bluuebunny/crossref_metadata_2025 huggingface dataset:
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

So 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

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

ThermpA2gene, 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

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

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

Susceptibility 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

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

TheEscherichia 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

Transcriptional 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

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

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.
Postdivisional Synthesis of theSporosarcina ureaeDNA Translocase SpoIIIE either in the Mother Cell or in the Prespore EnablesBacillus subtilisTo Translocate DNA from the Mother Cell to the Prespore
Authors: Chary Vasant K. , Piggot Patrick J.
Published: January 2003

The differentiation of vegetative cells ofBacillus subtilisinto spores involves asymmetric cell division, which precedes complete chromosome partitioning. The DNA translocase SpoIIIE is required to translocate the origin distal 70% of the chromosome from the larger mother cell into the smaller prespore, the two cells that result from the division. We have tested the effect of altering the time and location of SpoIIIE synthesis on spore formation. We have expressed thespoIIIEhomologue fromSporosarcina ureaeinB. subtilisunder the control of different promoters. Expression from either a weak mother cell-specific (σE) promoter or a weak prespore-specific (σF) promoter partly complemented the sporulation defect of aspoIIIE36mutant; however, expression from a strong prespore-specific (σF) promoter did not. DNA translocation from the mother cell to the prespore was assayed usingspoIIQ-lacZinserted atthrC; transcription ofspoIIQoccurs only in the prespore. Translocation ofthrC::spoIIQ-lacZinto the prespore occurred efficiently whenspoIIIESuwas expressed from the weak σE- or σF-controlled promoters but not when it was expressed from the strong σF-controlled promoter. It is speculated that the mechanism directing SpoIIIE insertion into the septum in the correct orientation may accommodate slow postseptational, prespore-specific SpoIIIE synthesis but may be swamped by strong prespore-specific synthesis.
Requirements for Cu
A
and Cu-S Center Assembly of Nitrous Oxide Reductase Deduced from Complete Periplasmic Enzyme Maturation in the Nondenitrifier
Pseudomonas putida
Authors: Wunsch Patrick , Herb Margitta , Wieland Hagen , Schiek Ulrike M. , Zumft Walter G.
Published: January 2003

Bacterial nitrous oxide (N
2
O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N
2
O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu
A
site in the periplasm. There are seven putative maturation factors, encoded by
nosA
,
nosD
,
nosF
,
nosY
,
nosL
,
nosX
, and
sco
. We wanted to determine the indispensable proteins by expressing
nos
genes from
Pseudomonas stutzeri
in the nondenitrifying organism
Pseudomonas putida
. An in silico study of denitrifying bacteria revealed that
nosL
,
nosX
(or a homologous gene,
apbE
), and
sco
, but not
nosA
, coexist consistently with the N
2
O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with
nosRZ
in
trans
was sufficient to produce catalytically active holo-N
2
O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with
P. stutzeri
, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu
A
metallation. Both of these were found to be dispensable elements for N
2
O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N
2
O reductase maturation.
Plant Lectin-Like Bacteriocin from a Rhizosphere-Colonizing
Pseudomonas
Isolate
Authors: Parret Annabel H. A. , Schoofs Geert , Proost Paul , De Mot René
Published: January 2003

Rhizosphere isolate
Pseudomonas
sp. strain BW11M1, which belongs to the
Pseudomonas putida
cluster, secretes a heat- and protease-sensitive bacteriocin which kills
P. putida
GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a Tn
Mod
mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into
Escherichia coli
cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in
E. coli
. This gene was designated
llp
A (
l
ectin-
l
ike
p
utidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the
llpA
gene also conveyed bacteriocin production to several
Pseudomonas fluorescens
strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described
Pseudomonas
bacteriocins.
Heme-Responsive Transcriptional Activation of
Bordetella bhu
Genes
Authors: Vanderpool Carin K. , Armstrong Sandra K.
Published: January 2003

Bordetella pertussis
and
Bordetella bronchiseptica
, gram-negative respiratory pathogens of mammals, possess a heme iron utilization system encoded by the
bhuRSTUV
genes. Preliminary evidence suggested that expression of the BhuR heme receptor was stimulated by the presence of heme under iron-limiting conditions. The
hurIR
(heme uptake regulator) genes were previously identified upstream of the
bhuRSTUV
gene cluster and are predicted to encode homologs of members of the iron starvation subfamily of extracytoplasmic function (ECF) regulators. In this study,
B. pertussis
and
B. bronchiseptica
Δ
hurI
mutants, predicted to lack an ECF σ factor, were constructed and found to be deficient in the utilization of hemin and hemoglobin. Genetic complementation of Δ
hurI
strains with plasmid-borne
hurI
restored wild-type levels of heme utilization.
B. bronchiseptica
Δ
hurI
mutant BRM23 was defective in heme-responsive production of the BhuR heme receptor;
hurI
in
trans
restored heme-inducible BhuR expression to the mutant and resulted in BhuR overproduction. Transcriptional analyses with
bhuR-lacZ
fusion plasmids confirmed that
bhuR
transcription was activated in iron-starved cells in response to heme compounds. Heme-responsive
bhuR
transcription was not observed in mutant BRM23, indicating that
hurI
is required for positive regulation of
bhu
gene expression. Furthermore,
bhuR
was required for heme-inducible
bhu
gene activation, supporting the hypothesis that positive regulation of
bhuRSTUV
occurs by a surface signaling mechanism involving the heme-iron receptor BhuR.
Recombination Activity of a Distinctive Integron-Gene Cassette System Associated withPseudomonas stutzeriPopulations in Soil
Authors: Holmes Andrew J. , Holley Marita P. , Mahon Andrew , Nield Blair , Gillings Michael , Stokes H. W.
Published: January 2003

Class 1 integrons have strongly influenced the evolution of multiple antibiotic resistance. Diverse integrons have recently been detected directly in a range of natural environments. In order to characterize the properties of these environmental integrons, we sought to isolate organisms containing integrons from soils, which resulted in the isolation ofPseudomonas stutzeristrain Q. Further isolation efforts targeted at this species resulted in recovery of two other strains (P and BAM). 16S rRNA sequences and chromosome mapping showed that these three strains are very closely related clonal variants in a single genomovar ofP. stutzeri. Only strains Q and BAM were found to contain an integron and an associated gene cassette array. TheintIandattIcomponents of these strains showed 99 and 90% identity, respectively. The structure of these integrons and their associated gene cassettes was similar to that reported previously for other integron classes. The two integrons contained nonoverlapping sets of cassette-associated genes. In contrast, many of the cassette-associated recombination sites in the two integrons were similar and were considered to constitute a distinct subfamily consisting of 59-base element (59-be) recombination sites (thePseudomonassubfamily). The recombination activity ofP. stutzeriintegron components was tested in cointegrate assays. IntIPstQ was shown to catalyze site-specific recombination between its cognateattIsite and 59-be sites from antibiotic resistance gene cassettes. While IntIPstQ did not efficiently mediate recombination between members of thePseudomonas59-be subfamily and other 59-be types, the former sites were functional when they were tested with IntI1. We concluded that integrons present inP. stutzeripossess recombination activity and represent a hot spot for genomic diversity in this species.
In Vivo Analysis of HPr Reveals a Fructose-Specific Phosphotransferase System That Confers High-Affinity Uptake inStreptomyces coelicolor
Authors: Nothaft Harald , Parche Stephan , Kamionka Annette , Titgemeyer Fritz
Published: January 2003

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organismStreptomyces coelicolor, the encoding gene,ptsH, was deleted. TheptsHmutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparentKmof 2 μM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putativefruKAgene locus led to identification of the fructose-specific enzyme II permease encoded by thefruAgene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis ofptsHrevealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in aptsHbackground, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS inS. coelicolor.
Characterization of a Bifunctional Archaeal Acyl Coenzyme A Carboxylase
Authors: Chuakrut Songkran , Arai Hiroyuki , Ishii Masaharu , Igarashi Yasuo
Published: January 2003

Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from
Acidianus brierleyi
. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an α
4
β
4
γ
4
subunit structure. The optimum temperature for the enzyme was 60 to 70°C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent

K
m

for acetyl-CoA was 0.17 ± 0.03 mM, with a
V
max
of 43.3 ± 2.8 U mg
−1
, and the

K
m

for propionyl-CoA was 0.10 ± 0.008 mM, with a
V
max
of 40.8 ± 1.0 U mg
−1
. This result showed that
A. brierleyi
acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of
Bacteria
than to those of
Eucarya
. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit.
A SecE Mutation That Modulates SecY-SecE Translocase Assembly, Identified as a Specific Suppressor of SecY Defects
Authors: Mori Hiroyuki , Akiyama Yoshinori , Ito Koreaki
Published: January 2003

The SecY39(Cs) (cold-sensitive) alteration of Arg357 results in a defect of translocation initiation. As a means to dissect the Sec translocation machinery, we isolated mutations that act as suppressors of the
secY39
defect. A specific
secE
mutation, designated
secE105
, was thus isolated. This mutation proved to be identical with the
prlG2
mutation and to suppress a number of cold-sensitive
secY
mutations. However, other
prlG
mutations did not effectively suppress the
secY
defects. Evidence indicates that the Ser105-to-Pro alteration in the C-terminal transmembrane segment of SecE weakens SecY-SecE association. In vitro analyses showed that the SecE(S105P) alteration preferentially stimulates the initial phase of translocation. It is suggested that the S105P alteration affects the SecYEG channel such that it is more prone to open and to accept the translocation initiation domain of a preprotein molecule.
Characterization of the Stringent Response and

rel
Bbu

Expression in
Borrelia burgdorferi
Authors: Bugrysheva Julia , Dobrikova Elena Y. , Sartakova Marina L. , Caimano Melissa J. , Daniels Thomas J. , Radolf Justin D. , Godfrey Henry P. , Cabello Felipe C.
Published: January 2003

The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious
Borrelia burgdorferi
strain B31 and infectious
B. burgdorferi
strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in
B. burgdorferi
under these conditions. Here we report that (p)ppGpp levels in
B. burgdorferi
growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of
B. burgdorferi
(

rel
Bbu

) was produced by B31 as by N40 during in vitro growth (2 ± 0.5 and 4 ± 0.8 fg of

rel
Bbu

mRNA/ng of total
Borrelia
RNA, respectively). Although the amounts of N40

rel
Bbu

mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in

rel
Bbu

mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to
Bacillus subtilis
Rel
Bsu
were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in

rel
Bbu

promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40

rel
Bbu

ATG start codon and identical σ
70
-like promoters were identified by primer extension and sequencing analysis.

rel
Bbu

complemented an
Escherichia coli
CF1693
relA spoT
double mutant for growth on M9 minimal medium, and the transformed cells produced

rel
Bbu

mRNA. These results indicate that

rel
Bbu

is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in
B. burgdorferi
, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in
E. coli
and that (p)ppGpp may not be responsible for growth rate control.
Rates and Consequences of Recombination between rRNA Operons
Authors: Hashimoto Joel G. , Stevenson Bradley S. , Schmidt Thomas M.
Published: January 2003

A mutant strain of
Escherichia coli
was created by inserting a cassette encoding sucrose sensitivity and neomycin resistance (
sacB-neo
) into the small-subunit rRNA-encoding gene
rrs
in the
rrnB
operon. During growth in a complex medium, the cassette was lost from the population, and a complete
rrs
gene was restored at a rate of 5 × 10
−9
per cell division. Repair of this lesion required flanking regions of DNA that were similar to the six remaining intact rRNA operons and reestablished the full complement of seven rRNA operons. The relative fitness of strains with restored
rrnB
operons was 1 to 2% higher than that of the mutant strain. The
rrnB
operon normally contains a spacer region between the 16S and 23S rRNA-encoding genes that is similar in length and tRNA gene content to the spacer in
rrnC
, -
E
, and -
G
. In 2 of the 14 strains in which
rrnB
was restored, the spacer region had the same length as the spacer region in
rrnA
, -
D
, and -
H
. The requirement for flanking regions of nearly identical DNA and the replication of the spacer region from other rRNA operons during the repair of
rrnB
suggest that the restoration was accomplished via gene conversion. The rate of gene conversion was 10-fold less than the fixation of point mutations in the same region of the chromosome but was apparently sufficient to homogenize the sequences of rRNA genes in
E. coli
. These findings are discussed in the context of a conceptual model describing the presence of sequence heterogeneity in coevolving rRNA genes.
The Absence of FtsH Metalloprotease Activity Causes Overexpression of the σ
W
-Controlled
pbpE
Gene, Resulting in Filamentous Growth of
Bacillus subtilis
Authors: Zellmeier Stephan , Zuber Ulrich , Schumann Wolfgang , Wiegert Thomas
Published: January 2003

FtsH is a membrane-bound and energy-dependent metalloprotease in bacteria which is involved in the posttranslational control of the activity of a variety of important transcription factors and in the degradation of uncomplexed integral membrane proteins. For
Bacillus subtilis
, little is known about the target proteins of FtsH protease. Its gene is not essential, but knockout strains display a pleiotropic phenotype including sensitivity toward salt and heat stress, defects in sporulation and competence, and largely filamentous growth. Comparison of the intracellular proteomes of wild-type and
ftsH
knockout strains revealed that at least nine proteins accumulated in the absence of
ftsH
, four of which could be identified. Two of these proteins turned out to be members of the σ
W
regulon. Accumulation of one of these σ
W
-controlled proteins, the penicillin-binding protein PBP4*, was analyzed in more detail. We could show that PBP4* is not a proteolytic substrate of FtsH and that its overproduction is due to the enhanced transcription of its gene (
pbpE
) in
ftsH
null mutants. The filamentous growth phenotype of Δ
ftsH
strains was abolished in a Δ
ftsH
Δ
pbpE
double knockout. In
ftsH
wild-type strains with the
pbpE
gene under regulatable control,
pbpE
overexpression caused filamentation of the cells. DNA macroarray analysis revealed that most genes of the σ
W
regulon are transcribed at elevated levels in an
ftsH
mutant. The influence of FtsH on σ
W
-controlled genes is discussed.
Escherichia coliEndoribonucleases Involved in Cleavage of Bacteriophage T4 mRNAs
Authors: Otsuka Yuichi , Ueno Hiroyuki , Yonesaki Tetsuro
Published: January 2003

Thedmdmutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA. Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon. Other cleavages are independent of translation. In the present study, by introducing plasmids carrying varioussocalleles, we could detect cleavages ofsocRNA in uninfected cells identical to those found indmdmutant-infected cells. We isolated fiveEscherichia colimutant strains in which thedmdmutant was able to grow. One of these strains completely suppressed thedmdmutant-specific cleavages ofsocRNA. The loci of theE. colimutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing thedmdmutant-specific mRNA degradation is attributable to a novel RNase. In addition, we present evidence that 5′-truncatedsocRNA, a stable form in T4-infected cells regardless of the presence of admdmutation, is generated by RNase E.
Characterization of SrgA, a
Salmonella enterica
Serovar Typhimurium Virulence Plasmid-Encoded Paralogue of the Disulfide Oxidoreductase DsbA, Essential for Biogenesis of Plasmid-Encoded Fimbriae
Authors: Bouwman C. W. , Kohli M. , Killoran A. , Touchie G. A. , Kadner R. J. , Martin N. L.
Published: January 2003

Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of
Salmonella enterica
serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example,
Escherichia coli
, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family
Enterobacteriaceae
. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmid-encoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the
S. enterica
serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not.
pefA
and
srgA
were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in
pef
gene expression are also involved in
srgA
expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of
S. enterica
serovar Typhimurium.
Dynamic crushing of auxetic open-cell polyurethane foam
Authors: Scarpa F , Yates J R , Ciffo L G , Patsias S
Published: January 2003

A high strain rate compression test with a constant speed of 1.5 m/s has been performed on samples of negative Poisson's ratio and normal open-cell polyurethane foam. The tests show that the transformation of the normal foam into the auxetic phase greatly increases the crashworthiness qualities of the open-cell foam.
Failure of an internally threaded structure: The valve centre spool
Authors: Akour S N , Nayfeh J F
Published: January 2003

The failure of the internally threaded centre spool of large valves is investigated. Brittle fracture occurred at the root of the internally threaded area. Several visual tests were performed. Visual investigation shows that the failure occurs on the last thread engaged with the bolt. Primary finite element method analysis shows that the last thread root has the highest stress concentration and opening mode of fracture mechanics. Redesign optimization and a parametric study are carried out. Reduction of the stress concentration is achieved and so the fracture is prevented.
Analysis of a flexural wave due to a laser heating pulse: The two-layer assembly case
Authors: Yilbas B S , Hyder S J
Published: January 2003

In the present study laser ablation of a two-layer assembly with a cantilever arrangement is considered. The recoil pressure generated at the vapour front-workpiece interface is formulated and the resulting pressure force (loading force) is computed. The flexural motion of the workpiece due to the loading force is formulated. The first layer of the workpiece is Inconel 625 with a thickness on the order of 200 μm, which may resemble the HVOF (high-velocity oxygen fuel) coating, while the second layer is 1 mm thick stainless steel plate. The simulations are repeated for four values of the first layer thickness and the relation between the magnitude of the displacement and the first layer thickness is discussed. It is found that the displacement of the workpiece reaches as high as 2 μm in the region close to the free end, while the equivalent stress level is high in the region close to the fixed end. The temporal behaviour of the equivalent stress almost follows the displacement behaviour of the workpiece. As the first layer thickness increases, the displacement reduces. The variation of displacement with the first layer thickness is almost linear and the slope of this variation attains high values for certain time periods in the flexural motion.
Geometric synthesis of spatial parallel manipulators with fewer than six degrees of freedom
Authors: Zhao T S , Dai J S , Huang Z
Published: January 2003

Manipulators with fewer than six degrees of freedom meet specific tasks and have the advantage of reducing structural complexity, design redundancy and cost. In order to construct parallel manipulators for given tasks, this paper develops an algebraic approach to type synthesis of spatial parallel mechanisms with fewer than six degrees of freedom based on the screw theory. With the proposed steps (i.e. describing restraining screws, identifying basic kinematic pair (KP) screws reciprocal to the restraining screws, linearly transforming the basic KP screws to obtain equivalent serial limbs and allocating the serial limbs) new parallel mechanisms can be constructed. The approach converts a mechanism design into a screw algebra operation, in which screws describe kinematic pairs and constraints between links. As examples, synthesis procedures of parallel mechanisms with four degrees of freedom are given, from which five novel parallel mechanisms result.
Numerical study on the propulsive performance of a wiggling blade in bubbly flow
Authors: Uchiyama T
Published: January 2003

In order to search for an efficient propulsion mechanism in an air-water bubbly flow, the propulsive performance of a blade wiggling in the bubbly flow is analysed by a two-dimensional numerical method. The blade, whose geometry is similar to an NACA65–010 hydrofoil, is set in a straight conduit, in which the bubbly mixture flows. The wiggling motion is expressed by a progressive wave with reference to the swimming motions of fish. The bubbly flow is calculated by an incompressible two-fluid model in conjunction with the finite element method proposed by the author in an earlier paper. The calculations reveal the effects of a progressive waveform and volumetric fraction of air upstream of the blade on the propulsive performance of the blade. The time variations of the flow properties around the blade are also discussed in relation to the blade motion and propulsive performance.
Analysis of flowrates as a basis for the simulation of dry-running rotational positive displacement pumps
Authors: Kauder K , Wenderott D
Published: January 2003

New applications improve the market situation of dry-running positive displacement pumps. The mostly empirically based design of these pumps has to take into account partly competing viewpoints. These viewpoints are energetic process optimization, on the one hand, and operational reliability, on the other hand. A simulation system can be used to solve this problem. The simulation system uses an energy and a mass balance in order to simulate the operational behaviour of the vacuum pumps. Therefore knowledge of the different states of flow through clearances in a vacuum is essential. The experimental examination of the flow is done by flood curve measurements, to describe the mass flow integrally using the characteristic number of the standardized mass flow. The results for some possible plain clearance shapes are discussed.