bluuebunny/crossref_metadata_2025_split huggingface dataset:
Striking Complexity of Lipopolysaccharide Defects in a Collection of
Sinorhizobium meliloti
Mutants
Authors: Campbell Gordon R. O. , Sharypova Larissa A. , Scheidle Heiko , Jones Kathryn M. , Niehaus Karsten , Becker Anke , Walker Graham C. 
Published: June 2003

ABSTRACT

Although the role that lipopolysaccharide (LPS) plays in the symbiosis between
Sinorhizobium meliloti
and alfalfa has been studied for over a decade, its function in this process remains controversial and poorly understood. This is largely due to a lack of mutants affected by its synthesis. In one of the definitive studies concerning this issue, Clover et al. (R. H. Clover, J. Kieber, and E. R. Signer, J. Bacteriol. 171:3961-3967, 1989) identified a series of mutants with putative LPS defects, judged them to be symbiotically proficient on
Medicago sativa
, and concluded that LPS might not have a symbiotic function in
S. meliloti
. The mutations in these strains were never characterized at the molecular level nor was the LPS from most of them analyzed. We have transduced these mutations from the Rm2011 background from which they were originally isolated into the sequenced strain Rm1021 and have characterized the resulting strains in greater detail. We found the LPS from these mutants to display a striking complexity of phenotypes on polyacrylamide electrophoresis gels, including additional rough LPS bands and alterations in the molecular weight distribution of the smooth LPS. We found that some of the mutants contain insertions in genes that are predicted to be involved in the synthesis of carbohydrate components of LPS, including
ddhB
,
lpsB
,
lpsC
, and
lpsE
. The majority, however, code for proteins predicted to be involved in a wide variety of functions not previously recognized to play a role in LPS synthesis, including a possible transcription elongation factor (GreA), a possible queuine synthesis protein, and a possible chemotaxis protein. Furthermore, using more extensive assays, we have found that most of these strains have symbiotic deficiencies. These results support more recent findings that alterations in LPS structure can affect the ability of
S. meliloti
to form an effective symbiosis.
Functional Characterization of Cysteine Residues in GlpT, the Glycerol 3-Phosphate Transporter of
Escherichia coli
Authors: Fann Mon-Chou , Busch Anne , Maloney Peter C. 
Published: June 2003

ABSTRACT

In
Escherichia coli
, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate. Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration ≈ 35 μM) by the hydrophilic thiol-specific agent
p
-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes,
N
-maleimidylpropionylbiocytin (MPB) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), have no effect. Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS. The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution. To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop. In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes. Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway.
Suppressor Mutations in the Study of Photosystem I Biogenesis:
sll0088
Is a Previously Unidentified Gene Involved in Reaction Center Accumulation in
Synechocystis
sp. Strain PCC 6803
Authors: Yu Jianping , Shen Gaozhong , Wang Tao , Bryant Donald A. , Golbeck John H. , McIntosh Lee 
Published: June 2003

ABSTRACT

In previous work, some members of our group isolated mutant strains of
Synechocystis
sp. strain PCC 6803 in which point mutations had been inserted into the
psaC
gene to alter the cysteine residues to the F
A
and F
B
iron-sulfur clusters in the PsaC subunit of photosystem I (J. P. Yu, I. R. Vassiliev, Y. S. Jung, J. H. Golbeck, and L. McIntosh, J. Biol. Chem.
272:
8032-8039, 1997). These mutant strains did not grow photoautotrophically due to suppressed levels of chlorophyll
a
and photosystem I. In the results described here, we show that suppressor mutations produced strains that are capable of photoautotrophic growth at moderate light intensity (20 μmol m
−2
s
−1
). Two separate suppressor strains of C14S
PsaC
, termed C14S
PsaC
-R62 and C14S
PsaC
-R18, were studied and found to have mutations in a previously uncharacterized open reading frame of the
Synechocystis
sp. strain PCC 6803 genome named
sll0088
. C14S
PsaC
-R62 was found to substitute Pro for Arg at residue 161 as the result of a G482→C change in
sll0088
, and C14S
PsaC
-R18 was found to have a three-amino-acid insertion of Gly-Tyr-Phe following Cys231 as the result of a TGGTTATTT duplication at T690 in
sll0088
. These suppressor strains showed near-wild-type levels of chlorophyll
a
and photosystem I, yet the serine oxygen ligand to F
B
was retained as shown by the retention of the
S
≥ 3/2 spin state of the [4Fe-4S] cluster. The inactivation of
sll0088
by insertion of a kanamycin resistance cartridge in the primary C14S
PsaC
mutant produced an engineered suppressor strain capable of photoautotrophic growth. There was no difference in
psaC
gene expression or in the amount of PsaC protein assembled in thylakoids between the wild type and an
sll0088
deletion mutant. The
sll0088
gene encodes a protein predicted to be a transcriptional regulator with sequence similarities to transcription factors in other prokaryotic and eukaryotic organisms, including
Arabidopsis thaliana
. The protein contains a typical helix-turn-helix DNA-binding motif and can be classified as a negative regulator by phylogenetic analysis. This suggests that the product of
sll0088
has a role in regulating the biogenesis of photosystem I.
PAV1, the First Virus-Like Particle Isolated from a Hyperthermophilic Euryarchaeote, “
Pyrococcus abyssi
”
Authors: Geslin C. , Le Romancer M. , Erauso G. , Gaillard M. , Perrot G. , Prieur D. 
Published: June 2003

ABSTRACT

We describe the first virus-like particle of a hyperthermophilic euryarchaeote which was discovered in a strain of “
Pyrococcus abyssi
” previously characterized in our laboratory. This particle, named PAV1, is lemon-shaped (120 nm × 80 nm), with a short tail terminated by fibers, and resembles the virus SSV1, the type member of the
Fuselloviridae
, isolated from
Sulfolobus shibatae
. Sensitivity of the virus-like particle to organic solvents and detergents suggested that the envelope of PAV1 may contain lipids in addition to proteins. It contains a double-stranded circular DNA of 18 kb which is also present in high copy number in a free form in the host cytoplasm. No integrated form of the PAV1 genome could be detected in the host chromosome. Under standard growth conditions, the host cells continuously release PAV1 particles into the culture supernatant without spontaneous lysis, with a maximum reached in the late stationary phase. UV, gamma irradiation, treatment with mitomycin C, and various physiological stresses had no effect on PAV1 production. Screening of a large number of
Thermococcales
isolates did not permit to find a sensitive host. These results suggest that PAV1 persists in the host strain in a stable carrier state rather than a prophage.
Expression of the Nitroarene Dioxygenase Genes inComamonassp. Strain JS765 andAcidovoraxsp. Strain JS42 Is Induced by Multiple Aromatic Compounds
Authors: Lessner Daniel J. , Parales Rebecca E. , Narayan Shakti , Gibson David T. 
Published: June 2003

ABSTRACTThis work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) inComamonassp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) inAcidovoraxsp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes inPseudomonas putidaG7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from alacZreporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42ntdRnull mutant. Complementation with the closely related regulators NagR (fromRalstoniasp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes inPseudomonas putidaG7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.
Expression of
spoIIIJ
in the Prespore Is Sufficient for Activation of σ
G
and for Sporulation in
Bacillus subtilis
Authors: Serrano Mónica , Côrte Luísa , Opdyke Jason , Moran, Charles P. , Henriques Adriano O. 
Published: June 2003

ABSTRACT

During sporulation in
Bacillus subtilis
, the prespore-specific developmental program is initiated soon after asymmetric division of the sporangium by the compartment-specific activation of RNA polymerase sigma factor σ
F
. σ
F
directs transcription of
spoIIIG
, encoding the late forespore-specific regulator σ
G
. Following synthesis, σ
G
is initially kept in an inactive form, presumably because it is bound to the SpoIIAB anti-sigma factor. Activation of σ
G
occurs only after the complete engulfment of the prespore by the mother cell. Mutations in
spoIIIJ
arrest sporulation soon after conclusion of the engulfment process and prevent activation of σ
G
. Here we show that σ
G
accumulates but is mostly inactive in a
spoIIIJ
mutant. We also show that expression of the
spoIIIGE155K
allele, encoding a form of σ
G
that is not efficiently bound by SpoIIAB in vitro, restores σ
G
-directed gene expression to a
spoIIIJ
mutant. Expression of
spoIIIJ
occurs during vegetative growth. However, we show that expression of
spoIIIJ
in the prespore is sufficient for σ
G
activation and for sporulation. Mutations in the mother cell-specific
spoIIIA
locus are known to arrest sporulation just after completion of the engulfment process. Previous work has also shown that σ
G
accumulates in an inactive form in
spoIIIA
mutants and that the need for
spoIIIA
expression for σ
G
activation can be circumvented by the
spoIIIGE155K
allele. However, in contrast to the case for
spoIIIJ
, we show that expression of
spoIIIA
in the prespore does not support efficient sporulation. The results suggest that the activation of σ
G
at the end of the engulfment process involves the action of
spoIIIA
from the mother cell and of
spoIIIJ
from the prespore.
Archaeal Homolog of Bacterial Type IV Prepilin Signal Peptidases with Broad Substrate Specificity
Authors: Albers Sonja-Verena , Szabó Zalán , Driessen Arnold J. M. 
Published: June 2003

ABSTRACT

A large number of secretory proteins in the thermoacidophile
Sulfolobus solfataricus
are synthesized as a precursor with an unusual leader peptide that resembles bacterial type IV prepilin signal sequences. This set of proteins includes the flagellin subunit but also various solute binding proteins. Here we describe the identification of the
S. solfataricus
homolog of bacterial type IV prepilin peptidases, termed PibD. PibD is an integral membrane protein that is phylogenetically related to the bacterial enzymes. When heterologously expressed in
Escherichia coli
, PibD is capable of processing both the flagellin and glucose-binding protein (GlcS) precursors. Site-directed mutagenesis of the GlcS signal peptide shows that the substrate specificity of PibD is consistent with the variations found in proteins with type IV prepilin-like signal sequences of
S. solfataricus
. We conclude that PibD is responsible for the processing of these secretory proteins in
S. solfataricus
.
Whole-Genome DNA Microarray Analysis of a Hyperthermophile and an Archaeon:
Pyrococcus furiosus
Grown on Carbohydrates or Peptides
Authors: Schut Gerrit J. , Brehm Scott D. , Datta Susmita , Adams Michael W. W. 
Published: June 2003

ABSTRACT

The first complete-genome DNA microarray was constructed for a hyperthermophile or a nonhalophilic archaeon by using the 2,065 open reading frames (ORFs) that have been annotated in the genome of
Pyrococcus furiosus
(optimal growth temperature, 100°C). This was used to determine relative transcript levels in cells grown at 95°C with either peptides or a carbohydrate (maltose) used as the primary carbon source. Approximately 20% (398 of 2065) of the ORFs did not appear to be significantly expressed under either growth condition. Of the remaining 1,667 ORFs, the expression of 125 of them (8%) differed by more than fivefold between the two cultures, and 82 of the 125 (65%) appear to be part of operons, indicating extensive coordinate regulation. Of the 27 operons that are regulated, 5 of them encode (conserved) hypothetical proteins. A total of 18 operons are up-regulated (greater than fivefold) in maltose-grown cells, including those responsible for maltose transport and for the biosynthesis of 12 amino acids, of ornithine, and of citric acid cycle intermediate products. A total of nine operons are up-regulated (greater than fivefold) in peptide-grown cells, including those encoding enzymes involved in the production of acyl and aryl acids and 2-ketoacids, which are used for energy conservation. Analyses of the spent growth media confirmed the production of branched-chain and aromatic acids during growth on peptides. In addition, six nonlinked enzymes in the pathways of sugar metabolism were regulated more than fivefold—three in maltose-grown cells that are unique to the unusual glycolytic pathway and three in peptide-grown cells that are unique to gluconeogenesis. The catalytic activities of 16 metabolic enzymes whose expression appeared to be highly regulated in the two cell types correlated very well with the microarray data. The degree of coordinate regulation revealed by the microarray data was unanticipated and shows that
P. furiosus
can readily adapt to a change in its primary carbon source.
Characterization and Functional Complementation of a Nonlethal Deletion in the Chromosome of a β-Glycosidase Mutant of
Sulfolobus solfataricus
Authors: Bartolucci Simonetta , Rossi Mosè , Cannio Raffaele 
Published: June 2003

ABSTRACT

LacS
−
mutants of
Sulfolobus solfataricus
defective in β-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the β-glycosidase gene (
lacS
). Fine mapping performed in comparison to the genomic sequence of
S. solfataricus
P2 indicated an extended deletion of ∼13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC
1058
. In order to complement the LacS
−
phenotype, an expression vector was constructed by inserting the
lacS
coding sequence with its 5′ and 3′ flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the
lacTr
(lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the β-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-β-
d
-galactopyranoside).
Physiological Responses of the Hyperthermophilic Archaeon “
Pyrococcus abyssi
” to DNA Damage Caused by Ionizing Radiation
Authors: Jolivet Edmond , Matsunaga Fujihiko , Ishino Yoshizumi , Forterre Patrick , Prieur Daniel , Myllykallio Hannu 
Published: June 2003

ABSTRACT

The mechanisms by which hyperthermophilic
Archaea
, such as “
Pyrococcus abyssi
” and
Pyrococcus furiosus
, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of “
P. abyssi
” chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated “
P. abyssi
” cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated “
P. abyssi
” cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that “
P. abyssi
” contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.
Purification and Characterization of a Monooxygenase Involved in the Biosynthetic Pathway of the Antitumor Drug Mithramycin
Authors: Rodríguez David , Quirós Luis M. , Braña Alfredo F. , Salas José A. 
Published: June 2003

ABSTRACT

A monooxygenase encoded by the
mtmOIV
gene from the mithramycin gene cluster of
Streptomyces argillaceus
was purified 21-fold by a three-step purification procedure. This monooxygenase catalyzes the oxidative cleavage of the fourth ring of premithramycin B. The enzyme was dependent on NADPH and flavin adenine dinucleotide for activity with optimal pH at 9.5, and the

K
m

values for NADPH and premithramycin B were 269.22 and 23.35 μM, respectively. The reaction catalyzed by MtmOIV yields two possible isomers of the same basic shortened aliphatic chain molecule. One of the reaction products showed important biological activity, thus highlighting the importance of the cleavage of the fourth ring of the aglycon for biological activity.
Genome Analysis of a Novel Shiga Toxin 1 (Stx1)-Converting Phage Which Is Closely Related to Stx2-Converting Phages but Not to Other Stx1-Converting Phages
Authors: Sato Toshio , Shimizu Takeshi , Watarai Masahisa , Kobayashi Midori , Kano Shigeyuki , Hamabata Takashi , Takeda Yoshifumi , Yamasaki Shinji 
Published: June 2003

ABSTRACT

Two Stx-converting phages, designated Stx1φ and Stx2φ-II, were isolated from an
Escherichia coli
O157:H7 strain, Morioka V526, and their entire nucleotide sequences were determined. The genomes of both phages were similar except for the
stx
gene-flanking regions. Comparing these phages to other known Stx-converting phages, we concluded that Stx1φ is a novel Stx1-converting phage closely related to Stx2-converting phages so far reported.
The
dinB
Operon and Spontaneous Mutation in
Escherichia
coli
Authors: McKenzie Gregory J. , Magner Daniel B. , Lee Peter L. , Rosenberg Susan M. 
Published: June 2003

ABSTRACT

Apparently conflicting data regarding the role of SOS-inducible, error-prone DNA polymerase IV (DinB) in spontaneous mutation are resolved by the finding that mutation is reduced by a polar allele with which
dinB
and neighboring
yafN
are deleted but not by two nonpolar
dinB
alleles. We demonstrate the existence of a
dinB
operon that contains four genes,
dinB
-
yafN
-
yafO
-
yafP
. The results imply a role for
yafN
,
yafO
, and/or
yafP
in spontaneous mutation.
The ATPase FliI Can Interact with the Type III Flagellar Protein Export Apparatus in the Absence of Its Regulator, FliH
Authors: Minamino Tohru , González-Pedrajo Bertha , Kihara May , Namba Keiichi , Macnab Robert M. 
Published: June 2003

ABSTRACT

Salmonella
FliI is the ATPase that drives flagellar protein export. It normally exists as a complex together with the regulatory protein FliH. A
fliH
null mutant was slightly motile, with overproduction of FliI resulting in substantial improvement of its motility. Mutations in the cytoplasmic domains of FlhA and FlhB, which are integral membrane components of the type III flagellar export apparatus, also resulted in substantially improved motility, even at normal FliI levels. Thus, FliH, though undoubtedly important, is not essential.
Review of evaluations of educational approaches to promote safe storage of firearms: Table 1
Authors: McGee K S , Coyne-Beasley T , Johnson R M 
Published: June 2003

Objective:To systematically review evaluation studies of educational interventions promoting safe firearm storage.Methods:Medline, ERIC, PsycINFO, Criminal Justice Periodicals Index, Cumulative Index of Nursing and Allied Health Literature, and Sociofile were searched. The references from each potentially eligible study were checked, and experts in the field were contacted for additional reports. In addition, an internet search was performed to identify programs not published in the conventional literature. Sources relevant to safe firearm storage promotion were selected and evaluated.Results:Seven studies met inclusion criteria: adult subjects, program description was included, and firearm storage outcomes were measured. One was a randomized controlled trial and the other six were one group pre-test and/or post-test. The studies were classified into the following categories based on the intervention strategies used: (1) counseling and firearm safety materials (n=3); (2) counseling/educational message (n=3); and (3) firearm safety materials distribution (n=1).The outcomes were safe firearms storage (firearms locked up and unloaded or removal from home) after intervention. Four studies, three using counseling and materials distribution, reported improved storage after the interventions.Conclusions:It is not yet clear what types of interventions, or which specific intervention components, prompt gun owners to securely store their weapons. Increased understanding of gun storage behaviors and stronger evaluation designs will aid further understanding of this important issue.