bluuebunny/crossref_metadata_2025_split huggingface dataset:
Demonstration and Characterization of a Specific Interaction between Gonococcal Transferrin Binding Protein A and TonB
Authors: Kenney Christopher D. , Cornelissen Cynthia Nau 
Published: October 2002

ABSTRACTIron scavenging byNeisseria gonorrhoeaeis accomplished by the expression of receptors that are specific for host iron-binding proteins, such as transferrin and lactoferrin. Efficient transferrin-iron acquisition is dependent on the combined action of two proteins, designated TbpA and TbpB. TbpA is a TonB-dependent outer membrane receptor, whereas TbpB is lipid modified and serves to increase the efficiency of transferrin-iron uptake. Both proteins, together or separately, can be isolated from the gonococcal outer membrane by using affinity chromatography techniques. In the present study, we identified an additional protein in transferrin-affinity preparations, which had an apparent molecular mass of 45 kDa. The ability to copurify this protein by transferrin affinity was dependent upon the presence of TbpA and not TbpB. The amino-terminal sequence of the 45-kDa protein was identical to the amino terminus of gonococcal TonB, indicating that TbpA stably interacted with TonB, without the addition of chemical cross-linkers. Using immunoprecipitation, we could recover TbpA-TonB complexes without the addition of transferrin, suggesting that ligand binding was not a necessary prerequisite for TonB interaction. In contrast, a characterized TonB box mutant of TbpA did not facilitate interaction between these two proteins such that complexes could be isolated. We generated an in-frame deletion of gonococcal TonB, which removed 35 amino acids, including aNeisseria-specific, glycine-rich domain. This mutant protein, like the parental TonB, energized TbpA to enable growth on transferrin. Consistent with the functionality of this deletion derivative, TbpA-TonB complexes could be recovered from this strain. The results of the present study thus begin to define the requirements for a functional interaction between gonococcal TbpA and TonB.
Proteins Released byHelicobacter pyloriIn Vitro
Authors: Kim Nayoung , Weeks David L. , Shin Jai Moo , Scott David R. , Young Mary K. , Sachs George 
Published: October 2002

ABSTRACTSecretion of proteins byHelicobacter pylorimay contribute to gastric inflammation and epithelial damage. An in vitro analysis was designed to identify proteins released by mechanisms other than nonspecific lysis. The radioactivity of proteins in the supernatant was compared with that of the intact organism by two-dimensional gel phosphorimaging following a 4-h pulse-chase. The ratio of the amount of UreB, a known cytoplasmic protein, in the supernatant to that in the pellet was found to be 0.25, and this was taken as an index of lysis during the experiments (n= 6). Ratios greater than that of UreB were used to distinguish proteins that were selectively released into the medium. Thus, proteins enriched more than 10-fold in the supernatant compared to UreB were identified by mass spectrometry. Sixteen such proteins were present in the supernatant: VacA; a conserved secreted protein (HP1286); putative peptidylcis-transisomerase (HP0175); six proteins encoded by HP0305, HP0231, HP0973, HP0721, HP0129, and HP0902; thioredoxin (HP1458); single-stranded-DNA-binding 12RNP2 precursor (HP0827); histone-like DNA-binding protein HU (HP0835); ribosomal protein L11 (HP1202); a putative outer membrane protein (HP1564); and outer membrane proteins Omp21 (HP0913) and Omp20 (HP0912). All except HP0902, thioredoxin, HP0827, HP0835, and HP1202 had a signal peptide. When nalidixic acid, a DNA synthesis inhibitor, was added to inhibit cell division but not protein synthesis, to decrease possible contamination due to outer membrane shedding, two outer membrane proteins (Omp21 and Omp20) disappeared from the supernatant, and the amount of VacA also decreased. Thus, 13 proteins were still enriched greater than 10-fold in the medium after nalidixic acid treatment, suggesting these were released specifically, possibly by secretion. These proteins may be implicated inH. pylori-induced effects on the gastric epithelium.
Involvement of Two Putative Alternative Sigma Factors in Stress Response of the Radioresistant Bacterium
Deinococcus radiodurans
Authors: Schmid Amy K. , Lidstrom Mary E. 
Published: October 2002

ABSTRACT

Two genes bearing similarity to alternative sigma factors were identified in the
Deinococcus radiodurans
genome sequence and designated
sig1
and
sig2
. These genes were cloned and inactivated, and both were found to be important for survival during heat and ethanol stress, although the
sig1
mutants displayed a more severe phenotype than the
sig2
mutants. Reporter gene fusions to the
groESL
and
dnaKJ
operons transformed into these mutant backgrounds indicated that
sig1
is required for the heat shock induction of
groESL
and
dnaKJ
, whereas
sig2
mutants show a more moderate defect in
dnaKJ
induction and are not impaired for
groESL
induction. Essentiality tests suggested that neither
sig1
nor
sig2
is essential under all conditions. Sequence comparisons demonstrated that the
sig1
gene product is classed distinctly with extracytoplasmic function (ECF) sigma factors, whereas Sig2 appears to be a more divergent sigma factor ortholog. These results suggest that
sig1
encodes the major ECF-derived heat shock sigma factor in
D. radiodurans
and that it plays a central role in the positive regulation of heat shock genes.
sig2
, in contrast, appears to play a more minor role in heat shock protection and may serve to modulate the expression of some heat protective genes.
Inversions over the Terminus Region inSalmonellaandEscherichia coli: IS200s as the Sites of Homologous Recombination Inverting the Chromosome ofSalmonella entericaSerovar Typhi
Authors: Alokam Suneetha , Liu Shu-Lin , Said Kamal , Sanderson Kenneth E. 
Published: October 2002

ABSTRACTGenomic rearrangements (duplications and inversions) in enteric bacteria such asSalmonella entericaserovar Typhimurium LT2 andEscherichia coliK12 are frequent (10−3to 10−5) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences fromS. entericaserovar Typhimurium LT2,S. entericaserovar Typhi CT18,E. coliK12, andE. coliO157:H7 revealed genomic inversions spanning the TER region. Assuming thatS. entericaserovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200elements and an inversion of about 700 kb inE. coliK12 andE. coliO157:H7. In addition, there is another inversion of 500 kb inE. coliO157:H7 compared withE. coliK12. PCR analysis confirmed that allS. entericaserovar Typhi strains tested, but not strains of otherSalmonellaserovars, have an inversion at the exact site of the IS200insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.
Effects of Environmental Changes on Expression of the Oligopeptide Permease (
opp
) Genes of
Borrelia burgdorferi
Authors: Wang Xing-Guo , Lin Bo , Kidder J. Michael , Telford Samuel , Hu Linden T. 
Published: October 2002

ABSTRACT

We analyzed expression of a putative oligopeptide permease (Opp) of
Borrelia burgdorferi.
Unlike the
opp
operons of other bacteria for which there is a single substrate binding protein,
B. burgdorferi
codes for three substrate binding proteins (OppA-I to -III) in its
opp
operon and an additional two homologs on plasmids (OppA-IV and -V). Instead of a single promoter region regulating transcription of the entire operon, as seen in other bacterial
opp
operons, it appears that among
oppA-I
,
-II
, and -
III
, as well as
oppA-IV
and -
V
, each has a potential upstream promoter region. We tested the function of these putative promoter sequences by fusion to a promoterless β-galactosidase reporter gene in pCB182. Each of the promoter regions was found to be active. The level of activity in the reporter constructs closely paralleled the level of expression of each gene in in vitro-grown
B. burgdorferi
. Changes in carbon and nitrogen availability differentially affected individual promoters, but no changes in promoter activity were seen when
Escherichia coli
bacteria (with the promoter constructs) were grown in various concentrations of phosphate and leucine and changes in pH. Expression of specific
oppA
genes with
B. burgdorferi
varied significantly between its mouse and fed and unfed tick hosts. Differences in regulation of
opp
gene expression suggest a potential role in environmental response by the organism.
The IrrE Protein of
Deinococcus radiodurans
R1 Is a Novel Regulator of
recA
Expression
Authors: Earl Ashlee M. , Mohundro Michael M. , Mian I. Saira , Battista John R. 
Published: October 2002

ABSTRACT

IRS24 is a DNA damage-sensitive strain of
Deinococcus radiodurans
strain 302 carrying a mutation in an uncharacterized locus designated
irrE
. Five overlapping cosmids capable of restoring ionizing radiation resistance to IRS24 were isolated from a genomic library. The ends of each cloned insert were sequenced, and these sequences were used to localize
irrE
to a 970-bp region on chromosome I of
D. radiodurans
R1. The
irrE
gene corresponds to coding sequence DR0167 in the R1 genome. The
irrE
gene encodes a 35,000-Da protein that has no similarity to any previously characterized peptide. The
irrE
locus of R1 was also inactivated by transposon mutagenesis, and this strain was sensitive to ionizing radiation, UV light, and mitomycin C. Preliminary findings indicate that IrrE is a novel regulatory protein that stimulates transcription of the
recA
gene following exposure to ionizing radiation.
Bacterial Outer Membrane Ushers Contain Distinct Targeting and Assembly Domains for Pilus Biogenesis
Authors: Thanassi David G. , Stathopoulos Christos , Dodson Karen , Geiger Dominik , Hultgren Scott J. 
Published: October 2002

ABSTRACT

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic
Escherichia coli.
Structural analysis indicated PapC folds as a β-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal
fim
gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.
Helicobacter pyloriExpresses an Autolytic Enzyme: Gene Identification, Cloning, and Theoretical Protein Structure
Authors: Marsich Eleonora , Zuccato Pierfrancesco , Rizzi Sonia , Vetere Amedeo , Tonin Enrico , Paoletti Sergio 
Published: October 2002

ABSTRACTHelicobacter pyloriis an important pathogen of the gastric system. The clinical outcome of infection is thought to be correlated with some genetic features of the bacterium. However, due to the extreme genetic variability of this organism, it is hard to draw definitive conclusions concerning its virulence factors. Here we describe a novelH. pylorigene which expresses an autolytic enzyme that is also capable of degrading the cell walls of both gram-positive and gram-negative bacteria. We designated this genelys. We found this gene and observed its expression in a number of unrelated clinical strains, a fact that suggests that it is well conserved in the species. A comparison of the nucleotide sequences oflysand the hypothetical geneHP0339fromH. pyloristrain ATCC 26695 revealed almost total identity, except for the presence of an insertion consisting of 24 nucleotides in thelyssequence. The coding sequences oflysandHP0339show a high degree of homology with the coding sequence of bacteriophage T4 lysozyme. Because of this similarity, it was possible to model the three-dimensional structures of both thelysandHP0339products.
Functional Analysis by Site-Directed Mutagenesis of the NAD
+
-Reducing Hydrogenase from
Ralstonia eutropha
Authors: Burgdorf Tanja , De Lacey Antonio L. , Friedrich Bärbel 
Published: October 2002

ABSTRACT

The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of
Ralstonia eutropha
couples the oxidation of hydrogen to the reduction of NAD
+
under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H
2
-oxidizing activity. In one of these isolates (HoxH I64A), H
2
binding was impaired. Class II mutants revealed a high D
2
/H
+
exchange rate relative to a low H
2
-oxidizing activity. A representative (HoxH H16L) displayed D
2
/H
+
exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O
2
-sensitive growth on hydrogen due to an O
2
-sensitive SH protein.
Characterization of the Two-Component Abortive Phage Infection Mechanism AbiT from
Lactococcus lactis
Authors: Bouchard Julie D. , Dion Eric , Bissonnette Frédéric , Moineau Sylvain 
Published: October 2002

ABSTRACT

During the production of fermented dairy products, virulent bacteriophages infecting
Lactococcus lactis
can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from
L. lactis
. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10
−5
and 10
−7
) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle.
Functional Analysis of TraA, the Sex Pheromone Receptor Encoded by pPD1, in a Promoter Region Essential for the Mating Response in
Enterococcus faecalis
Authors: Horii Takaaki , Nagasawa Hiromichi , Nakayama Jiro 
Published: October 2002

ABSTRACT

Conjugative transfer of a bacteriocin plasmid, pPD1, of
Enterococcus faecalis
is induced in response to a peptide sex pheromone, cPD1, secreted from plasmid-free recipient cells. cPD1 is taken up by a pPD1 donor cell and binds to an intracellular receptor, TraA. Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells. In this study, we discuss how TraA transduces the signal of cPD1 to the mating response. Gel mobility shift assays indicated that TraA is bound to a
traA
-
ipd
intergenic region, which is essential for cPD1 response. DNase I footprinting analysis suggested the presence of one strong (
tab1
) and two weak (
tab2
and
tab3
) TraA-binding sites in the intergenic region. Primer extension analysis implied that the transcriptional initiation sites of
traA
and
ipd
were located in the intergenic region. Northern analysis showed that cPD1 upregulated and downregulated transcription of
ipd
and
traA
, respectively. The circular permutation assay showed that TraA bent a DNA fragment corresponding to the
tab1
region, and its angle was changed in the presence of cPD1 or iPD1. From these data, we propose a model that TraA changes the conformation of the
tab1
region in response to cPD1 and upregulates the transcription of
ipd
, which may lead to expression of genes required for the mating response.
Enterococcus faecalis
Heme-Dependent Catalase
Authors: Frankenberg Lena , Brugna Myriam , Hederstedt Lars 
Published: October 2002

ABSTRACT

Enterococcus faecalis
cells cannot synthesize porphyrins and do not rely on heme for growth but can take up heme and use it to synthesize heme proteins. We recently described a cytochrome
bd
in
E. faecalis
strain V583 and here report the identification of a chromosomal gene,
katA
, encoding a heme-containing cytoplasmic catalase. The 54-kDa KatA polypeptide shows sequence similarity to members of the family of monofunctional catalases. A hexahistidyl-tagged version of the catalase was purified, and major characteristics of the enzyme were determined. It contains one protoheme IX group per KatA polypeptide. Catalase activity was detected only in
E. faecalis
cells grown in the presence of heme in the medium; about 2 and 10 μM hemin was required for half-maximal and maximal production of catalase, respectively. Our finding of a catalase whose synthesis is dependent on the acquisition of heme in the opportunistic pathogen
E. faecalis
might be of clinical importance. Studies of cellular heme transport and heme protein assembly and in vivo synthesis of metalloprotein analogs for biotechnological applications are impeded by the lack of experimental systems. We conclude that the
E. faecalis
cell potentially provides such a desired system.
Evolution and Virulence of Serogroup 6 Pneumococci on a Global Scale
Authors: Robinson D. Ashley , Briles David E. , Crain Marilyn J. , Hollingshead Susan K. 
Published: October 2002

ABSTRACT

To study the evolution and virulence of pneumococcal populations, we used multilocus sequence typing to identify the major clones among 212 carriage and invasive isolates expressing capsular serogroup 6 from 39 countries. The global population consisted of 8 major complexes and 6 minor complexes of related clones and 32 clones of diverse origin. Surprisingly, serotype 6A clones evolved by mutation nearly as often as by recombination, whereas serotype 6B clones evolved almost exclusively by recombination (
P
= 0.0029). This is the first report of population genetic differences among serotypes of this species. The largest clonal complex was associated with invasive disease (
P
= 0.019) and included a common ancestor for five previously identified drug-resistant clones. The putative ancestors of the major clonal complexes were represented by a greater proportion of carriage isolates than were their descendents (
P
= 0.001), and the ancestors tended to be less virulent than their descendents in a mouse model of infection. These data suggested that virulent serogroup 6 clones have evolved multiple times from less-virulent ancestral clones.
A Proline-Rich Region with a Highly Periodic Sequence in Streptococcal β Protein Adopts the Polyproline II Structure and Is Exposed on the Bacterial Surface
Authors: Areschoug Thomas , Linse Sara , Stålhammar-Carlemalm Margaretha , Hedén Lars-Olof , Lindahl Gunnar 
Published: October 2002

ABSTRACTProline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the β protein of group B streptococci. The proline-rich region in β, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a β mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the β protein. Comparison of the XPZ region in different β proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen.
Structural Heterogeneity of the Streptococcal C5a Peptidase Gene inStreptococcus pyogenes
Authors: Koroleva Irina V. , Efstratiou Androulla , Suvorov Alexander N. 
Published: October 2002

ABSTRACTThe 3′ ends of the genes for the C-terminal region of C5a peptidase from 15Streptococcus pyogenesisolates were analyzed by PCR. Amplicons were found to differ in size. DNA sequence analysis revealed that the differences between PCR fragment sizes accorded with the number of R repeats in the C5a peptidase gene.
Protein Splicing of the
Deinococcus radiodurans
Strain R1 Snf2 Intein
Authors: Southworth Maurice W. , Perler Francine B. 
Published: October 2002

ABSTRACT

Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for
Deinococcus radiodurans
R1, leading to the speculation that a frameshift was required for splicing or that
trans
splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues.
Bex, theBacillus subtilisHomolog of the EssentialEscherichia coliGTPase Era, Is Required for Normal Cell Division and Spore Formation
Authors: Minkovsky Natalie , Zarimani Arash , Chary Vasant K. , Johnstone Brian H. , Powell Bradford S. , Torrance Pamela D. , Court Donald L. , Simons Robert W. , Piggot Patrick J. 
Published: October 2002

ABSTRACTTheBacillus subtilis bexgene complemented the defect in anEscherichia coli eramutant. The Bex protein showed 39% identity and 67% similarity to theE. coliEra GTPase. In contrast toera,bexwas not essential in all strains.bexmutant cells were elongated and filled with diffuse nucleoid material. They grew slowly and exhibited severely impaired spore formation.