bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Phenanthrene Dioxygenase from
Nocardioides
sp. Strain KP7: Expression in
Escherichia coli
Authors: Saito Atsushi , Iwabuchi Tokuro , Harayama Shigeaki 
Published: July 2002

ABSTRACT

Nocardioides
sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate,
o
-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to
o
-phthalate (
phd
) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight
phd
genes was cloned and sequenced. The
phdA
,
phdB
,
phdC
, and
phdD
genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster,
phdAB
, was located 8.3 kb downstream of the previously characterized
phdK
gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The
phdCD
gene cluster was located 2.9 kb downstream of the
phdB
gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The
phdABCD
genes were expressed poorly in
Escherichia coli
, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the
phdABCD
genes improved their expression in
E. coli. E. coli
cells carrying
phdBCD
or
phdACD
exhibited no phenanthrene-degrading activity, and those carrying
phdABD
or
phdABC
exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the
phdABCD
genes. It was thus concluded that all of the
phdABCD
genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the
phd
genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in
Nocardioides
sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.
Multiple Hexose Transporters ofSchizosaccharomyces pombe
Authors: Heiland Sylvia , Radovanovic Nada , Höfer Milan , Winderickx Joris , Lichtenberg Hella 
Published: July 2002

ABSTRACTWe have identified a family of six hexose transporter genes (Ght1toGht6) in the fission yeastSchizosaccharomyces pombe. Sequence homology toSaccharomyces cerevisiaeand mammalian hexose transporters (Hxtp and GLUTp, respectively) and secondary-structure predictions of 12 transmembrane domains for each of the Ght proteins place them into the sugar porter subfamily within the major facilitator superfamily. Interestingly, among this sugar porter family, the emergingS. pombehexose transporter family clusters are separate from monosaccharide transporters of other yeasts (S. cerevisiae,Kluyveromyces lactis, andCandida albicans) and of humans, suggesting that these proteins form a distinct structural family of hexose transporters. Expression of theGht1,Ght2,Ght5, andGht6genes in theS. cerevisiaemutant RE700A may functionally complement itsd-glucose uptake-deficient phenotype. Northern blot analysis and reverse transcription-PCR showed that among allGht's ofS. pombe,Ght5is the most prominently expressed hexose transporter. Ght1p, Ght2p, and Ght5p displayed significantly higher specificities ford-glucose than ford-fructose. Analysis of the previously describedS. pombed-glucose transport-deficient mutant YGS-5 revealed that this strain is defective in theGht1,Ght5, andGht6genes. Based on an analysis of threeS. pombestrains bearing single or double mutations inGht3andGht4, we conclude that the Ght3p function is required ford-gluconate transport inS. pombe. The function of Ght4p remains to be clarified. Ght6p exhibited a slightly higher affinity tod-fructose than tod-glucose, and among the Ght's it is the transporter with the highest specificity ford-fructose.
Recombinational Repair Is Critical for Survival of
Escherichia coli
Exposed to Nitric Oxide
Authors: Spek Erik J. , Wright Teresa L. , Stitt Molly S. , Taghizadeh Nazbeh R. , Tannenbaum Steven R. , Marinus Martin G. , Engelward Bevin P. 
Published: July 2002

ABSTRACT

Nitric oxide (NO
⋅
) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO
⋅
-induced DNA damage on
Escherichia coli
. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO
⋅
-induced toxicity was determined.
E. coli
strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [
alkA tag
strain], oxidative damage [
fpg nei nth
strain], and deaminated cytosine [
ung
strain]) showed essentially wild-type levels of NO
⋅
resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (
xth nfo
strain) were very sensitive to killing by NO
⋅
, which indicates that normal processing of abasic sites is critical for defense against NO
⋅
. In addition,
recA
mutant cells were exquisitely sensitive to NO
⋅
-induced killing. Both SOS-deficient (
lexA3
) and Holliday junction resolvase-deficient (
ruvC
) cells were very sensitive to NO
⋅
, indicating that both SOS and recombinational repair play important roles in defense against NO
⋅
. Furthermore, strains specifically lacking double-strand end repair (
recBCD
strains) were very sensitive to NO
⋅
, which suggests that NO
⋅
exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO
⋅
induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO
⋅
, it is also important to consider recombination events among the spectrum of genetic changes that NO
⋅
can induce. Furthermore, the importance of recombinational repair for cellular survival of NO
⋅
exposure reveals a potential susceptibility factor for invading microbes.
Glucose-Induced Monoubiquitination of the
Saccharomyces cerevisiae
Galactose Transporter Is Sufficient To Signal Its Internalization
Authors: Horak Jaroslav , Wolf Dieter H. 
Published: July 2002

ABSTRACT

In
Saccharomyces cerevisiae
, the addition of glucose to cells growing on galactose induces internalization of the galactose transporter Gal2p and its subsequent proteolysis in the vacuole. Here we report that the essential step in Gal2p down-regulation is its ubiquitination through the Ubc1p-Ubc4p-Ubc5p triad of ubiquitin-conjugating enzymes and Npi1/Rsp5p ubiquitin-protein ligase. Moreover, Gal2p appears to be stabilized in mutant cells defective in the ubiquitin-hydrolase Npi2p/Doa4p, and the mutant phenotype can be reversed by overexpression of ubiquitin. An analysis of the fate of Gal2p in cells overexpressing wild-type ubiquitin as well as its variants incompetent to form polyubiquitin chains showed that monoubiquitination of Gal2p is sufficient to signal internalization of the protein into the endocytic pathway.
Tagging Morphogenetic Genes by Insertional Mutagenesis in the Yeast
Yarrowia lipolytica
Authors: Richard Mathias , Quijano Raymundo Rosas , Bezzate Samira , Bordon-Pallier Florence , Gaillardin Claude 
Published: July 2002

ABSTRACT

The yeast
Yarrowia lipolytica
is distantly related to
Saccharomyces cerevisiae
, can be genetically modified, and can grow in both haploid and diploid states in either yeast, pseudomycelial, or mycelial forms, depending on environmental conditions. Previous results have indicated that the
STE
and
RIM
pathways, which mediate cellular switching in other dimorphic yeasts, are not required for
Y. lipolytica
morphogenesis. To identify the pathways involved in morphogenesis, we mutagenized a wild-type strain of
Y. lipolytica
with a Tn
3
derivative. We isolated eight tagged mutants, entirely defective in hyphal formation, from a total of 40,000 mutants and identified seven genes homologous to
S. cerevisiae CDC25, RAS2, BUD6, KEX2, GPI7, SNF5
, and
PPH21
. We analyzed their abilities to invade agar and to form pseudomycelium or hyphae under inducing conditions and their sensitivity to temperature and to Calcofluor white. Chitin staining was used to detect defects in their cell walls. Our results indicate that a functional Ras-cyclic AMP pathway is required for the formation of hyphae in
Y. lipolytica
and that perturbations in the processing of extracellular, possibly parietal, proteins result in morphogenetic defects.
Transcriptional Pattern of Genes Coding for the Proteolytic System of
Lactococcus lactis
and Evidence for Coordinated Regulation of Key Enzymes by Peptide Supply
Authors: Guédon Eric , Renault Pierre , Ehrlich S. Dusko , Delorme Christine 
Published: July 2002

ABSTRACT

The transcription of 16 genes encoding 12 peptidases (
pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM
, and
pepO1
), P
I
and P
III
proteinases (
prtP1
and
prtP3
), and three transport systems (
dtpT, dtpP,
and
opp-pepO1
) of
Lactococcus lactis
MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37°C, and peptide supply on the transcription of these genes. Only transcription of the
pepP
gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes.
prtP1, prtP3, pepC, pepN, pepX,
and the
opp-pepO1
operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription of
prtP1, prtP3, pepC, pepN
, and the
opp-pepO1
operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of
pepDA2
might also be repressed by the peptide sources, but this effect is not observed on the regulation of
dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM
, and the
dtpP
operon. The significance of these results with respect to the functions of different components of the proteolytic system in
L. lactis
are discussed.
Plasmid-Encoded Phthalate Catabolic Pathway in
Arthrobacter keyseri
12B
Authors: Eaton Richard W. 
Published: July 2002

ABSTRACT

Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown
Arthrobacter keyseri
(formerly
Micrococcus
sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant
Escherichia coli
strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp
Pst
I fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units:
tnpR, pcm
operon,
ptr
genes,
pehA, norA
fragment, and
pht
operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant
E. coli
strains.
Role of a Ferredoxin Gene Cotranscribed with the
nifHDK
Operon in N
2
Fixation and Nitrogenase “Switch-Off” of
Azoarcus
sp. Strain BH72
Authors: Egener Tanja , Martin Dietmar E. , Sarkar Abhijit , Reinhold-Hurek Barbara 
Published: July 2002

ABSTRACT

The endophytic diazotroph
Azoarcus
sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (
nif
) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (
nifHDK
) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of
nifHDK
, a ferredoxin gene (
fdxN
; 59% amino acid sequence identity to
R. capsulatus
FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to
nifY
of
Pseudomonas stutzeri
A15 and ORF1 of
Azotobacter vinelandii
were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a ς
54
-type promoter. Shorter transcripts sequentially missing genes of the 3′ part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of
fdxN
resulted in reduced growth rates (59% ± 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an
fdxN
mutant which carried a
nifH
promoter-driven
fdxN
gene in
trans
. Also, in coculture with the ascomycete
Acremonium alternatum
, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the
fdxN
deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the
fdxN
deletion also had an effect on the rapid “switch-off” of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with
fdxN
in
trans
showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.
Glycerol 3-Phosphate Inhibits Swarming and Aggregation of
Myxococcus xanthus
Authors: Moraleda-Muñoz Aurelio , Carrero-Lérida Juana , Extremera Antonio L. , Arias José M. , Muñoz-Dorado José 
Published: July 2002

ABSTRACT

We have cloned a gene of
Myxococcus xanthus
with similarities to the permease for glycerol 3-phosphate (G3P) of other bacteria. Expression of the gene increased significantly during the first hours of starvation. Swarming of the wild-type strain was inhibited and aggregation was delayed by G3P. Conversely, a Δ
glpT
strain aggregated even on rich medium. These results indicate that G3P may function to regulate the timing of aggregation in
M. xanthus
.
Complementation of the Essential Peptidoglycan Transpeptidase Function of Penicillin-Binding Protein 2 (PBP2) by the Drug Resistance Protein PBP2A in
Staphylococcus aureus
Authors: Pinho Mariana G. , Filipe Sérgio R. , de Lencastre Hermı́nia , Tomasz Alexander 
Published: July 2002

ABSTRACT

The essential function of penicillin-binding protein 2 (PBP2) in methicillin-susceptible
Staphylococcus aureus
RN4220 was clearly established by placing the
pbp2
gene under control of the inducible P
spac
promoter; the resulting bacteria were unable to grow in the absence of inducer. In contrast, the deficit in PBP2 caused by inhibition of transcription of the
pbp2
gene did not block growth of a methicillin-resistant
S. aureus
strain expressing the extra penicillin-binding protein PBP2A, a protein of extraspecies origin that is central to the mechanism of methicillin resistance. Several lines of evidence indicate that the essential function of PBP2 that can be compensated for by PBP2A is the transpeptidase activity. This provides direct genetic evidence that PBP2A has transpeptidase activity.
Identification of a Genomic Island Present in the Majority of Pathogenic Isolates of
Pseudomonas aeruginosa
Authors: Liang Xiaoyou , Pham Xuan-Quynh T. , Olson Maynard V. , Lory Stephen 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic
P. aeruginosa
strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from
P. aeruginosa
strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human
P. aeruginosa
isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for “
P. aeruginosa
genomic island 1”). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of
P. aeruginosa
isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow
P. aeruginosa
to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the
P. aeruginosa
chromosome, which provides a selective advantage for strains that have acquired this genomic island.
Purification of the RelB and RelE Proteins of
Escherichia coli
: RelE Binds to RelB and to Ribosomes
Authors: Galvani Cheryl , Terry Jefferson , Ishiguro Edward E. 
Published: July 2002

ABSTRACT

The direct interaction of the
Escherichia coli
cytotoxin RelE with its specific antidote, RelB, was demonstrated in two ways: (i) copurification of the two proteins and (ii) a positive yeast two-hybrid assay involving the
relB
and
relE
genes. In addition, the purified RelE protein exhibited ribosome-binding activity in an in vitro assay, supporting previous observations suggesting that it is an inhibitor of translation.
Streptococcus salivarius
Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure
Authors: Lévesque Céline , Vadeboncoeur Christian , Chandad Fatiha , Frenette Michel 
Published: July 2002

ABSTRACT

Streptococcus salivarius
, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of
S. salivarius
fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of
S. salivarius
ATCC 25975) with glass beads. Preliminary experiments showed that
S. salivarius
fimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 10
6
to 40 × 10
6
Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that
S. salivarius
fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species,
Streptococcus mitis
and
Streptococcus constellatus
.
The Active Component of the Bioemulsifier Alasan from
Acinetobacter radioresistens
KA53 Is an OmpA-Like Protein
Authors: Toren Amir , Orr Elisha , Paitan Yossi , Ron Eliora Z. , Rosenberg Eugene 
Published: July 2002

ABSTRACT

The bioemulsifier of
Acinetobacter radioresistens
KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from
Acinetobacter
spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in
Escherichia coli
. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of
E. coli
OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 μg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein.
E. coli
OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.