acid (DPA). Nutrients bind receptors in the spore, and this binding triggers events in the spore core, including DPA excretion and rehydration, and also activates hydrolysis of the surrounding cortex through mechanisms that are largely unknown. As Ca
2+
-DPA does not require receptors to induce spore germination, we asked if this process utilizes other proteins, such as the putative cortex-lytic enzymes SleB and CwlJ, that are involved in nutrient-induced germination. We found that Ca
2+
-DPA triggers germination by first activating CwlJ-dependent cortex hydrolysis; this mechanism is different from nutrient-induced germination where cortex hydrolysis is not required for the early germination events in the spore core. Nevertheless, since nutrients can induce release of the spore's DPA before cortex hydrolysis, we examined if the DPA excreted from the core acts as a signal to activate CwlJ in the cortex. Indeed, endogenous DPA is required for nutrient-induced CwlJ activation and this requirement was partially remedied by exogenous Ca
2+
-DPA. Our findings thus define a mechanism for Ca
2+
-DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.
Different Physiological Roles of ATP- and PPi-Dependent Phosphofructokinase Isoenzymes in the Methylotrophic ActinomyceteAmycolatopsis methanolica
Authors: Alves A. M. C. R. , Euverink G. J. W. , Santos H. , Dijkhuizen L.
Published: July 2002

Cells of the actinomyceteAmycolatopsis methanolicagrown on glucose possess only a single, exclusively PPi-dependent phosphofructokinase (PPi-PFK) (A. M. C. R. Alves, G. J. W. Euverink, H. J. Hektor, J. van der Vlag, W. Vrijbloed, D.H.A. Hondmann, J. Visser, and L. Dijkhuizen, J. Bacteriol. 176:6827–6835, 1994). When this methylotrophic bacterium is grown on one-carbon (C1) compounds (e.g., methanol), an ATP-dependent phosphofructokinase (ATP-PFK) activity is specifically induced, completely replacing the PPi-PFK. The twoA. methanolicaPFK isoenzymes have very distinct functions, namely, in the metabolism of C6and C1carbon substrates. This is the first report providing biochemical evidence for the presence and physiological roles of PPi-PFK and ATP-PFK isoenzymes in a bacterium. The novel ATP-PFK enzyme was purified to homogeneity and characterized in detail at the biochemical and molecular levels. TheA. methanolicaATP-PFK and PPi-PFK proteins possess a low level of amino acid sequence similarity (24%), clearly showing that the two proteins are not the result of a gene duplication event. PPi-PFK is closely related to other (putative) actinomycete PFK enzymes. Surprisingly, theA. methanolicaATP-PFK is most similar to ATP-PFK from the protozoonTrypanosoma bruceiand PPi-PFK proteins from the bacteriaBorrelia burgdorferiandTreponema pallidum, both spirochetes, very distinct from actinomycetes. The data thus suggest thatA. methanolicaobtained the ATP-PFK-encoding gene via a lateral gene transfer event.
Prevalence of Antibodies to Hepatitis E Virus in Veterinarians Working with Swine and in Normal Blood Donors in the United States and Other Countries
Authors: Meng X. J. , Wiseman B. , Elvinger F. , Guenette D. K. , Toth T. E. , Engle R. E. , Emerson S. U. , Purcell R. H.
Published: July 2002

Hepatitis E virus (HEV) is endemic in many developing and some industrialized countries. It has been hypothesized that animals may be the source of infection. The recent identification of swine HEV in U.S. pigs and the demonstration of its ability to infect across species have lent credence to this hypothesis. To assess the potential risk of zoonotic HEV infection, we tested a total of 468 veterinarians working with swine (including 389 U.S. swine veterinarians) and 400 normal U.S. blood donors for immunoglobulin G anti-HEV. Recombinant capsid antigens from a U.S. strain of swine HEV and from a human HEV strain (Sar-55) were each used in an enzyme-linked immunosorbent assay. The anti-HEV prevalence assayed with the swine HEV antigen showed 97% concordance with that obtained with the human HEV antigen (κ = 92%). Among the 295 swine veterinarians tested from the eight U.S. states (Minnesota, Indiana, Nebraska, Iowa, Illinois, Missouri, North Carolina, and Alabama) from which normal blood donor samples were available, 26% were positive with Sar-55 antigen and 23% were positive with swine HEV antigen. In contrast, 18% of the blood donors from the same eight U.S. states were positive with Sar-55 antigen and 17% were positive with swine HEV antigen. Swine veterinarians in the eight states were 1.51 times more likely when tested with swine HEV antigen (95% confidence interval, 1.03 to 2.20) and 1.46 times more likely when tested with Sar-55 antigen (95% confidence interval, 0.99 to 2.17) to be anti-HEV positive than normal blood donors. We did not find a difference in anti-HEV prevalence between veterinarians who reported having had a needle stick or cut and those who had not or between those who spent more time (≥80% of the time) and those who spent less time (≤20% of the time) working with pigs. Similarly, we did not find a difference in anti-HEV prevalence according to four job categories (academic, practicing, student, and industry veterinarians). There was a difference in anti-HEV prevalence in both swine veterinarians and blood donors among the eight selected states, with subjects from Minnesota six times more likely to be anti-HEV positive than those from Alabama. Age was not a factor in the observed differences from state to state. Anti-HEV prevalence in swine veterinarians and normal blood donors was age specific and paralleled increasing age. The results suggest that swine veterinarians may be at somewhat higher risk of HEV infection than are normal blood donors.
The Human Factors YY1 and LSF Repress the Human Immunodeficiency Virus Type 1 Long Terminal Repeat via Recruitment of Histone Deacetylase 1
Authors: Coull Jason J. , Romerio Fabio , Sun Jian-Min , Volker Janet L. , Galvin Katherine M. , Davie James R. , Shi Yang , Hansen Ulla , Margolis David M.
Published: July 2002

Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.
Induction of Necrotic-Like Cell Death by Tumor Necrosis Factor Alpha and Caspase Inhibitors: Novel Mechanism for Killing Virus-Infected Cells
Authors: Li Ming , Beg Amer A.
Published: July 2002

Induction of apoptotic cell death generally requires the participation of cysteine proteases belonging to the caspase family. However, and similar to most cell types, mouse fibroblasts are normally resistant to tumor necrosis factor alpha (TNF-α)-induced apoptosis. Surprisingly, TNF-α treatment of vaccinia virus-infected mouse fibroblasts resulted in necrotic-like cell death, which was significantly reduced in cells infected with a vaccinia virus mutant lacking the caspase inhibitor B13R. Furthermore, TNF-α also induced necrotic-like cell death of fibroblasts in the presence of peptidyl caspase inhibitors. In both cases, necrosis was accompanied by generation of superoxide species. Caspase inhibitors also sensitized fibroblasts to killing by double-stranded RNA and gamma interferon. In all cases, cell death was efficiently blocked by antioxidants or mitochondrial respiratory chain inhibitors. These results define a new mitochondrion-dependent mechanism which may be important in the killing of cells infected with viruses encoding caspase inhibitors.