bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Residues Involved in Catalytic Activity of the Inverting Glycosyl Transferase WbbE from
Salmonella enterica
Serovar Borreze
Authors: Keenleyside Wendy J. , Clarke Anthony J. , Whitfield Chris 
Published: July 2002

ABSTRACT

Synthesis of the O:54 O antigen of
Salmonella enterica
is initiated by the nonprocessive glycosyl transferase WbbE, assigned to family 2 of the glycosyl transferase enzymes (GT2). GT2 enzymes possess a characteristic N-terminal domain, domain A. Based on structural data from the GT2 representative SpsA (S. J. Charnock and G. J. Davies, Biochemistry 38:6380–6385, 1999), this domain is responsible for nucleotide binding. It possesses two invariant Asp residues, the first forming a hydrogen bond to uracil and the second coordinating a Mn
2+
ion. Site-directed replacement of Asp41 (D41A) of WbbE, the analogue of the first Asp residue of SpsA, revealed that this is not required for activity. WbbE possesses three Asp residues near the position analogous to the second conserved residue. Whereas D95A reduced WbbE activity, activity in D93A and D96A mutants was abrogated, suggesting that either D93 or D96 may coordinate the Mn
2+
ion. Our studies also identified a C-terminal region of sequence conservation in 22 GT2 members, including WbbE. SpsA was not among these. This region is characterized by an ED(Y) motif. The Glu and Asp residues of this motif were individually replaced in WbbE. E180D in WbbE had greatly reduced activity, and an E180Q replacement completely abrogated activity; however, D181E had no effect. E180 is predicted to reside on a turn. Combined with the alignment of the motif with potential catalytic residues in the GT2 enzymes ExoM and SpsA, we speculate that E180 is the catalytic residue of WbbE. Sequence and predicted structural divergence in the catalytic region of GT2 members suggests that this is not a homogeneous family.
SpoVID Guides SafA to the Spore Coat in
Bacillus subtilis
Authors: Ozin Amanda J. , Samford Craig S. , Henriques Adriano O. , Moran Charles P. 
Published: July 2002

ABSTRACT

Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases
Bacillus subtilis
spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.
ChvD, a Chromosomally Encoded ATP-Binding Cassette Transporter-Homologous Protein Involved in Regulation of Virulence Gene Expression in
Agrobacterium tumefaciens
Authors: Liu Zhenying , Jacobs Mark , Schaff Dennis A. , McCullen Colleen A. , Binns Andrew N. 
Published: July 2002

ABSTRACT

A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the
Agrobacterium tumefaciens
VirB8 protein was carried out. This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon screen as a potential regulator of
virG
expression,
chvD
. In this report, the cloning of the entire
chvD
gene is described and the gene is sequenced and characterized. Insertion of a promoterless
lacZ
gene into the
chvD
locus greatly attenuated virulence and
vir
gene expression. Compared to that of the wild-type strain, growth of the
chvD
mutant was reduced in rich, but not minimal, medium. Expression of
chvD
, as monitored by expression of β-galactosidase activity from the
chvD-lacZ
fusion, occurred in both rich and minimal media as well as under conditions that induce virulence gene expression. The ChvD protein is highly homologous to a family of ATP-binding cassette transporters involved in antibiotic export from bacteria and has two complete Walker box motifs. Molecular genetic analysis demonstrated that disruption of either Walker A box, singly, does not inactivate this protein's effect on virulence but that mutations in both Walker A boxes renders it incapable of complementing a
chvD
mutant strain. Constitutive expression of
virG
in the
chvD
mutant strain restored virulence, supporting the hypothesis that ChvD controls virulence through effects on
virG
expression.
Constitutive Septal Murein Synthesis in
Escherichia coli
with Impaired Activity of the Morphogenetic Proteins RodA and Penicillin-Binding Protein 2
Authors: de Pedro Miguel A. , Donachie William D. , Höltje Joachim-Volker , Schwarz Heinz 
Published: July 2002

ABSTRACT

The pattern of peptidoglycan (murein) segregation in cells of
Escherichia coli
with impaired activity of the morphogenetic proteins penicillin-binding protein 2 and RodA has been investigated by the
d
-cysteine–biotin immunolabeling technique (M. A. de Pedro, J. C. Quintela, J.-V. Höltje, and H. Schwarz, J. Bacteriol. 179:2823–2834, 1997). Inactivation of these proteins either by amdinocillin treatment or by mutations in the corresponding genes,
pbpA
and
rodA
, respectively, leads to the generation of round, osmotically stable cells. In normal rod-shaped cells, new murein precursors are incorporated all over the lateral wall in a diffuse manner, being mixed up homogeneously with preexisting material, except during septation, when strictly localized murein synthesis occurs. In contrast, in rounded cells, incorporation of new precursors is apparently a zonal process, localized at positions at which division had previously taken place. Consequently, there is no mixing of new and old murein. Old murein is preserved for long periods of time in large, well-defined areas. We propose that the observed patterns are the result of a failure to switch off septal murein synthesis at the end of septation events. Furthermore, the segregation results confirm that round cells of
rodA
mutants do divide in alternate, perpendicular planes as previously proposed (K. J. Begg and W. D. Donachie, J. Bacteriol. 180:2564–2567, 1998).
Localization of c-di-GMP-Binding Protein with the Linear Terminal Complexes of
Acetobacter xylinum
Authors: Kimura Satoshi , Chen He Ping , Saxena Inder M. , Brown R. Malcolm , Itoh Takao 
Published: July 2002

ABSTRACT

Specific labeling of a single row of cellulose-synthesizing complexes (terminal complexes, TC subunits, TCs, or TC arrays) in
Acetobacter xylinum
by antibodies raised against a 93-kDa protein (the cyclic dignanylic acid-binding protein) has been demonstrated by using the sodium dodecyl sulfate (SDS)–freeze-fracture labeling (FRL) technique. The antibodies to the 93-kDa protein specifically recognized the TC subunits on the protoplasmic fracture (PF) face of the outer membrane in
A. xylinum
; however, nonlabeled TCs were also observed. Two types of TC subunits (particles or pits) are observed on the PF face of the outer membrane: (i) immunogold-labeled TCs showing a line of depressions (pits) with an indistinct particle array and (ii) nonlabeled TC subunits with a distinct single row of particle arrays. The evidence indicates that the labeling patterns differ with respect to the presence or absence of certain TC subunits remaining attached to the replica after SDS treatment. This suggests the presence of at least two TC components, one in the outer membrane and the other in the cytoplasmic membrane. If the TC component in the outer membrane is preferentially fractured and remains attached to the ectoplasmic fracture face (or outer leaflet) of the outer membrane, subsequent replica formation reveals a pit or depression with positive antibody labeling on the PF face of the outer membrane. If the TC component in the outer membrane remains with the PF face (or inner leaflet) of the outer membrane, the innermost TC component is removed during SDS treatment and labeling does not occur. SDS-FRL of TCs in
A. xylinum
has enabled us to provide the first topological molecular analysis of component proteins in a cellulose-synthesizing TC structure in a prokaryotic organism.
Regulation of Potassium-Dependent Kdp-ATPase Expression in the Nitrogen-Fixing Cyanobacterium
Anabaena torulosa
Authors: Alahari Anuradha , Ballal Anand , Apte Shree Kumar 
Published: July 2002

ABSTRACT

The KdpB polypeptides in the cyanobacterium
Anabaena torulosa
were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K
+
deficiency and repressed or degraded upon readdition of K
+
. In both
Anabaena
and
Escherichia coli
strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation.
Novel Posttranslational Activation of the
LYS2-
Encoded α-Aminoadipate Reductase for Biosynthesis of Lysine and Site-Directed Mutational Analysis of Conserved Amino Acid Residues in the Activation Domain of
Candida albicans
Authors: Guo Shujuan , Evans Sarah A. , Wilkes Mindy B. , Bhattacharjee J. K. 
Published: July 2002

ABSTRACT

The α-aminoadipate pathway for lysine biosynthesis is present only in fungi. The α-aminoadipate reductase (AAR) of this pathway catalyzes the conversion of α-aminoadipic acid to α-aminoadipic-δ-semialdehyde by a complex mechanism involving two gene products, Lys2p and Lys5p. The
LYS2
and
LYS5
genes encode, respectively, a 155-kDa inactive AAR and a 30-kDa phosphopantetheinyl transferase (PPTase) which transfers a phosphopantetheinyl group from coenzyme A (CoA) to Lys2p for the activation of Lys2p and AAR activity. In the present investigation, we have confirmed the posttranslational activation of the 150-kDa Lys2p of
Candida albicans
, a pathogenic yeast, in the presence of CoA and
C. albicans lys2
mutant (CLD2) extract as a source of PPTase (Lys5p). The recombinant Lys2p or CLD2 mutant extract exhibited no AAR activity with or without CoA. However, the recombinant 150-kDa Lys2p, when incubated with CLD2 extract and CoA, exhibited significant AAR activity compared to that of wild-type
C. albicans
CAI4 extract. The PPTase in the CLD2 extract was required only for the activation of Lys2p and not for AAR reaction. Site-directed mutational analysis of G882 and S884 of the Lys2p activation domain (LGGHSI) revealed no AAR activity, indicating that these two amino acids are essential for the activation. Replacement of other amino acid residues in the domain resulted in partial or full AAR activity. These results demonstrate the posttranslational activation and the requirement of specific amino acid residues in the activation domain of the AAR of
C. albicans
.
The
lytB
Gene of
Escherichia coli
Is Essential and Specifies a Product Needed for Isoprenoid Biosynthesis
Authors: McAteer Sean , Coulson Andrew , McLennan Neil , Masters Millicent 
Published: July 2002

ABSTRACT

LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional
Escherichia coli lytB
mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of
E. coli lytB
mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.
Role for
draTG
and
rnf
Genes in Reduction of 2,4-Dinitrophenol by
Rhodobacter capsulatus
Authors: Sáez Lara P. , Garcı́a Patricia , Martı́nez-Luque Manuel , Klipp Werner , Blasco Rafael , Castillo Francisco 
Published: July 2002

ABSTRACT

The phototrophic bacterium
Rhodobacter capsulatus
is able to reduce 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol enzymatically and thus can grow in the presence of this uncoupler. DNP reduction was switched off by glutamine or ammonium, but this short-term regulation did not take place in a
draTG
deletion mutant. Nevertheless, the target of DraTG does not seem to be the nitrophenol reductase itself since the ammonium shock did not inactivate the enzyme. In addition to this short-term regulation, ammonium or glutamine repressed the DNP reduction system. Mutants of
R. capsulatus
affected in
ntrC
or
rpoN
exhibited a 10-fold decrease in nitroreductase activity in vitro but almost no DNP activity in vivo. In addition, mutants affected in
rnfA
or
rnfC
, which are also under NtrC control and encode components involved in electron transfer to nitrogenase, were unable to metabolize DNP. These results indicate that NtrC regulates dinitrophenol reduction in
R. capsulatus
, either directly or indirectly, by controlling expression of the Rnf proteins. Therefore, the Rnf complex seems to supply electrons for both nitrogen fixation and DNP reduction.
TFE, an Archaeal Transcription Factor in
Methanobacterium thermoautotrophicum
Related to Eucaryal Transcription Factor TFIIEα
Authors: Hanzelka Brian L. , Darcy Trevor J. , Reeve John N. 
Published: July 2002

ABSTRACT

In the archaeon
Methanobacterium thermoautotrophicum
, MTH1669 encodes a protein with a sequence related to the N-terminal sequences of the α-subunits of eucaryal general transcription factor TFIIE. The recombinant MTH1669 gene product has been purified and shown to stimulate transcription in vitro from
M. thermoautotrophicum
promoters that were almost inactive or much less active in reaction mixtures that contained only
M. thermoautotrophicum
RNA polymerase, TATA-binding protein and transcription factor B. As all complete archaeal genome sequences contain an MTH1669 homolog, the protein encoded by this gene is apparently the first characterized example of a transcription activator, here designated TFE, that may be universally present in the
Archaea
.
Nitric Oxide Signaling and Transcriptional Control of Denitrification Genes inPseudomonas stutzeri
Authors: Vollack Kai-Uwe , Zumft Walter G. 
Published: July 2002

ABSTRACTThe expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus ofPseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in anirFstrain, where NO generation from nitrite is prevented because of a defect in heme D1biosynthesis. NO-triggered responses were monitored from thenirSTBoperon (encoding cytochromecd1nitrite reductase), thenorCBoperon (encoding NO reductase),nosZ(encoding nitrous oxide reductase), andnosR(encoding a putative regulator). Transcription ofnirSTBandnorCBwas activated by 5 to 50 nM NO, whereas thenosZpromoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell the transient appearance ofnosZandnosRtranscripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N2O. NO at 250 nM stimulated in this cell type the expression ofnosgenes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression ofdnrDin an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of thednrDoperon by inducing the transcription ofdnrNanddnrO, located upstream ofdnrD. Insertional mutagenesis ofdnrNaltered the kinetic response of thenirSTBoperon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification inP. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by thenarGHJIoperon.
Mechanisms Causing Rapid and Parallel Losses of Ribose Catabolism in Evolving Populations ofEscherichia coliB
Authors: Cooper Vaughn S. , Schneider Dominique , Blot Michel , Lenski Richard E. 
Published: July 2002

ABSTRACTTwelve populations ofEscherichia coliB all lostd-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs−mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs+progenitor. These Rbs−mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 × 10−5per cell generation, of mutation from Rbs+to Rbs−, which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbsgenes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150element located immediately upstream of therbsoperon. The deletions apparently involved transposition into various locations within therbsoperon; recombination between the new IS150copy and the one upstream of therbsoperon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of therbsoperon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functionalrbsoperon to two Rbs−mutants, and we constructed another Rbs−strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs−mutants have a competitive advantage relative to their Rbs+counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs−mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.
pfs-Dependent Regulation of Autoinducer 2 Production inSalmonella entericaSerovar Typhimurium
Authors: Beeston Anne L. , Surette Michael G. 
Published: July 2002

ABSTRACTBacterial intercellular communication provides a mechanism for signal-dependent regulation of gene expression to promote coordinated population behavior.Salmonella entericaserovar Typhimurium produces a non-homoserine lactone autoinducer in exponential phase as detected by aVibrio harveyireporter assay for autoinducer 2 (AI-2) (M. G. Surette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). TheluxSgene product mediates the production of AI-2 (M. G. Surette, M. B. Miller, and B. L. Bassler, Proc. Natl. Acad. Sci. USA 96:1639-1644, 1999). Environmental cues such as rapid growth, the presence of preferred carbon sources, low pH, and/or high osmolarity were found to influence the production of AI-2 (M. G. Surette and B. L. Bassler, Mol. Microbiol. 31:585-595, 1999). In addition to LuxS, thepfsgene product (Pfs) is required for AI-2 production, as well asS-adenosylhomocysteine (SAH) (S. Schauder, K. Shokat, M. G. Surette, and B. L. Bassler, Mol. Microbiol. 41:463-476, 2001). In bacterial cells, Pfs exhibits both 5′-methylthioadenosine (MTA) and SAH nucleosidase functions. Pfs is involved in methionine metabolism, regulating intracellular MTA and SAH levels (elevated levels of MTA and SAH are potent inhibitors of polyamine synthetases and S-adenosylmethionine dependent methyltransferase reactions, respectively). To further investigate regulation of AI-2 production inSalmonella, we constructedpfsandluxSpromoter fusions to aluxCDABEreporter in a low-copy-number vector, allowing an examination of transcription of the genes in the pathway for signal synthesis. Here we report thatluxSexpression is constitutive but that the transcription ofpfsis tightly correlated to AI-2 production inSalmonellaserovar Typhimurium 14028. NeitherluxSnorpfsexpression appears to be regulated by AI-2. These results suggest that AI-2 production is regulated at the level of LuxS substrate availability and not at the level ofluxSexpression. Our results indicate that AI-2-dependent signaling is a reflection of metabolic state of the cell and not cell density.
Chromosomal Locus That Affects Pathogenicity ofRhodococcus fascians
Authors: Vereecke Danny , Cornelis Karen , Temmerman Wim , Jaziri Mondher , Van Montagu Marc , Holsters Marcelle , Goethals Koen 
Published: July 2002

ABSTRACTThe gram-positive plant pathogenRhodococcus fasciansprovokes leafy gall formation on a wide range of plants through secretion of signal molecules that interfere with the hormone balance of the host. Crucial virulence genes are located on a linear plasmid, and their expression is tightly controlled. A mutant with a mutation in a chromosomal locus that affected virulence was isolated. The mutation was located in genevicA, which encodes a malate synthase and is functional in the glyoxylate shunt of the Krebs cycle. VicA is required for efficient in planta growth in symptomatic, but not in normal, plant tissue, indicating that the metabolic requirement of the bacteria or the nutritional environment in plants or both change during the interaction. We propose that induced hyperplasia on plants represents specific niches for the causative organisms as a result of physiological alterations in the symptomatic tissue. Hence, such interaction could be referred to as metabolic habitat modification.