bluuebunny/crossref_metadata_2025 huggingface dataset:
Experimental Study on the Role of Entropy Waves in Low-Frequency Oscillations in a RQL Combustor
Authors: Eckstein J. , Freitag E. , Hirsch C. , Sattelmayer T.
Published: April 2006

“Rumble” is a self-excited combustion instability, usually occurring at the start-up of aero-engines with fuel-spray atomizers at sub-idle and idle conditions, and exhibiting low limit frequencies in the range of 50Hzto150Hz. Entropy waves at the (nearly) choked combustor outlet are supposed to be the key feedback mechanism for the observed self-excited pressure oscillations. The experimental study presented here aims at clarifying the role of entropy waves in the occurrence of rumble. A generic air-blast atomizer with a design prone to self-excitation has been incorporated into a thermoacoustic combustor test rig with variable outlet conditions. The thermoacoustic response of the flame was characterized by recording the OH* chemiluminescence, the dynamic pressures, the dynamic temperatures, and by applying PIV. The measurements have shown the occurrence of periodic hot spots traveling with the mean flow with considerable dispersion. Measurements have been conducted with an open-ended resonance tube in order to eliminate the impact of entropy waves on the mechanism of self-excitation. The oscillation obtained, comparable in amplitude and frequency, proved that self-excitation primarily depends on convective time delays of the droplets in the primary zone and thus on the atomization characteristics of the nozzle.
Desulfovibrio gigasFlavodiiron Protein Affords Protection against Nitrosative Stress In Vivo
Authors: Rodrigues Rute , Vicente João B. , Félix Rute , Oliveira Solange , Teixeira Miguel , Rodrigues-Pousada Claudina
Published: April 2006

Desulfovibrio gigasflavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O2. Functional complementation studies of anEscherichia colimutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using aD. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agentS-nitrosoglutathione (GSNO), which effectively impairs the growth of theD. gigasΔroostrain.roois constitutively expressed inD. gigasunder all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role ofD. gigasROO as an NO reductase combined with the O2reductase activity reveals a versatility which appears to afford protection toD. gigasat the onset of both oxidative and nitrosative stresses.
The Riboflavin Transporter RibU in
Lactococcus lactis
: Molecular Characterization of Gene Expression and the Transport Mechanism
Authors: Burgess Catherine M. , Slotboom Dirk Jan , Geertsma Eric R. , Duurkens Ria H. , Poolman Bert , van Sinderen Douwe
Published: April 2006

This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium
Lactococcus lactis
subsp.
cremoris
NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that
ribU
transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An
L. lactis
strain carrying a mutated
ribU
gene exhibits altered transcriptional control of the riboflavin biosynthesis operon
ribGBAH
in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the
ribU
mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the
ribGBAH
operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.
A Database of Bacterial Lipoproteins (DOLOP) with Functional Assignments to Predicted Lipoproteins
Authors: Babu M. Madan , Priya M. Leena , Selvan A. Tamil , Madera Martin , Gough Julian , Aravind L. , Sankaran K.
Published: April 2006

Lipid modification of the N-terminal Cys residue (
N
-acyl-
S
-diacylglyceryl-Cys) has been found to be an essential, ubiquitous, and unique bacterial posttranslational modification. Such a modification allows anchoring of even highly hydrophilic proteins to the membrane which carry out a variety of functions important for bacteria, including pathogenesis. Hence, being able to identify such proteins is of great value. To this end, we have created a comprehensive database of bacterial lipoproteins, called DOLOP, which contains information and links to molecular details for about 278 distinct lipoproteins and predicted lipoproteins from 234 completely sequenced bacterial genomes. The website also features a tool that applies a predictive algorithm to identify the presence or absence of the lipoprotein signal sequence in a user-given sequence. The experimentally verified lipoproteins have been classified into different functional classes and more importantly functional domain assignments using hidden Markov models from the SUPERFAMILY database that have been provided for the predicted lipoproteins. Other features include the following: primary sequence analysis, signal sequence analysis, and search facility and information exchange facility to allow researchers to exchange results on newly characterized lipoproteins. The website, along with additional information on the biosynthetic pathway, statistics on predicted lipoproteins, and related figures, is available at
http://www.mrc-lmb.cam.ac.uk/genomes/dolop/
.
DNA Replication during Aggregation Phase Is Essential for
Myxococcus xanthus
Development
Authors: Tzeng Linfong , Ellis Terri N. , Singer Mitchell
Published: April 2006

Previous studies have demonstrated that fruiting body-derived
Myxococcus xanthus
myxospores contain two fully replicated copies of its genome, implying developmental control of chromosome replication and septation. In this study, we employ DNA replication inhibitors to determine if chromosome replication is essential to development and the exact time frame in which chromosome replication occurs within the developmental cycle. Our results show that DNA replication during the aggregation phase is essential for developmental progression, implying the existence of a checkpoint that monitors chromosome integrity at the end of the aggregation phase.
A Novel Three-Protein Two-Component System Provides a Regulatory Twist on an Established Circuit To Modulate Expression of thecbbIRegion ofRhodopseudomonas palustrisCGA010
Authors: Romagnoli Simona , Tabita F. Robert
Published: April 2006

A novel two-component system has been identified in thecbbIregion of the nonsulfur purple photosynthetic bacteriumRhodopseudomonas palustris. Genes encoding this system, here designatedcbbRRS, are juxtaposed between the divergently transcribed transcription activator gene,cbbR, and the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes,cbbLS. The three genes of thecbbRRSsystem represent a variation of the well-known two-component signal transduction systems, as there are a transmembrane hybrid sensor kinase and two response regulators, with no apparent DNA binding domain associated with any of the three proteins encoded by these genes. In this study, we showed that the membrane-bound full-length kinase undergoes autophosphorylation and transfers phosphate to both response regulators. A soluble, truncated version of the kinase was subsequently prepared and found to catalyze phosphorylation of response regulator 1 but not response regulator 2, implying that conformational changes and/or sequence-specific regions of the kinase are important for discriminating between the two response regulators. Analyses indicated that a complex network of control of gene expression must occur, with CbbR required for the expression of thecbbLSgenes but dispensable for the synthesis of form II RubisCO (encoded bycbbM). The CbbRRS proteins specifically affected the activity and accumulation of form I RubisCO (CbbLS), as revealed by analyses of nonpolar, unmarked gene deletions. A tentative model of regulation suggested that changes in the phosphotransfer activity of the sensor kinase, possibly in response to a redox metabolic signal, cause modulation of the activity and synthesis of form I RubisCO.
DNA Microarray and Proteomic Analyses of the RpoS Regulon in
Geobacter sulfurreducens
Authors: Núñez Cinthia , Esteve-Núñez Abraham , Giometti Carol , Tollaksen Sandra , Khare Tripti , Lin Winston , Lovley Derek R. , Methé Barbara A.
Published: April 2006

The regulon of the sigma factor RpoS was defined in
Geobacter sulfurreducens
by using a combination of DNA microarray expression profiles and proteomics. An
rpoS
mutant was examined under steady-state conditions with acetate as an electron donor and fumarate as an electron acceptor and with additional transcriptional profiling using Fe(III) as an electron acceptor. Expression analysis revealed that RpoS acts as both a positive and negative regulator. Many of the RpoS-dependent genes determined play roles in energy metabolism, including the tricarboxylic acid cycle, signal transduction, transport, protein synthesis and degradation, and amino acid metabolism and transport. As expected, RpoS activated genes involved in oxidative stress resistance and adaptation to nutrient limitation. Transcription of the cytochrome
c
oxidase operon, necessary for
G. sulfurreducens
growth using oxygen as an electron acceptor, and expression of at least 13
c
-type cytochromes, including one previously shown to participate in Fe(III) reduction (MacA), were RpoS dependent. Analysis of a subset of the
rpoS
mutant proteome indicated that 15 major protein species showed reproducible differences in abundance relative to those of the wild-type strain. Protein identification using mass spectrometry indicated that the expression of seven of these proteins correlated with the microarray data. Collectively, these results indicate that RpoS exerts global effects on
G. sulfurreducens
physiology and that RpoS is vital to
G. sulfurreducens
survival under conditions typically encountered in its native subsurface environments.
Assembly of the Type III Secretion Apparatus of Enteropathogenic
Escherichia coli
Authors: Ogino Tomoaki , Ohno Ryuta , Sekiya Kachiko , Kuwae Asaomi , Matsuzawa Takeshi , Nonaka Takashi , Fukuda Hiroyuki , Imajoh-Ohmi Shinobu , Abe Akio
Published: April 2006

Enteropathogenic
Escherichia coli
(EPEC) secretes many Esps (
E. coli
-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione
S
-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore,
escC
,
escD
, and
escJ
mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.
Differences in Resolution of
mwr
-Containing Plasmid Dimers Mediated by the
Klebsiella pneumoniae
and
Escherichia coli
XerC Recombinases: Potential Implications in Dissemination of Antibiotic Resistance Genes
Authors: Bui Duyen , Ramiscal Judianne , Trigueros Sonia , Newmark Jason S. , Do Albert , Sherratt David J. , Tolmasky Marcelo E.
Published: April 2006

Xer-mediated dimer resolution at the
mwr
site of the multiresistance plasmid pJHCMW1 is osmoregulated in
Escherichia coli
containing either the
Escherichia coli
Xer recombination machinery or Xer recombination elements from
K. pneumoniae
. In the presence of
K. pneumoniae
XerC (XerC
Kp
), the efficiency of recombination is lower than that in the presence of the
E. coli
XerC (XerC
Ec
) and the level of dimer resolution is insufficient to stabilize the plasmid, even at low osmolarity. This lower efficiency of recombination at
mwr
is observed in the presence of
E. coli
or
K. pneumoniae
XerD proteins. Mutagenesis experiments identified a region near the N terminus of XerC
Kp
responsible for the lower level of recombination catalyzed by XerC
Kp
at
mwr
. This region encompasses the second half of the predicted α-helix B and the beginning of the predicted α-helix C. The efficiencies of recombination at other sites such as
dif
or
cer
in the presence of XerC
Kp
or XerC
Ec
are comparable. Therefore, XerC
Kp
is an active recombinase whose action is impaired on the
mwr
recombination site. This characteristic may result in restriction of the host range of plasmids carrying this site, a phenomenon that may have important implications in the dissemination of antibiotic resistance genes.
Morphology of Isolated Gli349, a Leg Protein Responsible for
Mycoplasma mobile
Gliding via Glass Binding, Revealed by Rotary Shadowing Electron Microscopy
Authors: Adan-Kubo Jun , Uenoyama Atsuko , Arata Toshiaki , Miyata Makoto
Published: April 2006

Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species,
Mycoplasma mobile
, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed. Gel filtration analysis showed that the isolated Gli349 protein is monomeric. Rotary shadowing electron microscopy revealed that the molecular structure resembles the symbol for an eighth note in music. It contains an oval foot 14 nm long in axis. From this foot extend three rods in tandem of 43, 20, and 20 nm, in that order. The hinge connecting the first and second rods is flexible, while the next hinge has a distinct preference in its angle, near 90 degrees. Molecular images revealed that a monoclonal antibody that can bind to the position at one-third of the total peptide length from the N terminus bound to a position two-thirds from the foot end, suggesting that the foot corresponds to the C-terminal region. The amino acid sequence was assigned to the molecular image, and the topology of the molecule in the gliding machinery is discussed.
A Novel FtsZ-Like Protein Is Involved in Replication of the Anthrax Toxin-Encoding pXO1 Plasmid inBacillus anthracis
Authors: Tinsley Eowyn , Khan Saleem A.
Published: April 2006

Plasmid pXO1 encodes the tripartite anthrax toxin, which is the major virulence factor ofBacillus anthracis. In spite of the important role of pXO1 in anthrax pathogenesis, very little is known about its replication and maintenance inB. anthracis. We cloned a 5-kb region of the pXO1 plasmid into anEscherichia colivector and showed that this plasmid can replicate when introduced intoB. anthracis. Mutational analysis showed that open reading frame 45 (repX) of pXO1 was required for the replication of the miniplasmid inB. anthracis. Interestingly,repXshowed limited homology to bacterial FtsZ proteins that are involved in cell division. A mutation in the predicted GTP binding domain of RepX abolished its replication activity. Genes almost identical torepXare contained on several megaplasmids in members of theBacillus cereusgroup, including aB. cereusstrain that causes an anthrax-like disease. Our results identify a novel group of FtsZ-related initiator proteins that are required for the replication of virulence plasmids inB. anthracisand possibly in related organisms. Such replication proteins may provide novel drug targets for the elimination of plasmids encoding the anthrax toxin and other virulence factors.
Identification of Lactaldehyde Dehydrogenase in
Methanocaldococcus jannaschii
and Its Involvement in Production of Lactate for F
420
Biosynthesis
Authors: Grochowski Laura L. , Xu Huimin , White Robert H.
Published: April 2006

One of the early steps in the biosynthesis of coenzyme F
420
in
Methanocaldococcus jannaschii
requires generation of 2-phospho-
l
-lactate, which is formed by the phosphorylation of
l
-lactate. Preliminary studies had shown that
l
-lactate in
M. jannaschii
is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that
l
-lactate is formed by the NAD
+
-dependent oxidation of
l
-lactaldehyde by the MJ1411 gene product. The lactaldehyde, in turn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-1-phosphate by fuculose-1-phosphate aldolase, the MJ1418 gene product.
Transcriptional Heat Shock Response in the Smallest Known Self-Replicating Cell,Mycoplasma genitalium
Authors: Musatovova Oxana , Dhandayuthapani Subramanian , Baseman Joel B.
Published: April 2006

Mycoplasma genitaliumis a human bacterial pathogen linked to urethritis and other sexually transmitted diseases as well as respiratory and joint pathologies. Though its complete genome sequence is available, little is understood about the regulation of gene expression in this smallest known, self-replicating cell, as its genome lacks orthologues for most of the conventional bacterial regulators. Still, the transcriptional repressor HrcA (heatregulation atCIRCE [controllinginvertedrepeat ofchaperoneexpression]) is predicted in theM. genitaliumgenome as well as three copies of its corresponding regulatory sequence CIRCE. We investigated the transcriptional response ofM. genitaliumto elevated temperatures and detected the differential induction of fourhspgenes. Three of the up-regulated genes, which encode DnaK, ClpB, and Lon, possess CIRCE within their promoter regions, suggesting that the HrcA-CIRCE regulatory mechanism is functional. Additionally, one of three DnaJ-encoding genes was up-regulated, even though no known regulatory sequences were found in the promoter region. Transcript levels returned to control values after 1 h of incubation at 37°C, reinforcing the transient nature of the heat shock transcriptional response. Interestingly, neither of thegroESLoperon genes, which encode the GroEL chaperone and its cochaperone GroES, responded to heat shock. These data suggest thatM. genitaliumselectively regulates a limited number of genes in response to heat shock.
Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE
Authors: Ito Yasuko , Matsuzawa Hitomi , Matsuyama Shin-ichi , Narita Shin-ichiro , Tokuda Hajime
Published: April 2006

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of
Escherichia coli
. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of
E. coli
. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal
lolD
+
background. We then selected suppressor mutations of the
lolC
and
lolE
genes that correct the growth defect caused by the LolD mutations. Mutations of the
lolC
suppressors were mainly located in the periplasmic loop, whereas ones of
lolE
suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.
Comparison of OG1RF and an IsogenicfsrBDeletion Mutant by Transcriptional Analysis: the Fsr System ofEnterococcus faecalisIs More than the Activator of Gelatinase and Serine Protease
Authors: Bourgogne Agathe , Hilsenbeck Susan G. , Dunny Gary M. , Murray Barbara E.
Published: April 2006

The FsrABC system ofEnterococcus faecaliscontrols the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on theE. faecalisstrain V583 sequence, to compare theE. faecalisstrain OG1RF with its isogenic mutant, TX5266, anfsrBdeletion mutant. We found that the presence of an intactfsrBinfluences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus thefsrBdeletion mutant, expression of ef1617 to -1634 (eut-pduorthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects.
Functional Analysis ofluxSinStaphylococcus aureusReveals a Role in Metabolism but Not Quorum Sensing
Authors: Doherty Neil , Holden Matthew T. G. , Qazi Saara N. , Williams Paul , Winzer Klaus
Published: April 2006

The function of AI-2 in many bacteria and the physiological role of LuxS, the enzyme responsible for its production, remain matters of debate. Here, we show that inStaphylococcus aureustheluxSgene forms a monocistronic transcriptional unit under the control of a σ70-dependent promoter. The gene was transcribed throughout growth under a variety of conditions, including intracellular growth in MAC-T cells. AI-2 was produced in rich media under aerobic and anaerobic conditions, peaking during the transition to stationary phase, but was hardly detectable in a sulfur-limited defined medium. In the presence of glucose or under anaerobic conditions, cultures retained considerable AI-2 activity after entry into stationary phase. Inactivation ofluxSin variousS. aureusstrains did not affect virulence-associated traits, such as production of hemolysins and extracellular proteases, biofilm formation, and theagrsignaling system. Conversely, AI-2 production remained unchanged in anagrmutant. However,luxSmutants grown in a sulfur-limited defined medium exhibited a growth defect. When grown together with the wild type in mixed culture,luxSmutants of variousS. aureusstrains showed reduced ability to compete for growth under these conditions. In contrast, a complementedluxSmutant grew as well as the parent strain, suggesting that the observed growth defect was of an intracellular nature and had not been caused by either second-site mutations or the lack of a diffusible factor. However, the LuxS/AI-2 system does not appear to contribute to the overall fitness ofS. aureusRN6390B during intracellular growth in epithelial cells: the wild type and aluxSmutant showed very similar growth patterns after their internalization by MAC-T cells.
TheppuI-rsaL-ppuRQuorum-Sensing System Regulates Biofilm Formation ofPseudomonas putidaPCL1445 by Controlling Biosynthesis of the Cyclic Lipopeptides Putisolvins I and II
Authors: Dubern Jean-Frédéric , Lugtenberg Ben J. J. , Bloemberg Guido V.
Published: April 2006

Pseudomonas putidastrain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of severalPseudomonasspp., includingP. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed ofppuI,rsaL, andppuRand shows very high similarity with gene clusters ofP. putidastrains IsoF and WCS358. Strains with mutations inppuIandppuRshowed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in appuIbackground showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C10-N-acyl homoserine lactone (3-oxo-C10-AHL) or 3-oxo-C12-AHL to the medium. AnrsaLmutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed thatppuIandppuRmutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas anrsaLmutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation byPseudomonas putida.
The DtxR Regulon ofCorynebacterium glutamicum
Authors: Wennerhold Julia , Bott Michael
Published: April 2006

Previous studies withCorynebacterium diphtheriaeandMycobacteriumspecies revealed that the transcriptional regulator DtxR and its ortholog IdeR play a central role in the control of iron metabolism. In the present work, we used genome-based approaches to determine the DtxR regulon ofCorynebacterium glutamicum, a nonpathogenic relative ofC. diphtheriae. First, global gene expression of adtxRdeletion mutant was compared with that of the wild type using DNA microarrays. Second, we used a computer-based approach to identify 117 putative DtxR binding sites in theC. glutamicumgenome. In the third step, 74 of the corresponding genome regions were amplified by PCR, 51 of which were shifted by the DtxR protein. Finally, we analyzed which of the genes preceded by a functional DtxR binding site showed altered mRNA levels in the transcriptome comparison. Fifty-one genes organized in 27 putative operons displayed an increased mRNA level in theΔdtxRmutant and thus are presumably repressed by DtxR. The majority of these genes are obviously involved in iron acquisition, three encode transcriptional regulators, e.g., the recently identified repressor of iron proteins RipA, and the others encode proteins of diverse or unknown functions. Thirteen genes showed a decreased mRNA level in theΔdtxRmutant and thus might be activated by DtxR. This group included thesufoperon, whose products are involved in the formation and repair of iron-sulfur clusters, and several genes for transcriptional regulators. Our results clearly establish DtxR as the master regulator of iron-dependent gene expression inC. glutamicum.
Aerobic Benzoyl-Coenzyme A (CoA) Catabolic Pathway in
Azoarcus evansii
: Conversion of Ring Cleavage Product by 3,4-Dehydroadipyl-CoA Semialdehyde Dehydrogenase
Authors: Gescher Johannes , Ismail Wael , Ölgeschläger Ellen , Eisenreich Wolfgang , Wörth Jürgen , Fuchs Georg
Published: April 2006

Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde was studied in the β-proteobacterium
Azoarcus evansii
. Cell extracts contained a benzoate-induced, NADP
+
-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in
Escherichia coli
, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.
Green Fluorescent Chimeras Indicate Nonpolar Localization of Pullulanase Secreton Components PulL and PulM
Authors: Buddelmeijer Nienke , Francetic Olivera , Pugsley Anthony P.
Published: April 2006

TheKlebsiella oxytocapullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components inEscherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. InK. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to theirVibrio choleraeEps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.
Transcriptional Control of the MycobacterialembCABOperon by PknH through a Regulatory Protein, EmbR, In Vivo
Authors: Sharma Kirti , Gupta Meetu , Pathak Monika , Gupta Nidhi , Koul Anil , Sarangi Smilona , Baweja Renu , Singh Yogendra
Published: April 2006

EmbR, a putative transcriptional regulator fromMycobacterium tuberculosis, is homologous to the OmpR class of transcriptional regulators that possess winged helix-turn-helix DNA binding motifs. In contrast to other OmpR-like response regulators that are usually phosphorylated and controlled by histidine kinases, EmbR was recently shown to be phosphorylated by the cognate mycobacterial serine/threonine kinase PknH. Despite the in vitro evidence of phosphorylation and interaction between the kinase and regulator, the physiological function of the PknH-EmbR pair is still unknown. We identify theembCABoperon encoding arabinosyltransferases inM. tuberculosisas the cellular target of EmbR. Phosphorylation of EmbR enhances its DNA binding activity towards promoter regions ofembCABgenes. In vivo studies involving expression of PknH inMycobacterium smegmatisestablished its positive regulatory effect on transcription of theembCABoperon via phosphorylation of EmbR. Interestingly, increased transcription ofembC, catalyzing arabinosylation of lipomannan (LM) to lipoarabinomannan (LAM), results in a high LAM/LM ratio, which in turn is a crucial factor in mycobacterial virulence. The PknH-mediated increase in the transcription ofembABgenes significantly alters resistance to ethambutol, a frontline antituberculosis drug known to targetembABgenes. These findings and in vivo upregulation of PknH inside the host macrophages suggest a functionally relevant signaling mechanism involving the PknH-EmbR-embCABsystem.
Transcription of the ContiguoussigB,dtxR, andgalEGenes inCorynebacterium diphtheriae: Evidence for Multiple Transcripts and Regulation by Environmental Factors
Authors: Oram Diana Marra , Jacobson Andrew D. , Holmes Randall K.
Published: April 2006

The iron-dependent transcriptional regulator DtxR fromCorynebacterium diphtheriaeis the prototype for a family of metal-dependent regulators found in diverse bacterial species. The structure of DtxR and its action as a repressor have been extensively characterized, but little is known about expression ofdtxR. In the current study, we investigated transcription ofdtxRas well as thesigBandgalEgenes located immediately upstream and downstream fromdtxR, respectively. We identified two promoters that determine transcription ofdtxR.The first, located upstream ofsigB, appears to be controlled by an extracytoplasmic function σ factor. The second, located in the intergenic region betweensigBanddtxR, is similar to promoters used by the primary vegetative σ factors in other actinomycete species. Using quantitative real-time assays, we demonstrated that the number of transcripts initiated upstream fromsigBis affected by several environmental factors. In contrast, the presence of sodium dodecyl sulfate was the only factor tested that conclusively affects the number of transcripts initiated in thesigB-dtxRintergenic region. Additionally, we provided evidence for the existence of transcripts that containsigB,dtxR, andgalE. Our studies provide the first quantitative transcriptional analysis of a gene encoding a DtxR family regulator and give new insights into transcriptional regulation inC. diphtheriae.
Acetylornithine Transcarbamylase: a Novel Enzyme in Arginine Biosynthesis
Authors: Morizono Hiroki , Cabrera-Luque Juan , Shi Dashuang , Gallegos Rene , Yamaguchi Saori , Yu Xiaolin , Allewell Norma M. , Malamy Michael H. , Tuchman Mendel
Published: April 2006

Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In
Xanthomonas campestris
, the protein annotated as ornithine transcarbamylase, and encoded by the
argF
gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this
X. campestris
gene in
Escherichia coli
and show that it catalyzes the formation of
N
-acetyl-l-citrulline from
N
-acetyl-l-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The

K
m

values for
N
-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads,
Cytophaga
, and
Bacteroidetes
as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor,
N
α
-acetyl-
N
δ
-phosphonoacetyl-
l
-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases.
Influence of BrpA on Critical Virulence Attributes ofStreptococcus mutans
Authors: Wen Zezhang T. , Baker Henry V. , Burne Robert A.
Published: April 2006

Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. ThebrpAgene codes for a predicted surface-associated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P< 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation inS. mutans, which are key virulence attributes of the organism.
Role Of MinD-Membrane Association in Min Protein Interactions
Authors: Taghbalout Aziz , Ma Luyan , Rothfield Lawrence
Published: April 2006

Division site placement in
Escherichia coli
involves interactions of the MinD protein with MinC and MinE and with other MinD molecules to form membrane-associated polymeric structures. In this work, as part of a study of these interactions, we established that heterologous membrane-associated proteins such as MinD can be targeted to the yeast nuclear membrane, dependent only on the presence of a membrane-binding domain and a nuclear targeting sequence. Targeting to the nuclear membrane was equally effective using the intrinsic MinD membrane-targeting domain or the completely unrelated membrane-targeting domain of cytochrome
b
5
. The chimeric proteins differing in their membrane-targeting sequences were then used to establish the roles of membrane association and specificity of the membrane anchor in MinD interactions, using the yeast two-hybrid system. The chimeric proteins were also used to show that the membrane association of MinD and MinE in
E. coli
cells had no specificity for the membrane anchor, whereas formation of MinDE polar zones and MinE rings required the presence of the native MinD membrane-targeting sequence.
Differential and Dynamic Localization of Topoisomerases in
Bacillus subtilis
Authors: Tadesse Serkalem , Graumann Peter L.
Published: April 2006

Visualization of topoisomerases in live
Bacillus subtilis
cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcelluar topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the
B. subtilis
nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.
Comparative Genomics of NAD Biosynthesis in Cyanobacteria
Authors: Gerdes Svetlana Y. , Kurnasov Oleg V. , Shatalin Konstantin , Polanuyer Boris , Sloutsky Roman , Vonstein Veronika , Overbeek Ross , Osterman Andrei L.
Published: April 2006

Biosynthesis of NAD(P) cofactors is of special importance for cyanobacteria due to their role in photosynthesis and respiration. Despite significant progress in understanding NAD(P) biosynthetic machinery in some model organisms, relatively little is known about its implementation in cyanobacteria. We addressed this problem by a combination of comparative genome analysis with verification experiments in the model system ofSynechocystissp. strain PCC 6803. A detailed reconstruction of the NAD(P) metabolic subsystem using the SEED genomic platform (http://theseed.uchicago.edu/FIG/index.cgi) helped us accurately annotate respective genes in the entire set of 13 cyanobacterial species with completely sequenced genomes available at the time. Comparative analysis of operational variants implemented in this divergent group allowed us to elucidate both conserved (de novo and universal pathways) and variable (recycling and salvage pathways) aspects of this subsystem. Focused genetic and biochemical experiments confirmed several conjectures about the key aspects of this subsystem. (i) The product of the slr1691 gene, a homolog ofEscherichia coligenenadEcontaining an additional nitrilase-like N-terminal domain, is a NAD synthetase capable of utilizing glutamine as an amide donor in vitro. (ii) The product of the sll1916 gene, a homolog ofE. coligenenadD, is a nicotinic acid mononucleotide-preferring adenylyltransferase. This gene is essential for survival and cannot be compensated for by an alternative nicotinamide mononucleotide (NMN)-preferring adenylyltransferase (slr0787 gene). (iii) The product of the slr0788 gene is a nicotinamide-preferring phosphoribosyltransferase involved in the first step of the two-step nondeamidating utilization of nicotinamide (NMN shunt). (iv) The physiological role of this pathway encoded by a conserved gene cluster, slr0787-slr0788, is likely in the recycling of endogenously generated nicotinamide, as supported by the inability of this organism to utilize exogenously provided niacin. Positional clustering and the cooccurrence profile of the respective genes across a diverse collection of cellular organisms provide evidence of horizontal transfer events in the evolutionary history of this pathway.
Functional Domains of theBacillus subtilisTranscription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding
Authors: Franco Irina Saraiva , Mota Luís Jaime , Soares Cláudio Manuel , de Sá-Nogueira Isabel
Published: April 2006

TheBacillus subtilisAraR transcription factor represses at least 13 genes required for the extracellular degradation of arabinose-containing polysaccharides, transport of arabinose, arabinose oligomers, xylose, and galactose, intracellular degradation of arabinose oligomers, and further catabolism of this sugar. AraR exhibits a chimeric organization comprising a small N-terminal DNA-binding domain that contains a winged helix-turn-helix motif similar to that seen with the GntR family and a larger C-terminal domain homologous to that of the LacI/GalR family. Here, a model for AraR was derived based on the known crystal structures of the FadR and PurR regulators fromEscherichia coli. We have used random mutagenesis, deletion, and construction of chimeric LexA-AraR fusion proteins to map the functional domains of AraR required for DNA binding, dimerization, and effector binding. Moreover, predictions for the functional role of specific residues were tested by site-directed mutagenesis. In vivo analysis identified particular amino acids required for dimer assembly, formation of the nucleoprotein complex, and composition of the sugar-binding cleft. This work presents a structural framework for the function of AraR and provides insight into the mechanistic mode of action of this modular repressor.