bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Amino Acid Residues of the T-Cell Epitope ofMycobacterium tuberculosisα Antigen Critical for Vβ11+Th1 Cells
Authors: Kariyone Ai , Higuchi Kazue , Yamamoto Shigeki , Nagasaka-Kametaka Ai , Harada Mamoru , Takahashi Akio , Harada Nobuyuki , Ogasawara Kazumasa , Takatsu Kiyoshi
Published: December 2019

Stimulation ofMycobacterium tuberculosis-primed lymph node cells from C57BL/6 mice with α antigen (also known as antigen 85B and MPT59) induced cell proliferation, production of interleukin 2 and gamma interferon, and expansion of Vβ11+CD4+T cells in conjunction with antigen-presenting cells in an I-Ab-restricted manner. Using a series of 15-amino-acid peptides that overlapped each other by 5 amino acids and spanned the mature α antigen, we identified the antigenic epitope for α antigen-specific Vβ11+Th1 cells. That peptide (peptide-25), which corresponds to amino acid residues 240 to 254 of α antigen, contains a motif that is conserved in I-Aband requires processing by antigen-presenting cells. Using peptide-25-reactive Vβ11+T-cell clones and substituted peptide-25 mutants, we determined which amino acid residues within peptide-25 were critical for T-cell receptor (TCR) recognition. Our results showed that the amino acid residues at positions 245, 246, 248, 250, and 251 are important for recognition of TCRVβ11 and that residues at positions 244, 247, 249, and 252 are I-Abcontact residues. We also observed that active immunization of C57BL/6 mice with peptide-25 can lead to decreased bacterial load in the lungs ofM. tuberculosisH37Rv-infected mice. These results should provide us with a useful tool for delineating the regulation of Vβ11+Th1-cell development duringM. tuberculosisinfection and for developing a vaccine inducing a Th1-dominant immune response.
Detection of Intrastrain Antigenic Variation of
Bacteroides fragilis
Surface Polysaccharides by Monoclonal Antibody Labelling
Authors: Patrick Sheila , Gilpin Deirdre , Stevenson Leanne
Published: December 2019

Bacteroides fragilis
is a constituent of the normal resident microbiota of the human intestine and is the gram-negative obligately anaerobic bacterium most frequently isolated from clinical infection. Surface polysaccharides are implicated as potential virulence determinants. We present evidence of within strain immunochemical variation of surface polysaccharides in populations that are noncapsulate by light microscopy as determined by monoclonal antibody labelling. Expression of individual epitopes can be enriched from a population of an individual strain by use of immunomagnetic beads. Also, individual colonies in which either >94% or <7% of the bacteria carry an individual epitope retain this level of expression when subcultured into broth. In broth cultures where >94% of the bacteria carry a given epitope, there is no enrichment for other epitopes recognized by different polysaccharide-specific monoclonal antibodies. This intrastrain variation has important implications for the development of potential vaccines or immunodiagnostic tests.
Truncated Surface Protective Antigen (SpaA) of
Erysipelothrix rhusiopathiae
Serotype 1a Elicits Protection against Challenge with Serotypes 1a and 2b in Pigs
Authors: Imada Yumiko , Goji Noriko , Ishikawa Hitoshi , Kishima Masato , Sekizaki Tsutomu
Published: December 2019

Erysipelothrix rhusiopathiae
is a causal agent of swine erysipelas, which is of economic importance in the swine industry by virtue of causing acute septicemia, chronic arthritis, and endocarditis. However, little is known about the genetic properties of its protective antigens. Recently, a surface protective antigen (SpaA) gene was identified from serotype 2 in a mouse model. We cloned
spaA
from virulent strain Fujisawa (serotype 1a) and determined that the N-terminal 342 amino acids without C-terminal repeats of 20 amino acids have the ability to elicit protection in mice. Fusions of 342 amino acids of Fujisawa SpaA and histidine hexamer (HisSpa1.0) protected pigs against challenge with both serotype 1 and serotype 2, the most important serotypes in the swine industry. Pigs immunized with HisSpa1.0 reacted well with both HisSpa1.0 and intact SpaA by enzyme-linked immunosorbent assay and immunoblotting. Serum collected at the time of challenge from a pig immunized with HisSpa1.0 markedly enhanced the in vitro phagocytic and killing activity of pig neutrophils against the bacteria. DNA sequences of protective regions of
spaA
genes from five strains of serotypes 1 and 2 were almost identical. The full DNA sequences also seemed to be conserved among strains of all 12 serotype reference strains harboring the
spaA
gene by restriction fragment length polymorphism analysis of PCR products. These results indicates that SpaA is a common protective antigen of serotypes 1 and 2 of
E. rhusiopathiae
in swine and will be a useful tool for development of new types of vaccines and diagnostic tools for effective control of the disease.
Down-Regulation of Th2 Responses byBrucella abortus, a Strong Th1 Stimulus, Correlates with Alterations in the B7.2-CD28 Pathway
Authors: Agranovich Inna , Scott Dorothy E. , Terle Douglas , Lee Katherine , Golding Basil
Published: December 2019

Down-regulation of the Th2-like response induced by ovalbumin-alum (OVA/alum) immunization by heat-killedBrucella abortuswas not reversed by anti-IL-12 antibody treatment or in gamma interferon (IFN-γ) knockout mice, suggesting that induction of Th1 cytokines was not the only mechanism involved in theB. abortus-mediated inhibition of the Th2 response to OVA/alum. The focus of this study was to determine whether an alternative pathway involves alteration in expression of costimulatory molecules. First we show that the Th2-like response to OVA/alum is dependent on B7.2 interaction with ligand since it can be abrogated by anti-B7.2 treatment. Expression of costimulatory molecules was then studied in mice immunized with OVA/alum in the absence or presence ofB. abortus. B7.2, but not B7.1, was up-regulated on mouse non-T and T cells following immunization withB. abortus. Surprisingly,B. abortusinduced down-regulation of CD28 and up-regulation of B7.2 on murine CD4+and CD8+T cells. These effects on T cells were maximal for CD28 and B7.2 at 40 to 48 h and were not dependent on interleukin-12 (IL-12) or IFN-γ. On the basis of these results, we propose that the IL-12/IFN-γ-independent inhibition of Th2 responses to OVA/alum is secondary to the effects ofB. abortuson expression of costimulatory molecules on T cells. We suggest that down-regulation of CD28 following activation inhibits subsequent differentiation of Th0 into Th2 cells. In addition, decreased expression of CD28 and increased expression of B7.2 on T cells would favor B7.2 interaction with CTLA-4 on T cells, and this could provide a negative signal to developing Th2 cells.
Sulfated Polysaccharide-Directed Recruitment of Mammalian Host Proteins: a Novel Strategy in Microbial Pathogenesis
Authors: Duensing Thomas D. , Wing Jenny S. , van Putten Jos P. M.
Published: December 2019

Fundamental to the virulence of microbial pathogens is their capacity for adaptation and survival within variable, and often hostile, environments encountered in the host. We describe a novel, extragenomic mechanism of surface modulation which may amplify the adaptive and pathogenic potential of numerous bacterial species, includingStaphylococcus,Yersinia, and pathogenicNeisseriaspecies, as well asHelicobacter pyloriandStreptococcus pyogenes. The mechanism involves specific bacterial recruitment of heparin, glycosaminoglycans, or related sulfated polysaccharides, which in turn serve as universal binding sites for a diverse array of mammalian heparin binding proteins, including adhesive glycoproteins (vitronectin and fibronectin), inflammatory (MCP-3, PF-4, and MIP-1α) and immunomodulatory (gamma interferon) intermediates, and fibroblast growth factor. This strategy impacts key aspects of microbial pathogenicity as exemplified by increased bacterial invasion of epithelial cells and inhibition of chemokine-induced chemotaxis. Our findings illustrate a previously unrecognized form of parasitism that complements classical virulence strategies encoded within the microbial genome.
Stress-Induced Membrane Association of the
Streptococcus mutans
GTP-Binding Protein, an Essential G Protein, and Investigation of Its Physiological Role by Utilizing an Antisense RNA Strategy
Authors: Baev Didi , England Reg , Kuramitsu Howard K.
Published: December 2019

SGP (for
Streptococcus
GTP-binding protein) is a
Streptococcus mutans
essential GTPase which has significant sequence identity to the previously identified
Escherichia coli
Era protein and to numerous other prokaryotic GTPase proteins of unknown function. Recent studies in our laboratory have addressed the possible role of SGP in the stress response of the oral pathogen
S. mutans
. Here we report that during growth in the early stationary phase, and in response to elevated temperatures or acidic pH, the distribution of SGP between the cytoplasm and the membranes of
S. mutans
cells varies. Immunoblot analysis of soluble and membrane protein fractions collected from the mid-log and early stationary growth phases of bacterial populations grown at normal temperature (37°C) and at the elevated temperature of 43°C, or at acidic pH, demonstrated that the total amount of SGP increased with the age of the bacterial culture, elevated temperature, or acidic pH. Furthermore, it was established that a substantial amount of SGP is associated with the membrane fraction under stress conditions. In order to investigate the physiological role of SGP, we constructed an
S. mutans
strain capable of chromosomal
sgp
antisense RNA expression, which interferes with the normal information processing of the
sgp
gene. Utilizing this strain, we determined conditions whereby the streptococcal cells can be depleted of SGP, thus avoiding the problem of constructing a conditional lethal system. From the results of measurements of the nucleotide pools extracted from the antisense strain and its isogenic counterpart, we propose that one of the physiological roles of SGP is regulation and modulation of the GTP/GDP ratio under different growth conditions. Moreover, we observed that in SGP-depleted cells the levels of glucan-binding protein A (GbpA) substantially increased, suggesting that GbpA may have stress response-related physiological functions. Finally, the potential applications of the antisense RNA approach that we employed are discussed.
Polarization of the Immune Response to the Single Immunodominant Epitope of p38, a MajorSchistosoma mansoniEgg Antigen, Generates Th1- or Th2-Type Cytokines and Granulomas
Authors: Chen Yiguang , Boros Dov L.
Published: December 2019

In schistosomiasis mansoni, helminth eggs secrete soluble egg antigens (SEA) that induce T-cell-mediated granulomatous tissue responses. The cloned 38-kDa peptide (p38) of SEA was shown to induce and elicit Th1-type responsiveness inH-2kmice. Subsequently, the immunodominant T-cell epitope (P4) of p38 was shown to elicit pulmonary granuloma formation and Th1-type cytokine production in sensitized or infected mice. Here, we report that the immune response to p38 or P4 can be polarized to a Th1 or Th2 profile when the peptides are presented intraperitoneally in soluble recombinant interleukin-12 (IL-12) or alum adjuvant, respectively. The Th1 or Th2 profile was verified by cytokine secretion, enzyme-linked spot assay, and antibody isotype characterization. Importantly, the polarized immune response generated two types of pulmonary granulomas around injected P4-coated beads. The type 1 granulomas were smaller and contained mononuclear cells and occasional thin strands of deposited collagen. In contrast, the type 2 lesions were larger and contained mononuclear cells, large numbers of eosinophils, and several thick bands of deposited collagen. By reverse transcription-PCR cytokine, message in the type 1 granuloma-bearing lungs was found for gamma interferon, tumor necrosis factor alpha, and inducible nitric oxide synthase but not for IL-4 or IL-5. Conversely, lungs with type 2 granulomas had message only for IL-4 and IL-5. These results show that in the proper cytokine environment, the response to a strong Th1 inducer peptide can be deviated to a Th2 profile.
Pure Botulinum Neurotoxin Is Absorbed from the Stomach and Small Intestine and Produces Peripheral Neuromuscular Blockade
Authors: Maksymowych Andrew B. , Reinhard Marco , Malizio Carl J. , Goodnough Michael C. , Johnson Eric A. , Simpson Lance L.
Published: December 2019

Clostridium botulinum
serotype A produces a neurotoxin composed of a 100-kDa heavy chain and a 50-kDa light chain linked by a disulfide bond. This neurotoxin is part of a ca. 900-kDa complex, formed by noncovalent association with a single nontoxin, nonhemagglutinin subunit and a family of hemagglutinating proteins. Previous work has suggested, although never conclusively demonstrated, that neurotoxin alone cannot survive passage through the stomach and/or cannot be absorbed from the gut without the involvement of auxiliary proteins in the complex. Therefore, this study compared the relative absorption and toxicity of three preparations of neurotoxin in an in vivo mouse model. Equimolar amounts of serotype A complex with hemagglutinins, complex without hemagglutinins, and purified neurotoxin were surgically introduced into the stomach or into the small intestine. In some experiments, movement of neurotoxin from the site of administration was restricted by ligation of the pylorus. Comparison of relative toxicities demonstrated that at adequate doses, complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin can be absorbed from the stomach. The potency of neurotoxin in complex was greater than that of pure neurotoxin, but the magnitude of this difference diminished as the dosage of neurotoxin increased. Qualitatively similar results were obtained when complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin were placed directly into the intestine. This work establishes that pure botulinum neurotoxin serotype A is toxic when administered orally. This means that pure neurotoxin does not require hemagglutinins or other auxiliary proteins for absorption from the gastrointestinal system into the general circulation.
Identification of Abundantly Expressed Novel and Conserved Genes from the Infective Larval Stage ofToxocara canisby an Expressed Sequence Tag Strategy
Authors: Tetteh Kevin K. A. , Loukas Alex , Tripp Cindy , Maizels Rick M.
Published: December 2019

Larvae ofToxocara canis, a nematode parasite of dogs, infect humans, causing visceral and ocular larva migrans. In noncanid hosts, larvae neither grow nor differentiate but endure in a state of arrested development. Reasoning that parasite protein production is orientated to immune evasion, we undertook a random sequencing project from a larval cDNA library to characterize the most highly expressed transcripts. In all, 266 clones were sequenced, most from both 3′ and 5′ ends, and similarity searches against GenBank protein and dbEST nucleotide databases were conducted. Cluster analyses showed that 128 distinct gene products had been found, all but 3 of which represented newly identified genes. Ninety-five genes were represented by a single clone, but seven transcripts were present at high frequencies, each composing >2% of all clones sequenced. These high-abundance transcripts include a mucin and a C-type lectin, which are both major excretory-secretory antigens released by parasites. Four highly expressed novel gene transcripts, termedant(abundant novel transcript) genes, were found. Together, these four genes comprised 18% of all cDNA clones isolated, but no similar sequences occur in theCaenorhabditis elegansgenome. While the coding regions of the four genes are dissimilar, their 3′ untranslated tracts have significant homology in nucleotide sequence. The discovery of these abundant, parasite-specific genes of newly identified lectins and mucins, as well as a range of conserved and novel proteins, provides defined candidates for future analysis of the molecular basis of immune evasion byT. canis.
Molecular Cloning and Characterization of Rat Genes Encoding Homologues of Human β-Defensins
Authors: Jia Hong Peng , Mills Jesse N. , Barahmand-Pour Fariba , Nishimura Darryl , Mallampali Rama K. , Wang Guoshun , Wiles Kerry , Tack Brian F. , Bevins Charles L. , McCray Paul B.
Published: December 2019

β-Defensins are cationic peptides with broad-spectrum antimicrobial activity that may play a role in mucosal defenses of several organs. They have been isolated in several species, and in humans, two β-defensins have been identified. Here, we report the identification of two genes encoding β-defensin homologues in the rat. Partial cDNAs were found by searching the expressed-sequence-tag database, and primers were designed to generate full-length mRNA coding sequences. One gene was highly similar to the human β-defensin-1 (HBD-1) gene and mouse β-defensin-1 gene at both the nucleic acid and amino acid levels and was termed rat β-defensin-1 (RBD-1). The other gene, named RBD-2, was homologous to the HBD-2 and bovine tracheal antimicrobial peptide (TAP) genes. The predicted prepropeptides were strongly cationic, were 69 and 63 residues in length for RBD-1 and RBD-2, respectively, and contained the six-cysteine motif characteristic of β-defensins. The β-defensin genes mapped closely on rat chromosome 16 and were closely linked to the α-defensins genes, suggesting that they are part of a gene cluster, similar to the organization reported for humans. Northern blot analysis showed that both RBD-1 and RBD-2 mRNA transcripts were ∼0.5 kb in length; RBD-1 mRNA was abundantly transcribed in the rat kidney, while RBD-2 was prevalent in the lung. Reverse transcription-PCR indicated that RBD-1 and RBD-2 mRNAs were distributed in a variety of other tissues. In the lung, RBD-1 mRNA expression localized to the tracheal epithelium while RBD-2 was expressed in alveolar type II cells. In conclusion, we characterized two novel β-defensin homologues in the rat. The rat may be a useful model to investigate the function and contribution of β-defensins to host defense in the lung, kidney, and other tissues.
Inhibitory Effects of Protamines on Proteolytic and Adhesive Activities of
Porphyromonas gingivalis
Authors: Kontani Masanori , Amano Atsuo , Nakamura Takayuki , Nakagawa Ichiro , Kawabata Shigetada , Hamada Shigeyuki
Published: December 2019

Protamines (salmine prepared from sperm DNA of salmon and clupeine from herring sperm), which are basic peptides rich in arginine, were found to inhibit the proteolytic activity of arginine-specific cysteine protease (RC-protease) from
Porphyromonas gingivalis
. Lineweaver-Burk plot analysis revealed that the protamines competitively inhibited proteolytic activity with cleavage of benzoyl-
l
-arginine-
p
-nitroanilide, a synthetic substrate of RC-protease. Furthermore, the protamines were capable of binding strongly to
P. gingivalis
fimbriae and inhibited fimbrial interaction with immobilized fibronectin. These results clearly show that protamines are potent inhibitors of the proteolytic and adhesive activities of
P. gingivalis.
Bactericidal and Cross-Protective Activities of a Monoclonal Antibody Directed againstNeisseria meningitidisNspA Outer Membrane Protein
Authors: Cadieux Nathalie , Plante Martin , Rioux Clément R. , Hamel Josée , Brodeur Bernard R. , Martin Denis
Published: December 2019

The cross-bactericidal and cross-protective activities of a monoclonal antibody (MAb) named Me-7, which is directed against an antigenically highly conserved epitope on the meningococcal NspA protein, were studied. This MAb efficiently killed in vitro, in the presence of rabbit or human serum, 13 of 14 meningococcal strains tested, including 9 of 9, 2 of 3, and 2 of 2 strains of serotypes B, A, and C, respectively. MAb Me-7 also significantly reduced by more than 75% the levels of bacteremia recorded for mice challenged with 10 of 11 meningococcal strains tested. Analysis of the predicted amino acid sequence of the NspA protein from the meningococcal strain MCH88 (A:4:P1.10), which was not killed by MAb Me-7, indicated the presence of an additional glutamine residue at position 73, compared to the three other NspA sequences. The data presented in this study suggest that antibodies directed against this highly conserved outer membrane protein could protect against meningococcal infections.
Experimental Transmission ofNeisseria gonorrhoeaefrom Pregnant Rat to Fetus
Authors: Nowicki Stella , Selvarangan Rangaraj , Anderson Garland
Published: December 2019

Sprague-Dawley rats were infected on day 20 of pregnancy by intraperitoneal inoculation withNeisseria gonorrhoeae. Disseminated gonococcal infection (DGI) and pelvic inflammatory disease (PID) strains in the presence of C1q but not in the presence of bovine serum albumin (BSA) were able to spread from the pregnant rat to the fetus and resulted in fetal mortality. Transmission of DGI and PID strains that are serum resistant (serr) andsac-4positive but not of a local infection strain that is sersandsac-4negative was facilitated by the C1q-dependent mechanism. This study provides the first experimental model that may mimic the transmission of gonococcal infection from mother to the fetus during pregnancy.
Defects in Assembly of the Extracellular Matrix Are Responsible for Altered Morphogenesis of a
Candida albicans phr1
Mutant
Authors: Popolo Laura , Vai Marina
Published: December 2019

Analysis of
Candida albicans
cells using antibodies directed against Gas1p/Ggp1p,
Saccharomyces cerevisiae
homolog of Phr1p, revealed that Phr1p is a glycoprotein of about 88 kDa whose accumulation increases with the rise of external pH. This polypeptide is present both in the yeast form and during germ tube induction. In the Phr1
−
cells at pH 8 the solubility of glucans in alkali is greatly affected. In the parental strain the alkali-soluble/-insoluble glucan ratio shows a 50% decrease at pH 8 with respect to pH 4.5, whereas in the null mutant it is unchanged, indicating the lack of a polymer cross-linker activity induced by the rise of pH. The mutant has a sixfold increase in chitin level and is hypersensitive to calcofluor. Consistently with a role of chitin in strengthening the cell wall, Phr1
−
cells are more sensitive to nikkomycin Z than the parental strain.
Stability of the
Escherichia coli
Division Inhibitor Protein MinC Requires Determinants in the Carboxy-Terminal Region of the Protein
Authors: Sen Mita , Rothfield Lawrence I.
Published: December 2019

Certain mutations in the C-terminal region of the
Escherichia coli
division inhibitor protein MinC cause loss of function of the division inhibitor by making MinC more sensitive to degradation by Lon protease, implying a possible role for the C-terminal region in regulating the stability and cellular concentration of MinC.
Molecular Characterization of IS
1541
Insertions in the Genome of
Yersinia pestis
Authors: Odaert Monique , Devalckenaere Annie , Trieu-Cuot Patrick , Simonet Michel
Published: December 2019

The genome of
Yersinia pestis
, the causative agent of plague, contains at least 30 copies of an element, designated IS
1541
, which is structurally related to IS
200
(85% identity). One such element is inserted within the chromosomal
inv
gene (M. Simonet, B. Riot, N. Fortineau, and P. Berche, Infect. Immun. 64:375–379, 1996). We characterized other IS
1541
insertions by cloning 14 different
Y. pestis
6/69M loci carrying a single copy of this insertion sequence (IS) into
Escherichia coli
and, for each element, sequencing 250 bp of both flanking regions. In no case was this IS element inserted into large open reading frames; however, in eight cases, it was detected downstream (17 to 139 bp) of genes thought to be transcribed monocistronically or which constituted the last gene of an operon, and in only one case was it detected upstream (37 bp) of the first gene of an operon. Sequence analysis revealed stem-loop structures (Δ
G
, <−10 kcal) resembling rho-independent transcription terminators in 8 of the 14 insertion sites. These motifs might constitute hot spots for insertion of this IS
1541
element within the
Y. pestis
genome.
RimM and RbfA Are Essential for Efficient Processing of 16S rRNA in
Escherichia coli
Authors: Bylund Göran O. , Wipemo L. Charlotta , Lundberg L. A. Carina , Wikström P. Mikael
Published: December 2019

The
trmD
operon is located at 56.7 min on the genetic map of the
Escherichia coli
chromosome and contains the genes for ribosomal protein (r-protein) S16, a 21-kDa protein (RimM, formerly called 21K), the tRNA (m
1
G37)methyltransferase (TrmD), and r-protein L19, in that order. Previously, we have shown that strains from which the
rimM
gene has been deleted have a sevenfold-reduced growth rate and a reduced translational efficiency. The slow growth and translational deficiency were found to be partly suppressed by mutations in
rpsM
, which encodes r-protein S13. Further, the RimM protein was shown to have affinity for free ribosomal 30S subunits but not for 30S subunits in the 70S ribosomes. Here we have isolated several new suppressor mutations, most of which seem to be located close to or within the
nusA
operon at 68.9 min on the chromosome. For at least one of these mutations, increased expression of the ribosome binding factor RbfA is responsible for the suppression of the slow growth and translational deficiency of a Δ
rimM
mutant. Further, the RimM and RbfA proteins were found to be essential for efficient processing of 16S rRNA.
Molecular and Genetic Analysis of Two Closely Linked Genes That Encode, Respectively, a Protein Phosphatase 1/2A/2B Homolog and a Protein Kinase Homolog in the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Zhang Cheng-Cai , Friry Aline , Peng Ling
Published: December 2019

Reversible protein phosphorylation plays important roles in signal transduction. One gene,
prpA
, encoding a protein similar to eukaryotic types of phosphoprotein phosphatases PP1, PP2A, and PP2B, was cloned from the nitrogen-fixing cyanobacterium
Anabaena
sp. strain PCC 7120. Interestingly, a eukaryotic-type protein kinase gene,
pknE
, was found 301 bp downstream of
prpA
. This unusual genetic arrangement provides the opportunity for study about how the balance between protein phosphorylation and dephosphorylation can regulate cellular activities. Both proteins were overproduced in
Escherichia coli
and used to raise polyclonal antibodies. Immunodetection and RNA/DNA hybridization experiments suggest that these two genes are unlikely to be coexpressed, despite their close genetic linkage. PrpA is expressed constitutively under different nitrogen conditions, while PknE expression varies according to the nature of the nitrogen source. Inactivation analysis in vivo suggests that PrpA and PknE function to ensure a correct level of phosphorylation of the targets in order to regulate similar biological processes such as heterocyst structure formation and nitrogen fixation.
The
l
-Isoaspartyl Protein Repair Methyltransferase Enhances Survival of Aging
Escherichia coli
Subjected to Secondary Environmental Stresses
Authors: Visick Jonathan E. , Cai Hui , Clarke Steven
Published: December 2019

Like its homologs throughout the biological world, the
l
-isoaspartyl protein repair methyltransferase of
Escherichia coli
, encoded by the
pcm
gene, can convert abnormal
l
-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues. Mutations in
pcm
were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl. Acad. Sci. USA 89:9885–9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation in
rpoS
, which encodes a stationary-phase-specific sigma factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158–4163, 1997). We now show that the
rpoS
mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes. We further demonstrate that a new
pcm
mutant lacks these phenotypes. Interestingly, the newly constructed
pcm
mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42°C. The
pcm
mutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functional
pcm
gene integrated elsewhere in the chromosome. These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging
E. coli
and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo.
An rRNA Fragment and Its Antisense Can Alter Decoding of Genetic Information
Authors: Arkov Alexey L. , Mankin Alexander , Murgola Emanuel J.
Published: December 2019

rRNA plays a central role in protein synthesis and is intimately involved in the initiation, elongation, and termination stages of translation. However, the mode of its participation in these reactions, particularly as to the decoding of genetic information, remains elusive. In this paper, we describe a new approach that allowed us to identify an rRNA segment whose function is likely to be related to translation termination. By screening an expression library of random rRNA fragments, we identified a fragment of the
Escherichia coli
23S rRNA (nucleotides 74 to 136) whose expression caused readthrough of UGA nonsense mutations in certain codon contexts in vivo. The antisense RNA fragment produced a similar effect, but in neither case was readthrough of UAA or UAG observed. Since termination at UGA in
E. coli
specifically requires release factor 2 (RF2), our data suggest that the fragments interfere with RF2-dependent termination.
Penicillin-Binding Protein 1 of
Staphylococcus aureus
Is Essential for Growth
Authors: Wada Akihito , Watanabe Haruo
Published: December 2019

pbpA
, a gene encoding penicillin-binding protein (PBP) 1 of
Staphylococcus aureus
, was cloned in an
Escherichia coli
MC1061 transformant which grew on a plate containing 512 μg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of
pbpA
could be disrupted only when RN4220, a methicillin-sensitive
S. aureus
strain, had additional copies of
pbpA
in its episome. Furthermore, these episomal copies of
pbpA
could not be eliminated by an incompatible plasmid when the chromosomal copy of
pbpA
was disrupted beforehand. Based on these observations, we concluded that
pbpA
is essential for the growth of methicillin-sensitive
S. aureus.
Adaptive Mutagenesis at
ebgR
Is Regulated by PhoPQ
Authors: Hall Barry G.
Published: December 2019

Adaptive mutations are mutations that occur in nondividing or very slowly dividing microbial cells during prolonged nonlethal selection and that are specific to the challenge of the selection in the sense that the only mutations that can be detected are those that provide a growth advantage to the cell. The
phoPQ
genes encode a two-component positively acting regulatory system that controls expression of at least 25 to 30 genes in
Escherichia coli
and
Salmonella typhimurium
. PhoPQ responds to a variety of environmental stress signals including Mg
2+
starvation and nutritional deprivation. Here I show that disruption of
phoP
or
phoQ
by Tn
10
d
C
am significantly reduces the adaptive mutation rate to
ebgR
, indicating that the adaptive mutagenesis machinery is regulated, directly or indirectly, by
phoPQ
. The finding that it is regulated implies that adaptive mutagenesis does not simply result from a failure of various error correction mechanisms during prolonged starvation.
Novel Temperature-Sensitive Mutants of
Escherichia coli
That Are Unable To Grow in the Absence of Wild-Type tRNA
6
Leu
Authors: Nakayashiki Toru , Inokuchi Hachiro
Published: December 2019

Escherichia coli
has only a single copy of a gene for tRNA
6
Leu
(Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to
O
2
-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA
4
Leu
, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-
O
2
-methyluridine) as its anticodon, tRNA
6
Leu
is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA
6
Leu
with a change from CAA to CUA, which results in the amber suppressor activity of this strain (
supP
, Su
+
6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA
6
Leu
. These tRNA
6
Leu
-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the
miaA
gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA
4
Leu
restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA
4
Leu
poorly recognizes the UUG codon at 42°C and that the wild-type tRNA
6
Leu
is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the
gidA
gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA
6
Leu
remains to be investigated.
A Regulatory Mutant of
Hansenula polymorpha
Exhibiting Methanol Utilization Metabolism and Peroxisome Proliferation in Glucose
Authors: Parpinello Giuseppinia , Berardi Enrico , Strabbioli Rosanna
Published: December 2019

Mutant LGM-128 of
Hansenula polymorpha
harbors the recessive mutation
glr2-1
which confers a complex pleiotropic phenotype, the major feature of which is the metabolically unnecessary induction of methanol utilization metabolism (C
1
metabolism) during growth on glucose, whether or not methanol is in the medium. Therefore, in this mutant, peroxisomes are formed and proliferate upon cultivation in glucose-containing media. In these media, LGM-128 shows induction levels of C
1
metabolism that are similar to those observed in methanol-containing media. This indicates that
GLR2
controls the repression-derepression process stimulated by glucose and that the induction process triggered by methanol plays only a minor role in activating C
1
metabolism. Cultivating LGM-128 in methanol and then transferring it to glucose media revealed that active degradative processes occur, leading to the disappearance of C
1
metabolism. This observation suggests that, although stimulated by glucose, the two processes are controlled by elements which are, at least in part, distinct. Finally,
glr2-1
does not affect ethanol repression, suggesting that in
H. polymorpha
the two repressing circuits are separated.
Phylogeny of Dissimilatory Sulfite Reductases Supports an Early Origin of Sulfate Respiration
Authors: Wagner Michael , Roger Andrew J. , Flax Jodi L. , Brusseau Gregory A. , Stahl David A.
Published: December 2019

Microorganisms that use sulfate as a terminal electron acceptor for anaerobic respiration play a central role in the global sulfur cycle. Here, we report the results of comparative sequence analysis of dissimilatory sulfite reductase (DSR) genes from closely and distantly related sulfate-reducing organisms to infer the evolutionary history of DSR. A 1.9-kb DNA region encoding most of the α and β subunits of DSR could be recovered only from organisms capable of dissimilatory sulfate reduction with a PCR primer set targeting highly conserved regions in these genes. All DNA sequences obtained were highly similar to one another (49 to 89% identity), and their inferred evolutionary relationships were nearly identical to those inferred on the basis of 16S rRNA. We conclude that the high similarity of bacterial and archaeal DSRs reflects their common origin from a conserved DSR. This ancestral DSR was either present before the split between the domains
Bacteria
,
Archaea
, and
Eucarya
or laterally transferred between
Bacteria
and
Archaea
soon after domain divergence. Thus, if the physiological role of the DSR was constant over time, then early ancestors of
Bacteria
and
Archaea
already possessed a key enzyme of sulfate and sulfite respiration.
A Tn
7
-Like Transposon Is Present in the
glmUS
Region of the Obligately Chemoautolithotrophic Bacterium
Thiobacillus ferrooxidans
Authors: Oppon Joseph C. , Sarnovsky Robert J. , Craig Nancy L. , Rawlings Douglas E.
Published: December 2019

The region downstream of the
Thiobacillus ferrooxidans
ATCC 33020
atp
operon was examined, and the genes encoding
N
-acetylglucosamine-1-uridyltransferase (
glmU
) and glucosamine synthetase (
glmS
) were found. This
atpEFHAGDC-glmUS
gene order is identical to that of
Escherichia coli
. The
T. ferrooxidans glmS
gene was shown to complement
E. coli glmS
mutants for growth on minimal medium lacking glucosamine. A Tn
7
-like transposon, Tn
5468
, was found inserted into the region immediately downstream of the
glmS
gene in a manner similar to the site-specific insertion of transposon Tn
7
within the termination region of the
E. coli glmS
gene. Tn
5468
was sequenced, and Tn
7
-like terminal repeat sequences as well as several open reading frames which are related to the Tn
7
transposition genes
tnsA
,
tnsB
,
tnsC
, and
tnsD
were found. Tn
5468
is the closest relative of Tn
7
to have been characterized to date. Southern blot hybridization indicated that a similar or identical transposon was present in three
T. ferrooxidans
strains isolated from different parts of the world but not in two
Thiobacillus thiooxidans
strains or a
Leptospirillum ferrooxidans
strain. Since
T. ferrooxidans
is an obligately acidophilic autotroph and
E. coli
is a heterotroph, ancestors of the Tn
7
-like transposons must have been active in a variety of physiologically different bacteria so that their descendants are now found in bacteria that occupy very different ecological niches.
In Vivo Analysis of Sequence Requirements for Processing and Degradation of the Colicin A Lysis Protein Signal Peptide
Authors: Howard S. Peter , Lindsay Lisa
Published: December 2019

The lipid modification and processing of a number of colicin lysis proteins take place exceedingly slowly and result in the release of a stable signal peptide. It is possible that this peptide or the presence of lipid-modified precursors which result from the slow processing plays a role in the release of colicins and in the quasilysis that occurs in induced colicinogenic cultures. We used in vitro mutagenesis and pulse-chase radiolabeling and immunoprecipitation to examine the reasons for the slow processing and signal peptide degradation reactions for the colicin A lysis protein (Cal). In one mutant, isoleucine 13 was replaced with serine, and in another, alanine 18, the last residue of the signal peptide, was replaced with glycine. In each case, the mutation caused a striking increase in the rate of maturation of the precursor, and in the case of the serine 13 derivative, the mutation also destabilized the signal peptide. A precursor containing both of these mutations was completely matured and its signal sequence degraded within seconds of its synthesis. The release of colicin A and the quasilysis of producing cultures were unchanged for each of these mutants, indicating that neither the stable signal peptide nor lipid-modified processing intermediates of Cal are required for either of these events in wild-type cells.
Multidomain Structure and Cellulosomal Localization of the
Clostridium thermocellum
Cellobiohydrolase CbhA
Authors: Zverlov Vladimir V. , Velikodvorskaya Galina V. , Schwarz Wolfgang H. , Bronnenmeier Karin , Kellermann Josef , Staudenbauer Walter L.
Published: December 2019

The nucleotide sequence of the
Clostridium thermocellum
F7
cbhA
gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like β-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.
Transcriptional Regulation ofStreptomyces coelicolorPathway-Specific Antibiotic Regulators by theabsAandabsBLoci
Authors: Aceti David J. , Champness Wendy C.
Published: December 2019

The four antibiotics produced byStreptomyces coelicolorare all affected by mutations in theabsAandabsBloci. TheabsAlocus encodes a putative two-component signal transduction system, and theabsBlocus encodes a homolog ofEscherichia coliRNase III. We assessed whether these loci control synthesis of the antibiotics actinorhodin and undecylprodigiosin by regulating transcript abundance from the biosynthetic and regulatory genes specific for each antibiotic. Strains that were Abs−(for antibiotic synthesis deficient) due to mutations inabsAorabsBwere examined. In the Abs−absAmutant strain, transcripts for the actinorhodin biosynthetic genesactVI-ORF1 andactI, and for the pathway-specific regulatory geneactII-ORF4, were substantially lower in abundance than in the parent strain. The level of the transcript for the undecylprodigiosin pathway-specific regulatory generedDwas similarly reduced in this mutant. Additionally, a strain that exhibits precocious hyperproduction of antibiotics (Pha phenotype) due to disruption of theabsAlocus contained elevated levels of theactVI-ORF1,actII-ORF4, andredDtranscripts. In theabsBmutant strain,actVI-ORF1,actI,actII-ORF4, andredDtranscript levels were also substantially lower than in the parent strain. These results establish that theabsgenes affect production of antibiotics through regulation of expression of the antibiotic-specific regulatory genes inS. coelicolor.
Different Modes of Diaminopimelate Synthesis and Their Role in Cell Wall Integrity: a Study with
Corynebacterium glutamicum
Authors: Wehrmann Axel , Phillipp Bodo , Sahm Hermann , Eggeling Lothar
Published: December 2019

In eubacteria, there are three slightly different pathways for the synthesis of
m
-diaminopimelate (m-DAP), which is one of the key linking units of peptidoglycan. Surprisingly, for unknown reasons, some bacteria use two of these pathways together. An example is
Corynebacterium glutamicum
, which uses both the succinylase and dehydrogenase pathways for m-DAP synthesis. In this study, we clone
dapD
and prove by enzyme experiments that this gene encodes the succinylase (
M
r
= 24082), initiating the succinylase pathway of m-DAP synthesis. By using gene-directed mutation,
dapD
, as well as
dapE
encoding the desuccinylase, was inactivated, thereby forcing
C. glutamicum
to use only the dehydrogenase pathway of m-DAP synthesis. The mutants are unable to grow on organic nitrogen sources. When supplied with low ammonium concentrations but excess carbon, their morphology is radically altered and they are less resistant to mechanical stress than the wild type. Since the succinylase has a high affinity toward its substrate and uses glutamate as the nitrogen donor, while the dehydrogenase has a low affinity and incorporates ammonium directly, the m-DAP synthesis is another example of twin activities present in bacteria for access to important metabolites such as the well-known twin activities for the synthesis of glutamate or for the uptake of potassium.
Regulation of
agr
-Dependent Virulence Genes in
Staphylococcus aureus
by RNAIII from Coagulase-Negative Staphylococci
Authors: Tegmark Karin , Morfeldt Eva , Arvidson Staffan
Published: December 2019

Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in
Staphylococcus aureus
are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, including
hla
(alpha-toxin),
saeB
(enterotoxin B),
tst
(toxic shock syndrome toxin 1), and
ssp
(serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like
spa
(protein A) and
fnb
(fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e.,
Staphylococcus epidermidis
,
Staphylococcus simulans
, and
Staphylococcus warneri
. RNAIII from these CoNS turned out to be very similar to that of
S. aureus
and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5′ half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficient
S. aureus
mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of
S. aureus
RNAIII, suggesting that these regulatory functions are independent. By creating
S. epidermidis-S. aureus
RNAIII hybrids, we could also show that both the 5′ and 3′ halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in
S. aureus.