bluuebunny/crossref_metadata_2025_split huggingface dataset:
Substitutions in the Periplasmic Domain of Low-Abundance Chemoreceptor Trg That Induce or Reduce Transmembrane Signaling: Kinase Activation and Context Effects
Authors: Beel Bryan D. , Hazelbauer Gerald L. 
Published: July 2002

ABSTRACT

We extended characterization of mutational substitutions in the ligand-binding region of Trg, a low-abundance chemoreceptor of
Escherichia coli
. Previous investigations using patterns of adaptational methylation in vivo led to the suggestion that one class of substitutions made the receptor insensitive, reducing ligand-induced signaling, and another mimicked ligand occupancy, inducing signaling in the absence of ligand. We tested these deductions with in vitro assays of kinase activation and found that insensitive receptors activated the kinase as effectively as wild-type receptors and that induced-signaling receptors exhibited the low level of kinase activation characteristic of occupied receptors. Differential activation by the two mutant classes was not dependent on high-abundance receptors. Cellular context can affect the function of low-abundance receptors. Assays of chemotactic response and adaptational modification in vivo showed that increasing cellular dosage of mutant forms of Trg to a high-abundance level did not significantly alter phenotypes, nor did the presence of high-abundance receptors significantly correct phenotypic defects of reduced-signaling receptors. In contrast, defects of induced-signaling receptors were suppressed by the presence of high-abundance receptors. Grafting the interaction site for the adaptational-modification enzymes to the carboxyl terminus of induced-signaling receptors resulted in a similar suppression of phenotypic defects of induced-signaling receptors, implying that high-abundance receptors could suppress defects in induced-signaling receptors by providing their natural enzyme interaction sites in
trans
in clusters of suppressing and suppressed receptors. As in the case of cluster-related functional assistance provided by high-abundance receptors for wild-type low-abundance receptors, suppression by high-abundance receptors of phenotypic defects in induced-signaling forms of Trg involved assistance in adaptation, not signaling.
Genetic and Physiological Characterization of
ohr
, Encoding a Protein Involved in Organic Hydroperoxide Resistance in
Pseudomonas aeruginosa
Authors: Ochsner Urs A. , Hassett Daniel J. , Vasil Michael L. 
Published: July 2002

ABSTRACT

The
ohr
(organic hydroperoxide resistance) gene product of
Pseudomonas aeruginosa
was essential for optimal resistance to organic hydroperoxides (OHPs) but not to hydrogen peroxide or paraquat. A Δ
ohr
mutant was hypersusceptible to OHPs in disk inhibition assays and showed enhanced killing by OHPs in liquid culture. The
ohr
gene product was demonstrated to contribute to the decomposition of OHPs. Transcription of
ohr
was induced up to 15-fold upon exposure to OHPs, and this induction was independent of OxyR. Somewhat enhanced
ohr-lacZ
activity was detected in mutant strains affected in
ohr
,
ahpC
, and
oxyR
, and this phenotype correlated with hypersusceptibility to OHPs, suggesting overlapping or compensatory functions of the
ohr
and
ahpC
gene products. A single transcriptional start site for
ohr
was determined, and
ohr
transcripts were abundant in cells treated with a sublethal dose of OHPs but not in cells treated with paraquat. An 84-bp portion upstream of the
ohr
mRNA start site was sufficient for
ohr
induction by OHPs. Thus, the
ohr
gene appears to encode an antioxidant enzyme that is not part of the OxyR regulon yet is specifically induced by OHPs.
Nonspecific Adherence and Fibril Biogenesis by
Actinobacillus actinomycetemcomitans
: TadA Protein Is an ATPase
Authors: Bhattacharjee Mrinal K. , Kachlany Scott C. , Fine Daniel H. , Figurski David H. 
Published: July 2002

ABSTRACT

Cells of
Actinobacillus actinomycetemcomitans
, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in
flp-1
, which encodes the major subunit of the pili, or any of seven downstream
tad
genes (
tadABCDEFG
) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the
tad
genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the
tadA
gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The
tadA
gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of
A. actinomycetemcomitan
s
tadA
mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of
A. actinomycetemcomitan
s cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement
tadA
mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of
A. actinomycetemcomitans
.
Two Class A High-Molecular-Weight Penicillin-Binding Proteins of
Bacillus subtilis
Play Redundant Roles in Sporulation
Authors: McPherson Derrell C. , Driks Adam , Popham David L. 
Published: July 2002

ABSTRACT

The four class A penicillin-binding proteins (PBPs) of
Bacillus subtilis
appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls. The
ywhE
product was shown to bind penicillin, so the gene and gene product were renamed
pbpG
and PBP2d, respectively. Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis. A
pbpG
null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core. In a
pbpF pbpG
double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles. A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore. The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic δ-lactam and
l
-alanine side chains and a slight increase in cross-linking. Sporulation gene expression in the
pbpF pbpG
double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG. We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.
Presence of Prokaryotic and Eukaryotic Species in All Subgroups of the PP
i
-Dependent Group II Phosphofructokinase Protein Family
Authors: Müller Miklós , Lee Jennifer A. , Gordon Paul , Gaasterland Terry , Sensen Christoph W. 
Published: July 2002

ABSTRACT

Inorganic pyrophosphate-dependent phosphofructokinase (PP
i
-PFK) of the amitochondriate eukaryote
Mastigamoeba balamuthi
was sequenced and showed about 60% identity to PP
i
-PFKs from two eubacteria,
Propionibacterium freudenreichii
and
Sinorhizobium meliloti
. These gene products represent a newly recognized lineage of PFKs. All four lineages of group II PFKs, as defined by phylogenetic analysis, contained both prokaryotic and eukaryotic species, underlining the complex evolutionary history of this enzyme.
Elevated Temperature Differentially Affects Virulence, VirB Protein Accumulation, and T-Pilus Formation in Different
Agrobacterium tumefaciens
and
Agrobacterium vitis
Strains
Authors: Baron Christian , Domke Natalie , Beinhofer Michael , Hapfelmeier Siegfried 
Published: July 2002

ABSTRACT

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in
Agrobacterium tumefaciens
strain C58 at different temperatures ranging from 20°C to growth-inhibitory 37°C. Incubation at 28°C but not at 26°C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20°C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28°C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26°C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20°C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37°C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different
A. tumefaciens
and
Agrobacterium vitis
strains grown at 20 and 28°C. Levels of many virulence proteins were strongly reduced at 28°C compared to 20°C, and T-pilus assembly did not occur in all strains except “temperature-resistant” Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.
Isolation and Characterization of
Escherichia coli tolC
Mutants Defective in Secreting Enzymatically Active Alpha-Hemolysin
Authors: Vakharia Hema , German Greg J. , Misra Rajeev 
Published: July 2002

ABSTRACT
This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed α-helical domain, while the remaining two mapped within the outer membrane-embedded β-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.
Cooperativity between KorB and TrbA Repressors of Broad-Host-Range Plasmid RK2
Authors: Zatyka Malgorzata , Bingle Lewis , Jones Anthony C. , Thomas Christopher M. 
Published: July 2002

ABSTRACT

The KorB and TrbA proteins of broad-host-range plasmid RK2 are key regulators of the plasmid genes required for conjugative transfer.
trbBp
is the primary promoter responsible for expression of mating pair formation genes. We show that despite the targets for KorB and TrbA at
trbBp
being about 165 bp apart, 189 bp upstream of the transcription start point and overlapping the −10 region, respectively, these two proteins show up to 10-fold cooperativity for the repression of
trbBp
. Deletion analysis of TrbA showed that the C-terminal domain (CTD), which has a high degree of sequence conservation with the CTD of KorA, is required for this cooperativity with KorB. Western blotting demonstrated that the apparently mutual enhancement of repression is not due simply to elevation of repressor level by the presence of the second protein, suggesting that the basis for cooperativity is interaction between KorB and TrbA bound at their respective operators.
Multiple Catalase Genes Are Differentially Regulated in
Aspergillus nidulans
Authors: Kawasaki Laura , Aguirre Jesús 
Published: July 2002

ABSTRACT

Detoxification of hydrogen peroxide is a fundamental aspect of the cellular antioxidant responses in which catalases play a major role. Two differentially regulated catalase genes,
catA
and
catB
, have been studied in
Aspergillus nidulans
. Here we have characterized a third catalase gene, designated
catC
, which predicts a 475-amino-acid polypeptide containing a peroxisome-targeting signal. With a molecular mass of 54 kDa, CatC shows high similarity to other small-subunit monofunctional catalases and is most closely related to catalases from other fungi,
Archaea
, and animals. In contrast, the CatA (∼84 kDa) and CatB (∼79 kDa) enzymes belong to a family of large-subunit catalases, constituting a unique fungal and bacterial group. The
catC
gene displayed a relatively constant pattern of expression, not being induced by oxidative or other types of stress. Targeted disruption of
catC
eliminated a constitutive catalase activity not detected previously in zymogram gels. However, a catalase activity detected in
catA catB
mutant strains during late stationary phase was still present in
catC
and
catABC
null mutants, thus demonstrating the presence of a fourth catalase, here named catalase D (CatD). Neither
catC
nor
catABC
triple mutants showed any developmental defect, and both mutants grew as well as wild-type strains in H
2
O
2
-generating substrates, such as fatty acids, and/or purines as the sole carbon and nitrogen sources, respectively. CatD activity was induced during late stationary phase by glucose starvation, high temperature, and, to a lesser extent, H
2
O
2
treatment. The existence of at least four differentially regulated catalases indicates a large and regulated capability for H
2
O
2
detoxification in filamentous fungi.
Amino Acids in the
Bacillus subtilis
Morphogenetic Protein SpoIVA with Roles in Spore Coat and Cortex Formation
Authors: Catalano Francesca A. , Meador-Parton Jennifer , Popham David L. , Driks Adam 
Published: July 2002

ABSTRACT

Bacterial spores are protected from the environment by a proteinaceous coat and a layer of specialized peptidoglycan called the cortex. In
Bacillus subtilis
, the attachment of the coat to the spore surface and the synthesis of the cortex both depend on the spore protein SpoIVA. To identify functionally important amino acids of SpoIVA, we generated and characterized strains bearing random point mutations of
spoIVA
that result in defects in coat and cortex formation. One mutant resembles the null mutant, as sporulating cells of this strain lack the cortex and the coat forms a swirl in the surrounding cytoplasm instead of a shell around the spore. We identified a second class of six mutants with a partial defect in spore assembly. In sporulating cells of these strains, we frequently observed swirls of mislocalized coat in addition to a coat surrounding the spore, in the same cell. Using immunofluorescence microscopy, we found that in two of these mutants, SpoIVA fails to localize to the spore, whereas in the remaining strains, localization is largely normal. These mutations identify amino acids involved in targeting of SpoIVA to the spore and in attachment of the coat. We also isolated a large set of mutants producing spores that are unable to maintain the dehydrated state. Analysis of one mutant in this class suggests that spores of this strain accumulate reduced levels of peptidoglycan with an altered structure.
Global Impact of
sdiA
Amplification Revealed by Comprehensive Gene Expression Profiling of
Escherichia coli
Authors: Wei Yan , Lee Jian-Ming , Smulski Dana R. , LaRossa Robert A. 
Published: July 2002

ABSTRACT

In
Escherichia coli
the amplification of
sdiA
, a positive activator of
ftsQAZ
, genes that are essential for septation, results in mitomycin C resistance. To help us understand this resistance phenotype, genes whose expression was altered by increased
sdiA
dosage were identified using a DNA microarray-based, comprehensive transcript profiling method. The expression of 62 genes was reduced by more than threefold; of these, 41 are involved in motility and chemotaxis. Moreover, the expression of 75 genes, 36 of which had been previously characterized, was elevated at least threefold. As expected, increased
sdiA
dosage led to significantly elevated
sdiA
and ′
ddlB-ftsQAZ-lpxC
operon expression. Transcription of two genes,
uvrY
and
uvrC
, located downstream of
sdiA
and oriented in the same direction, was elevated about 10-fold, although the intervening gene,
yecF
, of opposite polarity was unaffected by increased
sdiA
dosage. Three genes (
mioC
and
gidAB
) flanking the replication origin,
oriC
, were transcribed more often when
sdiA
dosage was high, as were 12 genes within 1 min of a terminus of replication,
terB
. Transcription of the
acrABDEF
genes, mapping in three widely spaced loci, was elevated significantly, while several genes involved in DNA repair and replication (e.g.,
nei, recN, mioC
, and
mcrC
) were moderately elevated in expression. Such global analysis provides a link between septation and the response to DNA-damaging agents.
A Functional
myo
-Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of
Sinorhizobium fredii
USDA191 To Nodulate Soybean (
Glycine max
[L.] Merr.)
Authors: Jiang Guoqiao , Krishnan Ammulu Hari , Kim Yong-Woong , Wacek Thomas J. , Krishnan Hari B. 
Published: July 2002

ABSTRACT

Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds.
Rhizobium
strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the
myo
-inositol dehydrogenase gene (
idhA
) of
Sinorhizobium fredii
USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of
Sinorhizobium meliloti
IdhA and MocA;
Bacillus subtilis
IolG, YrbE, and YucG; and
Streptomyces griseus
StrI.
S. fredii
USDA191
idhA
mutants revealed no detectable
myo
-inositol dehydrogenase activity and failed to grow on
myo
-inositol as a sole carbon source. Northern blot analysis and
idhA
-
lacZ
fusion expression studies indicate that
idhA
is inducible by
myo
-inositol.
S. fredii
USDA191
idhA
mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by
S. fredii
USDA191. In addition, the
idhA
mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the
idhA
mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional
idhA
gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by
S. fredii
USDA191.
A Quorum-Sensing Signaling System Essential for Genetic Competence in
Streptococcus mutans
Is Involved in Biofilm Formation
Authors: Li Yung-Hua , Tang Nan , Aspiras Marcelo B. , Lau Peter C. Y. , Lee Janet H. , Ellen Richard P. , Cvitkovitch Dennis G. 
Published: July 2002

ABSTRACT

In a previous study, a quorum-sensing signaling system essential for genetic competence in
Streptococcus mutans
was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of
S. mutans
to initiate biofilm formation. To test this hypothesis,
S. mutans
wild-type strain NG8 and its knockout mutants defective in
comC
,
comD
,
comE
, and
comX
, as well as a
comCDE
deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The
comC
mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the
comD
and
comE
mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type
comC
gene into the cultures restored the wild-type biofilm architecture of
comC
mutants but showed no effect on the
comD
,
comE
, or
comX
mutant biofilms. The fact that biofilms formed by
comC
mutants differed from the
comD
,
comE
, and
comX
mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type
comC
gene on a shuttle vector into the
comCDE
deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in
S. mutans
is important for the formation of biofilms by this gram-positive organism.
Formation of dTDP-Rhamnose Is Essential for Growth of Mycobacteria
Authors: Ma Yufang , Pan Fei , McNeil Michael 
Published: July 2002

ABSTRACT

It was determined that the dTDP-rhamnose synthesis gene,
rmlD
, could be inactivated in
Mycobacterium smegmatis
only in the presence of a rescue plasmid carrying functional
rmlD.
Hence, dTDP-rhamnose biosynthesis is essential for the growth of mycobacteria and the targeting of dTDP-rhamnose synthesis for new tuberculosis drugs is supported.