bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of the Length of the Lipooligosaccharide α Chain on Its Sialylation in
Neisseria meningitidis
Authors: Tsai Chao-Ming , Kao George , Zhu Peixuan
Published: July 2002

The sialylation of lipooligosaccharide (LOS) in
Neisseria meningitidis
plays a role in the resistance of the organism to killing by normal human serum. The length of the α chain extending out from the heptose I [Hep (I)] moiety of LOS influenced sialylation of
N. meningitidis
LOS in vitro and in vivo. The α chain required a terminal Gal and a trisaccharide or longer oligosaccharide to serve as an acceptor for sialylation. The disaccharide lactose (Galβ1-4Glc) in the α chain of immunotype L8 LOS could not function as an acceptor for the sialyltransferase, probably due to steric hindrance imposed by the neighboring Hep (II) with phosphorylethanolamine and another group attached.
Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A
Authors: Masri Heather P. , Cornelissen Cynthia Nau
Published: July 2002

The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in
Escherichia coli
, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.
Induction ofdnaKthrough Its Native Heat Shock Promoter Is Necessary for Intramacrophagic Replication ofBrucella suis
Authors: Köhler Stephan , Ekaza Euloge , Paquet Jean-Yves , Walravens Karl , Teyssier Jacques , Godfroid Jacques , Liautard Jean-Pierre
Published: July 2002

The heat shock protein DnaK is essential for intramacrophagic replication ofBrucella suis. The replacement of the stress-inducible, nativednaKpromoter ofB. suisby the promoter of the constitutively expressedblagene resulted in temperature-independent synthesis of DnaK. In contrast to adnaKnull mutant, this strain grew at 37°C, with a thermal cutoff at 39°C. However, the constitutivednaKmutant, which showed high sensitivity to H2O2-mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction ofdnaKis a crucial event in the intracellular replication ofB. suis.
Randomized, Controlled Human Challenge Study of the Safety, Immunogenicity, and Protective Efficacy of a Single Dose of Peru-15, a Live Attenuated Oral Cholera Vaccine
Authors: Cohen Mitchell B. , Giannella Ralph A. , Bean Judy , Taylor David N. , Parker Susan , Hoeper Amy , Wowk Stephen , Hawkins Jennifer , Kochi Sims K. , Schiff Gilbert , Killeen Kevin P.
Published: July 2002

Peru-15 is a live attenuated oral vaccine derived from a
Vibrio cholerae
O1 El Tor Inaba strain by a series of deletions and modifications, including deletion of the entire CT genetic element. Peru-15 is also a stable, motility-defective strain and is unable to recombine with homologous DNA. We wished to determine whether a single oral dose of Peru-15 was safe and immunogenic and whether it would provide significant protection against moderate and severe diarrhea in a randomized, double-blind, placebo-controlled human volunteer cholera challenge model. A total of 59 volunteers were randomly allocated to groups to receive either 2 × 10
8
CFU of reconstituted, lyophilized Peru-15 vaccine diluted in CeraVacx buffer or placebo (CeraVacx buffer alone). Approximately 3 months after vaccination, 36 of these volunteers were challenged with approximately 10
5
CFU of virulent
V. cholerae
O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Among vaccinees, 98% showed at least a fourfold increase in vibriocidal antibody titers. After challenge, 5 (42%) of the 12 placebo recipients and none (0%) of the 24 vaccinees had moderate or severe diarrhea (≥3,000 g of diarrheal stool) (
P
= 0.002; protective efficacy, 100%; lower one-sided 95% confidence limit, 75%). A total of 7 (58%) of the 12 placebo recipients and 1 (4%) of the 24 vaccinees had any diarrhea (
P
< 0.001; protective efficacy, 93%; lower one-sided 95% confidence limit, 62%). The total number of diarrheal stools, weight of diarrheal stools, incidence of fever, and peak stool
V. cholerae
excretion among vaccinees were all significantly lower than in placebo recipients. Peru-15 is a well-tolerated and immunogenic oral cholera vaccine that affords protective efficacy against life-threatening cholera diarrhea in a human volunteer challenge model. This vaccine may therefore be a safe and effective tool to prevent cholera in travelers and is a strong candidate for further evaluation to prevent cholera in an area where cholera is endemic.
Role of Tumor Necrosis Factor Alpha inHelicobacter pyloriGastritis in Tumor Necrosis Factor Receptor 1-Deficient Mice
Authors: Thalmaier Ulrike , Lehn Norbert , Pfeffer Klaus , Stolte Manfred , Vieth Michael , Schneider-Brachert Wulf
Published: July 2002

Increased gastric production of interleukin 8 and tumor necrosis factor alpha (TNF-α) has been implicated in the pathogenesis ofHelicobacter pylori-associated gastroduodenal disease. In the present study we used a mouse model to demonstrate whether loss of the tumor necrosis factor receptor 1 (TNF-R1) function leads to differences in gastric inflammation or the systemic immune response inH. pyloriinfection. Six different clinical isolates ofH. pylori(three cytotoxin-positive and three cytotoxin-negative strains) were adapted to C57BL/6 mice. TNF-R1-deficient (TNF-R1−/−) mice (n= 19) and isogenetic controls (n= 24) were infected and sacrificed after 4 weeks of infection. Inflammation of the stomach and the humoral immune response toH. pyloriwere evaluated by histological, immunohistochemical, and serological methods. There was no detectable difference in the grade or activity of gastritis in TNF-R1−/−mice when they were compared with wild-type mice, but the number of lymphoid aggregates was slightly reduced in the gastric mucosa of TNF-R1−/−mice. Interestingly, total immunoglobulin G (IgG), as well as IgG1, IgG2b, and IgG3,H. pylori-specific antibody titers were significantly higher in wild-type mice. As revealed by immunoblot analysis, the difference in reactivity againstH. pyloriantigens was not based on a failure to recognize singleH. pyloriantigens in TNF-R1−/−mice. We therefore suggest that TNF-R1-mediated TNF-α signals might support a systemic humoral immune response againstH. pyloriand that the gastric inflammatory response toH. pyloriinfection seems to be independent of TNF-R1-mediated signals.
Modulation of C5aR Expression on Human Neutrophils by Encapsulated and AcapsularCryptococcus neoformans
Authors: Monari Claudia , Kozel Thomas R. , Bistoni Francesco , Vecchiarelli Anna
Published: July 2002

Cryptococcus neoformansand cryptococcal surface polysaccharides influenced C5aR expression on human polymorphonuclear neutrophils (PMN). Encapsulated and acapsular strains produced dramatically different effects. Treatment of PMN with acapsular cryptococci up-regulated C5aR expression; whereas treatment with encapsulated cells suppressed C5aR expression. Glucuronoxylomannan (GXM), the principal constituent of the cryptococcal capsule, was responsible for such inhibition. Increased C5aR expression following treatment with acapsular cryptococci was accompanied by increased binding of C5a to PMN, increased superoxide production in response to stimulation with C5a, and an increased chemotactic response to C5a. Conversely, decreased C5aR expression following treatment with encapsulated cryptococci or acapsular cryptococci that had been pretreated with GXM was accompanied by decreased binding of C5a to PMN and a decreased chemotactic response to C5a. Our results raise the possibility that the down-regulation of C5aR expression by encapsulated cryptococci might alter PMN function at the site of cryptococcal infection.
Natural Diversity in the N Terminus of the Mature Vacuolating Cytotoxin of
Helicobacter pylori
Determines Cytotoxin Activity
Authors: Letley D. P. , Atherton J. C.
Published: July 2002

Naturally occurring noncytotoxic
vacA
type s2 strains of
Helicobacter pylori
have a 12-residue extension to the vacuolating cytotoxin (VacA) compared with cytotoxic type s1 strains. We show that adding the region encoding this extension to type s1
vacA
completely abolishes vacuolating cytotoxin activity but has no effect on VacA production.
Control of Sporulation Gene Expression in
Bacillus subtilis
by the Chromosome Partitioning Proteins Soj (ParA) and Spo0J (ParB)
Authors: Quisel John D. , Grossman Alan D.
Published: July 2002

Two chromosome partitioning proteins, Soj (ParA) and Spo0J (ParB), regulate the initiation of sporulation in
Bacillus subtilis
. In a
spo0J
null mutant, sporulation is inhibited by the action of Soj. Soj negatively regulates expression of several sporulation genes by binding to the promoter regions and inhibiting transcription. All of the genes known to be inhibited by Soj are also activated by the phosphorylated form of the transcription factor Spo0A (Spo0A∼P). We found that, in a
spo0J
null mutant, Soj affected sporulation, in part, by decreasing the level of Spo0A protein. Soj negatively regulated transcription of
spo0A
and associated with the
spo0A
promoter region in vivo. Expression of
spo0A
from a heterologous promoter in a
spo0J
null mutant restored Spo0A levels and partly bypassed the sporulation and gene expression defects. Soj did not appear to significantly affect phosphorylation of Spo0A. Thus, in the absence of Spo0J, Soj inhibits sporulation and sporulation gene expression by inhibiting accumulation of the activator protein Spo0A and by acting downstream of Spo0A to inhibit gene expression directly.
Substrate Specificity and Signal Transduction Pathways in the Glucose-Specific Enzyme II (EII
Glc
) Component of the
Escherichia coli
Phosphotransferase System
Authors: Notley-McRobb Lucinda , Ferenci Thomas
Published: July 2002

Escherichia coli
adapted to glucose-limited chemostats contained mutations in
ptsG
resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EII
Glc
) and resulting in increased transport of glucose and methyl-α-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increased
ptsG
transcription in the absence of glucose. Increased expression of wild-type EII
Glc
was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EII
Glc
. The conformation of EII
Glc
elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of
pts
genes.
Expression of a Constitutively Active Cdc42 Homologue Promotes Development of Sclerotic Bodies but Represses Hyphal Growth in the Zoopathogenic Fungus
Wangiella
(
Exophiala
)
dermatitidis
Authors: Ye Xiangcang , Szaniszlo Paul J.
Published: July 2002

In contrast to the
CDC42
homologues of
Saccharomyces cerevisiae
and
Schizosaccharomyces pombe
, the
WdCDC42
gene in the human pathogenic fungus
Wangiella
(
Exophiala
)
dermatitidis
was found to be nonessential for cell viability. Expression of the constitutively active allele
wdcdc42
G14V
at 37°C induced nonpolarized growth that led to cell enlargement and multiple nucleation. The swollen cells subsequently converted into planate divided bicellular forms or multiply septated sclerotic bodies in post-log phase, when the G14V-altered protein was diminished. The
wdcdc42
G14V
mutation also strongly repressed filamentous growth both in the wild-type strain and in the temperature-sensitive hyphal-form mutant Hf1. In contrast, overexpression of the dominant negative alleles
wdcdc42
T19N
and
wdcdc42
D120A
had no obvious effect on fungal-cell polarization. These results suggested that WdCdc42p plays a unique regulatory role in cellular morphogenesis in
W. dermatitidis
. Activation of this protein in response to extracellular or intracellular signals seems to commit its yeast-like cells to a phenotype transition that produces sclerotic bodies while repressing hyphal development.
Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface of
Bacteroides thetaiotaomicron
Authors: Shipman Joseph A. , Berleman James E. , Salyers Abigail A.
Published: July 2002

Bacteroides thetaiotaomicron
, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of
14
C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusF's role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.
Role of TnrA in Nitrogen Source-Dependent Repression of
Bacillus subtilis
Glutamate Synthase Gene Expression
Authors: Belitsky Boris R. , Wray Lewis V. , Fisher Susan H. , Bohannon Dian E. , Sonenshein Abraham L.
Published: July 2002

Synthesis of glutamate, the cell's major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism. In
Bacillus subtilis
, the
gltAB
operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression. In addition, the
gltAB
operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation. TnrA was found to bind directly to a site immediately downstream of the
gltAB
promoter. As is true for other genes, the activity of TnrA at the
gltAB
promoter was antagonized by glutamine synthetase under certain growth conditions.
The Mammalian Neuroendocrine Hormone Norepinephrine Supplies Iron for Bacterial Growth in the Presence of Transferrin or Lactoferrin
Authors: Freestone Primrose P. E. , Lyte Mark , Neal Christopher P. , Maggs Anthony F. , Haigh Richard D. , Williams Peter H.
Published: July 2002

Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum. Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of
Escherichia coli
. Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth. Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels. Norepinephrine also caused the loss of iron from lactoferrin. The pharmacologically inactive metabolite norepinephrine 3-
O
-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium. Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin. Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine. Norepinephrine-stimulated growth in medium containing
55
Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells. Moreover, norepinephrine-stimulated growth in medium containing [
3
H]norepinephrine indicated concomitant uptake of norepinephrine. In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity (
55
Fe or
3
H) associated with bacterial cells. A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed.
Spore Photoproduct (SP) Lyase from
Bacillus subtilis
Specifically Binds to and Cleaves SP (5-Thyminyl-5,6-Dihydrothymine) but Not Cyclobutane Pyrimidine Dimers in UV-Irradiated DNA
Authors: Slieman Tony A. , Rebeil Roberto , Nicholson Wayne L.
Published: July 2002

The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the
splB
gene in
Bacillus subtilis
. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant
B. subtilis
spores and shown to efficiently cleave SP but not cyclobutane
cis
,
syn
thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an
Escherichia coli
overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py◊Py) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py◊Py-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the
32
P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3′ to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.
The Spirochete FlaA Periplasmic Flagellar Sheath Protein Impacts Flagellar Helicity
Authors: Li Chunhao , Corum Linda , Morgan David , Rosey Everett L. , Stanton Thaddeus B. , Charon Nyles W.
Published: July 2002

Spirochete periplasmic flagella (PFs), including those from
Brachyspira
(
Serpulina
),
Spirochaeta
,
Treponema
, and
Leptospira
spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using
Brachyspira hyodysenteriae
as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed
flaA
::
cat
,
flaA
::
kan
, and
flaB1
::
kan
mutants and newly constructed
flaB2
::
cat
and
flaB3
::
cat
mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with
flaA
::
cat
,
flaA
::
kan
, and
flaB1
::
kan
mutants,
flaB2
::
cat
and
flaB3
::
cat
mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a
flaA
::
cat
mutant were significantly thinner (19.6 nm) than those of the wild-type strain and
flaB1
::
kan
,
flaB2
::
cat
, and
flaB3
::
cat
mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that
flaA
::
cat
and
flaA
::
kan
mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and
flaB
mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.
Ralstonia eutropha
TF93 Is Blocked in Tat-Mediated Protein Export
Authors: Bernhard Michael , Friedrich Bärbel , Siddiqui Roman A.
Published: July 2002

Ralstonia eutropha
(formerly
Alcaligenes eutrophus
) TF93 is pleiotropically affected in the translocation of redox enzymes synthesized with an N-terminal signal peptide bearing a twin arginine (S/T-R-R-X-F-L-K) motif. Immunoblot analyses showed that the catalytic subunits of the membrane-bound [NiFe] hydrogenase (MBH) and the molybdenum cofactor-binding periplasmic nitrate reductase (Nap) are mislocalized to the cytoplasm and to the inner membrane, respectively. Moreover, physiological studies showed that the copper-containing nitrous oxide reductase (NosZ) was also not translocated to the periplasm in strain TF93. The cellular localization of enzymes exported by the general secretion system was unaffected. The translocation-arrested MBH and Nap proteins were enzymatically active, suggesting that twin-arginine signal peptide-dependent redox enzymes may have their cofactors inserted prior to transmembrane export. The periplasmic destination of MBH, Nap, and NosZ was restored by heterologous expression of
Azotobacter chroococcum tatA
mobilized into TF93.
tatA
encodes a bacterial Hcf106-like protein, a component of a novel protein transport system that has been characterized in thylakoids and shown to translocate folded proteins across the membrane.
Succinate:Quinol Oxidoreductases in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803: Presence and Function in Metabolism and Electron Transport
Authors: Cooley Jason W. , Howitt Crispin A. , Vermaas Wim F. J.
Published: July 2002

The open reading frames
sll
1625 and
sll
0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium
Synechocystis
sp. strain PCC 6803. When the organic acid content in the Δ
sll
1625 and Δ
sll
0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the Δ
sll
1625 Δ
sll
0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the
sll
1625 and
sll
0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.
Transposition of IS
1397
in the Family
Enterobacteriaceae
and First Characterization of ISKpn
1
, a New Insertion Sequence Associated with
Klebsiella pneumoniae
Palindromic Units
Authors: Wilde Caroline , Bachellier Sophie , Hofnung Maurice , Clément Jean-Marie
Published: July 2002

IS
1397
and ISKpn
1
are IS
3
family members which are specifically inserted into the loop of palindromic units (PUs). IS
1397
is shown to transpose into PUs with sequences close or identical to the
Escherichia coli
consensus, even in other enterobacteria (
Salmonella enterica
serovar Typhimurium,
Klebsiella pneumoniae
, and
Klebsiella oxytoca
). Moreover, we show that homologous intergenic regions containing PUs constitute IS
1397
transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn
1
, described here for the first time, is specific for PUs from
K. pneumoniae
, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.
Reconstruction of
Escherichia coli mrcA
(PBP 1a) Mutants Lacking Multiple Combinations of Penicillin Binding Proteins
Authors: Meberg Bernadette M. , Sailer Frances C. , Nelson David E. , Young Kevin D.
Published: July 2002

Previously, we constructed a set of mutants from which eight penicillin binding protein (PBP) genes were deleted in 192 combinations from
Escherichia coli
(S. A. Denome, P. K. Elf, T. A. Henderson, D. E. Nelson, and K. D. Young, J. Bacteriol. 181:3981-3993, 1999). Although these mutants were constructed correctly as determined by restriction mapping and the absence of relevant protein products, we recently discovered by PCR mapping that strains from which
mrcA
(PBP 1a) was deleted were also missing two neighboring genes of unknown function (
yrfE
and
yrfF
). We created a new deletion mutation in
mrcA
and reconstructed 63 strains lacking PBP 1a and other PBP mutant combinations. The new
mrcA
mutants do not exhibit mucoidy, phage resistance, temperature sensitivity, growth rate defects, or antibiotic resistance, suggesting that these phenotypes require the loss of either
yrfE
or
yrfF
alone or in combination with the absence of multiple PBPs.
Integron Integrases Possess a Unique Additional Domain Necessary for Activity
Authors: Messier Nancy , Roy Paul H.
Published: July 2002

Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion).
RNA Expression Analysis Using an Antisense
Bacillus subtilis
Genome Array
Authors: Lee Jian-Ming , Zhang Shehui , Saha Soumitra , Santa Anna Sonia , Jiang Can , Perkins John
Published: July 2002

We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in
Bacillus subtilis
by using Affymetrix technology. Quality control tests of the
B. subtilis
GeneChip were performed to ascertain the quality of the array. These tests included optimization of the labeling and hybridization conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression patterns of known vitamin biosynthetic genes. In minimal medium, we detected transcripts for approximately 70% of the known open reading frames (ORFs). In addition, we were able to monitor the transcript level of known biosynthetic genes regulated by riboflavin, biotin, or thiamine. Moreover, novel transcripts were also detected within intergenic regions and on the opposite coding strand of known ORFs. Several of these novel transcripts were subsequently correlated to new coding regions.
Demonstration that the Group II Intron from the Clostridial Conjugative Transposon Tn
5397
Undergoes Splicing In Vivo
Authors: Roberts Adam P. , Braun Veit , von Eichel-Streiber Christoph , Mullany Peter
Published: July 2002

Previous work has identified the conjugative transposon Tn
5397
from
Clostridium difficile
. This element was shown to contain a group II intron. Tn
5397
can be conjugatively transferred from
C. difficile
to
Bacillus subtilis
. In this work we show that the intron is spliced in both these hosts and that nonspliced RNA is also present. We constructed a mutation in the open reading frame within the intron, and this prevented splicing but did not prevent the formation of the circular form of the conjugative transposon (the likely transposition intermediate) or decrease the frequency of intergeneric transfer of Tn
5397
. Therefore, the intron is spliced, but splicing is not required for conjugation of Tn
5397
.
Genetic Footprinting in Bacteria
Authors: Hare Roberta S. , Walker Scott S. , Dorman Thomas E. , Greene Jonathan R. , Guzman Luz-Maria , Kenney Teresa J. , Sulavik Mark C. , Baradaran Khandan , Houseweart Chad , Yu Haiying , Foldes Zuzana , Motzer Anna , Walbridge Michael , Shimer George H. , Shaw Karen Joy
Published: July 2002

In vivo genetic footprinting was developed in the yeast
Saccharomyces cerevisiae
to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for
Escherichia coli
, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of
E. coli
under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn
10
transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn
10
transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage λ transposon delivery system.
Occurrence of Transsulfuration in Synthesis of
l
-Homocysteine in an Extremely Thermophilic Bacterium,
Thermus thermophilus
HB8
Authors: Yamagata Shuzo , Ichioka Kazuhito , Goto Koji , Mizuno Yasuko , Iwama Tomonori
Published: July 2002

A cell extract of an extremely thermophilic bacterium,
Thermus thermophilus
HB8, cultured in a synthetic medium catalyzed cystathionine γ-synthesis with
O
-acetyl-
l
-homoserine and
l
-cysteine as substrates but not β-synthesis with
dl
-homocysteine and
l
-serine (or
O
-acetyl-
l
-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cysthathionine β-lyase (EC
4.4.1.8
) and
O
-acetyl-
l
-serine sulfhydrylase (EC
4.2.99.8
) were markedly repressed by
l
-methionine supplemented to the medium.
l
-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of
O
-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of
O
-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of
O
-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine γ-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine γ-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of
O
-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited
O
-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of
O
-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having
O
-acetylhomoserine sulfhydrylase activity, one of which is cystathionine γ-synthase. Although it has been reported that homocysteine is directly synthesized in
T. thermophilus
HB27 by the catalysis of
O
-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271–279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in
T. thermophilus
HB8.
Characterization of
Brucella suis clpB
and
clpAB
Mutants and Participation of the Genes in Stress Responses
Authors: Ekaza Euloge , Teyssier Jacques , Ouahrani-Bettache Safia , Liautard Jean-Pierre , Köhler Stephan
Published: July 2002

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in
Brucella suis
, a gene highly homologous to
Escherichia coli clpB
was isolated from
Brucella suis
, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of
B. suis
was induced, and an isogenic
B. suis clpB
mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of
clpA
and
clpB
resulted in a mutant that was sensitive to oxidative stress. In
B. suis
expressing
gfp
, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42°C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.
Identification of a Lycopene β-Cyclase Required for Bacteriorhodopsin Biogenesis in the ArchaeonHalobacterium salinarum
Authors: Peck Ronald F. , Johnson Eric A. , Krebs Mark P.
Published: July 2002

Biogenesis of the light-driven proton pump bacteriorhodopsin in the archaeonHalobacterium salinarumrequires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor. As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified anH. salinarumgene required for conversion of lycopene to β-carotene, a retinal precursor. The gene, designatedcrtY, is predicted to encode an integral membrane protein homologous to lycopene β-cyclases identified in bacteria and fungi. To testcrtYfunction, we constructedH. salinarumstrains with in-frame deletions in the gene. In the deletion strains, bacteriorhodopsin, retinal, and β-carotene were undetectable, whereas lycopene accumulated to high levels (≈1.3 nmol/mg of total cell protein). Heterologous expression ofH. salinarum crtYin a lycopene-producingEscherichia colistrain resulted in β-carotene production. These results indicate thatH. salinarum crtYencodes a functional lycopene β-cyclase required for bacteriorhodopsin biogenesis. Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.
Codenitrification and Denitrification Are Dual Metabolic Pathways through Which Dinitrogen Evolves from Nitrate inStreptomyces antibioticus
Authors: Kumon Yasuyuki , Sasaki Yasuyuki , Kato Isao , Takaya Naoki , Shoun Hirofumi , Beppu Teruhiko
Published: July 2002

We screened actinomycete strains for dinitrogen (N2)-producing activity and discovered thatStreptomyces antibioticusB-546 evolves N2and some nitrous oxide (N2O) from nitrate (NO3−). Most of the N2that evolved from the heavy isotope ([15N]NO3−) was15N14N, indicating that this nitrogen species consists of two atoms, one arising from NO3−and the other from different sources. This phenomenon is similar to codenitrification in fungi. The strain also evolved less, but significant, amounts of15N15N from [15N]NO3−in addition to15N15NO with concomitant cell growth. Prior to the production of N2and N2O, NO3−was rapidly reduced to nitrite (NO2−) accompanied by distinct cell growth, showing that the actinomycete strain is a facultative anaerobe that depends on denitrification and nitrate respiration for anoxic growth. The cell-free activities of denitrifying enzymes could be reconstituted, supporting the notion that the15N15N and15N15NO species are produced by denitrification from NO3−via NO2−. We therefore demonstrated a unique system in an actinomycete that produces gaseous nitrogen (N2and N2O) through both denitrification and codenitrification. The predominance of codenitrification over denitrification along with oxygen tolerance is the key feature of nitrate metabolism in this actinomycete.
Postexponential Regulation of
sin
Operon Expression in
Bacillus subtilis
Authors: Shafikhani Sasha H. , Mandic-Mulec Ines , Strauch Mark A. , Smith Issar , Leighton Terrance
Published: July 2002

The expression of many gene products required during the early stages of
Bacillus subtilis
sporulation is regulated by
sinIR
operon proteins. Transcription of
sinIR
from the P1 promoter is induced at the end of exponential growth. In vivo transcription studies suggest that P1 induction is repressed by the transition-state regulatory protein Hpr and is induced by the phosphorylated form of Spo0A. In vitro DNase I footprinting studies confirmed that Hpr, AbrB, and Spo0A are
trans
-acting transcriptional factors that bind to the P1 promoter region of
sinIR
. We have also determined that the P1 promoter is transcribed in vitro by the major vegetative sigma factor, ς
A
, form of RNA polymerase.
Localization of the Cortex Lytic Enzyme CwlJ in Spores of
Bacillus subtilis
Authors: Bagyan Irina , Setlow Peter
Published: July 2002

The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of
Bacillus subtilis
. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca
2+
-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in
cotE
spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and
cotE
spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.
Effect of Different Concentrations of H-NS Protein on Chromosome Replication and the Cell Cycle in
Escherichia coli
Authors: Atlung T. , Hansen F. G.
Published: July 2002

Flow cytometric analysis showed that the
hns205
and
hns206
mutants, lacking the abundant nucleoid-associated protein H-NS, have decreased origin concentration, as well as a low number of origins per cell (ploidy). The most striking observation was that the low ploidy was due to a very short replication time, e.g., at 30°C it was halved compared to that of the

hns
+

strain. The decreased origin concentration was not caused by a decreased
dnaA
gene expression, and the
hns206
mutant had normal DnaA protein concentrations. The replication phenotypes of the
hns206
mutant were independent of RpoS. Cells overproducing H-NS from a LacI-controlled plasmid had a normal origin concentration, indicating that H-NS is not controlling initiation. A wild-type H-NS concentration is, however, required to obtain a wild-type origin concentration, since cells with an intermediate H-NS concentration had an intermediate origin concentration. Two lines of evidence point to an indirect effect of H-NS on initiation. First, H-NS did not show high-affinity binding to any part of
oriC
, and H-NS had no effect on transcription entering
oriC
from the
mioC
promoter. Second, in a shift experiment with the
hns206
mutant, when H-NS protein was induced to wild-type levels within 10 min, it took more than one generation before the origin concentration started to increase.
Use of the
Caulobacter crescentus
Genome Sequence To Develop a Method for Systematic Genetic Mapping
Authors: West Lisandra , Yang Desiree , Stephens Craig
Published: July 2002

The functional analysis of sequenced genomes will be facilitated by the development of tools for the rapid mapping of mutations. We have developed a systematic approach to genetic mapping in
Caulobacter crescentus
that is based on bacteriophage-mediated transduction of strategically placed antibiotic resistance markers. The genomic DNA sequence was used to identify sites distributed evenly around the chromosome at which plasmids could be nondisruptively integrated. DNA fragments from these sites were amplified by PCR and cloned into a kanamycin-resistant (Kan
r
) suicide vector. Delivery of these plasmids into
C
.
crescentus
resulted in integration via homologous recombination. A set of 41 strains containing Kan
r
markers at 100-kb intervals was thereby generated. These strains serve as donors for generalized transduction using bacteriophage φCr30, which can transduce at least 120 kb of DNA. Transductants are selected with kanamycin and screened for loss of the mutant phenotype to assess linkage between the marker and the site of the mutation. The dependence of cotransduction frequency on sequence distance was evaluated using several markers and mutant strains. With these data as a standard, previously unmapped mutations were readily localized to DNA sequence intervals equivalent to less than 1% of the genome. Candidate genes within the interval were then examined further by subcloning and complementation analysis. Mutations resulting in sensitivity to ampicillin, in nutritional auxotrophies, or temperature-sensitive growth were mapped. This approach to genetic mapping should be applicable to other bacteria with sequenced genomes for which generalized transducing phage are available.
Fluorescent Amplified-Fragment Length Polymorphism Subtyping of the S
almonella enterica
Serovar Enteritidis Phage Type 4 Clone Complex
Authors: Desai Meeta , Threlfall E. John , Stanley John
Published: July 2002

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis, a high-resolution PCR-based genome fingerprinting method, was used to subtype
Salmonella enterica
serovar Enteritidis phage type 4. This single phage type is responsible for the majority of salmonellosis in Europe. Twenty strains isolated from nine outbreaks, five isolates from sporadic cases of human infection, four strains of poultry origin, and one laboratory-derived strain were comparatively studied by pulsed-field gel electrophoresis (PFGE) and FAFLP analysis. Following macrorestriction with
Xba
I, PFGE classified 73% of PT4 strains as a single type. FAFLP analysis was carried out with the primer pair
Eco
RI+0 and
Mse
I+C, by simultaneously sampling 170 to 190 loci throughout the PT4 genome. Twenty-three FAFLP profiles, with 1 to 61 amplified-fragment differences, were found among the 30 strains. The index of discriminatory power of FAFLP analysis was 0.98, compared to 0.47 for PFGE. FAFLP analysis assigned genotypes to each PT4 outbreak, as well as sporadic PT4 infections, a significant development for the epidemiology and control of this zoonotic enteric pathogen.