Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques

PROTEOMICS ◽  
2008 ◽  
Vol 8 (10) ◽  
pp. 2062-2076 ◽  
Author(s):  
Annette Dreisbach ◽  
Andreas Otto ◽  
Dörte Becher ◽  
Elke Hammer ◽  
Alexander Teumer ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Membrane Proteome ◽  
In Vivo Labeling

scholarly journals The PrpC Serine-Threonine Phosphatase and PrkC Kinase Have Opposing Physiological Roles in Stationary-Phase Bacillus subtilis Cells

2002 ◽  
Vol 184 (22) ◽  
pp. 6109-6114 ◽  
Author(s):  
Tatiana A. Gaidenko ◽  
Tae-Jong Kim ◽  
Chester W. Price
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Cell Viability ◽  
Cell Density ◽  
Elongation Factor ◽  
Pleiotropic Effects ◽  
General Stress ◽  
Factor G

ABSTRACT Loss of the PrpC serine-threonine phosphatase and the associated PrkC kinase of Bacillus subtilis were shown to have opposite effects on stationary-phase physiology by differentially affecting cell density, cell viability, and accumulation of β-galactosidase from a general stress reporter fusion. These pleiotropic effects suggest that PrpC and PrkC have important regulatory roles in stationary-phase cells. Elongation factor G (EF-G) was identified as one possible target of the PrpC and PrkC pair in vivo, and purified PrpC and PrkC manifested the predicted phosphatase and kinase activities against EF-G in vitro.

scholarly journals Direct Regulation of Bacillus subtilis phoPR Transcription by Transition State Regulator ScoC

2010 ◽  
Vol 192 (12) ◽  
pp. 3103-3113 ◽  
Author(s):  
Bindiya Kaushal ◽  
Salbi Paul ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Transition State ◽  
Stationary Phase ◽  
Promoter Region ◽  
Regulatory Protein ◽  
Cellular Growth ◽  
Loss Of Function ◽  
Wild Type ◽  
Promoter Fusion

ABSTRACT Induction of the Pho response in Bacillus subtilis occurs when the Pi concentrations in the growth medium fall below 0.1 mM, a condition which results in slowed cellular growth followed by entry into stationary phase. The phoPR promoter region contains three σA-responsive promoters; only promoter PA4 is PhoP autoregulated. Expression of the phoPR operon is postexponential, suggesting the possibility of a repressor role for a transition-state-regulatory protein(s). Expression of a phoPR promoter-lacZ fusion in a scoC loss-of-function mutant strain grown in low-phosphate defined medium was significantly higher than expression in the wild-type strain during exponential growth or stationary phase. Derepression in the scoC strain from a phoP promoter fusion containing a mutation in the CcpA binding site (cre1) was further elevated approximately 1.4-fold, indicating that the repressor effects of ScoC and CcpA on phoP expression were cumulative. DNase I footprinting showed protection of putative binding sites by ScoC, which included the −10 and/or −35 elements of five (PB1, PE2, PA3, PA4, and PA6) of the six promoters within the phoPR promoter region. PA6 was expressed in vivo from the phoP cre1 promoter fusion in both wild-type and scoC strains. Evidence for ScoC repression in vivo was shown by primer extension for PA4 and PA3 from the wild-type promoter and for PA4 and PE2 from the phoP cre1 promoter. The latter may reflect ScoC repression of sporulation that indirectly affects phoPR transcription. ScoC was shown to repress PA6, PA4, PE2, and PB1 in vitro.

scholarly journals Two ResD-Controlled Promoters RegulatectaA Expression in Bacillus subtilis

2001 ◽  
Vol 183 (10) ◽  
pp. 3237-3246 ◽  
Author(s):  
Salbi Paul ◽  
Xiaohui Zhang ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Binding Site ◽  
Binding Sites ◽  
Promoter Activity ◽  
In Vitro Transcription ◽  
Promoter Deletion Analysis ◽  
Transcription Assays

ABSTRACT The Bacillus subtilis ResDE two-component system plays a positive role in global regulation of genes involved in aerobic and anaerobic respiration. ctaA is one of the several genes involved in aerobic respiration that requires ResD for in vivo expression. The ctaAB-divergent promoter regulatory region has three ResD binding sites; A1, A2, and A3. The A2 site is essential for in vivo promoter activity, while binding sites A2 and A3 are required for full ctaA promoter activity. In this study, we demonstrate the role of ResD∼P in the activation of thectaA promoter using an in vitro transcription system. The results indicate that the ctaA promoter (binding sites A2 and A3) has two transcriptional start sites. Binding site A2 was sufficient for weak transcription of the upstream promoter (Pv) by EςA, transcription which was enhanced approximately 1.5-fold by ResD and 5-fold by ResD∼P. The downstream promoter (Ps) required both binding sites A2 and A3 and was not transcribed by EςA with or without ResD∼P. RNA polymerase (RNAP) isolated from B. subtilis when cells were at the end of exponential growth (T0) or 3, 4, or 5 h into the stationary phase (T3, T4, or T 5, respectively) was used in in vitro transcription assays. Maximal transcription from Ps required T4 RNAP plus ResD∼P. RNAP isolated from a spo0A or a sigE mutant strain was not capable of Ps transcription. Comparison of the Ps promoter sequence with the SigE binding consensus suggests that thectaA Ps promoter may be a SigE promoter. The collective data from ResD footprinting, in vivo promoter deletion analysis, and in vitro transcription assays suggest that ctaA is transcribed during late exponential to early stationary phases of growth from the Pv promoter, which requires ResD binding site A2, EςA, and ResD∼P, and during later stationary phase from Ps, which requires binding sites A2 and A3, ResD∼P, and EςE or a sigma factor whose transcription is dependent on SigE.

Bioavailability of Vitamin B12

2010 ◽  
Vol 80 (45) ◽  
pp. 330-335 ◽  
Author(s):  
Lindsay Helen Allen
Keyword(s):  
Vitamin B12 ◽  
Vitamin B12 Deficiency ◽  
The Elderly ◽  
Radioactive Isotopes ◽  
Animal Source Foods ◽  
Dietary Requirements ◽  
In Vivo Labeling ◽  
B12 Deficiency ◽  
Vitamin B12 Malabsorption

Vitamin B12 deficiency is common in people of all ages who consume a low intake of animal-source foods, including populations in developing countries. It is also prevalent among the elderly, even in wealthier countries, due to their malabsorption of B12 from food. Several methods have been applied to diagnose vitamin B12 malabsorption, including Schilling’s test, which is now used rarely, but these do not quantify percent bioavailability. Most of the information on B12 bioavailability from foods was collected 40 to 50 years ago, using radioactive isotopes of cobalt to label the corrinoid ring. The data are sparse, and the level of radioactivity required for in vivo labeling of animal tissues can be prohibitive. A newer method under development uses a low dose of radioactivity as 14C-labeled B12, with measurement of the isotope excreted in urine and feces by accelerator mass spectrometry. This test has revealed that the unabsorbed vitamin is degraded in the intestine. The percent bioavailability is inversely proportional to the dose consumed due to saturation of the active absorption process, even within the range of usual intake from foods. This has important implications for the assessment and interpretation of bioavailability values, setting dietary requirements, and interpreting relationships between intake and status of the vitamin.

scholarly journals Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ςS-Dependent Transcription in Pseudomonas putida

2000 ◽  
Vol 182 (23) ◽  
pp. 6707-6713 ◽  
Author(s):  
Eve-Ly Ojangu ◽  
Andres Tover ◽  
Riho Teras ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Deficient Mutant ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Silent Genes ◽  
Rna Polymerase Holoenzyme

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of ςS on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

scholarly journals Non-B DNA-Forming Motifs Promote Mfd-Dependent Stationary-Phase Mutagenesis in Bacillus subtilis

2021 ◽  
Vol 9 (6) ◽  
pp. 1284
Author(s):  
Tatiana Ermi ◽  
Carmen Vallin ◽  
Ana Gabriela Regalado García ◽  
Moises Bravo ◽  
Ismaray Fernandez Cordero ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Genome Instability ◽  
Mutagenic Effect ◽  
Genetic Changes ◽  
Dna Structures ◽  
Coding Regions ◽  
G4 Dna ◽  
Stressed Cells ◽  
Growing Conditions

Transcription-induced mutagenic mechanisms limit genetic changes to times when expression happens and to coding DNA. It has been hypothesized that intrinsic sequences that have the potential to form alternate DNA structures, such as non-B DNA structures, influence these mechanisms. Non-B DNA structures are promoted by transcription and induce genome instability in eukaryotic cells, but their impact in bacterial genomes is less known. Here, we investigated if G4 DNA- and hairpin-forming motifs influence stationary-phase mutagenesis in Bacillus subtilis. We developed a system to measure the influence of non-B DNA on B. subtilis stationary-phase mutagenesis by deleting the wild-type argF at its chromosomal position and introducing IPTG-inducible argF alleles differing in their ability to form hairpin and G4 DNA structures into an ectopic locus. Using this system, we found that sequences predicted to form non-B DNA structures promoted mutagenesis in B. subtilis stationary-phase cells; such a response did not occur in growing conditions. We also found that the transcription-coupled repair factor Mfd promoted mutagenesis at these predicted structures. In summary, we showed that non-B DNA-forming motifs promote genetic instability, particularly in coding regions in stressed cells; therefore, non-B DNA structures may have a spatial and temporal mutagenic effect in bacteria. This study provides insights into mechanisms that prevent or promote mutagenesis and advances our understanding of processes underlying bacterial evolution.

Metabolic in Vivo Labeling Highlights Differences of Metabolically Active Microbes from the Mucosal Gastrointestinal Microbiome between High-Fat and Normal Chow Diet

2017 ◽  
Vol 16 (4) ◽  
pp. 1593-1604 ◽  
Author(s):  
Andreas Oberbach ◽  
Sven-Bastiaan Haange ◽  
Nadine Schlichting ◽  
Marco Heinrich ◽  
Stefanie Lehmann ◽  
...  
Keyword(s):  
Chow Diet ◽  
High Fat ◽  
Gastrointestinal Microbiome ◽  
In Vivo Labeling ◽  
Normal Chow ◽  
Normal Chow Diet

scholarly journals In Bacillus subtilis, the Sirtuin Protein Deacetylase, Encoded by the srtN Gene (Formerly yhdZ), and Functions Encoded by the acuABC Genes Control the Activity of Acetyl Coenzyme A Synthetase

2009 ◽  
Vol 191 (6) ◽  
pp. 1749-1755 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
New Information ◽  
Low Concentrations ◽  
Dependent Protein ◽  
Protein Deacetylase

ABSTRACT This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a NAD+-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the acuA + acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA.

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.

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