bluuebunny/crossref_metadata_2025 huggingface dataset:
A Region of the Transmembrane Regulatory Protein ToxR That Tethers the Transcriptional Activation Domain to the Cytoplasmic Membrane Displays Wide Divergence among
Vibrio
Species
Authors: Osorio Carlos R. , Klose Karl E.
Published: July 2002

The virulence regulatory protein ToxR of
Vibrio cholerae
is unique in that it contains a cytoplasmic DNA-binding–transcriptional activation domain, a transmembrane domain, and a periplasmic domain. Although ToxR and other transmembrane transcriptional activators have been discovered in other bacteria, little is known about their mechanism of activation. Utilizing degenerate oligonucleotides and PCR, we have amplified internal
toxR
gene sequences from seven
Vibrio
and
Photobacterium
species and subspecies, demonstrating that
toxR
is an ancestral gene of the family
Vibrionaceae
. Sequence alignment of all available ToxR amino acid sequences revealed a region between the transcriptional activation and transmembrane domains that displays wide divergence among
Vibrio
species. We hypothesize that this region merely tethers the transcriptional activation domain to the cytoplasmic membrane and thus can tolerate wide divergence and multiple insertions and deletions. The divergence in the tether region at the nucleotide level may provide a useful tool for the distinction of
Vibrio
and
Photobacterium
species.
Leafy Gall Formation Is Controlled by
fasR
, an AraC-Type Regulatory Gene in
Rhodococcus fascians
Authors: Temmerman Wim , Vereecke Danny , Dreesen Rozemarijn , Van Montagu Marc , Holsters Marcelle , Goethals Koen
Published: July 2002

Rhodococcus fascians
can interact with many plant species and induce the formation of either leafy galls or fasciations. To provoke symptoms,
R. fascians
strain D188 requires pathogenicity genes that are located on a linear plasmid, pFiD188. The
fas
genes are essential for virulence and constitute an operon that encodes, among other functions, a cytokinin synthase gene. Expression of the
fas
genes is induced by extracts of infected plant tissue only. We have isolated an AraC-type regulatory gene,
fasR
, located on pFiD188, which is indispensable for pathogenesis and for
fas
gene expression. The combined results of our experiments show that in vitro expression of the
fas
genes in a defined medium is strictly regulated and that several environmental factors (pH, carbon and nitrogen sources, phosphate and oxygen content, and cell density) and regulatory proteins are involved. We further show that expression of the
fas
genes is controlled at both the transcriptional and the translational levels. The complex expression pattern probably reflects the necessity of integrating a multitude of signals and underlines the importance of the
fas
operon in the pathogenicity of
R. fascians
.
Sequence of the Genome of
Salmonella
Bacteriophage P22
Authors: Byl Carolyn Vander , Kropinski Andrew M.
Published: July 2002

The sequence of the nonredundant region of the
Salmonella enterica
serovar Typhimurium temperate, serotype-converting bacteriophage P22 has been completed. The genome is 41,724 bp with an overall moles percent GC content of 47.1%. Numerous examples of potential integration host factor and C1-binding sites were identified in the sequence. In addition, five potential rho-independent terminators were discovered. Sixty-five genes were identified and annotated. While many of these had been described previously, we have added several new ones, including the genes involved in serotype conversion and late control. Two of the serotype conversion gene products show considerable sequence relatedness to GtrA and -B from
Shigella
phages SfII, SfV, and SfX. We have cloned the serotype-converting cassette (
gtrABC
) and demonstrated that it results in
Salmonella
serovar Typhimurium LT2 cells which express antigen O1. Many of the putative proteins show sequence relatedness to proteins from a great variety of other phages, supporting the hypothesis that this phage has evolved through the recombinational exchange of genetic information with other viruses.
Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins
Authors: Enz Sabine , Mahren Susanne , Stroeher Uwe H. , Braun Volkmar
Published: July 2002

InEscherichia coli, transcription of the ferric citrate transport genesfecABCDEis controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)10-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)6] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)10-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA1–79interacts with FecR101–317and FecR1–85interacts with FecI1–173. These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.
Bacteriophage T4 Self-Assembly: Localization of gp3 and Its Role in Determining Tail Length
Authors: Vianelli A. , Wang G. R. , Gingery M. , Duda R. L. , Eiserling F. A. , Goldberg E. B.
Published: July 2002

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.
Polar Clustering of the Chemoreceptor Complex in
Escherichia coli
Occurs in the Absence of Complete CheA Function
Authors: Skidmore J. M. , Ellefson D. D. , McNamara B. P. , Couto M. M. P. , Wolfe A. J. , Maddock J. R.
Published: July 2002

Bacterial chemotaxis requires a phosphorelay system initiated by the interaction of a ligand with its chemoreceptor and culminating in a change in the directional bias of flagellar rotation. Chemoreceptor-CheA-CheW ternary complexes mediate transduction of the chemotactic signal. In vivo, these complexes cluster predominantly in large groups at the cell poles. The function of chemoreceptor clustering is currently unknown. To gain insight into the relationship between signaling and chemoreceptor clustering, we examined these properties in several
Escherichia coli
mutant strains that produce CheA variants altered in their ability to mediate chemotaxis, autophosphorylate, or bind ATP. We show here that polar clustering of chemoreceptor complexes does not require functional CheA protein, although maximal clustering occurred only in chemotactically competent cells. Surprisingly, in cells containing a minimum of 13 gold particles at the cell pole, a significant level of clustering was observed in the absence of CheA, demonstrating that CheA is not absolutely essential for chemoreceptor clustering. Nonchemotactic cells expressing only CheA
S
, a C-terminal CheA deletion, or CheA bearing a mutation in the ATP-binding site mediated slightly less than maximal chemoreceptor clustering. Cells expressing only full-length CheA (CheA
L
) from either a chromosomal or a plasmid-encoded allele displayed a methyl-accepting chemotaxis protein localization pattern indistinguishable from that of strains carrying both CheA
L
and CheA
S
, demonstrating that CheA
L
alone can mediate polar clustering.
Role of Cell Shape in Determination of the Division Plane in
Schizosaccharomyces pombe
: Random Orientation of Septa in Spherical Cells
Authors: Sipiczki M. , Yamaguchi M. , Grallert A. , Takeo K. , Zilahi E. , Bozsik A. , Miklos I.
Published: July 2002

The establishment of growth polarity in
Schizosaccharomyces pombe
cells is a combined function of the cytoplasmic cytoskeleton and the shape of the cell wall inherited from the mother cell. The septum that divides the cylindrical cell into two siblings is formed midway between the growing poles and perpendicularly to the axis that connects them. Since the daughter cells also extend at their ends and form their septa at right angles to the longitudinal axis, their septal (division) planes lie parallel to those of the mother cell. To gain a better understanding of how this regularity is ensured, we investigated septation in spherical cells that do not inherit morphologically predetermined cell ends to establish poles for growth. We studied four mutants (defining four novel genes), over 95% of whose cells displayed a completely spherical morphology and a deficiency in mating and showed a random distribution of cytoplasmic microtubules, Tea1p, and F-actin, indicating that the cytoplasmic cytoskeleton was poorly polarized or apolar. Septum positioning was examined by visualizing septa and division scars by calcofluor staining and by the analysis of electron microscopic images. Freeze-substitution, freeze-etching, and scanning electron microscopy were used. We found that the elongated bipolar shape is not essential for the determination of a division plane that can separate the postmitotic nuclei. However, it seems to be necessary for the maintenance of the parallel orientation of septa over the generations. In the spherical cells, the division scars and septa usually lie at angles to each other on the cell surface. We hypothesize that the shape of the cell indirectly affects the positioning of the septum by directing the extension of the spindle.
Cellular Responses to Postsegregational Killing by Restriction-Modification Genes
Authors: Handa Naofumi , Ichige Asao , Kusano Kohji , Kobayashi Ichizo
Published: July 2002

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the
Eco
RI restriction modification gene complex carried by a temperature-sensitive plasmid in an
Escherichia coli
strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in
recA
,
ruvAB
,
ruvC
,
recG
, and
recN
mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted “nonself” DNA and repair restricted “self” DNA.
Cloning and Sequence Analysis of the Mercury Resistance Operon of
Streptomyces
sp. Strain CHR28 Reveals a Novel Putative Second Regulatory Gene
Authors: Ravel Jacques , DiRuggiero Jocelyne , Robb Frank T. , Hill Russell T.
Published: July 2002

A DNA library of pRJ28, a large linear plasmid encoding mercury resistance, was constructed, and the mercury resistance genes were cloned. The 5,921-bp sequence was analyzed and showed a high degree of similarity to the
Streptomyces lividans
1326 mercury resistance operon. Genes
merR
,
merT
,
merP
, and
orfIV
were found in a similar order and in a single transcription unit.
merA
and
merB
were found to be transcribed in the opposite direction to genes
merR
,
merT
,
merP
, and
orfIV
, as in
S. lividans
1326. A novel putative regulatory gene,
orfX
, was found 22 bp downstream of
merA. orfX
encodes a 137-amino acid protein with a potential helix-turn-helix motif in the N-terminal domain, characteristic of the MerR family of transcriptional regulators. Transcriptional studies showed that
orfX
is cotranscribed with
merA
and
merB
. It is hypothesized that
orfX
plays a role in the regulation of the mercury resistance operon, probably by binding at the MerR operator site.
Equivalence of Lauric Acid and Glycerol Monolaurate as Inhibitors of Signal Transduction in
Staphylococcus aureus
Authors: Ruzin Alexey , Novick Richard P.
Published: July 2002

Glycerol monolaurate (GML) inhibits the expression of virulence factors in
Staphylococus aureus
and the induction of vancomycin resistance in
Enterococcus faecalis
, presumably by blocking signal transduction. Although GML is rapidly hydrolyzed by bacteria, one of the products, lauric acid, has identical inhibitory activity and is metabolized much more slowly. At least four distinct GML-hydrolyzing activities are identified in
S. aureus
: the secreted Geh lipase, residual supernatant activity in a
geh
-null mutant strain, a novel membrane-bound esterase, and a cytoplasmic activity.
Promoter Cloning in the Radioresistant Bacterium
Deinococcus radiodurans
Authors: Meima Rob , Rothfuss Heather M. , Gewin Lindy , Lidstrom Mary E.
Published: July 2002

Deinococcus radiodurans
is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in
D. radiodurans
were developed. These vectors are based on
Escherichia coli
replicons that are unable to replicate autonomously in
D. radiodurans
and carry homologous sequences for replacement recombination in the
D. radiodurans
chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the
D. radiodurans
R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the −10 and −35 regions of these promoters resembled the ς
70
consensus sequence of
E. coli
.
Role of Acid Metabolism in
Streptomyces coelicolor
Morphological Differentiation and Antibiotic Biosynthesis
Authors: Viollier Patrick H. , Minas Wolfgang , Dale Glenn E. , Folcher Marc , Thompson Charles J.
Published: July 2002

Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in
Streptomyces coelicolor
. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent
K

m

values of CitA for acetyl coenzyme A (acetyl-CoA) (32 μM) and oxaloacetate (17 μM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD
+
, NADH, ATP, ADP, isocitrate, or α-ketoglutarate). The corresponding gene (
citA
) was cloned and sequenced, allowing construction of a
citA
mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that
citA
encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the
citA
mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains,
citA
and
bldJ
mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that
citA
defines a new stage of the
Streptomyces
developmental cascade.
Specificity and Topology of the
Escherichia coli
Xanthosine Permease, a Representative of the NHS Subfamily of the Major Facilitator Superfamily
Authors: Nørholm Morten H. H. , Dandanell Gert
Published: July 2002

The specificity of XapB permease was compared with that of the known nucleoside transporters NupG and NupC. XapB-mediated xanthosine uptake is abolished by 2,4-dinitrophenol and exhibits saturation kinetics with an apparent

K
m

of 136 μM. A 12-transmembrane-segment model was confirmed by translational fusions to alkaline phosphatase and the α fragment of β-galactosidase.
High-Density Microarray-Mediated Gene Expression Profiling of
Escherichia coli
Authors: Wei Yan , Lee Jian-Ming , Richmond Craig , Blattner Frederick R. , Rafalski J. Antoni , LaRossa Robert A.
Published: July 2002

A nearly complete collection of 4,290
Escherichia coli
open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from
E. coli
cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-β-
d
-thiogalactopyranoside (IPTG) treatment elevated
lacZ
,
lacY
, and
lacA
transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between
E. coli
cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species. Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of
E. coli
physiology.
Iron-Sulfur Flavoprotein (Isf) from
Methanosarcina thermophila
Is the Prototype of a Widely Distributed Family
Authors: Zhao Tong , Cruz Francisco , Ferry James G.
Published: July 2002

A total of 35 homologs of the iron-sulfur flavoprotein (Isf) from
Methanosarcina thermophila
were identified in databases. All three domains were represented, and multiple homologs were present in several species. An unusually compact cysteine motif ligating the 4Fe-4S cluster in Isf is conserved in all of the homologs except two, in which either an aspartate or a histidine has replaced the second cysteine in the motif. A phylogenetic analysis of Isf homologs identified four subgroups, two of which were supported by bootstrap data. Three homologs from metabolically and phylogenetically diverse species in the
Bacteria
and
Archaea
domains (Af3 from
Archaeoglobus fulgidus
, Cd1 from
Clostridium difficile
, and Mj2 from
Methanococcus jannaschii
) were overproduced in
Escherichia coli
. Each homolog purified as a homodimer, and the UV-visible absorption spectra were nearly identical to that of Isf. After reconstitution with iron, sulfide, and flavin mononucleotide (FMN) the homologs contained six to eight nonheme iron atoms and 1.6 to 1.7 FMN molecules per dimer, suggesting that two 4Fe-4S or 3Fe-4S clusters and two FMN cofactors were bound to each dimer, which is consistent with Isf data. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by cell extract of acetate-grown
M. thermophila
, but Cd1 was not. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by
A. fulgidus
and
M. jannaschii
cell extracts. Cell extract of
Clostridium thermoaceticum
catalyzed CO reduction of Cd1. Our database sequence analyses and biochemical characterizations indicate that Isf is the prototype of a family of iron-sulfur flavoproteins that occur in members of all three domains.
relA
-Independent Amino Acid Starvation Response Network of
Streptococcus pyogenes
Authors: Steiner Kerstin , Malke Horst
Published: July 2002

Streptococcus pyogenes
(group A streptococcus [GAS]), a multiple-amino-acid-auxotrophic human pathogen, may face starvation for essential amino acids during various stages of the infection process. Since the response of GAS to such conditions is likely to influence pathogenetic processes, we set out to identify by transcriptional analyses genes and operons that are responsive to amino acid starvation and examined whether functionally meaningful response patterns can be ascertained. We discovered that GAS are capable of mounting a
relA
-independent amino acid starvation response that involves transcriptional modulation of a wide array of housekeeping genes as well as accessory and dedicated virulence genes. Housekeeping genes that were upregulated during starvation of both wild-type and
relA
mutant strains included the newly identified T-box members of the aminoacyl-tRNA synthetase genes, the genes for components of the tmRNA-mediated peptide tagging and proteolysis system for abnormal proteins (
ssrA
,
smpB
,
clpP
, and
clpC
), and the operons for the
dnaK
and
groE
groups of molecular chaperones. In addition to upregulation of the genes for oligopeptide permease (
opp
), intracellular peptidase (
pepB
), and the two-component regulator
covRS
reported previously (K. Steiner and H. Malke, Mol. Microbiol. 38:1004–1016, 2000), amino acid starvation stimulated the transcription of the growth phase-associated, virulence-regulatory
fas
operon, the streptolysin S operon (
sag
), and the gene for autoinducer-2 production protein (
luxS
). A prominent feature of operons exhibiting internal transcriptional termination (
opp
,
fas
, and
sag
) was starvation-promoted full-length transcription, a mechanism that improves the efficacy of these systems by increasing the level of coordinate transcription of functionally related genes. Based on these results, a regulatory network with feedback mechanisms is proposed that counteracts the stringent response, links the levels of key rate-limiting enzymes to virulence gene expression, and enables the organism in a dynamic way to take advantage of protein-rich environments provided by its human host. As several of the affected target genes are controlled by more than one regulator, fine modulation may result in accordance with the demands imposed by ecologically different colonization sites upon the adaptive capacity of the pathogen.
Molecular Characterization ofDesulfovibrio gigasNeelaredoxin, a Protein Involved in Oxygen Detoxification in Anaerobes
Authors: Silva Gabriela , LeGall Jean , Xavier António V. , Teixeira Miguel , Rodrigues-Pousada Claudina
Published: July 2002

Desulfovibrio gigasneelaredoxin is an iron-containing protein of 15 kDa, having a single iron site with a His4Cys coordination. Neelaredoxins and homologous proteins are widespread in anaerobic prokaryotes and have superoxide-scavenging activity. To further understand its role in anaerobes, its genomic organization and expression inD. gigaswere studied and its ability to complementEscherichia colisuperoxide dismutase deletion mutant was assessed. InD. gigas, neelaredoxin is transcribed as a monocistronic mRNA of 500 bases as revealed by Northern analysis. Putative promoter elements resembling ς70recognition sequences were identified. Neelaredoxin is abundantly and constitutively expressed, and its expression is not further induced during treatment with O2or H2O2. The neelaredoxin gene was cloned by PCR and expressed inE. coli, and the protein was purified to homogeneity. The recombinant neelaredoxin has spectroscopic properties identical to those observed for the native one. Mutations of Cys-115, one of the iron ligands, show that this ligand is essential for the activity of neelaredoxin. In an attempt to elucidate the function of neelaredoxin within the cell, it was expressed in anE. colimutant deficient in cytoplasmic superoxide dismutases (sodA sodB). Neelaredoxin suppresses the deleterious effects produced by superoxide, indicating that it is involved in oxygen detoxification in the anaerobeD. gigas.
SecG Function and Phospholipid Metabolism in
Escherichia coli
Authors: Flower Ann M.
Published: July 2002

SecG is an auxiliary protein in the Sec-dependent protein export pathway of
Escherichia coli
. Although the precise function of SecG is unknown, it stimulates translocation activity and has been postulated to enhance the membrane insertion-deinsertion cycle of SecA. Deletion of
secG
was initially reported to result in a severe export defect and cold sensitivity. Later results demonstrated that both of these phenotypes were strain dependent, and it was proposed that an additional mutation was required for manifestation of the cold-sensitive phenotype. The results presented here demonstrate that the cold-sensitive
secG
deletion strain also contains a mutation in
glpR
that causes constitutive expression of the
glp
regulon. Introduction of both the
glpR
mutation and the
secG
deletion into a wild-type strain background produced a cold-sensitive phenotype, confirming the hypothesis that a second mutation (
glpR
) contributes to the cold-sensitive phenotype of
secG
deletion strains. It was speculated that the
glpR
mutation causes an intracellular depletion of glycerol-3-phosphate due to constitutive synthesis of GlpD and subsequent channeling of glycerol-3-phosphate into metabolic pathways. In support of this hypothesis, it was demonstrated that addition of glycerol-3-phosphate to the growth medium ameliorated the cold sensitivity, as did introduction of a
glpD
mutation. This depletion of glycerol-3-phosphate is predicted to limit phospholipid biosynthesis, causing an imbalance in the levels of membrane phospholipids. It is hypothesized that this state of phospholipid imbalance imparts a dependence on SecG for proper function or stabilization of the translocation apparatus.
Endophytic Colonization of Rice by a Diazotrophic Strain of
Serratia marcescens
Authors: Gyaneshwar Prasad , James Euan K. , Mathan Natarajan , Reddy Pallavolu M. , Reinhold-Hurek Barbara , Ladha Jagdish K.
Published: July 2002

Six closely related N
2
-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as
Serratia marcescens
by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of
15
N
2
incorporation and by sequence analysis of the PCR-amplified fragment of
nifH
. To examine its interaction with rice, strain IRBG500 was marked with
gusA
fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.
Characterization of Outer Membrane Proteins in
Chlamydia trachomatis
LGV Serovar L2
Authors: Tanzer Regina J. , Hatch Thomas P.
Published: July 2002

We used a photoactivatable, lipophilic reagent, 3′-(trifluoromethyl)-3-(
m
-[
125
I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies of
Chlamydia trachomatis
LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.
Degradation of Aromatics and Chloroaromatics byPseudomonassp. Strain B13: Cloning, Characterization, and Analysis of Sequences Encoding 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase
Authors: Göbel Markus , Kassel-Cati Kerstin , Schmidt Eberhard , Reineke Walter
Published: July 2002

3-Oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway. This report describes the characterization of DNA fragments with the overall length of 5.9 kb fromPseudomonassp. strain B13 that encode these enzymes. DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one. ORF1, of unknown function, has a length of 414 bp. ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813. ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661. CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a. ORF4, termedcatF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity. The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp betweencatIandcatJand of 4 bp betweencatJandcatF. Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF. ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase. An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found. The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.
Yersinia enterocolitica
Type III Secretion: Mutational Analysis of the
yopQ
Secretion Signal
Authors: Ramamurthi Kumaran S. , Schneewind Olaf
Published: July 2002

Pathogenic
Yersinia
spp. secrete Yop proteins via the type III pathway.
yopQ
codons 1 to 15 were identified as a signal necessary and sufficient for the secretion of a fused reporter protein. Frameshift mutations that alter codons 2 to 15 with little alteration of
yopQ
mRNA sequence do not abolish type III transport, suggesting a model in which
yopQ
mRNA may provide a signal for secretion (D. M. Anderson and O. Schneewind, Mol. Microbiol. 31:1139-1148, 2001). In a recent study, the
yopE
signal was truncated to codons 1 to 12. All frameshift mutations introduced within the first 12 codons of
yopE
abolished secretion. Also, multiple synonymous mutations that changed the mRNA sequence of
yopE
codons 1 to 12 without altering the amino acid sequence did not affect secretion. These results favor a model whereby an N-terminal signal peptide initiates YopE into the type III pathway (S. A. Lloyd et al., Mol. Microbiol. 39:520-531, 2001). It is reported here that codons 1 to 10 of
yopQ
act as a minimal secretion signal. Further truncation of
yopQ
, either at codon 10 or at codon 2, abolished secretion. Replacement of
yopQ
AUG with either of two other start codons, UUG or GUG, did not affect secretion. However, replacement of AUG with CUG or AAA and initiating translation at the fusion site with
npt
did not permit Npt secretion, suggesting that the translation of
yopQ
codons 1 to 15 is a prerequisite for secretion. Frameshift mutations of
yopQ
codons 1 to 10, 1 to 11, and 1 to 12 abolished secretion signaling, whereas frameshift mutations of
yopQ
codons 1 to 13, 1 to 14, and 1 to 15 did not. Codon changes at
yopQ
positions 2 and 10 affected secretion signaling when placed within the first 10 codons but had no effect when positioned in the larger fusion of
yopQ
codons 1 to 15. An mRNA mutant of
yopQ
codons 1 to 10, generated by a combination of nine synonymous mutations, was defective in secretion signaling, suggesting that the YopQ secretion signal is not proteinaceous. A model is discussed whereby the initiation of YopQ polypeptide into the type III pathway is controlled by properties of
yopQ
mRNA.
Yop Fusions to Tightly Folded Protein Domains and Their Effects on
Yersinia enterocolitica
Type III Secretion
Authors: Lee Vincent T. , Schneewind Olaf
Published: July 2002

Yersinia enterocolitica
organisms secrete Yop proteins via the type III pathway. Translational fusion of
yop
genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted. The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops.
ClpXP Protease Regulates the Signal Peptide Cleavage of Secretory Preproteins inBacillus subtiliswith a Mechanism Distinct from That of the Ecs ABC Transporter
Authors: Pummi Tiina , Leskelä Soile , Wahlström Eva , Gerth Ulf , Tjalsma Harold , Hecker Michael , Sarvas Matti , Kontinen Vesa P.
Published: July 2002

Identification and characterization of a suppressor mutation,sup-15, which partially restored secretion in the protein secretion-deficientBacillus subtilis ecsA26mutant, led us to discover a novel function of Clp protease. Inactivation of ClpP improved the processing of the precursor of AmyQ α-amylase exposed on the outer surface of the cytoplasmic membrane. A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex. In the absence of ClpXP, the transcription of thesipS,sipT,sipV, andlspsignal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation ofecsmutations. Specific overproduction of SipT enhanced the secretion. These findings extend the regulatory roles of ClpXP to protein secretion. ClpXP also influenced the processing of the lipoprotein PrsA. A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested. In contrast, Ecs did not affect transcription of thesipgenes, pointing to a different mechanism of secretion regulation.
Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism byPseudomonas butanovora
Authors: Vangnai Alisa S. , Arp Daniel J. , Sayavedra-Soto Luis A.
Published: July 2002

The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization inPseudomonas butanovora(ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption ofbdhorbohaffected adversely, but did not eliminate, butane utilization byP. butanovora. TheP. butanovoramutant with both genesbohandbdhinactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of theP. butanovorastrain withbdhdisrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD+-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway ofP. butanovora.
Perfringolysin O Expression in
Clostridium perfringens
Is Independent of the Upstream
pfoR
Gene
Authors: Awad Milena M. , Rood Julian I.
Published: July 2002

The pathogenesis of
Clostridium perfringens
-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with
Escherichia coli
suggested that the perfringolysin O structural gene,
pfoA
, was positively regulated by the product of the upstream
pfoR
gene. In an attempt to confirm this hypothesis in
C
.
perfringens
, a
pfoR-pfoA
deletion mutant was complemented with isogenic

pfoA
+

shuttle plasmids that varied only in their ability to encode an intact
pfoR
gene. No difference in the ability to produce perfringolysin O was observed for
C
.
perfringens
strains carrying these plasmids. In addition, chromosomal
pfoR
mutants were constructed by homologous recombination in
C
.
perfringens
. Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of
pfoR
, it was concluded that the
pfoR
gene product is unlikely to have a role in the regulation of
pfoA
expression in
C
.
perfringens
.
Differential Recognition of Surface Proteins inStreptococcus pyogenesby Two Sortase Gene Homologs
Authors: Barnett Timothy C. , Scott June R.
Published: July 2002

The interaction ofStreptococcus pyogenes(group A streptococcus [GAS]) with its human host requires several surface proteins. In this study, we isolated mutations in a gene required for the surface localization of protein F by transposon mutagenesis of the M6 strain JRS4. This gene (srtA) encodes a protein homologous toStaphylococcus aureussortase, which covalently links proteins containing an LPXTG motif to the cell wall. The GASsrtAmutant was defective in anchoring the LPXTG-containing proteins M6, protein F, ScpA, and GRAB to the cell surface. This phenotype was complemented when a wild-typesrtAgene was provided intrans. The surface localization of T6, however, was unaffected by thesrtAmutation. The M1 genome sequence contains a second open reading frame with a motif characteristic of sortase proteins. Inactivation of this gene (designatedsrtB) in strain JRS4 affected the surface localization of T6 but not M6, protein F, ScpA, or GRAB. This phenotype was complemented bysrtBintrans. AnsrtAprobe hybridized with DNA from all GAS strains tested (M types 1, 3, 4, 5, 6, 18, 22, and 50 and nontypeable strain 64/14) and from streptococcal groups C and G, whilesrtBhybridized with DNA from only a few GAS strains. We conclude thatsrtAandsrtBencode sortase enzymes required for anchoring different subsets of proteins to the cell wall. It seems likely that the multiple sortase homologs in the genomes of other gram-positive bacteria have a similar substrate-specific role.
Simple and Rational Approach to the Identification of
Mycobacterium tuberculosis
,
Mycobacterium avium
Complex Species, and Other Commonly Isolated Mycobacteria
Authors: Wong Derek A. , Yip Peter C. W. , Cheung Danny T. L. , Kam Kai Man
Published: July 2002

A novel PCR-restriction fragment length polymorphism analysis of the
hsp65
gene was developed. The restriction patterns for
Mycobacterium tuberculosis
and
Mycobacterium avium
complex (MAC) species were designed to be highly distinct, and the overall number of restriction patterns was designed to be limited. Four hundred specimens (17 reference strains and 383 clinical isolates) were tested, of which 98 were
M. tuberculosis
and 132 were MAC species. The assay was virtually 100% sensitive and specific for
M. tuberculosis
and MAC species. Moreover, it gave highly concordant results for other mycobacterial species other than
M. terrae
complex species. This assay can be completed in one day and is user-friendly and robust. Therefore, it is highly suitable for large-scale use in a clinical laboratory.
Performance of Candida ID, a New Chromogenic Medium for Presumptive Identification of
Candida
Species, in Comparison to CHROMagar Candida
Authors: Willinger Birgit , Hillowoth Cornelia , Selitsch Brigitte , Manafi Mammad
Published: July 2002

Candida ID agar allows identification of
Candida albicans
and differentiation of other
Candida
species. In comparison with CHROMagar Candida, we evaluated the performance of this medium directly from 596 clinical specimens. In particular, detection of
C. albicans
after 24 h of incubation was easier on Candida ID (sensitivity, 96.8%) than on CHROMagar (sensitivity, 49.6%).
Rapid and Accurate Determination of Genotypic Clarithromycin Resistance in Cultured
Helicobacter pylori
by Fluorescent In Situ Hybridization
Authors: Rüssmann Holger , Adler Kristin , Haas Rainer , Gebert Bettina , Koletzko Sibylle , Heesemann Jürgen
Published: July 2002

Phenotypic susceptibility testing for clarithromycin by E-test and disk diffusion of 109 cultured
Helicobacter pylori
isolates was compared with the genotypic susceptibility determination by fluorescent in situ hybridization (FISH). No discrepancies were found between these three methods. However, FISH has the advantage of providing results after 3 h.
Purification and Characterization of PCR-Inhibitory Components in Blood Cells
Authors: Al-Soud Waleed Abu , Rådström Peter
Published: July 2002

In a recent study, immunoglobulin G in human plasma was identified as a major inhibitor of diagnostic PCR (W. Abu Al-Soud, L. J. Jönsson, and P. Rådström. J. Clin. Microbiol. 38:345–350, 2000). In this study, two major PCR inhibitors in human blood cells were purified using size exclusion and anion-exchange chromatographic procedures. Based on N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptides, hemoglobin and lactoferrin were identified as PCR-inhibitor components in erythrocytes and leukocytes, respectively. When different concentrations of hemoglobin or lactoferrin were added to PCR mixtures of 25 μl containing 10 different thermostable DNA polymerases and 1 ng ofListeria monocytogenesDNA as template DNA, AmpliTaqGold,Pwo, andUltmawere inhibited in the presence of ≤1.3 μg of hemoglobin and ≤25 ng of lactoferrin, whilerTthandTliwere found to resist inhibition of at least 100 μg of hemoglobin. In addition, the quantitative effects of seven low-molecular-mass inhibitors, present in blood samples or degradation products of hemoglobin, on real-time DNA synthesis ofrTthusing the LightCycler Instrument were investigated. A reaction system based on a single-stranded poly(dA) template with an oligo(dT) primer annealed to the 3′ end was used. It was found that the addition of 0.25 to 0.1 mg of bile per ml, 2.5 mM CaCl2, 0.25 mM EDTA, 5 μM FeCl3, and 0.01 IU of heparin per ml reduced the fluorescence to approximately 76, 70, 46, 17, and 51%, respectively. Finally, the effects of nine amplification facilitators were studied in the presence of hemoglobin and lactoferrin. Bovine serum albumin (BSA) was the most efficient amplification facilitator, so that the addition of 0.4% (wt/vol) BSA allowed AmpliTaqGold to amplify DNA in the presence of 20 instead of 1 μg of hemoglobin and 500 instead of 5 ng of lactoferrin. Including 0.02% (wt/vol) gp32, a single-stranded-DNA binding protein, in the reaction mixture of AmpliTaqGold was also found to reduce the inhibitory effects of hemoglobin and lactoferrin.
Normalized Quantification by Real-Time PCR of Epstein-Barr Virus Load in Patients at Risk for Posttransplant Lymphoproliferative Disorders
Authors: Jabs Wolfram J. , Hennig Holger , Kittel Michael , Pethig Klaus , Smets Françoise , Bucsky Peter , Kirchner Holger , Wagner Hans J.
Published: July 2002

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.
Analysis of
Clostridium difficile
Isolates from Nosocomial Outbreaks at Three Hospitals in Diverse Areas of Japan
Authors: Kato Haru , Kato Naoki , Watanabe Kunitomo , Yamamoto Toshinobu , Suzuki Kanzo , Ishigo Shiomi , Kunihiro Seiko , Nakamura Isao , Killgore George E. , Nakamura Shinichi
Published: July 2002

Clostridium difficile
isolates recovered from patients with
C. difficile
-associated diarrhea (CDAD) at three hospitals located in diverse areas of Japan were analyzed by three typing systems, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), and Western immunoblotting. At the three hospitals examined, a single PCR ribotype strain (type smz) was predominant and accounted for 22 (65%) of 34, 18 (64%) of 28, and 11 (44%) of 25 isolates, respectively. All of the 51 isolates that represented PCR ribotype smz were nontypeable by PFGE because of DNA degradation. Since the type smz strain did not react with any of the antisera against 10 different serogroups (A, B, C, D, F, G, H, I, K, and X), we prepared a new antiserum against a type smz isolate. All 51 type smz isolates presented identical banding patterns, reacting with the newly prepared antiserum (designated subserogroup JP-0 of serogroup JP). These results were compared with those of a strain from a hospital outbreak that occurred in New York, which has been identified as type J9 by restriction enzyme analysis and type 01/A by arbitrarily primed PCR but was nontypeable by PFGE because of DNA degradation. This strain was reported to be epidemic at multiple hospitals in the United States. The J9 strain represented a PCR ribotype pattern different from that of a type smz strain and was typed as subserogroup G-1 of serogroup G by immunoblot analysis. A single outbreak type causing nosocomial CDAD in Japan was found to be different from the strain causing multiple outbreaks in the United States, even though the outbreak strains from the two countries were nontypeable by PFGE because of DNA degradation.