bluuebunny/crossref_metadata_2025 huggingface dataset:
The YadA Protein ofYersinia pseudotuberculosisMediates High-Efficiency Uptake into Human Cells under Environmental Conditions in Which Invasin Is Repressed
Authors: Eitel Julia , Dersch Petra
Published: October 2002

The YadA protein is a major adhesin ofYersinia pseudotuberculosisthat promotes tight adhesion to mammalian cells by binding to extracellular matrix proteins. In this study, we first addressed the possibility of competitive interference of YadA and the major invasive factor invasin and found that expression of YadA in the presence of invasin affected neither the export nor the function of invasin in the outer membrane. Furthermore, expression of YadA promoted both bacterial adhesion and high-efficiency invasion entirely independently of invasin. Antibodies against fibronectin and β1integrins blocked invasion, indicating that invasion occurs via extracellular-matrix-dependent bridging between YadA and the host cell β1integrin receptors. Inhibitor studies also demonstrated that tyrosine and Ser/Thr kinases, as well as phosphatidylinositol 3-kinase, are involved in the uptake process. Further expression studies revealed thatyadAis regulated in response to several environmental parameters, including temperature, ion and nutrient concentrations, and the bacterial growth phase. In complex medium, YadA production was generally repressed but could be induced by addition of Mg2+. Maximal expression ofyadAwas obtained in exponential-phase cells grown in minimal medium at 37°C, conditions under which the invasin gene is repressed. These results suggest that YadA ofY. pseudotuberculosisconstitutes another independent high-level uptake pathway that might complement other cell entry mechanisms (e.g., invasin) at certain sites or stages during the infection process.
Streptococcal Inhibitor of Complement Inhibits Two Additional Components of the Mucosal Innate Immune System: Secretory Leukocyte Proteinase Inhibitor and Lysozyme
Authors: Fernie-King Barbara A. , Seilly David J. , Davies Alexandra , Lachmann Peter J.
Published: October 2002

Streptococcal inhibitor of complement (SIC) is a 31-kDa extracellular protein of a few, very virulent, strains of
Streptococcus pyogenes
(particularly M1 strains). It is secreted in large quantities (about 5 mg/liter) and inhibits complement lysis by blocking the membrane insertion site on C5b67. We describe investigations into the interaction of SIC with three further major components of the innate immune system found in airway surface liquid, namely, secretory leukocyte proteinase inhibitor (SLPI), lysozyme, and lactoferrin. Enzyme-linked immunosorbent assays showed that SIC binds to SLPI and to both human and hen egg lysozyme (HEL) but not to lactoferrin. Studies using
125
I-labeled proteins showed that SIC binds approximately two molecules of SLPI and four molecules of lysozyme. SLPI binding shows little temperature dependence and a small positive enthalpy, suggesting that the binding is largely hydrophobic. By contrast, lysozyme binding shows strong temperature dependence and a substantial negative enthalpy, suggesting that the binding is largely ionic. Lysozyme is precipitated from solution by SIC. Further studies examined the ability of SIC to block the biological activities of SLPI and lysozyme. An M1 strain of group A streptococci was killed by SLPI, and the antibacterial activity of this protein was inhibited by SIC. SIC did not inhibit the antiproteinase activity of SLPI, implying that there is specific inhibition of the antibacterial domain. The antibacterial and enzymatic activities of lysozyme were also inhibited by SIC. SIC is the first biological inhibitor of the antibacterial action of SLPI to be described and may prove to be an important tool for investigating this activity in vivo. Inhibition of the antibacterial actions of SLPI and lysozyme would be advantageous to
S. pyogenes
in establishing colonization on mucosal surfaces, and we propose that this is the principal function of SIC.
Pneumococcal Infections in Humans Are Associated with Increased Apoptosis and Trafficking of Type 1 Cytokine-Producing T Cells
Authors: Kemp Kåre , Bruunsgaard Helle , Skinhøj Peter , Klarlund Pedersen Bente
Published: October 2002

Streptococcus pneumoniae
infections are associated with considerable morbidity and mortality throughout the world. The immunopathology is characterized by an intense inflammatory reaction, including a strong acute-phase response and increased numbers of neutrophils in the circulation. However, little is known regarding the T-cell response during in vivo infections in humans. The purpose of this study was to test the hypothesis that activated T cells producing type 1 cytokines were engaged in the host response to pneumococcal infections. The phenotype and function of T cells were studied in 22 patients at admission to a department of infectious diseases and after antibiotic treatment for 1 week compared with an age-matched, healthy control group. Pneumococcal infections induced lymphopenia in the circulation due to the disappearance of activated T lymphocytes with a type 1 cytokine profile. In contrast, the numbers of naive T cells and interleukin-4-producing T cells did not change. Activated type 1 cytokine-producing cells reappeared in the circulation in relation to the treatment and clinical improvement. The underlying mechanisms during infection may include sequestration in the peripheral tissues and/or apoptosis. In fact, increased activation-induced apoptosis in the remaining peripheral lymphocytes and elevated levels of soluble Fas ligand were detected at admission to the hospital. In conclusion, these data suggest that activated T lymphocytes with a type 1 cytokine profile are highly engaged in the in vivo immune response to
S. pneumoniae
.
Nitric Oxide InhibitsCoxiella burnetiiReplication and Parasitophorous Vacuole Maturation
Authors: Howe Dale , Barrows Lorraine F. , Lindstrom Nicole M. , Heinzen Robert A.
Published: October 2002

Nitric oxide is a recognized cytotoxic effector against facultative and obligate intracellular bacteria. This study examined the effect of nitric oxide produced by inducible nitric oxide synthase (iNOS) up-regulated in response to cytokine stimulation, or by a synthetic nitric oxide donor, on replication of obligately intracellularCoxiella burnetiiin murine L-929 cells. Immunoblotting and nitrite assays revealed thatC. burnetiiinfection of L-929 cells augments expression of iNOS up-regulated in response to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Infection in the absence of cytokine stimulation did not result in demonstrable up-regulation of iNOS expression or in increased nitrite production. Nitrite production by cytokine-treated cells was significantly inhibited by the iNOS inhibitorS-methylisothiourea (SMT). Treatment of infected cells with IFN-γ and TNF-α or the synthetic nitric oxide donor 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NONOate) had a bacteriostatic effect onC. burnetiireplication. Inhibition of replication was reversed upon addition of SMT to the culture medium of cytokine-treated cells. Microscopic analysis of infected cells revealed that nitric oxide (either cytokine induced or donor derived) inhibited formation of the mature (large) parasitophorous vacuole that is characteristic ofC. burnetiiinfection of host cells. Instead, exposure of infected cells to nitric oxide resulted in the formation of multiple small, acidic vacuoles usually containing oneC. burnetiicell. Removal of nitrosative stress resulted in the coalescence of small vacuoles to form a large vacuole harboring multipleC. burnetiicells. These experiments demonstrate that nitric oxide reversibly inhibits replication ofC. burnetiiand formation of the parasitophorous vacuole.
Increased Host Resistance againstPneumocystis cariniiPneumonia in γδ T-Cell-Deficient Mice: Protective Role of Gamma Interferon and CD8+T Cells
Authors: Steele Chad , Zheng Mingquan , Young Erana , Marrero Luis , Shellito Judd E. , Kolls Jay K.
Published: October 2002

Although a clear relationship between αβ T-cell receptor-positive (αβ-TCR+) CD4+T cells and susceptibility toPneumocystis cariniiinfection exists, the role of other T-cell subsets is less clearly defined. Previous studies have shown that γδ-TCR+T cells infiltrate into the lung duringP. cariniipneumonia. Therefore, the present study examined the role of γδ-TCR+T cells in host defense againstP. cariniipneumonia. C57BL/6 (control) and B6.129P2-Tcrdtm1Mom(γδ-TCR+T-cell-deficient) mice were inoculated intratracheally withP. carinii. At specific time points, mice were sacrificed and analyzed forP. cariniiburden, T-cell subsets, and cytokine levels in lung tissue. Analysis ofP. cariniiburden showed a more rapid and complete resolution of infection in γδ-TCR+T-cell-deficient mice than in C57BL/6 controls. This augmented resolution was associated with elevated gamma interferon (IFN-γ) levels in bronchoalveolar lavage fluid predominantly produced by CD8+T cells, as well as an increased recruitment of CD8+T cells in general. In separate experiments, neutralization of IFN-γ or depletion of CD8+T cells early during infection abolished the augmented resolution previously observed in γδ-TCR+T-cell-deficient mice. These results show that the presence of γδ-TCR+T cells modulates host susceptibility toP. cariniipneumonia through interactions with pulmonary CD8+T cells and tissue production of IFN-γ.
Campylobacter jejunifrom Patients with Guillain-Barré Syndrome Preferentially Expresses a GD1a-Like Epitope
Authors: Nachamkin Irving , Liu Jirong , Li Ming , Ung Huong , Moran Anthony P. , Prendergast Martina M. , Sheikh Kazim
Published: October 2002

GM1- and GD1a-like ganglioside mimicry inCampylobacter jejunilipooligosaccharide (LOS) is considered to be involved in the pathogenesis ofCampylobacter-induced Guillain-Barré syndrome (GBS). Compared with gastroenteritis-related isolates, GBS-relatedC. jejuniisolates were strongly associated with the expression of GD1a-like mimicry. The presence of a few genes involved in LOS ganglioside mimicry,cst-II,cgtA, andcgtB, was also associated with GBS-related strains. GD1a-like epitope expression may be an important virulence phenotype associated with the risk of developing GBS following campylobacter infection.
Salmonella enterica
Serovar Typhimurium Periplasmic Superoxide Dismutases SodCI and SodCII Are Required for Protection against the Phagocyte Oxidative Burst
Authors: Sly Laura M. , Guiney Donald G. , Reiner Neil E.
Published: October 2002

Vitamin D
3
(1,25-dihydroxycholecalciferol) induced the phagocyte oxidative burst and intracellular killing of
Salmonella enterica
serovar Typhimurium in a phosphatidylinositol 3-kinase-dependent manner. The antimicrobial effect was more pronounced for
Salmonella
SodCI and SodCII mutants, confirming the role of the phagocyte oxidase in the vitamin D
3
effect. The results for an in vitro system with human THP-1 cells correlate with in vivo virulence data for mice and show that both the SodCI and SodCII enzymes are required to protect against the oxidative burst.
The Human Immune Response to
Plasmodium falciparum
Includes Both Antibodies That Inhibit Merozoite Surface Protein 1 Secondary Processing and Blocking Antibodies
Authors: Nwuba Roseangela I. , Sodeinde Olugbemiro , Anumudu Chiaka I. , Omosun Yusuf O. , Odaibo Alexander B. , Holder Anthony A. , Nwagwu Mark
Published: October 2002

Malaria merozoite surface protein 1 (MSP1) is cleaved in an essential step during erythrocyte invasion. The responses of children to natural malaria infection included antibodies that inhibit this cleavage and others that block the binding of these inhibitory antibodies. There was no correlation between the titer of the antibody to the 19-kDa fragment of MSP1 and its inhibitory activity. These findings have implications for the design of MSP1-based vaccines.
Chlamydia pneumoniae
and Hyperlipidemia Are Co-Risk Factors for Atherosclerosis: Infection Prior to Induction of Hyperlipidemia Does Not Accelerate Development of Atherosclerotic Lesions in C57BL/6J Mice
Authors: Blessing Erwin , Campbell Lee Ann , Rosenfeld Michael E. , Kuo Cho-chou
Published: October 2002

Chlamydia pneumoniae
has been shown to accelerate atherosclerotic lesion development in hyperlipidemic animals. This study showed that
C. pneumoniae
did not accelerate lesion development in mice if a high-fat/high-cholesterol diet was started after infection, indicating that
C. pneumoniae
is a co-risk factor with hyperlipidemia for cardiovascular disease.
Yersinia
High-Pathogenicity Island Contributes to Virulence in
Escherichia coli
Causing Extraintestinal Infections
Authors: Schubert Sören , Picard Bertrand , Gouriou Stéphanie , Heesemann Jürgen , Denamur Erick
Published: October 2002

The
Yersinia
high-pathogenicity island (HPI) encodes an iron uptake system mediated by the siderophore yersiniabactin (Ybt) and confers the virulence of highly pathogenic
Yersinia
species. This HPI is also widely distributed in human pathogenic members of the family of
Enterobacteriaceae
, above all in extraintestinal pathogenic
Escherichia coli
(ExPEC). In the present study we demonstrate a highly significant correlation of a functional HPI and extraintestinal virulence in
E. coli
. Moreover, using a mouse infection model, we show for the first time that the HPI contributes to the virulence of ExPEC.
The
N
-Acetyltransferase RimJ Responds to Environmental Stimuli To Repress
pap
Fimbrial Transcription in
Escherichia coli
Authors: White-Ziegler Christine A. , Black Alia M. , Eliades Stacie H. , Young Sarah , Porter Kimberly
Published: October 2002

In uropathogenic
Escherichia coli
, P pili (Pap) facilitate binding to host epithelial cells and subsequent colonization. Whereas P pili can be produced at 37°C, the expression of these fimbriae is suppressed at 23°C. Previously, insertion mutations in
rimJ
, a gene encoding the N-terminal acetyltransferase of ribosomal protein S5, were shown to disrupt this thermoregulatory response, allowing
papBA
transcription at low temperature. In this study, we created an in-frame deletion of
rimJ
. This deletion relieved the repressive effects not only of low temperature but also of rich (Luria-Bertani [LB]) medium and glucose on
papBA
transcription, indicating that RimJ modulates
papBA
transcription in response to multiple environmental stimuli.
papI
transcription was also shown to be regulated by RimJ.
papBA
transcription is also controlled by a phase variation mechanism. We demonstrated that the regulators necessary to establish a phase ON state—PapI, PapB, Dam, Lrp, and cyclic AMP-CAP-are still required for
papBA
transcription in a
rimJ
mutant strain.
rimJ
mutations increase the rate at which bacteria transition into the phase ON state, indicating that RimJ inhibits the phase OFF→ON transition. A Δ
rimJ hns651
mutant is viable on LB medium but not on minimal medium. This synthetic lethality, along with transcriptional analyses, indicates that RimJ and H-NS work through separate pathways to control
papBA
transcription. Mutations in
rimJ
do not greatly influence the transcription of the
fan
,
daa
, or
fim
operon, suggesting that RimJ may be a
pap
-specific regulator. Overexpression of
rimJ
under conditions repressive for
papBA
transcription complements the Δ
rimJ
mutation but has little effect on transcription under activating conditions, indicating that the ability of RimJ to regulate transcription is environmentally controlled.
Trehalose-Mediated Inhibition of the Plasma Membrane H
+
-ATPase from
Kluyveromyces lactis
: Dependence on Viscosity and Temperature
Authors: Sampedro José G. , Muñoz-Clares Rosario A. , Uribe Salvador
Published: October 2002

The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H
+
-ATPase from
Kluyveromyces lactis
was studied at different temperatures. At 20°C, increasing concentrations of trehalose (0.2 to 0.8 M) decreased
V
max
and increased
S
0.5
(substrate concentration when initial velocity equals 0.5
V
max
), mainly at high trehalose concentrations (0.6 to 0.8 M). The quotient
V
max
/
S
0.5
decreased from 5.76 μmol of ATP mg of protein
−1
min
−1
mM
−1
in the absence of trehalose to 1.63 μmol of ATP mg of protein
−1
min
−1
mM
−1
in the presence of 0.8 M trehalose. The decrease in
V
max
was linearly dependent on solution viscosity (η), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramer's theory for reactions in solution. In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted. In the absence of trehalose, increasing the temperature up to 40°C resulted in an exponential increase in
V
max
and a decrease in enzyme cooperativity (
n
), while
S
0.5
was not modified. As temperature increased, the effect of trehalose on
V
max
decreased to become negligible at 40°C, in good correlation with the temperature-mediated decrease in viscosity. The trehalose-mediated increase in
S
0.5
was similar at all temperatures tested, and thus, trehalose effects on
V
max
/
S
0.5
were always observed. Trehalose increased the activation energy for ATP hydrolysis. Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock.
Role of the C-Terminal Domain of the Alpha Subunit of RNA Polymerase in LuxR-Dependent Transcriptional Activation of the
lux
Operon during Quorum Sensing
Authors: Finney Angela H. , Blick Robert J. , Murakami Katsuhiko , Ishihama Akira , Stevens Ann M.
Published: October 2002

During quorum sensing in
Vibrio fischeri
, the luminescence, or
lux
, operon is regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule [
N
-(3-oxohexanoyl) homoserine lactone]. LuxR, which binds to the
lux
operon promoter at a position centered at −42.5 relative to the transcription initiation site, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the α-subunit C-terminal domain (αCTD) of RNAP in LuxR-dependent transcriptional activation of the
lux
operon promoter has been investigated. The effects of 70 alanine substitution variants of the α subunit were determined in vivo by measuring the rate of transcription of the
lux
operon via luciferase assays in recombinant
Escherichia coli
. The mutant RNAPs from strains exhibiting at least twofold-increased or -decreased activity in comparison to the wild type were further examined by in vitro assays. Since full-length LuxR has not been purified, an autoinducer-independent N-terminally truncated form of LuxR, LuxRΔN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRΔN, and 14 alanine substitutions in the αCTD were identified as having negative effects on the rate of transcription from the
lux
operon promoter. Five of these 14 α variants were also involved in the mechanisms of both LuxR- and LuxRΔN-dependent activation in vivo. The positions of these residues lie roughly within the 265 and 287 determinants in α that have been identified through studies of the cyclic AMP receptor protein and its interactions with RNAP. This suggests a model where residues 262, 265, and 296 in α play roles in DNA recognition and residues 290 and 314 play roles in α-LuxR interactions at the
lux
operon promoter during quorum sensing.
Genome-Wide Transcriptional Profiling in a Histidine Kinase Mutant of
Helicobacter pylori
Identifies Members of a Regulon
Authors: Forsyth Mark H. , Cao Ping , Garcia Preston P. , Hall Joshua D. , Cover Timothy L.
Published: October 2002

To identify putative members of a regulon controlled by the
H. pylori
sensory histidine kinase HP0164 (HK0164), we constructed HK0164 null mutant
H. pylori
strains and analyzed bacterial gene transcription using DNA arrays. Seven genes were differentially expressed in multiple HK0164 mutant strains compared to their expression in control strains. Strain-dependent variations in differential expression were also detected. These results indicate that the signal transduction circuit utilizing HK0164 controls the transcription of at least seven genes in
H. pylori
.
Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-α Subunit in a Heterologous Host
Authors: Tooley Aaron J. , Glazer Alexander N.
Published: October 2002

The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-α subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in
Escherichia coli.
Cyanobacterial genes encoding enzymes required for the conversion of heme to 3
Z
-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3
Z-
phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid
trp-lac
(
trc
) promoter. Genes for the apo-phycoerythrocyanin α subunit (
pecA
) and the heterodimeric lyase/isomerase (
pecE
and
pecF
), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the
trc
promoter on a second plasmid. Upon induction
,
recombinant
E. coli
used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered
E. coli
strain that lacks
pecE
and
pecF.
By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
SalmonellaType III Secretion-Associated Protein InvE Controls Translocation of Effector Proteins into Host Cells
Authors: Kubori Tomoko , Galán Jorge E.
Published: October 2002

Salmonella entericaencodes a type III secretion system (TTSS) within a pathogenicity island located at centisome 63 (SPI-1), which is essential for its pathogenicity. This system mediates the transfer of a battery of bacterial proteins into the host cell with the capacity to modulate cellular functions. The transfer process is dependent on the function of protein translocases SipB, SipC, and SipD. We report here thatSalmonellaprotein InvE, which is also encoded within SPI-1, is essential for the translocation of bacterial proteins into host cells. AnS. entericaserovar Typhimurium mutant carrying a loss-of-function mutation ininvEshows reduced secretion of SipB, SipC, and SipD while exhibiting increased secretion of other TTSS effector proteins. We also demonstrate that InvE interacts with a protein complex formed by SipB, SipC, and their cognate chaperone, SicA. We propose that InvE controls protein translocation by regulating the function of the Sip protein translocases.
Palindromic Unit-Independent Transposition of IS1397inYersinia pestis
Authors: Wilde Caroline , Bachellier Sophie , Hofnung Maurice , Carniel Elisabeth , Clément Jean-Marie
Published: October 2002

Palindromic units (PUs) are intergenic repeated sequences scattered over the chromosomes ofEscherichia coliand several other enterobacteria. In the latter, IS1397, anE. coliinsertion sequence specific to PUs, transposes into PUs with sequences close to theE. coliconsensus. Reasons for this insertion specificity can relate to either a direct recognition of the target (by its sequence or its structure) by the transposase or an interaction between a specific host protein and the PU target DNA sequence. In this study, we show that forYersinia pestis, a species deprived of PUs, IS1397can transpose onto its chromosome, with transpositional hot spots. Our results are in favor of a direct recognition of target DNA by IS1397transposase.
Stochastic Assembly of Two-Component Staphylococcal γ-Hemolysin into Heteroheptameric Transmembrane Pores with Alternate Subunit Arrangements in Ratios of 3:4 and 4:3
Authors: Sugawara-Tomita Noriko , Tomita Toshio , Kamio Yoshiyuki
Published: October 2002

Self-assembling, pore-forming toxins from
Staphylococcus aureus
are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins. On the basis of previous studies, we have shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca. 200 kDa. Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure. When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3. The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione
S
-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively.
Site-Specific Recombination System Encoded by Toluene Catabolic Transposon Tn4651
Authors: Genka Hiroyuki , Nagata Yuji , Tsuda Masataka
Published: October 2002

The 56-kb class II toluene catabolic transposon Tn4651fromPseudomonas putidaplasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kbtnpT-res-tnpSregion is 48 kb apart from thetnpAgene (M. Tsuda, K.-I. Minegishi, and T. Iino, J. Bacteriol. 171:1386-1393, 1989). Detailed analysis of the 2.4-kb region revealed that thetnpSandtnpTgenes encoding the putative 323- and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bpressite. The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS. In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination. Analysis of site-specific recombination under theEscherichia coli recAcells indicated that (i) the site-specific resolution between the two copies of theressite on a single molecule was catalyzed by TnpS, (ii) the functionalressite was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution. It was also found that TnpS catalyzed the site-specific recombination between theressites located at two different molecules to form a cointegrate molecule. Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination.
Membrane Protein Degradation by FtsH Can Be Initiated from Either End
Authors: Chiba Shinobu , Akiyama Yoshinori , Ito Koreaki
Published: October 2002

FtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins inEscherichia coli. In this paper we investigated how membrane-embedded substrates are recognized by this enzyme. We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence. Subsequent proteolysis should involve dislocation of the substrates into the cytosol. We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail. This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain. These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction. Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously. These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH.
Factors Affecting Start Site Selection at the
Escherichia coli fis
Promoter
Authors: Walker Kimberly A. , Osuna Robert
Published: October 2002

Transcription initiation with CTP is an uncommon feature among
Escherichia coli
σ
70
promoters. The
fis
promoter (
fis
P), which is subject to growth phase-dependent regulation, is among the few that predominantly initiate transcription with CTP. Mutations in this promoter that cause a switch from utilization of CTP to either ATP or GTP as the initiation nucleotide drastically alter its growth phase regulation pattern, suggesting that the choice of the primary initiating nucleotide can significantly affect its regulation. To better understand what factors influence this choice in
fis
P, we made use of a series of promoter mutations that altered the nucleotide or position used for initiation. Examination of these promoters indicates that start site selection is determined by a combination of factors that include preference for a nucleotide distance from the −10 region (8 > 7 > 9 ≫ 6 ≫ 10 > 11), initiation nucleotide preference (A = G ≫ CTP ≥ UTP), the DNA sequence surrounding the initiation region, the position of the −35 region, and changes in the intracellular nucleoside triphosphate pools. We describe the effects that each of these factors has on start site selection in the
fis
P and discuss the interplay between position and nucleotide preference in this important process.
Structure and Function of Hib Pili fromHaemophilus influenzaeType b
Authors: Mu Xiang-Qi , Egelman Edward H. , Bullitt Esther
Published: October 2002

Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding ofHaemophilus influenzaetype b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 Å in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 Å along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenicEscherichia coliwere used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence ofH. influenzaetype b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.
Imbroglios of Viral Taxonomy: Genetic Exchange and Failings of Phenetic Approaches
Authors: Lawrence Jeffrey G. , Hatfull Graham F. , Hendrix Roger W.
Published: October 2002

The practice of classifying organisms into hierarchical groups originated with Aristotle and was codified into nearly immutable biological law by Linnaeus. The heart of taxonomy is the biological species, which forms the foundation for higher levels of classification. Whereas species have long been established among sexual eukaryotes, achieving a meaningful species concept for prokaryotes has been an onerous task and has proven exceedingly difficult for describing viruses and bacteriophages. Moreover, the assembly of viral “species” into higher-order taxonomic groupings has been even more tenuous, since these groupings were based initially on limited numbers of morphological features and more recently on overall genomic similarities. The wealth of nucleotide sequence information that catalyzed a revolution in the taxonomy of free-living organisms necessitates a reevaluation of the concept of viral species, genera, families, and higher levels of classification. Just as microbiologists discarded dubious morphological traits in favor of more accurate molecular yardsticks of evolutionary change, virologists can gain new insight into viral evolution through the rigorous analyses afforded by the molecular phylogenetics of viral genes. For bacteriophages, such dissections of genomic sequences reveal fundamental flaws in the Linnaean paradigm that necessitate a new view of viral evolution, classification, and taxonomy.
Membrane Association and Kinase-Like Motifs of the RamC Protein of
Streptomyces coelicolor
Authors: Hudson Michael E. , Zhang Dachuan , Nodwell Justin R.
Published: October 2002

The protein RamC is required for the production of the spore-forming cells called aerial hyphae by the filamentous bacterium
Streptomyces coelicolor.
We showed that RamC, which contains several weakly predicted membrane-spanning sequences, is located exclusively in the
S. coelicolor
membrane. By constructing site-directed mutants in the cloned
ramC
gene and complementing a
ramC
null mutant, we showed that protein kinase-like sequence motifs in the amino-terminal half of the protein are required for function in vivo
.
These data suggest that RamC is a membrane-associated receptor kinase.
Comparative Proteome Analysis ofBrucella melitensisVaccine Strain Rev 1 and a Virulent Strain, 16M
Authors: Eschenbrenner Michel , Wagner Mary Ann , Horn Troy A. , Kraycer Jo Ann , Mujer Cesar V. , Hagius Sue , Elzer Philip , DelVecchio Vito G.
Published: October 2002

The genusBrucellaconsists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the differentBrucellaspecies,Brucella melitensisis considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence inB. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the twoB. melitensisproteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.
Purification and Polar Localization of Pneumococcal LytB, a Putative Endo-β-
N
-Acetylglucosaminidase: the Chain-Dispersing Murein Hydrolase
Authors: De Las Rivas Blanca , García José L. , López Rubens , García Pedro
Published: October 2002

The DNA region encoding the mature form of a pneumococcal murein hydrolase (LytB) was cloned and expressed in
Escherichia coli
. LytB was purified by affinity chromatography, and its activity was suggested to be the first identified endo-β-
N
-acetylglucosaminidase of
Streptococcus pneumoniae
. LytB can remove a maximum of only 25% of the radioactivity from [
3
H]choline-labeled pneumococcal cell walls in in vitro assays. Inactivation of the
lytB
gene of wild-type strain R6 (R6B mutant) led to the formation of long chains but did not affect either total cell wall hydrolytic activity at the stationary phase of growth or development of genetic competence. Longer chains were formed when the
lytB
mutation was introduced into the M31 strain (M31B mutant), which harbors a complete deletion of
lytA
, which codes for the major autolysin. Furthermore, the use of this mutant revealed that LytB is the first nonautolytic murein hydrolase of pneumococcus. Purified LytB added to pneumococcal cultures of R6B or M31B was capable of dispersing, in a dose-dependent manner, the long chains characteristic of these mutants into diplococci or short chains, the typical morphology of R6 and M31 strains, respectively. In vitro acetylation of purified pneumococcal cell walls did not affect the activity of LytB, whereas that of the LytA amidase was drastically reduced. On the other hand, the use of a translational fusion between the gene (
gfp
) coding for the green fluorescent protein (GFP) and
lytB
supports the notion that LytB accumulates in the cell poles of either the wild-type R6,
lytB
mutants, or ethanolamine-containing cells (EA cells). The GFP-LytB fusion protein was also able to unchain the
lytB
mutants but not the EA cells. In contrast, translational fusion protein GFP-LytA preferentially bound to the equatorial regions of choline-containing cells but did not affect their average chain length. These observations suggest the existence of specific receptors for LytB that are positioned at the polar region on the pneumococcal surface, allowing localized peptidoglycan hydrolysis and separation of the daughter cells.
Mycobacterium smegmatisl-Alanine Dehydrogenase (Ald) Is Required for Proficient Utilization of Alanine as a Sole Nitrogen Source and Sustained Anaerobic Growth
Authors: Feng Zhengyu , Cáceres Nancy E. , Sarath Gautam , Barletta Raúl G.
Published: October 2002

NAD(H)-dependentl-alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination ofl-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald inMycobacterium smegmatis, we cloned thealdgene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence ofl- ord-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore,M. smegmatis aldnull mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. Thealdmutants grew poorly in minimal medium withl-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by theM. smegmatis aldmutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.
Regulatory Circuitry of the CsrA/CsrB and BarA/UvrY Systems ofEscherichia coli
Authors: Suzuki Kazushi , Wang Xin , Weilbacher Thomas , Pernestig Anna-Karin , Melefors Öjar , Georgellis Dimitris , Babitzke Paul , Romeo Tony
Published: October 2002

The global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation inEscherichia coli. CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex. CsrA indirectly activatescsrBtranscription, in an apparent autoregulatory mechanism. In the present study, we elucidate the intermediate regulatory circuitry of this system. Mutations affecting the BarA/UvrY two-component signal transduction system decreasedcsrBtranscription but did not affectcsrA′-′lacZexpression. TheuvrYdefect was severalfold more severe than that ofbarA. BothcsrAanduvrYwere required for optimalbarAexpression. The latter observation suggests an autoregulatory loop for UvrY. Ectopic expression ofuvrYsuppressed thecsrB-lacZexpression defects caused byuvrY,csrA, orbarAmutations;csrAsuppressedcsrAorbarAdefects; andbarAcomplemented only thebarAmutation. Purified UvrY protein stimulatedcsrB-lacZexpression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY oncsrBtranscription. Disruption ofsdiA, which encodes a LuxR homologue, decreased the expression ofuvrY′-′lacZandcsrB-lacZfusions but did not affectcsrA′-′lacZ. The BarA/UvrY system activated biofilm formation. Ectopic expression ofuvrYstimulated biofilm formation by acsrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development. Collectively, these results demonstrate thatuvrYresides downstream fromcsrAin a signaling pathway forcsrBand that CsrA stimulates UvrY-dependent activation ofcsrBexpression by BarA-dependent and -independent mechanisms.
Conidial Viability Assay for Rapid Susceptibility Testing of
Aspergillus
Species
Authors: Balajee S. Arunmozhi , Marr Kieren A.
Published: October 2002

Antifungal susceptibility testing of filamentous fungi has become more important given the recognition of drug-resistant organisms and the availability of therapies other than amphotericin B (AMB). As current microdilution and E-test methods are limited by a 2 to 3 day incubation time required to obtain results, a more rapid method for susceptibility testing of fungi is needed. We report here a flow cytometric assay that relies on conidial metabolism of the viability dye FUN-1. Conidia are incubated in media containing increasing concentrations of AMB for 3 h, exposed to FUN-1, and then analyzed by flow cytometry. Relative susceptibility to AMB can be measured both by forward and side scatter characteristics of the conidial population and by mean fluorescence intensity (MFI) of the dye. MIC, calculated as the concentration of AMB to yield 90% reduction in MFI relative to growth controls, was determined for 27 clinical isolates
Aspergillus
species and correlated well with the standard (i.e., NCCLS) method. The results of these studies illustrate a method by which AMB susceptibility can be rapidly and reproducibly determined by measuring conidial viability.
Multiple
Mycobacterium tuberculosis
Strains in Early Cultures from Patients in a High-Incidence Community Setting
Authors: Richardson Madalene , Carroll Nora M. , Engelke Erica , van der Spuy Gian D. , Salker Faeeza , Munch Zahn , Gie Robert P. , Warren Robin M. , Beyers Nulda , van Helden Paul D.
Published: October 2002

In an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled.
Mycobacterium tuberculosis
strains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple
M. tuberculosis
strains in the context of the molecular epidemiology of the study setting.
Evaluation of a New Etest for Detecting Metallo-β-Lactamases in Routine Clinical Testing
Authors: Walsh Timothy R. , Bolmström Anne , Qwärnström Anette , Gales Ana
Published: October 2002

Several Etest (AB BIODISK, Solna, Sweden) gradient formats were developed for detection of metallo-β-lactamases based on the reduction of imipenem (IP) or ceftazidime (TZ) MICs in the presence of EDTA or 2-mercaptopropionic acid (MPA). The Etest metallo-β-lactamase (Etest MBL) strips consisted of a double-sided seven-dilution range of IP or TZ (4 to 256 μg/ml) and IP or TZ (1 to 64 μg/ml) overlaid with a constant concentration of EDTA or MPA. The prototype strips were evaluated on several agar media (brain heart infusion agar, Isosensitest agar, nutrient agar, and Mueller-Hinton agar for aerobes and brucella blood agar for anaerobes) with 138 challenge strains:
Acinetobacter
spp. (
n
= 9),
Aeromonas
spp. (
n
= 8),
Chryseobacterium
spp. (
n
= 28),
Escherichia coli
(
n
= 1),
Klebsiella pneumoniae
(
n
= 4),
Pseudomonas aeruginosa
(
n
= 14),
Proteus mirabilis
(
n
= 3),
Serratia
spp. (
n
= 10),
Stenotrophomonas maltophilia
(
n
= 43),
Sphingobacterium
spp. (
n
= 3), and
Bacteroides fragilis
group (
n
= 15). PCR analysis using specific primers for IMP-1, L1, CcrA, and
bla
B/C
confirmed the presence of the metallo-β-lactamase genes. Enzyme assays were also performed with IP as an indicator substrate followed by EDTA inhibition profiles. EDTA was found to be a better inhibitor of metallo-β-lactamases, especially for anaerobes. IP was a better than TZ. Mueller-Hinton agar was the preferred medium, particularly when compared to Isosensitest agar, which frequently produced falsely low MICs for IP. Etest IP plus IP-EDTA with Mueller-Hinton agar had a sensitivity of 94% (79 of 84) and specificity of 95% (124 of 130). The Etest MBL strip appears to be an acceptable diagnostic reagent to detect metallo-β-lactamase phenotypes in the clinical microbiology laboratory.
Evaluation of Three Techniques for Detection of Low-Level Methicillin-Resistant
Staphylococcus aureus
(MRSA): a Disk Diffusion Method with Cefoxitin and Moxalactam, the Vitek 2 System, and the MRSA-Screen Latex Agglutination Test
Authors: Felten Annie , Grandry Bernadette , Lagrange Philippe Henri , Casin Isabelle
Published: October 2002

Very-low-level methicillin-resistant
Staphylococcus aureus
(MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible
S. aureus
(MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the
mecA
gene by PCR was considered to be the “gold standard.” We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-μg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter
−1
for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of
S. aureus
studied were divided into 69 MSSA (
mecA
-negative) and 83 MRSA (
mecA
-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-μg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-μg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA.