bluuebunny/crossref_metadata_2025 huggingface dataset:
The
rpoZ
Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in
Streptomyces kasugaensis
Authors: Kojima Ikuo , Kasuga Kano , Kobayashi Masayuki , Fukasawa Akira , Mizuno Satoshi , Arisawa Akira , Akagawa Hisayoshi
Published: November 2002

The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type
Streptomyces kasugaensis
A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that
orf5
, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that
orf5
was a homolog of
rpoZ
, the gene that is known to encode RNA polymerase subunit omega (ω), thus leading to the conclusion that
orf5
was
rpoZ
in
S. kasugaensis
. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the
rpoZ
region of mutant KSB, which probably resulted in a truncated, incomplete ω of 47 amino acids. Furthermore,
rpoZ
-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn
5 aphII
into the middle of the
rpoZ
-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When
rpoZ
of
S. kasugaensis
and
Streptomyces coelicolor
, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that
rpoZ
is required for kasugamycin production and aerial mycelium formation in
S. kasugaensis
and responsible for pleiotropy.
Substrate Specificity of the AmpG Permease Required for Recycling of Cell Wall Anhydro-Muropeptides
Authors: Cheng Qiaomei , Park James T.
Published: November 2002

AmpG
was originally identified as a gene required for induction of β-lactamase. Subsequently, we found AmpG to be a permease required for recycling of murein tripeptide and uptake of anhydro-muropeptides. We have now studied the specificity of the AmpG permease. The principal requirement is for the presence of the disaccharide,
N
-acetylglucosaminyl-β-1,4-anhydro-
N
-acetylmuramic acid (GlcNAc-anhMurNAc). These unique substrates for AmpG, which contain murein peptides linked to GlcNAc-anhMurNAc, are produced by turnover of the cell wall during logarithmic growth. AmpG permease is sensitive to carbonylcyanide
m
-chlorophenylhydrazone, demonstrating that AmpG permease is a single-component permease and that transport is dependent on the proton motive force.
Impact of Phosphorylation of Specific Residues in the Tyrosine Autokinase, Wzc, on Its Activity in Assembly of Group 1 Capsules inEscherichia coli
Authors: Paiment Anne , Hocking Jennifer , Whitfield Chris
Published: November 2002

WzcCPSis a tyrosine autokinase essential for the assembly of a high-molecular-weight (HMW) group 1 capsular polysaccharide (CPS) inEscherichia coli. Homologues of Wzc participate in the formation of CPS and exopolysaccharides in a variety of gram-positive and gram-negative bacteria. Phosphorylation of tyrosine residues in the WzcCPSC terminus is essential for HMW CPS assembly. Overexpression of WzbCPS(phosphatase) in a wild-type background caused a 3.7-fold decrease in the amount of cell-associated K30 CPS produced, confirming the importance of WzcCPSphosphorylation for capsule assembly. In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for WzcCPSphosphorylation and/or capsule expression. Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of WzcCPSin vivo or for HMW CPS expression. Furthermore, no single tyrosine residue is essential for phosphorylation or capsule assembly, since removal of any one tyrosine residue has no detectable effect. Altering combinations of tyrosine residues (from two to five) led to WzcCPSderivatives that were still competent for phosphorylation but that could not support assembly of HMW CPS, showing that phosphorylation of Wzc per se is not an accurate measure of its ability to function in capsule assembly. One interpretation of these data is that the overall level of phosphorylation in this region, rather than the precise combination of residues accessible to phosphorylation, is important for the activity of WzcCPS. Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of WzcCAfromE. coliK-12, was also mutated. The derivative with this mutation still functioned in capsule assembly. Quantitation of K30CPSfrom this mutant revealed no difference in the amount of polymer produced. Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that WzcCPSforms complexes in vivo, independent of the phosphorylation state of the protein.
Structural Analysis and Evidence for Dynamic Emergence of
Bacillus anthracis
S-Layer Networks
Authors: Couture-Tosi Evelyne , Delacroix Hervé , Mignot Tâm , Mesnage Stéphane , Chami Mohamed , Fouet Agnès , Mosser Gervaise
Published: November 2002

Surface layers (S-layers), which form the outermost layers of many
Bacteria
and
Archaea
, consist of protein molecules arranged in two-dimensional crystalline arrays.
Bacillus anthracis
, a gram-positive, spore-forming bacterium, responsible for anthrax, synthesizes two abundant surface proteins: Sap and EA1. Regulatory studies showed that EA1 and Sap appear sequentially at the surface of the parental strain. Sap and EA1 can form arrays. The structural parameters of S-layers from mutant strains (EA1
−
and Sap
−
) were determined by computer image processing of electron micrographs of negatively stained regular S-layer fragments or deflated whole bacteria. Sap and EA1 projection maps were calculated on a
p1
symmetry basis. The unit cell parameters of EA1 were
a
= 69 Å,
b
= 83 Å, and γ = 106°, while those of Sap were
a
= 184 Å,
b
= 81 Å, and γ = 84°. Freeze-etching experiments and the analysis of the peripheral regions of the cell suggested that the two S-layers have different settings. We characterized the settings of each network at different growth phases. Our data indicated that the scattered emergence of EA1 destabilizes the Sap S-layer.
Aquifex aeolicus
PilT, Homologue of a Surface Motility Protein, Is a Thermostable Oligomeric NTPase
Authors: Herdendorf Timothy J. , McCaslin Darrell R. , Forest Katrina T.
Published: November 2002

Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium
Aquifex aeolicus
was cloned, overexpressed, and purified.
A. aeolicus
PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein
.
This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a ≈5- to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed.
Autolysis and Autoaggregation in
Pseudomonas aeruginosa
Colony Morphology Mutants
Authors: D'Argenio David A. , Calfee M. Worth , Rainey Paul B. , Pesci Everett C.
Published: November 2002

Two distinctive colony morphologies were noted in a collection of
Pseudomonas aeruginosa
transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the
Pseudomonas
quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of
P. aeruginosa
between survival of the many and persistence of the few.
Substrate Specificity of the RND-Type Multidrug Efflux Pumps AcrB and AcrD of
Escherichia coli
Is Determined Predominately by Two Large Periplasmic Loops
Authors: Elkins Christopher A. , Nikaido Hiroshi
Published: November 2002

AcrAB-TolC is a constitutively expressed, tripartite efflux transporter complex that functions as the primary resistance mechanism to lipophilic drugs, dyes, detergents, and bile acids in
Escherichia coli
. TolC is an outer membrane channel, and AcrA is an elongated lipoprotein that is hypothesized to span the periplasm and coordinate efflux of such substrates by AcrB and TolC. AcrD is an efflux transporter of
E. coli
that provides resistance to aminoglycosides as well as to a limited range of amphiphilic agents, such as bile acids, novobiocin, and fusidic acid. AcrB and AcrD belong to the resistance nodulation division superfamily and share a similar topology, which includes a pair of large periplasmic loops containing more than 300 amino acid residues each. We used this knowledge to test several plasmid-encoded chimeric constructs of
acrD
and
acrB
for substrate specificity in a
marR1
Δ
acrB
Δ
acrD
host. AcrD chimeras were constructed in which the large, periplasmic loops between transmembrane domains 1 and 2 and 7 and 8 were replaced with the corresponding loops of AcrB. Such constructs provided resistance to AcrB substrates at levels similar to native AcrB. Conversely, AcrB chimeras containing both loops of AcrD conferred resistance only to the typical substrates of AcrD. These results cannot be explained by simply assuming that AcrD, not hitherto known to interact with AcrA, acquired this ability by the introduction of the loop regions of AcrB, because (i) both AcrD and AcrA were found, in this study, to be required for the efflux of amphiphilic substrates, and (ii) chemical cross-linking in intact cells efficiently produced complexes between AcrD and AcrA. Since AcrD can already interact with AcrA, the alterations in substrate range accompanying the exchange of loop regions can only mean that substrate recognition (and presumably binding) is determined largely by the two periplasmic loops.
Chimeric Analysis of the Multicomponent Multidrug Efflux Transporters from Gram-Negative Bacteria
Authors: Tikhonova Elena B. , Wang Quiju , Zgurskaya Helen I.
Published: November 2002

Many multidrug transporters from gram-negative bacteria belong to the resistance-nodulation-cell division (RND) superfamily of transporters. RND-type multidrug transporters have an extremely broad substrate specificity and protect bacterial cells from the actions of antibiotics on both sides of the cytoplasmic membrane. They usually function as three-component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of gram-negative bacteria. The structural determinants of RND transporters responsible for multidrug recognition and complex assembly remain unknown. We constructed chimeric RND transporters composed of N-terminal residues of AcrB and C-terminal residues of MexB, the major RND-type transporters from
Escherichia coli
and
Pseudomonas aeruginosa
, respectively. The assembly of complexes and multidrug efflux activities of chimeric transporters were determined by coexpression of hybrid genes either with AcrA, the periplasmic component of the AcrAB transporter from
E. coli
, or with MexA and OprM, the accessory proteins of the MexAB-OprM pump from
P. aeruginosa
. We found that the specificity of interaction with the corresponding periplasmic component is encoded in the T60-V612 region of transporters. Our results also suggest that the large periplasmic loops of RND-type transporters are involved in multidrug recognition and efflux.
Long-Chain Acyl-Homoserine Lactone Quorum-Sensing Regulation of
Rhodobacter capsulatus
Gene Transfer Agent Production
Authors: Schaefer Amy L. , Taylor Terumi A. , Beatty J. Thomas , Greenberg E. P.
Published: November 2002

Many proteobacteria use acyl-homoserine lactones as quorum-sensing signals. Traditionally, biological detection systems have been used to identify bacteria that produce acyl-homoserine lactones, although the specificities of these detection systems can limit discovery. We used a sensitive approach that did not require a bioassay to detect production of long-acyl-chain homoserine lactone production by
Rhodobacter capsulatus
and
Paracoccus denitrificans
. These long-chain acyl-homoserine lactones are not readily detected by standard bioassays. The most abundant acyl-homoserine lactone was
N
-hexadecanoyl-homoserine lactone. The long-chain acyl-homoserine lactones were concentrated in cells but were also found in the culture fluid. An
R. capsulatus
gene responsible for long-chain acyl-homoserine lactone synthesis was identified. A mutation in this gene, which we named
gtaI
, resulted in decreased production of the
R. capsulatus
gene transfer agent, and gene transfer agent production was restored by exogenous addition of
N
-hexadecanoyl-homoserine lactone. Thus, long-chain acyl-homoserine lactones serve as quorum-sensing signals to enhance genetic exchange in
R. capsulatus
.
Programmed Translational Frameshift in the Bacteriophage P2
FETUD
Tail Gene Operon
Authors: Christie Gail E. , Temple Louise M. , Bartlett Becky A. , Goodwin Tina S.
Published: November 2002

The major structural components of the P2 contractile tail are encoded in the
FETUD
tail gene operon. The sequences of genes
F
I
and
F
II
, encoding the major tail sheath and tail tube proteins, have been reported previously (L. M. Temple, S. L. Forsburg, R. Calendar, and G. E. Christie, Virology 181:353-358, 1991). Sequence analysis of the remainder of this operon and the locations of amber mutations
E
am
30
,
T
am
5
,
T
am
64
,
T
am
215
,
U
am
25
,
U
am
77
,
U
am
92
, and
D
am
6
and missense mutation
E
ts
55
identified the coding regions for genes
E
,
T
,
U
, and
D
, completing the sequence determination of the P2 genome. Inspection of the DNA sequence revealed a new open reading frame overlapping the end of the essential tail gene
E
. Lack of an apparent translation initiation site and identification of a putative sequence for a programmed translational frameshift within the
E
gene suggested that this new reading frame (
E
′) might be translated as an extension of gene
E
, following a −1 translational frameshift. Complementation analysis demonstrated that
E
′ was essential for P2 lytic growth. Analysis of fusion polypeptides verified that this reading frame was translated as a −1 frameshift extension of gpE, with a frequency of approximately 10%. The arrangement of these two genes within the tail gene cluster of phage P2 and their coupling via a translational frameshift appears to be conserved among P2-related phages. This arrangement shows a striking parallel to the organization in the tail gene cluster of phage lambda, despite a lack of amino acid sequence similarity between the tail gene products of these phage families.
Lactococcus lactis
Lytic Bacteriophages of the P335 Group Are Inhibited by Overexpression of a Truncated CI Repressor
Authors: Durmaz Evelyn , Madsen Søren M. , Israelsen Hans , Klaenhammer Todd R.
Published: November 2002

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage φ31 encodes a genetic switch region of
c
I and
cro
homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative
c
I repressor gene of phage φ31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host,
Lactococcus lactis
subsp.
lactis
NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length
c
I gene in
Lactococcus
in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the
c
I gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage φ31 was inhibited to an efficiency of plaquing (EOP) of 10
−6
in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the φ31 EOP to <10
−7
. Phage φ31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on
L. lactis
harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the φ31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for
L. lactis
NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.
Isolation of the Gene for the B
12
-Dependent Ribonucleotide Reductase from
Anabaena
sp. Strain PCC 7120 and Expression in
Escherichia coli
Authors: Gleason Florence K. , Olszewski Neil E.
Published: November 2002

The gene for ribonucleotide reductase from
Anabaena
sp. strain PCC 7120 was identified and expressed in
Escherichia coli
. This gene codes for a 1,172-amino-acid protein that contains a 407-amino-acid intein. The intein splices itself from the protein when it is expressed in
E. coli
, yielding an active ribonucleotide reductase of 765 residues. The mature enzyme was purified to homogeneity from
E. coli
extracts.
Anabaena
ribonucleotide reductase is a monomer with a molecular weight of approximately 88,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Superose 12 column chromatography. The enzyme reduces ribonucleotides at the triphosphate level and requires a divalent cation and a deoxyribonucleoside triphosphate effector. The enzyme is absolutely dependent on the addition of the cofactor, 5′-adenosylcobalamin. These properties are characteristic of the class II-type reductases. The cyanobacterial enzyme has limited sequence homology to other class II reductases; the greatest similarity (38%) is to the reductase from
Lactobacillus leichmannii
. In contrast, the
Anabaena
reductase shows over 90% sequence similarity to putative reductases found in genome sequences of other cyanobacteria, such as
Nostoc punctiforme, Synechococcus
sp. strain WH8102, and
Prochlorococcus marinus
MED4, suggesting that the cyanobacterial reductases form a closely related subset of the class II enzymes.
Gene Expression Profiling of the pH Response inEscherichia coli
Authors: Tucker Don L. , Tucker Nancy , Conway Tyrrell
Published: November 2002

Escherichia coliMG1655 acid-inducible genes were identified by whole-genome expression profiling. Cultures were grown to the mid-logarithmic phase on acidified glucose minimal medium, conditions that induce glutamate-dependent acid resistance (AR), while the other AR systems are either repressed or not induced. A total of 28 genes were induced in at least two of three experiments in which the gene expression profiles of cells grown in acid (pH 5.5 or 4.5) were compared to those of cells grown at pH 7.4. As expected, the genes encoding glutamate decarboxylase,gadAandgadB, were significantly induced. Interestingly, two acid-inducible genes code for small basic proteins with pIs of >10.5, and six code for small acidic proteins with pIs ranging from 5.7 to 4.0; the roles of these small basic and acidic proteins in acid resistance are unknown. The acid-induced genes represented only five functional grouping categories, including eight genes involved in metabolism, nine associated with cell envelope structures or modifications, two encoding chaperones, six regulatory genes, and six unknown genes. It is unlikely that all of these genes are involved in the glutamate-dependent AR. However, nine acid-inducible genes are clustered in thegadAregion, includinghdeA, which encodes a putative periplasmic chaperone, and four putative regulatory genes. One of these putative regulators,yhiE, was shown to significantly increase acid resistance when overexpressed in cells that had not been preinduced by growth at pH 5.5, and mutation ofyhiEdecreased acid resistance;yhiEcould therefore encode an activator of AR genes. Thus, the acid-inducible genes clustered in thegadAregion appear to be involved in glutatmate-dependent acid resistance, although their specific roles remain to be elucidated.
Functional Analysis of the Heat Shock Regulator HrcA ofChlamydia trachomatis
Authors: Wilson Adam C. , Tan Ming
Published: November 2002

HrcA is a regulator of bacterial heat shock gene expression that binds to acis-acting DNA element called CIRCE. It has been proposed that HrcA and CIRCE function as a repressor-operator pair. We have purified recombinant HrcA from the pathogenic bacteriumChlamydia trachomatisand have shown that it is a DNA-binding protein that functions as a negative regulator of transcription. HrcA bound specifically to the CIRCE element in a concentration-dependent manner. HrcA repressed the in vitro transcription of a chlamydial heat shock promoter, and this repression was promoter specific. HrcA-mediated repression appears to be dependent on the topological state of the promoter, as repression on a supercoiled promoter template was greater than that on a linearized template. These results provide direct support for the role of HrcA as a transcriptional repressor in bacteria. This is the first report of the in vitro reconstitution of transcriptional regulation inChlamydia.
Vibrio choleraePhage K139: Complete Genome Sequence and Comparative Genomics of Related Phages
Authors: Kapfhammer Dagmar , Blass Julia , Evers Stefan , Reidl Joachim
Published: November 2002

In this report, we characterize the complete genome sequence of the temperate phage K139, which morphologically belongs to theMyoviridaephage family (P2 and 186). The prophage genome consists of 33,106 bp, and the overall GC content is 48.9%. Forty-four open reading frames were identified. Homology analysis and motif search were used to assign possible functions for the genes, revealing a close relationship to P2-like phages. By Southern blot screening of aVibrio choleraestrain collection, two highly K139-related phage sequences were detected in non-O1, non-O139 strains. Combinatorial PCR analysis revealed almost identical genome organizations. One region of variable gene content was identified and sequenced. Additionally, the tail fiber genes were analyzed, leading to the identification of putative host-specific sequence variations. Furthermore, a K139-encoded Dam methyltransferase was characterized.
Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors fromEscherichia coliandBacillus stearothermophilus
Authors: Ghochikyan Anahit , Karaivanova Iovka Miltcheva , Lecocq Michèle , Vusio Patricia , Arnaud Marie-Claire , Snapyan Marina , Weigel Pierre , Guével Laetitia , Buckle Malcolm , Sakanyan Vehary
Published: November 2002

Bacillus stearothermophilusArgR binds efficiently to theEscherichia coli carABoperator, whereas theE. colirepressor binds very poorly to theargCooperator ofB. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying theE. coliDNA-binding domain and theB. stearothermophilusoligomerization domain showed sequence-nonspecific rather than sequence-specific interactions withargoperators. Chimeras carrying theB. stearothermophilusDNA-binding domain andE. colioligomerization domain exhibited a high DNA-binding affinity for theB. stearothermophilus argCoandE. coli carABoperators and repressed the reporter-gene transcription from theB. stearothermophilusPargCocontrol region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-typeB. stearothermophilusArgR and derivatives of it containing only the exchanged linker fromE. coliArgR or carrying theB. stearothermophilusDNA-binding domain along with the linker and the α4 regions were able to bindargCocontaining the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the α4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.
Regulation of the Hydrogenase-4 Operon of
Escherichia coli
by the σ
54
-Dependent Transcriptional Activators FhlA and HyfR
Authors: Skibinski David A. G. , Golby Paul , Chang Yung-Sheng , Sargent Frank , Hoffman Ralf , Harper R. , Guest John R. , Attwood Margaret M. , Berks Ben C. , Andrews Simon C.
Published: November 2002

The
hyf
locus (
hyfABCDEFGHIJ-hyfR-focB
) of
Escherichia coli
encodes a putative 10-subunit hydrogenase complex (hydrogenase-4 [Hyf]); a potential σ
54
-dependent transcriptional activator, HyfR (related to FhlA); and a putative formate transporter, FocB (related to FocA). In order to gain insight into the physiological role of the Hyf system, we investigated
hyf
expression by using a
hyfA-lacZ
transcriptional fusion. This work revealed that
hyf
is induced under fermentative conditions by formate at a low pH and in an FhlA-dependent fashion. Expression was σ
54
dependent and was inhibited by HycA, the negative transcriptional regulator of the formate regulon. Thus,
hyf
expression resembles that of the
hyc
operon. Primer extension analysis identified a transcriptional start site 30 bp upstream of the
hyfA
structural gene, with appropriately located −24 and −12 boxes indicative of a σ
54
-dependent promoter. No reverse transcriptase PCR product could be detected for
hyfJ-hyfR
, suggesting that
hyfR-focB
may be independently transcribed from the rest of the
hyf
operon. Expression of
hyf
was strongly induced (∼1,000-fold) in the presence of a multicopy plasmid expressing
hyfR
from a heterologous promoter. This induction was dependent on low pH, anaerobiosis, and postexponential growth and was weakly enhanced by formate. The
hyfR-
expressing plasmid increased
fdhF-lacZ
transcription just twofold but did not influence the expression of
hycB-lacZ
. Interestingly, inactivation of the chromosomal
hyfR
gene had no effect on
hyfA-lacZ
expression. Purified HyfR was found to specifically interact with the
hyf
promoter/operator region. Inactivation of the
hyf
operon had no discernible effect on growth under the range of conditions tested. No Hyf-derived hydrogenase or formate dehydrogenase activity could be detected, and no Ni-containing protein corresponding to HyfG was observed.
Differential Expression of the CO
2
Fixation Operons of
Rhodobacter sphaeroides
by the Prr/Reg Two-Component System during Chemoautotrophic Growth
Authors: Gibson Janet L. , Dubbs James M. , Tabita F. Robert
Published: November 2002

In
Rhodobacter sphaeroides
, the two
cbb
operons encoding duplicated Calvin-Benson Bassham (CBB) CO
2
fixation reductive pentose phosphate cycle structural genes are differentially controlled. In attempts to define the molecular basis for the differential regulation, the effects of mutations in genes encoding a subunit of Cbb3 cytochrome oxidase,
ccoP,
and a global response regulator,
prrA
(
regA
), were characterized with respect to CO
2
fixation (
cbb
) gene expression by using translational
lac
fusions to the
R. sphaeroides
cbb

I

and

cbb
II

promoters. Inactivation of the
ccoP
gene resulted in derepression of both promoters during chemoheterotophic growth, where
cbb
expression is normally repressed; expression was also enhanced over normal levels during phototrophic growth. The
prrA
mutation effected reduced expression of

cbb
I

and

cbb
II

promoters during chemoheterotrophic growth, whereas intermediate levels of expression were observed in a double
ccoP prrA
mutant.
PrrA
and
ccoP1 prrA
strains cannot grow phototrophically, so it is impossible to examine
cbb
expression in these backgrounds under this growth mode. In this study, however, we found that PrrA mutants of
R. sphaeroides
were capable of chemoautotrophic growth, allowing, for the first time, an opportunity to directly examine the requirement of PrrA for
cbb
gene expression in vivo under growth conditions where the CBB cycle and CO
2
fixation are required. Expression from the

cbb
II

promoter was severely reduced in the PrrA mutants during chemoautotrophic growth, whereas

cbb
I

expression was either unaffected or enhanced. Mutations in
ccoQ
had no effect on expression from either promoter. These observations suggest that the Prr signal transduction pathway is not always directly linked to Cbb3 cytochrome oxidase activity, at least with respect to
cbb
gene expression. In addition,
lac
fusions containing various lengths of the

cbb
I

promoter demonstrated distinct sequences involved in positive regulation during photoautotrophic versus chemoautotrophic growth, suggesting that different regulatory proteins may be involved. In
Rhodobacter capsulatus
, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) expression was not affected by
cco
mutations during photoheterotrophic growth, suggesting that differences exist in signal transduction pathways regulating
cbb
genes in the related organisms.
Gene Islands Integrated into tRNA
Gly
Genes Confer Genome Diversity on a
Pseudomonas aeruginosa
Clone
Authors: Larbig Karen D. , Christmann Andreas , Johann André , Klockgether Jens , Hartsch Thomas , Merkl Rainer , Wiehlmann Lutz , Fritz Hans-Joachim , Tümmler Burkhard
Published: November 2002

Intraclonal genome diversity of
Pseudomonas aeruginosa
was studied in one of the most diverse mosaic regions of the
P. aeruginosa
chromosome. The ca. 110-kb large hypervariable region located near the
lipH
gene in two members of the predominant
P. aeruginosa
clone C, strain C and strain SG17M, was sequenced. In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs. The gene islands are integrated into conserved tRNA
Gly
genes and have a bipartite structure. The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome
c
biosynthesis, or mercury resistance. The second part is made up mostly of ORFs of yet-unknown function. Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands. We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat.
A
Sinorhizobium meliloti
Lipopolysaccharide Mutant Altered in Cell Surface Sulfation
Authors: Keating David H. , Willits Michael G. , Long Sharon R.
Published: November 2002

The
Rhizobium
-legume symbiosis involves the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange for plant photosynthates. Nodule development requires a bacterial signal referred to as Nod factor, which in
Sinorhizobium meliloti
is a β-(1,4)-linked tetramer of
N
-acetylglucosamine containing
N
-acyl and
O
-acetyl modifications at the nonreducing end and a critical 6-
O
-sulfate at the reducing end. This sulfate modification requires the action of three gene products:
nodH
, which catalyzes the sulfonyl transfer, and
nodPQ
, which produce the activated form of sulfate, 3′-phosphoadenosine-5′-phosphosulfate. It was previously reported that
S. meliloti
cell surface polysaccharides are also covalently modified by sulfate in a reaction dependent on NodPQ. We have further characterized this unique form of bacterial carbohydrate modification. Our studies have determined that one of the
nodPQ
mutant strains used in the initial study of sulfation of cell surface harbored a second unlinked mutation. We cloned the gene affected by this mutation (referred to as
lps-212
) and found it to be an allele of
lpsL
, a gene previously predicted to encode a UDP-glucuronic acid epimerase. We demonstrated that
lpsL
encoded a UDP-glucuronic acid epimerase activity that was reduced in the
lps-212
mutant. The
lps-212
mutation resulted in an altered lipopolysaccharide structure that was reduced in sulfate modification in vitro and in vivo. Finally, we determined that the
lps-212
mutation resulted in a reduced ability to elicit the formation of plant nodules and by altered infection thread structures that aborted prematurely.
Evolution of the C
30
Carotenoid Synthase CrtM for Function in a C
40
Pathway
Authors: Umeno Daisuke , Tobias Alexander V. , Arnold Frances H.
Published: November 2002

The C
30
carotene synthase CrtM from
Staphylococcus aureus
and the C
40
carotene synthase CrtB from
Erwinia uredovora
were swapped into their respective foreign C
40
and C
30
biosynthetic pathways (heterologously expressed in
Escherichia coli
) and evaluated for function. Each displayed negligible ability to synthesize the natural carotenoid product of the other. After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C
40
carotenoids. In contrast, we failed to find a single variant of CrtB with detectable C
30
activity. Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C
40
pathway. These mutants showed significant variation in performance in their original C
30
pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity. We discovered that Phe 26 alone determines the specificity of CrtM. The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures.
Identification by RNA Profiling and Mutational Analysis of the Novel Copper Resistance Determinants CrdA (HP1326), CrdB (HP1327), and CzcB (HP1328) in
Helicobacter pylori
Authors: Waidner Barbara , Melchers Klaus , Ivanov Igor , Loferer Hannes , Bensch Klaus W. , Kist Manfred , Bereswill Stefan
Published: November 2002

Mechanisms involved in maintaining cytoplasmic metal ion homeostasis play a central role in the adaptation of
Helicobacter pylori
to the changing gastric environment. An investigation of the global regulatory responses to copper ions by using RNA profiling with a threshold factor of 4.0 revealed that copper induces transcription of 19
H. pylori
genes and that only the ferritin gene
pfr
is repressed. The 57-fold copper induction identified the HP1326 gene encoding an
H. pylori
-specific protein as a candidate for a novel copper resistance determinant. The HP1326 gene is expressed as a monocistronic unit, and two small HP1326 mRNAs are copper induced. The HP1326 protein is secreted and is required for copper resistance maintained by cytoplasmic copper homeostasis, as
H. pylori
HP1326 mutants were copper sensitive and displayed increased copper induction of HP1326 transcription as well as elevated copper repression of ferritin synthesis. The clear copper-sensitive phenotype displayed by
H. pylori
HP1327 and HP1328 mutants provides strong evidence that the HP1326 protein, together with the signal peptide site of the
H. pylori
-specific protein HP1327, whose gene is located downstream from that encoding HP1326, and the CzcB and CzcA metal efflux system component homologs HP1328 and HP1329, constitutes a novel type of copper efflux pump, as discussed below. The HP1329 gene could not be inactivated, but the 14-fold transcriptional copper induction determined by RNA profiling points towards a function of the encoded CzcA homolog in copper resistance. In summary, results from RNA profiling identified the novel
H. pylori
-specific copper resistance determinants CrdA (HP1326) and CrdB (HP1327), which are required for adaptation to copper-rich environmental conditions.
Isolation of Glucocardiolipins from
Geobacillus stearothermophilus
NRS 2004/3a
Authors: Schäffer Christina , Beckedorf Anke I. , Scheberl Andrea , Zayni Sonja , Peter-Katalinić Jasna , Messner Paul
Published: November 2002

Glucose-substituted cardiolipins account for about 4 mol% of total phospholipid extracted from exponentially grown cells of
Geobacillus stearothermophilus
NRS 2004/3a. Individual glucocardiolipin species exhibited differences in fatty acid substitution, with iso-C
15:0
and anteiso-C
17:0
prevailing. The compounds were purified to homogeneity by a novel protocol and precharacterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Up-Regulated Expression of the

cbb
I

and

cbb
II

Operons during Photoheterotrophic Growth of a Ribulose 1,5-Bisphosphate Carboxylase-Oxygenase Deletion Mutant of
Rhodobacter sphaeroides
Authors: Smith Stephanie A. , Tabita F. Robert
Published: November 2002

In a
Rhodobacter sphaeroides
ribulose 1,5-bisphosphate carboxylase-oxygenase deletion strain that requires an exogenous electron donor for photoheterotrophic growth, transcription of the genes of the Calvin-Benson-Bassham (CBB) cycle was increased. This finding pointed to a potential physiological effector that enhances the capability of the positive transcriptional activator CbbR to mediate
cbb
transcription. This effector is most likely ribulose 1,5-bisphosphate or a metabolite derived from this CBB pathway intermediate.
DNA Microarray Analysis of the Expression Profile of
Escherichia coli
in Response to Treatment with 4,5-Dihydroxy-2-Cyclopenten-1-One
Authors: Phadtare Sangita , Kato Ikunoshin , Inouye Masayori
Published: November 2002

We carried out DNA microarray-based global transcript profiling of
Escherichia coli
in response to 4,5-dihydroxy-2-cyclopenten-1-one to explore the manifestation of its antibacterial activity. We show that it has widespread effects in
E. coli
affecting genes encoding proteins involved in cell metabolism and membrane synthesis and functions. Genes belonging to the regulon involved in synthesis of Cys are upregulated. In addition,
rpoS
and RpoS-regulated genes responding to various stresses and a number of genes responding to oxidative stress are upregulated.
Low-Temperature-Induced Changes in Composition and Fluidity of Lipopolysaccharides in the Antarctic Psychrotrophic BacteriumPseudomonas syringae
Authors: Kumar G. Seshu , Jagannadham M. V. , Ray M. K.
Published: November 2002

The Antarctic psychrotrophic bacteriumPseudomonas syringaewas more sensitive to polymyxin B at a lower (4°C) temperature of growth than at a higher (22°C) temperature. The amount of hydroxy fatty acids in the lipopolysaccharides (LPS) also increased at the lower temperature. These changes correlated with the increase in fluidity of the hydrophobic phase of lipopolysaccharide aggregates in vitro.
In Vivo Imaging of Thrombin Activity in Experimental Thrombi With Thrombin-Sensitive Near-Infrared Molecular Probe
Authors: Jaffer Farouc A. , Tung Ching-Hsuan , Gerszten Robert E. , Weissleder Ralph
Published: November 2002

Objective—

Thrombin, a serine protease, plays an important role in thrombosis as well as other cellular and developmental processes. In this study, we investigated the ability of a novel thrombin-activatable molecular probe to provide in vivo images of thrombin activity in experimental thrombi.

Methods and Results—

The thrombin probe consists of a near-infrared (NIR) fluorochrome attached to a delivery vehicle via a thrombin-specific oligopeptide substrate. In human blood, endogenous thrombin activated the thrombin probe and increased the fluorescence signal by 18-fold (
P
=0.008). Hirudin, a specific thrombin inhibitor, suppressed probe activation by 82% (
P
=0.007). Imaging of in vivo thrombin activity was then investigated in acute experimental murine thrombosis models up to 12 hours. After systemic thrombin probe injection, focal NIR fluorescence signal enhancement was rapidly detected within acute and subacute thrombi. In contrast, no thrombosis signal enhancement was seen in similar experiments with a control NIR fluorochrome.

Conclusions—

Thrombin activity can be imaged in vivo by using a novel thrombin-activatable and thrombin-specific NIR molecular probe. The thrombin probe could enhance the understanding of the role of thrombin in thrombogenesis and other homeostatic and pathological conditions.
C-Reactive Protein Relaxes Human Vessels In Vitro
Authors: Sternik Leonid , Samee Saquib , Schaff Hartzel V. , Zehr Kenton J. , Lerman Lilach O. , Holmes David R. , Herrmann Joerg , Lerman Amir
Published: November 2002

Objective—

C-reactive protein (CRP) is a sensitive marker of inflammation and a prognostic marker in cardiovascular disease. Evidence suggests direct biological activities of CRP within the vascular wall. The study was designed to examine the vasoreactive effects of CRP.

Methods and Results—

Human internal mammary artery rings were obtained during cardiovascular bypass surgery and suspended in an organ bath chamber. The rings were precontracted with endothelin-1, and response to cumulative concentrations of CRP was obtained. Experiments were repeated after initial incubation with 20, 40, and 60 mmol/L KCl, the potassium channel blockers BaCl, tetraethylammonium chloride, and glibenclamide, and the NO synthase inhibitor
N
-monomethyl-
l
-arginine and also after removal of the endothelium. CRP caused dose-dependent relaxation of human internal mammary artery rings, which was not affected by preincubation with
N
-monomethyl-
l
-arginine or removal of the endothelium. Maximum relaxation response to CRP (79.5±10%) was attenuated by KCl (2.5±11.5%,
P
<0.001), BaCl (24.5±7.5%,
P
<0.001), and tetraethylammonium chloride (34.9±8.25%,
P
<0.01) but not by glibenclamide.

Conclusions–

The present study demonstrates that CRP exerts an endothelium-independent vasorelaxing effect via potassium channels. Thus, the study suggests a role of CRP in the regulation of vascular tone.
Detection of Superoxide in Vascular Tissue
Authors: Münzel Thomas , Afanas’ev Igor B. , Kleschyov Andrei L. , Harrison David G.
Published: November 2002

During the past decade, it has become apparent that reactive oxygen species play a critical role in the genesis of many vascular diseases. The superoxide anion is among the most important of these, not only because of its rapid reaction with NO but also because it serves as a progenitor for many other reactive oxygen species. Although there are many approaches to detecting and quantifying superoxide in chemical systems, its detection in intact tissues is more difficult. The validity of the most popular and frequently used assay for this purpose, lucigenin-enhanced chemiluminescence, has been recently questioned. It has been suggested that lucigenin itself, especially at high concentrations (>50 μmol/L), may act as a source for superoxide via redox cycling. Lower lucigenin concentrations (5 μmol/L) do not participate in redox cycling to an important extent in intact tissues and, therefore, provide an accurate assessment of the rate of superoxide production in such samples. Other useful assays for superoxide include those using the fluorescent dye dihydroethidine, 2-methyl-6-phenyl-3,7-dihydroimidazo(1,2-α)pyrazin-3-one (CLA), and 2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl) 8-benzylimidazo[1,2-α]pyrazin-3-one (coelenterazine). The chemiluminescent compound 5-amino-2,3-dihydroxy-1,4-phthalayineidone (luminol) may also be used to detect various reactive oxygen species and may be made specific for various oxidants, such as hydrogen peroxide, superoxide, and peroxynitrite, by altering the experimental conditions. Although each of these methods may be associated with potential artifacts, the use of ≥2 different techniques that yield similar results provides a reliable approach for the study of reactive oxygen species in intact vascular tissues.
Fluid Shear Stress Activates Proline-Rich Tyrosine Kinase via Reactive Oxygen Species–Dependent Pathway
Authors: Tai Lung-Kuo , Okuda Masanori , Abe Jun-ichi , Yan Chen , Berk Bradford C.
Published: November 2002

Objective—

Fluid shear stress (flow) modulates endothelial cell (EC) function via specific signal transduction events. Previously, we showed that flow-mediated tyrosine phosphorylation of p130 Crk-associated substrate (Cas) required calcium-dependent c-Src activation. Because flow increases reactive oxygen species (ROS) production in ECs and because H
2
O
2
increases tyrosine phosphorylation of proline-rich tyrosine kinase (PYK2), we hypothesized that flow may activate PYK2 via ROS.

Methods and Results—

Exposure of bovine aortic ECs to flow stimulated PYK2 phosphorylation rapidly, with a peak at 2 minutes. The activation of PYK2 and phosphorylation of Cas induced by flow were inhibited by pretreatment with the antioxidant
N
-acetylcysteine. Flow-induced PYK2 phosphorylation was inhibited by BAPTA-AM, an intracellular calcium chelator. Bovine aortic ECs transfected with kinase-inactive PYK2 showed attenuated flow-stimulated Cas tyrosine phosphorylation. Although flow-induced Cas phosphorylation was inhibited by kinase-inactive Src, PYK2 activation induced by flow was not inhibited by overexpression of kinase-inactive Src.

Conclusions—

These results show a redox-sensitive pathway for flow-mediated activation of nonreceptor tyrosine kinase activity that requires ROS and intracellular calcium, but not Src kinase.