bluuebunny/crossref_metadata_2025_split huggingface dataset:
Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides
Authors: Vignon Guillaume , Köhler Rolf , Larquet Eric , Giroux Stéphanie , Prévost Marie-Christine , Roux Pascal , Pugsley Anthony P. 
Published: May 2003

ABSTRACT

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown
Escherichia
coli
K-12 cells expressing the
Klebsiella
oxytoca
pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in
E
.
coli
or to PulG. For example, high-level expression of the endogenous
E
.
coli
gsp
secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic “pseudopilus” and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).
Cysteine-Scanning Analysis of the Dimerization Domain of EnvZ, an Osmosensing Histidine Kinase
Authors: Qin Ling , Cai Shengjian , Zhu Yan , Inouye Masayori 
Published: May 2003

ABSTRACT

EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in
Escherichia coli
. The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B. Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A. The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized. From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity. Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A. The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation. Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities. The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.
Open Reading Frame
sso2387
from the Archaeon
Sulfolobus solfataricus
Encodes a Polypeptide with Protein-Serine Kinase Activity
Authors: Lower Brian H. , Kennelly Peter J. 
Published: May 2003

ABSTRACT

The predicted polypeptide product of open reading frame
sso2387
from the archaeon
Sulfolobus solfataricus
, SsoPK2, displayed several of the sequence features conserved among the members of the “eukaryotic” protein kinase superfamily.
sso2387
was cloned, and its polypeptide product was expressed in
Escherichia coli
. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures ≥60°C, whereas phosphorylation of exogenous proteins was detectable at 37°C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser
548
, with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.
Quantitation of
mecA
Transcription in Oxacillin-Resistant
Staphylococcus aureus
Clinical Isolates
Authors: Rosato Adriana E. , Craig William A. , Archer Gordon L. 
Published: May 2003

ABSTRACT

The transcription of
mecA
, the gene required for oxacillin resistance in staphylococci, was quantified in a collection of 65 geographically and genetically diverse clinical and 8 defined laboratory
Staphylococcus aureus
isolates.
mecA
transcription was measured by real-time reverse transcription-PCR, confirmed by Northern blot analysis, and correlated with the presence and DNA sequence of the two
mecA
repressors,
mecI
and
blaI
. Isolates were first examined that contained
mecI
and/or
blaI
with wild-type sequence. BlaI provided significantly more repression of
mecA
transcription than did MecI, unrelated to
blaI
genetic location. Both together repressed
mecA
better than either one alone. In clinical isolates containing only wild-type
mecI
,
mecA
transcription repression was 10- to 25-fold less effective than that seen in previously studied constructs derived from strain N315. There was a difference in the
mecI
ribosomal binding site (RBS) between the clinical isolates (GGAA) and N315 (GGAG). The GGAA RBS was associated with 5.5- to 7.3-fold less
mecA
repression than GGAG in isogenic constructs. The values generated for wild-type repressors were compared to those in 26 isolates containing
mecI
mutations.
mecA
transcription appeared to be repressed only by BlaI in isolates with
mecI
nonsense and frameshift mutations. In contrast,
mecI
repression seemed to be partially or fully retained in many of the isolates with
mecI
and one isolate with
blaI
missense mutations, providing structure-function correlates with the site and type of mutation. We conclude that
mecA
repressor activity is highly variable in clinical
S. aureus
isolates due to
mecI
mutations, RBS polymorphisms, and unidentified genomic adaptations.
Conserved Eukaryotic Histone-Fold Residues Substituted into an Archaeal Histone Increase DNA Affinity but Reduce Complex Flexibility
Authors: Soares Divya J. , Marc Frédéric , Reeve John N. 
Published: May 2003

ABSTRACT
Although the archaeal and eukaryotic nucleosome core histones evolved from a common ancestor, conserved lysine residues are present at DNA-binding locations in all four eukaryotic histones that are not present in the archaeal histones. Introduction of lysine residues at the corresponding locations into an archaeal histone, HMfB, generated a variant with increased affinity for DNA that formed more compact complexes with DNA. However, these complexes no longer facilitated the circularization of short DNA molecules and had lost the flexibility to wrap DNA alternatively in either a negative or positive supercoil.
Tertiary Structure of Bacterial Murein: the Scaffold Model
Authors: Dmitriev Boris A. , Toukach Filip V. , Schaper Klaus-Jürgen , Holst Otto , Rietschel Ernst T. , Ehlers Stefan 
Published: May 2003

ABSTRACT
Although the chemical structure and physical properties of peptidoglycan have been elucidated for some time, the precise three-dimensional organization of murein has remained elusive. Earlier published computer simulations of the bacterial murein architecture modeled peptidoglycan strands in either a regular (D. Pink, J. Moeller, B. Quinn, M. Jericho, and T. Beveridge, J. Bacteriol. 182: 5925-5930, 2000) or an irregular (A. Koch, J. Theor. Biol. 204: 533-541, 2000) parallel orientation with respect to the plasma membrane. However, after integrating published experimental data on glycan chain length distribution and the degree of peptide side chain cross-linking into this computer simulation, we now report that the proposed planar network of murein appears largely dysfunctional. In contrast, a scaffold model of murein architecture, which assumes that glycan strands extend perpendicularly to the plasma membrane, was found to accommodate published experimental evidence and yield a viable stress-bearing matrix. Moreover, this model is in accordance with the well-established principle of murein assembly in vivo, i.e., sequential attachment of strands to the preexisting structure. For the first time, the phenomenon of division plane alternation in dividing bacteria can be reconciled with a computer model of the molecular architecture of murein.
Error-Prone Polymerase, DNA Polymerase IV, Is Responsible for Transient Hypermutation during Adaptive Mutation in
Escherichia coli
Authors: Tompkins Joshua D. , Nelson Jennifer L. , Hazel Jill C. , Leugers Stacy L. , Stumpf Jeffrey D. , Foster Patricia L. 
Published: May 2003

ABSTRACT

The frequencies of nonselected mutations among adaptive Lac
+
revertants of
Escherichia coli
strains with and without the error-prone DNA polymerase IV (Pol IV) were compared. This frequency was more than sevenfold lower in the Pol IV-defective strain than in the wild-type strain. Thus, the mutations that occur during hypermutation are due to Pol IV.
Genomic Structure of the
Salmonella enterica
Serovar Typhimurium DT 64 Bacteriophage ST64T: Evidence for Modular Genetic Architecture
Authors: Mmolawa Princess T. , Schmieger Horst , Tucker Carly P. , Heuzenroeder Michael W. 
Published: May 2003

ABSTRACT

The complete sequence of the double-stranded DNA genome of a serotype-converting temperate bacteriophage, ST64T, was determined. The 40,679-bp genomic sequence of ST64T has an overall GC content of 47.5% and was reminiscent of a number of lambdoid phages, in particular, P22. Inferred proteins of ST64T which exhibited a high degree of sequence similarity to P22 proteins (>90%) included the functional serotype conversion cassette, integrase, excisionase, Abc1, Abc2, early antitermination (gp24), NinD, NinH, NinZ, packaging (gp3 and gp2), head (with the exception of gp26, gp7, gp20, and gp16), and tail proteins. The putative immunity genes were highly related to those of
Salmonella enterica
serotype Typhimurium phage L, whereas the lysis genes were almost identical to those of
S. enterica
serovar Typhimurium PS3.
The Glycosyltransferase Gene Encoding the Enzyme Catalyzing the First Step of Mycothiol Biosynthesis (
mshA
)
Authors: Newton Gerald L. , Koledin Teresa , Gorovitz Batia , Rawat Mamta , Fahey Robert C. , Av-Gay Yossef 
Published: May 2003

ABSTRACT

Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1
d
-
myo
-inosityl 2-acetamido-2-deoxy-α-
d
-glucopyranoside (GlcNAc-Ins). A transposon mutant of
Mycobacterium smegmatis
shown to be GlcNAc-Ins and mycothiol deficient was sequenced to identify a putative glycosyltransferase gene designated
mshA
. The ortholog in
Mycobacterium tuberculosis
, Rv0486, was used to complement the mutant phenotype.
Synthesis and Localization of the
Salmonella
SPI-1 Type III Secretion Needle Complex Proteins PrgI and PrgJ
Authors: Sukhan Anand , Kubori Tomoko , Galán Jorge E. 
Published: May 2003

ABSTRACT

An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In
Salmonella enterica
, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different
S. enterica
serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient
invA
mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type
S. enterica
serovar Typhimurium, was absent from needle complexes obtained from an
invJ
mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.
Transplacental Transfer and Subsequent Neonate Utilization of Herpes Simplex Virus-Specific Immunity Are Resilient to Acute Maternal Stress
Authors: Yorty Jodi L. , Bonneau Robert H. 
Published: May 2003

ABSTRACT
Neonates are severely compromised in the ability to generate an immune response to pathogens and thus rely heavily on maternally derived immunity that is acquired by transplacental and transmammary means. The passive transfer of maternal herpes simplex virus (HSV)-specific antibody is critical in determining the outcome of neonatal HSV infection. In adults, psychological stress alters immune responsiveness via the increased level of corticosterone that is produced as a result of hypothalamic-pituitary-adrenal axis activation. Although the behavioral and neuroendocrine effects of pre- and postnatal stress-induced increases in corticosterone are well documented, the effects of maternal stress on the efficacy of prenatally transferred and neonatally developed viral immunity has yet to be addressed. By using a well-established prenatal restraint-and-light stress mouse model, we investigated the effects of increased maternal corticosterone on the passive transfer of total and HSV-specific immunoglobulin G (IgG) antibody and subsequent neonatal susceptibility to HSV infection. Serum corticosterone levels in pregnant mice were significantly increased in response to restraint-and-light stress, and fetuses derived from these stressed mice had significantly elevated levels of corticosterone. Despite the increases in corticosterone, the passive transfer of total and HSV-specific IgG antibody persisted and, in turn, protected the neonate from systemic viral spread. Therefore, prenatal stress did not increase the susceptibility of neonates to HSV type 2-associated mortality. These findings demonstrate the resiliency of the passive transfer of protective HSV-specific immunity under conditions of acute psychological stress.
Human Immunodeficiency Virus Type 1 Entry Inhibitors Selected on Living Cells from a Library of Phage Chemokines
Authors: Hartley Oliver , Dorgham Karim , Perez-Bercoff Danielle , Cerini Fabrice , Heimann Anouk , Gaertner Hubert , Offord Robin E. , Pancino Gianfranco , Debré Patrice , Gorochov Guy 
Published: May 2003

ABSTRACT
The chemokine receptors CCR5 and CXCR4 are promising non-virus-encoded targets for human immunodeficiency virus (HIV) therapy. We describe a selection procedure to isolate mutant forms of RANTES (CCL5) with antiviral activity considerably in excess of that of the native chemokine. The phage-displayed library of randomly mutated and N-terminally extended variants was screened by using live CCR5-expressing cells, and two of the selected mutants, P1 and P2, were further characterized. Both were significantly more potent HIV inhibitors than RANTES, with P2 being the most active (50% inhibitory concentration of 600 pM in a viral coat-mediated cell fusion assay, complete protection of target cells against primary HIV type 1 strains at a concentration of 10 nM). P2 resembles AOP-RANTES in that it is a superagonist of CCR5 and potently induces receptor sequestration. P1, while less potent than P2, has the advantage of significantly reduced signaling activity via CCR5 (30% of that of RANTES). Additionally, both P1 and P2 exhibit not only significantly increased affinity for CCR5 but also enhanced receptor selectivity, retaining only trace levels of signaling activity via CCR1 and CCR3. The phage chemokine approach that was successfully applied here could be adapted to other chemokine-chemokine receptor systems and used to further improve the first-generation mutants reported in this paper.
Protection against Recurrent Ocular Herpes Simplex Virus Type 1 Disease after Therapeutic Vaccination of Latently Infected Mice
Authors: Richards C. M. , Case R. , Hirst T. R. , Hill T. J. , Williams N. A. 
Published: May 2003

ABSTRACTThe potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinantEscherichia coliheat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.
Mechanisms of Avian Retroviral Host Range Extension
Authors: Rainey G. Jonah A. , Natonson Andrew , Maxfield Lori F. , Coffin John M. 
Published: May 2003

ABSTRACT
Alpharetroviruses provide a useful system for the study of the molecular mechanisms of host range and receptor interaction. These viruses can be divided into subgroups based on diverse receptor usage due to variability within the two host range determining regions, hr1 and hr2, in their envelope glycoprotein SU (gp85). In previous work, our laboratory described selection from a subgroup B avian sarcoma-leukosis virus of an extended-host-range variant (LT/SI) with two adjacent amino acid substitutions in hr1. This virus retains its ability to use the subgroup BD receptor but can also infect QT6/BD cells, which bear a related subgroup E receptor (R. A. Taplitz and J. M. Coffin, J. Virol 71:7814-7819, 1997). Here, we report further analysis of this unusual variant. First, one (L154S) of the two substitutions is sufficient for host range extension, while the other (T155I) does not alter host range. Second, these mutations extend host range to non-avian cell types, including human, dog, cat, mouse, rat, and hamster. Third, interference experiments imply that the mutants interact efficiently with the subgroup BD receptor and possibly the related subgroup E receptor, but they have another means of entry that is not dependent on these interactions. Fourth, binding studies indicate that the mutant SU proteins retain the ability to interact as monomers with subgroup BD and BDE receptors but only bind the subgroup E receptor in the context of an Env trimer. Further, the mutant SU proteins bind well to chicken cells but do not bind any better than wild-type subgroup B to QT6 or human cells, even though the corresponding viruses are capable of infecting these cells.
Tomato Yellow Leaf Curl Sardinia Virus
Rep-Derived Resistance to Homologous and Heterologous Geminiviruses Occurs by Different Mechanisms and Is Overcome if Virus-Mediated Transgene Silencing Is Activated
Authors: Lucioli Alessandra , Noris Emanuela , Brunetti Angela , Tavazza Raffaela , Ruzza Valentino , Castillo Araceli G. , Bejarano Eduardo R. , Accotto Gian Paolo , Tavazza Mario 
Published: May 2003

ABSTRACT

The replication-associated protein (Rep) of geminiviruses is involved in several biological processes brought about by the presence of distinct functional domains. Recently, we have exploited the multifunctional character of the
Tomato yellow leaf curl Sardinia virus
(TYLCSV) Rep to develop a molecular interference strategy to impair TYLCSV infection. We showed that transgenic expression of its N-terminal 210 amino acids (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication. We have now used biochemical and transgenic approaches to carry out a fuller investigation of the molecular resistance mechanisms in transgenic plants expressing Rep-210. We show that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by the ability of Rep-210 to tightly inhibit C1 gene transcription, while that to heterologous virus is due to the interacting property of the Rep-210 oligomerization domain. Furthermore, we present evidence that in Rep-210-expressing plants, the duration of resistance is related to the ability of the challenging virus to shut off transgene expression by a posttranscriptional homology-dependent gene silencing mechanism. A model of Rep-210-mediated geminivirus resistance that takes transgene- and virus-mediated mechanisms into account is proposed.