bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nitric Oxide-Mediated Proteasome-Dependent Oligonucleosomal DNA Fragmentation inLeishmania amazonensisAmastigotes
Authors: Holzmuller Philippe , Sereno Denis , Cavaleyra Mireille , Mangot Isabelle , Daulouede Sylvie , Vincendeau Philippe , Lemesre Jean-Loup 
Published: July 2002

ABSTRACTResistance to leishmanial infections depends on intracellular parasite killing by activated host macrophages through thel-arginine-nitric oxide (NO) metabolic pathway. Here we investigate the cell death process induced by NO for the intracellular protozoanLeishmania amazonensis. Exposure of amastigotes to moderate concentrations of NO-donating compounds (acidified sodium nitrite NaNO2or nitrosylated albumin) or to endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages resulted in a dramatic time-dependent cell death. The combined use of several standard DNA status analysis techniques (including electrophoresis ladder banding patterns, YOPRO-1 staining in flow cytofluorometry, and in situ recognition of DNA strand breaks by TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] assay) revealed a rapid and extensive fragmentation of nuclear DNA in both axenic and intracellular NO-treated amastigotes ofL. amazonensis. Despite some similarities to apoptosis, the nuclease activation responsible for characteristic DNA degradation was not under the control of caspase activity as indicated by the lack of involvement of cell-permeable inhibitors of caspases and cysteine proteases. In contrast, exposure of NO-treated amastigotes with specific proteasome inhibitors, such as lactacystin or calpain inhibitor I, markedly reduced the induction of the NO-mediated apoptosis-like process. These data strongly suggest that NO-induced oligonucleosomal DNA fragmentation inLeishmaniaamastigotes is, at least in part, regulated by noncaspase proteases of the proteasome. The determination of biochemical pathways leading up to cell death might ultimately allow the identification of new therapeutic targets.
Quantitative Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources: Comparative Analyses with
Corynebacterium glutamicum
during Growth on Acetate and/or Glucose
Authors: Wendisch Volker F. , de Graaf Albert A. , Sahm Hermann , Eikmanns Bernhard J. 
Published: July 2002

ABSTRACT

Growth of
Corynebacterium glutamicum
on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By
13
C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of
C. glutamicum
were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol · (mg of protein)
−1
· min
−1
, respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol · [mg of protein]
−1
· min
−1
) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol · [mg of protein]
−1
· min
−1
). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol · [mg of protein]
−1
· min
−1
) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of
C. glutamicum
, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.
Structural Elements Required for Replication and Incompatibility of the
Rhizobium etli
Symbiotic Plasmid
Authors: Ramírez-Romero Miguel A. , Soberón Nora , Pérez-Oseguera Angeles , Téllez-Sosa Juan , Cevallos Miguel A. 
Published: July 2002

ABSTRACT

The symbiotic plasmid of
Rhizobium etli
CE3 belongs to the RepABC family of plasmid replicons. This family is characterized by the presence of three conserved genes,
repA
,
repB
, and
repC
, encoded by the same DNA strand. A long intergenic sequence (
igs
) between
repB
and
repC
is also conserved in all members of the plasmid family. In this paper we demonstrate that (i) the
repABC
genes are organized in an operon; (ii) the RepC product is essential for replication; (iii) RepA and RepB products participate in plasmid segregation and in the regulation of plasmid copy number; (iv) there are two
cis
-acting incompatibility regions, one located in the
igs
(
inc
α) and the other downstream of
repC
(
inc
β) (the former is essential for replication); and (v) RepA is a
trans
-acting incompatibility factor. We suggest that
inc
α is a
cis
-acting site required for plasmid partitioning and that the origin of replication lies within
inc
β.
Integration and Excision of a
Bacteroides
Conjugative Transposon, CTnDOT
Authors: Cheng Qi , Paszkiet Brian J. , Shoemaker Nadja B. , Gardner Jeffrey F. , Salyers Abigail A. 
Published: July 2002

ABSTRACT

Bacteroides
conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most
Bacteroides
CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the
Bacteroides
CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF),
intDOT
, was necessary and sufficient for integration into the
Bacteroides
chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn
916
, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing
intDOT
and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of
intDOT
designated
orf2
. Thus, excision involves other genes besides
intDOT
and
orf2
. Both
intDOT
and
orf2
were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that
intDOT
was essential for excision but
orf2
was not. Despite its proximity to the integrase gene,
orf2
appears not to be essential for excision.
3-Deoxy-
d
-
manno
-oct-2-ulosonic Acid (Kdo) Transferase of
Legionella pneumophila
Transfers Two Kdo Residues to a Structurally Different Lipid A Precursor of
Escherichia coli
Authors: Brabetz Werner , Schirmer Christiane E. , Brade Helmut 
Published: July 2002

ABSTRACT

The 3-deoxy-
d
-
manno
-oct-2-ulosonic acid (Kdo) transferase gene of
Legionella pneumophila
was cloned and sequenced. Despite remarkable structural differences in lipid A, the gene complemented a corresponding
Escherichia coli
mutant and was shown to encode a bifunctional enzyme which transferred 2 Kdo residues to a lipid A acceptor of
E. coli.
Regulation of Stalk Elongation by Phosphate in
Caulobacter crescentus
Authors: Gonin Madeleine , Quardokus Ellen M. , O'Donnol Danielle , Maddock Janine , Brun Yves V. 
Published: July 2002

ABSTRACT

In
Caulobacter crescentus
, stalk biosynthesis is regulated by cell cycle cues and by extracellular phosphate concentration. Phosphate-starved cells undergo dramatic stalk elongation to produce stalks as much as 30 times as long as those of cells growing in phosphate-rich medium. To identify genes involved in the control of stalk elongation, transposon mutants were isolated that exhibited a long-stalk phenotype irrespective of extracellular phosphate concentration. The disrupted genes were identified as homologues of the high-affinity phosphate transport genes
pstSCAB
of
Escherichia coli
. In
E. coli
,
pst
mutants have a constitutively expressed phosphate (Pho) regulon. To determine if stalk elongation is regulated by the Pho regulon, the
Caulobacter phoB
gene that encodes the transcriptional activator of the Pho regulon was cloned and mutated. While
phoB
was not required for stalk synthesis or for the cell cycle timing of stalk synthesis initiation, it was required for stalk elongation in response to phosphate starvation. Both
pstS
and
phoB
mutants were deficient in phosphate transport. When a
phoB
mutant was grown with limiting phosphate concentrations, stalks only increased in length by an average of 1.4-fold compared to the average 9-fold increase in stalk length of wild-type cells grown in the same medium. Thus, the phenotypes of
phoB
and
pst
mutants were opposite.
phoB
mutants were unable to elongate stalks during phosphate starvation, whereas
pst
mutants made long stalks in both high- and low-phosphate media. Analysis of double
pst phoB
mutants indicated that the long-stalk phenotype of
pst
mutants was dependent on
phoB
. In addition, analysis of a
pstS-lacZ
transcriptional fusion showed that
pstS
transcription is dependent on
phoB
. These results suggest that the signal transduction pathway that stimulates stalk elongation in response to phosphate starvation is mediated by the Pst proteins and the response regulator PhoB.
Genetic and Biochemical Analysis of Dimer and Oligomer Interactions of the λ S Holin
Authors: Gründling Angelika , Bläsi Udo , Young Ry 
Published: July 2002

ABSTRACT

Bacteriophage λ uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis. λ S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from λ S
C51S
were tested for holin function. Most of the single-cysteine variants retained the ability to support lysis. Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains. Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless S
C51S
(i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contrast, the lysis-defective S
A52V
mutant dimerizes but does not form cross-linkable oligomers. Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective S
A52V/C51S
is found in dimers after treatment of the membrane with a cross-linking agent. Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for S
A52V
. Both S
A52V
(dominant negative) and S
A48V
(antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation. Thus, productive and unproductive heterodimer formation between the parental protein and the mutants S
A52V
and S
A48V
, respectively, may account for the dominant and antidominant lysis phenotypes. Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface. These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.
Metabolism of Acyl-Homoserine Lactone Quorum-Sensing Signals by
Variovorax paradoxus
Authors: Leadbetter Jared R. , Greenberg E. P. 
Published: July 2002

ABSTRACT

Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class
Proteobacteria
. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize
N
-(3-oxohexanoyl)-
l
-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of
Variovorax paradoxus
. The
V. paradoxus
isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of
V. paradoxus
degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by
V. paradoxus
.
Correction of the DNA Sequence of theregBGene ofRhodobacter capsulatuswith Implications for the Membrane Topology of the Sensor Kinase RegB
Authors: Chen Weizhen , Jäger Andreas , Klug Gabriele 
Published: July 2002

ABSTRACTWe corrected the previously published sequence for theregBgene, which encodes a histidine sensor kinase inRhodobacter capsulatus. The deduced RegB amino acid sequence has an additional putative transmembrane domain at the N terminus. Analysis of RegB-PhoA and RegB-LacZ fusion proteins supports a topology model for RegB with six membrane-spanning domains.
Barriers to Genetic Exchange between Bacterial Species:
Streptococcus pneumoniae
Transformation
Authors: Majewski Jacek , Zawadzki Piotr , Pickerill Paul , Cohan Frederick M. , Dowson Christopher G. 
Published: July 2002

ABSTRACT

Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In
Bacillus
and
Escherichia
, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between
Streptococcus pneumoniae
recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in
Bacillus
but less than that in
Escherichia
. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from
Bacillus
,
Escherichia
, and
Streptococcus
.
Transposon Insertions in the
Flavobacterium johnsoniae ftsX
Gene Disrupt Gliding Motility and Cell Division
Authors: Kempf Michael J. , McBride Mark J. 
Published: July 2002

ABSTRACT

Flavobacterium johnsoniae
is a gram-negative bacterium that exhibits gliding motility. To determine the mechanism of flavobacterial gliding motility, we isolated 33 nongliding mutants by Tn
4351
mutagenesis. Seventeen of these mutants exhibited filamentous cell morphology. The region of DNA surrounding the transposon insertion in the filamentous mutant CJ101-207 was cloned and sequenced. The transposon was inserted in a gene that was similar to
Escherichia coli ftsX
. Two of the remaining 16 filamentous mutants also carried insertions in
ftsX
. Introduction of the wild-type
F. johnsoniae ftsX
gene restored motility and normal cell morphology to each of the three
ftsX
mutants. CJ101-207 appears to be blocked at a late stage of cell division, since the filaments produced cross walls but cells failed to separate. In
E. coli
, FtsX is thought to function with FtsE in translocating proteins involved in potassium transport, and perhaps proteins involved in cell division, into the cytoplasmic membrane. Mutations in
F. johnsoniae ftsX
may prevent translocation of proteins involved in cell division and proteins involved in gliding motility into the cytoplasmic membrane, thus resulting in defects in both processes. Alternatively, the loss of gliding motility may be an indirect result of the defect in cell division. The inability to complete cell division may alter the cell architecture and disrupt gliding motility by preventing the synthesis, assembly, or functioning of the motility apparatus.
Probable Identification of a Membrane-Associated Repressor of
Bacillus subtilis
DNA Replication as the E2 Subunit of the Pyruvate Dehydrogenase Complex
Authors: Stein Andrew , Firshein William 
Published: July 2002

ABSTRACT

Two
Bacillus subtilis
lysogenic libraries were probed by an antibody specific for a previously described membrane-associated inhibitor of
B. subtilis
DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452–7456, 1988). Three clones that reacted strongly with the antibody contained an entire open reading frame. Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase. An AT-rich sequence in the origin region was identified initially as the site to which extracts from the R1-2 clone were bound. This sequence was almost identical to one detected in
Bacillus thuringiensis
that also bound the E2 subunit but which was involved in activating the Cry1 protoxin gene of the organism, not in inhibiting DNA replication (T. Walter and A. Aronson, J. Biol. Chem., 274:7901–7906, 1999). However, the exact sequence was not as important in
B. subtilis
as the AT-rich core region. Binding would occur as long as most of the AT character of the core remained. Purified E2 protein obtained by use of PCR and an expression vector reacted strongly with antibody prepared against the repressor protein and the protein in the R1-2 clone, but its specificity for the AT-rich region was altered. The purified E2 protein was capable of inhibiting membrane-associated DNA replication in vitro, but anti-E2 antibody was variable in its ability to rescue repression when added to the assay.
Roles of Horizontal Gene Transfer and Gene Integration in Evolution of 1,3-Dichloropropene- and 1,2-Dibromoethane-Degradative Pathways
Authors: Poelarends Gerrit J. , Kulakov Leonid A. , Larkin Michael J. , van Hylckama Vlieg Johan E. T. , Janssen Dick B. 
Published: July 2002

ABSTRACT

The haloalkane-degrading bacteria
Rhodococcus rhodochrous
NCIMB13064,
Pseudomonas pavonaceae
170, and
Mycobacterium
sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (
dhaA
). Here, we describe the extent of the conserved
dhaA
segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The
dhaA
gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (
invA
), a regulatory protein (
dhaR
), an alcohol dehydrogenase (
adhA
), and an aldehyde dehydrogenase (
aldA
). The latter two enzymes may catalyze the oxidative conversion of
n
-butanol, the hydrolytic product of 1-chlorobutane, to
n
-butyric acid, a growth substrate for many bacteria. The activity of the
dhaR
gene product was analyzed in
Pseudomonas
sp. strain GJ1, in which it appeared to function as a repressor of
dhaA
expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included
dhaR
,
dhaA
, and part of
invA
. A 12-nucleotide deletion in
dhaR
led to constitutive expression of
dhaA
in strain GP1, in contrast to the inducible expression of
dhaA
in strain NCIMB13064. The 1,3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the
dhaA
gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved
dhaA
segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1,3-dichloropropene and 1,2-dibromoethane.
Alternative Pathways for Siroheme Synthesis inKlebsiella aerogenes
Authors: Kolko M. Miriam , Kapetanovich Lori A. , Lawrence Jeffrey G. 
Published: July 2002

ABSTRACTSiroheme, the cofactor for sulfite and nitrite reductases, is formed by methylation, oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen III (Uro-III). The CysG protein performs all three steps of siroheme biosynthesis in the enteric bacteriaEscherichia coliandSalmonella enterica. In either taxon,cysGmutants cannot reduce sulfite to sulfide and require a source of sulfide or cysteine for growth. In addition, CysG-mediated methylation of Uro-III is required for de novo synthesis of cobalamin (coenzyme B12) inS. enterica. We have determined thatcysGmutants of the related enteric bacteriumKlebsiella aerogeneshave no defect in the reduction of sulfite to sulfide. These data suggest that an alternative enzyme allows for siroheme biosynthesis in CysG-deficient strains ofKlebsiella. However,Klebsiella cysGmutants fail to synthesize coenzyme B12, suggesting that the alternative siroheme biosynthetic pathway proceeds by a different route. GenecysF, encoding an alternative siroheme synthase homologous to CysG, has been identified by genetic analysis and lies within thecysFDNCoperon; thecysFgene is absent from theE. coliandS. entericagenomes. While CysG is coregulated with the siroheme-dependent nitrite reductase, thecysFgene is regulated by sulfur starvation. Models for alternative regulation of the CysF and CysG siroheme synthases inKlebsiellaand for the loss of thecysFgene from the ancestor ofE. coliandS. entericaare presented.