bluuebunny/crossref_metadata_2025 huggingface dataset:
Conjugative Junctions in RP4-Mediated Mating of
Escherichia coli
Authors: Samuels A. Lacey , Lanka Erich , Davies Julian E.
Published: July 2002

The physical association of bacteria during conjugation mediated by the IncPα plasmid RP4 was investigated.
Escherichia coli
mating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (
trbC
), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.
Role of the
Azotobacter vinelandii
Nitrogenase-Protective Shethna Protein in Preventing Oxygen-Mediated Cell Death
Authors: Maier R. J. , Moshiri F.
Published: July 2002

Azotobacter vinelandii
strains lacking the nitrogenase-protective Shethna protein lost viability upon carbon-substrate deprivation in the presence of oxygen. This viability loss was dependent upon the N
2
-fixing status of cultures (N
2
-fixing cells lost viability, while non-N
2
-fixing cells did not) and on the ambient O
2
level. Supra-atmosheric O
2
tensions (40% partial pressure) decreased the viable cell number of the mutant further, and the mutant had a slightly higher spontaneous mutation frequency than the wild type in the high-O
2
conditions. Iron starvation conditions, which resulted in fourfold-reduced superoxide dismutase levels, were also highly detrimental to the viability of the protective protein mutants, but these conditions did not affect the viability of the wild-type strain. Nitrogenase or other powerful reductants associated with N
2
fixation may be sources of damaging partially reduced oxygen species, and the production of such species are perhaps minimized by the Shethna protein.
Chromosomal and Extrachromosomal Synthesis of Exfoliative Toxin from
Staphylococcus hyicus
Authors: Sato Hisaaki , Watanabe Takao , Higuchi Kohichi , Teruya Kuniaki , Ohtake Ayumi , Murata Yasuko , Saito Hiroshi , Aizawa Chikara , Danbara Hirofumi , Maehara Nobutoshi
Published: July 2002

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by
Staphylococcus hyicus
(SHET) under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxins are distinct from each other is given. The molecular weights of SHET from plasmidless strain P-1 (SHETA) and from plasmid-carrying strains P-10 and P-23 (SHETB) were almost equal. Both of the serotypes of SHET exhibited exfoliation in 1-day-old chickens. The plasmid-cured (P
−
) substrains (P-23C1 and P-23C2) of
S. hyicus
P-23 did not cause exfoliation in 1-day-old chickens, whereas P
−
substrains (P-10C1 and P-10C2) of strain P-10 caused exfoliation, but they decreased their exfoliative activity. These findings suggest that SHETB was synthesized along with SHETA by strain P-10, whereas the P-23 strain synthesized SHETB alone. The plasmid-carrying strain (P-23) as well as the plasmidless strain (P-1) exhibited the typical clinical signs of exudative epidermitis in pigs. However, plasmid-cured (P
−
) substrains of P-23 (P23C1 and P23C2) did not exhibit the typical clinical signs of exudative epidermitis. These findings suggest that SHETA is synthesized under chromosomal control and SHETB is synthesized under plasmid control and that SHET-producing strains can be divided into three groups: SHETA-producing strains, SHETB-producing strains, and strains producing both toxins.
The
Escherichia coli
O111 and
Salmonella enterica
O35 Gene Clusters: Gene Clusters Encoding the Same Colitose-Containing O Antigen Are Highly Conserved
Authors: Wang Lei , Reeves Peter R.
Published: July 2002

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria.
Escherichia coli
and
Salmonella enterica
each have many forms of O antigen, but only three are common to the two species. It has been found that, in general, O-antigen genes are of low GC content. This deviation in GC content from that of typical
S. enterica
or
E. coli
genes (51%) is thought to indicate that the O-antigen DNA originated in species other than
S. enterica
or
E. coli
and was captured by lateral transfer. The O-antigen structure of
Salmonella enterica
O35 is identical to that of
E. coli
O111, commonly found in enteropathogenic
E. coli
strains. This O antigen, which has been shown to be a virulence factor in
E. coli
, contains colitose, a 3,6-dideoxyhexose found only rarely in the
Enterobacteriaceae
. Sequencing of the O35-antigen gene cluster of
S. enterica
serovar Adelaide revealed the same gene order and flanking genes as in
E. coli
O111. The divergence between corresponding genes of these two gene clusters at the nucleotide level ranges from 21.8 to 11.7%, within the normal range of divergence between
S. enterica
and
E. coli
. We conclude that the ancestor of
E. coli
and
S. enterica
had an O antigen identical to the O111 and O35 antigens, respectively, of these species and that the gene cluster encoding it has survived in both species.
An Operon for a Putative ATP-Binding Cassette Transport System Involved in Acetoin Utilization of
Bacillus subtilis
Authors: Yoshida Ken-Ichi , Fujita Yasutaro , Ehrlich S. Dusko
Published: July 2002

The
ytrABCDEF
operon of
Bacillus subtilis
was deduced to encode a putative ATP-binding cassette (ABC) transport system. YtrB and YtrE could be the ABC subunits, and YtrC and YtrD are highly hydrophobic and could form a channel through the cell membrane, while YtrF could be a periplasmic lipoprotein for substrate binding. Expression of the operon was examined in cells grown in a minimal medium. The results indicate that the expression was induced only early in the stationary phase. The six
ytr
genes form a single operon, transcribed from a putative ς
A
-dependent promoter present upstream of
ytrA
. YtrA, which possesses a helix-turn-helix motif of the GntR family, acts probably as a repressor and regulates its own transcription. Inactivation of the operon led to a decrease in maximum cell yield and less-efficient sporulation, suggesting its involvement in the growth in stationary phase and sporulation. It is known that
B. subtilis
produces acetoin as an external carbon storage compound and then reuses it later during stationary phase and sporulation. When either the entire
ytr
operon or its last gene,
ytrF
, was inactivated, the production of acetoin was not affected, but the reuse of acetoin became less efficient. We suggest that the Ytr transport system plays a role in acetoin utilization during stationary phase and sporulation.
Analysis of the Essential Cell Division GeneftsLofBacillus subtilisby Mutagenesis and Heterologous Complementation
Authors: Sievers Jörg , Errington Jeff
Published: July 2002

TheftsLgene is required for the initiation of cell division in a broad range of bacteria.Bacillus subtilis ftsLencodes a 13-kDa protein with a membrane-spanning domain near its N terminus. The external C-terminal domain has features of an α-helical leucine zipper, which is likely to be involved in the heterodimerization with another division protein, DivIC. To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis. Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon. It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect. Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function.ftsLhomologues cloned from three diverseBacillusspecies,Bacillus licheniformis,Bacillus badius, andBacillus circulans, could complement anftsLnull mutation inB. subtilis, even though up to 66% of the amino acid residues of the predicted proteins were different fromB. subtilisFtsL. However, theftsLgene fromStaphylococcus aureus(whose product has 73% of its amino acids different from those of theB. subtilis ftsLproduct) was not functional. We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.
Mutation of the Lipopolysaccharide Core Glycosyltransferase Encoded by
waaG
Destabilizes the Outer Membrane of
Escherichia coli
by Interfering with Core Phosphorylation
Authors: Yethon Jeremy A. , Vinogradov Evgeny , Perry Malcolm B. , Whitfield Chris
Published: July 2002

In
Escherichia coli
, phosphoryl substituents in the lipopolysaccharide core region are essential for outer membrane stability. Mutation of the core glucosyltransferase encoded by
waaG
(formerly
rfaG
) resulted in lipopolysaccharide truncated immediately after the inner core heptose residues, which serve as the sites for phosphorylation. Surprisingly, mutation of
waaG
also destabilized the outer membrane. Structural analyses of
waaG
mutant lipopolysaccharide showed that the cause for this phenotype was a decrease in core phosphorylation, an unexpected side effect of the
waaG
mutation.
Differential Processing of Propeptide Inhibitors of Rap Phosphatases in
Bacillus subtilis
Authors: Jiang Min , Grau Roberto , Perego Marta
Published: July 2002

In the phosphorelay signal transduction system for sporulation initiation in
Bacillus subtilis
, the opposing activities of histidine kinases and aspartyl phosphate phosphatases determine the cell's decision whether to continue with vegetative growth or to initiate the differentiation process. Regulated dephosphorylation of the Spo0A and Spo0F response regulators allows a variety of negative signals from physiological processes that are antithetical to sporulation to impact on the activation level of the phosphorelay. Spo0F∼P is the known target of two related phosphatases, RapA and RapB. In addition to RapA and RapB, a third member of the Rap family of phosphatases, RapE, specifically dephosphorylated the Spo0F∼P intermediate in response to competence development. RapE phosphatase activity was found to be controlled by a pentapeptide (SRNVT) generated from within the carboxy-terminal domain of the
phrE
gene product. A synthetic PhrE pentapeptide could (i) complement the sporulation deficiency caused by deregulated RapE activity of a
phrE
mutant and (ii) inhibit RapE-dependent dephosphorylation of Spo0F∼P in in vitro experiments. The PhrE pentapeptide did not inhibit the phosphatase activity of RapA and RapB. These results confirm previous conclusions that the specificity for recognition of the target phosphatase is contained within the amino acid sequence of the pentapeptide inhibitor.
Dimerization between the Holin and Holin Inhibitor of Phage λ
Authors: Gründling Angelika , Smith David L. , Bläsi Udo , Young Ry
Published: July 2002

Holins are integral membrane proteins that control the access of phage-encoded muralytic enzymes, or endolysins, to the cell wall by the sudden formation of an uncharacterized homo-oligomeric lesion, or hole, in the membrane, at a precisely defined time. The timing of λ-infected cell lysis depends solely on the 107 codon
S
gene, which encodes two proteins, S105 and S107, which are the holin and holin inhibitor, respectively. Here we report the results of biochemical and genetic studies on the interaction between the holin and the holin inhibitor. A unique cysteine at position 51, in the middle of the second transmembrane domain, is shown to cause the formation of disulfide-linked dimers during detergent membrane extraction. Forced oxidation of membranes containing S molecules also results in the formation of covalently linked dimers. This technique is used to demonstrate efficient dimeric interactions between S105 and S107. These results, coupled with the previous finding that the timing of lysis depends on the excess of the amount of S105 over S107, suggest a model in which the inhibitor functions by titrating out the effector in a stoichiometric fashion. This provides a basis for understanding two evolutionary advantages provided by the inhibitor system, in which the production of the inhibitor not only causes a delay in the timing of lysis, allowing the assembly of more virions, but also increases effective hole formation after triggering.
Characterization of the ς
B
Regulon in
Staphylococcus aureus
Authors: Gertz Silke , Engelmann Susanne , Schmid Roland , Ziebandt Anne-Kathrin , Tischer Karsten , Scharf Christian , Hacker Jörg , Hecker Michael
Published: July 2002

The ς
B
-dependent stress regulon in gram-positive bacteria might fulfill a physiological role in stress response and virulence similar to that of the ς
S
regulon in
Escherichia coli
and other gram-negative bacteria. In order to obtain evidence for the function of the ς
B
regulon of
Staphylococcus aureus
, especially in virulence control, ς
B
-dependent stress genes were identified. The two-dimensional protein pattern of wild-type cells of
S. aureus
COL was compared with that of an isogenic
sigB
mutant. By this approach, we found that the synthesis of about 27 cytoplasmic proteins seemed to be under the positive control of ς
B
. N-terminal sequencing of 18 proteins allowed the identification of their genes on the almost finished genome sequence of
S. aureus
COL and the analysis of the promoter structure. Transcriptional analyses of 11 of these genes confirmed their ς
B
dependency, and moreover, about 7 additional ς
B
-dependent genes were found which are cotranscribed with the newly detected genes, forming operons. Altogether, we identified 23 ς
B
-dependent genes and their corresponding proteins. Among them are proteins probably involved in the generation of NADH or in membrane transport mechanisms. Furthermore, at least one
clpC
-homologous gene was localized on the
S. aureus
sequence solely transcribed by ς
B
. In contrast, a second
clpC
-homologous gene in
S. aureus
forming an operon with
ctsR
,
yacH
, and
yacI
was ς
B
independently expressed.
TheAgrobacteriumT-DNA Transport Pore Proteins VirB8, VirB9, and VirB10 Interact with One Another
Authors: Das Anath , Xie Yong-Hong
Published: July 2002

The VirB proteins ofAgrobacterium tumefaciensform a transport pore to transfer DNA from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10. Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another. In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself. These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore. Fusions that contain small, defined segments of the proteins were used to define the interaction domains of VirB8 and VirB9. All interaction domains of both proteins mapped to the N-terminal half of the proteins. Two separate domains at the N- and C-terminal ends of VirB9 are involved in its homotypic interaction, suggesting that VirB9 forms a higher oligomer. We observed that the alteration of serine at position 87 of VirB8 to leucine abolished its DNA transfer function. Studies on the interaction of the mutant protein with the other VirB proteins showed that the VirB8S87L mutant is defective in interaction with VirB9. The mutant, however, interacted efficiently with VirB8 and VirB10, suggesting that the VirB8-VirB9 interaction is essential for DNA transfer.
Extracellular Glycanases of
Rhizobium leguminosarum
Are Activated on the Cell Surface by an Exopolysaccharide-Related Component
Authors: Zorreguieta Angeles , Finnie Christine , Downie J. Allan
Published: July 2002

Rhizobium leguminosarum
secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of
R. leguminosarum
, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS
+
strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to
Agrobacterium tumefaciens
enabled it to degrade CMC, but this was only observed if it was grown on a lawn of
R. leguminosarum
. This indicates that the surface of
A. tumefaciens
is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.
AcrD of
Escherichia coli
Is an Aminoglycoside Efflux Pump
Authors: Rosenberg Emiko Y. , Ma Dzwokai , Nikaido Hiroshi
Published: July 2002

AcrD, a transporter belonging to the resistance-nodulation-division family, was shown to participate in the efflux of aminoglycosides. Deletion of the
acrD
gene decreased the MICs of amikacin, gentamicin, neomycin, kanamycin, and tobramycin by a factor of two to eight, and
ΔacrD
cells accumulated higher levels of [
3
H]dihydrostreptomycin and [
3
H]gentamicin than did the parent strain.
Dephosphorylation of the
Escherichia coli
Transcriptional Antiterminator BglG by the Sugar Sensor BglF Is the Reversal of Its Phosphorylation
Authors: Chen Qing , Postma Pieter W. , Amster-Choder Orna
Published: July 2002

The
Escherichia coli
BglF protein catalyzes transport and phosphorylation of β-glucosides. In addition, BglF is a membrane sensor which reversibly phosphorylates the transcriptional regulator BglG, depending on β-glucoside availability. Therefore, BglF has three enzymatic activities: β-glucoside phosphotransferase, BglG phosphorylase, and phospho-BglG (BglG-P) dephosphorylase. Cys-24 of BglF is the active site which delivers the phosphoryl group either to the sugar or to BglG. To characterize the dephosphorylase activity, we asked whether BglG-P can give the phosphoryl group back to Cys-24 of BglF. Here we provide evidence which is consistent with the interpretation that Cys-24–P is an intermediate in the BglG-P dephosphorylation reaction. Hence, the dephosphorylation reaction catalyzed by BglF proceeds via reversal of the phosphorylation reaction.
Genome Plasticity among Related
Lactococcus
Strains: Identification of Genetic Events Associated with Macrorestriction Polymorphisms
Authors: Le Bourgeois Pascal , Daveran-Mingot Marie-Line , Ritzenthaler Paul
Published: July 2002

The genomic diversity of nine strains of the
Lactococcus lactis
subsp.
cremoris
(NCDO712, NCDO505, NCDO2031, NCDO763, MMS36, C2, LM0230, LM2301, and MG1363) was studied by macrorestriction enzyme analysis using pulsed-field gel electrophoresis. These strains were considered adequate for the investigation of genomic plasticity because they have been described as belonging to the same genetic lineage. Comparison of
Apa
I and
Sma
I genome fingerprints of each strain revealed the presence of several macrorestriction fragment length polymorphisms (RFLPs), despite a high degree of similarity of the generated restriction patterns. The physical map of the MG1363 chromosome was used to establish a genome map of the other strains and allocate the RFLPs to five regions. Southern hybridization analysis correlated the polymorphic regions with genetic events such as chromosomal inversion, integration of prophage DNA, and location of the transposon-like structures carrying conjugative factor or oligopeptide transport system.
Characterization of a Three-Component Vanillate
O
-Demethylase from
Moorella thermoacetica
Authors: Naidu Devendra , Ragsdale Stephen W.
Published: July 2002

The
Moorella thermoacetica
aromatic
O
-demethylase was characterized as an inducible three-component system with similarity to the methanogenic methanol, methylamine, and methanethiol methyltransferases and to the
O
-demethylase system from
Acetobacterium dehalogenans
. MtvB catalyzes methyl transfer from a phenylmethylether to the cobalt center of MtvC, a corrinoid protein. MtvA catalyzes transmethylation from MtvC to tetrahydrofolate, forming methyltetrahydrofolate. Cobalamin can substitute for MtvC.
Characterization of the Role of the ToxR-Modulated Outer Membrane Porins OmpU and OmpT in
Vibrio cholerae
Virulence
Authors: Provenzano Daniele , Lauriano Crystal M. , Klose Karl E.
Published: July 2002

ToxR, the transmembrane regulatory protein required for expression of virulence factors in the human diarrheal pathogen
Vibrio cholerae
, directly activates and represses the transcription of two outer membrane porins, OmpU and OmpT, respectively. In an attempt to dissect the role of the OmpU and OmpT porins in viability and virulence factor expression, in-frame chromosomal deletions were constructed in the coding sequences of
ompU
and
ompT
of
V. cholerae
. Two separate deletions were introduced into
ompU
; the first (small) deletion, Δ
ompU1
, removed the coding sequence for 84 internal amino acids (aa), while the second (large) deletion, Δ
ompU2
, removed the coding sequence for the entire amino-terminal 274 aa. The Δ
ompU1
strain had a growth defect that could not be complemented by episomal expression of full-length
ompU
. In contrast, a strain with Δ
ompU2
displayed wild-type growth kinetics in rich media, suggesting that this is the true phenotype of a strain lacking OmpU and that the truncated OmpU protein, rather than the absence of OmpU, may be the cause for the Δ
ompU1
phenotype. A large deletion removing the coding sequence for the entire N-terminal 273 aa of OmpT (Δ
ompT
) was also constructed in wild-type as well as Δ
toxR
and Δ
ompU2
strains, and these strains displayed wild-type growth kinetics in rich media. However, the Δ
ompU2
strain was deficient for growth in deoxycholate compared to wild-type, Δ
ompT
, and Δ
ompU2
Δ
ompT
strains, reinforcing a positive role for the OmpU porin and a negative role for the OmpT porin in
V. cholerae
resistance to anionic detergents. The Δ
ompU2
, Δ
ompT
, and Δ
ompU2
Δ
ompT
strains exhibited wild-type levels of in vitro virulence factor expression and resistance to polymyxin B and serum and in vivo colonization levels similar to a wild-type strain in the infant mouse intestine. Our results demonstrate that (i) OmpU and OmpT are not essential proteins, as was previously thought; (ii) these porins contribute to
V. cholerae
resistance to anionic detergents; and (iii) OmpU and OmpT are not essential for virulence factor expression in vitro or intestinal colonization in vivo.
Complex Regulation of the Organic Hydroperoxide Resistance Gene (
ohr
) from
Xanthomonas
Involves OhrR, a Novel Organic Peroxide-Inducible Negative Regulator, and Posttranscriptional Modifications
Authors: Sukchawalit Rojana , Loprasert Suvit , Atichartpongkul Sopapan , Mongkolsuk Skorn
Published: July 2002

Analysis of the sequence immediate upstream of
ohr
revealed an open reading frame, designated
ohrR,
with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression.
ohrR
was transcribed as bicistronic mRNA with
ohr,
while
ohr
mRNA was found to be 95% monocistronic and 5% bicistronic with
ohrR
. Expression of both genes was induced by
tert
-butyl hydroperoxide (tBOOH) treatment. High-level expression of
ohrR
negatively regulated
ohr
expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the
ohrR
promoter (P1) has organic peroxide-inducible, strong activity, while the
ohr
promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR.
ohr
primer extension results revealed three major primer extension products corresponding to the 5′ ends of
ohr
mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical
Xanthomonas
promoter. Instead, the regions can form a stem-loop secondary structure with the 5′ ends of
ohr
mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the
ohrR-ohr
operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of
ohr
mRNA, while
ohrR
mRNA is rapidly degraded.
The
licC
Gene of
Streptococcus pneumoniae
Encodes a CTP:Phosphocholine Cytidylyltransferase
Authors: Rock Charles O. , Heath Richard J. , Park Hee-Won , Jackowski Suzanne
Published: July 2002

The
licC
gene product of
Streptococcus pneumoniae
was expressed and characterized. LicC is a nucleoside triphosphate transferase family member and possesses CTP:phosphocholine cytidylyltransferase activity. Phosphoethanolamine is a poor substrate. The LicC protein plays a role in the biosynthesis of the phosphocholine-derivatized cell wall constituents that are critical for cell separation and pathogenesis.
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

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

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

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

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

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

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

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

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

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

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

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

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.