bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteomic Analysis Reveals the Participation of Energy- and Stress-Related Proteins in the Response of
Pseudomonas putida
DOT-T1E to Toluene
Authors: Segura Ana , Godoy Patricia , van Dillewijn Pieter , Hurtado Ana , Arroyo Nuria , Santacruz Simon , Ramos Juan-Luis 
Published: August 2005

ABSTRACT

Pseudomonas putida
DOT-T1E is tolerant to toluene and other toxic hydrocarbons through extrusion of the toxic compounds from the cell by means of three efflux pumps, TtgABC, TtgDEF, and TtgGHI. To identify other cellular factors that allow the growth of
P. putida
DOT-T1E in the presence of high concentrations of toluene, we performed two-dimensional gel analyses of proteins extracted from cultures grown on glucose in the presence and in the absence of the organic solvent. From a total of 531 spots, 134 proteins were observed to be toluene specific. In the absence of toluene, 525 spots were clearly separated and 117 proteins were only present in this condition. Moreover, 35 proteins were induced by at least twofold in the presence of toluene whereas 26 were repressed by at least twofold under these conditions. We reasoned that proteins that were highly induced could play a role in toluene tolerance. These proteins, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, were classified into four categories: 1, proteins involved in the catabolism of toluene; 2, proteins involved in the channeling of metabolic intermediates to the Krebs cycle and activation of purine biosynthesis; 3, proteins involved in sugar transport; 4, stress-related proteins. The set of proteins in groups 2 and 3 suggests that the high energy demand required for solvent tolerance is achieved via activation of cell metabolism. The role of chaperones that facilitate the proper folding of newly synthesized proteins under toluene stress conditions was analyzed in further detail. Knockout mutants revealed that CspA, XenA, and Tuf-1 play a role in solvent tolerance in
Pseudomonas
, although this role is probably not specific to toluene, as indicated by the fact that all mutants grew more slowly than the wild type without toluene.
Transcriptional Termination Control of a Novel ABC Transporter Gene Involved in Antibiotic Resistance in
Bacillus subtilis
Authors: Ohki Reiko , Tateno Kozue , Takizawa Teruaki , Aiso Toshiko , Murata Makiko 
Published: August 2005

ABSTRACT

In members of one of the subfamilies of the bacterial ATP binding cassette (ABC) transporters, the two nucleotide binding domains are fused as a single peptide and the proteins have no membrane-spanning domain partners. Most of the ABC efflux transporters of this subfamily have been characterized in actinomycetes, producing macrolide, lincosamide, and streptogramin antibiotics. Among 40 ABC efflux transporters of
Bacillus subtilis
, five proteins belong to this subfamily. None of these proteins has been functionally characterized. We examined macrolide, lincosamide, and streptogramin antibiotic resistance in insertional disruptants of the genes that encode these proteins. It was found that only a disruptant of
vmlR
(formerly named
expZ
) showed hypersensitivity to virginiamycin M and lincomycin. Expression of the
vmlR
gene was induced by the addition of these antibiotics in growth medium. Primer extension analysis revealed that transcription of the
vmlR
gene initiates at an adenosine residue located 225 bp upstream of the initiation codon. From the analysis of the
vmlR
and
lacZ
fusion genes, a 52-bp deletion from +159 to +211 resulted in constitutive expression of the
vmlR
gene. In this region, a typical ρ-independent transcriptional terminator was found. It was suggested that the majority of transcription ends at this termination signal in the absence of antibiotics, whereas under induced conditions, RNA polymerase reads through the terminator, and transcription continues to the downstream
vmlR
coding region, resulting in an increase in
vmlR
expression. No stabilization of
vmlR
mRNA occurred under the induced conditions.
The Heat Shock Genes
dnaK
,
dnaJ
, and
grpE
Are Involved in Regulation of Putisolvin Biosynthesis in
Pseudomonas putida
PCL1445
Authors: Dubern Jean-Frédéric , Lagendijk Ellen L. , Lugtenberg Ben J. J. , Bloemberg Guido V. 
Published: August 2005

ABSTRACT

Pseudomonas putida
PCL1445 produces two cyclic lipopeptides, putisolvins I and II, which possess surfactant activity and play an important role in biofilm formation and degradation. In order to identify genes and traits that are involved in the regulation of putisolvin production of PCL1445, a Tn
5luxAB
library was generated and mutants were selected for the lack of biosurfactant production using a drop-collapsing assay. Sequence analysis of the Tn5
luxAB
flanking region of one biosurfactant mutant, strain PCL1627, showed that the transposon had inserted in a
dnaK
homologue which is located downstream of
grpE
and upstream of
dnaJ
. Analysis of putisolvin production and expression studies indicate that
dnaK
, together with the
dnaJ
and
grpE
heat shock genes, takes part in the positive regulation (directly or indirectly) of putisolvin biosynthesis at the transcriptional level. Growth of PCL1445 at low temperature resulted in an increased level of putisolvins, and mutant analyses showed that this requires
dnaK
and
dnaJ
but not
grpE
. In addition, putisolvin biosynthesis of PCL1445 was found to be dependent on the GacA/GacS two-component signaling system. Expression analysis indicated that
dnaK
is positively regulated by GacA/GacS.
Species and Incompatibility Determination within the P1parFamily of Plasmid Partition Elements
Authors: Dabrazhynetskaya Alena , Sergueev Kirill , Austin Stuart 
Published: August 2005

ABSTRACTThe P1parfamily of active plasmid partition systems consists of at least six members, broadly distributed in a variety of plasmid types and bacterial genera. Each encodes two Par proteins and contains acis-actingparSsite. Individualparsystems can show distinct species specificities; the proteins from one type cannot function with theparSsite of another. P1par-versus-P7parspecificity resides within two hexamer BoxB repeats encoded byparSthat contact the ParB protein near the carboxy terminus. Here, we examine the species specificity differences betweenYersinia pestispMT1parSandEscherichia coliP1 and P7parS. pMT1parSsite specificity could be altered to that of either P1 or P7 by point mutation changes in the BoxB repeats. Just one base change in a single BoxB repeat sometimes sufficed. The BoxB sequence appears to be able to adopt a number of forms that define exclusive interactions with different ParB species. The loopedparSstructure may facilitate this repertoire of interaction specificities. Different P1parfamily members have different partition-mediated incompatibility specificities. This property defines whether two related plasmids can coexist in the same cell and is important in promoting the evolution of new plasmid species. BoxB sequence changes that switch species specificity between P1, P7, and pMT1 species switched partition-mediated plasmid incompatibility in concert. Thus, there is a direct mechanistic link between species specificity and partition-mediated incompatibility, and the BoxB-ParB interaction can be regarded as a special mechanism for facilitating plasmid evolution.
Induction of Manganese-Containing Superoxide Dismutase Is Required for Acid Tolerance inVibrio vulnificus
Authors: Kim Ju-Sim , Sung Moon-Hee , Kho Dhong-Hyo , Lee Jeong K. 
Published: August 2005

ABSTRACTThe manganese-containing superoxide dismutase (MnSOD) ofVibrio vulnificus, normally detected after the onset of the stationary phase, is expressed during the lag that immediately follows the transfer of cells grown exponentially to a fresh medium acidified to pH 5.0, whereas Fe-containing SOD is constitutively expressed. The signal triggering the growth lag and MnSOD induction therein is not low pH but intracellular superoxide accumulated under these conditions, since addition of a superoxide scavenger not only shortened the lag but also abrogated the MnSOD induction. If the lysine decarboxylase reaction proceeds in the presence of sufficient lysine, the broth is rapidly neutralized to abolish the generation of oxidative stress. Accordingly, the acid tolerance response was examined without the addition of lysine. SoxR regulates MnSOD induction. Lack of MnSOD caused by mutations insoxRorsodAresulted in low tolerance to low pH. Thefurmutant derepressing MnSOD showed better tolerance than the wild type. Thus, an increase in total cytosolic SOD activity through MnSOD induction is essential for the cell to withstand the acid challenge. The contribution of cuprozinc-containing SOD to acid tolerance is not significant compared with those of cytosolic SODs.
Cytochrome
c
Maturation and the Physiological Role of
c
-Type Cytochromes in
Vibrio cholerae
Authors: Braun Martin , Thöny-Meyer Linda 
Published: August 2005

ABSTRACT

Vibrio cholerae
lives in different habitats, varying from aquatic ecosystems to the human intestinal tract. The organism has acquired a set of electron transport pathways for aerobic and anaerobic respiration that enable adaptation to the various environmental conditions. We have inactivated the
V. cholerae ccmE
gene, which is required for cytochrome
c
biogenesis. The resulting strain is deficient of all
c
-type cytochromes and allows us to characterize the physiological role of these proteins. Under aerobic conditions in rich medium,
V. cholerae
produces at least six
c
-type cytochromes, none of which is required for growth. Wild-type
V. cholerae
produces active fumarate reductase, trimethylamine
N
-oxide reductase,
cbb
3
oxidase, and nitrate reductase, of which only the fumarate reductase does not require maturation of
c
-type cytochromes. The reduction of nitrate in the medium resulted in the accumulation of nitrite, which is toxic for the cells. This suggests that
V. cholerae
is able to scavenge nitrate from the environment only in the presence of other nitrite-reducing organisms. The phenotypes of cytochrome
c
-deficient
V. cholerae
were used in a transposon mutagenesis screening to search for additional genes required for cytochrome
c
maturation. Over 55,000 mutants were analyzed for nitrate reductase and
cbb
3
oxidase activity. No transposon insertions other than those within the
ccm
genes for cytochrome
c
maturation and the
dsbD
gene, which encodes a disulphide bond reductase, were found. In addition, the role of a novel CcdA-like protein in
cbb
3
oxidase assembly is discussed.
Heterocyst-Specific Excision of the
Anabaena
sp. Strain PCC 7120
hupL
Element Requires
xisC
Authors: Carrasco Claudio D. , Holliday Scott D. , Hansel Alfred , Lindblad Peter , Golden James W. 
Published: August 2005

ABSTRACT

In nitrogen-limiting conditions, approximately 10% of the vegetative cells in filaments of the cyanobacterium
Anabaena
(
Nostoc
) sp. strain PCC 7120 differentiate into nitrogen-fixing heterocysts. During the late stages of heterocyst differentiation, three DNA elements, each embedded within an open reading frame, are programmed to excise from the chromosome by site-specific recombination. The DNA elements are named after the genes that they interrupt:
nifD
,
fdxN
, and
hupL
. The
nifD
and
fdxN
elements each contain a gene,
xisA
or
xisF
, respectively, that encodes the site-specific recombinase required for programmed excision of the element. Here, we show that the
xisC
gene (alr0677), which is present at one end of the 9,435-bp
hupL
element, is required for excision of the
hupL
element. A strain in which the
xisC
gene was inactivated showed no detectable excision of the
hupL
element.
hupL
encodes the large subunit of uptake hydrogenase. The
xisC
mutant forms heterocysts and grows diazotrophically, but unlike the wild type, it evolved hydrogen gas under nitrogen-fixing conditions. Overexpression of
xisC
from a plasmid in a wild-type background caused a low level of
hupL
rearrangement even in nitrogen-replete conditions. Expression of
xisC
in
Escherichia coli
was sufficient to produce rearrangement of an artificial substrate plasmid bearing the
hupL
element recombination sites. Sequence analysis indicated that XisC is a divergent member of the phage integrase family of recombinases. Site-directed mutagenesis of
xisC
showed that the XisC recombinase has functional similarity to the phage integrase family.
Phosphoenolpyruvate Carboxykinase Is an Acid-Induced, Chromosomally Encoded Virulence Factor in
Agrobacterium tumefaciens
Authors: Liu Pu , Wood Derek , Nester Eugene W. 
Published: August 2005

ABSTRACT

The
pckA
gene, encoding phosphoenolpyruvate carboxykinase, catalyzes the reversible decarboxylation and phosphorylation of oxaloacetate to form phosphoenolpyruvate. Located on the circular chromosome of
Agrobacterium
, this locus is adjacent to the loci
chvG
and
chvI
, encoding a two-component regulatory system that has been shown to be important in virulence. Using a reporter gene fusion, studies showed that the
pckA
gene is induced by acidic pH but not by acetosyringone. This acid induction is regulated by the
chvG-chvI
regulatory system, which controls acid-inducible genes. A
pckA
mutant had no demonstrable PckA enzyme activity and grew on AB minimal medium with glucose but did not grow on the same medium with succinate as the sole carbon source and was more inhibited in its growth than the wild-type strain by an acidic environment. A
pckA
mutant was highly attenuated in tumor-inducing ability on tobacco leaf disks and was severely attenuated in
vir
gene expression. Although
vir
gene induction was completely restored when a constitutive
virG
gene was introduced into the mutant strain, virulence was only partially restored. These results suggest that avirulence may be due to a combination of the inhibition of this mutant in the acidic plant wound environment and the poor induction of the
vir
genes.
PtrB of
Pseudomonas aeruginosa
Suppresses the Type III Secretion System under the Stress of DNA Damage
Authors: Wu Weihui , Jin Shouguang 
Published: August 2005

ABSTRACT

In a search for regulatory genes of the type III secretion system (TTSS) in
Pseudomonas aeruginosa
, transposon (Tn
5
) insertional mutants of the
prtR
gene were found defective in the TTSS. PrtR is an inhibitor of
prtN
, which encodes a transcriptional activator for pyocin synthesis genes. In
P. aeruginosa
, pyocin synthesis is activated when PrtR is degraded during the SOS response. Treatment of a wild-type
P. aeruginosa
strain with mitomycin C, a DNA-damaging agent, resulted in the inhibition of TTSS activation. A
prtR
/
prtN
double mutant had the same TTSS defect as the
prtR
mutant, and complementation by a
prtR
gene but not by a
prtN
gene restored the TTSS function. Also, overexpression of the
prtN
gene in wild-type PAK had no effect on the TTSS; thus, PrtN is not involved in the repression of the TTSS. To identify the PrtR-regulated TTSS repressor, another round of Tn mutagenesis was carried out in the background of a
prtR
/
prtN
double mutant. Insertion in a small gene, designated
ptrB
, restored the normal TTSS activity. Expression of
ptrB
is specifically repressed by PrtR, and mitomycin C-mediated suppression of the TTSS is also abolished in a
ptrB
mutant strain. Therefore, PtrB is a new TTSS repressor that coordinates TTSS repression and pyocin synthesis under the stress of DNA damage.
MtdC, a Novel Class of Methylene Tetrahydromethanopterin Dehydrogenases
Authors: Vorholt Julia A. , Kalyuzhnaya Marina G. , Hagemeier Christoph H. , Lidstrom Mary E. , Chistoserdova Ludmila 
Published: August 2005

ABSTRACT

Novel methylene tetrahydromethanopterin (H
4
MPT) dehydrogenase enzymes, named MtdC, were purified after expressing in
Escherichia coli
genes from, respectively,
Gemmata
sp. strain Wa1-1 and environmental DNA originating from unidentified microbial species. The MtdC enzymes were shown to possess high affinities for methylene-H
4
MPT and NADP but low affinities for methylene tetrahydrofolate or NAD. The substrate range and the kinetic properties revealed by MtdC enzymes distinguish them from the previously characterized bacterial methylene-H
4
MPT dehydrogenases, MtdA and MtdB. While revealing higher sequence similarity to MtdA enzymes, MtdC enzymes appear to fulfill a function homologous to the function of MtdB, as part of the H
4
MPT-linked pathway for formaldehyde oxidation/detoxification.
An Amino-Terminal Secretion Signal Is Required for YplA Export by the Ysa, Ysc, and Flagellar Type III Secretion Systems ofYersinia enterocoliticaBiovar 1B
Authors: Warren Sasha M. , Young Glenn M. 
Published: August 2005

ABSTRACTYersinia enterocoliticabiovar 1B maintains three distinct type III secretion (TTS) systems, which independently operate to target proteins to extracellular sites. The Ysa and Ysc systems are prototypical contact-dependent TTS systems that translocate toxic effectors to the cytosols of targeted eukaryotic host cells during infection. The flagellar TTS system is utilized during the assembly of the flagellum and is required for secretion of the virulence-associated phospholipase YplA to the bacterial milieu. When ectopically produced, YplA is also a secretion substrate for the Ysa and Ysc TTS systems. In this study, we define elements that allow YplA recognition and export by the Ysa, Ysc, and flagellar TTS systems. Fusion of various amino-terminal regions of YplA toEscherichia colialkaline phosphatase (PhoA) lacking its native secretion signal demonstrated that the first 20 amino acids or corresponding mRNA codons of YplA were sufficient for export of YplA-PhoA chimeras by each TTS system. Export of native YplA by each of the three TTS systems was also found to depend on the integrity of its amino terminus. Introduction of a frameshift mutation or deletion ofyplAsequences encoding the amino-terminal 20 residues negatively impacted YplA secretion. Deletion of otheryplAregions was tolerated, including that resulting in the removal of amino acid residues 30 through 40 of the polypeptide and removal of the 5′ untranslated region of the mRNA. This work supports a model in which independent and distantly related TTS systems ofY. enterocoliticarecognize protein substrates by a similar mechanism.
Identification of theMycobacterium tuberculosisSUF Machinery as the Exclusive Mycobacterial System of [Fe-S] Cluster Assembly: Evidence for Its Implication in the Pathogen's Survival
Authors: Huet Gaëlle , Daffé Mamadou , Saves Isabelle 
Published: August 2005

ABSTRACTThe worldwide recrudescence of tuberculosis and widespread antibiotic resistance have strengthened the need for the rapid development of new antituberculous drugs targeting essential functions of its etiologic agent,Mycobacterium tuberculosis. In our search for new targets, we found that theM. tuberculosis pps1gene, which contains an intein coding sequence, belongs to a conserved locus of seven open reading frames. In silico analyses indicated that the mature Pps1 protein is orthologous to the SufB protein of many organisms, a highly conserved component of the [Fe-S] cluster assembly and repair SUF (mobilization of sulfur) machinery. We showed that the mycobacterialpps1locus constitutes an operon which encodes Suf-like proteins. Interactions between these proteins were demonstrated, supporting the functionality of theM. tuberculosisSUF system. The noticeable absence of any alternative [Fe-S] cluster assembly systems in mycobacteria is in agreement with the apparent essentiality of thesufoperon inMycobacterium smegmatis. Altogether, these results establish that Pps1, as a central element of the SUF system, could play an essential function forM. tuberculosissurvival virtually through its implication in the bacterial resistance to iron limitation and oxidative stress. As such, Pps1 may represent an interesting molecular target for new antituberculous drugs.
Immobilization ofEscherichia coliRNA Polymerase and Location of Binding Sites by Use of Chromatin Immunoprecipitation and Microarrays
Authors: Herring Christopher D. , Raffaelle Marni , Allen Timothy E. , Kanin Elenita I. , Landick Robert , Ansari Aseem Z. , Palsson Bernhard Ø. 
Published: August 2005

ABSTRACTThe genome-wide location of RNA polymerase binding sites was determined inEscherichia coliusing chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the β′ subunit. The DNA was amplified and hybridized to “tiled” oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known σ70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors inE. coliand an important evaluation of the chIP-chip technique.
Role of Disulfide Bridges in the Activity and Stability of a Cold-Active α-Amylase
Authors: Siddiqui Khawar Sohail , Poljak Anne , Guilhaus Michael , Feller Georges , D'Amico Salvino , Gerday Charles , Cavicchioli Ricardo 
Published: August 2005

ABSTRACT

The cold-adapted α-amylase from
Pseudoalteromonas haloplanktis
unfolds reversibly and cooperatively according to a two-state mechanism at 30°C and unfolds reversibly and sequentially with two transitions at temperatures below 12°C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with β-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity.
Implications of Physiological Studies Based on Genomic Sequences:
Streptococcus pneumoniae
TIGR4 Synthesizes a Functional LytC Lysozyme
Authors: Moscoso Miriam , López Elena , García Ernesto , López Rubens 
Published: August 2005

ABSTRACT

The
Streptococcus pneumoniae
LytC lysozyme is responsible for autolysis at 30°C (a temperature close to that of the upper respiratory tract), promotes DNA release in competent cultures, and participates in nasopharyngeal colonization. We show that the virulent pneumococcal TIGR4 strain encodes an active LytC enzyme, in contrast with genome-based predictions.