scholarly journals Decay of ermC mRNA in a Polynucleotide Phosphorylase Mutant of Bacillus subtilis

1998 ◽  
Vol 180 (22) ◽  
pp. 5968-5977 ◽  
Author(s):  
David H. Bechhofer ◽  
Wei Wang
Keyword(s):  
Bacillus Subtilis ◽  
Rna Structure ◽  
Northern Blot Analysis ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
Gene Coding ◽  
In The Wild ◽  
Rna Fragments ◽  
Specific Mrna

ABSTRACT ermC mRNA decay was examined in a mutant ofBacillus subtilis that has a deleted pnpA gene (coding for polynucleotide phosphorylase). 5′-proximal RNA fragments less than 400 nucleotides in length were abundant in thepnpA strain but barely detectable in the wild type. On the other hand, the patterns of 3′-proximal RNA fragments were similar in the wild-type and pnpA strains. Northern blot analysis with different probes showed that the 5′ end of the decay intermediates was the native ermC 5′ end. For one prominent ermCRNA fragment, in particular, it was shown that formation of its 3′ end was directly related to the presence of a stalled ribosome. 5′-proximal decay intermediates were also detected for transcripts encoded by theyybF gene. These results suggest that PNPase activity, which may be less sensitive to structures or sequences that block exonucleolytic decay, is required for efficient decay of specific mRNA fragments. However, it was shown that even PNPase activity could be blocked in vivo at a particular RNA structure.

scholarly journals Stability of Proteins Out of Service: the GapB Case of Bacillus subtilis

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Ulf Gerth ◽  
Eleonora Krieger ◽  
Daniela Zühlke ◽  
Alexander Reder ◽  
Uwe Völker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Arginine Kinase ◽  
Adaptor Protein ◽  
Glycolytic Enzyme ◽  
Dependent Manner ◽  
Wild Type ◽  
Recognition Mechanism ◽  
Content Type ◽  
In The Wild

ABSTRACT Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type. IMPORTANCE GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for B. subtilis under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in B. subtilis, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.

scholarly journals In Vivo Role of Catalase-Peroxidase inSynechocystis sp. Strain PCC 6803

1999 ◽  
Vol 181 (6) ◽  
pp. 1875-1882 ◽  
Author(s):  
Martin Tichy ◽  
Wim Vermaas
Keyword(s):  
Electron Transport ◽  
Photosynthetic Electron Transport ◽  
Protective Role ◽  
Scavenging Activity ◽  
Wild Type ◽  
Gene Coding ◽  
In The Wild ◽  
Catalase Peroxidase ◽  
Pcc 6803

ABSTRACT The katG gene coding for the only catalase-peroxidase in the cyanobacterium Synechocystis sp. strain PCC 6803 was deleted in this organism. Although the rate of H2O2 decomposition was about 30 times lower in the ΔkatG mutant than in the wild type, the strain had a normal phenotype and its doubling time as well as its resistance to H2O2 and methyl viologen were indistinguishable from those of the wild type. The residual H2O2-scavenging capacity was more than sufficient to deal with the rate of H2O2production by the cell, estimated to be less than 1% of the maximum rate of photosynthetic electron transport in vivo. We propose that catalase-peroxidase has a protective role against environmental H2O2 generated by algae or bacteria in the ecosystem (for example, in mats). This protective role is most apparent at a high cell density of the cyanobacterium. The residual H2O2-scavenging activity in the ΔkatG mutant was a light-dependent peroxidase activity. However, neither glutathione peroxidase nor ascorbate peroxidase accounted for a significant part of this H2O2-scavenging activity. When a small thiol such as dithiothreitol was added to the medium, the rate of H2O2 decomposition in the ΔkatG mutant increased more than 10-fold, indicating that a thiol-specific peroxidase, for which thioredoxin may be the physiological electron donor, is present. Oxidized thioredoxin is likely to be reduced again by photosynthetic electron transport. Therefore, under laboratory conditions, there are only two enzymatic mechanisms for H2O2 decomposition present inSynechocystis sp. strain PCC 6803. One is catalyzed by a catalase-peroxidase, and the other is catalyzed by thiol-specific peroxidase.

scholarly journals MyD88-Dependent Signaling Affects the Development of Meningococcal Sepsis by Nonlipooligosaccharide Ligands

2006 ◽  
Vol 74 (6) ◽  
pp. 3538-3546 ◽  
Author(s):  
Laura Plant ◽  
Hong Wan ◽  
Ann-Beth Jonsson
Keyword(s):  
Inflammatory Responses ◽  
Innate Immune ◽  
Meningococcal Sepsis ◽  
Wild Type ◽  
Microbial Infections ◽  
In The Wild ◽  
Myeloid Differentiation Factor ◽  
Dependent Pathway ◽  
Meningococcal Strain

ABSTRACT The Toll-like receptors (TLRs) and the adaptor myeloid differentiation factor 88 (MyD88) are important in the innate immune defenses of the host to microbial infections. Meningococcal ligands signaling via TLRs control inflammatory responses, and stimulation can result in fulminant meningococcal sepsis. In this study, we show that the responses to nonlipooligosaccharide (non-LOS) ligands of meningococci are MyD88 dependent. An isogenic LOS-deficient mutant of the serogroup C meningococcal strain FAM20 caused fatal disease in wild type C57BL/6 mice that was not observed in MyD88−/− mice. Fatality correlated with high proinflammatory cytokine and C5a levels in serum, high neutrophil numbers in blood, and increased bacteremia at 24 h postinfection in the wild-type mice. Infection with the parent strain FAM20 resulted in fatality in 100% of the wild-type mice and 50% of the MyD88−/− mice. We conclude that both LOS and another neisserial ligand cause meningococcal sepsis in an in vivo mouse model and confirm that meningococcal LOS can act via both the MyD88- dependent and -independent pathways, while the non-LOS meningococcal ligand(s) acts only via the MyD88-dependent pathway.

Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family

2021 ◽  
Vol 85 (2) ◽  
pp. 297-306
Author(s):  
Zui Fujimoto ◽  
Le Thi Thu Hong ◽  
Naomi Kishine ◽  
Nobuhiro Suzuki ◽  
Keitarou Kimura
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
X Ray Crystallography ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Single Amino Acid Mutation

ABSTRACT Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

Effect offurmutation on acid-tolerance response and in vivo virulence of avian septicemicEscherichia coli

2002 ◽  
Vol 48 (5) ◽  
pp. 458-462 ◽  
Author(s):  
Chengru Zhu ◽  
Musangu Ngeleka ◽  
Andrew A Potter ◽  
Brenda J Allan
Keyword(s):  
Parent Strain ◽  
Acid Tolerance ◽  
Wild Type ◽  
Acid Tolerance Response ◽  
E Coli ◽  
Regulator Protein ◽  
In The Wild ◽  
Tolerance Response ◽  
Wild Type Parent

The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria. In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined. The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain. However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation. These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E. coli, as detected by the ability to cause septicemia in our experimental infection.Key words: E. coli, fur, acid-tolerance response.

scholarly journals A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Daniele Peterle ◽  
Giulia Pontarollo ◽  
Stefano Spada ◽  
Paola Brun ◽  
Luana Palazzi ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Human Plasma ◽  
Amyloid Fibrils ◽  
Wild Type ◽  
Senile Systemic Amyloidosis ◽  
Amyloid Deposits ◽  
Gut Mucosa ◽  
Standard Composition ◽  
Human Wild Type

AbstractAggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1–3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59–127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.

scholarly journals Addition of Poly(A) and Heteropolymeric 3′ Ends in Bacillus subtilis Wild-Type and Polynucleotide Phosphorylase-Deficient Strains

2005 ◽  
Vol 187 (14) ◽  
pp. 4698-4706 ◽  
Author(s):  
Juan Campos-Guillén ◽  
Patricia Bralley ◽  
George H. Jones ◽  
David H. Bechhofer ◽  
Gabriela Olmedo-Alvarez
Keyword(s):  
Bacillus Subtilis ◽  
Bacterial Species ◽  
Synthetic Activity ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
Mutant Strains ◽  
Mean Size ◽  
Identical Nucleotide ◽  
End Sequences ◽  
Polymerase I

ABSTRACT Polyadenylation plays a role in decay of some bacterial mRNAs, as well as in the quality control of stable RNA. In Escherichia coli, poly(A) polymerase I (PAP I) is the main polyadenylating enzyme, but the addition of 3′ tails also occurs in the absence of PAP I via the synthetic activity of polynucleotide phosphorylase (PNPase). The nature of 3′-tail addition in Bacillus subtilis, which lacks an identifiable PAP I homologue, was studied. Sizing of poly(A) sequences revealed a similar pattern in wild-type and PNPase-deficient strains. Sequencing of 152 cloned cDNAs, representing 3′-end sequences of nontranslated and translated RNAs, revealed modified ends mostly on incomplete transcripts, which are likely to be decay intermediates. The 3′-end additions consisted of either short poly(A) sequences or longer heteropolymeric ends with a mean size of about 40 nucleotides. Interestingly, multiple independent clones exhibited complex heteropolymeric ends of very similar but not identical nucleotide sequences. Similar polyadenylated and heteropolymeric ends were observed at 3′ ends of RNA isolated from wild-type and pnpA mutant strains. These data demonstrated that, unlike the case of some other bacterial species and chloroplasts, PNPase of Bacillus subtilis is not the major enzyme responsible for the addition of nucleotides to RNA 3′ ends.

scholarly journals Replication Properties of Clade A/C Chimeric Feline Immunodeficiency Viruses and Evaluation of Infection Kinetics in the Domestic Cat

2008 ◽  
Vol 82 (16) ◽  
pp. 7953-7963 ◽  
Author(s):  
Sohela de Rozìeres ◽  
Jesse Thompson ◽  
Magnus Sundstrom ◽  
Julia Gruber ◽  
Debora S. Stump ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Domestic Cat ◽  
Fine Tuning ◽  
Surface Glycoprotein ◽  
Wild Type ◽  
Coding Region ◽  
Mild Disease ◽  
Clade C ◽  
In The Wild

ABSTRACT Feline immunodeficiency virus (FIV) causes progressive immunodeficiency in domestic cats, with clinical course dependent on virus strain. For example, clade A FIV-PPR is predominantly neurotropic and causes a mild disease in the periphery, whereas clade C FIV-C36 causes fulminant disease with CD4+ T-cell depletion and neutropenia but no significant pathology in the central nervous system. In order to map pathogenic determinants, chimeric viruses were prepared between FIV-C36 and FIV-PPR, with reciprocal exchanges involving (i) the 3′ halves of the viruses, including the Vif, OrfA, and Env genes; (ii) the 5′ end extending from the 5′ long terminal repeat (LTR) to the beginning of the capsid (CA)-coding region; and (iii) the 3′ LTR and Rev2-coding regions. Ex vivo replication rates and in vivo replication and pathologies were then assessed and compared to those of the parental viruses. The results show that FIV-C36 replicates ex vivo and in vivo to levels approximately 20-fold greater than those of FIV-PPR. None of the chimeric FIVs recapitulated the replication rate of FIV-C36, although most replicated to levels similar to those of FIV-PPR. The rates of chloramphenicol acetyltransferase gene transcription driven by the FIV-C36 and FIV-PPR LTRs were identical. Furthermore, the ratios of surface glycoprotein (SU) to capsid protein (CA) in the released particles were essentially the same in the wild-type and chimeric FIVs. Tests were performed in vivo on the wild-type FIVs and chimeras carrying the 3′ half of FIV-C36 or the 3′ LTR and Rev2 regions of FIV-C36 on the PPR background. Both chimeras were infectious in vivo, although replication levels were lower than for the parental viruses. The chimera carrying the 3′ half of FIV-C36 demonstrated an intermediate disease course with a delayed peak viral load but ultimately resulted in significant reductions in neutrophil and CD4+ T cells, suggesting potential adaptation in vivo. Taken together, the findings suggest that the rapid-growth phenotype and pathogenicity of FIV-C36 are the result of evolutionary fine tuning throughout the viral genome, rather than being properties of any one constituent.

Influence of double-stranded RNAs on growth, sporulation, pathogenicity, and survival of Chalara elegans

1995 ◽  
Vol 73 (7) ◽  
pp. 1001-1009 ◽  
Author(s):  
Zamir K. Punja
Keyword(s):  
Plant Pathogen ◽  
Dry Weight ◽  
Complete Loss ◽  
Thielaviopsis Basicola ◽  
Wild Type ◽  
C Elegans ◽  
Chalara Elegans ◽  
In The Wild ◽  
Rna Fragments ◽  
Type Strains

Three strains of Chalara elegans from diverse geographical areas that contained multiple (4 or 5) double-stranded RNA fragments were compared with spontaneously derived cultures from these strains that were either partially cured or completely free of dsRNA. In the wild-type strains, presence of the dsRNAs was found to significantly enhance phialospore production and pigmentation of colonies, whereas radial growth and mycelial dry weight accumulation were reduced. The rate and overall percentage of phialospore germination on 1% Noble water agar were also significantly reduced by the presence of the dsRNAs. In two partially cured strains (only one 2.8-kb fragment remaining), pathogenicity to various plant tissues was significantly enhanced when compared with the wild-type strains containing multiple dsRNA. However, survival in field soil was enhanced in one strain and reduced in the other. In the completely cured strain, the loss of multiple dsRNA fragments was associated with enhanced growth, reduced phialospore production, and a complete loss of pathogenicity and capability for survival in soil. These results indicate that the effects of dsRNAs in C. elegans vary with the strain. In general, the presence of multiple dsRNAs in this fungus enhanced sporulation, altered colony morphology, and reduced growth and pathogenicity. However, since the complete loss of dsRNA was found to eliminate pathogenicity and reduce survival, it suggests that some dsRNA fragments in C. elegans may confer an advantage to this soil-borne facultative plant pathogen. Key words: black root rot, soil-borne plant pathogen, Thielaviopsis basicola.

scholarly journals Pyruvate Formate Lyase Is Required for Pneumococcal Fermentative Metabolism and Virulence

2009 ◽  
Vol 77 (12) ◽  
pp. 5418-5427 ◽  
Author(s):  
Hasan Yesilkaya ◽  
Francesca Spissu ◽  
Sandra M. Carvalho ◽  
Vanessa S. Terra ◽  
Karen A. Homer ◽  
...  
Keyword(s):  
Wild Type ◽  
Product Analysis ◽  
Acid Fermentation ◽  
Pyruvate Formate Lyase ◽  
Growth And Survival ◽  
Mixed Acid Fermentation ◽  
Mixed Acid ◽  
Fermentative Metabolism ◽  
In The Wild

ABSTRACT Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pfl A genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.

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