bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of DNA Mismatch Repair and Double-Strand Break Repair in Genome Stability and Antifungal Drug Resistance in
Candida albicans
Authors: Legrand Melanie , Chan Christine L. , Jauert Peter A. , Kirkpatrick David T. 
Published: October 2007

ABSTRACT

Drug resistance has become a major problem in the treatment of
Candida albicans
infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (
MSH2
and
PMS1
) or double-strand break repair (
MRE11, RAD50, RAD52
, and
YKU80
). We show that the
mre11
Δ/
mre11
Δ,
rad50
Δ/
rad50
Δ, and
rad52
Δ/
rad52
Δ mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The
msh2
Δ/
msh2
Δ,
pms1
Δ/
pms1
Δ, and
yku80
Δ/
yku80
Δ mutants exhibit none of these phenotypes. We observed an increase in genome instability in
mre11
Δ/
mre11
Δ and
rad50
Δ/
rad50
Δ mutants by using a
GAL1
/
URA3
marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of
mre11
Δ/
mre11
Δ,
rad50
Δ/
rad50
Δ, or
rad52
Δ/
rad52
Δ leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both
msh2
Δ/
msh2
Δ and
pms1
Δ/
pms1
Δ (MMR mutants) and
rad50
Δ/
rad50
Δ (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in
C. albicans
strains lacking mismatch repair proteins or proteins central to double-strand break repair.
Kinetics of Polynucleotide Phosphorylase: Comparison of Enzymes from
Streptomyces
and
Escherichia coli
and Effects of Nucleoside Diphosphates
Authors: Chang Samantha A. , Cozad Madeline , Mackie George A. , Jones George H. 
Published: October 2007

ABSTRACT

We examined the activity of polynucleotide phosphorylase (PNPase) from
Streptomyces coelicolor
,
Streptomyces antibioticus
, and
Escherichia coli
in phosphorolysis using substrates derived from the
rpsO-pnp
operon of
S. coelicolor
. The
Streptomyces
and
E. coli
enzymes were both able to digest a substrate with a 3′ single-stranded tail although
E. coli
PNPase was more effective in digesting this substrate than were the
Streptomyces
enzymes. The
k
cat
for the
E. coli
enzyme was ca. twofold higher than that observed with the
S. coelicolor
enzyme.
S. coelicolor
PNPase was more effective than its
E. coli
counterpart in digesting a substrate possessing a 3′ stem-loop structure, and the

K
m

for the
E. coli
enzyme was ca. twice that of the
S. coelicolor
enzyme. Electrophoretic mobility shift assays revealed an increased affinity of
S. coelicolor
PNPase for the substrate possessing a 3′ stem-loop structure compared with the
E. coli
enzyme. We observed an effect of nucleoside diphosphates on the activity of the
S. coelicolor
PNPase but not the
E. coli
enzyme. In the presence of a mixture of 20 μM ADP, CDP, GDP, and UDP, the

K
m

for the phosphorolysis of the substrate with the 3′ stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of
E. coli
PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.
The Metalloprotease ofListeria monocytogenesIs Activated by Intramolecular Autocatalysis
Authors: Bitar Alan Pavinski , Cao Min , Marquis Hélène 
Published: October 2007

ABSTRACTThe metalloprotease (Mpl) ofListeria monocytogenesis a thermolysin-like protease that mediates the maturation of a broad-range phospholipase C, whose function contributes to the ability of this food-borne bacterial pathogen to survive intracellularly. Mpl is made as a proprotein that undergoes maturation by proteolytic cleavage of a large N-terminal prodomain. In this study, we identified the N terminus of mature Mpl and generated Mpl catalytic mutants to investigate the mechanism of Mpl maturation. We observed that Mpl activity was a prerequisite for maturation, suggesting a mechanism of autocatalysis. Furthermore, using a strain ofL. monocytogenesexpressing both the wild-type form and a catalytic mutant form of Mpl simultaneously, we determined that in vivo maturation of Mpl occurs exclusively by an intramolecular autocatalysis mechanism.
Effects of Single-Strand DNases ExoI, RecJ, ExoVII, and SbcCD on Homologous Recombination of
recBCD
+
Strains of
Escherichia coli
and Roles of SbcB15 and XonA2 ExoI Mutant Enzymes
Authors: Thoms Brigitte , Borchers Inka , Wackernagel Wilfried 
Published: October 2007

ABSTRACT

To assess the contributions of single-strand DNases (ssDNases) to recombination in a
recBCD
+
background, we studied 31 strains with all combinations of null alleles of exonuclease I (Δ
xon
), exonuclease VII (
xseA
), RecJ DNase (
recJ
), and SbcCD DNase (
sbcCD
) and exonuclease I mutant alleles
xonA2
and
sbcB15
. The
xse recJ sbcCD
Δ
xon
and
xse recJ sbcCD sbcB15
quadruple mutants were cold sensitive, while the quadruple mutant with
xonA2
was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with
sbcB15
were generally like the wild type. We determined that the
sbcB15
mutation caused an A183V exchange in exonuclease motif III and identified
xonA2
as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon
+
), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3′ DNA overhangs, giving protection against Xon
+
. In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in
recBC
strains, contributes independently of RecF to recombination in
recBCD
+
cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with
sbcB15
.
Interactions of the Energy Transducer TonB with Noncognate Energy-Harvesting Complexes
Authors: Brinkman Kerry K. , Larsen Ray A. 
Published: October 2007

ABSTRACTThe TonB and TolA proteins are energy transducers that couple the ion electrochemical potential of the cytoplasmic membrane to support energy-dependent processes at the outer membrane of the gram-negative envelope. The transfer of energy to these transducers is facilitated by energy-harvesting complexes, which are heteromultimers of cytoplasmic membrane proteins with homologies to proton pump proteins of the flagellar motor. Although the cognate energy-harvesting complex best services each transducer, components of the complexes (for TonB, ExbB and ExbD; for TolA, TolQ and TolR) are sufficiently similar that each complex can imperfectly replace the other. Previous investigations of this molecular cross talk considered energy-harvesting complex components expressed from multicopy plasmids in strains in which the corresponding genes were interrupted by insertions, partially absent due to polarity, or missing due to a larger deletion. These questions were reexamined here using strains in which individual genes were removed by precise deletions and, where possible, components were expressed from single-copy genes with native promoters. By more closely approximating natural stoichiometries between components, this study provided insight into the roles of energy-harvesting complexes in both the energization and the stabilization of TonB. Further, the data suggest a distinct role for ExbD in the TonB energy transduction cycle.
Roles of Ring-Hydroxylating Dioxygenases in Styrene and Benzene Catabolism inRhodococcus jostiiRHA1
Authors: Patrauchan Marianna A. , Florizone Christine , Eapen Shawn , Gómez-Gil Leticia , Sethuraman Bhanu , Fukuda Masao , Davies Julian , Mohn William W. , Eltis Lindsay D. 
Published: October 2007

ABSTRACTProteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene inRhodococcus jostiiRHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAaandetbAa1::cmrA etbAa2::aphIImutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion ofpcaLof the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded viameta- andortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.
Phosphoketolase Pathway Dominates in
Lactobacillus reuteri
ATCC 55730 Containing Dual Pathways for Glycolysis
Authors: Årsköld Emma , Lohmeier-Vogel Elke , Cao Rong , Roos Stefan , Rådström Peter , van Niel Ed W. J. 
Published: October 2007

ABSTRACT

Metabolic flux analysis indicated that the heterofermentative
Lactobacillus reuteri
strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of
L. reuteri
ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro
31
P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.
A New TetR Family Transcriptional Regulator Required for Morphogenesis inStreptomyces coelicolor
Authors: Hillerich Brandan , Westpheling Janet 
Published: October 2007

ABSTRACTBoth morphogenesis and antibiotic production in the streptomycetes are initiated in response to starvation, and these events are coupled. We previously described a transposon-generated mutant inStreptomyces coelicolor, SE293, that resulted in abldstrain that overproduced the antibiotic actinorhodin. The SCO1135 open reading frame identified by the insertion encodes a member of the TetR family of transcriptional regulators. Here we show that a constructed deletion of the SCO1135 open reading frame resulted in the same morphological and antibiotic production phenotype as the insertion mutant. The constructed deletion also resulted in constitutive expression of SCO1135 transcript, as well as that of the gene cluster immediately adjacent to it, SCO1134-1132, which encodes a putative molybdopterin binding complex. A His6-tagged version of the SCO1135 protein product was shown to bind the intergenic region between SCO1135 and SCO1134, which contains the apparent transcription start sites for each gene mapped by primer extension analysis. Increased expression of the SCO1134-1132 transcript in the SCO1135 deletion mutant also resulted in increased expression of xanthine dehydrogenase activity, confirming the predictions about these open reading framed based on protein similarity. We have designated the SCO1134-1142 gene clusterxdhABCand the regulator encoded by SCO1135xdhR. We speculate that the inappropriate expression of xanthine dehydrogenase affects purine salvaging pathways at the onset of development, creating artificially high concentrations of both GTP and ppGpp and perturbing the pathways these molecules participate in for the initiation of morphogenesis and antibiotic production.
The Two Chorismate Mutases from both
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
: Biochemical Analysis and Limited Regulation of Promoter Activity by Aromatic Amino Acids
Authors: Schneider Cristopher Z. , Parish Tanya , Basso Luiz A. , Santos Diógenes S. 
Published: October 2007

ABSTRACT

Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, plants, and apicomplexan parasites. Since this enzyme is absent from mammals, it represents a promising target for the development of new antimycobacterial drugs, which are needed to combat
Mycobacterium tuberculosis
, the causative agent of tuberculosis. Until recently, two putative open reading frames (ORFs), Rv0948c and Rv1885c, showing low sequence similarity to CMs have been described as “conserved hypothetical proteins” in the
M. tuberculosis
genome. However, we and others demonstrated that these ORFs are in fact monofunctional CMs of the AroQ structural class and that they are differentially localized in the mycobacterial cell. Since homologues to the
M. tuberculosis
enzymes are also present in
Mycobacterium smegmatis
, we cloned the coding sequences corresponding to ORFs MSMEG5513 and MSMEG2114 from the latter. The CM activities of both ORFs was determined, as well as their translational start sites. In addition, we analyzed the promoter activities of three
M. tuberculosis
loci related to phenylalanine and tyrosine biosynthesis under a variety of conditions using
M. smegmatis
as a surrogate host. Our results indicate that the
aroQ
(Rv0948c), *
aroQ
(Rv1885c), and
fbpB
(Rv1886c) genes from
M. tuberculosis
are constitutively expressed or subjected to minor regulation by aromatic amino acids levels, especially tryptophan.
Transcriptome Dynamics during the Transition from Anaerobic Photosynthesis to Aerobic Respiration in
Rhodobacter sphaeroides
2.4.1
Authors: Arai Hiroyuki , Roh Jung Hyeob , Kaplan Samuel 
Published: October 2007

ABSTRACT

Rhodobacter sphaeroides
2.4.1 is a facultative photosynthetic anaerobe that grows by anoxygenic photosynthesis under anaerobic-light conditions. Changes in energy generation pathways under photosynthetic and aerobic respiratory conditions are primarily controlled by oxygen tensions. In this study, we performed time series microarray analyses to investigate transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration. Major changes in gene expression profiles occurred in the initial 15 min after the shift from anaerobic-light to aerobic-dark conditions, with changes continuing to occur up to 4 hours postshift. Those genes whose expression levels changed significantly during the time series were grouped into three major classes by clustering analysis. Class I contained genes, such as that for the
aa
3
cytochrome oxidase, whose expression levels increased after the shift. Class II contained genes, such as those for the photosynthetic apparatus and Calvin cycle enzymes, whose expression levels decreased after the shift. Class III contained genes whose expression levels temporarily increased during the time series. Many genes for metabolism and transport of carbohydrates or lipids were significantly induced early during the transition, suggesting that those endogenous compounds were initially utilized as carbon sources. Oxidation of those compounds might also be required for maintenance of redox homeostasis after exposure to oxygen. Genes for the repair of protein and sulfur groups and uptake of ferric iron were temporarily upregulated soon after the shift, suggesting they were involved in a response to oxidative stress. The flagellar-biosynthesis genes were expressed in a hierarchical manner at 15 to 60 min after the shift. Numerous transporters were induced at various time points, suggesting that the cellular composition went through significant changes during the transition from anaerobic photosynthesis to aerobic respiration. Analyses of these data make it clear that numerous regulatory activities come into play during the transition from one homeostatic state to another.
Two Segments in Bacterial Antizyme P22 Are Essential for Binding and Enhance Degradation of Lysine/Ornithine Decarboxylase in
Selenomonas ruminantium
Authors: Yamaguchi Yoshihiro , Takatsuka Yumiko , Kamio Yoshiyuki 
Published: October 2007

ABSTRACT

In
Selenomonas ruminantium
, a strictly anaerobic and gram-negative bacterium, the degradation of lysine/ornithine decarboxylase (LDC/ODC) by ATP-requiring protease(s) is accelerated by the binding of P22, which is a ribosomal protein of this strain. Amino acid sequence alignment of
S. ruminantium
P22 with the L10 ribosomal proteins of gram-positive and -negative bacteria showed that P22 has a 5-residue K
101
NKLD
105
segment and an 11-residue G
160
VIRNAVYVLD
170
segment, both of which are lacking in L10 in any other gram-positive and gram-negative bacteria reported. To elucidate whether the two segments are involved in P22 function, a series of mutant genes of P22 were constructed and expressed in
Escherichia coli
. The proteins were isolated and assayed for their function with respect to
S. ruminantium
LDC/ODC and mouse ODC. The results indicated that the two segments of P22 are crucial for P22 binding to both enzymes and also accelerated degradation of both decarboxylases.
Two Closely Related ABC Transporters in
Streptococcus mutans
Are Involved in Disaccharide and/or Oligosaccharide Uptake
Authors: Webb Alexander J. , Homer Karen A. , Hosie Arthur H. F. 
Published: October 2007

ABSTRACT

Streptococcus mutans
has a large number of transporters apparently involved in the uptake of carbohydrates. At least two of these, the multiple sugar metabolism transporter, MsmEFGK, and the previously uncharacterized MalXFGK, are members of the ATP-binding cassette (ABC) superfamily. Mutation analysis revealed that the MsmEFGK and MalXFGK transporters are principally involved in the uptake of distinct disaccharides and/or oligosaccharides. Furthermore, the data also indicated an unusual protein interaction between the components of these two related transporters. Strains lacking
msmE
(which encodes a solute binding protein) can no longer utilize raffinose or stachyose but grow normally on maltodextrins in the absence of MalT, a previously characterized EII
mal
phosphotransferase system component. In contrast, a mutant of
malX
(which encodes a solute binding protein) cannot utilize maltodextrins but grows normally on raffinose or stachyose. Radioactive uptake assays confirmed that MalX, but not MsmE, is required for uptake of [U-
14
C]maltotriose and that MalXFGK is principally involved in the uptake of maltodextrins with as many as 7 glucose units. Surprisingly, inactivation of the corresponding ATPase components did not result in an equivalent abolition of growth: the
malK
mutant can grow on maltotetraose as a sole carbon source, and the
msmK
mutant can utilize raffinose. We propose that the ATPase domains of these ABC transporters can interact with either their own or the alternative transporter complex. Such unexpected interaction of ATPase subunits with distinct membrane components to form complete multiple ABC transporters may be widespread in bacteria.
p300 expression repression by hypermethylation associated with tumour invasion and metastasis in oesophageal squamous cell carcinoma
Authors: Zhang Changsong , Li Ke , Wei Lixin , Li Zhengyou , Yu Ping , Teng Lijuan , Wu Kusheng , Zhu Jin 
Published: October 2007

Background:Aberrant promoter methylation is an important mechanism for gene silencing.Aims:To evaluate the promoter methylation status of p300 gene in patients with oesophageal squamous cell carcinoma (OSCC).Methods:The methylation status of p300 promoter was analysed by methylation-specific PCR (MSP) in 50 OSCC tissues and the matching non-cancerous tissues. Oesophageal cancer cell lines (ECa-109 and TE-10) were treated with the demethylation agent 5-aza-2′-deoxycytidine (5-Aza-CdR), and p300 mRNA expression was detected by RT-PCR.Results:p300 methylation was found in 42% (21/50) of the OSCC tissues, but in only 20% (10/50) of the corresponding non-cancerous tissues (p = 0.017). In OSCC samples, 65% of those with deep tumour invasion (adventitia) and 63% samples with metastasis revealed p300 promoter methylation (p<0.05). p300 mRNA expression was observed in 19.0% (4/21) of methylated tumours and 58.6% (17/29) of unmethylated tumours (p = 0.005). In addition, p300 mRNA expression was observed in 40% (4/10) of methylated non-neoplastic tissues and 87.5% (35/40) of unmethylated non-tumours (p = 0.001). The demethylation caused by 5-Aza-CdR increased the p300 mRNA expression levels in oesophageal cancer cell lines.Conclusions:p300 transcription silenced by promoter hypermethylation could play a role in the pathogenesis of oesophageal squamous cell carcinoma.