bluuebunny/crossref_metadata_2025 huggingface dataset:
An α-Proteobacterium Converts Linear Alkylbenzenesulfonate Surfactants into Sulfophenylcarboxylates and Linear Alkyldiphenyletherdisulfonate Surfactants into Sulfodiphenylethercarboxylates
Authors: Schleheck David , Dong Wenbo , Denger Karin , Heinzle Elmar , Cook Alasdair M.
Published: July 2002

The surfactant linear alkylbenzenesulfonate (LAS; 0.5 mM) or linear monoalkyldiphenyletherdisulfonate (LADPEDS; 0.5 mM) in salts medium was easily degraded in laboratory trickling filters, whereas carbon-limited, aerobic enrichment cultures in suspended culture with the same inocula did not grow. We took portions of the trickling filters which degraded LADPEDS, shook the organisms from the solid support (polyester), and found that growth in suspended culture in LADPEDS-salts medium occurred only in the presence of some solid support (polyester fleece or glass wool), though little biomass was immobilized on the support. The end products in suspended culture were identical with those from the trickling filters. There was low plating efficiency of LADPEDS-grown cultures on complex medium, and no picked colony or mixture of colonies grew in LADPEDS-salts-glass wool medium. However, selective plates containing LADPEDS-salts medium solidified with agarose yielded LADPEDS-dependent, pinpoint colonies which could be picked singly and subcultured in selective liquid medium. Isolate DS-1 was a bacterium which showed 93% sequence homology (16S ribosomal DNA) to its nearest phylogenetic neighbor, an α-proteobacterium. Strain DS-1 grew heterotrophically in LADPEDS-salts-glass wool medium and converted the set of aryl-substituted alkanes to the corresponding aryl-substituted carboxylic acids of shorter chain length. Similarly, strain DS-1 grew heterotrophically with commercial LAS, converting it to a set of sulfophenylcarboxylates. Growth with a single isomer of LAS [3-(4-sulfophenyl)dodecane] was concomitant with excretion of 4-(4-sulfophenyl)hexanoate, which was identified by matrix-assisted laser desorption ionization mass spectrometry. The growth yield (6.4 g of protein/mol of C) indicated mass balance, which, with the specific growth rate (0.05 h−1), indicated a specific utilization rate of LAS of 2.2 mkat/kg of protein.
Phylogenetic Relationships of
Cryptosporidium
Parasites Based on the 70-Kilodalton Heat Shock Protein (HSP70) Gene
Authors: Sulaiman Irshad M. , Morgan Una M. , Thompson R. C. Andrew , Lal Altaf A. , Xiao Lihua
Published: July 2002

We have characterized the nucleotide sequences of the 70-kDa heat shock protein (HSP70) genes of
Cryptosporidium baileyi
,
C. felis
,
C. meleagridis
,
C. muris
,
C. serpentis
,
C. wrairi
, and
C. parvum
from various animals. Results of the phylogenetic analysis revealed the presence of several genetically distinct species in the genus
Cryptosporidium
and eight distinct genotypes within the species
C. parvum
. Some of the latter may represent cryptic species. The phylogenetic tree constructed from these sequences is in agreement with our previous results based on the small-subunit rRNA genes of
Cryptosporidium
parasites. The
Cryptosporidium
species formed two major clades: isolates of
C. muris
and
C. serpentis
formed the first major group, while isolates of
C. felis
,
C. meleagridis
,
C. wrairi
, and eight genotypes of
C. parvum
formed the second major group. Sequence variations were also observed between
C. muris
isolates from ruminants and rodents. The HSP70 gene provides another useful locus for phylogenetic analysis of the genus
Cryptosporidium
.
Cyt1A from
Bacillus thuringiensis
Lacks Toxicity to Susceptible and Resistant Larvae of Diamondback Moth (
Plutella xylostella
) and Pink Bollworm (
Pectinophora gossypiella
)
Authors: Meyer Susan K. , Tabashnik Bruce E. , Liu Yong-Biao , Wirth Margaret C. , Federici Brian A.
Published: July 2002

We tested Cyt1Aa, a cytolytic endotoxin of
Bacillus thuringiensis
, against susceptible and Cry1A-resistant larvae of two lepidopteran pests, diamondback moth (
Plutella xylostella
) and pink bollworm (
Pectinophora gossypiella
). Unlike previous results obtained with mosquito and beetle larvae, Cyt1Aa alone or in combination with Cry toxins was not highly toxic to the lepidopteran larvae that we examined.
Differential Effects of pH on the Pore-Forming Properties of
Bacillus thuringiensis
Insecticidal Crystal Toxins
Authors: Tran Le Binh , Vachon Vincent , Schwartz Jean-Louis , Laprade Raynald
Published: July 2002

The effect of pH on the pore-forming ability of two
Bacillus thuringiensis
toxins, Cry1Ac and Cry1C, was examined with midgut brush border membrane vesicles isolated from the tobacco hornworm,
Manduca sexta
, and a light-scattering assay. In the presence of Cry1Ac, membrane permeability remained high over the entire pH range tested (6.5 to 10.5) for KCl and tetramethylammonium chloride, but was much lower at pH 6.5 than at higher pHs for potassium gluconate, sucrose, and raffinose. On the other hand, the Cry1C-induced permeability to all substrates tested was much higher at pH 6.5, 7.5, and 8.5 than at pH 9.5 and 10.5. These results indicate that the pores formed by Cry1Ac are significantly smaller at pH 6.5 than under alkaline conditions, whereas the pore-forming ability of Cry1C decreases sharply above pH 8.5. The reduced activity of Cry1C at high pH correlates well with the fact that its toxicity for
M. sexta
is considerably weaker than that of Cry1Aa, Cry1Ab, and Cry1Ac. However, Cry1E, despite having a toxicity comparable to that of Cry1C, formed channels as efficiently as the Cry1A toxins at pH 10.5. These results strongly suggest that although pH can influence toxin activity, additional factors also modulate toxin potency in the insect midgut.
Molecular Diversity of
Lactobacillus
spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA
Authors: Heilig Hans G.H.J. , Zoetendal Erwin G. , Vaughan Elaine E. , Marteau Philippe , Akkermans Antoon D.L. , de Vos Willem M.
Published: July 2002

A
Lactobacillus
group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera
Leuconostoc
,
Pediococcus
, and
Weissella.
Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in
Lactobacillus
-like sequences, of which ca. 28% were most similar to the 16S rDNA of
Lactobacillus ruminis
. Moreover, four sequences of
Leuconostoc
species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using
Lactobacillus
-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The
Lactobacillus
community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the
Lactobacillus
community was observed during the first 5 months of an infant’s life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a
Lactobacillus
strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.
Identification of a Gene Essential for Sheathed Structure Formation in
Sphaerotilus natans
, a Filamentous Sheathed Bacterium
Authors: Suzuki Toshihiko , Kanagawa Takahiro , Kamagata Yoichi
Published: July 2002

Sphaerotilus natans
, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concentration for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn
5
mutagenesis. Of the approximately 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn
5
-flanking regions showed that five of the seven Tn
5
insertions converged at the same open reading frame, designated
sthA
. The deduced amino acids encoded by
sthA
were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that
sthA
is a crucial component responsible for sheath formation.
Calcium Phosphate Nanoparticle Adjuvant
Authors: He Qing , Mitchell Alaina R. , Johnson Stacy L. , Wagner-Bartak Claus , Morcol Tulin , Bell Steve J. D.
Published: July 2002

Vaccination to protect against human infectious diseases may be enhanced by using adjuvants that can selectively stimulate immunoregulatory responses. In a murine model, a novel nanoparticulate adjuvant composed of calcium phosphate (CAP) was compared with the commonly used aluminum (alum) adjuvants for its ability to induce immunity to herpes simplex virus type 2 (HSV-2) and Epstein-Barr virus (EBV) infections. Results indicated that CAP was more potent as an adjuvant than alum, elicited little or no inflammation at the site of administration, induced high titers of immunoglobulin G2a (IgG2a) antibody and neutralizing antibody, and facilitated a high percentage of protection against HSV-2 infection. Additional benefits of CAP include (i) an insignificant IgE response, which is an important advantage over injection of alum compounds, and (ii) the fact that CAP is a natural constituent of the human body. Thus, CAP is very well tolerated and absorbed. These studies were performed with animal models. By virtue of the potency of this CAP adjuvant and the relative absence of side effects, we believe that this new CAP formulation has great potential for use as an adjuvant in humans.
DNA Sequence and Comparison of Virulence Plasmids from
Rhodococcus equi
ATCC 33701 and 103
Authors: Takai Shinji , Hines Stephen A. , Sekizaki Tsutomu , Nicholson Vivian M. , Alperin Debra A. , Osaki Makoto , Takamatsu Daisuke , Nakamura Mutsu , Suzuki Kayo , Ogino Nobuko , Kakuda Tsutomu , Dan Hanhong , Prescott John F.
Published: July 2002

The virulence plasmids of the equine virulent strains
Rhodococcus equi
ATCC 33701 and 103 were sequenced, and their genetic structure was analyzed. p33701 was 80,610 bp in length, and p103 was 1 bp shorter; their sequences were virtually identical. The plasmids contained 64 open reading frames (ORFs), 22 of which were homologous with genes of known function and 3 of which were homologous with putative genes of unknown function in other species. Putative functions were assigned to five ORFs based on protein family characteristics. The most striking feature of the virulence plasmids was the presence of a 27,536-bp pathogenicity island containing seven virulence-associated protein (
vap
) genes, including
vapA
. These
vap
genes have extensive homology to
vapA
, which encodes a thermoregulated and surface-expressed protein. The pathogenicity island contained a LysR family transcriptional regulator and a two-component response regulator upstream of six of the
vap
genes. The
vap
genes were present as a cluster of three (
vapA
,
vapC
, and
vapD
), as a pair (
vapE
and
vapF
), or individually (
vapG
;
vapH
). A region of extensive direct repeats of unknown function, possibly associated with thermoregulation, was present immediately upstream of the clustered and the paired genes but not the individual
vap
genes. There was extensive homology among the C-terminal halves of all
vap
genes but not generally among the N-terminal halves. The remainder of the plasmid consisted of a large region which appears to be associated with conjugation functions and a large region which appears to be associated with replication and partitioning functions.
Nonopsonic Binding of Type III Group B Streptococci to Human Neutrophils Induces Interleukin-8 Release Mediated by the p38 Mitogen-Activated Protein Kinase Pathway
Authors: Albanyan Esam A. , Vallejo Jesus G. , Smith C. Wayne , Edwards Morven S.
Published: July 2002

Nonopsonic interaction of host immune cells with pathogens is an important first line of defense. We hypothesized that nonopsonic recognition between type III group B streptococcus and human neutrophils would occur and that the interaction would be sufficient to trigger neutrophil activation. By using a serum-free system, it was found that heat-killed type III group B streptococci bound to neutrophils in a rapid, stable, and inoculum-dependent manner that did not result in ingestion. Transposon-derived type III strain COH1-13, which lacks capsular polysaccharide, and strain COH1-11 with capsular polysaccharide lacking terminal sialic acid demonstrated increased neutrophil binding, suggesting that capsular polysaccharide masks an underlying binding site. Experiments using monoclonal antibodies to complement receptor 1 and to the I domain or lectin site of complement receptor 3 did not inhibit binding, indicating that the complement receptors used for ingestion of opsonized group B streptococci were not required for nonopsonic binding. Nonopsonic binding resulted in rapid activation of cellular p38 and p44/42 mitogen-activated protein kinases. This interaction was not an effective trigger for superoxide production but did promote release of the proinflammatory cytokine interleukin-8. The release of interleukin-8 was markedly suppressed by the p38 mitogen-activated protein kinase inhibitor SB203580 but was only minimally suppressed by the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD98059. Thus, nonopsonic binding of type III group B streptococci to neutrophils is sufficient to initiate intracellular signaling pathways and could serve as an arm of innate immunity of particular importance to the immature host.
Identification of a Human Lactoferrin-Binding Protein in
Gardnerella vaginalis
Authors: Jarosik Gregory P. , Land Carol Beth
Published: July 2002

Previous studies have shown that
Gardnerella vaginalis
can utilize iron-loaded human lactoferrin as a sole source of iron. In this study,
G. vaginalis
cells were shown to bind digoxigenin (DIG)-labeled human lactoferrin in a dot blot assay. Using the DIG-labeled human lactoferrin, a 120-kDa human lactoferrin-binding protein was detected by Western blot analysis of
G. vaginalis
proteins. The lactoferrin-binding activity of this protein was found to be heat stable. Competition studies indicated that this binding activity was specific for human lactoferrin. Treatment of
G. vaginalis
cells with proteases suggested that this protein was surface exposed. An increase in lactoferrin binding by the 120-kDa protein was observed in
G. vaginalis
cells grown under iron-restrictive conditions, suggesting that this activity may be iron regulated.
Gamma Interferon Is Essential for Clearing
Mycobacterium genavense
Infection
Authors: Ehlers Stefan , Richter Elvira
Published: July 2002

Factors determining the in vivo replication of the opportunistic pathogen
Mycobacterium genavense
are largely unknown. Following intravenous injection of a patient isolate,
M. genavense
could not be recovered by culture or detected by PCR in the livers or spleens of infected BALB/c mice. In contrast,
M. genavense
was found to chronically persist and multiply in the livers and spleens of intravenously infected syngeneic gamma-interferon-gene-deficient (GKO) mice as evidenced by acid-fast stains of infected tissues and recovery by both PCR and liquid culture following organ homogenization. In GKO mice,
M. genavense
elicited a chronic inflammatory response, resulting in marked splenomegaly and extensive lymphadenopathy. Granulomatous lesions in the livers of GKO mice were diffuse, were composed of monocytes, neutrophils, and CD3
+
cells, and were histochemically negative for inducible nitric oxide synthase.
Identification of a Novel Gene Cluster Encoding Staphylococcal Exotoxin-Like Proteins: Characterization of the Prototypic Gene and Its Protein Product, SET1
Authors: Williams Rachel J. , Ward John M. , Henderson Brian , Poole Stephen , O'Hara Bernard P. , Wilson Michael , Nair Sean P.
Published: July 2002

We report the discovery of a novel genetic locus withinStaphylococcus aureusthat encodes a cluster of at least five exotoxin-like proteins. Designated the staphylococcal exotoxin-like genes 1 to 5 (set1toset5), these open reading frames have between 38 and 53% homology to each other. All five proteins contain consensus sequences that are found in staphylococcal and streptococcal exotoxins and toxic shock syndrome toxin 1 (TSST-1). However, the SETs have only limited overall sequence homology to the enterotoxins and TSST-1 and thus represent a novel family of exotoxin-like proteins. The prototypic gene in this cluster,set1, has been cloned and expressed. Recombinant SET1 stimulated the production of interleukin-1β, interleukin-6, and tumor necrosis factor alpha by human peripheral blood mononuclear cells. PCR analysis revealed thatset1was distributed among other strains ofS. aureusbut not in the other staphylococcal species examined. Sequence analysis of theset1genes from different strains revealed at least three allelic variants. The protein products of these allelic variants displayed a 100-fold difference in their cytokine-inducing potency. The distribution of allelic variants of thesetgenes among strains ofS. aureusmay contribute to differences in the pathogenic potential of this bacterium.
Evaluation of Phase Variation of Nontypeable
Haemophilus influenzae
Lipooligosaccharide during Nasopharyngeal Colonization and Development of Otitis Media in the Chinchilla Model
Authors: Tong H. H. , Blue L. E. , James M. A. , Chen Y. P. , DeMaria T. F.
Published: July 2002

Nontypeable
Haemophilus influenzae
(NTHI) has four loci,
lic-1
to
lic-3
and
lgtC
, that generate phase-variable lipooligosaccharide (LOS) structures.
lic-1
, which is required for the expression of phosphorylcholine (ChoP), is the best characterized and is associated with an enhanced ability of
H. influenzae
to persist within the nasopharynges of infant rats. Recent data indicate that LOS impacts various aspects of NTHI virulence in the chinchilla model of nasopharyngeal colonization and otitis media (OM). In this study the effects of ChoP expression and the sequences of
lic-1
to
lic-3
and
lgtC
of NTHI strain 2019 were evaluated in the chinchilla OM model. Nasopharyngeal colonization data showed that a switch from the ChoP
−
to the ChoP
+
phenotype was observed as early as day 3 after intranasal inoculation. Chinchillas colonized by strains with the ChoP
+
phenotype demonstrated a significantly higher level of NTHI 2019 per milliliter of nasal lavage fluid than chinchillas colonized with predominantly the ChoP
−
variant (
P
< 0.05). The concentration of cells with the ChoP
+
phenotype in the middle ear was 3 log units higher than that of cells with the ChoP
−
variant (
P
< 0.01). There was a statistically significant association between ChoP
+
expression in the nasal lavage and the development of OM with culture-positive middle ear fluids in this model. These data suggest that expression of the ChoP
+
phenotype promotes enhanced nasopharyngeal colonization and development of OM.
Role of Mannoprotein in Induction and Regulation of Immunity toCryptococcus neoformans
Authors: Pietrella Donatella , Cherniak Robert , Strappini Carla , Perito Stefano , Mosci Paolo , Bistoni Francesco , Vecchiarelli Anna
Published: July 2002

Our previous observations showed that mannoprotein (MP) induces early and massive production of interleukin-12 (IL-12) in vitro. This study was designed to investigate whether this phenomenon could be applied in vivo and to determine the biological significance of MP inCryptococcus neoformansinfection. The results reported here show that MP treatment induces IL-12 secretion by splenic macrophages and IL-12 p40 mRNA in the brain. DuringC. neoformansinfection, MP reinforced IL-12 and IFN-γ secretion that coincided with enhanced antifungal activity of natural effector cells, early resolution of the inflammatory process, and clearance of fungal load from the brain. These studies show that MP is a key inflammatory mediator that induces a protective immune response againstC. neoformansinfection. This information can be used to facilitate the design of a rational approach to manipulate the immune response toC. neoformans.
Ligand-Binding Properties of the Carboxyl-Terminal Repeat Domain of
Streptococcus mutans
Glucan-Binding Protein A
Authors: Haas Wolfgang , Banas Jeffrey A.
Published: July 2002

Streptococcus mutans
glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote
S. mutans
attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypothesis and to quantitate the ligand-binding specificities of the GbpA GBD, several fusion proteins were generated and tested by affinity electrophoresis or by precipitation of protein-ligand complexes, allowing the determination of binding constants. It was determined that the 16 YG repeats in GbpA comprise its GBD and that GbpA has a greater affinity for dextran (a water-soluble form of glucan) than for mutan (a water-insoluble form of glucan). Placement of the GBD at the carboxyl terminus was necessary for maximum glucan binding, and deletion of as few as two YG repeats from either end of the GBD reduced the affinity for dextran by over 10-fold. Interestingly, the binding constant of GbpA for dextran was 34-fold higher than that calculated for the GBDs of two
S. mutans
GTFs, one of which catalyzes the synthesis of water-soluble glucan and the other of which catalyzes the synthesis of water-insoluble glucan.
Autophosphorylation of Phosphoglucosamine Mutase from
Escherichia coli
Authors: Jolly Laure , Pompeo Frédérique , van Heijenoort Jean , Fassy Florence , Mengin-Lecreulx Dominique
Published: July 2002

Phosphoglucosamine mutase (GlmM) catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, an essential step in the pathway for UDP-
N
-acetylglucosamine biosynthesis in bacteria. This enzyme must be phosphorylated to be active and acts according to a ping-pong mechanism involving glucosamine-1,6-diphosphate as an intermediate (L. Jolly, P. Ferrari, D. Blanot, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx, Eur. J. Biochem. 262:202–210, 1999). However, the process by which the initial phosphorylation of the enzyme is achieved in vivo remains unknown. Here we show that the phosphoglucosamine mutase from
Escherichia coli
autophosphorylates in vitro in the presence of [
32
P]ATP. The same is observed with phosphoglucosamine mutases from other bacterial species, yeast
N
-acetylglucosamine-phosphate mutase, and rabbit muscle phosphoglucomutase. Labeling of the
E. coli
GlmM enzyme with [
32
P]ATP requires the presence of a divalent cation, and the label is subsequently lost when the enzyme is incubated with either of its substrates. Analysis of enzyme phosphorylation by high-pressure liquid chromatography and coupled mass spectrometry confirms that only one phosphate has been covalently linked to the enzyme. Only phosphoserine could be detected after acid hydrolysis of the labeled protein, and site-directed mutagenesis of serine residues located in or near the active site identifies the serine residue at position 102 as the site of autophosphorylation of
E. coli
GlmM.
A Novel Spore Peptidoglycan Hydrolase of
Bacillus cereus
: Biochemical Characterization and Nucleotide Sequence of the Corresponding Gene,
sleL
Authors: Chen Yinghua , Fukuoka Satoshi , Makino Shio
Published: July 2002

The exudate of germinated spores of
B. cereus
IFO 13597 in 0.15 M KCl–50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has substrate specificity for fragmented spore cortex from wild-type organisms (cortical-fragment-lytic enzyme [CFLE]), in addition to a previously characterized germination-specific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme [SCLE]) (R. Moriyama, S. Kudoh, S. Miyata, S. Nonobe, A. Hattori, and S. Makino, J. Bacteriol. 178:5330–5332, 1996). CFLE was not capable of degrading isolated cortical fragments from spores of
Bacillus subtilis
ADD1, which lacks muramic acid δ-lactam. This suggests that CFLE cooperates with SCLE in cortex hydrolysis during germination. CFLE was purified in an active form and identified as a 48-kDa protein which functions as an
N
-acetylglucosaminidase. Immunochemical studies suggested that the mature enzyme is localized on a rather peripheral region of the dormant spore, probably the exterior of the cortex layer. A gene encoding the enzyme,
sleL
, was cloned in
Escherichia coli
, and the nucleotide sequence was determined. The gene encodes a protein of 430 amino acids with a deduced molecular weight of 48,136. The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins. Inspection of the data banks showed no similarity of CFLE with
N
-acetylglucosaminidases found so far, suggesting that CFLE is a novel type of
N
-acetylglucosaminidase. The
B. subtilis
genome sequence contains genes,
yaaH
and
ydhD
, which encode putative proteins showing similarity to SleL.
Activities of
virE1
and the VirE1 Secretion Chaperone in Export of the Multifunctional VirE2 Effector via an
Agrobacterium
Type IV Secretion Pathway
Authors: Zhao Zhenming , Sagulenko Evgeniy , Ding Zhiyong , Christie Peter J.
Published: July 2002

Agrobacterium tumefaciens
uses a type IV secretion system to deliver oncogenic nucleoprotein particles and effector proteins, such as the multifunctional VirE2 protein, to plant cells. In this study, we examined the function of
virE1
and its product, the VirE1 secretion chaperone, in mediating VirE2 export. A nonpolar
virE1
null mutant accumulated low levels of VirE2, and
trans
expression of
virE1
in this mutant only partially restored VirE2 abundance. Deletion of
virE1
did not affect transcription but decreased translation of
virE2
, as shown by analysis of
lacZ
transcriptional and translational fusions. VirE2 was stable for a prolonged period, more than 6 h, when it was expressed in
cis
with
virE1
, and it exhibited half-lives of about 2 h when it was expressed in
trans
with
virE1
and less than 10 min when it was expressed in the absence of
virE1
, as shown by pulse-chase experiments. VirE1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synthesized in
A. tumefaciens
. VirE1 self-association was demonstrated by using bacteriophage λ cI repressor fusion and pull-down assays, and evidence of VirE1 homomultimerization in vivo was obtained by native polyacrylamide gel electrophoresis and gel filtration chromatography. A putative VirE1-VirE2 complex with a molecular mass of about 70 to 80 kDa was detected by gel filtration chromatography of extracts from wild-type cells, whereas higher-order VirE2 complexes or aggregates were detected in extracts from a
virE1
mutant. Taken together, our findings show that
virE1
contributes in several ways to VirE2 export:(i)
virE1
regulates efficient
virE2
translation in the context of expression from the native

P
virE

promoter; (ii) the VirE1 secretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a predicted 2:1 stoichiometry that inhibits assembly of higher-order VirE2 complexes or aggregates.
Diversity of
Streptococcus salivarius ptsH
Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System
Authors: Thomas Suzanne , Brochu Denis , Vadeboncoeur Christian
Published: July 2002

In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His∼P), HPr(Ser-P), and HPr(Ser-P)(His∼P), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of
Streptococcus salivarius ptsH
mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two
S. salivarius
mutants that produced approximately two- and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of
ptsH
reduced
ptsI
expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for α-galactosidase, β-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of α-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and the cellular levels of HPr(Ser-P).
Heat Shock Proteins Do Not Influence Wet Heat Resistance of
Bacillus subtilis
Spores
Authors: Melly Elizabeth , Setlow Peter
Published: July 2002

Spores of
Bacillus subtilis
are significantly more resistant to wet heat than are their vegetative cell counterparts. Analysis of the effects of mutations in and the expression of fusions of a coding gene for a thermostable β-galactosidase to a number of heat shock genes has shown that heat shock proteins play no significant role in the wet heat resistance of
B. subtilis
spores.
Involvement of Two Distinct Catabolite-Responsive Elements in Catabolite Repression of the
Bacillus subtilis myo
-Inositol (
iol
) Operon
Authors: Miwa Yasuhiko , Fujita Yasutaro
Published: July 2002

The
Bacillus subtilis
inositol operon (
iolABCDEFGHIJ
) is involved in
myo
-inositol catabolism. Glucose repression of the
iol
operon induced by inositol is exerted through catabolite repression mediated by CcpA and the
iol
induction system mediated by IolR. In this study, we identified two
iol
catabolite-responsive elements (
cre
's), to which CcpA complexed with P-Ser-HPr or P-Ser-Crh probably binds. One is located in
iolB
(
cre
-
iolB
, nucleotides +2397 to +2411; +1 is the transcription initiation nucleotide), which was the only
cre
-
iol
found in the previous
cre
search of the
B. subtilis
genome using a query sequence of WTGNAANCGNWNNCW (W stands for A or T, and N stands for any base). Deletion and base substitution analysis of the
iol
region indicated that
cre
-
iolB
functions even if it is located far downstream of the
iol
promoter. Further deletion and base substitution analysis revealed another
cre
located between the
iol
promoter and the
iolA
gene (
cre
-i
iolA
, nucleotides +86 to +100); the prefix “i” indicates a location in the intergenic region. Both
cre
-i
iolA
and
cre
-
iolB
appeared to be recognized to almost the same extent by CcpA complexed with either P-Ser-HPr or P-Ser-Crh. Sequence alignment of the six known
cre
's, including
cre
-i
iolA
, which were not revealed in the previous
cre
search, exhibited another consensus sequence of WTGAAARCGYTTWWN (R stands for A or G, and Y stands for C or T); the right two thymines (TT) were found to be essential for the function of
cre
-i
iolA
by means of base substitution analysis. A
cre
search with this query sequence led to the finding of 14 additional putative
cre
's.
Recombination in theompAGene but Not theomcBGene ofChlamydiaContributes to Serovar-Specific Differences in Tissue Tropism, Immune Surveillance, and Persistence of the Organism
Authors: Millman Kim L. , Tavaré Simon , Dean Deborah
Published: July 2002

Sequences of the major outer membrane protein (MOMP) gene (ompA) and the outer membrane complex B protein gene (omcB) fromChlamydia trachomatis, Chlamydia pneumoniae, andChlamydia psittaciwere analyzed for evidence of intragenic recombination and for linkage equilibrium. The Sawyer runs test, compatibility matrices, and index of association analyses provided substantial evidence that there has been a history of intragenic recombination atompAincluding one instance of interspecies recombination between theC. trachomatismouse pneumonitis strain and theC. pneumoniaehorse N16 strain. Although none of these methods detected intragenic recombination withinomcB, differences in divergence reported in earlier studies suggested that there has been intergenic recombination involvingomcB, and the analyses presented in this study are consistent with this. ForC. trachomatis, index-of-association analyses suggested a higher degree of recombination for C class than for B class strains and a higher degree of recombination in the downstream half ofompA. In concordance with these findings, many significant breakpoints were found in variable segments 3 and 4 of MOMP for the recombinant strains D/B120, G/UW-57, E/Bour, and LGV-98 identified in this study. We provide examples of how genetic diversity generated by repeated recombination in these regions may be associated with evasion of immune surveillance, serovar-specific differences in tissue tropism, and persistence.
Diel Expression of Cell Cycle-Related Genes in Synchronized Cultures of
Prochlorococcus
sp. Strain PCC 9511
Authors: Holtzendorff J. , Partensky F. , Jacquet S. , Bruyant F. , Marie D. , Garczarek L. , Mary I. , Vaulot D. , Hess W. R.
Published: July 2002

The cell cycle of the chlorophyll
b
-possessing marine cyanobacterium
Prochlorococcus
is highly synchronized under natural conditions. To understand the underlying molecular mechanisms we cloned and sequenced
dnaA
and
ftsZ
, two key cell cycle-associated genes, and studied their expression. An axenic culture of
Prochlorococcus
sp. strain PCC 9511 was grown in a turbidostat with a 12 h–12 h light-dark cycle for 2 weeks. During the light periods, a dynamic light regimen was used in order to simulate the natural conditions found in the upper layers of the world's oceans. This treatment resulted in strong cell cycle synchronization that was monitored by flow cytometry. The steady-state mRNA levels of
dnaA
and
ftsZ
were monitored at 4-h intervals during four consecutive division cycles. Both genes exhibited clear diel expression patterns with mRNA maxima during the replication (S) phase. Western blot experiments indicated that the peak of FtsZ concentration occurred at night, i.e., at the time of cell division. Thus, the transcript accumulation of genes involved in replication and division is coordinated in
Prochlorococcus
sp. strain PCC 9511 and might be crucial for determining the timing of DNA replication and cell division.
Cloning and Characterization of the
pnb
Genes, Encoding Enzymes for 4-Nitrobenzoate Catabolism in
Pseudomonas putida
TW3
Authors: Hughes Michelle A. , Williams Peter A.
Published: July 2002

Pseudomonas putida
strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate [PCA]) to central metabolites. We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA. In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the oxidation of 2 mol of NADH per mol of 4NBen, a reductase-like enzyme (Orf1) which appears to have no function in the pathway, a regulator protein (PnbR) of the LysR family, a 4-hydroxylaminobenzoate lyase (PnbB) which catalyzes the conversion of 4-hydroxylaminobenzoate to PCA and ammonium, and a second lyase-like enzyme (Orf2) which is closely associated with
pnbB
but appears to have no function in the pathway. The central
pnbR
gene is transcribed in the opposite direction to the other four genes. These genes complete the characterization of the whole pathway of 4-nitrotoluene catabolism to the ring cleavage substrate PCA in
P. putida
strain TW3.
In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated ς
54
-Dependent Po Promoter
Authors: Sze Chun Chau , Laurie Andrew D. , Shingler Victoria
Published: July 2002

Transcription from the
Pseudomonas
CF600-derived ς
54
-dependent promoter Po is controlled by the aromatic-responsive activator DmpR. Here we examine the mechanism(s) by which integration host factor (IHF) stimulates DmpR-activated transcriptional output of the Po promoter both in vivo and in vitro. In vivo, the Po promoter exhibits characteristics that typify many ς
54
-dependent promoters, namely, a phasing-dependent tolerance with respect to the distance from the regulator binding sites to the distally located RNA polymerase binding site, and a strong dependence on IHF for optimal promoter output. IHF is shown to affect transcription via structural repercussions mediated through binding to a single DNA signature located between the regulator and RNA polymerase binding sites. In vitro, using DNA templates that lack the regulator binding sites and thus bypass a role of IHF in facilitating physical interaction between the regulator and the transcriptional apparatus, IHF still mediates a DNA binding-dependent stimulation of Po transcription. This stimulatory effect is shown to be independent of previously described mechanisms for the effects of IHF at ς
54
promoters such as aiding binding of the regulator or recruitment of ς
54
-RNA polymerase via UP element-like DNA. The effect of IHF could be traced to promotion and/or stabilization of open complexes within the nucleoprotein complex that may involve an A+T-rich region of the IHF binding site and promoter-upstream DNA. Mechanistic implications are discussed in the context of a model in which IHF binding results in transduction of DNA instability from an A+T-rich region to the melt region of the promoter.
Proteome and Transcriptome Analysis of the Virulence Genes Regulated by the VirR/VirS System in
Clostridium perfringens
Authors: Shimizu Takeshi , Shima Kensuke , Yoshino Ken-ichi , Yonezawa Kazuyoshi , Shimizu Tohru , Hayashi Hideo
Published: July 2002

The proteins under the control of the two-component system VirR/VirS in
Clostridium perfringens
were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the
virR
mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of
C. perfringens
.
Genome-Wide Dynamic Transcriptional Profiling of the Light-to-Dark Transition inSynechocystissp. Strain PCC 6803
Authors: Gill Ryan T. , Katsoulakis Eva , Schmitt William , Taroncher-Oldenburg Gaspar , Misra Jatin , Stephanopoulos Gregory
Published: July 2002

We report the results of whole-genome transcriptional profiling of the light-to-dark transition with the model photosynthetic prokaryoteSynechocystissp. strain PCC 6803 (Synechocystis). Experiments were conducted by growingSynechocystiscultures to mid-exponential phase and then exposing them to two cycles of light/dark conditions, during which RNA samples were obtained. These samples were probed with a full-genome DNA microarray (3,169 genes, 20 samples) as well as a partial-genome microarray (88 genes, 29 samples). We concluded that (i) 30-min sampling intervals accurately captured transcriptional dynamics throughout the light/dark transition, (ii) 25% of theSynechocystisgenes (783 genes) responded positively to the presence of light, and (iii) the response dynamics varied greatly for individual genes, with a delay of up to 120 to 150 min for some genes. Four classes of genes were identified on the basis of their dynamic gene expression profiles: class I (108 genes, 30-min response time), class II (279 genes, 60 to 90 min), class III (258 genes, 120 to 150 min), and class IV (138 genes, 180 min). The dynamics of several transcripts from genes involved in photosynthesis and primary energy generation are discussed. Finally, we applied Fisher discriminant analysis to better visualize the progression of the overall transcriptional program throughout the light/dark transition and to determine those genes most indicative of the lighting conditions during growth.
The Extracellular Transport Signal of the
Vibrio cholerae
Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555
Authors: Folster Jason P. , Connell Terry D.
Published: July 2002

ChiA, an 88-kDa endochitinase encoded by the
chiA
gene of the gram-negative enteropathogen
Vibrio cholerae,
is secreted via the
eps
-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of
Escherichia coli
and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(
chiA
::Kan
r
), a
chiA
-deficient but secretion-competent mutant of
V. cholerae
. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type
V. cholerae
but not by an
epsD
mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of
V. cholerae
. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.
Validity of Earlier Positivity of Central Venous Blood Cultures in Comparison with Peripheral Blood Cultures for Diagnosing Catheter-Related Bacteremia in Cancer Patients
Authors: Malgrange Véronique Bussy , Escande Marie Christine , Theobald Serge
Published: July 2002

We carried out a prospective study in two French Comprehensive Cancer Centers (95 and 184 beds, respectively) to assess the validity of a test based on the earlier positivity of central venous blood cultures in comparison with peripheral blood cultures for predicting catheter-related bacteremia. The differences between the times to positivity for the 21 patients with clinical catheter-related bacteremia and the differences between the times to positivity for the nine patients with bacteremia due to another source were compared by the median test. The difference between the median values was significant (
P
= 0.0003). A receiver operating characteristic curve was constructed to determine the optimum threshold of the test, which appeared to be at the cutoff point of ≥+3 h, with 100% specificity and 81% sensitivity. The positive and negative predictive values obtained with this cutoff point confirmed the efficacy of the test for predicting the presence or absence of catheter-related bacteremia in cancer patients. The cutoff point was then used to post-classify the 68 episodes of bacteremia from an unknown source. The characteristics and clinical course of both the positive and negative post-classified episodes did not show that the test was clearly useful for a large number of clinical presentations. We therefore suggest restricting it to febrile neutropenic cancer patients for whom clinical signs of infection are slight or absent and when the test is positive.
First Report of Methicillin-Resistant
Staphylococcus aureus
with Reduced Susceptibility to Vancomycin in Thailand
Authors: Trakulsomboon Suwanna , Danchaivijitr Somwang , Rongrungruang Yong , Dhiraputra Chertsak , Susaemgrat Wattanachai , Ito Teruyo , Hiramatsu Keiichi
Published: July 2002

To investigate whether there are methicillin-resistant
Staphylococcus aureus
(MRSA) strains with reduced susceptibility to vancomycin in Thailand, a total of 155 MRSA strains isolated from patients hospitalized between 1988 and 1999 in university hospitals in Thailand were tested for glycopeptide susceptibility. All the strains were classified as susceptible to vancomycin and teicoplanin when judged by NCCLS criteria for glycopeptide susceptibility using the agar dilution MIC determination. Vancomycin MICs at which 50 and 90% of the isolates tested were inhibited (MIC
50
and MIC
90
, respectively) were 0.5 and 1 μg/ml, respectively, with a range of 0.25 to 2 μg/ml. For teicoplanin, MIC
50
and MIC
90
were 2 μg/ml, with a range of 0.5 to 4 μg/ml. However, one-point population analysis identified three MRSA strains, MR135, MR187, and MR209, which contained subpopulations of cells that could grow in 4 μg of vancomycin per ml. The proportions of the subpopulations were 2 × 10
−4
, 1.5 × 10
−6
, and 4 × 10
−7
, respectively. The subsequent performance of a complete population analysis and testing for the emergence of mutants with reduced susceptibility to vancomycin (MIC ≥ 8 μg/ml) confirmed that these strains were heterogeneously resistant to vancomycin. Two of these strains caused infection that was refractory to vancomycin therapy. Pulsed-field gel electrophoresis showed that the two strains had identical
Sma
I macrorestriction patterns and that they were one of the common types of MRSA isolated in the hospital. This is the first report of heterogeneous resistance to vancomycin in Thailand and an early warning for the possible emergence of vancomycin resistance in
S. aureus
in Southeast Asia.
Validation of the String Test for the Recovery of
Helicobacter pylori
from Gastric Secretions and Correlation of Its Results with Urea Breath Test Results, Serology, and Gastric pH Levels
Authors: Torres Javier , Camorlinga Margarita , Pérez-Peréz Guillermo , Gonzalez Gerardo , Muñoz Onofre
Published: July 2002

The efficacy of the string culture test to isolate
Helicobacter pylori
from gastric secretions of 28 volunteers was studied. With the urea breath test (UBT) as the “gold standard,” the string culture test showed a sensitivity of 75% and a specificity of 100%. The results of string culture did not correlate with the UBT results, with serum antibody levels, or with the pH levels of gastric secretions.