bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Determinants Required To Target Penicillin-Binding Protein 3 to the Septum ofEscherichia coli
Authors: Piette André , Fraipont Claudine , den Blaauwen Tanneke , Aarsman Mirjam E. G. , Pastoret Soumya , Nguyen-Distèche Martine 
Published: September 2004

ABSTRACTInEscherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.
An SOS Response Induced by High Pressure inEscherichia coli
Authors: Aertsen Abram , Van Houdt Rob , Vanoirbeek Kristof , Michiels Chris W. 
Published: September 2004

ABSTRACTAlthough pressure is an important environmental parameter in microbial niches such as the deep sea and is furthermore used in food preservation to inactivate microorganisms, the fundamental understanding of its effects on bacteria remains fragmentary. Our group recently initiated differential fluorescence induction screening to search for pressure-inducedEscherichia colipromoters and has already reported induction of the heat shock regulon. Here the screening was continued, and we report for the first time that pressure induces a bona fide SOS response inE. coli, characterized by the RecA and LexA-dependent expression ofuvrA,recA, andsulA. Moreover, it was shown that pressure is capable of triggering lambda prophage induction inE. colilysogens. The remnant lambdoid e14 element, however, could not be induced by pressure, as opposed to UV irradiation, indicating subtle differences between the pressure-induced and the classical SOS response. Furthermore, the pressure-induced SOS response seems not to be initiated by DNA damage, sinceΔrecAandlexA1(Ind−) mutants, which are intrinsically hypersensitive to DNA damage, were not sensitized or were only very slightly sensitized for pressure-mediated killing and since pressure treatment was not found to be mutagenic. In light of these findings, the current knowledge of pressure-mediated effects on bacteria is discussed.
RsbV-Independent Induction of the SigB-Dependent General Stress Regulon ofBacillus subtilisduring Growth at High Temperature
Authors: Holtmann Gudrun , Brigulla Matthias , Steil Leif , Schütz Alexandra , Barnekow Karsta , Völker Uwe , Bremer Erhard 
Published: September 2004

ABSTRACTGeneral stress proteins protectBacillus subtiliscells against a variety of environmental insults. This adaptive response is particularly important for nongrowing cells, to which it confers a multiple, nonspecific, and preemptive stress resistance. Induction of the general stress response relies on the alternative transcription factor, SigB, whose activity is controlled by a partner switching mechanism that also involves the anti-sigma factor, RsbW, and the antagonist protein, RsbV. Recently, the SigB regulon has been shown to be continuously induced and functionally important in cells actively growing at low temperature. With the exception of this chill induction, all SigB-activating stimuli identified so far trigger a transient expression of the SigB regulon that depends on RsbV. Through a proteome analysis and Northern blot and gene fusion experiments, we now show that the SigB regulon is continuously induced in cells growing actively at 51°C, close to the upper growth limit ofB. subtilis. This heat induction of SigB-dependent genes requires the environmental stress-responsive phosphatase RsbU, but not the metabolic stress-responsive phosphatase RsbP. RsbU dependence of SigB activation by heat is overcome in mutants that lack RsbV. In addition, loss of RsbV alone or in combination with RsbU triggers a hyperactivation of the general stress regulon exclusively at high temperatures detrimental for cell growth. These new facets of heat induction of the SigB regulon indicate that the current view of the complex genetic and biochemical regulation of SigB activity is still incomplete and that SigB perceives signals independent of the RsbV-mediated signal transduction pathways under heat stress conditions.
The Small Noncoding DsrA RNA Is an Acid Resistance Regulator inEscherichia coli
Authors: Lease Richard A. , Smith Dorie , McDonough Kathleen , Belfort Marlene 
Published: September 2004

ABSTRACTDsrA RNA is a small (87-nucleotide) regulatory RNA ofEscherichia colithat acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (σs), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenicE. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes ofE. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in thehdeAB,gadAX, andgadBCoperons.E. coliK-12 strains, as well as pathogenicE. coliO157:H7, exhibited compromised acid resistance indsrAmutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitivedsrAmutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenicE. coli.
New Protein-Protein Interactions Identified for the Regulatory and Structural Components and Substrates of the Type III Secretion System of the PhytopathogenXanthomonas axonopodisPathovar citri
Authors: Alegria Marcos C. , Docena Cassia , Khater Leticia , Ramos Carlos H. I. , da Silva Ana C. R. , Farah Chuck S. 
Published: September 2004

ABSTRACTWe have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogenXanthomonas axonopodispv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by thehrp(for hypersensitive response and pathogenicity),hrc(forhrpconserved), andhpa(forhrpassociated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression ofX. axonopodispv. citrihrpoperons, and XAC0095, a previously uncharacterized protein encountered only inXanthomonasspp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlyingXanthomonaspathogenicity.
Novel Xylose Dehydrogenase in the Halophilic Archaeon
Haloarcula marismortui
Authors: Johnsen Ulrike , Schönheit Peter 
Published: September 2004

ABSTRACT

During growth of the halophilic archaeon
Haloarcula marismortui
on
d
-xylose, a specific
d
-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP
+
and NAD
+
as cosubstrates with 10-fold higher affinity for NADP
+
. In addition to
d
-xylose,
d
-ribose was oxidized at similar kinetic constants, whereas
d
-glucose was used with about 70-fold lower catalytic efficiency (
k
cat
/

K
m

). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)—coding for a 39.9-kDA protein—was identified in the partially sequenced genome of
H. marismortui
. The function of the ORF as the gene designated
xdh
and coding for xylose dehydrogenase was proven by its functional overexpression in
Escherichia coli
. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from
Zymomonas mobilis
and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of
H. marismortui
, and the genes that encode them,
xylA
and
xylB
, were not found in the genome of
H. marismortui
. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by
H. marismortui
.
The hFbpABC Transporter from
Haemophilus influenzae
Functions as a Binding-Protein-Dependent ABC Transporter with High Specificity and Affinity for Ferric Iron
Authors: Anderson Damon S. , Adhikari Pratima , Nowalk Andrew J. , Chen Cheng Y. , Mietzner Timothy A. 
Published: September 2004

ABSTRACT

Pathogenic
Haemophilus influenzae
,
Neisseria
spp. (
Neisseria gonorrhoeae
and
N. meningitidis
),
Serratia marcescens
, and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the
H. influenzae
FbpABC transporter in a siderophore-deficient
Escherichia coli
background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe
3+
transport assay, we established an apparent

K
m

= 0.9 μM and
V
max
= 1.8 pmol/10
7
cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide
m
-chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe
3+
and selectivity for trivalent metals, including Ga
3+
and Al
3+
, over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards
E. coli
. Significantly, gallium-induced toxicity was specific only to
E. coli
expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity

K
d

= 5.2 × 10
−4
M
−1
, ∼14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, ∼35% of wild-type transport, with

K
m

= 1.2 μM and
V
max
= 0.5 pmol/10
7
cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe
3+
) binding and membrane transport.
Mutational Analysis of
Xanthomonas
Harpin HpaG Identifies a Key Functional Region That Elicits the Hypersensitive Response in Nonhost Plants
Authors: Kim Jung-Gun , Jeon Eunkyung , Oh Jonghee , Moon Jae Sun , Hwang Ingyu 
Published: September 2004

ABSTRACT

HpaG is a type III-secreted elicitor protein of
Xanthomonas axonopodis
pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and site-directed mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying
hpaG
was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were obtained, including 51 single missense mutants and 1 double missense mutant. The HR elicitation activity was abolished in the two missense mutants [HpaG(L50P) and HpaG(L43P/L50P)]. Seven single missense mutants showed reduced activity, and the HR elicitation activity of the rest of the mutants was similar to that of wild-type HpaG. Mutational and deletion analyses narrowed the region essential for elicitor activity to the 23-amino-acid peptide (H
2
N-NQGISEKQLDQLLTQLIMALLQQ-COOH). A synthetic peptide of this sequence possessed HR elicitor activity at the same concentration as the HpaG protein. This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from
Pseudomonas
and
Erwinia
spp. The secondary structure of the peptide is predicted to be an α-helix, as is the HrpW region that is homologous to HpaG. The predicted α-helix of HpaG is probably critical for the elicitation of the HR in tobacco plants. In addition, mutagenesis of a
xopA
gene yielded two gain-of-function mutants: XopA(F48L) and XopA(F48L/M52L). These results indicate that the 12 amino acid residues between L39 and L50 of HpaG have critical roles in HR elicitation in tobacco plants.
Complementation of an
Escherichia coli
DnaK Defect by Hsc70-DnaK Chimeric Proteins
Authors: Suppini Jean-Philippe , Amor Mouna , Alix Jean-Hervé , Ladjimi Moncef M. 
Published: September 2004

ABSTRACT

Escherichia coli
DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein (Hsp70) family that show strong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones. We show here that, while the DnaK protein is, as expected, able to complement an
E. coli dnaK
mutant strain for growth at high temperatures and λ phage propagation, Hsc70 protein is not. However, an Hsc70 in which the peptide-binding domain has been replaced by that of DnaK is able to complement this strain for both phenotypes, suggesting that the peptide-binding domain of DnaK is essential to fulfill the specific functions of this protein necessary for growth at high temperatures and for λ phage replication. The implications of these findings on the functional specificities of the Hsp70s and the role of protein-protein interactions in the DnaK chaperone system are discussed.
Transcriptional Repressor CopR: Use of SELEX To Study the
copR
Operator Indicates that Evolution Was Directed at Maximal Binding Affinity
Authors: Freede Peggy , Brantl Sabine 
Published: September 2004

ABSTRACT

CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the
repR
mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the
repR
promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5′-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 μM, respectively. In this work, we used a SELEX procedure to study
copR
operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated
copR
operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3′ positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.
A Novel Mutation within the CentralListeria monocytogenesRegulator PrfA That Results in Constitutive Expression of Virulence Gene Products
Authors: Wong Kendy K. Y. , Freitag Nancy E. 
Published: September 2004

ABSTRACTThe PrfA protein ofListeria monocytogenesfunctions as a key regulatory factor for the coordinated expression of many virulence genes during bacterial infection of host cells. PrfA activity is controlled by multiple regulatory mechanisms, including an apparent requirement for either the presence of a cofactor or some form of posttranslational modification that regulates the activation of PrfA. In this study, we describe the identification and characterization of a novel PrfA mutation that results in constitutive activation of the PrfA protein. The PrfA L140F mutation was found to confer high-level expression of PrfA-regulated genes and to be functionally dominant over the wild-type allele. The presence of the PrfA L140F mutation resulted in the aggregation ofL. monocytogenesin broth culture and, unlike previously describedprfAmutations, appeared to be slightly toxic to the bacteria. High-level PrfA-dependent gene expression showed no additional increase inL. monocytogenesstrains containing an additional copy ofprfAL140F despite a >4-fold increase in PrfA protein levels. In contrast, the introduction of multiple copies of the wild-typeprfAallele toL. monocytogenesresulted in a corresponding increase in PrfA-dependent gene expression, although overall expression levels remained far below those observed for PrfA L140F strains. These results suggest a hierarchy of PrfA regulation, such that the relative levels of PrfA protein present within the cell correlate with the levels of PrfA-dependent gene expression when the protein is not in its fully activated state; however, saturating levels of the protein are then quickly reached when PrfA is converted to its active form. Regulation of the PrfA activation status must be an important facet ofL. monocytogenessurvival, as mutations that result in constitutive PrfA activation may have deleterious consequences for bacterial physiology.
Amino Acid Contacts between Sigma 70 Domain 4 and the Transcription Activators RhaS and RhaR
Authors: Wickstrum Jason R. , Egan Susan M. 
Published: September 2004

ABSTRACT

The RhaS and RhaR proteins are transcription activators that respond to the availability of
l
-rhamnose and activate transcription of the operons in the
Escherichia coli
l
-rhamnose catabolic regulon. RhaR activates transcription of
rhaSR
, and RhaS activates transcription of the operon that encodes the
l
-rhamnose catabolic enzymes,
rhaBAD
, as well as the operon that encodes the
l
-rhamnose transport protein,
rhaT
. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the −35 hexamers of the promoters they regulate, suggesting they may contact the σ
70
subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and σ
70
residue R599. In the present study, we first identified two positively charged amino acids in σ
70
, K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of the
rhaSR
operon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and σ
70
R599. Finally, previous results from our lab separately showed that RhaS D250A and σ
70
K593A were defective at the
rhaBAD
promoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and σ
70
.
Transcription by
Methanothermobacter thermautotrophicus
RNA Polymerase In Vitro Releases Archaeal Transcription Factor B but Not TATA-Box Binding Protein from the Template DNA
Authors: Xie Yunwei , Reeve John N. 
Published: September 2004

ABSTRACT

Transcription initiation in
Archaea
requires the assembly of a preinitiation complex containing the TATA- box binding protein (TBP), transcription factor B (TFB), and RNA polymerase (RNAP). The results reported establish the fate of
Methanothermobacter thermautotrophicus
TBP and TFB following transcription initiation by
M. thermautotrophicus
RNAP in vitro. TFB is released after initiation, during extension of the transcript from 4 to 24 nucleotides, but TBP remains bound to the template DNA. Regulation of archaeal transcription initiation by a repressor competition with TBP for TATA-box region binding must accommodate this observation.
mRNA Secondary Structure Modulates Translation of Tat-Dependent Formate Dehydrogenase N
Authors: Punginelli Claire , Ize Bérengère , Stanley Nicola R. , Stewart Valley , Sawers Gary , Berks Ben C. , Palmer Tracy 
Published: September 2004

ABSTRACT

Formate dehydrogenase N (FDH-N) of
Escherichia coli
is a membrane-bound enzyme comprising FdnG, FdnH, and FdnI subunits organized in an (αβγ)
3
configuration. The FdnG subunit carries a Tat-dependent signal peptide, which localizes the protein complex to the periplasmic side of the membrane. We noted that substitution of the first arginine (R
5
) in the twin arginine signal sequence of FdnG for a variety of other amino acids resulted in a dramatic (up to 60-fold) increase in the levels of protein synthesized. Bioinformatic analysis suggested that the mRNA specifying the first 17 codons of
fdnG
forms a stable stem-loop structure. A detailed mutational analysis has demonstrated the importance of this mRNA stem-loop in modulating FDH-N translation.
Bacterial Thymidylate Synthase with Intein, Group II Intron, and Distinctive ThyX Motifs
Authors: Liu Xiang-Qin , Yang Jing 
Published: September 2004

ABSTRACTThe ThyX class of thymidylate synthases was previously characterized by a common ThyX motif, RHRX7S. We report bacterial ThyX sequences having distinctive ThyX motifs, suggesting a more general ThyX motif, R/THRX7-8S. One ThyX sequence has an intein in its ThyX motif that was shown to do protein splicing and a group II intron in its gene, suggesting a hot spot for these self-splicing mobile elements.
Recombinant Cyclophilins Lack Nuclease Activity
Authors: Manteca Angel , Sanchez Jesus 
Published: September 2004

ABSTRACT

Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host
Escherichia coli
endonuclease and is not an intrinsic property of these proteins.