bluuebunny/crossref_metadata_2025_split huggingface dataset:
1-Methylguanosine-Deficient tRNA of
Salmonella enterica
Serovar Typhimurium Affects Thiamine Metabolism
Authors: Björk Glenn R. , Nilsson Kristina 
Published: January 2003

ABSTRACT

In
Salmonella enterica
serovar Typhimurium a mutation in the
purF
gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a
purF
mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from
S. enterica
specific for leucine, proline, and arginine contain 1-methylguanosine (m
1
G37) adjacent to and 3′ of the anticodon (position 37). The formation of m
1
G37 is catalyzed by the enzyme tRNA(m
1
G37)methyltransferase, which is encoded by the
trmD
gene. Mutations in this gene, which result in an m
1
G37 deficiency in the tRNA, in a
purF
mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m
1
G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m
1
G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.
Agrobacterium tumefaciens
Twin-Arginine-Dependent Translocation Is Important for Virulence, Flagellation, and Chemotaxis but Not Type IV Secretion
Authors: Ding Zhiyong , Christie Peter J. 
Published: January 2003

ABSTRACT

This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen
Agrobacterium tumefaciens
. In contrast to wild-type strain A348, a
tatC
null mutant failed to export the green fluorescent protein fused to the trimethylamine
N
-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth. The
tatC
mutant displayed defects in growth rate and cell division but not in cell viability, and it also released abundant levels of several proteins into the culture supernatant when grown in rich medium or in
vir
induction minimal medium. Nearly all A348 cells were highly motile in both rich and minimal media. By contrast, approximately 0.1% of the
tatC
mutant cells were motile in rich medium, and <0.01% were motile in
vir
induction medium. Nonmotile
tatC
mutant cells lacked detectable flagella, whereas motile
tatC
mutant cells collected from the edge of a motility halo possessed flagella but not because of reversion to a functional TAT system. Motile
tatC
cells failed to exhibit chemotaxis toward sugars under aerobic conditions or towards nitrate under anaerobic conditions. The
tatC
mutant was highly attenuated for virulence, only occasionally (∼15% of inoculations) inciting formation of small tumors on plants after a prolonged incubation period of 6 to 8 weeks. However, an enriched subpopulation of motile
tatC
mutants exhibited enhanced virulence compared to the nonmotile variants. Finally, the
tatC
mutant transferred T-DNA and protein effectors to plant cells and a mobilizable IncQ plasmid to agrobacterial recipients at wild-type levels. Together, our findings establish that, in addition to its role in secretion of folded cofactor-bound enzymes functioning in alternative respiration, the TAT system of
A. tumefaciens
is an important virulence determinant. Furthermore, this secretion pathway contributes to flagellar biogenesis and chemotactic responses but not to sensory perception of plant signals or the assembly of a type IV secretion system.
Development of a Gene Knockout System for the Halophilic ArchaeonHaloferax volcaniiby Use of thepyrEGene
Authors: Bitan-Banin Gili , Ortenberg Ron , Mevarech Moshe 
Published: January 2003

ABSTRACTSo far, the extremely halophilic archaeonHaloferax volcaniihas the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development ofpyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. TheH. volacanii pyrE1andpyrE2genes were isolated, and thepyrE2gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A ΔpyrE2strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both thepyrE2wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.
Sizing the Holin Lesion with an Endolysin-β-Galactosidase Fusion
Authors: Wang Ing-Nang , Deaton John , Young Ry 
Published: January 2003

ABSTRACT
Double-stranded DNA phages require two proteins for efficient host lysis: the endolysin, a muralytic enzyme, and the holin, a small membrane protein. In an event that defines the end of the vegetative cycle, the λ holin S acts suddenly to permeabilize the membrane. This permeabilization enables the R endolysin to attack the cell wall, after which cell lysis occurs within seconds. A C-terminal fusion of the R endolysin with full-length β-galactosidase (β-Gal) was tested for lytic competence in the context of the late-gene expression system of an induced λ lysogen. Under these conditions, the hybrid R-β-Gal product, an active tetrameric β-Gal greater than 480 kDa in mass, was fully functional in lysis mediated by the S holin. Western blot analysis demonstrated that the lytic competence was not due to the proteolytic release of the endolysin domain of the R-β-Gal fusion protein. The ability of this massive complex to be released by the S holin suggests that S causes a generalized membrane disruption rather than a regular oligomeric membrane pore. Similar results were obtained with an early lysis variant of the S holin and also in parallel experiments with the T4 holin, T, in an identical lambda context. However, premature holin lesions triggered by depolarization of the membrane were nonpermissive for the hybrid endolysin, indicating that these premature lesions constituted less-profound damage to the membrane. Finally, a truncated T holin functional in lysis with the endolysin is completely incompetent for lysis with the hybrid endolysin. A model for the formation of the membrane lesion within homo-oligomeric rafts of holin proteins is discussed.
RmpA2, an Activator of Capsule Biosynthesis inKlebsiella pneumoniaeCG43, Regulates K2cpsGene Expression at the Transcriptional Level
Authors: Lai Yi-Chyi , Peng Hwei-Ling , Chang Hwan-You 
Published: January 2003

ABSTRACTThermpA2gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid ofKlebsiella pneumoniaeCG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression ofcpsgenes that are responsible for CPS biosynthesis. TwoluxABtranscriptional fusions, each containing a putative promoter region of theK. pneumoniaeK2cpsgenes, were constructed and were found to be activated in the presence of multicopyrmpA2. The activation is likely due to direct binding of RmpA2 to thecpsgene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in aK. pneumoniaestrain deficient in RcsB, a regulator forcpsgene expression, could be recovered by complementing the strain with a multicopy plasmid carryingrmpA2. The CPS production in Lon protease-deficientK. pneumoniaesignificantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of thermpA2gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3element located upstream of thermpA2was required for the full activation of thermpA2promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis inK. pneumoniaeCG43 by RmpA2 can be attributed to its transcriptional activation of K2cpsgenes, and the expression level ofrmpA2is autoregulated and under the control of Lon protease.
Role for both DNA and RNA in GTP Hydrolysis by the
Neisseria gonorrhoeae
Signal Recognition Particle Receptor
Authors: Frasz Cody , Arvidson Cindy Grove 
Published: January 2003

ABSTRACT

The prokaryotic signal recognition particle (SRP) targeting system is a complex of two proteins, FtsY and Ffh, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic membrane cotranslationally. We previously showed that
Neisseria gonorrhoeae
PilA is the gonococcal FtsY homolog. In this work, we isolated the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and characterized the interactions among the three SRP components by using gel retardation and nitrocellulose filter-binding assays and enzymatic analyses of the two proteins. In the current model of prokaryotic SRP function, based on studies of the
Escherichia coli
and mammalian systems, Ffh binds to 4.5S RNA and the Ffh-4.5S RNA complex binds to the signal sequence of nascent peptides and then docks with FtsY at the membrane. GTP is hydrolyzed by both proteins synergistically, and the nascent peptide is transferred to the translocon. We present evidence that the in vitro properties of the gonococcal SRP differ from those of previously described systems. GTP hydrolysis by PilA, but not that by Ffh, was stimulated by 4.5S RNA, suggesting a direct interaction between PilA and 4.5S RNA that has not been reported in other systems. This interaction was confirmed by gel retardation analyses in which PilA and Ffh, both alone and together, bound to 4.5S RNA. An additional novel finding was that P

pilE

DNA, previously shown by us to bind PilA in vitro, also stimulates PilA GTP hydrolysis. On the basis of these data, we hypothesize that DNA may play a role in targeting proteins via the SRP.
Quorum Sensing in
Rhizobium
sp. Strain NGR234 Regulates Conjugal Transfer (
tra
) Gene Expression and Influences Growth Rate
Authors: He Xuesong , Chang William , Pierce Deanne L. , Seib Laura Ort , Wagner Jennifer , Fuqua Clay 
Published: January 2003

ABSTRACT

Rhizobium
sp. strain NGR234 forms symbiotic, nitrogen-fixing nodules on a wide range of legumes via functions largely encoded by the plasmid pNGR234a. The pNGR234a sequence revealed a region encoding plasmid replication (
rep
) and conjugal transfer (
tra
) functions similar to those encoded by the
rep
and
tra
genes from the tumor-inducing (Ti) plasmids of
Agrobacterium tumefaciens
, including homologues of the Ti plasmid quorum-sensing regulators TraI, TraR, and TraM. In
A. tumefaciens
, TraI, a LuxI-type protein, catalyzes synthesis of the acylated homoserine lactone (acyl-HSL)
N
-3-oxo-octanoyl-
l
-homoserine lactone (3-oxo-C8-HSL). TraR binds 3-oxo-C8-HSL and activates expression of Ti plasmid
tra
and
rep
genes, increasing conjugation and copy number at high population densities. TraM prevents this activation under noninducing conditions. Although the pNGR234a TraR, TraI, and TraM appear to function similarly to their
A. tumefaciens
counterparts, the TraR and TraM orthologues are not cross-functional, and the quorum-sensing systems have differences. NGR234 TraI synthesizes an acyl-HSL likely to be 3-oxo-C8-HSL, but
traI
mutants and a pNGR234a-cured derivative produce low levels of a similar acyl-HSL and another, more hydrophobic signal molecule. TraR activates expression of several pNGR234a
tra
operons in response to 3-oxo-C8-HSL and is inhibited by TraM. However, one of the pNGR234a
tra
operons is not activated by TraR, and conjugal efficiency is not affected by TraR and 3-oxo-C8-HSL. The growth rate of NGR234 is significantly decreased by TraR and 3-oxo-C8-HSL through functions encoded elsewhere in the NGR234 genome.
β-Lactamase Genes of the Penicillin-SusceptibleBacillus anthracisSterne Strain
Authors: Chen Yahua , Succi Janice , Tenover Fred C. , Koehler Theresa M. 
Published: January 2003

ABSTRACTSusceptibility to penicillin and other β-lactam-containing compounds is a common trait ofBacillus anthracis. β-lactam agents, particularly penicillin, have been used worldwide to treat anthrax in humans. Nonetheless, surveys of clinical and soil-derived strains reveal penicillin G resistance in 2 to 16% of isolates tested. Bacterial resistance to β-lactam agents is often mediated by production of one or more types of β-lactamases that hydrolyze the β-lactam ring, inactivating the antimicrobial agent. Here, we report the presence of two β-lactamase (bla) genes in the penicillin-susceptible Sterne strain ofB. anthracis. We identifiedbla1by functional cloning withEscherichia coli. bla1is a 927-nucleotide (nt) gene predicted to encode a protein with 93.8% identity to the type I β-lactamase gene ofBacillus cereus. A second gene,bla2, was identified by searching the unfinishedB. anthracischromosome sequence database of The Institute for Genome Research for open reading frames (ORFs) predicted to encode β-lactamases. We found a partial ORF predicted to encode a protein with significant similarity to the carboxy-terminal end of the type II β-lactamase ofB. cereus. DNA adjacent to the 5′ end of the partial ORF was cloned using inverse PCR.bla2is a 768-nt gene predicted to encode a protein with 92% identity to theB. cereustype II enzyme. Thebla1andbla2genes confer ampicillin resistance toE. coliandBacillus subtiliswhen cloned individually in these species. The MICs of various antimicrobial agents for theE. coliclones indicate that the two β-lactamase genes confer different susceptibility profiles toE. coli;bla1is a penicillinase, whilebla2 appears to be a cephalosporinase. The β-galactosidase activities ofB. cereusgroup species harboringblapromoter-lacZtranscriptional fusions indicate thatbla1is poorly transcribed inB. anthracis,B. cereus, andB. thuringiensis.Thebla2gene is strongly expressed inB. cereusandB. thuringiensisand weakly expressed inB. anthracis. Taken together, these data indicate that thebla1andbla2genes of theB. anthracisSterne strain encode functional β-lactamases of different types, but gene expression is usually not sufficient to confer resistance to β-lactam agents.
Elucidation of the
Vibrio anguillarum
Genetic Response to the Potential Fish Probiont
Pseudomonas fluorescens
AH2, Using RNA-Arbitrarily Primed PCR
Authors: Holmstrøm Kim , Gram Lone 
Published: January 2003

ABSTRACT

The antagonistic interaction between a potential fish probiont,
Pseudomonas fluorescens
strain AH2, and its target organism,
Vibrio anguillarum
, was investigated by studying the genetic response of the target organism when it was exposed to the antagonist. We compared the differential display of arbitrarily PCR-amplified gene transcripts in
V. anguillarum
serotype O1 when it was exposed to AH2 supernatant with the display of transcripts in nonexposed control cultures. Growth of
V. anguillarum
was immediately arrested when the organism was exposed to 50% (vol/vol) AH2 supernatant. A total of 10 potentially differentially expressed transcripts were identified. Among these we identified a gene homologous to
rpoS
that was induced in a dose-dependent manner when
V. anguillarum
was cultured in media supplemented with sterile filtered supernatant from AH2.
rpoS
was also induced when growth was arrested with the iron chelator 2,2-dipyridyl. A chromosomal transcript homologous to
vibE
that participates in vibriobactin synthesis in
Vibrio cholerae
was also upregulated during AH2 exposure. This transcript could represent a functionally active gene in
V. anguillarum
involved in biosynthesis of anguibactin or another
V. anguillarum
siderophore. On the pJM1 plasmid of
V. anguillarum
serotype O1, a pseudogene designated open reading frame E (ORF E) that contains a frameshift mutation was previously identified. The gene homologous to
vibE
identified in this study, interestingly, also has significant homology to ORF E on the amino acid level and does not possess the frameshift mutation. Thus, the chromosomally encoded
vibE
homologue could fulfil the role of the inactive plasmid-encoded ORF E pseudogene. Addition of Fe
3+
to the system eliminated the growth arrest, and the genes homologous to
rpoS
and
vibE
were not induced. To our knowledge, this is the first study linking
rpoS
induction to iron starvation. Taken together, the results of this study suggest that a major part of the antagonistic property exhibited by strain AH2 is caused by the ability of siderophores in the supernatant to efficiently chelate iron, which results in instant iron deprivation of the pathogen
V. anguillarum
and complete growth arrest.
TheEscherichia coliBarA-UvrY Two-Component System Is Needed for Efficient Switching between Glycolytic and Gluconeogenic Carbon Sources
Authors: Pernestig Anna-Karin , Georgellis Dimitris , Romeo Tony , Suzuki Kazushi , Tomenius Henrik , Normark Staffan , Melefors Öjar 
Published: January 2003

ABSTRACTTheEscherichia coliBarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene,barA, or the response regulator gene,uvrY, in uropathogenicE. colidrastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment.
CcpA-Independent Regulation of Expression of the Mg2+-Citrate Transporter GenecitMby Arginine Metabolism inBacillus subtilis
Authors: Warner Jessica B. , Magni Christian , Lolkema Juke S. 
Published: January 2003

ABSTRACTTranscriptional regulation of the Mg2+-citrate transporter, CitM, the main citrate uptake system ofBacillus subtilis, was studied during growth in rich medium. Citrate in the growth medium was required for induction under all growth conditions. In Luria-Bertani medium containing citrate,citMexpression was completely repressed during the exponential growth phase, marginally expressed in the transition phase, and highly expressed in the stationary growth phase. The repression was relieved when the cells were grown in spent Luria-Bertani medium. The addition of a mixture of 18 amino acids restored repression.l-Arginine in the mixture appeared to be solely responsible for the repression, and ornithine appeared to be an equally potent repressor ofcitMexpression. Studies of mutant strains deficient in RocR and SigL, proteins required for the expression of the enzymes of the arginase pathway, confirmed that uptake into the cell and, most likely, conversion of arginine to ornithine were required for repression. Arginine-mediated repression was independent of a functional CcpA, the global regulator protein in carbon catabolite repression (CCR). Nevertheless, CCR-mediated repression was the major mechanism controlling the expression during exponential growth, while the newly described, CcpA-independent arginine-mediated repression was specifically apparent during the transition phase of growth.
2,5-Dialkylresorcinol Biosynthesis in
Pseudomonas aurantiaca
: Novel Head-to-Head Condensation of Two Fatty Acid-Derived Precursors
Authors: Nowak-Thompson Brian , Hammer Philip E. , Hill D. Steven , Stafford Jill , Torkewitz Nancy , Gaffney Thomas D. , Lam Stephen T. , Molnár István , Ligon James M. 
Published: January 2003

ABSTRACT

2-Hexyl-5-propylresorcinol is the predominant analog of several dialkylresorcinols produced by
Pseudomonas aurantiaca
(
Pseudomonas fluorescens
BL915). We isolated and characterized three biosynthetic genes that encode an acyl carrier protein, a β-ketoacyl-acyl carrier protein synthase III, and a protein of unknown function, all of which collectively allow heterologous production of 2-hexyl-5-propylresorcinol in
Escherichia coli.
Two regulatory genes exhibiting similarity to members of the AraC family of transcriptional regulators are also present in the identified gene cluster. Based on the deduced functions of the proteins encoded by the gene cluster and the observed incorporation of labeled carbons from octanoic acid into 2-hexyl-5-propylresorcinol, we propose that dialkylresorcinols are derived from medium-chain-length fatty acids by an unusual head-to-head condensation of β-ketoacyl thioester intermediates. Genomic evidence suggests that there is a similar pathway for the biosynthesis of the flexirubin-type pigments in certain bacteria belonging to the order
Cytophagales
.
Characterization, Expression, and Mutation of the
Lactococcus lactis galPMKTE
Genes, Involved in Galactose Utilization via the Leloir Pathway
Authors: Grossiord Benoît P. , Luesink Evert J. , Vaughan Elaine E. , Arnaud Alain , de Vos Willem M. 
Published: January 2003

ABSTRACT

A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in
Lactococcus lactis
subsp.
cremoris
MG1363 was cloned and characterized. The order of the genes is
galPMKTE
, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the
galP
,
galK
,
galT
, and
galE
genes was shown by complementation studies performed with both
Escherichia coli
and
L. lactis
mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using
galP
disruption mutant strains of
L. lactis
MG1363. A
galK
deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the
galE
gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the
galE
mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.