bluuebunny/crossref_metadata_2025_split huggingface dataset:
Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B12and Factor A
Authors: Hoffmann Bernd , Oberhuber Michael , Stupperich Erhard , Bothe Harald , Buckel Wolfgang , Konrat Robert , Kräutler Bernhard 
Published: July 2002

ABSTRACTThe corrinoids from the obligate anaerobeClostridium cochleariumwere extracted as a mixture of Coβ-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 μmol) of B12derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample ofC. cochleariumby a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B12(Coβ-cyano-7"-adeninylcobamide) (60%) and factor A (Coβ-cyano-7"-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B12was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B12and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional1H,13C-, and15N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-[N6-methyl]-adeninylcobamide, a previously unknown corrinoid.C. cochleariumthus biosynthesizes as its native “complete” B12cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.
Inactivation of the
ampDE
Operon Increases Transcription of
algD
and Affects Morphology and Encystment of
Azotobacter vinelandii
Authors: Núñez Cinthia , Moreno Soledad , Cárdenas Luis , Soberón-Chávez Gloria , Espín Guadalupe 
Published: July 2002

ABSTRACT

Transcription of
algD
, encoding GDP-mannose dehydrogenase, the key enzyme in the alginate biosynthetic pathway, is highly regulated in
Azotobacter vinelandii
. We describe here the characterization of a Tn
5
insertion mutant (AC28) which shows a higher level of expression of an
algD
::
lacZ
fusion. AC28 cells were morphologically abnormal and unable to encyst. The cloning and nucleotide sequencing of the Tn
5
-disrupted locus in AC28 revealed an operon homologous to the
Escherichia coli ampDE
operon. Tn
5
was located within the
ampD
gene, encoding a cytosolic
N
-acetyl-anhydromuramyl-
l
-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments. The
ampE
gene encodes a transmembrane protein, but the function of the protein is not known. We constructed strains carrying
ampD
or
ampE
mutations and one with an
ampDE
deletion. The strain with a deletion of the
ampDE
operon showed a phenotype similar to that of mutant AC28. The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.
Glycine Betaine Transport in the Obligate Halophilic Archaeon
Methanohalophilus portucalensis
Authors: Lai Mei-Chin , Hong Tong-Yung , Gunsalus Robert P. 
Published: July 2002

ABSTRACT

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe
Methanohalophilus portucalensis
(strain FDF1), since solute uptake for this class of obligate halophilic methanogenic
Archaea
has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed

K
t

of 23 μM and a
V
max
of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [
14
C]betaine uptake. The proton-conducting uncoupler 2,4-dinitrophenol and the ATPase inhibitor
N
,
N
-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 μM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, α-glutamate, β-glutamine, and
N
ɛ
-acetyl-β-lysine. These investigations demonstrate that this model halophilic methanogen,
M. portucalensis
strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.
Early Cephamycin Biosynthetic Genes Are Expressed from a Polycistronic Transcript in
Streptomyces clavuligerus
Authors: Alexander Dylan C. , Brumlik Michael J. , Lee Linda , Jensen Susan E. 
Published: July 2002

ABSTRACT

A polycistronic transcript that is initiated at the
lat
promoter has been implicated in the expression of the genes involved in early steps of cephamycin C biosynthesis in
Streptomyces clavuligerus. pcbC
is also expressed as a monocistronic transcript from its own promoter. However, an alternative interpretation involving expression via three separate yet interdependent transcripts has also been proposed. To distinguish between these possibilities, mutants lacking the
lat
promoter and containing a transcription terminator within the
lat
gene (Δ
lat
::
tsr
/term mutants) were created. This mutation eliminated the production of lysine-ɛ-aminotransferase (the
lat
gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of δ-(
l
-α-aminoadipyl)-
l
-cysteinyl-
d
-valine synthetase (ACVS) (the
pcbAB
gene product) was eliminated in Δ
lat
::
tsr
/term mutants, while production of isopenicillin N synthase (IPNS) (the
pcbC
gene product) was greatly reduced. The provision of α-aminoadipate to the Δ
lat
::
tsr
/term mutants, either via exogenous feeding or via
lat
gene complementation, did not restore production of ACVS or IPNS. Analysis of RNA isolated from the Δ
lat
::
tsr
/term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic
pcbC
transcript levels were greatly decreased. In contrast, Δ
lat
mutants created by in-frame internal deletion of
lat
maintained the polycistronic transcript and allowed production of wild-type levels of both ACVS and IPNS.
Transcriptional Control of the Sulfur-Regulated
cysH
Operon, Containing Genes Involved in
l
-Cysteine Biosynthesis in
Bacillus subtilis
Authors: Mansilla Maria Cecilia , Albanesi Daniela , de Mendoza Diego 
Published: July 2002

ABSTRACT

The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in
Bacillus subtilis
and other gram-positive bacteria. In this study we describe the expression pattern of the
B. subtilis cysH
operon in response to sulfur starvation. A 6.1-kb polycistronic transcript which includes the
cysH
,
cysP
,
ylnB
,
ylnC
,
ylnD
,
ylnE
, and
ylnF
genes was identified. Its synthesis was induced by sulfur limitation and strongly repressed by cysteine. The
cysH
operon contains a 5′ leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system. Here we show that induction of
B. subtilis cysH
operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription. Deletion of a 46-bp region adjacent to the −35 region of the
cysH
promoter led to high-level expression of the operon, even in the presence of cysteine. We also found that
O
-acetyl-
l
-serine (OAS), a direct precursor of cysteine, renders
cysH
transcription independent of sulfur starvation and insensitive to cysteine repression. We propose that transcription of the
cysH
operon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS. Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of
cysH
expression. These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.
Association of the Cytoplasmic Membrane Protein XpsN with the Outer Membrane Protein XpsD in the Type II Protein Secretion Apparatus of
Xanthomonas campestris
pv. Campestris
Authors: Lee Hsien-Ming , Wang Kuan-Cheng , Liu Yi-Ling , Yew Hsin-Yan , Chen Ling-Yun , Leu Wei-Ming , Chen David Chanhen , Hu Nien-Tai 
Published: July 2002

ABSTRACT

An
xps
gene cluster composed of 11 open reading frames is required for the type II protein secretion in
Xanthomonas campestris
pv. campestris. Immediately upstream of the
xpsD
gene, which encodes an outer membrane protein that serves as the secretion channel by forming multimers, there exists an open reading frame (previously designated ORF2) that could encode a protein of 261 amino acid residues. Its N-terminal hydrophobic region is a likely membrane-anchoring sequence. Antibody raised against this protein could detect in the wild-type strain of
X. campestris
pv. campestris a protein band with an apparent molecular mass of 36 kDa by Western blotting. Its aberrant slow migration in sodium dodecyl sulfate-polyacrylamide gels might be due to its high proline content. We designated this protein XpsN. By constructing a mutant strain with an in-frame deletion of the chromosomal
xpsN
gene, we demonstrated that it is required for the secretion of extracellular enzyme by
X. campestris
pv. campestris. Subcellular fractionation studies indicated that the XpsN protein was tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis revealed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated that the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)
6
-tagged XpsD protein from the metal affinity chromatography column. All observations suggested that the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with various truncated XpsD proteins revealed that the C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved in complex formation between the two.
A 90-Kilobase Conjugative Chromosomal Element Coding for Biphenyl and Salicylate Catabolism in
Pseudomonas putida
KF715
Authors: Nishi Akito , Tominaga Kiyomi , Furukawa Kensuke 
Published: July 2002

ABSTRACT

The biphenyl and salicylate metabolic pathways in
Pseudomonas putida
KF715 are chromosomally encoded. The
bph
gene cluster coding for the conversion of biphenyl to benzoic acid and the
sal
gene cluster coding for the salicylate
meta
-pathway were obtained from the KF715 genomic cosmid libraries. These two gene clusters were separated by 10-kb DNA and were highly prone to deletion when KF715 was grown in nutrient medium. Two types of deletions took place at the region including only the
bph
genes (ca. 40 kb) or at the region including both the
bph
and
sal
genes (ca. 70 kb). A 90-kb DNA region, including both the
bph
and
sal
genes (termed the
bph-sal
element), was transferred by conjugation from KF715 to
P. putida
AC30. Such transconjugants gained the ability to grow on biphenyl and salicylate as the sole sources of carbon. The
bph
and
sal
element was located on the chromosome of the recipient. The
bph-sal
element in strain AC30 was also highly prone to deletion; however, it could be mobilized to the chromosome of
P. putida
KT2440 and the two deletion mutants of KF715.
Type IV Pilus Genes
pilA
and
pilC
of
Pseudomonas stutzeri
Are Required for Natural Genetic Transformation, and
pilA
Can Be Replaced by Corresponding Genes from Nontransformable Species
Authors: Graupner Stefan , Frey Verena , Hashemi Rozita , Lorenz Michael G. , Brandes Gudrun , Wackernagel Wilfried 
Published: July 2002

ABSTRACT

Pseudomonas stutzeri
lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a
P. stutzeri
JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of
Pseudomonas aeruginosa
, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The
pilC
mutant had no pili and was defective in twitching motility. Further sequencing revealed that
pilC
is clustered in an operon with genes homologous to
pilB
and
pilD
of
P. aeruginosa
, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a
pilA
gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of
pilA
abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of
3
H-labeled
P. stutzeri
DNA that were bound to competent parental cells and taken up were strongly reduced in the
pilC
and
pilA
mutants. Remarkably, the cloned
pilA
genes from nontransformable organisms like
Dichelobacter nodosus
and the PAK and PAO strains of
P. aeruginosa
fully restored pilus formation and transformability of the
P. stutzeri pilA
mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium
P. stutzeri
function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.
Negative Regulation of Expression of the
Bacillus megaterium bmlP1
Gene by the
bmlP1
3′ Flanking Region
Authors: Shaw Gwo-Chyuan , Hsueh Yi-Huang , Sung Chi-Chang , Chen Yuan-Shou , Liu Chang-Hsiesh 
Published: July 2002

ABSTRACT

We report that the expression of the
Bacillus megaterium bmlP1
gene is subject to negative regulation by the
bmlP1
3′ flanking region. This repression occurred both in
B. megaterium
and in
Escherichia coli
. When the
bmlP1
promoter was replaced with a heterologous promoter or when the orientation of the
bmlP1
3′ flanking region was reversed, the inhibitory effect was still observed. However, the
bmlP1
3′ flanking region was unable to exert repression on a heterologous gene when fused downstream in either orientation, and it was incapable of acting in
trans
. Dot blot and Northern blot analyses revealed that the repression occurred at the RNA level. Deletion analysis showed that the regulatory site responsible for the repression is located within a 116-bp region immediately following the
bmlP1
gene. Possible mechanisms for this repression are discussed.
pepA
, a Gene Mediating pH Regulation of Virulence Genes in
Vibrio cholerae
Authors: Behari Jaideep , Stagon Lisa , Calderwood Stephen B. 
Published: July 2002

ABSTRACT

ToxT, a member of the AraC family of transcriptional regulators, controls the expression of several virulence factors in
Vibrio cholerae
. In the classical biotype of
V. cholerae
, expression of
toxT
is regulated by the same environmental conditions that control expression of the virulence determinants cholera toxin and the toxin coregulated pilus. Several genes that activate
toxT
expression have been identified. To identify genes that repress
toxT
expression in nonpermissive environmental conditions, a genetic screen was used to isolate mutations which alter the expression of a
toxT-gusA
transcriptional fusion. Several mutants were isolated, and the mutants could be divided into two classes. One class of mutants exhibited higher expression levels of
toxT-gusA
at both the nonpermissive pH and temperature, while the second class showed elevated
toxT-gusA
expression only at the nonpermissive pH. One mutant from the second class was chosen for further characterization. This mutant was found to carry a Tn
phoA
insertion in a homolog of the
Escherichia coli pepA
gene. Disruption of
pepA
in
V. cholerae
resulted in elevated levels of expression of cholera toxin,
tcpA
,
toxT
, and
tcpP
at the noninducing pH but not at the noninducing temperature. Elevated levels of expression of
toxT
and
tcpP
at the nonpermissive pH in the
pepA
mutant were abolished in
tcpP toxR
mutant and
aphB
mutant backgrounds, respectively. A putative binding site for PepA was identified in the
tcpPH-tcpI
intergenic region, suggesting that PepA may act at the level of
tcpPH
transcription. Disruption of
pepA
caused only partial deregulation at the noninducing pH, suggesting the involvement of additional factors in the pH regulation of virulence genes in
V. cholerae
.
Amino Acid Residues in LuxR Critical for Its Mechanism of Transcriptional Activation during Quorum Sensing in
Vibrio fischeri
Authors: Trott Amy E. , Stevens Ann M. 
Published: July 2002

ABSTRACT

PCR-based site-directed mutagenesis has been used to generate 38 alanine-substitution mutations in the C-terminal 41 amino acid residues of LuxR. This region plays a critical role in the mechanism of LuxR-dependent transcriptional activation of the
Vibrio fischeri lux
operon during quorum sensing. The ability of the variant forms of LuxR to activate transcription of the
lux
operon was examined by using in vivo assays in recombinant
Escherichia coli
. Eight recombinant strains produced luciferase at levels less than 50% of that of a strain expressing wild-type LuxR. Western immunoblotting analysis verified that the altered forms of LuxR were expressed at levels equivalent to those of the wild type. An in vivo DNA binding-repression assay in recombinant
E. coli
was subsequently used to measure the ability of the variant forms of LuxR to bind to the
lux
box, the binding site of LuxR at the
lux
operon promoter. All eight LuxR variants found to affect cellular luciferase levels were unable to bind to the
lux
box. An additional 11 constructs that had no effect on cellular luciferase levels were also found to exhibit a defect in DNA binding. None of the alanine substitutions in LuxR affected activation of transcription of the
lux
operon without also affecting DNA binding. These results support the conclusion that the C-terminal 41 amino acids of LuxR are important for DNA recognition and binding of the
lux
box rather than positive control of the process of transcription initiation.
Genes Involved in Control of Galactose Uptake in
Lactobacillus brevis
and Reconstitution of the Regulatory System in
Bacillus subtilis
Authors: Djordjevic Gordana M. , Tchieu Jason H. , Saier Milton H. 
Published: July 2002

ABSTRACT

The heterofermentative lactic acid bacterium
Lactobacillus brevis
transports galactose and the nonmetabolizable galactose analogue thiomethyl-β-galactoside (TMG) by a permease-catalyzed sugar:H
+
symport mechanism. Addition of glucose to
L. brevis
cells loaded with [
14
C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H
+
symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in
L. brevis
, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H
+
symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned
galP
gene was integrated into the chromosome of
Bacillus subtilis
, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in
L. brevis
operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H
+
symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.
Microbial Origin of Plant-Type 2-Keto-3-Deoxy-
d
-
arabino
-Heptulosonate 7-Phosphate Synthases, Exemplified by the Chorismate- and Tryptophan-Regulated Enzyme from
Xanthomonas campestris
Authors: Gosset Guillermo , Bonner Carol A. , Jensen Roy A. 
Published: July 2002

ABSTRACT

Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-
d
-
arabino
-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic
Escherichia coli
paralogs are AroA
I
proteins, but many members of the
Bacteria
possess the AroA
II
class of enzyme, sometimes in combination with AroA
I
proteins. AroA
II
DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the
Xanthomonas campestris
AroA
II
protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis.
X. campestris
AroA
II
was cloned in
E. coli
by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroA
II
protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector.
l
-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA
II
of higher plants,
X. campestris
AroA
II
was not hysteretically activated by dithiols. Compared to plant AroA
II
proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA
II
originated within the
Bacteria
domain, and it seems probable that higher-plant plastids acquired AroA
II
from a gram-negative bacterium via endosymbiosis. The
X. campestris
AroA
II
protein is suggested to exemplify a case of analog displacement whereby an ancestral
aroA
I
species was discarded, with the
aroA
II
replacement providing an alternative pattern of allosteric control. Three subgroups of AroA
II
proteins can be recognized: a large, central group containing the plant enzymes and that from
X. campestris
, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.