bluuebunny/crossref_metadata_2025_split huggingface dataset:
Repair of DNA Lesions Induced by Hydrogen Peroxide in the Presence of Iron Chelators in
Escherichia coli
: Participation of Endonuclease IV and Fpg
Authors: Galhardo Rodrigo S. , Almeida Carlos E. B. , Leitão Alvaro C. , Cabral-Neto Januário B. 
Published: July 2002

ABSTRACT

In
Escherichia coli
, the repair of lethal DNA damage induced by H
2
O
2
requires exonuclease III, the
xthA
gene product. Here, we report that both endonuclease IV (the
nfo
gene product) and exonuclease III can mediate the repair of lesions induced by H
2
O
2
under low-iron conditions. Neither the
xthA
nor the
nfo
mutants was sensitive to H
2
O
2
in the presence of iron chelators, while the
xthA nfo
double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both
xthA
and
nfo
mutants, but not the
xthA nfo
double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H
2
O
2
under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the
fpg
mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.
Contribution of Base Excision Repair, Nucleotide Excision Repair, and DNA Recombination to Alkylation Resistance of the Fission Yeast
Schizosaccharomyces pombe
Authors: Memisoglu Asli , Samson Leona 
Published: July 2002

ABSTRACT

DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated
Schizosaccharomyces pombe
strains mutant for the
mag1
3-methyladenine DNA glycosylase gene. We report that
S. pombe mag1
mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair (
rad13
) and recombinational repair (
rhp51
) are much more alkylation sensitive than
mag1
mutants. In addition,
S. pombe
mutant for the flap endonuclease
rad2
gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than
mag1
mutants. Further,
mag1
and
rad13
interact synergistically for alkylation resistance, and
mag1
and
rhp51
display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in
S. pombe
is discussed.
Events during Initiation of Archaeal Transcription: Open Complex Formation and DNA-Protein Interactions
Authors: Hausner Winfried , Thomm Michael 
Published: July 2002

ABSTRACT

Transcription in
Archaea
is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon
Methanococcus
, in contrast to polymerase II, does not require hydrolysis of the β-γ bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex spanned at least the DNA region from −11 to −1 at a tRNA
Val
promoter. The
Methanococcus
TBP-TFB promoter complex protected the DNA region from −40 to −14 on the noncoding DNA strand and the DNA segment from −36 to −17 on the coding DNA strand from DNase I digestion. This DNase I footprint was extended only to the downstream end by the addition of the RNA polymerase to position +17 on the noncoding strand and to position +13 on the coding DNA strand.
The Type IV Fimbrial Subunit Gene (
fimA
) of
Dichelobacter nodosus
Is Essential for Virulence, Protease Secretion, and Natural Competence
Authors: Kennan Ruth M. , Dhungyel Om P. , Whittington Richard J. , Egerton John R. , Rood Julian I. 
Published: July 2002

ABSTRACT

Dichelobacter nodosus
is the essential causative agent of footrot in sheep. The major
D. nodosus
-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the
fimA
gene, which encodes the fimbrial subunit protein, from the virulent type G
D. nodosus
strain VCS1703A. Detailed analysis of two independently derived
fimA
mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream
fimB
gene because insertionally inactivated
fimB
mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the
fimA
mutants were avirulent, providing evidence that the
fimA
gene is an essential
D. nodosus
virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.
Genome Size Determination and Coding Capacity of
Sodalis glossinidius
, an Enteric Symbiont of Tsetse Flies, as Revealed by Hybridization to
Escherichia coli
Gene Arrays
Authors: Akman Leyla , Rio Rita V. M. , Beard Charles B. , Aksoy Serap 
Published: July 2002

ABSTRACT

Recent molecular characterization of various microbial genomes has revealed differences in genome size and coding capacity between obligate symbionts and intracellular pathogens versus free-living organisms. Multiple symbiotic microorganisms have evolved with tsetse fly, the vector of African trypanosomes, over long evolutionary times. Although these symbionts are indispensable for tsetse fecundity, the biochemical and molecular basis of their functional significance is unknown. Here, we report on the genomic aspects of the secondary symbiont
Sodalis glossinidius
. The genome size of
Sodalis
is approximately 2 Mb. Its DNA is subject to extensive methylation and based on some of its conserved gene sequences has an A+T content of only 45%, compared to the typically AT-rich genomes of endosymbionts. Sodalis also harbors an extrachromosomal plasmid about 134 kb in size. We used a novel approach to gain insight into
Sodalis
genomic contents, i.e., hybridizing its DNA to macroarrays developed for
Escherichia coli
, a closely related enteric bacterium. In this analysis we detected 1,800 orthologous genes, corresponding to about 85% of the
Sodalis
genome. The
Sodalis
genome has apparently retained its genes for DNA replication, transcription, translation, transport, and the biosynthesis of amino acids, nucleic acids, vitamins, and cofactors. However, many genes involved in energy metabolism and carbon compound assimilation are apparently missing, which may indicate an adaptation to the energy sources available in the only nutrient of the tsetse host, blood. We present gene arrays as a rapid tool for comparative genomics in the absence of whole genome sequence to advance our understanding of closely related bacteria.
Membrane Topology of theStreptomyces lividansType I Signal Peptidases
Authors: Geukens Nick , Lammertyn Elke , Van Mellaert Lieve , Schacht Sabine , Schaerlaekens Kristien , Parro Victor , Bron Sierd , Engelborghs Yves , Mellado Rafael P. , Anné Jozef 
Published: July 2002

ABSTRACTMost bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. ForStreptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These threeS. lividansSPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast,S. lividansSipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for theS. lividansSPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.
Distribution of the
Ssu
DAT1I Restriction-Modification System among Different Serotypes of
Streptococcus suis
Authors: Sekizaki Tsutomu , Osaki Makoto , Takamatsu Daisuke , Shimoji Yoshihiro 
Published: July 2002

ABSTRACT

The
Ssu
DAT1I restriction-modification (R-M) system, which contains two methyltransferases and two restriction endonucleases with recognition sequence 5′-GATC-3′, was first found in a field isolate of
Streptococcus suis
serotype 2. Isoschizomers of the R-M system were found in the same locus between
purH
and
purD
in a field isolate of serotype 1/2 and the reference strains of serotypes 3, 7, 23, and 26 among 29 strains of different serotypes examined in this study. The R-M gene sequences in serotypes 1/2, 3, 7, and 23 were very similar to those of
Ssu
DAT1I, whereas those in serotype 26 were less similar. These results indicate intraspecies recombination among them and genetic divergence through their evolution.
Autophosphorylation of Archaeal Cdc6 Homologues Is Regulated by DNA
Authors: Grabowski Beatrice , Kelman Zvi 
Published: July 2002

ABSTRACTThe initiator protein Cdc6 (Cdc18 in fission yeast) plays an essential role in the initiation of eukaryotic DNA replication. In yeast the protein is expressed before initiation of DNA replication and is thought to be essential for loading of the helicase onto origin DNA. The biochemical properties of the protein, however, are largely unknown. Using three archaeal homologues of Cdc6, it was found that the proteins are autophosphorylated on Ser residues. The winged-helix domain at the C terminus of Cdc6 interacts with DNA, which apparently regulates the autophosphorylation reaction. Yeast Cdc18 was also found to autophosphorylate, suggesting that this function of Cdc6 may play a widely conserved and essential role in replication initiation.
Properties of a Thermostable Nitrate Reductase from the Hyperthermophilic Archaeon
Pyrobaculum aerophilum
Authors: Afshar Sepideh , Johnson Eric , de Vries Simon , Schröder Imke 
Published: July 2002

ABSTRACT

The nitrate reductase of the hyperthermophilic archaeon
Pyrobaculum aerophilum
was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome
b
(0.49-mol/mol complex) as cofactors. The
P. aerophilum
nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (
V
max
with nitrate, 1,162 s
−1
(326 U/mg);
V
max
with chlorate, 1,348 s
−1
(378 U/mg) [assayed at 75°C]). The

K
m

values for nitrate and chlorate were 58 and 140 μM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.
Identification of the Partitioning Site within the
repABC-
Type Replicon of the Composite
Paracoccus versutus
Plasmid pTAV1
Authors: Bartosik Dariusz , Szymanik Michal , Wysocka Edyta 
Published: July 2002

ABSTRACT

The replicator region of composite plasmid pTAV1 of
Paracoccus versutus
(included in mini-replicon pTAV320) belongs to the family of
repABC
replicons commonly found in plasmids harbored by
Agrobacterium
and
Rhizobium
spp. The
repABC
replicons encode three genes clustered in an operon, which are involved in partitioning (
repA
and
repB
) and replication (
repC
). In order to localize the partitioning site of pTAV320, the two identified incompatibility determinants of this mini-replicon (
inc1
, located in the intergenic sequence between
repB
and
repC
; and
inc2
, situated downstream of the
repC
gene) were PCR amplified and used together with purified RepB fusion protein (homologous to the type B partitioning proteins binding to the partitioning sites) in an electrophoretic mobility shift assay. The protein bound only
inc2
, forming two complexes in a protein concentration-dependent manner. The
inc2
region contains two long (14-bp) repeated sequences (R1 and R2). Disruption of these sequences completely eliminates RepB binding ability. R1 and R2 have sequence similarities with analogous repeats of another
repABC
replicon of plasmid pPAN1 of
Paracoccus pantotrophus
DSM 82.5 and with centromeric sequences of the
Bacillus subtilis
chromosome. Excess RepB protein resulted in destabilization of the
inc2
-containing plasmid in
Escherichia coli
. On the other hand, the
inc2
region could stabilize another unstable replicon in
P. versutus
when RepA and RepB were delivered in
trans
, proving that this region has centromere-like activity. Thus, it was demonstrated that
repA
,
repB
, and
inc2
constitute a functional system for active partitioning of pTAV320.
Contributions of UP Elements and the Transcription Factor FIS to Expression from the Seven
rrn
P1 Promoters in
Escherichia coli
Authors: Hirvonen Christine A. , Ross Wilma , Wozniak Christopher E. , Marasco Erin , Anthony Jennifer R. , Aiyar Sarah E. , Newburn Vanessa H. , Gourse Richard L. 
Published: July 2002

ABSTRACT

The high activity of the
rrnB
P1 promoter in
Escherichia coli
results from a
cis
-acting DNA sequence, the UP element, and a
trans
-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six
rrn
P1 promoters. We find that UP elements are present at all of the
rrn
P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven
rrn
P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each
rrn
P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most
rrn
P1 promoters compared to
rrnB
P1. Our studies indicate that the overall activities of the seven
rrn
P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.
TOR Modulates
GCN4
-Dependent Expression of Genes Turned on by Nitrogen Limitation
Authors: Valenzuela Lourdes , Aranda Cristina , González Alicia 
Published: July 2002

ABSTRACT

In
Saccharomyces cerevisiae
, the rapamycin-sensitive TOR signaling pathway plays an essential role in up-regulating translation initiation and cell cycle progression in response to nutrient availability. One of the mechanisms by which TOR regulates cell proliferation is by excluding the
GLN3
transcriptional activator from the nucleus and, in consequence, preventing its transcriptional activation therein. We examined the possibility that the TOR cascade could also control the transcriptional activity of Gcn4p, which is known to respond to amino acid availability. The results presented in this paper indicate that
GCN4
plays a role in the rapamycin-sensitive signaling pathway, regulating the expression of genes involved in the utilization of poor nitrogen sources, a previously unrecognized role for Gcn4p, and that the TOR pathway controls
GCN4
activity by regulating the translation of
GCN4
mRNA. This constitutes an additional TOR-dependent mechanism which modulates the action of transcriptional activators.
Sinorhizobium meliloti
Plasmid pRm1132f Replicates by a Rolling-Circle Mechanism
Authors: Barran L. R. , Ritchot N. , Bromfield E. S. P. 
Published: July 2002

ABSTRACT

pRm1132f isolated from
Sinorhizobium meliloti
is a group III rolling-circle-replicating (RCR) plasmid. At least seven of eight open reading frames in the nucleotide sequence represented coding regions. The minimal replicon contained a
rep
gene and single- and double-stranded origins of replication. Detection of single-stranded plasmid DNA confirmed that pRm1132f replicated via an RCR mechanism.
Regulation of thenfsAGene inEscherichia coliby SoxS
Authors: Paterson E. Suzanne , Boucher Sherri E. , Lambert I. B. 
Published: July 2002

ABSTRACTInEscherichia coli,the response to oxidative stress due to elevated levels of superoxide is mediated, in part, by thesoxRSregulon. One member of thesoxRSregulon,nfsA,encodes the major oxygen-insensitive nitroreductase inEscherichia coliwhich catalyzes the reduction of nitroaromatic and nitroheterocyclic compounds by NADPH. In this study we investigate the regulation ofnfsAin response to the superoxide generating compound paraquat. The transcription start site (TSS) ofnfsAwas located upstream of theybjCgene, a small open reading frame of unknown function located directly upstream ofnfsA,suggesting that these two genes form an operon. The activity of the promoter associated with this TSS was confirmed withlacZfusions and was shown to be inducible by paraquat. Footprinting and band shift analysis showed that purified His-tagged SoxS protein binds to a 20-base sequence 10 bases upstream of the −35 promoter sequence in the forward orientation, suggesting that theybjC-nfsApromoter is a class I SoxS-dependent promoter.
ArgR-Independent Induction and ArgR-Dependent Superinduction of theastCADBEOperon inEscherichia coli
Authors: Kiupakis Alexandros K. , Reitzer Larry 
Published: July 2002

ABSTRACTForEscherichia coli, growth in the absence of ammonia is termed nitrogen limited and results in the induction of genes that assimilate other nitrogen sources, a response mediated by σ54and nitrogen regulator I (NRI, also called NtrC). TheastCADBEoperon, which is required for growth with arginine as the sole nitrogen source, is moderately expressed during general nitrogen limitation and maximally expressed in the presence of arginine. The operon is also induced in stationary phase. Primer extension analysis ofE. colirevealed the presence of a σ54-dependent promoter utilized in exponential phase during nitrogen limitation and a σS-dependent promoter active during stationary phase. We used anast-lacZfusion to show that arginine stimulates expression, that ArgR, the arginine repressor, enhances expression from both promoters but is not essential, and that transcription by the two forms of the RNA polymerase is competitive and mutually exclusive. We demonstrated the binding of RNA polymerase holoenzymes, NRI, and ArgR to the promoter region in vitro. We also reconstituted transcription from both promoters with purified components, which confirmed the accessory role of ArgR for the σ54-dependent promoter. Thus, theastoperon exhibits nitrogen source-specific induction that is unique for an NRI-dependent gene. The transcriptional regulation of theastoperon inE. colidiffers from that inSalmonella entericaserovar Typhimurium, in which ArgR is required forastoperon expression.
The N Terminus of the
Escherichia coli
Transcription Activator MalT Is the Domain of Interaction with MalY
Authors: Schlegel Anja , Danot Olivier , Richet Evelyne , Ferenci Thomas , Boos Winfried 
Published: July 2002

ABSTRACT

The maltose system of
Escherichia coli
consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins. The system is positively regulated by MalT, its transcriptional activator. MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins. MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity. By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK. This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain. Mutants in MalT causing elevated
mal
gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors. The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them. Some of them became nearly completely resistant to Aes while still being sensitive to MalY. These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT.
Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters
Authors: Goffrini Paola , Ferrero Iliana , Donnini Claudia 
Published: July 2002

ABSTRACT

In many yeast species, including
Kluyveromyces lactis
, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained
K. lactis
strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for
K. lactis
cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in
K. lactis
and probably in many other yeasts.