bluuebunny/crossref_metadata_2025 huggingface dataset:
vimA
Gene Downstream of
recA
Is Involved in Virulence Modulation in
Porphyromonas gingivalis
W83
Authors: Abaibou Hafid , Chen Zhuo , Olango G. Jon , Liu Yi , Edwards Jessica , Fletcher Hansel M.
Published: July 2002

A 0.9-kb open reading frame encoding a unique 32-kDa protein was identified downstream of the
recA
gene of
Porphyromonas gingivalis
. Reverse transcription-PCR and Northern blot analysis showed that both the
recA
gene and this open reading frame are part of the same transcriptional unit. This cloned fragment was insertionally inactivated using the
ermF-ermAM
antibiotic resistance cassette to create a defective mutant by allelic exchange. When plated on
Brucella
blood agar, the mutant strain, designated
P. gingivalis
FLL92, was non-black pigmented and showed significant reduction in beta-hemolysis compared with the parent strain,
P. gingivalis
W83. Arginine- and lysine-specific cysteine protease activities, which were mostly soluble, were approximately 90% lower than that of the parent strain. Expression of the
rgpA
,
rgpB
, and
kgp
protease genes was the same in
P. gingivalis
FLL92 as in the wild-type strain. In contrast to the parent strain,
P. gingivalis
FLL92 showed increased autoaggregration in addition to a significant reduction in hemagglutinating and hemolysin activities. In in vivo experiments using a mouse model,
P. gingivalis
FLL92 was dramatically less virulent than the parent strain. A molecular survey of this mutant and the parent strain using all known
P. gingivalis
insertion sequence elements as probes suggested that no intragenomic changes due to the movement of these elements have occurred in
P. gingivalis
FLL92. Taken together, these results suggest that the
recA
downstream gene, designated
vimA
(virulence-modulating gene), plays an important role in virulence modulation in
P. gingivalis
W83, possibly representing a novel posttranscriptional or translational regulation of virulence factors in
P. gingivalis
.
Microtubule- and Dynein-Mediated Movement of
Orientia tsutsugamushi
to the Microtubule Organizing Center
Authors: Kim Sang-Wook , Ihn Kyung-Soo , Han Seung-Hoon , Seong Seung-Yong , Kim Ik-Sang , Choi Myung-Sik
Published: July 2002

The host cell microfilaments and microtubules (MTs) are known to play a critical role in the life cycles of several pathogenic intracellular microbes by providing for successful invasion and promoting movement of the pathogen once inside the host cell cytoplasm.
Orientia tsutsugamushi
, an obligate intracellular bacterium, enters host cells by induced phagocytosis, escapes to the cytosol, and then replicates in the cytosol. ECV304 cells infected with
O. tsutsugamushi
revealed the colocalization of the MT organizing center (MTOC) and cytosolic orientiae by indirect immunofluorescence assay. Using immunofluorescence microscopy in the presence and absence of MT-depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of dynamitin [also called p50], which is known to associate with dynein-dependent movement), the movement of
O. tsutsugamushi
to the MTOC was also mediated by dynein, the minus-end-directed MT-related motor. Although the significance of this movement in the life cycle of
O. tsutsugamushi
was not proven, we propose that the cytosolic
O. tsutsugamushi
bacteria use MTs and dyneins to propel themselves from the cell periphery to the MTOC.
Resistance of T-Cell Receptor δ-Chain-Deficient Mice to ExperimentalCandidaalbicansVaginitis
Authors: Wormley Floyd L. , Steele Chad , Wozniak Karen , Fujihashi Kohtaro , McGhee Jerry R. , Fidel Paul L.
Published: July 2002

Conditions consistent with tolerance or immunoregulation have been observed in experimentalCandida albicansvaginal infections. The present study investigated the role of γ/δ T cells in experimental vaginal candidiasis. Results showed that T-cell receptor δ-chain-knockout mice had significantly less vaginal fungal burden when compared to wild-type mice, suggesting an immunoregulatory role for γ/δ T cells inCandidavaginitis.
Interleukin-4 Receptor–Stat6 Signaling in Murine Infections with a Tissue-Dwelling Nematode Parasite
Authors: Spencer L. , Shultz L. , Rajan T. V.
Published: July 2002

Interleukin-4 (IL-4) has been shown to be crucial in parasite expulsion in several gastrointestinal nematode infection models. Data from both epidemiological studies with humans and experimental infections in animals imply a critical role for the type II helper response, dominated by IL-4, in host protection. Here we utilized inbred mice on two distinct backgrounds to document the involvement of IL-4 in the clearance of a primary infection ofBrugiafrom the murine host. Our data from infections of IL-4 receptor−/−and Stat6−/−mice further indicate that IL-4 exerts its effects by activating the Stat6 molecule in host target cells, a finding which links clearance requirements of a gastrointestinal tract-dwelling nematode with those of a tissue-dwelling nematode. Additionally, we show that the requirements for IL-4 receptor binding and Stat6 activation extend to accelerated clearance of a secondary infection as well. The data shown here, including analysis of cell populations at the site of infection and infection of immunoglobulin E (IgE)−/−mice, lead us to suggest that deficiencies in eosinophil recruitment and isotype switching to IgE production may be at least partially responsible for slower parasite clearance in the absence of IL-4.
Pretreatment with Recombinant Flt3 Ligand Partially Protects against Progressive Cutaneous Leishmaniasis in Susceptible BALB/c Mice
Authors: Kremer Inger B. , Gould Meetha P. , Cooper Kevin D. , Heinzel Frederick P.
Published: July 2002

Dendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-γ)-producing Th1-type CD4+T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneousLeishmania majorinfection. Pretreatment of disease-susceptible BALB/c mice with 10 μg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alterL. major-induced IFN-γ and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection withL. majorreduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.
Systemic and Mucosal Immunizations with Fibronectin-Binding Protein FBP54 Induce Protective Immune Responses againstStreptococcus pyogenesChallenge in Mice
Authors: Kawabata Shigetada , Kunitomo Eiji , Terao Yutaka , Nakagawa Ichiro , Kikuchi Ken , Totsuka Kyo-ichi , Hamada Shigeyuki
Published: July 2002

The purpose of this study was to examine the suitability of fibronectin-binding protein FBP54 as a putative vaccine forStreptococcus pyogenesinfections. When the distribution of thefbp54gene among the clinical isolates representing various M serotypes was tested by PCR and Southern blot assays, it was found that all of the strains possess this gene. Furthermore, a significant increase in immunoglobulin G (IgG) antibody titers against FBP54 was observed in sera from patients withS. pyogenesinfections compared with those from healthy volunteers (P< 0.005). Mice were immunized with FBP54 subcutaneously, orally, or nasally. An enzyme-linked immunosorbent assay revealed that antigen-specific IgG antibodies were induced in the sera of immunized mice, while high salivary levels of IgA antibodies were detected after oral and nasal immunizations. Mice subcutaneously or orally immunized with FBP54 survived significantly longer following the challenge withS. pyogenesthan did nonimmunized mice (P< 0.001). These results indicate that FBP54 is a promising vaccine for the prevention ofS. pyogenesinfections.
Neutralization of Shiga Toxins Stx1, Stx2c, and Stx2e by Recombinant Bacteria Expressing Mimics of Globotriose and Globotetraose
Authors: Paton Adrienne W. , Morona Renato , Paton James C.
Published: July 2002

Strains of
Escherichia coli
producing Shiga toxins Stx1, Stx2, Stx2c, and Stx2d cause gastrointestinal disease and the hemolytic-uremic syndrome in humans. We have recently constructed a recombinant bacterium which displays globotriose (the receptor for these toxins) on its surface and adsorbs and neutralizes these Shiga toxins with very high efficiency. This agent has great potential for the treatment of humans with such infections.
E. coli
strains which cause edema disease in pigs produce a variant toxin, Stx2e, which has a different receptor specificity from that for the other members of the Stx family. We have now modified the globotriose-expressing bacterium such that it expresses globotetraose (the preferred receptor for Stx2e) by introducing additional genes encoding a
N
-acetylgalactosamine transferase and a UDP-
N
-acetylgalactosamine-4-epimerase. This bacterium had a reduced capacity to neutralize Stx1 and Stx2c in vitro, but remarkably, its capacity to bind Stx2e was similar to that of the globotriose-expressing construct; both constructs neutralized 98.4% of the cytotoxicity in lysates of
E. coli
JM109 expressing cloned
stx
2e
. These data suggest that either globotriose- or globotetraose-expressing constructs may be suitable for treatment and/or prevention of edema disease in pigs.
Inhibition of Hydrophobic Protein-MediatedCandida albicansAttachment to Endothelial Cells during Physiologic Shear Flow
Authors: Glee Pati M. , Cutler Jim E. , Benson Evelyn E. , Bargatze Robert F. , Hazen Kevin C.
Published: July 2002

Adhesion interactions during hematogenous dissemination ofCandida albicanslikely involve a complex array of host and fungal factors. PossibleC. albicansfactors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1β activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phaseC. albicansyeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. HydrophobicC. albicanscells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobicC. albicanscompared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocksC. albicansbinding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked duringC. albicansdissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.
In Vivo-Expressed Genes ofPasteurella multocida
Authors: Hunt Meredith L. , Boucher David J. , Boyce John D. , Adler Ben
Published: July 2002

Pasteurella multocidais the causative agent of infectious diseases of economic importance such as fowl cholera, bovine hemorrhagic septicemia, and porcine atrophic rhinitis. However, knowledge of the molecular mechanisms and determinants thatP. multocidarequires for virulence and pathogenicity is still limited. To address this issue, we developed a genetic expression system, based on the in vivo expression technology approach first described by Mahan et al. (Science 259:686–688, 1993), to identify in vivo-expressed genes ofP. multocida. Numerous genes, such as those encoding outer membrane lipoproteins, metabolic and biosynthetic enzymes, and a number of hypothetical proteins, were identified. These may prove to be useful targets for attenuating mutation and/or warrant further investigation for their roles in immunity and/or pathogenesis.
Selection for Urease Activity during
Helicobacter pylori
Infection of Rhesus Macaques (
Macaca mulatta
)
Authors: Hansen Lori M. , Solnick Jay V.
Published: July 2002

Helicobacter pylori
strain J166 recovered from experimentally inoculated rhesus monkeys had up to a 250-fold-increased urease activity over that before inoculation. This was found to result from the selection of urease positive J166 clones from a heterogenous inoculum, which was predominantly urease negative due to a 1-bp insertion in the
ureA
gene. These results confirm the importance of urease for
H. pylori
colonization. Strain J166 is particularly well adapted to the rhesus monkey, since it colonized preferentially despite the fact that less than 0.1% of the inoculum was urease positive.
Major Outer Membrane Protein Omp25 ofBrucella suisIs Involved in Inhibition of Tumor Necrosis Factor Alpha Production during Infection of Human Macrophages
Authors: Jubier-Maurin Véronique , Boigegrain Rose-Anne , Cloeckaert Axel , Gross Antoine , Alvarez-Martinez Maria-Teresa , Terraza Annie , Liautard Janny , Köhler Stephan , Rouot Bruno , Dornand Jacques , Liautard Jean Pierre
Published: July 2002

Brucellaspp. can establish themselves and cause disease in humans and animals. The mechanisms by whichBrucellaspp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-α) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria have been shown to modulate macrophage functions, including cytokine production. Thus, we have analyzed the effects of two major OMPs (Omp25 and Omp31) ofBrucella suis1330 (wild-type [WT]B. suis) on TNF-α production. For this purpose,omp25andomp31null mutants ofB. suis(Δomp25 B. suisand Δomp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-α. We showed that, in contrast to WTB. suisor Δomp31 B. suis, Δomp25 B. suisinduced TNF-α production when phagocytosed by human macrophages. The complementation of Δomp25 B. suiswith WTomp25(Δomp25-omp25 B. suismutant) significantly reversed this effect: Δomp25-omp25 B. suis-infected macrophages secreted significantly less TNF-α than did macrophages infected with the Δomp25 B. suismutant. Furthermore, pretreatment of WTB. suiswith an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-α production during macrophage infection. These observations demonstrated that Omp25 ofB. suisis involved in the negative regulation of TNF-α production upon infection of human macrophages.
Identification and Characterization of a Novel Secreted Immunoglobulin Binding Protein from Group A Streptococcus
Authors: Fagan Peter K. , Reinscheid Dieter , Gottschalk Birgit , Chhatwal Gursharan S.
Published: July 2002

Immunoglobulin binding proteins are one of several pathogenicity factors which have been associated with invasive disease caused by group A streptococci. The surface-bound M and M-like proteins ofStreptococcus pyogenesare the most characterized of these immunoglobulin binding proteins, and in most cases they bind only a single antibody class. Here we report the identification of a novel non-M-type secreted protein, designated SibA (for secreted immunoglobulin binding protein from group A streptococcus), which binds all immunoglobulin G (IgG) subclasses, the Fc and Fab fragments, and also IgA and IgM. SibA has no significant sequence homology to any M-related proteins, is not found in thevirregulon, and contains none of the characteristic M-protein regions, such as the A or C repeats. Like M proteins, however, SibA does have relatively high levels of alanine, lysine, glutamic acid, leucine, and glycine. SibA and M proteins also share an alpha-helical N-terminal secondary structure which has been previously implicated in immunoglobulin binding in M proteins. Evidence presented here indicates that this is also the case for SibA. SibA also has regions of local similarity with other coiled-coil proteins such asListeria monocytogenesP45 autolysin, human myosin heavy chain, macrogolgin, andSchistoma mansoniparamyosin, some of which are of potential significance since cross-reactive antibodies between myosin proteins and M proteins have been implicated in the development of the autoimmune sequelae of streptococcal disease.
Role of Bcl-2 Family Members in Caspase-Independent Apoptosis duringChlamydiaInfection
Authors: Perfettini Jean-Luc , Reed John C. , Israël Nicole , Martinou Jean-Claude , Dautry-Varsat Alice , Ojcius David M.
Published: July 2002

Infection with an obligate intracellular bacterium, theChlamydia trachomatislymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar ofChlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains ofChlamydiais independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria inC. psittaci-infected cells.C. psittaci-induced apoptosis is inhibited in host cells overexpressing Bax inhibitor-1 and is inhibited through overexpression of Bcl-2, which blocks both caspase-dependent and -independent apoptosis. As Bax and mitochondria are ideally located to sense stress-related metabolic changes emanating from the interior of an infected cell, it is likely that Bax-dependent apoptosis may also be observed in cells infected with other intracellular pathogens.
Physical Mapping of
bchG
,
orf427
, and
orf177
in the Photosynthesis Gene Cluster of
Rhodobacter sphaeroides
: Functional Assignment of the Bacteriochlorophyll Synthetase Gene
Authors: Addlesee Hugh A. , Fiedor Leszek , Hunter C. Neil
Published: July 2002

The purple photosynthetic bacterium
Rhodobacter sphaeroides
has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs,
bchG
, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that
bchG
is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to
bchG
was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in
Escherichia coli
, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in
R. sphaeroides
, a model organism for the study of structure-function relationships in photosynthesis. A second gene,
orf177
, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene,
orf427
, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF,
bchP
, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248–7255, 1999). An operonal arrangement of the genes is proposed.
Genetic and Phenotypic Analyses of the
rdx
Locus of
Rhodobacter sphaeroides
2.4.1
Authors: Roh Jung Hyeob , Kaplan Samuel
Published: July 2002

Previously, we reported that
rdxB
, encoding a likely membrane-bound two [4Fe-4S]-containing center, is involved in the aerobic regulation of photosystem gene expression in
Rhodobacter sphaeroides
2.4.1. To further investigate the role of
rdxB
as well as other genes of the
rdxBHIS
operon on photosystem gene expression, we constructed a series of nonpolar, in-frame deletion mutations in each of the
rdx
genes. Using both
puc
and
puf
operon
lacZ
fusions to monitor photosystem gene expression, under aerobic conditions, in each of the mutant strains revealed significant increased photosynthesis gene expression. In the case of mutations in either
rdxH
,
rdxI
, or
rdxS
, the aerobic induction of photosystem gene expression is believed to be indirect by virtue of a posttranscriptional effect on
cbb
3
cytochrome oxidase structure and integrity. For RdxB, we suggest that this redox protein has a more direct effect on photosystem gene expression by virtue of its interaction with the
cbb
3
oxidase. An associated phenotype, involving the enhanced conversion of the carotenoid spheroidene to spheroidenone, is also observed in the RdxB, -H, -I, and -S mutant strains. This phenotype is also suggested to be the result of the role of the
rdxBHIS
locus in
cbb
3
oxidase activity and/or structure. RdxI is suggested to be a new class of metal transporter of the CPx-type ATPases.
Genetic and Environmental Factors Affecting T-Pilin Export and T-Pilus Biogenesis in Relation to Flagellation of
Agrobacterium tumefaciens
Authors: Lai Erh-Min , Chesnokova Olga , Banta Lois M. , Kado Clarence I.
Published: July 2002

The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from
Agrobacterium tumefaciens
to plant cells. Although the
virB2
gene of the 11-gene
virB
operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role of each
virB
gene in T-pilin synthesis and transport and T-pilus biogenesis remained undefined. In the present study, each
virB
gene was examined and was found to be unessential for T-pilin biosynthesis, except
virB2
, but was determined to be essential for the export of the T-pilin subunits and for T-pilus formation. We also find that the genes of the
virD
operon are neither involved in T-pilin export nor T-pilus formation. Critical analysis of three different
virD4
mutants also showed that they are not involved in T-pilus biogenesis irrespective of the
A. tumefaciens
strains used. With respect to the environmental effects on T-pilus biogenesis, we find that T pili are produced both on agar and in liquid culture and are produced at one end of the
A. tumefaciens
rod-shaped cell in a polar manner. We also report a novel phenomenon whereby flagellum production is shut down under conditions which turn on T-pilus formation. These conditions are the usual induction with acetosyringone at pH 5.5 of Ti-plasmid
vir
genes. A search of the
vir
genes involved in controlling this biphasic reaction in induced
A. tumefaciens
cells revealed that
virA
on the Ti plasmid is involved and that neither
virB
nor
virD
genes are needed for this reaction. The biphasic reaction therefore appears to be mediated through a two-component signal transducing system likely involving an unidentified
vir
gene in
A. tumefaciens
.
Regulation of the
furA
and
catC
Operon, Encoding a Ferric Uptake Regulator Homologue and Catalase-Peroxidase, Respectively, in
Streptomyces coelicolor
A3(2)
Authors: Hahn Ji-Sook , Oh So-Young , Roe Jung-Hye
Published: July 2002

We isolated the
catC
gene, encoding catalase-peroxidase in
Streptomyces coelicolor
, using sequence homology with the
katG
gene from
Escherichia coli
. Upstream of the
catC
gene, an open reading frame (
furA
) encoding a homologue of ferric uptake regulator (Fur) was identified. S1 mapping analysis indicated that the
furA
gene was cotranscribed with the
catC
gene. The transcriptional start site of the
furA-catC
mRNA was mapped to the translation start codon ATG of the
furA
gene. The putative promoter contains consensus −10 and −35 elements similar to those recognized by ς
HrdB
, the major sigma factor of
S. coelicolor
. The transcripts were produced maximally at late-exponential phase and decreased at the stationary phase in liquid culture. The change in the amount of mRNA was consistent with that of CatC protein and enzyme activity. When the
furA
gene was introduced into
S. lividans
on a multicopy plasmid, the increased production of
catC
transcripts and protein product at late growth phase was inhibited, implying a role for FurA as the negative regulator of the
furA-catC
operon. FurA protein bound to its own promoter region between −59 and −39 nucleotides from the transcription start site. The binding affinity of FurA increased under reducing conditions and in the presence of metals such as Ni
2+
, Mn
2+
, Zn
2+
, or Fe
2+
. Addition of these metals to the growth medium decreased the production of CatC protein, consistent with the role of FurA as a metal-dependent repressor.
ς
54
Promoters Control Expression of Genes Encoding the Hook and Basal Body Complex in
Rhodobacter sphaeroides
Authors: Poggio Sebastian , Aguilar Carlos , Osorio Aurora , González-Pedrajo Bertha , Dreyfus Georges , Camarena Laura
Published: July 2002

Gene expression of the flagellar system is tightly controlled by external stimuli or intracellular signals. A general picture of this regulation has been obtained from studies of
Salmonella enterica
serovar Typhimurium. However, these regulatory mechanisms do not apply to all bacterial groups. In this study, we have investigated regulation of the flagellar genetic system in
Rhodobacter sphaeroides
. Deletion analysis, site-directed mutagenesis, and 5′-end mapping were conducted in order to identify the
fliO
promoter. Our results indicate that this promoter is recognized by the factor ς
54
. Additionally, 5′-end mapping of the
flgB
and
fliK
transcripts suggests that these mRNAs are also transcribed from ς
54
promoters. Finally, we showed evidence that suggests that
fliC
transcription is not entirely dependent on the presence of a complete basal body-hook structure. Our results are discussed in the context of a possible regulatory hierarchy controlling flagellar gene expression in
R. sphaeroides
.
Myxococcus xanthus dif
Genes Are Required for Biogenesis of Cell Surface Fibrils Essential for Social Gliding Motility
Authors: Yang Zhaomin , Ma Xiaoyuan , Tong Leming , Kaplan Heidi B. , Shimkets Lawrence J. , Shi Wenyuan
Published: July 2002

Myxococcus xanthus
social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the
dif
genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to
M. xanthus
S motility. We have demonstrated here that
M. xanthus dif
genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of
dif
mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the
pilGHIJ
genes of the twitching bacterium
Pseudomonas aeruginosa
, the
M. xanthus dif
genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the
dif
genes are linked to the
M. xanthus dsp
region, a locus known to be crucial for
M. xanthus
fibril biogenesis and S gliding.
The UspA1 Protein and a Second Type of UspA2 Protein Mediate Adherence of
Moraxella catarrhalis
to Human Epithelial Cells In Vitro
Authors: Lafontaine Eric R. , Cope Leslie D. , Aebi Christoph , Latimer Jo L. , McCracken George H. , Hansen Eric J.
Published: July 2002

The UspA1 and UspA2 proteins of
Moraxella catarrhalis
are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of
uspA1
and
uspA2
mutants of
M. catarrhalis
strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113–3119, 1998). In the present study, isogenic
uspA1
,
uspA2
, and
uspA1 uspA2
mutations were constructed in three additional
M. catarrhalis
strains: 012E, TTA37, and 046E. The
uspA1
mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the
uspA1
gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the
uspA2
gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the
uspA2
genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this “hybrid” protein was designated
uspA2H
. PCR-based analysis revealed that approximately 20% of
M. catarrhalis
strains apparently possess a
uspA2H
gene instead of a
uspA2
gene. The
M. catarrhalis uspA1
,
uspA2
, and
uspA2H
genes were cloned and expressed in
Haemophilus influenzae
cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.
Cpx Two-Component Signal Transduction in
Escherichia coli
: Excessive CpxR-P Levels Underlie CpxA* Phenotypes
Authors: De Wulf Peter , Lin E. C. C.
Published: July 2002

In
Escherichia coli
, the CpxA-CpxR two-component signal transduction system and the ς
E
and ς
32
response pathways jointly regulate gene expression in adaptation to adverse conditions. These include envelope protein distress, heat shock, oxidative stress, high pH, and entry into stationary phase. Certain mutant versions of the CpxA sensor protein (CpxA* proteins) exhibit an elevated ratio of kinase to phosphatase activity on CpxR, the cognate response regulator. As a result, CpxA* strains display numerous phenotypes, many of which cannot be easily related to currently known functions of the CpxA-CpxR pathway. It is unclear whether CpxA* phenotypes are caused solely by hyperphosphorylation of CpxR. We here report that all of the tested CpxA* phenotypes depend on elevated levels of CpxR-P and not on cross-signalling of CpxA* to noncognate response regulators.
Morphogenetic Proteins SpoVID and SafA Form a Complex during Assembly of the
Bacillus subtilis
Spore Coat
Authors: Ozin Amanda J. , Henriques Adriano O. , Yi Hong , Moran Charles P.
Published: July 2002

During endospore formation in
Bacillus subtilis
, over two dozen polypeptides are assembled into a multilayered structure known as the spore coat, which protects the cortex peptidoglycan (PG) and permits efficient germination. In the initial stages of coat assembly a protein known as CotE forms a ring around the forespore. A second morphogenetic protein, SpoVID, is required for maintenance of the CotE ring during the later stages, when most of proteins are assembled into the coat. Here, we report on a protein that appears to associate with SpoVID during the early stage of coat assembly. This protein, which we call SafA for SpoVID-associated factor A, is encoded by a locus previously known as
yrbA
. We confirmed the results of a previous study that showed
safA
mutant spores have defective coats which are missing several proteins. We have extended these studies with the finding that SafA and SpoVID were coimmunoprecipitated by anti-SafA or anti-SpoVID antiserum from whole-cell extracts 3 and 4 h after the onset of sporulation. Therefore, SafA may associate with SpoVID during the early stage of coat assembly. We used immunogold electron microscopy to localize SafA and found it in the cortex, near the interface with the coat in mature spores. SafA appears to have a modular design. The C-terminal region of SafA is similar to those of several inner spore coat proteins. The N-terminal region contains a sequence that is conserved among proteins that associate with the cell wall. This motif in the N-terminal region may target SafA to the PG-containing regions of the developing spore.
Two Nitrate/Nitrite Transporters Are Encoded within the Mobilizable Plasmid for Nitrate Respiration ofThermus thermophilusHB8
Authors: Ramírez Sandra , Moreno Renata , Zafra Olga , Castán Pablo , Vallés Cristina , Berenguer José
Published: July 2002

Thermus thermophilusHB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes,narK1andnarK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a doublenarK1 narK2mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed.
Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
and Its Role in Alanine Production
Authors: Ward Donald E. , Kengen Servé W. M. , van der Oost John , de Vos Willem M.
Published: July 2002

Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon
Pyrococcus furiosus
by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (
aat
) in the
P. furiosus
genome database. The gene was expressed in
Escherichia coli
, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from
P. furiosus
. The
k
cat
/

K
m

values for alanine and pyruvate formation were 41 and 33 s
−1
mM
−1
, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the
aat
gene. In addition, both the
aat
and
gdh
(encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the
aat
and
gdh
genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in
P. furiosus
.
Role of Arginines in Coenzyme A Binding and Catalysis by the Phosphotransacetylase from
Methanosarcina thermophila
Authors: Iyer Prabha P. , Ferry James G.
Published: July 2002

Phosphotransacetylase (EC
2.3.1.8
) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH
3
COOPO
3
2−
+ CoASH ⇆ CH
3
COSCoA + HPO
4
2−
. The role of arginine residues was investigated for the phosphotransacetylase from
Methanosarcina thermophila
. Kinetic analysis of a suite of variants indicated that Arg 87 and Arg 133 interact with the substrate CoA. Arg 87 variants were reduced in the ability to discriminate between CoA and the CoA analog 3′-dephospho-CoA, indicating that Arg 87 forms a salt bridge with the 3′-phosphate of CoA. Arg 133 is postulated to interact with the 5′-phosphate of CoA. Large decreases in
k
cat
and
k
cat
/

K
m

for all of the Arg 87 and Arg 133 variants indicated that these residues are also important, although not essential, for catalysis. Large decreases in
k
cat
and
k
cat
/

K
m

were also observed for the variants in which lysine replaced Arg 87 and Arg 133, suggesting that the bidentate interaction of these residues with CoA or their greater bulk is important for optimal activity. Desulfo-CoA is a strong competitive inhibitor of the enzyme, suggesting that the sulfhydryl group of CoA is important for the optimization of CoA-binding energy but not for tight substrate binding. Chemical modification of the wild-type enzyme by 2,3-butanedione and substrate protection by CoA indicated that at least one reactive arginine is in the active site and is important for activity. The inhibition pattern of the R87Q variant indicated that Arg 87 is modified, which contributes to the inactivation; however, at least one additional active-site arginine is modified leading to enzyme inactivation, albeit at a lower rate.
Isolation and Characterization of a
Shewanella putrefaciens
MR-1 Electron Transport Regulator
etrA
Mutant: Reassessment of the Role of EtrA
Authors: Maier Tamara M. , Myers Charles R.
Published: July 2002

Shewanella putrefaciens
MR-1 has emerged as a good model to study anaerobic respiration and electron transport-linked metal reduction. Its remarkable respiratory plasticity suggests the potential for a complex regulatory system to coordinate electron acceptor use in the absence of O
2
. It had previously been suggested that EtrA (electron transport regulator A), an analog of Fnr (fumarate nitrate regulator) from
Escherichia coli
, may regulate gene expression for anaerobic electron transport. An
etrA
knockout strain (ETRA-153) was isolated from MR-1 using a gene replacement strategy. Reverse transcription-PCR analysis of total RNA demonstrated the loss of the
etrA
mRNA in ETRA-153. ETRA-153 cells retained the ability to grow on all electron acceptors tested, including fumarate, trimethylamine
N
-oxide (TMAO), thiosulfate, dimethyl sulfoxide, ferric citrate, nitrate, and O
2
, as well as the ability to reduce ferric citrate, manganese(IV), nitrate, and nitrite. EtrA is therefore not necessary for growth on, or the reduction of, these electron acceptors. However, ETRA-153 had reduced initial growth rates on fumarate and nitrate but not on TMAO. The activities for fumarate and nitrate reductase were lower in ETRA-153, as were the levels of fumarate reductase protein and transcript. ETRA-153 was also deficient in one type of ubiquinone. These results are in contrast to those previously reported for the putative
etrA
mutant METR-1. Molecular analysis of METR-1 indicated that its
etrA
gene is not interrupted; its reported phenotype was likely due to the use of inappropriate anaerobic growth conditions.
Regulated Secretion of YopN by the Type III Machinery of
Yersinia enterocolitica
Authors: Cheng Luisa W. , Kay Olga , Schneewind Olaf
Published: July 2002

During infection,
Yersinia enterocolitica
exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 μM (low-calcium response). Yersiniae lacking
yopN (lcrE), yscB, sycN
, or
tyeA
do not inactivate the type III pathway even when the concentration of calcium is above 100 μM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of
yopN
gene sequences to the 5′ end of the
npt
reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of
yopN
. Expression of plasmid-borne
yopN
, but not of
yopN
1–294
-
npt
, complements the calcium-blind phenotype of
yopN
mutants. Surprisingly,
yopN
mutants respond to environmental changes in calcium concentration and secrete YopN
1–294
-Npt in the absence but not in the presence of calcium.
tyeA
is required for the low-calcium regulation of YopN
1–294
-Npt secretion, whereas
sycN
and
yscB
mutants fail to secrete YopN
1–294
-Npt in the presence of calcium. Experiments with
yopN-npt
fusions identified two other signals that regulate the secretion of YopN.
yopN
codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of
yscB
and
sycN
. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional
tyeA
. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.
Expression of
cspH
, Encoding the Cold Shock Protein in
Salmonella enterica
Serovar Typhimurium UK-1
Authors: Kim Bae Hoon , Bang Iel Soo , Lee Soo Youn , Hong Seong Karp , Bang Seong Ho , Lee In Soo , Park Yong Keun
Published: July 2002

Both
Salmonella enterica
serovar Typhimurium and
Escherichia coli
contain the
cspH
gene encoding CspH, one of the cold shock proteins (CSPs). In this study, we investigated the expression of
cspH
in
S. enterica
serovar Typhimurium and found that it was induced in response to a temperature downshift during exponential phase. The
cspH
promoter was activated at 37°C, and its mRNA was more stable than the other
csp
mRNAs at 37°C. Moreover,
lacZ
expression of the translational
cspH-lacZ
fusion was induced at that temperature. Interestingly, the
cspH
mRNA had a much shorter 5′-untranslated region than those in the other cold-shock-inducible genes, and the promoter sequence, which was only 55 bp, was sufficient for
cspH
expression. The 14-base downstream box located 12 bases downstream of the initiation codon of
cspH
mRNA was essential for its cold shock activation.
Transcriptional Activation of
Bordetella
Alcaligin Siderophore Genes Requires the AlcR Regulator with Alcaligin as Inducer
Authors: Brickman Timothy J. , Kang Ho Young , Armstrong Sandra K.
Published: July 2002

Genetic and biochemical studies have established that Fur and iron mediate repression of
Bordetella
alcaligin siderophore system (
alc
) genes under iron-replete nutritional growth conditions. In this study, transcriptional analyses using
Bordetella
chromosomal
alc-lacZ
operon fusions determined that maximal
alc
gene transcriptional activity under iron starvation stress conditions is dependent on the presence of alcaligin siderophore. Mutational analysis and genetic complementation confirmed that alcaligin-responsive transcriptional activation of
Bordetella
alcaligin system genes is dependent on AlcR, a Fur-regulated AraC-like positive transcriptional regulator encoded within the alcaligin gene cluster. AlcR-mediated transcriptional activation is remarkably sensitive to inducer, occurring at extremely low alcaligin concentrations. This positive autogenous control circuit involving alcaligin siderophore as the inducer for AlcR-mediated transcriptional activation of alcaligin siderophore biosynthesis and transport genes coordinates environmental and intracellular signals for maximal expression of these genes under conditions in which the presence of alcaligin in the environment is perceived.
ihfA
Gene of the Bacterium
Myxococcus xanthus
and Its Role in Activation of Carotenoid Genes by Blue Light
Authors: Moreno Alberto J. , Fontes Marta , Murillo Francisco J.
Published: July 2002

Myxococcus xanthus
responds to blue light by producing carotenoids. Several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes. We had already reported the isolation of a carotenoid-less, Tn
5
-induced strain (MR508), whose mutant site was unlinked to the indicated regulatory genes. Here, we show that ΩMR508::Tn
5
affects all known light-inducible promoters in different ways. It blocks the activation of two of them by light but makes the activity of a third one light independent. The ΩMR508 locus has been cloned and sequenced. The mutation had occurred at the promoter of a gene we propose is the
M. xanthus
ortholog of
ihfA
. This encodes the α subunit of the histone-like integration host factor protein. An in-frame deletion within
ihfA
causes the same effects as the ΩMR508::Tn
5
insertion. Like other IhfA proteins, the deduced amino acid sequence of
M. xanthus
IhfA shows much similarity to HU, another histone-like protein. Sequence comparison data, however, and the finding that the
M. xanthus
gene is preceded by gene
pheT
, as happens in other gram-negative bacteria, strongly argue for the proposed orthology relationship. The
M. xanthus ihfA
gene shows some unusual features, both from structural and physiological points of view. In particular, the protein is predicted to have a unique, long acidic extension at the carboxyl terminus, and it appears to be necessary for normal cell growth and even vital for a certain wild-type strain of
M. xanthus
.