bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proline Catabolism byPseudomonas putida: Cloning, Characterization, and Expression of theputGenes in the Presence of Root Exudates
Authors: Vílchez Susana , Molina Lázaro , Ramos Cayo , Ramos Juan L. 
Published: April 2009

ABSTRACTPseudomonas putidaKT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. AP. putidamutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5–Km mutagenesis. The mini-Tn5insertion was located at theputAgene, which is adjacent to and divergent from theputPgene. TheputAgene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein ofEscherichia coli,Salmonella entericaserovar Typhimurium,Rhodobacter capsulatus, and severalRhizobiumstrains. The central part ofP. putidaPutA showed homology to the proline dehydrogenase ofSaccharomyces cerevisiaeandDrosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase ofS. cerevisiaeand a number of aldehyde dehydrogenases. This suggests that inP. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. TheputPgene was homologous to theputPgenes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In aP. putida putA-deficient background, expression of bothputAandputPgenes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced theP. putida putgenes, as determined by using fusions of theputpromoters to ′lacZ. The induction ratio for theputApromoter (about 20-fold) was 6-fold higher than the induction ratio for theputPpromoter.
Novel Medium-Chain Prenyl Diphosphate Synthase from the Thermoacidophilic Archaeon
Sulfolobus solfataricus
Authors: Hemmi Hisashi , Ikejiri Satoru , Yamashita Satoshi , Nishino Tokuzo 
Published: April 2009

ABSTRACT

Two open reading frames which encode the homologues of (all-
E
) prenyl diphosphate synthase are found in the whole-genome sequence of
Sulfolobus solfataricus
, a thermoacidophilic archaeon. It has been suggested that one is a geranylgeranyl diphosphate synthase gene, but the specificity and biological significance of the enzyme encoded by the other have remained unclear. Thus, we isolated the latter by the PCR method, expressed the enzyme in
Escherichia coli
cells, purified it, and characterized it. The archaeal enzyme, 281 amino acids long, is highly thermostable and requires Mg
2+
and Triton X-100 for full activity. It catalyzes consecutive
E
-type condensations of isopentenyl diphosphate with an allylic substrate such as geranylgeranyl diphosphate and yields the medium-chain product hexaprenyl diphosphate. Despite such product specificity, phylogenetic analysis revealed that the archaeal medium-chain prenyl diphosphate synthase is distantly related to the other medium- and long-chain enzymes but is closely related to eucaryal short-chain enzymes.
The ClpXP ATP-Dependent Protease Regulates Flagellum Synthesis inSalmonella entericaSerovar Typhimurium
Authors: Tomoyasu Toshifumi , Ohkishi Tomiko , Ukyo Yoshifumi , Tokumitsu Akane , Takaya Akiko , Suzuki Masato , Sekiya Kachiko , Matsui Hidenori , Kutsukake Kazuhiro , Yamamoto Tomoko 
Published: April 2009

ABSTRACTThe ClpXP protease is a member of the ATP-dependent protease family and plays a dynamic role in the control of availability of regulatory proteins and the breakdown of abnormal and misfolded proteins. The proteolytic activity is rendered by the ClpP component, while the substrate specificity is determined by the ClpX component that has ATPase activity. We describe here a new role of the ClpXP protease inSalmonella entericaserovar Typhimurium in which ClpXP is involved in the regulation of flagellum synthesis. Cells deleted for ClpXP show “hyperflagellate phenotype,” exhibit overproduction of the flagellar protein, and show a fourfold increase in the rate of transcription of thefliCencoding flagellar filament. The assay for promoter activity of the genes responsible for expression of thefliCshowed that the depletion of ClpXP results in dramatic enhancement of the expression of thefliAencoding sigma factor ς28, leaving the expression level of theflhDmaster operon lying at the top of the transcription hierarchy of flagellar regulon almost normal. These results suggest that the ClpXP may be responsible for repressing the expression of flagellar regulon through the control of the FlhD/FlhC master regulators at the posttranscriptional and/or posttranslational levels. Proteome analysis of proteins secreted from the mutant cells deficient forflhDCandclpXPgenes demonstrated that the ΔflhDmutation abolished the enhanced effect by ΔclpXPmutation on the production of flagellar proteins, suggesting that the ClpXP possibly defines a regulatory pathway affecting the expression of flagellar regulon that is dependent on FlhD/FlhC master regulators.
Characterization of Indigoidine Biosynthetic Genes in
Erwinia chrysanthemi
and Role of This Blue Pigment in Pathogenicity
Authors: Reverchon Sylvie , Rouanet Carine , Expert Dominique , Nasser William 
Published: April 2009

ABSTRACT

In the plant-pathogenic bacterium
Erwinia chrysanthemi
production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain,
E. chrysanthemi ind
mutants deficient in indigoidine synthesis were isolated by screening a library of Tn
5
-B21 insertions in a
pecS
mutant. These
ind
mutations were localized close to the regulatory
pecS-pecM
locus, immediately downstream of
pecM
. Sequence analysis of this DNA region revealed three open reading frames,
indA
,
indB
, and
indC
, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of
ind
genes is strongly derepressed in the
pecS
background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses
indA
and
indC
expression by binding to
indA
and
indC
promoter regions. The regulatory link, via
pecS
, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in
E. chrysanthemi
pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted
Saintpaulia ionantha
. Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response.
Nested Evolution of a tRNA
Leu
(UAA) Group I Intron by both Horizontal Intron Transfer and Recombination of the Entire tRNA Locus
Authors: Rudi Knut , Fossheim Tonje , Jakobsen Kjetill S. 
Published: April 2009

ABSTRACT

The origin and evolution of bacterial introns are still controversial issues. Here we present data on the distribution and evolution of a recently discovered divergent tRNA
Leu
(UAA) intron. The intron shows a higher sequence affiliation with introns in tRNA
Ile
(CAU) and tRNA
Arg
(CCU) genes in α- and β-proteobacteria, respectively, than with other cyanobacterial tRNA
Leu
(UAA) group I introns. The divergent tRNA
Leu
(UAA) intron is sporadically distributed both within the
Nostoc
and the
Microcystis
radiations. The complete tRNA gene, including flanking regions and intron from
Microcystis aeruginosa
strain NIVA-CYA 57, was sequenced in order to elucidate the evolutionary pattern of this intron. Phylogenetic reconstruction gave statistical evidence for different phylogenies for the intron and exon sequences, supporting an evolutionary model involving horizontal intron transfer. The distribution of the tRNA gene, its flanking regions, and the introns were addressed by Southern hybridization and PCR amplification. The tRNA gene, including the flanking regions, were absent in the intronless stains but present in the intron-containing strains. This suggests that the sporadic distribution of this intron within the
Microcystis
genus cannot be attributed to intron mobility but rather to an instability of the entire tRNA
Leu
(UAA) intron-containing genome region. Taken together, the complete data set for the evolution of this intron can best be explained by a model involving a nested evolution of the intron, i.e., wherein the intron has been transferred horizontally (probably through a single or a few events) to a tRNA
Leu
(UAA) gene which is located within a unstable genome region.
Novel Pathway for Alcoholic Fermentation of δ-Gluconolactone in the YeastSaccharomyces bulderi
Authors: van Dijken Johannes P. , van Tuijl Arjen , Luttik Marijke A. H. , Middelhoven Wouter J. , Pronk Jack T. 
Published: April 2009

ABSTRACTUnder anaerobic conditions, the yeastSaccharomyces bulderirapidly ferments δ-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for δ-gluconolactone fermentation operates in this yeast. In this pathway, δ-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in δ-gluconolactone fermentation inS. bulderiis supported by several experimental observations. (i) Fermentation of δ-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as δ-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP+-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, δ-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on δ-gluconolactone, CO2production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO2. (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in δ-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in δ-gluconolactone metabolism.
Analysis of
ftsQ
Mutant Alleles in
Escherichia coli
: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins
Authors: Chen Joseph C. , Minev Michael , Beckwith Jon 
Published: April 2009

ABSTRACT

FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium
Escherichia coli
. To define residues in FtsQ critical for function, we performed random mutagenesis on the
ftsQ
gene and identified four alleles (
ftsQ2
,
ftsQ6
,
ftsQ15
, and
ftsQ65
) that fail to complement the
ftsQ1
(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an
ftsQ1
(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.
High-Salinity-Induced Iron Limitation in
Bacillus subtilis
Authors: Hoffmann Tamara , Schütz Alexandra , Brosius Margot , Völker Andrea , Völker Uwe , Bremer Erhard 
Published: April 2009

ABSTRACT

Proteome analysis of
Bacillus subtilis
cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively. Most of these protein spots were identified by mass spectrometry. Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin. These proteins are encoded by the
dhbACEBF
operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation. We found that iron limitation and high salinity derepressed
dhb
expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant. DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron. Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed
B. subtilis
cultures. Taken together, these findings strongly suggest that
B. subtilis
cells grown at high salinity experience iron limitation. In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress. The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of
B. subtilis
in natural settings, in which bioavailable iron is usually scarce.
Regulation of
mutY
and Nature of Mutator Mutations in
Escherichia coli
Populations under Nutrient Limitation
Authors: Notley-McRobb Lucinda , Pinto Rachel , Seeto Shona , Ferenci Thomas 
Published: April 2009

ABSTRACT

Previous analysis of aerobic, glucose-limited continuous cultures of
Escherichia coli
revealed that G:C-to-T:A (G:C→T:A) transversions were the most commonly occurring type of spontaneous mutation. One possible explanation for the preponderance of these mutations was that nutrient limitation repressed MutY-dependent DNA repair, resulting in increased proportions of G:C→T:A transversions. The regulation of the
mutY
-dependent DNA repair system was therefore studied with a transcriptional
mutY-lacZ
fusion recombined into the chromosome. Expression from the
mutY
promoter was fourfold higher under aerobic conditions than under anaerobic conditions. But
mutY
expression was higher in glucose- or ammonia-limited chemostats than in nutrient-excess batch culture, so
mutY
was not downregulated by nutrient limitation. An alternative explanation for the frequency of G:C→T:A transversions was the common appearance of
mutY
mutator mutations in the chemostat populations. Of 11 chemostat populations screened in detail, six contained mutators, and the mutator mutation in four cultures was located in the region of
mutY
at 66 min on the chromosome. The spectrum of mutations and rate of mutation in these isolates were fully consistent with a
mutY
-deficiency in each strain. Based on PCR analysis of the region within and around
mutY
, isolates from three individual populations contained deletions extending at least 2 kb upstream of
mutY
and more than 5 kb downstream. In the fourth population, the deletion was even longer, extending at least 5 kb upstream and 5 kb downstream of
mutY
. The isolation of
mutY
mutator strains from four independent populations with extensive chromosomal rearrangements suggests that
mutY
inactivation by deletion is a means of increasing mutation rates under nutrient limitation and explains the observed frequency of G:C→T:A mutations in glucose-limited chemostats.
Rubrivivax gelatinosus acsF
(Previously
orf358
) Codes for a Conserved, Putative Binuclear-Iron-Cluster-Containing Protein Involved in Aerobic Oxidative Cyclization of Mg-Protoporphyrin IX Monomethylester
Authors: Pinta Violaine , Picaud Martine , Reiss-Husson Françoise , Astier Chantal 
Published: April 2009

ABSTRACT

This study describes the characterization of
orf358
, an open reading frame of previously unidentified function, in the purple bacterium
Rubrivivax gelatinosus
. A strain in which
orf358
was disrupted exhibited a phenotype similar to the wild type under photosynthesis or low-aeration respiratory growth conditions. In contrast, under highly aerated respiratory growth conditions, the wild type still produced bacteriochlorophyll
a
(Bchl
a
), while the disrupted strain accumulated a compound that had the same absorption and fluorescence emission spectra as Mg-protoporphyrin but was less polar, suggesting that it was Mg-protoporphyrin monomethylester (MgPMe). These data indicated a blockage in Bchl
a
synthesis at the oxidative cyclization stage and implied the coexistence of two different mechanisms for MgPMe cyclization in
R. gelatinosus
, an anaerobic mechanism active under photosynthesis or low oxygenation and an aerobic mechanism active under high-oxygenation growth conditions. Based on these results as well as on sequence analysis indicating the presence of conserved putative binuclear-iron-cluster binding motifs, the designation of
orf358
as
acsF
(for aerobic cyclization system Fe-containing subunit) is proposed. Several homologs of AcsF were found in a wide range of photosynthetic organisms, including
Chlamydonomas reinhardtii
Crd1 and
Pharbitis nil
PNZIP, suggesting that this aerobic oxidative cyclization mechanism is conserved from bacteria to plants.
Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain
Authors: Rashid Naeem , Cornista Joel , Ezaki Satoshi , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki 
Published: April 2009

ABSTRACT

A gene encoding a cyclodextrin glucanotransferase (CGTase) from
Thermococcus kodakaraensis
KOD1 (CGT

Tk

) was identified and characterized. The gene (
cgt

Tk

) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the α-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80°C and 5.5 to 6.0, respectively. The presence of Ca
2+
enhanced the enzyme activity and elevated the optimum temperature to 85 to 90°C. With the addition of Ca
2+
, the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85°C, and a half-life of 20 min at 100°C. CGT

Tk

could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT

Tk

harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with β-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT

Tk

ΔC) with 23 C-terminal amino acid residues deleted. CGT

Tk

ΔC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT

Tk

. In contrast, the cyclization activity of CGT

Tk

ΔC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT

Tk

to properly catalyze the cyclization reaction.
Galactose and Lactose Genes from the Galactose-Positive Bacterium
Streptococcus salivarius
and the Phylogenetically Related Galactose-Negative Bacterium
Streptococcus thermophilus
: Organization, Sequence, Transcription, and Activity of the
gal
Gene Products
Authors: Vaillancourt Katy , Moineau Sylvain , Frenette Michel , Lessard Christian , Vadeboncoeur Christian 
Published: April 2009

ABSTRACT

Streptococcus salivarius
is a lactose- and galactose-positive bacterium that is phylogenetically closely related to
Streptococcus thermophilus
, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the
S. salivarius
and
S. thermophilus gal-lac
gene clusters. The clusters have the same organization with the order
galR
(codes for a transcriptional regulator and is transcribed in the opposite direction),
galK
(galactokinase),
galT
(galactose-1-P uridylyltransferase),
galE
(UDP-glucose 4-epimerase),
galM
(galactose mutarotase),
lacS
(lactose transporter), and
lacZ
(β-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from
galR
, the
gal
operon,
galM
, and the
lac
operon of
S. salivarius
. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the
S. thermophilus gal
-
lac
gene cluster. An additional putative internal promoter at the 3′ end of
galT
was found in
S. thermophilus
but not in
S. salivarius
. The results clearly indicated that the
gal-lac
gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of
galT
and
galE
were identical in both species, whereas the ribosome binding site of
S. thermophilus galK
differed from that of
S. salivarius
by two nucleotides, suggesting that the
S. thermophilus galK
gene might be poorly translated. This was confirmed by measurements of enzyme activities.