bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ramachandran plot on the web
Authors: Sheik S.S. , Sundararajan P. , Hussain A.S.Z. , Sekar K. 
Published: November 2002

Abstract
Summary: A graphics package has been developed to display the main chain torsion angles phi, psi (φ, Ψ); (Ramachandran angles) in a protein of known structure. In addition, the package calculates the Ramachandran angles at the central residue in the stretch of three amino acids having specified the flanking residue types. The package displays the Ramachandran angles along with a detailed analysis output. This software is incorporated with all the protein structures available in the Protein Databank.
Availability: This package is available over the world wide web at http://144.16.71.146/rp/ or http://dicsoft1.physics.iisc.ernet.in/rp.
Contact: sekar@physics.iisc.ernet.in
* To whom correspondence should be addressed.
† This work is dedicated to Late Professor G. N. Ramachandran.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACTWzcCPSis 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTEscherichia 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

ABSTRACTHrcA 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.