bluuebunny/crossref_metadata_2025_split huggingface dataset:
M+Group A Streptococci Are Phagocytized and Killed in Whole Blood by C5a-Activated Polymorphonuclear Leukocytes
Authors: DeMaster Eric , Schnitzler Norbert , Cheng Qi , Cleary Patrick 
Published: July 2002

ABSTRACTHistorically, resistance to phagocytosis has been determined by incubating group A streptococci in human blood and comparing the numbers of CFU before and after incubation. Utilizing a flow cytometry-based technique, we have investigated the phagocytosis of M+group A streptococci by polymorphonuclear leukocytes (PMNs) in heparinized human peripheral whole blood. Intracellular labeling of streptococci with a nontoxic fluorescent dye allowed us to quantify the association and phagocytosis of M+streptococci by PMNs in whole blood in the presence or absence of C5a, a physiologically important chemotactic activator of PMNs. We found that wild-type strains of group A streptococci that are resistant to phagocytosis (determined by the classical Lancefield method) readily associate with C5a-activated whole-blood PMNs. In the absence of opsonizing M-type-specific antibodies, the M+streptococci associated with PMNs are phagocytized and killed. In addition, blockade of the β2integrin, CD11b/CD18, with anti-human CD11b monoclonal antibody inhibited association between M+streptococci and C5a-activated PMNs. These findings establish a new relationship between M+streptococci and PMNs, in which C5a-activated PMNs have the capacity to kill M+streptococci in whole blood through a receptor-mediated phagocytic mechanism.
p53-Independent Expression of p21CIP1/WAF1in Plasmacytic Cells during G2Cell Cycle Arrest Induced byActinobacillus actinomycetemcomitansCytolethal Distending Toxin
Authors: Sato Tsuyoshi , Koseki Takeyoshi , Yamato Kenji , Saiki Keitarou , Konishi Kiyoshi , Yoshikawa Masanosuke , Ishikawa Isao , Nishihara Tatsuji 
Published: July 2002

ABSTRACTThe cytolethal distending toxin (CDT) fromActinobacillus actinomycetemcomitanshas been shown to induce cell cycle arrest in the G2/M phase in HeLa cells. In the present study, the mechanism of CDT-induced cell cycle arrest was investigated by using HS-72 cells, a murine B-cell hybridoma cell line. Using flow cytometric analysis, we found that the recombinant CDT (rCDT) fromA. actinomycetemcomitansinduced G2cell cycle arrest in HS-72 cells and that rCDT upregulated expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1and the tumor suppressor protein p53. HS-72 cells transfected with the E6/E7 gene of human papillomavirus type 16, which lacked rCDT-induced accumulation of p53, exhibited expression of p21CIP1/WAF1or G2cell cycle arrest upon exposure to rCDT. Furthermore, ectopic expression of a dominant negative p53 mutant did not inhibit rCDT-mediated p21CIP1/WAF1expression or G2cell cycle arrest in HS-72 cells. These results suggest that the CDT fromA. actinomycetemcomitansinduces p21CIP1/WAF1expression and G2cell cycle arrest in B-lineage cells by p53-independent pathways. Together with additional observations made with HeLa cells and COS-1 cells cultured with the rCDT fromA. actinomycetemcomitans,the results of this study indicate that CDT-induced p53 accumulation may not be required for G2cell cycle arrest and that an increased level of p21CIP1/WAF1may be important for sustaining G2cell cycle arrest in several mammalian cells.
Membrane Localization of the S1 Subunit of Pertussis Toxin in
Bordetella pertussis
and Implications for Pertussis Toxin Secretion
Authors: Farizo Karen M. , Fiddner Stefanie , Cheung Anissa M. , Burns Drusilla L. 
Published: July 2002

ABSTRACT

Pertussis toxin is secreted from
Bordetella pertussis
with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type
B. pertussis
, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of
B. pertussis
that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of
B. pertussis
. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of
Escherichia coli
(J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of
B. pertussis.
Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.
Virulence of
Streptococcus pneumoniae
: PsaA Mutants Are Hypersensitive to Oxidative Stress
Authors: Tseng Hsing-Ju , McEwan Alastair G. , Paton James C. , Jennings Michael P. 
Published: July 2002

ABSTRACT

psaA
encodes a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II) transport complex.
Streptococcus pneumoniae
D39
psaA
mutants have previously been shown to be significantly less virulent than wild-type D39, but the mechanism underlying the attenuation has not been resolved. In this study, we have shown that
psaA
and
psaD
mutants are highly sensitive to oxidative stress, i.e., to superoxide and hydrogen peroxide, which might explain why they are less virulent than the wild-type strain. Our investigations revealed altered expression of the key oxidative-stress response enzymes superoxide dismutase and NADH oxidase in
psaA
and
psaD
mutants, suggesting that PsaA and PsaD may play important roles in the regulation of expression of oxidative-stress response enzymes and intracellular redox homeostasis.
Immunization with the RgpA-Kgp Proteinase-Adhesin Complexes ofPorphyromonas gingivalisProtects against Periodontal Bone Loss in the Rat Periodontitis Model
Authors: Rajapakse P. Sunethra , O'Brien-Simpson Neil M. , Slakeski Nada , Hoffmann Brigitte , Reynolds Eric C. 
Published: July 2002

ABSTRACTA major virulence factor ofPorphyromonas gingivalisis the extracellular noncovalently associated complexes of Arg-X- and Lys-X-specific cysteine proteinases and adhesins designated the RgpA-Kgp complexes. In this study we investigated the ability of RgpA-Kgp as an immunogen to protect againstP. gingivalis-induced periodontal bone loss in the rat. Specific-pathogen-free Sprague-Dawley rats were immunized with either formalin-killed wholeP. gingivalisATCC 33277 cells with incomplete Freund's adjuvant, RgpA-Kgp with incomplete Freund's adjuvant, or incomplete Freund's adjuvant alone. The animals were then challenged by oral inoculation with liveP. gingivalisATCC 33277 cells. Marked periodontal bone loss was observed in animals immunized with incomplete Freund's adjuvant alone; this bone loss was significantly (P< 0.05) greater than that detected in animals immunized with formalin-killed whole cells or RgpA-Kgp or in unchallenged animals. There was no significant difference in periodontal bone loss between animals immunized with formalin-killed whole cells and those immunized with RgpA-Kgp. The bone loss in these animals was also not significantly different from that in unchallenged animals. DNA probe analysis of subgingival plaque samples showed that 100% of the animals immunized with incomplete Freund's adjuvant alone and challenged withP. gingivalisATCC 33277 were positive for the bacterium. However,P. gingivalisATCC 33277 could not be detected in subgingival plaque samples from animals immunized with formalin-killed whole cells or with RgpA-Kgp. Immunization with formalin-killed whole cells or RgpA-Kgp induced a high-titer serum immunoglobulin G2a response. Western blot analysis of RgpA-Kgp using pooled protective antisera taken from rats immunized with RgpA-Kgp revealed immunodominant bands at 44, 39, and 27 kDa. In conclusion, immunization with RgpA-Kgp restricted colonization byP. gingivalisand periodontal bone loss in the rat.
Point Mutations in a Peptidoglycan Biosynthesis Gene Cause Competence Induction in
Haemophilus influenzae
Authors: Ma Caixia , Redfield Rosemary J. 
Published: July 2002

ABSTRACT

We have identified three new
Haemophilus influenzae
mutations causing cells to exhibit extreme hypercompetence at all stages of growth. The mutations are in
murE
, which encodes the
meso
-diaminopimelate-adding enzyme of peptidoglycan synthesis. All are point mutations causing nonconservative amino acid substitutions, two at a poorly conserved residue (G
435
→R and G
435
→W) and the third at a highly conserved leucine (L
361
→S). The mutant strains have very similar phenotypes and do not exhibit any defects in cell growth, permeability, or sensitivity to peptidoglycan antibiotics. Cells retain the normal specificity of DNA uptake for the
H. influenzae
uptake signal sequence. The mutations do not bypass genes known to be needed for competence induction but do dramatically increase expression of genes required for the normal pathway of DNA uptake. We conclude that the mutations do not act by increasing cell permeability but by causing induction of the normal competence pathway via a previously unsuspected signal.
Genetic Antagonism and Hypermutability in
Mycobacterium smegmatis
Authors: Karunakaran Ponniah , Davies Julian 
Published: July 2002

ABSTRACT

Multidrug-resistant strains of
Mycobacterium tuberculosis
are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and
M. tuberculosis
strains that are resistant to both streptomycin and rifampin contain mutations in the
rpsL
and
rpoB
genes, respectively. Coexisting mutations of this kind in
Escherichia coli
have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084–2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157–1161, 1977). We investigated this possibility in
Mycobacterium smegmatis
by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of
M. smegmatis
under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in
M. smegmatis
showed that the frequencies were enhanced up to 10
4
-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.
ComP, a Pilin-Like Protein Essential for Natural Competence in
Acinetobacter
sp. Strain BD413: Regulation, Modification, and Cellular Localization
Authors: Porstendörfer Dirk , Gohl Olivia , Mayer Frank , Averhoff Beate 
Published: July 2002

ABSTRACT

We recently identified a pilin-like competence factor, ComP, which is essential for natural transformation of the gram-negative soil bacterium
Acinetobacter
sp. strain BD413. Here we demonstrate that transcription and synthesis of the pilin-like competence factor ComP are maximal in the late stationary growth phase, whereas competence is induced immediately after inoculation of a stationary-phase culture into fresh medium. Western blot analyses revealed three forms of ComP, one with an apparent molecular mass of 15 kDa, which correlates with the molecular mass deduced from the DNA sequence, one 20-kDa form, which was found to be glycosylated, and one 23-kDa form. The glycosylation of ComP was not required for its function in DNA binding and uptake. The 20-kDa form was present in the cytoplasmic membrane, the periplasm, and the outer membrane, whereas the 23-kDa form was located in the outer membrane and might be due to a further modification. Immunological data suggest that ComP is not a subunit of the pilus structures. Possible functions of ComP in the DNA transformation machinery of
Acinetobacter
sp. strain BD413 are discussed.
Genetic Analysis of an Incomplete
mutS
Gene from
Pseudomonas putida
Authors: Kurusu Yasurou , Narita Tomoaki , Suzuki Makoto , Watanabe Taeko 
Published: July 2002

ABSTRACT

We genetically characterized the
Pseudomonas putida mutS
gene and found that it encodes a smaller MutS protein than do the genes of other bacteria. This gene is able to function in the
mutS
mutants of
Escherichia coli
and
Bacillus subtilis
. A
P. putida mutS
mutant has a mutation frequency 1,000-fold greater than that of the wild-type strain.
Identification of a Serine Hydrolase Which Cleaves the Alicyclic Ring of Tetralin
Authors: Hernáez M. J. , Andújar E. , Ríos J. L. , Kaschabek S. R. , Reineke W. , Santero E. 
Published: July 2002

ABSTRACT

A gene designated
thnD
, which is required for biodegradation of the organic solvent tetralin by
Sphingomonas macrogoltabidus
strain TFA, has been identified. Sequence comparison analysis indicated that
thnD
codes for a carbon-carbon bond serine hydrolase showing highest similarity to hydrolases involved in biodegradation of biphenyl. An insertion mutant defective in ThnD accumulates the ring fission product which results from the extradiol cleavage of the aromatic ring of dihydroxytetralin. The gene product has been purified and characterized. ThnD is an octameric thermostable enzyme with an optimum reaction temperature at 65°C. ThnD efficiently hydrolyzes the ring fission intermediate of the tetralin pathway and also 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the ring fission product of the biphenyl
meta
-cleavage pathway. However, it is not active towards the equivalent intermediates of
meta
-cleavage pathways of monoaromatic compounds which have small substituents in C-6. When ThnD hydrolyzes the intermediate in the tetralin pathway, it cleaves a C-C bond comprised within the alicyclic ring of tetralin instead of cleaving a linear C-C bond, as all other known hydrolases of
meta
-cleavage pathways do. The significance of this activity of ThnD for the requirement of other activities to mineralize tetralin is discussed.
Identification of a Copper-Responsive Two-Component System on the Chromosome of
Escherichia coli
K-12
Authors: Munson George P. , Lam Deborah L. , Outten F. Wayne , O'Halloran Thomas V. 
Published: July 2002

ABSTRACT

Using a genetic screen we have identified two chromosomal genes,
cusRS
(
ylcA ybcZ
), from
Escherichia coli
K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of
pcoE
, a plasmid-borne gene from the
E. coli
copper resistance operon
pco
. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the
pco
operon of
E. coli
; CopR and CopS from the
cop
operon, which provides copper resistance to
Pseudomonas syringae
; and SilR and SilS from the
sil
locus, which provides silver ion resistance to
Salmonella enterica
serovar Typhimurium. The genes
cusRS
are also required for the copper-dependent expression of at least one chromosomal gene, designated
cusC
(
ylcB
), which is allelic to the recently identified virulence gene
ibeB
in
E. coli
K1. The
cus
locus may comprise a copper ion efflux system, because the expression of
cusC
is induced by high concentrations of copper ions. Furthermore, the translation products of
cusC
and additional downstream genes are homologous to known metal ion antiporters.
Nonspecific Adherence byActinobacillus actinomycetemcomitansRequires Genes Widespread inBacteriaandArchaea
Authors: Kachlany Scott C. , Planet Paul J. , Bhattacharjee Mrinal K. , Kollia Evyenia , DeSalle Rob , Fine Daniel H. , Figurski David H. 
Published: July 2002

ABSTRACTThe gram-negative coccobacillus,Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates ofA. actinomycetemcomitansform tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence ofA. actinomycetemcomitansto surfaces. The recently developed transposon IS903φkanwas used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad−). Unlike wild-type cells, Tad−mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that thetadgenes are involved in secretion of factors required for tight adherence ofA. actinomycetemcomitans. Remarkably, complete and highly conservedtadgene clusters are present in the genomes of the bubonic plague bacillusYersinia pestisand the human and animal pathogenPasteurella multocida. Partialtadloci also occur in strikingly diverseBacteriaandArchaea. Our results show that thetadgenes are required for tight adherence ofA. actinomycetemcomitansto surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose thattad-like genes have a significant role in microbial colonization.
Vancomycin Resistance Is Associated with Serine-Containing Peptidoglycan in
Enterococcus gallinarum
Authors: Grohs Patrick , Gutmann Laurent , Legrand Raymond , Schoot Bernard , Mainardi Jean Luc 
Published: July 2002

ABSTRACT

In
Enterococcus gallinarum
SC1, a low-level vancomycin-resistant strain, only monomeric muropentapeptides with a C-terminal
d
-alanine were detected after growth without vancomycin. In contrast, in SC1 induced by vancomycin, as well as in AIB39, a constitutive vancomycin-resistant strain, monomeric and dimeric muropentapeptides with a C-terminal
d
-serine were detected.
Environmental Regulation of Exopolysaccharide Production in
Sinorhizobium meliloti
Authors: Mendrygal Kiprian E. , González Juan E. 
Published: July 2002

ABSTRACT

Exopolysaccharide production by
Sinorhizobium meliloti
is required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners.
S. meliloti
wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits. The reason for the production of two functional exopolysaccharides is not clear. Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021. We found that phosphate concentrations determine which exopolysaccharide is produced by
S. meliloti
. The low-phosphate conditions normally found in the soil (1 to 10 μM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan. Interestingly, the EPS II produced by
S. meliloti
in low-phosphate conditions does not allow the invasion of alfalfa nodules. We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion. An analysis of the function of PhoB in this differential exopolysaccharide production is presented.
Cloning and Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon
Pyrobaculum islandicum
Authors: Kähler Markus , Antranikian Garabed 
Published: July 2002

ABSTRACT

In order to extend the limited knowledge about crenarchaeal DNA polymerases, we cloned a gene encoding a family B DNA polymerase from the hyperthermophilic crenarchaeon
Pyrobaculum islandicum
. The enzyme shared highest sequence identities with a group of phylogenetically related DNA polymerases, designated B3 DNA polymerases, from members of the kingdom
Crenarchaeota
,
Pyrodictium occultum
and
Aeropyrum pernix
, and several members of the kingdom
Euryarchaeota
. Six highly conserved regions as well as a DNA-binding motif, indicative of family B DNA polymerases, were identified within the sequence. Furthermore, three highly conserved 3′-5′ exonuclease motifs were also found. The gene was expressed in
Escherichia coli
, and the DNA polymerase was purified to homogeneity by heat treatment and affinity chromatography. Activity staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an active polypeptide of approximately 90 kDa. For the recombinant DNA polymerase from
P. islandicum
, activated calf thymus DNA was used as a substrate rather than primed single-stranded DNA. The enzyme was strongly inhibited by monovalent cations and
N
-ethylmaleimide; it is moderately sensitive to aphidicolin and dideoxyribonucleoside triphosphates. The half-life of the enzyme at 100 and 90°C was 35 min and >5 h, respectively. Interestingly, the pH of the assay buffer had a significant influence on the 3′-5′ exonuclease activity of the recombinant enzyme. Under suitable assay conditions for PCR, the enzyme was able to amplify λ DNA fragments of up to 1,500 bp.
Insertion Mutagenesis of
wca
Reduces Acid and Heat Tolerance of Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Mao Ying , Doyle Michael P. , Chen Jinru 
Published: July 2002

ABSTRACT

Strains of enterohemorrhagic
Escherichia coli
(EHEC) serotype O157:H7 produce under stress copious amounts of exopolysaccharide (EPS) composed of colanic acid (CA). Studies were performed to evaluate the association of production of CA with survival of EHEC under adverse environmental conditions. A CA-deficient mutant, M4020, was obtained from a CA-proficient parental strain,
E. coli
O157:H7 W6-13, by inserting a kanamycin resistance gene cassette (
kan
) into
wcaD
and
wcaE
, 2 of the 21 genes required for CA biosynthesis. M4020 was defective in CA production as determined from the ratio of uronic acid to protein (UA/P) of cells grown from 1 to 4 days at 25°C on minimal glucose agar (MGA), MacConkey agar, and sorbitol-MacConkey agar, and by colony morphology on MGA. The results of stress treatment revealed that M4020 was substantially less tolerant to acid (pH 4.5 and 5.5) and heat (55 and 60°C) in comparison to W6-13, indicating that CA confers on
E. coli
O157:H7 a protective effect from the environmental stresses of acid and heat.