bluuebunny/crossref_metadata_2025 huggingface dataset:
PcrV Immunization Enhances Survival of Burned
Pseudomonas aeruginosa
-Infected Mice
Authors: Holder Ian Alan , Neely Alice N. , Frank Dara W.
Published: July 2002

Burned
Pseudomonas aeruginosa
-infected mice immunized against PcrV, a type III virulence system translocating protein, showed significantly enhanced survival compared to controls. Survival was non-O serotype specific and correlated with a reduced systemic microbial load. Infection with a high-level toxin A-producing strain required supplemental antitoxin treatment to enhance survival.
In Vivo Behavior of a
Helicobacter pylori
SS1
nixA
Mutant with Reduced Urease Activity
Authors: Nolan Kylie J. , McGee David J. , Mitchell Hazel M. , Kolesnikow Tassia , Harro Janette M. , O'Rourke Jani , Wilson John E. , Danon Stephen J. , Moss Nathan D. , Mobley Harry L. T. , Lee Adrian
Published: July 2002

Helicobacter pylori
mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The
nixA
gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A
nixA
mutant of the Sydney strain of
H. pylori
(SS1) was constructed by disruption of the
nixA
gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j (
n
= 75) and BALB/c (
n
= 75) mice were inoculated independently with the wild-type or the
nixA
strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the
nixA
strain in BALB/c mice were significantly lower compared with SS1 (
P
= 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the
nixA
strain in BALB/c mice. On the contrary, when C57BL/6j (
n
= 35) and BALB/c (
n
= 35) mice were coinfected with the wild-type and
nixA
strains simultaneously, the
nixA
mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the
nixA
gene for increasing the fitness of
H. pylori
for gastric colonization. Since
nixA
is required for full urease activity, the decreased fitness of the
nixA
mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.
Lactoferrin Inhibits the Lipopolysaccharide-Induced Expression and Proteoglycan-Binding Ability of Interleukin-8 in Human Endothelial Cells
Authors: Elass Elisabeth , Masson Maryse , Mazurier Joël , Legrand Dominique
Published: July 2002

Interleukin-8 (IL-8), a C-X-C chemokine bound to endothelium proteoglycans, initiates the activation and selective recruitment of leukocytes at inflammatory foci. We demonstrate that human lactoferrin, an antimicrobial lipopolysaccharide (LPS)-binding protein, decreases both IL-8 mRNA and protein expression induced by the complexEscherichia coli055:B5 LPS/sCD14 in human umbilical vein endothelial cells. The use of recombinant lactoferrins mutated in the LPS-binding sites indicates that this inhibitory effect is mediated by an interaction of lactoferrin with LPS and CD14s that suppresses the endotoxin biological activity. Furthermore, since dimeric IL-8 and lactoferrin are both proteoglycan-binding molecules, the competition between these proteins for heparin binding was investigated. Lactoferrin strongly inhibited the interaction of radiolabeled IL-8 to immobilized heparin, whereas a lactoferrin variant lacking the amino acid residues essential for heparin binding was not inhibitory. Moreover, this process is specific, since serum transferrin, a glycoprotein whose structure is close to that of lactoferrin, did not prevent the interaction of IL-8 with heparin. These results suggest that the anti-inflammatory properties of lactoferrin during septicemia are related, at least in part, to the regulation of IL-8 production and also to the ability of lactoferrin to compete with chemokines for their binding to proteoglycans.
Dendritic Cells as Effector Cells: Gamma Interferon Activation of Murine Dendritic Cells Triggers Oxygen-Dependent Inhibition ofToxoplasma gondiiReplication
Authors: Aline F. , Bout D. , Dimier-Poisson I.
Published: July 2002

Toxoplasma gondiiis an obligate intracellular parasite that infects a wide variety of nucleated cells in its numerous intermediate hosts, including humans. Much interest has focused on the ability of gamma interferon (IFN-γ)-activated macrophages to prevent intracellular replication, but some other cells (e.g., fibroblasts, endothelial cells, microglial cells, astrocytes, enterocytes and retinal pigment cells) can also be activated to induce this inhibition of proliferation. Dendritic cells are generally known to be involved in the induction of immune responses, but no previous study had investigated the possibility that dendritic cells may act as effector cells of this system. Our results show that IFN-γ-activation inhibits the replication ofT. gondiiin dendritic cells, with the inhibition being dose dependent. Neither nitrogen derivatives nor tryptophan starvation appears to be involved in the inhibition of parasite replication by IFN-γ. Experiments with oxygen scavengers indicate that intracellularT. gondiireplication is oxygen dependent. Our findings suggest that, in addition to their essential role in stimulating the immune system, dendritic cells probably act as effector cells in the first line of defense against pathogen invasion.
Rickettsia-Macrophage Interactions: Host Cell Responses toRickettsia akariandRickettsia typhi
Authors: Radulovic S. , Price P. W. , Beier M. S. , Gaywee J. , Macaluso J. A. , Azad A.
Published: July 2002

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akariand -Rickettsia typhiinteractions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated thatR. akariandR. typhicaused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6. Furthermore, macrophage infection withR. akariandR. typhiresulted in differential synthesis and expression of IL-β and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-γ), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed betweenR. akari-andR. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.
Chlamydia trachomatis
Disrupts N-Cadherin-Dependent Cell-Cell Junctions and Sequesters β-Catenin in Human Cervical Epithelial Cells
Authors: Prozialeck Walter C. , Fay Michael J. , Lamar Peter C. , Pearson Celeste A. , Sigar Ira , Ramsey Kyle H.
Published: July 2002

The cadherin/catenin complex serves as an important structural component of adherens junctions in epithelial cells. Under certain conditions, β-catenin can be released from this complex and interact with transcription factors in the nucleus to stimulate the expression of genes that regulate apoptosis and cell cycle control. While studying the effects of the bacterial pathogen
Chlamydia trachomatis
on human cervical epithelial cells in culture, we observed that
C. trachomatis
caused the epithelial cells to separate from each other without detaching from their growing surface. The objective of the present study was to determine if this effect might involve the disruption of the cadherin/catenin complex. Primary cultures of human cervical epithelial cells or HeLa cells were infected with
C. trachomatis
serovar E. Cadherin-like immunoreactive materials and β-catenin were visualized by immunofluorescence. Preliminary studies showed that N-cadherin was the primary cadherin expressed in both the primary cultures and the HeLa cells. In noninfected cells, N-cadherin and β-catenin were colocalized at the intercellular junctional complexes. By contrast, the infected cells showed a marked loss of both N-cadherin and β-catenin labeling from the junctional complexes and the concomitant appearance of intense β-catenin labeling associated with the chlamydial inclusion. The results of Western blot analyses of extracts of
C. trachomatis
showed no evidence of cross-reactivity with the β-catenin antibody. These results indicate that
C. trachomatis
causes the breakdown of the N-cadherin/β-catenin complex and that the organism can sequester β-catenin within the chlamydial inclusion. This could represent an important mechanism by which
C. trachomatis
alters epithelial cell function.
Role of Toll-Like Receptors in Changes in Gene Expression and NF-κB Activation in Mouse Hepatocytes Stimulated with Lipopolysaccharide
Authors: Liu Shubing , Gallo David J. , Green Angela M. , Williams Debra L. , Gong Xiaoyan , Shapiro Richard A. , Gambotto Andrea A. , Humphris Elisabeth L. , Vodovotz Yoram , Billiar Timothy R.
Published: July 2002

The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-κB activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.
Biosynthesis of Lipoteichoic Acid in
Lactobacillus rhamnosus
: Role of DltD in
d
-Alanylation
Authors: Debabov Dmitri V. , Kiriukhin Michael Y. , Neuhaus Francis C.
Published: July 2002

The
dlt
operon (
dltA
to
dltD
) of
Lactobacillus rhamnosus
7469 encodes four proteins responsible for the esterification of lipoteichoic acid (LTA) by
d
-alanine. These esters play an important role in controlling the net anionic charge of the poly (GroP) moiety of LTA.
dltA
and
dltC
encode the
d
-alanine–
d
-alanyl carrier protein ligase (Dcl) and
d
-alanyl carrier protein (Dcp), respectively. Whereas the functions of DltA and DltC are defined, the functions of DltB and DltD are unknown. To define the role of DltD, the gene was cloned and sequenced and a mutant was constructed by insertional mutagenesis of
dltD
from
Lactobacillus casei
102S. Permeabilized cells of a
dltD
::
erm
mutant lacked the ability to incorporate
d
-alanine into LTA. This defect was complemented by the expression of DltD from pNZ123/
dlt
. In in vitro assays, DltD bound Dcp for ligation with
d
-alanine by Dcl in the presence of ATP. In contrast, the homologue of Dcp, the
Escherichia coli
acyl carrier protein (ACP), involved in fatty acid biosynthesis, was not bound to DltD and thus was not ligated with
d
-alanine. DltD also catalyzed the hydrolysis of the mischarged
d
-alanyl–ACP. The hydrophobic N-terminal sequence of DltD was required for anchoring the protein in the membrane. It is hypothesized that this membrane-associated DltD facilitates the binding of Dcp and Dcl for ligation of Dcp with
d
-alanine and that the resulting
d
-alanyl–Dcp is translocated to the primary site of
d
-alanylation.
Growth Phase-Coupled Changes of the Ribosome Profile in Natural Isolates and Laboratory Strains of
Escherichia coli
Authors: Wada Akira , Mikkola Riitta , Kurland Charles G. , Ishihama Akira
Published: July 2002

The growth phase-dependent change in sucrose density gradient centrifugation patterns of ribosomes was analyzed for both laboratory strains of
Escherichia coli
and natural isolates from the ECOR collection. All of the natural isolates examined formed 100S ribosome dimers in the stationary phase, and ribosome modulation factor (RMF) was associated with the ribosome dimers in the ECOR strains as in the laboratory strain W3110. The ribosome profile (70S monomers versus 100S dimers) follows a defined pattern over time during lengthy culture in both the laboratory strains and natural isolates. There are four discrete stages: (i) formation of 100S dimers in the early stationary phase; (ii) transient decrease in the dimer level; (iii) return of dimers to the maximum level; and (iv) dissociation of 100S dimers into 70S ribosomes, which are quickly degraded into subassemblies. The total time for this cycle of ribosome profile change, however, varied from strain to strain, resulting in apparent differences in the ribosome profiles when observed at a fixed time point. A correlation was noted in all strains between the decay of 100S ribosomes and the subsequent loss of cell viability. Two types of
E. coli
mutants defective in ribosome dimerization were identified, both of which were unable to survive for a prolonged period in stationary phase. The W3110 mutant, with a disrupted
rmf
gene, has a defect in ribosome dimerization because of lack of RMF, while strain Q13 is unable to form ribosome dimers due to a ribosomal defect in binding RMF.
Fermentative Metabolism of
Bacillus subtilis
: Physiology and Regulation of Gene Expression
Authors: Cruz Ramos Hugo , Hoffmann Tamara , Marino Marco , Nedjari Hafed , Presecan-Siedel Elena , Dreesen Oliver , Glaser Philippe , Jahn Dieter
Published: July 2002

Bacillus subtilis
grows in the absence of oxygen using nitrate ammonification and various fermentation processes. Lactate, acetate, and 2,3-butanediol were identified in the growth medium as the major anaerobic fermentation products by using high-performance liquid chromatography. Lactate formation was found to be dependent on the
lctEP
locus, encoding lactate dehydrogenase and a putative lactate permease. Mutation of
lctE
results in drastically reduced anaerobic growth independent of the presence of alternative electron acceptors, indicating the importance of NADH reoxidation by lactate dehydrogenase for the overall anaerobic energy metabolism. Anaerobic formation of 2,3-butanediol via acetoin involves acetolactate synthase and decarboxylase encoded by the
alsSD
operon. Mutation of
alsSD
has no significant effect on anaerobic growth. Anaerobic acetate synthesis from acetyl coenzyme A requires phosphotransacetylase encoded by
pta
. Similar to the case for
lctEP
, mutation of
pta
significantly reduces anaerobic fermentative and respiratory growth. The expression of both
lctEP
and
alsSD
is strongly induced under anaerobic conditions. Anaerobic
lctEP
and
alsSD
induction was found to be partially dependent on the gene encoding the redox regulator Fnr. The observed
fnr
dependence might be the result of Fnr-induced
arfM
(
ywiD
) transcription and subsequent
lctEP
and
alsSD
activation by the regulator ArfM (YwiD). The two-component regulatory system encoded by
resDE
is also involved in anaerobic
lctEP
induction. No direct
resDE
influence on the redox regulation of
alsSD
was observed. The alternative electron acceptor nitrate represses anaerobic
lctEP
and
alsSD
transcription. Nitrate repression requires
resDE
- and
fnr
-dependent expression of
narGHJI
, encoding respiratory nitrate reductase. The gene
alsR
, encoding a regulator potentially responding to changes of the intracellular pH and to acetate, is essential for anaerobic
lctEP
and
alsSD
expression. In agreement with its known aerobic function, no obvious oxygen- or nitrate-dependent
pta
regulation was observed. A model for the regulation of the anaerobic fermentation genes in
B. subtilis
is proposed.
Evidence that SpoIVFB Is a Novel Type of Membrane Metalloprotease Governing Intercompartmental Communication during
Bacillus subtilis
Sporulation
Authors: Yu Yuen-Tsu Nicco , Kroos Lee
Published: July 2002

Processing of pro-ς
K
in the mother cell compartment of sporulating
Bacillus subtilis
involves SpoIVFB and is governed by a signal from the forespore. SpoIVFB has an HEXXH motif characteristic of metalloproteases embedded in one of its transmembrane segments. Several conservative single amino acid changes in the HEXXH motif abolished function. However, changing the glutamic acid residue to aspartic acid, or changing the isoleucine residue that precedes the motif to proline, permitted SpoIVFB function. Only one other putative metalloprotease, site 2 protease has been shown to tolerate aspartic acid rather than glutamic acid in its HEXXH sequence. Site 2 protease and SpoIVFB share a second region of similarity with a family of putative membrane metalloproteases. A conservative change in this region of SpoIVFB abolished function. Interestingly, SpoIVFA increased the accumulation of certain mutant SpoIVFB proteins but was unnecessary for accumulation of wild-type SpoIVFB.
Target Site Selection by Tn
7
:
att
Tn
7
Transcription and Target Activity
Authors: DeBoy Robert T. , Craig Nancy L.
Published: July 2002

The bacterial transposon Tn
7
inserts at high frequency into a specific site called
att
Tn
7
, which is present in the chromosomes of many bacteria. We show here that transcription of a nearby gene,
glmS
, decreases the frequency of Tn
7
insertion into
att
Tn
7
, thus providing a link between Tn
7
transposition and host cell metabolism.
Megaplasmid pRme2011a of
Sinorhizobium meliloti
Is Not Required for Viability
Authors: Oresnik Ivan J. , Liu Shu-Lin , Yost Christopher K. , Hynes Michael F.
Published: July 2002

We report the curing of the 1,360-kb megaplasmid pRme2011a from
Sinorhizobium meliloti
strain Rm2011. With a positive selection strategy that utilized Tn
5
B12-S containing the
sacB
gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.
Eight of Fourteen
gvp
Genes Are Sufficient for Formation of Gas Vesicles in Halophilic Archaea
Authors: Offner Sonja , Hofacker Annette , Wanner Gerhard , Pfeifer Felicitas
Published: July 2002

The minimal number of genes required for the formation of gas vesicles in halophilic archaea has been determined. Single genes of the 14
gvp
genes present in the p-vac region on plasmid pHH1 of
Halobacterium salinarum
(p-
gvpACNO
and p-
gvpDEFGHIJKLM
) were deleted, and the remaining genes were tested for the formation of gas vesicles in
Haloferax volcanii
transformants. The deletion of six
gvp
genes (p-
gvpCN
, p-
gvpDE
, and p-
gvpHI
) still enabled the production of gas vesicles in
H. volcanii
. The gas vesicles formed in some of these
gvp
gene deletion transformants were altered in shape (ΔI, ΔC) or strength (ΔH) but still functioned as flotation devices. A minimal p-vac region (minvac) containing the eight remaining genes (
gvpFGJKLM-gvpAO
) was constructed and tested for gas vesicle formation in
H. volcanii
. The minvac transformants did not form gas vesicles; however, minvac/gvpJKLM double transformants contained gas vesicles seen as light refractile bodies by phase-contrast microscopy. Transcript analyses demonstrated that minvac transformants synthesized regular amounts of
gvpA
mRNA, but the transcripts derived from
gvpFGJKLM
were mainly short and encompassed only
gvpFG
(
J
), suggesting that the
gvpJKLM
genes were not sufficiently expressed. Since
gvpAO
and
gvpFGJKLM
are the only
gvp
genes present in minvac/JKLM transformants containing gas vesicles, these
gvp
genes represent the minimal set required for gas vesicle formation in halophilic archaea. Homologs of six of these
gvp
genes are found in
Anabaena flos-aquae
, and homologs of all eight minimal halobacterial
gvp
genes are present in
Bacillus megaterium
and in the genome of
Streptomyces coelicolor
.
Elevated Levels of Synthesis of over 20 Proteins Results after Mutation of the
Rhizobium leguminosarum
Exopolysaccharide Synthesis Gene
pssA
Authors: Guerreiro Nelson , Ksenzenko Vladimir N. , Djordjevic Michael A. , Ivashina Tanya V. , Rolfe Barry G.
Published: July 2002

The protein expression profiles of
Rhizobium leguminosarum
strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either
pssA
,
pssD
, or
pssE
of
R. leguminosarum
bv. viciae VF39 or in
pssA
of
R. leguminosarum
bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two
pssA
mutant strains. The differences identified in the
pssD
and
pssE
mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the
pssA
mutant. The
pssD
and
pssE
mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a
pssC
mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of
pssA
can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the
pssA
mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.
An Oligoribonuclease Gene in
Streptomyces griseus
Authors: Ohnishi Yasuo , Nishiyama Yoko , Sato Rie , Kameyama Shogo , Horinouchi Sueharu
Published: July 2002

In
Streptomyces griseus
, A-factor (2-isocapryloyl-3
R
-hydroxymethyl-γ-butyrolactone) serves as a microbial hormone that switches on many genes required for streptomycin production and morphological development. An open reading frame (Orf1) showing high sequence similarity to oligoribonucleases of various origins is present just downstream of
adpA
, one of the A-factor-dependent genes. Orf1 was named OrnA (oligoribonuclease A) because it showed 3′-to-5′ exo-oligoribonuclease activity, releasing [
32
P]CMP from ApCpC[
32
P]pC used as a substrate. Reverse transcription-PCR and S1 nuclease mapping analyses revealed that
ornA
was transcribed from two promoters; one was a developmentally regulated, A-factor-dependent promoter in front of
adpA
, and the other was a constitutive promoter in front of the
ornA
coding sequence. Transcription of
ornA
was thus additively enhanced at the initiation stage for secondary metabolism and aerial mycelium formation.
ornA
-disrupted strains grew slowly and scarcely formed aerial mycelium.
ornA
homologues were distributed in a wide variety of
Streptomyces
species, including
S. coelicolor
A3(2), as determined by Southern hybridization analysis. Disruption of the
ornA
homologue in
S. coelicolor
A3(2) also caused phenotypes similar to those of the
S. griseus ΔornA
strains. The OrnA oligoribonucleases in
Streptomyces
species are therefore not essential but play an important role in vegetative growth and in the initiation of differentiation.
Cloning and Characterization of the Glucooligosaccharide Catabolic Pathway β-Glucan Glucohydrolase and Cellobiose Phosphorylase in the Marine HyperthermophileThermotoga neapolitana
Authors: Yernool Dinesh A. , McCarthy James K. , Eveleigh Douglas E. , Bok Jin-Duck
Published: July 2002

Characterization inThermotoga neapolitanaof a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-β-d-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization inThermotogales. GghA purified fromT. neapolitanais a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95°C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers:kcat/Kmvalues are 155.2, 76.0, and 9.9 mM−1s−1for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. Withp-nitrophenyl-β-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning ofgghArevealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85°C. It has specific activity of 11.8 U/mg and aKmof 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and lowKmfor cellobiose (compared to GghA'sKmof 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose inThermotogaspp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.
The NosX and NirX Proteins of
Paracoccus denitrificans
Are Functional Homologues: Their Role in Maturation of Nitrous Oxide Reductase
Authors: Saunders Neil F. W. , Hornberg Jorrit J. , Reijnders Willem N. M. , Westerhoff Hans V. , de Vries Simon , van Spanning Rob J. M.
Published: July 2002

The
nos
(nitrous oxide reductase) operon of
Paracoccus denitrificans
contains a
nosX
gene homologous to those found in the
nos
operons of other denitrifiers. NosX is also homologous to NirX, which is so far unique to
P. denitrificans
. Single mutations of these genes did not result in any apparent phenotype, but a double
nosX nirX
mutant was unable to reduce nitrous oxide. Promoter-
lacZ
assays and immunoblotting against nitrous oxide reductase showed that the defect was not due to failure of expression of
nosZ
, the structural gene for nitrous oxide reductase. Electron paramagnetic resonance spectroscopy showed that nitrous oxide reductase in cells of the double mutant lacked the Cu
A
center. A twin-arginine motif in both NosX and NirX suggests that the NosX proteins are exported to the periplasm via the TAT translocon.
Characterization of
ssfR
and
ssgA
, Two Genes Involved in Sporulation of
Streptomyces griseus
Authors: Jiang Hao , Kendrick Kathleen E.
Published: July 2002

In the presence of cefoxitin, which inhibits septum formation during sporulation,
Streptomyces griseus
is unable to sporulate, retaining the sonication sensitivity of nonsporulating hyphae. Cefoxitin- and sonication-resistant mutant SKK2600 was isolated and showed many morphological differences from its parental strain. A 3.6-kb DNA fragment that complemented the mutations of SKK2600 contained two open reading frames (ORFs), either of which could complement SKK2600. One ORF, designated
ssfR
, encoded a protein containing a potential DNA-binding helix-turn-helix motif close to its N terminus. SsfR is similar to members of a large family of transcriptional regulators, particularly IclR of
Escherichia coli
. The second ORF was identified as
ssgA
, a previously described sporulation gene from
S. griseus
(S. Kawamoto and J. C. Ensign, Actinomycetology 9:136–151, 1995). A point mutation of C to T seven nucleotides upstream of the UGA stop codon of
ssfR
was responsible for the phenotype of isolated mutant strain SKK2600. Surprisingly, this mutation should not change the primary structure of SsfR. The
ssfR
and
ssgA
disruption mutants were constructed and showed the “white” mutant phenotype, with some growth medium dependence. In addition, the
ssfR
null mutant sporulated ectopically in phosphate starvation medium.
Isolation of Regulated Genes of the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 by Differential Display
Authors: Bhaya Devaki , Vaulot Daniel , Amin Pinky , Takahashi Akiko Watanabe , Grossman Arthur R.
Published: July 2002

Global identification of differentially regulated genes in prokaryotes is constrained because the mRNA does not have a 3′ polyadenylation extension; this precludes specific separation of mRNA from rRNA and tRNA and synthesis of cDNAs from the entire mRNA population. Knowledge of the entire genome sequence of
Synechocystis
sp. strain PCC 6803 has enabled us to develop a differential display procedure that takes advantage of a short palindromic sequence that is dispersed throughout the
Synechocystis
sp. strain PCC 6803 genome. This sequence, designated the HIP (highly iterated palindrome) element, occurs in approximately half of the
Synechocystis
sp. strain PCC 6803 genes but is absent in rRNA and tRNA genes. To determine the feasibility of exploiting the HIP element, alone or in combination with specific primer subsets, for analyzing differential gene expression, we used HIP-based primers to identify light intensity-regulated genes. Several gene fragments, including those encoding ribosomal proteins and phycobiliprotein subunits, were differentially amplified from RNA templates derived from cells grown in low light or exposed to high light for 3 h. One novel finding was that expression of certain genes of the
pho
regulon, which are under the control of environmental phosphate levels, were markedly elevated in high light. High-light activation of
pho
regulon genes correlated with elevated growth rates that occur when the cells are transferred from low to high light. These results suggest that in high light, the rate of growth of
Synechocystis
sp. strain PCC 6803 exceeds its capacity to assimilate phosphate, which, in turn, may trigger a phosphate starvation response and activation of the
pho
regulon.
Identification and Mapping of Sigma-54 Promoters in
Chlamydia trachomatis
Authors: Mathews Sarah A. , Timms Peter
Published: July 2002

The first ς
54
promoters in
Chlamydia trachomatis
L2 were mapped upstream of hypothetical proteins CT652.1 and CT683. Comparative genomics indicated that these ς
54
promoters and potential upstream activation binding sites are conserved in orthologous
C. trachomatis
D,
C. trachomatis
mouse pneumonitis strain, and
Chlamydia pneumoniae
(CWL029 and AR39) genes.
Role of the Carboxy Terminus of
Escherichia coli
FtsA in Self-Interaction and Cell Division
Authors: Yim Lucía , Vandenbussche Guy , Mingorance Jesús , Rueda Sonsoles , Casanova Mercedes , Ruysschaert Jean-Marie , Vicente Miguel
Published: July 2002

The role of the carboxy terminus of the
Escherichia coli
cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of the
ftsA
alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362–5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsAΔ1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsAΔ27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsAΔ27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.
Complementary Metal Ion Specificity of the Metal-Citrate Transporters CitM and CitH of
Bacillus subtilis
Authors: Krom Bastiaan P. , Warner Jessica B. , Konings Wil N. , Lolkema Juke S.
Published: July 2002

Citrate uptake in
Bacillus subtilis
is stimulated by a wide range of divalent metal ions. The metal ions were separated into two groups based on the expression pattern of the uptake system. The two groups correlated with the metal ion specificity of two homologous
B. subtilis
secondary citrate transporters, CitM and CitH, upon expression in
Escherichia coli
. CitM transported citrate in complex with Mg
2+
, Ni
2+
, Mn
2+
, Co
2+
, and Zn
2+
but not in complex with Ca
2+
, Ba
2+
, and Sr
2+
. CitH transported citrate in complex with Ca
2+
, Ba
2+
, and Sr
2+
but not in complex with Mg
2+
, Ni
2+
, Mn
2+
, Co
2+
, and Zn
2+
. Both transporters did not transport free citrate. Nevertheless, free citrate uptake could be demonstrated in
B. subtilis
, indicating the expression of at least a third citrate transporter, whose identity is not known. For both the CitM and CitH transporters it was demonstrated that the metal ion promoted citrate uptake and, vice versa, that citrate promoted uptake of the metal ion, indicating that the complex is the transported species. The results indicate that CitM and CitH are secondary transporters that transport complexes of divalent metal ions and citrate but with a complementary metal ion specificity. The potential physiological function of the two transporters is discussed.
Characterization of a Novel Outer Membrane Hemin-Binding Protein of
Porphyromonas gingivalis
Authors: Dashper S. G. , Hendtlass A. , Slakeski N. , Jackson C. , Cross K. J. , Brownfield L. , Hamilton R. , Barr I. , Reynolds E. C.
Published: July 2002

Porphyromonas gingivalis
is a gram-negative, anaerobic coccobacillus that has been implicated as a major etiological agent in the development of chronic periodontitis. In this paper, we report the characterization of a protein, IhtB (iron heme transport; formerly designated Pga30), that is an outer membrane hemin-binding protein potentially involved in iron assimilation by
P. gingivalis
. IhtB was localized to the cell surface of
P. gingivalis
by Western blot analysis of a Sarkosyl-insoluble outer membrane preparation and by immunocytochemical staining of whole cells using IhtB peptide-specific antisera. The protein, released from the cell surface, was shown to bind to hemin using hemin-agarose. The growth of heme-limited, but not heme-replete,
P. gingivalis
cells was inhibited by preincubation with IhtB peptide-specific antisera. The
ihtB
gene was located between an open reading frame encoding a putative TonB-linked outer membrane receptor and three open reading frames that have sequence similarity to ATP binding cassette transport system operons in other bacteria. Analysis of the deduced amino acid sequence of IhtB showed significant similarity to the
Salmonella typhimurium
protein CbiK, a cobalt chelatase that is structurally related to the ATP-independent family of ferrochelatases. Molecular modeling indicated that the IhtB amino acid sequence could be threaded onto the CbiK fold with the IhtB structural model containing the active-site residues critical for chelatase activity. These results suggest that IhtB is a peripheral outer membrane chelatase that may remove iron from heme prior to uptake by
P. gingivalis
.
Evidence for Horizontal Gene Transfer in Evolution of Elongation Factor Tu in Enterococci
Authors: Ke Danbing , Boissinot Maurice , Huletsky Ann , Picard François J. , Frenette Johanne , Ouellette Marc , Roy Paul H. , Bergeron Michel G.
Published: July 2002

The elongation factor Tu, encoded bytufgenes, is a GTP binding protein that plays a central role in protein synthesis. One to threetufgenes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only onetufgene. We have designed degenerate PCR primers derived from consensus sequences of thetufgene to amplify partialtufsequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two differenttufgenes (tufAandtufB) were found in 11 enterococcal species, includingEnterococcus avium,Enterococcus casseliflavus,Enterococcus dispar,Enterococcus durans,Enterococcus faecium,Enterococcus gallinarum,Enterococcus hirae,Enterococcus malodoratus,Enterococcus mundtii,Enterococcus pseudoavium, andEnterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum,Enterococcus columbae,Enterococcus faecalis,Enterococcus sulfureus,Enterococcus saccharolyticus, andEnterococcus solitarius), only thetufAgene was present. Based on 16S rRNA gene sequence analysis, the 11 species having twotufgenes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of thetufgene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis oftufsequences demonstrated that the enterococcaltufAgene branches with theBacillus,Listeria, andStaphylococcusgenera, while the enterococcaltufBgene clusters with the generaStreptococcusandLactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcaltufBgenes and thetufgenes of streptococci andLactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred atufgene to the common ancestor of the 11 enterococcal species which now carry twotufgenes.
Molecular Evolution of Virulence in Natural Field Strains of
Xanthomonas campestris
pv. vesicatoria
Authors: Gassmann Walter , Dahlbeck Douglas , Chesnokova Olga , Minsavage Gerald V. , Jones Jeffrey B. , Staskawicz Brian J.
Published: July 2002

The
avrBs2
avirulence gene of the bacterial plant pathogen
Xanthomonas campestris
pv. vesicatoria triggers disease resistance in pepper plants containing the
Bs2
resistance gene and contributes to bacterial virulence on susceptible host plants. We studied the effects of the pepper
Bs2
gene on the evolution of
avrBs2
by characterizing the molecular basis for virulence of 20
X. campestris
pv. vesicatoria field strains that were isolated from disease spots on previously resistant
Bs2
pepper plants. All field strains tested were complemented by a wild-type copy of
avrBs2
in their ability to trigger disease resistance on
Bs2
plants. DNA sequencing revealed four mutant alleles of
avrBs2
, two of which consisted of insertions or deletions of 5 nucleotides in a repetitive region of
avrBs2
. The other two
avrBs2
alleles were characterized by point mutations with resulting single amino acid changes (R403P or A410D). We generated isogenic
X. campestris
pv. vesicatoria strains by chromosomal
avrBs2
gene exchange to study the effects of these mutations on the dual functions of
avrBs2
in enhancing bacterial virulence and inducing plant resistance by in planta bacterial growth experiments. The deletion of 5 nucleotides led to loss of
avrBs2
-induced resistance on
Bs2
pepper plants and abolition of
avrBs2
-mediated enhancement of fitness on susceptible plants. Significantly, the point mutations led to minimal reduction in virulence function of
avrBs2
on susceptible pepper plants, with either minimal (R403P allele) or an intermediate level of (A410D allele) triggering of resistance on
Bs2
plants. Consistent with the divergent selection pressures on
avrBs2
exerted by the
Bs2
resistance gene, our results show that
avrBs2
is evolving to decrease detection by the
Bs2
gene while at the same time maintaining its virulence function.
Sensitive Genetic Screen for Protease Activity Based on a Cyclic AMP Signaling Cascade in
Escherichia coli
Authors: Dautin Nathalie , Karimova Gouzel , Ullmann Agnes , Ladant Daniel
Published: July 2002

We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in
Escherichia coli
. This genetic test is based on the specific proteolysis of a signaling enzyme, the adenylate cyclase (AC) of
Bordetella pertussis
. As a model system we used the human immunodeficiency virus (HIV) protease. When an HIV protease processing site, p5, was inserted in frame into the AC polypeptide, the resulting ACp5 protein retained enzymatic activity and, when expressed in an
E. coli cya
strain, restored the Cya
+
phenotype. The HIV protease coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya
−
. When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the resulting AC-HIV-Pr fusion protein, expressed in
E. coli cya
, was autoproteolysed and inactivated: the cells displayed Cya
−
phenotype. In the presence of the protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cells were Cya
+
. Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya
−
phenotype in the presence of these inhibitors. This genetic test could represent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients undergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.
Transcription Activation by a Variety of AraC/XylS Family Activators Does Not Depend on the Class II-Specific Activation Determinant in the N-Terminal Domain of the RNA Polymerase Alpha Subunit
Authors: Egan Susan M. , Pease Andrew J. , Lang Jeffrey , Li Xiang , Rao Vydehi , Gillette William K. , Ruiz Raquel , Ramos Juan L. , Wolf Richard E.
Published: July 2002

The N-terminal domain of the RNA polymerase α subunit (α-NTD) was tested for a role in transcription activation by a variety of AraC/XylS family members. Based on substitutions at residues 162 to 165 and an extensive genetic screen we conclude that α-NTD is not an activation target for these activators.
A Novel Operon Encoding Formaldehyde Fixation: the Ribulose Monophosphate Pathway in the Gram-Positive Facultative Methylotrophic Bacterium
Mycobacterium gastri
MB19
Authors: Mitsui Ryoji , Sakai Yasuyoshi , Yasueda Hisashi , Kato Nobuo
Published: July 2002

A 4.2-kb
Pst
I fragment harboring the gene cluster of the ribulose monophosphate (RuMP) pathway for formaldehyde fixation was identified in the chromosome of a gram-positive, facultative methylotroph,
Mycobacterium gastri
MB19, by using the coding region of 3-hexulose-6-phosphate synthase (HPS) as the hybridization probe. The
Pst
I fragment contained three complete open reading frames (ORFs) which encoded from the 5′ end, a DNA-binding regulatory protein (
rmpR
), 6-phospho-3-hexuloisomerase (PHI;
rmpB
), and HPS (
rmpA
). Sequence analysis suggested that
rmpA
and
rmpB
constitute an operon, and Northern blot analysis of RNA extracted from bacteria grown under various conditions suggested that the expression of the two genes is similarly regulated at the transcriptional level. A similarity search revealed that the proteins encoded by
rmpA
and
rmpB
in
M. gastri
MB19 show high similarity to the unidentified proteins of nonmethylotrophic prokaryotes, including bacteria and anaerobic archaea. The clusters in the phylogenetic tree of the HPS protein of
M. gastri
MB19 and those in the phylogenetic tree of the PHI protein were nearly identical, which implies that these two formaldehyde-fixing genes evolved as a pair. These findings give new insight into the acquisition of the formaldehyde fixation pathway during the evolution of diverse microorganisms.
Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of
Sinorhizobium meliloti
on PHB Cycle Intermediates
Authors: Cai Guo-qin , Driscoll Brian T. , Charles Trevor C.
Published: July 2002

We have identified two
Sinorhizobium meliloti
chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene
acsA
, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the
acsA
nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown
acsA
::Tn
5
mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the
S. meliloti
poly-3-hydroxybutyrate cycle. The second locus was identified as
phbC
, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.