bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Antibodies againstPlasmodium falciparumLiver-Stage Antigen 3 Cross-React withPlasmodium yoeliiPreerythrocytic-Stage Epitopes and Inhibit Sporozoite Invasion In Vitro and In Vivo
Authors: Brahimi Karima , Badell Edgar , Sauzet Jean-Pierre , BenMohamed Lbachir , Daubersies Pierre , Guérin-Marchand Claudine , Snounou George , Druilhe Pierre
Published: July 2002

ThePlasmodium falciparumliver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes inPlasmodium yoeliipreerythrocytic stages. On sporozoites theP. yoeliiprotein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting withP. yoeliiwere also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found inPlasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion ofP. yoeliisporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest thatP. yoeliicould be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.
Structural and Genetic Analyses of O Polysaccharide from
Actinobacillus actinomycetemcomitans
Serotype f
Authors: Kaplan Jeffrey B. , Perry Malcolm B. , MacLean Leann L. , Furgang David , Wilson Mark E. , Fine Daniel H.
Published: July 2002

The oral bacterium
Actinobacillus actinomycetemcomitans
is implicated as a causative agent of localized juvenile periodontitis (LJP).
A. actinomycetemcomitans
is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from
A. actinomycetemcomitans
CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of
l
-rhamnose and 2-acetamido-2-deoxy-
d
-galactose (molar ratio, 2:1) with the structure →2)-α-
l
-Rha
p
-(1–3)-2-
O
-(β-
d
-Gal
p
NAc)-α-
l
-Rha
p
-(1→. O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known
A. actinomycetemcomitans
serotypes. Strain CU1000 was mutagenized with transposon IS
903
φ
kan
, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of
A. actinomycetemcomitans
serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known
A. actinomycetemcomitans
serotypes. These data indicate that strain CU1000 should be assigned to a new
A. actinomycetemcomitans
serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored
A. actinomycetemcomitans
strains carrying the serotype f gene cluster. The finding of an
A. actinomycetemcomitans
serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.
Cytokine Responses to Group B Streptococci Induce Nitric Oxide Production in Respiratory Epithelial Cells
Authors: Goodrum Kenneth J. , Poulson-Dunlap Jane
Published: July 2002

Streptococcus agalactiae
(group B streptococcus [GBS]) is a leading cause of neonatal pneumonia, sepsis, and meningitis. Early-onset GBS pneumonia is characterized by marked pulmonary epithelial and endothelial cell injury. Innate proinflammatory responses to GBS infection that may contribute to the respiratory pathology include the synthesis and release of cytokines, prostaglandins, and nitric oxide (NO). The hypothesis that NO is directly induced in lung epithelial cells by invading GBS or indirectly induced by cytokines released by GBS-infected mononuclear cells was tested. A549 transformed human respiratory epithelial cells were directly cultured with GBS, cocultured with GBS-infected human mononuclear cells or purified macrophages, or exposed to conditioned culture medium from human mononuclear cells infected by GBS. The culture medium of A549 cultures was assayed for NO secretion, and the cell lysates were tested for presence of inducible nitric oxide synthase (iNOS) mRNA by reverse transcriptase PCR (RT-PCR). GBS-treated A549 cells neither secreted detectable NO nor expressed iNOS mRNA. GBS interaction with human mononuclear cells, however, stimulated release of soluble factors that readily induced iNOS mRNA expression and NO secretion by A549 cells. Inflammatory mediator-induced nitric oxide (NO) production by alveolar epithelium may exceed that of other lung cell types such as macrophages, and induction during GBS infection may play a significant role in pulmonary defense or free-radical-mediated lung injury.
Comparison of Biofilms Formed byCandidaalbicansandCandidaparapsilosison Bioprosthetic Surfaces
Authors: Kuhn D. M. , Chandra J. , Mukherjee P. K. , Ghannoum M. A.
Published: July 2002

Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by differentCandidaspecies, particularlyCandidaalbicansandC.parapsilosis, was evaluated by using a clinically relevant model ofCandidabiofilm on medical devices.Candidabiofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasiveC.albicansisolates were seen, significant differences between invasive and noninvasive isolates (P< 0.001) were noted.C.albicansisolates produced more biofilm thanC.parapsilosis,C.glabrata, andC.tropicalisisolates, as determined by DW assays (Pwas <0.001 for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy,C.albicansbiofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast,C.parapsilosisbiofilms had less volume thanC.albicansbiofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells,Candidabiofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 μg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show thatC.albicansproduces quantitatively larger and qualitatively more complex biofilms than other species, in particular,C.parapsilosis.
Shigella flexneriInteractions with the Basolateral Membrane Domain of Polarized Model Intestinal Epithelium: Role of Lipopolysaccharide in Cell Invasion and in Activation of the Mitogen-Activated Protein Kinase ERK
Authors: Köhler Henrik , Rodrigues Sonia P. , McCormick Beth A.
Published: July 2002

An early step governingShigella flexneripathogenesis is the invasion of the colonic epithelium from the basolateral surface followed by disruption of the colonic epithelial barrier. Despite recent insight intoS. flexneri-host interactions, much remains to be determined regarding the nature of the initial contact betweenS. flexneriand the host epithelial basolateral membrane domain. Since the lipopolysaccharide (LPS) is located at the outermost part of the bacterial membrane, we considered that this component might be used byS. flexnerito attach to the basolateral surface of the intestinal epithelium and promote a proinflammatory response. Therefore, polarized human T84 intestinal epithelial cells were infected from the basolateral surface with either wild-typeS. flexnerior one of its isogenic LPS-defective strains with mutations in eitherrfc,rfaL, orgalU. We found that both adherence to and internalization into the basolateral surface of a polarized intestinal epithelium withS. flexneriwere highly dependent on the length of the LPS (i.e.,rfc>rfaL>galU). Furthermore, the addition of the anti-inflammatory LPS (RsDPLA) considerably decreased the invasion profile of wild-typeS. flexneriby nearly 50%. Since LPS is associated with host inflammation, we further examined whether this molecule was involved inShigella-induced inflammatory events. We found thatS. flexneriLPS plays an important role in mediating epithelial-derived signaling, which leads to directed migration of polymorphonuclear leukocytes across model intestinal epithelium. This signaling most likely involves the activation of the mitogen-activated protein kinase extracellular regulated kinase. Thus, our findings have important implications on the understanding of the mechanisms by whichS. flexnerican elicit mucosal inflammation.
Characterization of Humoral and Cellular Immune Responses Elicited by Meningococcal Carriage
Authors: Robinson K. , Neal K. R. , Howard C. , Stockton J. , Atkinson K. , Scarth E. , Moran J. , Robins A. , Todd I. , Kaczmarski E. , Gray S. , Muscat I. , Slack R. , Ala'Aldeen D. A. A.
Published: July 2002

In order to study the immune response elicited by asymptomatic carriage ofNeisseria meningitidis, samples of serum, peripheral blood mononuclear cells (PBMCs), and saliva were collected from a cohort of more than 200 undergraduate students in Nottingham, United Kingdom, who were subject to high rates of acquisition and carriage of meningococci. Serum immunoglobulin G levels were elevated following increases in the rate of carriage, and these responses were specific for the colonizing strains. In order to investigate T-cell responses, PBMCs from 15 individuals were stimulated with a whole-cell lysate of the H44/76 meningococcal strain (B:15:P1.7,16), stained to detect cell surface markers and intracellular cytokines, and examined by flow cytometry. The cells were analyzed for expression of CD69 (to indicate activation), gamma interferon (IFN-γ) (a representative T-helper 1 subset [Th1]-associated cytokine), and interleukin-5 (IL-5) (a Th2-associated cytokine). Following a brief meningococcal stimulation, the numbers of CD69+IFN-γ+CD56/16+NK cells were much higher than cytokine-positive CD4+events. Both IFN-γ+and IL-5+events were detected among the CD69+CD4+population, leading to the conclusion that an unbiased T-helper subset response was elicited by meningococcal carriage.
HLA-B27-Transfected (SalmonellaPermissive) and HLA-A2-Transfected (SalmonellaNonpermissive) Human Monocytic U937 Cells Differ in Their Production of Cytokines
Authors: Ekman Päivi , Saarinen Marja , He Qiushui , Gripenberg-Lerche Christel , Grönberg Alvar , Arvilommi Heikki , Granfors Kaisa
Published: July 2002

The cytokine secretion of theSalmonella-permissive, HLA-B27-positive U937 cells was examined, as it was previously shown that these cells killSalmonellaless efficiently than controls.Salmonella-permissive U937 cells showed upregulated production of interleukin 10 and to a lesser extent tumor necrosis factor alpha. HLA-B27-associated modulation of cytokine responses may have importance in the pathogenesis of reactive arthritis.
Lipoteichoic Acids fromLactobacillus johnsoniiStrain La1 andLactobacillus acidophilusStrain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria
Authors: Vidal Karine , Donnet-Hughes Anne , Granato Dominique
Published: July 2002

Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS fromEscherichia coliorSalmonella enteritidis, neither the gram-positive bacteriaLactobacillus johnsoniistrain La1 andLactobacillus acidophilusstrain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.
Evaluation of Receptor Binding Specificity ofEscherichia coliK88 (F4) Fimbrial Adhesin Variants Using Porcine Serum Transferrin and Glycosphingolipids as Model Receptors
Authors: Grange Philippe A. , Mouricout Michèle A. , Levery Steven B. , Francis David H. , Erickson Alan K.
Published: July 2002

Diarrheal disease caused by enterotoxigenicEscherichia coliexpressing the K88 (F4) fimbrial adhesin (K88 ETEC) is a significant source of mortality and morbidity among newborn and weaned piglets. K88 fimbrial adhesins are filamentous surface appendages whose lectin (carbohydrate-binding) activity allows K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells. There are three variants of K88 adhesin (K88ab, K88ac, and K88ad), which possess different, yet related, carbohydrate-binding specificities. We used porcine serum transferrin (pSTf) and purified glycosphingolipids (GSL) to begin to define the minimal recognition sequence for K88 adhesin variants. We found that K88ab adhesin binds with high affinity to pSTf (dissociation constant, 75 μM), while neither K88ac nor K88ad adhesin recognizes pSTf. Degradation of the N-glycan on pSTf by extensive metaperiodate treatment abolished its interaction with the K88ab adhesin, indicating that the K88ab adhesin binds to the single N-glycan found on pSTf. Using exoglycosidase digestion of the pSTf glycan, we demonstrated that K88ab adhesin recognizesN-acetylglucosamine (GlcNAc) residues in the core of the N-glycan on pSTf. All three K88 variants were found to bind preferentially to GSL containing a β-linkedN-acetylhexosamine (HexNAc), either GlcNAc orN-acetylgalactosamine, in the terminal position or, alternatively, in the penultimate position with galactose in the terminal position. Considering the results from pSTf and GSL binding studies together, we propose that the minimal recognition sequence for the K88 adhesin variants contains a β-linked HexNAc. In addition, the presence of a terminal galactose β-linked to this HexNAc residue enhances K88 adhesin binding.
rgf
Encodes a Novel Two-Component Signal Transduction System of
Streptococcus agalactiae
Authors: Spellerberg Barbara , Rozdzinski Eva , Martin Simone , Weber-Heynemann Josefine , Lütticken Rudolf
Published: July 2002

The adhesion of gram-positive bacteria to extracellular matrix (ECM) proteins is regarded as an important determinant of pathogenicity. A comparison of the adhesion of
Streptococcus agalactiae
strain O90R to different ECM proteins showed that the most pronounced binding could be observed for immobilized fibrinogen. To investigate the genetic determinants of
S. agalactiae
fibrinogen binding, a pGhost9:IS
S1
mutant library was screened for mutants displaying reduced agglutination of fibrinogen-coated latex beads. A putative two-component signal transduction system was identified and designated
rgfBDAC
. It comprises genes encoding a putative response regulator of 218 amino acids and a putative histidine kinase of 426 amino acids. Comparison of the deduced proteins with the GenBank database revealed a significant similarity to quorum-sensing systems of gram-positive pathogens. Transcription analysis of the
rgf
locus showed that the encoding genes are located on one transcript. To further characterize the influence of the putative histidine kinase encoded in the
rgf
locus on the adhesion of
S. agalactiae
to immobilized fibrinogen, a targeted mutant of
rgfC
was generated. In comparison to the wild-type strain this mutant demonstrated altered fibrinogen binding capacities depending on bacterial cell density. Transcription analysis of secreted and surface-localized
S. agalactiae
proteins in the wild type and the
rgfC
mutant strain revealed that mRNA levels of the C5a peptidase gene
scpB
were increased in the mutant strain while the transcription of the secreted CAMP factor gene
cfb
was unaffected by this mutation. Based on these results, we hypothesize that
rgf
regulates the expression of bacterial cell surface components.
Enhanced Disease Severity inHelicobacter pylori-Infected Mice Deficient in Fas Signaling
Authors: Jones Nicola L. , Day Andrew S. , Jennings Hilary , Shannon Patrick T. , Galindo-Mata Esther , Sherman Philip M.
Published: July 2002

Recent evidence suggests that immune-mediated gastric epithelial cell apoptosis through Fas-Fas ligand interactions participates inHelicobacter pyloridisease pathogenesis. To define the role of Fas signaling in vivo,H. pyloristrain SS1 infection in C57BL/6 mice was compared to that in mice deficient in the Fas ligand (gld).gldmice had a degree of gastritis similar to that of C57BL/6 mice after 6 weeks (gastritis score, 5.2 ± 0.6 [mean ± standard error] versus 3.5 ± 0.8) and 12 weeks (4.0 ± 0.7 versus 3.4 ± 0.5) of infection. Bacterial colonization was comparable in each group of mice at 12 weeks of infection (2.1 ± 0.3 versus 1.6 ± 0.3 forgldand C57BL/6, respectively; the difference is not significant). Sixty-seven percent ofH. pylori-infectedgldmice displayed atrophic changes in the gastric mucosa, compared with 37% of infected C57BL/6 mice, at 12 weeks. In addition, atrophic changes were more severe inH. pylori-infectedgldmice (P< 0.05). Splenocytes isolated fromH. pylori-infected C57BL/6 mice had a twofold increase in production of the Th1 cytokine gamma interferon (IFN-γ) in response toH. pyloriantigens at both 6 and 12 weeks compared to controls (143 ± 65 versus 69 ±26 pg/ml and 336 ± 73 versus 172 ± 60, respectively). In contrast, there was a lack of detectable IFN-γ ingldmice infected with the bacterium.H. pylori-infected C57BL/6 mice had increased epithelial cell apoptosis compared with sham-infected C57BL/6 mice (35.0 ± 8.9 versus 12.3 ± 6.9;P< 0.05). Epithelial cell apoptosis did not differ betweenH. pylori-infected and controlgldmice (5.2 ± 1.6 versus 6.5 ± 2.9 [not significant]). These data demonstrate that mice with mutations in the Fas ligand develop more severe premalignant mucosal changes in response to infection withH. pyloriin association with both an impaired gastric epithelial cell apoptotic response and IFN-γ production. The Fas death pathway modulates disease pathophysiology following murine infection withH. pylori. Deregulation of the Fas pathway could be involved in the transition from gastritis to gastric cancers duringH. pyloriinfection.
Characterization of an Extracellular Lipase Encoded by
LIP2
in
Yarrowia lipolytica
Authors: Pignède Georges , Wang Huijie , Fudalej Franck , Gaillardin Claude , Seman Michel , Nicaud Jean-Marc
Published: July 2002

We isolated the
LIP2
gene from the lipolytic yeast
Yarrowia lipolytica
. It was found to encode a 334-amino-acid precursor protein. The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC
3.1.1.3
). The Lip2p precursor protein is processed by the
KEX2
-like endoprotease encoded by
XPR6
. Deletion of the
XPR6
gene resulted in the secretion of an active but less stable proenzyme. Thus, the pro region does not inhibit lipase secretion and activity. However, it does play an essential role in the production of a stable enzyme. Processing was found to be correct in
LIP2
A
(multiple
LIP2
copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that
XPR6
maturation was not limiting. No extracellular lipase activity was detected with the
lip2
knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the
LIP2
gene. Nevertheless, the
lip2
KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in
Y. lipolytica
.
Salmonella enterica
Serovar Typhimurium Peptidase B Is a Leucyl Aminopeptidase with Specificity for Acidic Amino Acids
Authors: Mathew Zacharia , Knox Tina M. , Miller Charles G.
Published: July 2002

Peptidase B (PepB) of
Salmonella enterica
serovar Typhimurium is one of three broad-specificity aminopeptidases found in this organism. We have sequenced the
pepB
gene and found that it encodes a 427-amino-acid (46.36-kDa) protein, which can be unambiguously assigned to the leucyl aminopeptidase (LAP) structural family. PepB has been overexpressed and purified. The active enzyme shows many similarities to other members of the LAP family: it is a heat-stable (70°C; 20 min) hexameric (∼270-kDa) metallopeptidase with a pH optimum of 8.5 to 9.5. A detailed study of the substrate specificity of the purified protein shows that it differs from other members of the family in its ability to hydrolyze peptides with N-terminal acidic residues. The preferred substrates for PepB are peptides with N-terminal Asp or Glu residues. Comparison of the amino acid sequence of PepB with those of other LAPs leads to the conclusion that PepB is the prototype of a new LAP subfamily with representatives in several other eubacterial species and to the prediction that the members of this family share the ability to hydrolyze peptides with N-terminal acidic residues. Site-directed mutagenesis has been used to show that this specificity appears to be determined by a single Lys residue present in a sequence motif conserved in all members of the subfamily.
Identification of an Archaeal 2-Hydroxy Acid Dehydrogenase Catalyzing Reactions Involved in Coenzyme Biosynthesis in Methanoarchaea
Authors: Graupner Marion , Xu Huimin , White Robert H.
Published: July 2002

Two putative malate dehydrogenase genes, MJ1425 and MJ0490, from
Methanococcus jannaschii
and one from
Methanothermus fervidus
were cloned and overexpressed in
Escherichia coli
, and their gene products were tested for the ability to catalyze pyridine nucleotide-dependent oxidation and reduction reactions of the following α-hydroxy–α-keto acid pairs: (
S
)-sulfolactic acid and sulfopyruvic acid; (
S
)-α-hydroxyglutaric acid and α-ketoglutaric acid; (
S
)-lactic acid and pyruvic acid; and 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid and 1-oxo-1,3,4,6-hexanetetracarboxylic acid. Each of these reactions is involved in the formation of coenzyme M, methanopterin, coenzyme F
420
, and methanofuran, respectively. Both the MJ1425-encoded enzyme and the MJ0490-encoded enzyme were found to function to different degrees as malate dehydrogenases, reducing oxalacetate to (
S
)-malate using either NADH or NADPH as a reductant. Both enzymes were found to use either NADH or NADPH to reduce sulfopyruvate to (
S
)-sulfolactate, but the
V
max
/

K
m

value for the reduction of sulfopyruvate by NADH using the MJ1425-encoded enzyme was 20 times greater than any other combination of enzymes and pyridine nucleotides. Both the
M. fervidus
and the MJ1425-encoded enzyme catalyzed the NAD
+
-dependent oxidation of (
S
)-sulfolactate to sulfopyruvate. The MJ1425-encoded enzyme also catalyzed the NADH-dependent reduction of α-ketoglutaric acid to (
S
)-hydroxyglutaric acid, a component of methanopterin. Neither of the enzymes reduced pyruvate to (
S
)-lactate, a component of coenzyme F
420
. Only the MJ1425-encoded enzyme was found to reduce 1-oxo-1,3,4,6-hexanetetracarboxylic acid, and this reduction occurred only to a small extent and produced an isomer of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid that is not involved in the biosynthesis of methanofuran c. We conclude that the MJ1425-encoded enzyme is likely to be involved in the biosynthesis of both coenzyme M and methanopterin.
New Substrates for the Dicarboxylate Transport System of
Sinorhizobium meliloti
Authors: Yurgel Svetlana , Mortimer Michael W. , Rogers Kimberly N. , Kahn Michael L.
Published: July 2002

The dicarboxylate transport (Dct) system of
Sinorhizobium meliloti
, which is essential for a functional nitrogen-fixing symbiosis, has been thought to transport only dicarboxylic acids. We show here that the permease component of the Dct system, DctA, can transport orotate, a monocarboxylic acid, with an apparent

K
m

of 1.7 mM and a
V
max
of 163 nmol min
−1
per mg of protein in induced cells. DctA was not induced by the presence of orotate. The transport of orotate was inhibited by several compounds, including succinamic acid and succinamide, which are not dicarboxylic acids. The dicarboxylic acid maleate (
cis
-butenedioic acid) was not an inhibitor of orotate transport, which suggests that it was not recognized by DctA. However, maleate was an excellent inducer of DctA expression. Our evaluation of 17 compounds as inducers and inhibitors of transport suggests that substrates recognized by
S. meliloti
DctA must have appropriately spaced carbonyl groups and an extended conformation, while good inducers are more likely to have a curved conformation.
Developmental Regulation of the Cell Division Protein FtsZ in
Anabaena
sp. Strain PCC 7120, a Cyanobacterium Capable of Terminal Differentiation
Authors: Kuhn Isabelle , Peng Ling , Bedu Sylvie , Zhang Cheng-Cai
Published: July 2002

Heterocysts are terminally differentiated cells devoted to nitrogen fixation in the filamentous cyanobacterium
Anabaena
sp. strain PCC 7120. We show here that the cell division protein FtsZ is present in vegetative cells but undetectable in heterocysts. These results provide a first rational explanation for the inability of mature heterocysts to undergo cell division.
Genetic Analysis of a Gene Cluster for Cyclohexanol Oxidation in
Acinetobacter
sp. Strain SE19 by In Vitro Transposition
Authors: Cheng Qiong , Thomas Stuart M. , Kostichka Kristy , Valentine James R. , Nagarajan Vasantha
Published: July 2002

Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an
Acinetobacter
sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from
Acinetobacter
sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant
Escherichia coli
carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from
Acinetobacter
sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in
E. coli
. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.
Native Corrinoids fromClostridium cochleariumAre Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B12and Factor A
Authors: Hoffmann Bernd , Oberhuber Michael , Stupperich Erhard , Bothe Harald , Buckel Wolfgang , Konrat Robert , Kräutler Bernhard
Published: July 2002

The corrinoids from the obligate anaerobeClostridium cochleariumwere extracted as a mixture of Coβ-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 μmol) of B12derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample ofC. cochleariumby a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B12(Coβ-cyano-7"-adeninylcobamide) (60%) and factor A (Coβ-cyano-7"-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B12was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B12and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional1H,13C-, and15N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-[N6-methyl]-adeninylcobamide, a previously unknown corrinoid.C. cochleariumthus biosynthesizes as its native “complete” B12cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.
Inactivation of the
ampDE
Operon Increases Transcription of
algD
and Affects Morphology and Encystment of
Azotobacter vinelandii
Authors: Núñez Cinthia , Moreno Soledad , Cárdenas Luis , Soberón-Chávez Gloria , Espín Guadalupe
Published: July 2002

Transcription of
algD
, encoding GDP-mannose dehydrogenase, the key enzyme in the alginate biosynthetic pathway, is highly regulated in
Azotobacter vinelandii
. We describe here the characterization of a Tn
5
insertion mutant (AC28) which shows a higher level of expression of an
algD
::
lacZ
fusion. AC28 cells were morphologically abnormal and unable to encyst. The cloning and nucleotide sequencing of the Tn
5
-disrupted locus in AC28 revealed an operon homologous to the
Escherichia coli ampDE
operon. Tn
5
was located within the
ampD
gene, encoding a cytosolic
N
-acetyl-anhydromuramyl-
l
-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments. The
ampE
gene encodes a transmembrane protein, but the function of the protein is not known. We constructed strains carrying
ampD
or
ampE
mutations and one with an
ampDE
deletion. The strain with a deletion of the
ampDE
operon showed a phenotype similar to that of mutant AC28. The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.
Glycine Betaine Transport in the Obligate Halophilic Archaeon
Methanohalophilus portucalensis
Authors: Lai Mei-Chin , Hong Tong-Yung , Gunsalus Robert P.
Published: July 2002

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe
Methanohalophilus portucalensis
(strain FDF1), since solute uptake for this class of obligate halophilic methanogenic
Archaea
has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed

K
t

of 23 μM and a
V
max
of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [
14
C]betaine uptake. The proton-conducting uncoupler 2,4-dinitrophenol and the ATPase inhibitor
N
,
N
-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 μM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, α-glutamate, β-glutamine, and
N
ɛ
-acetyl-β-lysine. These investigations demonstrate that this model halophilic methanogen,
M. portucalensis
strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.
Early Cephamycin Biosynthetic Genes Are Expressed from a Polycistronic Transcript in
Streptomyces clavuligerus
Authors: Alexander Dylan C. , Brumlik Michael J. , Lee Linda , Jensen Susan E.
Published: July 2002

A polycistronic transcript that is initiated at the
lat
promoter has been implicated in the expression of the genes involved in early steps of cephamycin C biosynthesis in
Streptomyces clavuligerus. pcbC
is also expressed as a monocistronic transcript from its own promoter. However, an alternative interpretation involving expression via three separate yet interdependent transcripts has also been proposed. To distinguish between these possibilities, mutants lacking the
lat
promoter and containing a transcription terminator within the
lat
gene (Δ
lat
::
tsr
/term mutants) were created. This mutation eliminated the production of lysine-ɛ-aminotransferase (the
lat
gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of δ-(
l
-α-aminoadipyl)-
l
-cysteinyl-
d
-valine synthetase (ACVS) (the
pcbAB
gene product) was eliminated in Δ
lat
::
tsr
/term mutants, while production of isopenicillin N synthase (IPNS) (the
pcbC
gene product) was greatly reduced. The provision of α-aminoadipate to the Δ
lat
::
tsr
/term mutants, either via exogenous feeding or via
lat
gene complementation, did not restore production of ACVS or IPNS. Analysis of RNA isolated from the Δ
lat
::
tsr
/term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic
pcbC
transcript levels were greatly decreased. In contrast, Δ
lat
mutants created by in-frame internal deletion of
lat
maintained the polycistronic transcript and allowed production of wild-type levels of both ACVS and IPNS.
Transcriptional Control of the Sulfur-Regulated
cysH
Operon, Containing Genes Involved in
l
-Cysteine Biosynthesis in
Bacillus subtilis
Authors: Mansilla Maria Cecilia , Albanesi Daniela , de Mendoza Diego
Published: July 2002

The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in
Bacillus subtilis
and other gram-positive bacteria. In this study we describe the expression pattern of the
B. subtilis cysH
operon in response to sulfur starvation. A 6.1-kb polycistronic transcript which includes the
cysH
,
cysP
,
ylnB
,
ylnC
,
ylnD
,
ylnE
, and
ylnF
genes was identified. Its synthesis was induced by sulfur limitation and strongly repressed by cysteine. The
cysH
operon contains a 5′ leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system. Here we show that induction of
B. subtilis cysH
operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription. Deletion of a 46-bp region adjacent to the −35 region of the
cysH
promoter led to high-level expression of the operon, even in the presence of cysteine. We also found that
O
-acetyl-
l
-serine (OAS), a direct precursor of cysteine, renders
cysH
transcription independent of sulfur starvation and insensitive to cysteine repression. We propose that transcription of the
cysH
operon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS. Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of
cysH
expression. These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.
Association of the Cytoplasmic Membrane Protein XpsN with the Outer Membrane Protein XpsD in the Type II Protein Secretion Apparatus of
Xanthomonas campestris
pv. Campestris
Authors: Lee Hsien-Ming , Wang Kuan-Cheng , Liu Yi-Ling , Yew Hsin-Yan , Chen Ling-Yun , Leu Wei-Ming , Chen David Chanhen , Hu Nien-Tai
Published: July 2002

An
xps
gene cluster composed of 11 open reading frames is required for the type II protein secretion in
Xanthomonas campestris
pv. campestris. Immediately upstream of the
xpsD
gene, which encodes an outer membrane protein that serves as the secretion channel by forming multimers, there exists an open reading frame (previously designated ORF2) that could encode a protein of 261 amino acid residues. Its N-terminal hydrophobic region is a likely membrane-anchoring sequence. Antibody raised against this protein could detect in the wild-type strain of
X. campestris
pv. campestris a protein band with an apparent molecular mass of 36 kDa by Western blotting. Its aberrant slow migration in sodium dodecyl sulfate-polyacrylamide gels might be due to its high proline content. We designated this protein XpsN. By constructing a mutant strain with an in-frame deletion of the chromosomal
xpsN
gene, we demonstrated that it is required for the secretion of extracellular enzyme by
X. campestris
pv. campestris. Subcellular fractionation studies indicated that the XpsN protein was tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis revealed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated that the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)
6
-tagged XpsD protein from the metal affinity chromatography column. All observations suggested that the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with various truncated XpsD proteins revealed that the C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved in complex formation between the two.
A 90-Kilobase Conjugative Chromosomal Element Coding for Biphenyl and Salicylate Catabolism in
Pseudomonas putida
KF715
Authors: Nishi Akito , Tominaga Kiyomi , Furukawa Kensuke
Published: July 2002

The biphenyl and salicylate metabolic pathways in
Pseudomonas putida
KF715 are chromosomally encoded. The
bph
gene cluster coding for the conversion of biphenyl to benzoic acid and the
sal
gene cluster coding for the salicylate
meta
-pathway were obtained from the KF715 genomic cosmid libraries. These two gene clusters were separated by 10-kb DNA and were highly prone to deletion when KF715 was grown in nutrient medium. Two types of deletions took place at the region including only the
bph
genes (ca. 40 kb) or at the region including both the
bph
and
sal
genes (ca. 70 kb). A 90-kb DNA region, including both the
bph
and
sal
genes (termed the
bph-sal
element), was transferred by conjugation from KF715 to
P. putida
AC30. Such transconjugants gained the ability to grow on biphenyl and salicylate as the sole sources of carbon. The
bph
and
sal
element was located on the chromosome of the recipient. The
bph-sal
element in strain AC30 was also highly prone to deletion; however, it could be mobilized to the chromosome of
P. putida
KT2440 and the two deletion mutants of KF715.
Type IV Pilus Genes
pilA
and
pilC
of
Pseudomonas stutzeri
Are Required for Natural Genetic Transformation, and
pilA
Can Be Replaced by Corresponding Genes from Nontransformable Species
Authors: Graupner Stefan , Frey Verena , Hashemi Rozita , Lorenz Michael G. , Brandes Gudrun , Wackernagel Wilfried
Published: July 2002

Pseudomonas stutzeri
lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a
P. stutzeri
JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of
Pseudomonas aeruginosa
, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The
pilC
mutant had no pili and was defective in twitching motility. Further sequencing revealed that
pilC
is clustered in an operon with genes homologous to
pilB
and
pilD
of
P. aeruginosa
, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a
pilA
gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of
pilA
abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of
3
H-labeled
P. stutzeri
DNA that were bound to competent parental cells and taken up were strongly reduced in the
pilC
and
pilA
mutants. Remarkably, the cloned
pilA
genes from nontransformable organisms like
Dichelobacter nodosus
and the PAK and PAO strains of
P. aeruginosa
fully restored pilus formation and transformability of the
P. stutzeri pilA
mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium
P. stutzeri
function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.
Negative Regulation of Expression of the
Bacillus megaterium bmlP1
Gene by the
bmlP1
3′ Flanking Region
Authors: Shaw Gwo-Chyuan , Hsueh Yi-Huang , Sung Chi-Chang , Chen Yuan-Shou , Liu Chang-Hsiesh
Published: July 2002

We report that the expression of the
Bacillus megaterium bmlP1
gene is subject to negative regulation by the
bmlP1
3′ flanking region. This repression occurred both in
B. megaterium
and in
Escherichia coli
. When the
bmlP1
promoter was replaced with a heterologous promoter or when the orientation of the
bmlP1
3′ flanking region was reversed, the inhibitory effect was still observed. However, the
bmlP1
3′ flanking region was unable to exert repression on a heterologous gene when fused downstream in either orientation, and it was incapable of acting in
trans
. Dot blot and Northern blot analyses revealed that the repression occurred at the RNA level. Deletion analysis showed that the regulatory site responsible for the repression is located within a 116-bp region immediately following the
bmlP1
gene. Possible mechanisms for this repression are discussed.
pepA
, a Gene Mediating pH Regulation of Virulence Genes in
Vibrio cholerae
Authors: Behari Jaideep , Stagon Lisa , Calderwood Stephen B.
Published: July 2002

ToxT, a member of the AraC family of transcriptional regulators, controls the expression of several virulence factors in
Vibrio cholerae
. In the classical biotype of
V. cholerae
, expression of
toxT
is regulated by the same environmental conditions that control expression of the virulence determinants cholera toxin and the toxin coregulated pilus. Several genes that activate
toxT
expression have been identified. To identify genes that repress
toxT
expression in nonpermissive environmental conditions, a genetic screen was used to isolate mutations which alter the expression of a
toxT-gusA
transcriptional fusion. Several mutants were isolated, and the mutants could be divided into two classes. One class of mutants exhibited higher expression levels of
toxT-gusA
at both the nonpermissive pH and temperature, while the second class showed elevated
toxT-gusA
expression only at the nonpermissive pH. One mutant from the second class was chosen for further characterization. This mutant was found to carry a Tn
phoA
insertion in a homolog of the
Escherichia coli pepA
gene. Disruption of
pepA
in
V. cholerae
resulted in elevated levels of expression of cholera toxin,
tcpA
,
toxT
, and
tcpP
at the noninducing pH but not at the noninducing temperature. Elevated levels of expression of
toxT
and
tcpP
at the nonpermissive pH in the
pepA
mutant were abolished in
tcpP toxR
mutant and
aphB
mutant backgrounds, respectively. A putative binding site for PepA was identified in the
tcpPH-tcpI
intergenic region, suggesting that PepA may act at the level of
tcpPH
transcription. Disruption of
pepA
caused only partial deregulation at the noninducing pH, suggesting the involvement of additional factors in the pH regulation of virulence genes in
V. cholerae
.
Amino Acid Residues in LuxR Critical for Its Mechanism of Transcriptional Activation during Quorum Sensing in
Vibrio fischeri
Authors: Trott Amy E. , Stevens Ann M.
Published: July 2002

PCR-based site-directed mutagenesis has been used to generate 38 alanine-substitution mutations in the C-terminal 41 amino acid residues of LuxR. This region plays a critical role in the mechanism of LuxR-dependent transcriptional activation of the
Vibrio fischeri lux
operon during quorum sensing. The ability of the variant forms of LuxR to activate transcription of the
lux
operon was examined by using in vivo assays in recombinant
Escherichia coli
. Eight recombinant strains produced luciferase at levels less than 50% of that of a strain expressing wild-type LuxR. Western immunoblotting analysis verified that the altered forms of LuxR were expressed at levels equivalent to those of the wild type. An in vivo DNA binding-repression assay in recombinant
E. coli
was subsequently used to measure the ability of the variant forms of LuxR to bind to the
lux
box, the binding site of LuxR at the
lux
operon promoter. All eight LuxR variants found to affect cellular luciferase levels were unable to bind to the
lux
box. An additional 11 constructs that had no effect on cellular luciferase levels were also found to exhibit a defect in DNA binding. None of the alanine substitutions in LuxR affected activation of transcription of the
lux
operon without also affecting DNA binding. These results support the conclusion that the C-terminal 41 amino acids of LuxR are important for DNA recognition and binding of the
lux
box rather than positive control of the process of transcription initiation.