bluuebunny/crossref_metadata_2025_split huggingface dataset:
pING Family of Conjugative Plasmids from the Extremely Thermophilic Archaeon
Sulfolobus islandicus
: Insights into Recombination and Conjugation in Crenarchaeota
Authors: Stedman Kenneth M. , She Qunxin , Phan Hien , Holz Ingelore , Singh Harpreet , Prangishvili David , Garrett Roger , Zillig Wolfram 
Published: July 2002

ABSTRACT

A novel family of conjugative plasmids from
Sulfolobus
comprising the active variants pING1, -4, and -6 and the functionally defective variants pING2 and -3, which require the help of an active variant for spreading, has been extensively characterized both functionally and molecularly. In view of the sparse similarity between bacterial and archaeal conjugation and the lack of a practical genetic system for
Sulfolobus
, we compared the functions and sequences of these variants and the previously described archaeal conjugative plasmid pNOB8 in order to identify open reading frames (ORFs) and DNA sequences that are involved in conjugative transfer and maintenance of these plasmids in
Sulfolobus
. The variants pING4 and -6 are reproducibly derived from pING1 in vivo by successive transpositions of an element from the
Sulfolobus
genome. The small defective but mobile variants pING2 and -3, which both lack a cluster of highly conserved ORFs probably involved in plasmid transfer, were shown to be formed in vivo by recombinative deletion of the larger part of the genomes of pING4 and pING6, respectively. The efficient occurrence of these recombination processes is further evidence for the striking plasticity of the
Sulfolobus
genome.
Purification and Characterization of a Catalase from the Facultatively Psychrophilic Bacterium
Vibrio rumoiensis
S-1
T
Exhibiting High Catalase Activity
Authors: Yumoto Isao , Ichihashi Daisen , Iwata Hideaki , Istokovics Anita , Ichise Nobutoshi , Matsuyama Hidetoshi , Okuyama Hidetoshi , Kawasaki Kosei 
Published: July 2002

ABSTRACT

Catalase from the facultatively psychrophilic bacterium
Vibrio rumoiensis
S-1
T
, which was isolated from an environment exposed to H
2
O
2
and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U · mg of protein
−1
under standard reaction conditions at 40°C, 1.5 and 4.3 times faster, respectively, than those of the
Micrococcus luteus
and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1
T
is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1
T
catalase and
M. luteus
and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1
T
should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1
T
is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.
Role of Trehalose in Growth at High Temperature of
Salmonella enterica
Serovar Typhimurium
Authors: Cánovas David , Fletcher Susanne A. , Hayashi Mikachi , Csonka Laszlo N. 
Published: July 2002

ABSTRACT

Moderate osmolality can stimulate bacterial growth at temperatures near the upper limit for growth. We investigated the mechanism by which high osmolality enhances the thermotolerance of
Salmonella enterica
serovar Typhimurium, by isolating bacteriophage MudI1734-induced insertion mutations that blocked the growth-stimulatory effect of 0.2 M NaCl at 45°C. One of these mutations proved to be in the
seqA
gene (a regulator of initiation of DNA synthesis). Because this gene is cotranscribed with
pgm
(which encodes phosphoglucomutase)
,
it is likely to be polar on the expression of the
pgm
gene. Pgm catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate during growth on glucose, and therefore loss of Pgm results in a deficiency in a variety of cellular constituents derived from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the
seqA
::MudI1734 mutant at high temperature in medium of high osmolality is due to the block in trehalose synthesis, we determined the effect of an
otsA
mutation, which inactivates the first step of the trehalose biosynthetic pathway. The
otsA
mutation caused a growth defect at 45°C in minimal medium containing 0.2 M NaCl that was similar to that caused by the
pgm
mutation, but
otsA
did not affect growth rate in this medium at 37°C. These results suggest that the growth defect of the
seqA
-
pgm
mutant at high temperature could be a consequence of the block in trehalose synthesis. We found that, in addition to the well-known osmotic control, there is a temperature-dependent control of trehalose synthesis such that, in medium containing 0.2 M NaCl, cells grown at 45°C had a fivefold higher trehalose pool size than cells grown at 30°C. Our observations that trehalose accumulation is thermoregulated and that mutations that block trehalose synthesis cause a growth defect at high temperature in media of high osmolality suggested that this disaccharide is crucial for growth at high temperature either for turgor maintenance or for protein stabilization.
The
Bordetella bhu
Locus Is Required for Heme Iron Utilization
Authors: Vanderpool Carin K. , Armstrong Sandra K. 
Published: July 2002

ABSTRACT

Bordetella pertussis
and
Bordetella bronchiseptica
are capable of obtaining iron from hemin and hemoglobin. Genes encoding a putative bacterial heme iron acquisition system (
bhu
, for
Bordetella
heme utilization) were identified in a
B. pertussis
genomic sequence database, and the corresponding DNA was isolated from a virulent strain of
B. pertussis
. A
B. pertussis bhuR
mutant, predicted to lack the heme outer membrane receptor, was generated by allelic exchange. In contrast to the wild-type strain,
bhuR
mutant PM5 was incapable of acquiring iron from hemin and hemoglobin; genetic complementation of PM5 with the cloned
bhuRSTUV
genes restored heme utilization to wild-type levels. In parallel studies,
B. bronchiseptica bhu
sequences were also identified and a
B. bronchiseptica bhuR
mutant was constructed and confirmed to be defective in heme iron acquisition. The wild-type
B. bronchiseptica
parent strain grown under low-iron conditions produced the presumptive BhuR protein, which was absent in the
bhuR
mutant. Furthermore, production of BhuR by iron-starved
B. bronchiseptica
was markedly enhanced by culture in hemin-supplemented medium, suggesting that these organisms sense and respond to heme in the environment. Analysis of the genetic region upstream of the
bhu
cluster identified open reading frames predicted to encode homologs of the
Escherichia coli
ferric citrate uptake regulators FecI and FecR. These putative
Bordetella
regulators may mediate heme-responsive positive transcriptional control of the
bhu
genes.
Multiple Domains Are Required for the Toxic Activity of
Pseudomonas aeruginosa
ExoU
Authors: Finck-Barbançon Viviane , Frank Dara W. 
Published: July 2002

ABSTRACT

Expression of ExoU by
Pseudomonas aeruginosa
is correlated with acute cytotoxicity in a number of epithelial and macrophage cell lines. In vivo, ExoU is responsible for epithelial injury. The absence of a known motif or significant homology with other proteins suggests that ExoU may possess a new mechanism of toxicity. To study the intracellular effects of ExoU, we developed a transient-transfection system in Chinese hamster ovary cells. Transfection with full-length but not truncated forms of ExoU inhibited reporter gene expression. Inhibition of reporter activity after cotransfection with ExoU-encoding constructs was correlated with cellular permeability and death. The toxicity of truncated versions of ExoU could be restored by coexpression of the remainder of the molecule from separate plasmids in
trans
. This strategy was used to map N- and C-terminal regions of ExoU that are necessary but not sufficient for toxicity. Disruption of a middle region of the protein reduces toxicity. This portion of the molecule is postulated to allow the N- and C-terminal regions to functionally complement one another. In contrast to ExoS and ExoT, native and recombinant ExoU molecules do not oligomerize or form aggregates. The complex domain structure of ExoU suggests that, like other
P. aeruginosa
-encoded type III effectors (ExoS and ExoT), ExoU toxicity may result from a molecule that possesses more than one activity.
Evolution of an Autotransporter: Domain Shuffling and Lateral Transfer from Pathogenic
Haemophilus
to
Neisseria
Authors: Davis Jeamelia , Smith Arnold L. , Hughes William R. , Golomb Miriam 
Published: July 2002

ABSTRACT

The genomes of pathogenic
Haemophilus influenzae
strains are larger than that of Rd KW20 (Rd), the nonpathogenic laboratory strain whose genome has been sequenced. To identify potential virulence genes, we examined genes possessed by Int1, an invasive nonencapsulated isolate from a meningitis patient, but absent from Rd. Int1 was found to have a novel gene termed
lav
, predicted to encode a member of the AIDA-I/VirG/PerT family of virulence-associated autotransporters (ATs). Associated with
lav
are multiple repeats of the tetranucleotide GCAA, implicated in translational phase variation of surface molecules. Laterally acquired by
H. influenzae, lav
is restricted in distribution to a few pathogenic strains, including
H. influenzae
biotype aegyptius and Brazilian purpuric fever isolates. The DNA sequence of
lav
is surprisingly similar to that of a gene previously described for
Neisseria meningitidis
. Sequence comparisons suggest that
lav
was transferred relatively recently from
Haemophilus
to
Neisseria
, shortly before the divergence of
N. meningitidis
and
Neisseria gonorrhoeae
. Segments of
lav
predicted to encode passenger and β-domains differ sharply in G+C base content, supporting the idea that AT genes have evolved by fusing domains which originated in different genomes. Homology and base sequence comparisons suggest that a novel biotype aegyptius AT arose by swapping an unrelated sequence for the passenger domain of
lav
. The unusually mobile
lav
locus joins a growing list of genes transferred from
H. influenzae
to
Neisseria
. Frequent gene exchange suggests a common pool of hypervariable contingency genes and may help to explain the origin of invasiveness in certain respiratory pathogens.
Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme
Authors: Johnson Hope A. , Pelletier Dale A. , Spormann Alfred M. 
Published: July 2002

ABSTRACT

The first step in anaerobic ethylbenzene mineralization in denitrifying
Azoarcus
sp. strain EB1 is the oxidation of ethylbenzene to (
S
)-(−)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (αβγ) with subunit masses of 100 kDa (α), 35 kDa (β), and 25 kDa (γ). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that
ebdA
encodes the α subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The
ebdB
gene encodes the β subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The
ebdC
gene encodes the γ subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.
Insertion-Duplication Mutagenesis of
Neisseria
: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island
Authors: Hamilton Holly L. , Schwartz Kevin J. , Dillard Joseph P. 
Published: July 2002

ABSTRACT

We created plasmids for use in insertion-duplication mutagenesis (IDM) of
Neisseria gonorrhoeae
. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene,
ermC
, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved
ermC
, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization of
traG
and
traH
mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.
Changes in rRNA Levels during Stress Invalidates Results from mRNA Blotting: Fluorescence In Situ rRNA Hybridization Permits Renormalization for Estimation of Cellular mRNA Levels
Authors: Hansen Martin C. , Nielsen Allan K. , Molin Søren , Hammer Karin , Kilstrup Mogens 
Published: July 2002

ABSTRACT

Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium
Lactococcus lactis
. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the
hrcA-grpE-dnaK
operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.
Cohesin-Dockerin Interactions of Cellulosomal Subunits of
Clostridium cellulovorans
Authors: Park Jae-Seon , Matano Yutaka , Doi Roy H. 
Published: July 2002

ABSTRACT

The cellulosome of
Clostridium cellulovorans
consists of three major subunits: CbpA, EngE, and ExgS. The
C. cellulovorans
scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.
The Alternative Sigma Factor SigH Regulates Major Components of Oxidative and Heat Stress Responses inMycobacterium tuberculosis
Authors: Raman Sahadevan , Song Taeksun , Puyang Xiaoling , Bardarov Stoyan , Jacobs William R. , Husson Robert N. 
Published: July 2002

ABSTRACTMycobacterium tuberculosisis a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyteMycobacterium smegmatis. In this work we investigated the role ofsigHin theM.tuberculosisresponse to heat and oxidative stress. We determined that aM.tuberculosis sigHmutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genestrxB2/trxCand a gene of unknown function, Rv2466c, is regulated bysigHvia expression from promoters directly recognized by SigH. We also determined that thesigHmutant is more susceptible to heat stress and that inducible expression of the heat shock genesdnaKandclpBis positively regulated bysigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role ofsigHin theM.tuberculosisstress response, we investigated the regulation of the stress-responsive sigma factor genessigEandsigB. We determined that inducible expression ofsigEis regulated bysigHand that basal and inducible expression ofsigBis dependent onsigEandsigH. These data indicate thatsigHplays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important inM.tuberculosispathogenesis.
An Archaeal Photosignal-Transducing Module Mediates Phototaxis in
Escherichia coli
Authors: Jung Kwang-Hwan , Spudich Elena N. , Trivedi Vishwa D. , Spudich John L. 
Published: July 2002

ABSTRACT

Halophilic archaea, such as
Halobacterium salinarum
and
Natronobacterium pharaonis
, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs). In
N. pharaonis
, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII. Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria. A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for
H. salinarum
, showing that the fusion does not inhibit functional receptor-transducer interactions. We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria. Purification of the fusion protein from
H. salinarum
and Tar fusion chimera from
Escherichia coli
membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes. Their photochemical reaction cycles in
H. salinarum
and
E. coli
membranes, respectively, are similar to those of native NpSRII in
N. pharaonis
. These fusion chimeras mediate retinal-dependent phototaxis responses by
Escherichia coli
, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes. This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.
Functions of the Mismatch Repair Gene
mutS
from
Acinetobacter
sp. Strain ADP1
Authors: Young David M. , Ornston L. Nicholas 
Published: July 2002

ABSTRACT

The genus
Acinetobacter
encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges.
Acinetobacter
sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned
mutS
from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of
mutS
resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of
mutS
also abolished the marker-specific variations in transforming efficiency seen in
mutS
+
recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual
Acinetobacter
strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the
Acinetobacter
amino acid sequences.