bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Amino Terminus of Bacteriophage λ Integrase Is Involved in Protein-Protein Interactions during Recombination
Authors: Jessop Lea , Bankhead Troy , Wong David , Segall Anca M. 
Published: July 2002

ABSTRACT

Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment (
att
) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the
att
sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from
Haemophilus influenzae
phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for recombination.
Distribution of Twelve Linear Extrachromosomal DNAs in Natural Isolates of Lyme Disease Spirochetes
Authors: Palmer Nanette , Fraser Claire , Casjens Sherwood 
Published: July 2002

ABSTRACT

We have analyzed a panel of independent North American isolates of the Lyme disease agent spirochete,
Borrelia burgdorferi
(sensu stricto), for the presence of linear plasmids with sequence similarities to the 12 linear plasmids present in the
B. burgdorferi
type strain, isolate B31. The frequency of similarities to probes from each of the 12 B31 plasmids varied from 13 to 100% in the strain panel examined, and these similarities usually reside on plasmids similar in size to the cognate B31 plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in all of the isolates examined, and >66% of the panel members hybridized to probes from 4 other plasmids. Sequences similar to most of the
B. burgdorferi
B31 plasmid-derived DNA probes used were also found on linear plasmids in the related Eurasian Lyme agents
Borrelia garinii
and
Borrelia afzelii
; however, some of these plasmids had uniform but substantially different sizes from their
B. burgdorferi
counterparts.
Generation of Dominant Selectable Markers for Resistance to Pseudomonic Acid by Cloning and Mutagenesis of the
ileS
Gene from the Archaeon
Methanosarcina barkeri
Fusaro
Authors: Boccazzi Paolo , Zhang Jun Kai , Metcalf William W. 
Published: July 2002

ABSTRACT

Currently, only one selectable marker is available for genetic studies in the archaeal genus
Methanosarcina
. Here we report the generation of selectable markers that encode resistance to pseudomonic acid (PA
r
) in
Methanosarcina
species by mutagenesis of the isoleucyl-tRNA synthetase gene (
ileS
) from
Methanosarcina barkeri
Fusaro. The
M. barkeri ileS
gene was obtained by screening of a genomic library for hybridization to a PCR fragment. The complete 3,787-bp DNA sequence surrounding and including the
ileS
gene was determined. As expected,
M. barkeri
IleS is phylogenetically related to other archaeal IleS proteins. The
ileS
gene was cloned into a
Methanosarcina-Escherichia coli
shuttle vector and mutagenized with hydroxylamine. Nine independent PA
r
clones were isolated after transformation of
Methanosarcina acetivorans
C2A with the mutagenized plasmids. Seven of these clones carry multiple changes from the wild-type sequence. Most mutations that confer PA
r
were shown to alter amino acid residues near the KMSKS consensus sequence of class I aminoacyl-tRNA synthetases. One particular mutation (G594E) was present in all but one of the PA
r
clones. The MIC of pseudomonic acid for
M. acetivorans
transformed with a plasmid carrying this single mutation is 70 μg/ml of medium (for the wild type, the MIC is 12 μg/ml). The highest MICs (560 μg/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PA
r
based on the mutant
ileS
alleles are described. Finally, the implications of the specific mutations we isolated with respect to binding of pseudomonic acid by IleS are discussed.
Twofold Reduction of Phosphofructokinase Activity in
Lactococcus lactis
Results in Strong Decreases in Growth Rate and in Glycolytic Flux
Authors: Andersen Heidi W. , Solem Christian , Hammer Karin , Jensen Peter R. 
Published: July 2002

ABSTRACT

Two mutant strains of
Lactococcus lactis
in which the promoter of the
las
operon, harboring
pfk, pyk
, and
ldh
, were replaced by synthetic promoters were constructed. These
las
mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions. One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two- to fourfold in the
las
mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.
Analysis of the PilQ Secretin from
Neisseria meningitidis
by Transmission Electron Microscopy Reveals a Dodecameric Quaternary Structure
Authors: Collins Richard F. , Davidsen Linn , Derrick Jeremy P. , Ford Robert C. , Tønjum Tone 
Published: July 2002

ABSTRACT

PilQ is a member of the secretin family of outer membrane proteins and is specifically involved in secretion of type IV pili in
Neisseria meningitidis, Neisseria gonorrhoeae
, and
Pseudomonas aeruginosa
. The quaternary structure of PilQ from
N. meningitidis
was analyzed by transmission electron microscopy by using a negative stain. Single particle averaging was carried out with a total data set of 650 individual particles, which produced a projection map generated from 296 particles at an estimated resolution of 2.6 nm. Oligomeric PilQ adopts a donut-like structure with an external ring that is 16.5 nm in diameter surrounding a central cavity that is 6.5 nm in diameter. Self-rotation and power spectrum analysis demonstrated the presence of 12-fold rotational symmetry, showing that PilQ is organized as a ring of 12 identical subunits. A model of the type IV meningococcal pilus fiber, based on the X-ray crystal structure of the
N. gonorrhoeae
pilin subunit, fitted neatly into the cavity, demonstrating how PilQ could serve as a channel for the growing pilus fiber.
Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of
Synechocystis
sp. Strain PCC 6803: Capacity Comparisons and Physiological Function
Authors: Cooley Jason W. , Vermaas Wim F. J. 
Published: July 2002

ABSTRACT
Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.
The
gdhB
Gene of
Pseudomonas aeruginosa
Encodes an Arginine-Inducible NAD
+
-Dependent Glutamate Dehydrogenase Which Is Subject to Allosteric Regulation
Authors: Lu Chung-Dar , Abdelal Ahmed T. 
Published: July 2002

ABSTRACT

The NAD
+
-dependent glutamate dehydrogenase (NAD-GDH) from
Pseudomonas aeruginosa
PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding
gdhB
gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from
P. aeruginosa
with the GenBank database showed the highest homology with hypothetical polypeptides from
Pseudomonas putida
,
Mycobacterium tuberculosis
,
Rickettsia prowazakii
,
Legionella pneumophila
,
Vibrio cholerae
,
Shewanella putrefaciens
,
Sinorhizobium meliloti
, and
Caulobacter crescentus.
A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from
Saccharomyces cerevisiae
or
Neurospora crassa
. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of
gdhB
is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of
P. aeruginosa
and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.
Surface Display of Recombinant Proteins onBacillus subtilisSpores
Authors: Isticato Rachele , Cangiano Giuseppina , Tran Hoa T. , Ciabattini Annalisa , Medaglini Donata , Oggioni Marco R. , De Felice Maurilio , Pozzi Gianni , Ricca Ezio 
Published: July 2002

ABSTRACTWe developed a novel surface display system based on the use of bacterial spores. A protein of theBacillus subtilisspore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 × 103TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record ofB. subtilisspores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.
Identification of a Plasmid-Encoded Gene from
Haemophilus ducreyi
Which Confers NAD Independence
Authors: Martin Paul R. , Shea Robin J. , Mulks Martha H. 
Published: July 2002

ABSTRACT

Members of the family
Pasteurellaceae
are classified in part by whether or not they require an NAD supplement for growth on laboratory media. In this study, we demonstrate that this phenotype can be determined by a single gene,
nadV
, whose presence allows NAD-independent growth of
Haemophilus influenzae
and
Actinobacillus pleuropneumoniae
. This gene was cloned from a 5.2-kb plasmid which was previously shown to be responsible for NAD independence in
Haemophilus ducreyi
. When transformed into
A. pleuropneumoniae
, this cloned gene allowed NAD-independent growth on complex media and allowed the utilization of nicotinamide in place of NAD on defined media. Sequence analysis revealed an open reading frame of 1,482 bp that is predicted to encode a protein with a molecular mass of 55,619 Da. Compared with the sequence databases, NadV was found to have significant sequence homology to the human pre-B-cell colony-enhancing factor PBEF and to predicted proteins of unknown function identified in the bacterial species
Mycoplasma genitalium, Mycoplasma pneumoniae, Shewanella putrefaciens, Synechocystis sp., Deinococcus radiodurans, Pasteurella multocida
, and
Actinobacillus actinomycetemcomitans. P. multocida
and
A. actinomycetemcomitans
are among the NAD-independent members of the
Pasteurellaceae
. Homologues of NadV were not found in the sequenced genome of
H. influenzae
, an NAD-dependent member of the
Pasteurellaceae
, or in species known to utilize a different pathway for synthesis of NAD, such as
Escherichia coli
. Sequence alignment of these nine homologues revealed regions and residues of complete conservation that may be directly involved in the enzymatic activity. Identification of a function for this gene in the
Pasteurellaceae
should help to elucidate the role of its homologues in other species.
Visualization of the Attachment Organelle and Cytadherence Proteins of
Mycoplasma pneumoniae
by Immunofluorescence Microscopy
Authors: Seto Shintaro , Layh-Schmitt Gerlinde , Kenri Tsuyoshi , Miyata Makoto 
Published: July 2002

ABSTRACT

A method was developed for protein localization in
Mycoplasma pneumoniae
by immunofluorescence microscopy. The P1 adhesin protein was revealed to be located at least at one cell pole in all adhesive cells, as has been observed by immunoelectron microscopy. Cell images were classified according to P1 localization and assigned by DNA content. Cells with a single P1 focus at one cell pole had a lower DNA content than cells with two foci, at least one of which was positioned at a cell pole. Those with one focus at each cell pole had the highest DNA content, suggesting that the nascent attachment organelle is formed next to the old one and migrates to the opposite cell pole before cell division. Double staining revealed that the accessory proteins for cytadherence—HMW1, HMW3, P30, P90, P40, and P65—colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was also examined in cytadherence-deficient mutant cells with a branched morphology. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, P1, and P30 were focused at the cell poles of short branches, and P65 showed no signal. In M7 mutant cells, which produce a truncated P30 protein, HMW1, HMW3, P1, P90, and P40 were focused, and P65 showed no signal. In M6 mutant cells, which express no HMW1 and a truncated P30 protein, the P1 adhesin was distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence proteins are sequentially assembled to the attachment organelle with HMW1 first, HMW3, P1, P30, P90, and P40 next, and P65 last.
Global Analysis of
Escherichia coli
Gene Expression during the Acetate-Induced Acid Tolerance Response
Authors: Arnold Carrie N. , McElhanon Justin , Lee Aaron , Leonhart Ryan , Siegele Deborah A. 
Published: July 2002

ABSTRACT

The ability of
Escherichia coli
to survive at low pH is strongly affected by environmental factors, such as composition of the growth medium and growth phase. Exposure to short-chain fatty acids, such as acetate, proprionate, and butyrate, at neutral or nearly neutral pH has also been shown to increase acid survival of
E. coli
and
Salmonella enterica
serovar Typhimurium. To investigate the basis for acetate-induced acid tolerance in
E. coli
O157:H7, genes whose expression was altered by exposure to acetate were identified using gene arrays. The expression of 60 genes was reduced by at least twofold; of these, 48 encode components of the transcription-translation machinery. Expression of 26 genes increased twofold or greater following treatment with acetate. This included six genes whose products are known to be important for survival at low pH. Five of these genes, as well as six other acetate-induced genes, are members of the
E. coli
RpoS regulon. RpoS, the stress sigma factor, is known to be required for acid tolerance induced by growth at nonlethal low pH or by entry into stationary phase. Disruption of the
rpoS
gene by a transposon insertion mutation also prevented acetate-induced acid tolerance. However, induction of RpoS expression did not appear to be sufficient to activate the acid tolerance response. Treatment with either NaCl or sodium acetate (pH 7.0) increased expression of an
rpoS
::
lacZ
fusion protein, but only treatment with acetate increased acid survival.
Mutually Exclusive Distribution of IS
1548
and GBSi1, an Active Group II Intron Identified in Human Isolates of Group B Streptococci
Authors: Granlund Margareta , Michel François , Norgren Mari 
Published: July 2002

ABSTRACT

The present study shows that active, self-splicing group II intron GBSi1 is located downstream of the C5a-peptidase gene,
scpB
, in some group B streptococcus (GBS) isolates that lack insertion sequence IS
1548
. IS
1548
was previously reported to be often present at the
scpB
locus in GBS isolated in association with endocarditis. Since none of 67 GBS isolates examined, 40 of which were of serotype III, harbored both IS
1548
and GBSi1, these two elements are suggested to be markers for different genetic lineages in GBS serotype III. The DNA region downstream of
scpB
in GBS isolates harboring either GBSi1, IS1548, or none of these mobile elements was found to encode the laminin binding protein, Lmb, which shows sequence similarities to a family of streptococcal adhesins. IS
1548
is inserted 9 bp upstream of the putative promoter for
lmb
, while the insertion site for GBSi1 is located 88 bp further upstream. Sequences highly similar to GBSi1 exist also in
Streptococcus pneumoniae
. An inverted repeat sequence, with features typical of transcription terminators, was identified immediately upstream of the insertion site for the group II intron both in the GBS and
S. pneumoniae
sequences. This motif is suggested to constitute a target for the GBS intron as well as for rather closely related introns in
Bacillus halodurans, Pseudomonas alcaligenes
, and
Pseudomonas putida
. When transcripts containing the GBSi1 intron were incubated at high concentrations of ammonium and magnesium, a major product with the expected length and sequence for the ligated exons was generated. Unlike, however, all members of group II investigated so far, the excised intron was in linear, rather than in a branched (lariat), form.
Biofilm Formation by
Staphylococcus epidermidis
Depends on Functional RsbU, an Activator of the
sigB
Operon: Differential Activation Mechanisms Due to Ethanol and Salt Stress
Authors: Knobloch Johannes K.-M. , Bartscht Katrin , Sabottke Axel , Rohde Holger , Feucht Heinz-Hubert , Mack Dietrich 
Published: July 2002

ABSTRACT

Staphylococcus epidermidis
is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the
icaADBC
gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn
917
insertions of two isogenic,
icaADBC
-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium
S. epidermidis
1457. The insertion site was the same in both of the mutants and was located in the first gene,
rsbU
, of an operon highly homologous to the
sigB
operons of
Staphylococcus aureus
and
Bacillus subtilis
. Supplementation of Trypticase soy broth with NaCl (TSB
NaCl
) or ethanol (TSB
EtOH
), both of which are known activators of
sigB
, led to increased biofilm formation and PIA synthesis by
S. epidermidis
1457. Insertion of Tn
917
into
rsbU
, a positive regulator of alternative sigma factor ς
B
, led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB
EtOH
, the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB
NaCl
, the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn
917
insertion of mutant M15 to the independent and biofilm-producing strain
S. epidermidis
8400. In 11 clinical
S. epidermidis
strains, a restriction fragment length polymorphism of the
sigB
operon was detected which was independent of the presence of the
icaADBC
locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.