bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA Class II Polymorphism Influences Onset and Severity of Pathology inSchistosoma mansoni-Infected Transgenic Mice
Authors: Angyalosi Gerhild , Neveu Raphaële , Wolowczuk Isabelle , Delanoye Anne , Herno Josiane , Auriault Claude , Pancré Véronique
Published: July 2002

Genetic factors that might influence susceptibility or resistance in naive individuals and early-stage pathology in schistosomiasis are difficult to study in clinical trials, since in areas where the disease is endemic the first contact with the parasite occurs most often at very early ages. Therefore, four strains (DR1.Aβ°, DR2.Aβ°, DQ8.Aβ°, and DQ6.Aβ°) of major histocompatibility complex class II-deficient mice (Aβ°), transgenic for different HLA alleles, have been used to evaluate the potential role of HLA class II polymorphism in the onset of the infection bySchistosoma mansoni. The survival rates and parasitological and immunological parameters after infection were evaluated and compared against the control values obtained with Aβ° mice. All four mouse strains used in this study were able to generate a specific immune response againstS. mansoniantigens (cytokine production and antibody production). However, only mice expressing DR alleles survived until the chronic stage of the infection and were able to mount protective granulomatous response avoiding hepatic damage, presenting predominant gamma interferon production. In contrast, strains expressing DQ alleles revealed an impairment in generating effective granulomas, resulting in earlier death, which was associated with an impaired hepatic granulomatous response and liquefactic necrosis, reflecting the influence of HLA polymorphism in the establishment of protective response in the early stage of infection.
Identification of a Promiscuous T-Cell Epitope inMycobacterium tuberculosisMce Proteins
Authors: Panigada Maddalena , Sturniolo Tiziana , Besozzi Giorgio , Boccieri Maria Giovanna , Sinigaglia Francesco , Grassi Giuliana Gialdroni , Grassi Fabio
Published: July 2002

The characterization ofMycobacterium tuberculosisantigens inducing CD4+T-cell responses could critically contribute to the development of subunit vaccines forM. tuberculosis.Here we performed computational analysis by using T-cell epitope prediction software (known as TEPITOPE) to predict promiscuous HLA-DR ligands in the products of themcegenes ofM. tuberculosis.The analysis of the proliferative responses of CD4+T cells from patients with pulmonary tuberculosis to selected peptides displaying promiscuous binding to HLA-DR in vitro led us to the identification of a peptide that induced proliferation of CD4+cells from 50% of the tested subjects. This study demonstrates that a systematic computational approach can be used to identify T-cell epitopes in proteins expressed by an intracellular pathogen.
Fibronectin Attachment Protein Is Necessary for Efficient Attachment and Invasion of Epithelial Cells by
Mycobacterium avium
subsp.
paratuberculosis
Authors: Secott T. E. , Lin T. L. , Wu C. C.
Published: July 2002

Attachment and ingestion of
Mycobacterium avium
subsp.
paratuberculosis
by two epithelial cell lines were enhanced by soluble fibronectin (FN). Peptide blocking of the FN attachment protein (FAP-P) inhibited the internalization of
M. avium
subsp.
paratuberculosis.
Disruption of FAP-P expression significantly reduced attachment and ingestion of
M. avium
subsp.
paratuberculosis
by T-24 and Caco-2 cells. The results indicate that the interaction between FN and FAP-P facilitates attachment and internalization of
M. avium
subsp.
paratuberculosis
by epithelial cells.
Macrophage-Induced Genes ofLegionella pneumophila:Protection from Reactive Intermediates and Solute Imbalance during Intracellular Growth
Authors: Rankin Susannah , Li Zhiru , Isberg Ralph R.
Published: July 2002

A promoter-probe strategy was devised to identify genes specifically expressed byLegionella pneumophiladuring growth within the macrophage. Random fragments from theL. pneumophilachromosome were inserted upstream of a promoterless phage T4tdgene, and fragments that led to complementation of thymine auxotrophy during intracellular growth of the bacterium were identified. Two different selection strategies were employed to eliminate promoters that were also active during extracellular growth of the bacterium. Some of these genes were identified independently by using both of the selection strategies. The factors identified include orthologs of efflux-mediated resistance determinants and transporters, a transporter involved in protection from osmotic stress, a stress response GTP-binding protein, a response regulator, a sensor kinase, and two systems that increase the reducing potential of the bacterium, one of which encodes theL. pneumophilaortholog ofahpC. Five of the clones analyzed here were fusions to promoters that were closely linked to genes encoding three-component chemiosmotic efflux pumps that export heavy metals or toxic organic compounds. Analysis ofahpCgene expression indicates that levels increased at least sevenfold during intracellular growth of the bacterium. Inactivation of several of the genes at their chromosomal loci had no effect on the intracellular growth rate ofL. pneumophilain cultured macrophages. This suggests that a number of genes with increased expression during intracellular growth may be part of redundant systems that allow survival and growth under the conditions encountered within host cells.
Identification of an Alternative Nucleoside Triphosphate: 5′-Deoxyadenosylcobinamide Phosphate Nucleotidyltransferase inMethanobacterium thermoautotrophicumΔH
Authors: Thomas Michael G. , Escalante-Semerena Jorge C.
Published: July 2002

Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5′-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU inSalmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of theS. enterica cobUgene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domainArchaea. The corresponding ORF open reading frame fromMethanobacterium thermoautotrophicumΔH (MTH1152; referred to ascobY) was amplified and cloned, and the CobY protein was expressed and purified fromEscherichia colias a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme ofS. enterica. NTP:adenosylcobinamide kinase activity was not detected inM. thermoautotrophicumΔH cell extract, suggesting that this organism may not have this activity. ThecobYgene complemented acobUmutant ofS. entericagrown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis.cobY, however, failed to restore adenosylcobalamin biosynthesis incobUmutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylcobinamide kinase activity is not required for de novo cobamide biosynthesis.
A Two-Dimensional Protein Gel Electrophoresis Study of the Heat Stress Response of
Bacillus subtilis
Cells during Sporulation
Authors: Movahedi Sara , Waites William
Published: July 2002

The heat resistance of spores of
Bacillus subtilis
formed at 30°C was enhanced by pretreatment at 48°C for 30 min, 60 min into sporulation, for all four strains examined. High-resolution two-dimensional gel electrophoresis showed the generation and/or overexpression of 60 proteins, 11 of which were specific to heat shock, concurrent to this acquired thermotolerance. The greatest number of new proteins was observed between 30 and 60 min after heat shock, and the longer the time between exponential growth and heat treatment, the fewer differences were observed on corresponding protein profiles. The time at which heating produced the maximum increase in spore resistance and the most new proteins on two-dimensional gels occurred before alkaline phosphatase and dipicolinic acid production and corresponded to stage I or II of sporulation. The stress proteins formed disappeared later in sporulation, suggesting that heat shock proteins increase spore heat resistance by altering spore structure rather than by repairing heat damage during germination and outgrowth.
IS
1675
, a Novel Lactococcal Insertion Element, Forms a Transposon-Like Structure Including the Lacticin 481 Lantibiotic Operon
Authors: Dufour Alain , Rincé Alain , Uguen Patricia , Le Pennec Jean-Paul
Published: July 2002

Two copies of IS
1675
, a novel lactococcal insertion element from the IS
4
family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon. The whole structure could be a composite transposon designated Tn
5721
. This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant. We identified five other IS
1675
copies not associated with the lacticin 481 operon. The conservation of IS
1675
flanking sequences suggested a 24-bp target site.
Membrane Topology of the
Bacillus subtilis
Pro-ς
K
Processing Complex
Authors: Green David H. , Cutting Simon M.
Published: July 2002

Activation of the final sporulation-specific transcription factor, ς
K
, is regulated by a signal emanating from the forespore which interacts with the pro-ς
K
processing complex, comprising SpoIVFA, BofA, and the pro-ς
K
processing protease, SpoIVFB. Mature ς
K
then directs late gene expression in the parental compartment of the developing sporangial cell. The nature of this complex and how it is activated to process pro-ς
K
are not understood. All three proteins are predicted to be integral membrane proteins. Here, we have analyzed the membrane topology of SpoIVFA and SpoIVFB by constructing chimeric forms of
spoIVFA
and
spoIVFB
with the complementary reporters
phoA
and
lacZ
and analyzing activity in
Escherichia coli
. SpoIVFA was found to have a single transmembrane-spanning domain, while SpoIVFB was shown to have six transmembrane-spanning domains (6-transmembrane configuration). Further, SpoIVFA is required to stabilize SpoIVFB in the membrane. SpoIVFB was shown to have a 4-transmembrane configuration when expressed on its own but was found to have a 6-transmembrane configuration when coexpressed with SpoIVFA, while BofA had a positive effect on the assembly of both SpoIVFA and SpoIVFB. The single transmembrane domain of SpoIVFA (approximately residues 73 to 90) was shown to be the principle determinant in stabilizing the 6-transmembrane configuration of SpoIVFB. Although the
bofB8
allele, which uncouples the ς
K
checkpoint, did not appear to promote a conformational change from a 6- to 4-transmembrane configuration of SpoIVFB (apparently ruling out a profound conformational change as the mechanism of activating SpoIVFB proteolytic activity), instability of SpoIVFB may be an important factor in SpoIVFB-mediated processing of pro-ς
K
.
Transport of C
4
-Dicarboxylates in
Wolinella succinogenes
Authors: Ullmann Roland , Gross Roland , Simon Jörg , Unden Gottfried , Kröger Achim
Published: July 2002

C
4
-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in
Wolinella succinogenes
, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane.
W. succinogenes
was found to transport C
4
-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 10
3
. The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na
+
, whereas the electrogenic antiport also operated in the absence of Na
+
. In the absence of Na
+
, no electrochemical proton potential (Δp) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (
dcuA
,
dcuB
, and
dctPQM
) encoding putative C
4
-dicarboxylate transporters were identified on the genome of
W. succinogenes
. The predicted gene products of
dcuA
and
dcuB
are similar to the Dcu transporters that are involved in the fumarate respiration of
Escherichia coli
with external C
4
-dicarboxylates. The genes
dctP
, -
Q
, and -
M
probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA
−
DcuB
−
) of
W. succinogenes
lacking the intact
dcuA
and
dcuB
genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA
−
, DcuB
−
, and DctQM
−
mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA
−
DcuB
−
mutant performed fumarate respiration without generating a proton potential even in the presence of Na
+
. This explains why the DcuA
−
DcuB
−
mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na
+
-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.
Transcription Initiation-Defective Forms of ς
54
That Differ in Ability To Function with a Heteroduplex DNA Template
Authors: Kelly Mary T. , Ferguson John A. , Hoover Timothy R.
Published: July 2002

Transcription by ς
54
-RNA polymerase holoenzyme requires an activator that catalyzes isomerization of the closed promoter complex to an open complex. We examined mutant forms of
Salmonella enterica
serovar Typhimurium ς
54
that were defective in transcription initiation but retained core RNA polymerase- and promoter-binding activities. Four of the mutant proteins allowed activator-independent transcription from a heteroduplex DNA template. One of these mutant proteins, L124P V148A, had substitutions in a sequence that had not been shown previously to participate in the prevention of activator-independent transcription. The remaining mutants did not allow efficient activator-independent transcription from the heteroduplex DNA template and had substitutions within a conserved 20-amino-acid segment (Leu-179 to Leu-199), suggesting a role for this sequence in transcription initiation.
Influence of Mutations in the
mexR
Repressor Gene on Expression of the MexA-MexB-OprM Multidrug Efflux System of
Pseudomonas aeruginosa
Authors: Srikumar Ramakrishnan , Paul Catherine J. , Poole Keith
Published: July 2002

Several
nalB
-type multidrug-resistant mutants of
Pseudomonas aeruginosa
overexpressed MexAB-OprM and carried mutations in the local regulatory gene,
mexR
. Others, dubbed
nalC
types, carried mutations elsewhere and overexpressed MexAB-OprM less extensively than the
nalB
strains. Available evidence showed that MexR acted solely as repressor. Disruption of the
mexR
gene at various places suggested that the 5′ end of
mexR
may be a part of the
mexAB-oprM
promoter.
Genetic Requirements of Phage λ Red-Mediated Gene Replacement in
Escherichia coli
K-12
Authors: Poteete Anthony R. , Fenton Anita C.
Published: July 2002

Recombination between short linear double-stranded DNA molecules and
Escherichia coli
chromosomes bearing the
red
genes of bacteriophage λ in place of
recBCD
was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the
cat
gene, with flanking
lac
sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac
−
chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced in
ruvAB
and
recQ
strains. In this genetic background, mutations in
recF
,
recO
, and
recR
had large effects on both cell viability and on recombination. In these cases, deletion of the
sulA
gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the
nin
region of phage λ partially complemented both the viability and recombination defects of the
recF
,
recO
, and
recR
mutants and the recombination defect of
ruvC
but not of
ruvAB
or
recQ
mutants.
The
yvaJ
Gene of
Bacillus subtilis
Encodes a 3′-to-5′ Exoribonuclease and Is Not Essential in a Strain Lacking Polynucleotide Phosphorylase
Authors: Oussenko Irina A. , Bechhofer David H.
Published: July 2002

Studies of
Bacillus subtilis
RNases that are involved in mRNA degradation reveal a different pattern from that of
Escherichia coli
. A strain lacking polynucleotide phosphorylase, the major 3′-to-5′ exoribonuclease activity in cell extracts, is viable. Here, we show that the
B. subtilis yvaJ
gene encodes a second 3′-to-5′ exoribonuclease. A strain lacking both of these RNases grows slowly but is viable. The existence of another, as yet unknown, 3′-to-5′ exoribonuclease in
B. subtilis
is suggested.
Mutational Analysis of the OprM Outer Membrane Component of the MexA-MexB-OprM Multidrug Efflux System of
Pseudomonas aeruginosa
Authors: Li Xian-Zhi , Poole Keith
Published: July 2002

OprM is the outer membrane component of the MexA-MexB-OprM efflux system of
Pseudomonas aeruginosa
. Multiple-sequence alignment of this protein and its homologues identified several regions of high sequence conservation that were targeted for site-directed mutagenesis. Of several deletions which were stably expressed, two, spanning residues G199 to A209 and A278 to N286 of the mature protein, were unable to restore antibiotic resistance in OprM-deficient strains of
P. aeruginosa
. Still, mutation of several conserved residues within these regions did not adversely affect OprM function. Mutation of the highly conserved N-terminal cysteine residue, site of acylation of this presumed lipoprotein, also did not affect expression or activity of OprM. Similarly, substitution of the OprM lipoprotein signal, including consensus lipoprotein box, with the signal peptide of OprF, the major porin of this organism, failed to impact on expression or activity. Apparently, acylation is not essential for OprM function. A large deletion at the N terminus, from A12 to R98, compromised OprM expression to some extent, although the deletion derivative did retain some activity. Several deletions failed to yield an OprM protein, including one lacking an absolutely conserved LGGGW sequence near the C terminus of the protein. The pattern of permissive and nonpermissive deletions was used to test a topology model for OprM based on the recently published crystal structure of the OprM homologue, TolC (V. Koronakis, A. Sharff, E. Koronakis, B. Luisi, and C. Hughes, Nature 405:914–919, 2000). The data are consistent with OprM monomer existing as a substantially periplasmic protein with four outer membrane-spanning regions.
The
CDC42
Homolog of the Dimorphic Fungus
Penicillium marneffei
Is Required for Correct Cell Polarization during Growth but Not Development
Authors: Boyce Kylie J. , Hynes Michael J. , Andrianopoulos Alex
Published: July 2002

The opportunistic human pathogenic fungus
Penicillium marneffei
is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in
Saccharomyces cerevisiae
is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved
CDC42
homolog from
P. marneffei
named
cflA.
By the generation of dominant-negative and dominant-activated
cflA
transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct
cflA
function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression of
cflA
alleles in
Aspergillus nidulans
prevented conidiation.
FhuA Barrel-Cork Hybrids Are Active Transporters and Receptors
Authors: Killmann Helmut , Braun Michael , Herrmann Christina , Braun Volkmar
Published: July 2002

The crystal structure of
Escherichia coli
FhuA reveals a β-barrel domain that is closed by a globular cork domain. It has been assumed that the proton motive force of the cytoplasmic membrane through the interaction of the TonB protein with the TonB box of the cork opens the FhuA channel. Yet, deletion of the cork results in an FhuA derivative, FhuAΔ5–160, that still displays TonB-dependent substrate transport and phage receptor activity. To investigate this unexpected finding further, we constructed FhuAΔ5–160 derivatives of FhuA proteins from
Salmonella paratyphi
B,
Salmonella enterica
serovar Typhimurium, and
Pantoea agglomerans
. The FhuAΔ5–160 proteins inserted correctly into the outer membrane, and with the exception of the
P. agglomerans
protein, transported ferrichrome and albomycin. FhuA hybrids consisting of the β-barrel of one strain and the cork of another strain were active and showed higher TonB-dependent ferrichrome transport rates than the corkless derivatives. Exceptions were the
E. coli
β-barrel/
Salmonella
serovar Typhimurium cork hybrid protein and the
Salmonella
serovar Typhimurium β-barrel/
P. agglomerans
cork hybrid protein, both of which were less active than the β-barrels alone. Each of the FhuA mutant proteins displayed activity for each of their ligands, except for phage T5, only when coupled to TonB. The hybrid FhuA proteins displayed a similar activity with the
E. coli
TonB protein as with their cognate TonB proteins. Sensitivity to phages T1, T5, and φ80, rifamycin CGP 4832, and colicin M was determined by the β-barrel, whereas sensitivity to phage ES18 and microcin J25 required both the β-barrel and cork domains. These results demonstrate that the β-barrel domain of FhuA confers activity and specificity and responds to TonB and that the cork domains of various FhuA proteins can be interchanged and contribute to the activities of the FhuA hybrids.
VirB7 Lipoprotein Is Exocellular and Associates with the
Agrobacterium tumefaciens
T Pilus
Authors: Sagulenko Vitaliya , Sagulenko Evgeniy , Jakubowski Simon , Spudich Elena , Christie Peter J.
Published: July 2002

Agrobacterium tumefaciens
transfers oncogenic T-DNA and effector proteins to plant cells via a type IV secretion pathway. This transfer system, assembled from the products of the
virB
operon, is thought to consist of a transenvelope mating channel and the T pilus. When screened for the presence of VirB and VirE proteins, material sheared from the cell surface of octopine strain A348 was seen to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer membrane lipoprotein. Material sheared from the cell surface of most
virB
gene deletion mutants also possessed VirB7, but not VirB2 or VirB5. During purification of the T pilus from wild-type cells, VirB2, VirB5, and VirB7 cofractionated through successive steps of gel filtration chromatography and sucrose density gradient centrifugation. A complex containing VirB2 and VirB7 was precipitated from a gel filtration fraction enriched for T pilus with both anti-VirB2 and anti-VirB7 antiserum. Both the exocellular and cellular forms of VirB7 migrated as disulfide-cross-linked dimers and monomers when samples were electrophoresed under nonreducing conditions. A mutant synthesizing VirB7 with a Ser substitution of the lipid-modified Cys15 residue failed to elaborate the T pilus, whereas a mutant synthesizing VirB7 with a Ser substitution for the disulfide-reactive Cys24 residue produced very low levels of T pilus. Together, these findings establish that the VirB7 lipoprotein localizes exocellularly, it associates with the T pilus, and both VirB7 lipid modification and disulfide cross-linking are important for T-pilus assembly. T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as a monomer. A strain synthesizing a VirB2 mutant with a Ser substitution for the reactive Cys64 residue elaborated T pilus but exhibited an attenuated virulence phenotype. Dithiothreitol-treated T pilus composed of native VirB2 pilin and untreated T pilus composed of the VirB2C64S mutant pilin distributed in sucrose gradients more predominantly in regions of lower sucrose density than untreated, native T pili. These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.
Involvement of Stringent Factor RelA in Expression of the Alkaline Protease Gene
aprE
in
Bacillus subtilis
Authors: Hata Michihiro , Ogura Mitsuo , Tanaka Teruo
Published: July 2002

Expression of
Bacillus subtilis aprE
, encoding an extracellular alkaline protease, is positively regulated by phosphorylated DegU, the regulator of a two-component regulatory system, DegS-DegU. We found that the expression of an
aprE′-′lacZ
fusion was greatly reduced in a disruption mutant with a mutation of
relA
, which encodes the stringent factor RelA. The level of DegU in the
relA
mutant was similar to that in the wild-type cell. A
relA degU
double mutation did not result in a further decrease of the
aprE′-′lacZ
level found in a
degU
single mutant. The expression of the
aprE′-′lacZ
fusion in the
relA
mutant was stimulated by multicopy
degR
or the
degU32
(Hy) and
degS200
(Hy) mutations that cause the stabilization of phosphorylated DegU. Furthermore, the expression of
sacB′-′lacZ
, which is also dependent on phosphorylated DegU, was stimulated by the
relA
mutation, and this stimulation was not seen in the
relA degU
double mutant. These results show that RelA (or its product guanosine-3′,5′-bisdiphosphate [pp Gpp]) does not affect the phosphorylation of DegU and suggest that it participates in the expression of
aprE
and
sacB
through the regulation of DegU-dependent transcription.
Proteolysis of the
Caulobacter
McpA Chemoreceptor Is Cell Cycle Regulated by a ClpX-Dependent Pathway
Authors: Tsai Jeng-Wen , Alley M. R. K.
Published: July 2002

Proteolysis is involved in cell differentiation and the progression through the cell cycle in
Caulobacter crescentus
. We have constitutively expressed the transmembrane chemoreceptor McpA from a multicopy plasmid to demonstrate that McpA degradation is modulated during the cell cycle. The level of McpA protein starts to decrease only when the swarmer cells differentiate into stalked cells. The reduction in McpA protein levels is maintained until the stalked cells develop into predivisional cells, at which point the level returns to that observed in swarmer cells. The cell-cycle-regulated degradation of McpA does not require the last 12 C-terminal amino acids, but it does require three amino acids (AAL) located 15 residues away from the C terminus. The ClpXP protease is essential in
C
.
crescentus
for viability, and thus, we tested McpA degradation in xylose conditional mutants. The effect on McpA degradation occurred within two generations from the start of ClpX depletion. The conditional mutants' growth rate was only slightly affected, suggesting that ClpX is directly involved in McpA proteolysis.
Glycopeptidolipid Acetylation Affects Sliding Motility and Biofilm Formation in
Mycobacterium smegmatis
Authors: Recht Judith , Kolter Roberto
Published: July 2002

The absence of glycopeptidolipids (GPLs) abolishes the ability of mycobacteria both to slide over the surface of motility plates and to form biofilms on polyvinyl chloride. In a screen for biofilm-defective mutants of
Mycobacterium smegmatis
mc
2
155, a new mutant was obtained that resulted in partial inhibition of both processes and also showed an intermediate rough colony morphology. The
mariner
transposon insertion mapped to a GPL biosynthesis gene (
atf1
) which encodes a putative acetyltranferase involved in the transfer of acetyl groups to the glycopeptide core. Physical characterization of the GPLs from the
atf1
mutant demonstrated that they were not acetylated.
Urea Utilization in the Phototrophic Bacterium
Rhodobacter capsulatus
Is Regulated by the Transcriptional Activator NtrC
Authors: Masepohl Bernd , Kaiser Björn , Isakovic Nazila , Richard Cynthia L. , Kranz Robert G. , Klipp Werner
Published: July 2002

The phototrophic nonsulfur purple bacterium
Rhodobacter capsulatus
can use urea as a sole source of nitrogen. Three transposon Tn
5
-induced mutations (Xan-9, Xan-10, and Xan-19), which led to a Ure
−
phenotype, were mapped to the
ureF
and
ureC
genes, whereas two other Tn
5
insertions (Xan-20 and Xan-22) were located within the
ntrC
and
ntrB
genes, respectively. As in
Klebsiella aerogenes
and other bacteria, the genes encoding urease (
ureABC
) and the genes required for assembly of the nickel metallocenter (
ureD
and
ureEFG
) are clustered in
R. capsulatus
(
ureDABC
-
orf136
-
ureEFG
). No homologues of Orf136 were found in the databases, and mutational analysis demonstrated that
orf136
is not essential for urease activity or growth on urea. Analysis of a
ureDA
-
lacZ
fusion showed that maximum expression of the
ure
genes occurred under nitrogen-limiting conditions (e.g., serine or urea as the sole nitrogen source), but
ure
gene expression was not substrate (urea) inducible. Expression of the
ure
genes was strictly dependent on NtrC, whereas ς
54
was not essential for urease activity. Expression of the
ure
genes was lower (by a factor of 3.5) in the presence of ammonium than under nitrogen-limiting conditions, but significant transcription was also observed in the presence of ammonium, approximately 10-fold higher than in an
ntrC
mutant background. Thus,
ure
gene expression in the presence of ammonium also requires NtrC. Footprint analyses demonstrated binding of NtrC to tandem binding sites upstream of the
ureD
promoter. Phosphorylation of NtrC increased DNA binding by at least eightfold. Although urea is effectively used as a nitrogen source in an NtrC-dependent manner, nitrogenase activity was not repressed by urea.
Bacillus subtilis
YxkJ Is a Secondary Transporter of the 2-Hydroxycarboxylate Transporter Family That Transports
l
-Malate and Citrate
Authors: Krom Bastiaan P. , Aardema Ronald , Lolkema Juke S.
Published: July 2002

The genome of
Bacillus subtilis
contains two genes that code for membrane proteins that belong to the 2-hydroxycarboxylate transporter family. Here we report the functional characterization of one of the two,
yxkJ
, which codes for a transporter protein named CimHbs. The gene was cloned and expressed in
Escherichia coli
and complemented the citrate-negative phenotype of wild-type
E. coli
and the malate-negative phenotype of the
E. coli
strain JRG4008, which is defective in malate uptake. Subsequent uptake studies in whole cells expressing CimHbs clearly demonstrated the citrate and malate transport activity of the protein. Immunoblot analysis showed that CimHbs is a 48-kDa protein that is well expressed in
E. coli
. Studies with right-side-out membrane vesicles demonstrated that CimHbs is an electroneutral proton-solute symporter. No indications were found for the involvement of Na
+
ions in the transport process. Inhibition of the uptake catalyzed by CimHbs by divalent metal ions, together with the lack of effect on transport by the chelator EDTA, showed that CimHbs translocates the free citrate and malate anions. Among a large set of substrates tested, only malate, citramalate, and citrate competitively inhibited citrate transport catalyzed by CimHbs. The transporter is strictly stereoselective, recognizing only the S enantiomers of malate and citramalate. Remarkably, though citramalate binds to the transporter, it is not translocated.
Production of Two Proteins Encoded by the
Bacteroides
Mobilizable Transposon NBU1 Correlates with Time-Dependent Accumulation of the Excised NBU1 Circular Form
Authors: Wang Jun , Wang Gui-Rong , Shoemaker Nadja B. , Salyers Abigail A.
Published: July 2002

NBU1 is a mobilizable transposon that excises from the
Bacteroides
chromosome to form a double-stranded circular transfer intermediate. Excision is triggered by exposure of the bacteria to tetracycline. Accordingly, we expected that the expression of NBU1 genes would be induced by tetracycline. To test this hypothesis, antibodies that recognized two NBU1-encoded proteins, PrmN1 and MobN1, were used to monitor production of these proteins. PrmN1 is essential for excision, and MobN1 is essential for transfer of the excised circular form. At first, expression of the genes encoding these two proteins appeared to be regulated by tetracycline, because the proteins were detectable on Western blots only after the cells were exposed to tetracycline. However, when the
prmN1
gene and/or the
mobN1
gene was cloned on a multicopy plasmid, production of the protein was constitutive. Initially, we assumed that the constitutive expression was due to loss of a repressor protein that was encoded by one of the other genes on NBU1. Deletions or insertions in the other genes (
orf2
and
orf3
) on NBU1 and various integrated NBU1 derivatives abolished production of PrmN1 and MobN1. This is the opposite of what should have happened if one or both of these genes encoded a repressor. A second possibility was that when NBU1 excised, it replicated transiently, increasing the gene dosage of
prmN1
and
mobN1
and thereby producing enough PrmN1 and MobN1 for these proteins to become detectable. In fact, after the cells entered late exponential phase the copy number of NBU1 increased to 2 to 3 copies per cell. Production of PrmN1 and MobN1 showed a similar pattern. Any mutation in NBU1 that decreased or prevented excision also prevented elevated production of these two proteins. Our results show that the apparent tetracycline dependence of the production of PrmN1 and MobN1 is due to a growth phase- or time-dependent increase in the number of copies of the NBU1 circular form.
Interactive Control of
Rhodobacter
capsulatus
Redox-Balancing Systems during Phototrophic Metabolism
Authors: Tichi Mary A. , Tabita F. Robert
Published: July 2002

In nonsulfur purple bacteria, redox homeostasis is achieved by the coordinate control of various oxidation-reduction balancing mechanisms during phototrophic anaerobic respiration. In this study, the ability of
Rhodobacter
capsulatus
to maintain a balanced intracellular oxidation-reduction potential was considered; in addition, interrelationships between the control of known redox-balancing systems, the Calvin-Benson-Bassham, dinitrogenase and dimethyl sulfoxide reductase systems, were probed in strains grown under both photoheterotrophic and photoautotrophic growth conditions. By using
cbb
I
(
cbb
form I operon)-,
cbb
II
-,
nifH
-, and
dorC
-reporter gene fusions, it was demonstrated that each redox-balancing system responds to specific metabolic circumstances under phototrophic growth conditions. In specific mutant strains of
R.
capsulatus
, expression of both the Calvin-Benson-Bassham and dinitrogenase systems was influenced by dimethyl sulfoxide respiration. Under photoheterotrophic growth conditions, coordinate control of redox-balancing systems was further manifested in ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase deletion strains. These findings demonstrated the existence of interactive control mechanisms that govern the diverse means by which
R.
capsulatus
maintains redox poise during photoheterotrophic and photoautotrophic growth.
Yeast PalA/AIP1/Alix Homolog Rim20p Associates with a PEST-Like Region and Is Required for Its Proteolytic Cleavage
Authors: Xu Wenjie , Mitchell Aaron P.
Published: July 2002

The
Saccharomyces cerevisiae
zinc finger protein Rim101p is activated by cleavage of its C-terminal region, which resembles PEST regions that confer susceptibility to proteolysis. Here we report that Rim20p, a member of the broadly conserved PalA/AIP1/Alix family, is required for Rim101p cleavage. Two-hybrid and coimmunoprecipitation assays indicate that Rim20p binds to Rim101p, and a two-hybrid assay shows that the Rim101p PEST-like region is sufficient for Rim20p binding. Rim101p-Rim20p interaction is conserved in
Candida albicans
, supporting the idea that interaction is functionally significant. Analysis of Rim20p mutant proteins indicates that some of its broadly conserved regions are required for processing of Rim101p and for stability of Rim20p itself but are not required for interaction with Rim101p. A recent genome-wide two-hybrid study (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569–4574, 2000) indicates that Rim20p interacts with Snf7p and that Snf7p interacts with Rim13p, a cysteine protease required for Rim101p proteolysis. We suggest that Rim20p may serve as part of a scaffold that places Rim101p and Rim13p in close proximity.
Activation of Silent
gal
Genes in the
lac-gal
Regulon of
Streptococcus thermophilus
Authors: Vaughan Elaine E. , van den Bogaard Patrick T. C. , Catzeddu Pasquale , Kuipers Oscar P. , de Vos Willem M.
Published: July 2002

Streptococcus thermophilus
strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with
Escherichia coli
demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order
galKTE
, which was preceded by a divergently transcribed regulator gene,
galR
, and followed by a
galM
gene and the lactose operon
lacSZ
. Results of Northern blot analysis showed that the structural
gal
genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the
galKTE
genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal
+
mutant NZ302G, the transcription of the
galR
gene was induced by growth on lactose. Disruption of
galR
indicated that it functioned as a transcriptional activator of both the
gal
and
lac
operons while negatively regulating its own expression. Sequence analysis of the
gal
promoter regions of NZ302G and nine other independently isolated Gal
+
mutants of CNRZ 302 revealed mutations at three positions in the
galK
promoter region, which included substitutions at positions −9 and −15 as well as a single-base-pair insertion at position −37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of
gusA
reporter gene fusions in strains containing the mutated promoters suggested that they were
gal
promoter-up mutations. We propose that poor expression of the
gal
genes in the galactose-negative
S. thermophilus
CNRZ 302 is caused by naturally occurring mutations in the
galK
promoter.
Structure-Function Analysis of the
Shigella
Virulence Factor IpaB
Authors: Guichon Andrea , Hersh David , Smith Mark R. , Zychlinsky Arturo
Published: July 2002

Infection by the gram-negative bacterium
Shigella flexneri
results in dysentery, an acute inflammatory disease of the colon. Essential events in the pathogenesis of
Shigella
infections include bacterial invasion of epithelial cells, escape from the phagosome, and induction of apoptosis in macrophages. The
Shigella
virulence factor invasion plasmid antigen B (IpaB) is required for all of these processes. Induction of apoptosis is dependent on IpaB binding to the cysteine protease caspase-1 (Casp-1). The activation of this enzyme triggers both apoptosis and release of the proinflammatory cytokine interleukin-1β. Several IpaB mutants were generated to correlate function with protein subdomains. We determined that the N-terminal portion of IpaB is necessary for stable expression of IpaB. A putative amphipathic α-helical domain preserves the structure of IpaB. We found 10 consecutive residues within the amino terminus of the hydrophobic region that play a critical role in invasion, phagosomal escape, and cytotoxicity. An IpaB mutant carrying a mutation in this region binds to Casp-1 yet is not cytotoxic, even following direct delivery to the macrophage cytoplasm. These results indicate that the association between IpaB and Casp-1 is only a step in the activation of macrophage apoptosis.
Phylogenetic Evidence for Horizontal Transfer of
mutS
Alleles among Naturally Occurring
Escherichia coli
Strains
Authors: Brown Eric W. , LeClerc J. Eugene , Li Baoguang , Payne William L. , Cebula Thomas A.
Published: July 2002

mutS
mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the
mutS
phenotype, the evolutionary structure of the
mutS
+
gene in
Escherichia coli
remains unclear. The physical proximity of
mutS
to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify
E. coli
strains in which
mutS
alleles have recombined. Comparison of
mutS
phylogeny against predicted
E. coli
“whole-chromosome” phylogenies (derived from multilocus enzyme electrophoresis and
mdh
sequences) revealed striking levels of phylogenetic discordance among
mutS
alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among
mutS
alleles. Examination of additional sites surrounding
mutS
also revealed incongruous distributions compared to
E. coli
strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min
mutS-rpoS
region is a recombinational hot spot within the
E. coli
chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by
mutS
mutators through rescue of defective
mutS
alleles with wild-type sequences.
Linear Plasmid in the Genome of
Clavibacter michiganensis
subsp.
sepedonicus
Authors: Brown Susan E. , Knudson Dennis L. , Ishimaru Carol A.
Published: July 2002

Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen
Clavibacter michiganensis
subsp.
sepedonicus
revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.
Characterization of the
Bacillus subtilis ywsC
Gene, Involved in γ-Polyglutamic Acid Production
Authors: Urushibata Yuji , Tokuyama Shinji , Tahara Yasutaka
Published: July 2002

The genes required for γ-polyglutamic acid (PGA) production were cloned from
Bacillus subtilis
IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the
ywsC
and
ywtABC
genes of
B. subtlis
168. Northern blot analysis showed that the four genes constitute an operon. Three genes,
ywsC
,
ywtA,
and
ywtB
, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in Δ
ywsC
and Δ
ywtA
strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the Δ
ywsC
strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the
ywsC
gene.
14
C-labeled PGA was synthesized by the purified proteins from
l
-[
14
C]-glutamate in the presence of ATP and MnCl
2
, through an acylphosphate intermediate, indicating that the
ywsC
gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.
raiIR
Genes Are Part of a Quorum-Sensing Network Controlled by
cinI
and
cinR
in
Rhizobium leguminosarum
Authors: Wisniewski-Dyé F. , Jones J. , Chhabra S. R. , Downie J. A.
Published: July 2002

Analysis of
N
-acyl-
l
-homoserine lactones (AHLs) produced by
Rhizobium leguminosarum
bv. viciae indicated that there may be a network of quorum-sensing regulatory systems producing multiple AHLs in this species. Using a strain lacking a symbiosis plasmid, which carries some of the quorum-sensing genes, we isolated mutations in two genes (
raiI
and
raiR
) that are required for production of AHLs. The
raiIR
genes are located adjacent to
dad
genes (involved in
d
-alanine catabolism) on a large indigenous plasmid. RaiR is predicted to be a typical LuxR-type quorum-sensing regulator and is required for
raiI
expression. The
raiR
gene was expressed at a low level, possibly from a constitutive promoter, and its expression was increased under the influence of the upstream
raiI
promoter. Using gene fusions and analysis of AHLs produced, we showed that expression of
raiI
is strongly reduced in strains carrying mutations in
cinI
or
cinR
, genes which determine a higher-level quorum-sensing system that is required for normal expression of
raiIR
. The product of CinI,
N
-(3-hydroxy-7-
cis
tetradecenoyl) homoserine lactone, can induce
raiR
-dependent
raiI
expression, although higher levels of expression are induced by other AHLs. Expression of
raiI
in a strain of
Agrobacterium
that makes no AHLs resulted in the identification of
N
-(3-hydroxyoctanoyl)-
l
-homoserine lactone (3OH,C
8
-HSL) as the major product of RaiI, although other AHLs that comigrate with
N
-hexanoyl-,
N
-heptanoyl-, and
N
-octanoyl-homoserine lactones were also made at low levels. The
raiI
gene was strongly induced by 3OH,C
8
-HSL (the product of RaiI) but could also be induced by other AHLs, suggesting that the
raiI
promoter can be activated by other quorum-sensing systems within a network of regulation which also involves AHLs determined by genes on the symbiotic plasmid. Thus, the
raiIR
and
cinIR
genes are part of a complex regulatory network that influences AHL biosynthesis in
R. leguminosarum
.