Metabolic Imbalance and Sporulation in an Isocitrate Dehydrogenase Mutant of Bacillus subtilis

ABSTRACT A Bacillus subtilis mutant with a deletion in thecitC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene (citZ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.

Download Full-text

Acetate metabolism in Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/m78-027 ◽

1978 ◽

Vol 24 (2) ◽

pp. 149-153 ◽

Cited By ~ 5

Author(s):

T. M. Lakshmi ◽

Robert B. Helling

Keyword(s):

Isocitrate Dehydrogenase ◽

Citrate Synthase ◽

Isocitrate Lyase ◽

Glyoxylate Cycle ◽

Acetate Metabolism ◽

Wild Type ◽

Intermediary Metabolites ◽

Lyase Activity ◽

Acetate Medium ◽

The Glyoxylate Cycle

Levels of several intermediary metabolites were measured in cells grown in acetate medium in order to test the hypothesis that the glyoxylate cycle is repressed by phosphoenolpyruvate (PEP). Wild-type cells had less PEP than either isocitrate dehydrogenase – deficient cells (which had greater isocitrate lyase activity than the wild type) or isocitrate dehydrogenase – deficient, citrate synthase – deficient cells (which are poorly inducible). Thus induction of the glyoxylate cycle is more complicated than a simple function of PEP concentration. No correlation between enzyme activity and the level of oxaloacetate, pyruvate, or citrate was found either. Citrate was synthesized in citrate synthase – deficient mutants, possibly via citrate lyase.

Download Full-text

Physiological and genetic characterization of the osmotic stress response in Bacillus subtilis

Canadian Journal of Microbiology ◽

10.1139/m94-022 ◽

1994 ◽

Vol 40 (2) ◽

pp. 140-144 ◽

Cited By ~ 10

Author(s):

Sandra M. Ruzal ◽

Carmen Sanchez-Rivas

Keyword(s):

Bacillus Subtilis ◽

Osmotic Stress ◽

Stationary Phase ◽

Resistant Mutant ◽

Wild Type ◽

Osmotic Stress Response ◽

Mutant Phenotypes ◽

Physiological Variations ◽

Growth Adaptation

Bacillus subtilis cultures submitted to an osmotic upshock (1.5 M NaCl) lysed unless stationary phase had been reached. Several physiological variations were observed, such as delayed growth (adaptation), a filamentous bacterial appearance, RecA-dependent osmoresistance (SOS), and cross-induction by a previous stress (heat shock). Osmoresistance and sporulation seem to share pathways of regulation such as inhibition in the presence of glucose and glutamine and derepression in a catabolite-resistant mutant such as degUh. However, spores were not obtained on hypertonic media. Mutants of later sporulation stages (spoII, spoIII) presented a response similar to that of the wild-type parent, indicating that both processes probably shared early controls. Null mutations in any of the known key modulators of sporulation (spoOA or degU) resulted in similar levels of osmosensitivity. Sensor mutations in kinA and degS also led to strains with altered responses, the kinA mutant being even more osmosensitive than the degS mutant. Several spoOA mutant phenotypes are due to this gene's control of abrB, a regulator of stationary-phase events, and an abrB mutation relieved the osmosensitivity of the spoOA-containing mutant but had no effect on a wild-type strain.Key words: Bacillus subtilis, osmotic stress, sporulation.

Download Full-text

Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

Journal of Bacteriology ◽

10.1128/jb.180.24.6493-6502.1998 ◽

1998 ◽

Vol 180 (24) ◽

pp. 6493-6502 ◽

Cited By ~ 29

Author(s):

Thomas Murray ◽

David L. Popham ◽

Christine B. Pearson ◽

Arthur R. Hand ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Cell Elongation ◽

Binding Protein ◽

Divalent Cations ◽

Cross Linking ◽

Penicillin Binding Protein ◽

Wild Type ◽

Spore Outgrowth ◽

Partial Redundancy

ABSTRACT The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect inpbpA spore outgrowth have shown that (i) outgrowingpbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpAspores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpAspores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.

Download Full-text

The N-terminal domain of the human eIF2β subunit and the CK2 phosphorylation sites are required for its function

Biochemical Journal ◽

10.1042/bj20050605 ◽

2006 ◽

Vol 394 (1) ◽

pp. 227-236 ◽

Cited By ~ 17

Author(s):

Franc Llorens ◽

Anna Duarri ◽

Eduard Sarró ◽

Nerea Roher ◽

Maria Plana ◽

...

Keyword(s):

Protein Kinase ◽

Hela Cells ◽

Mutant Cell ◽

Phosphorylation Site ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Mutant Form ◽

Wild Type ◽

Main Site

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2α evidenced the direct involvement of this protein kinase in eIF2β phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2β or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2β phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2β also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2β S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Δ2–138 N-terminal-truncated form of eIF2β (eIF2β-CT). Mutation at Ser2 and Ser67 did not affect eIF2β integrating into the eIF2 trimer or being able to complex with eIF5 and CK2α. The eIF2β-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2β slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2β S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2β-CT form, being detectable at doses where eIF2β and eIF2β S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2β for its function in mammals.

Download Full-text

Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity

Microbiology ◽

10.1099/mic.0.28345-0 ◽

2006 ◽

Vol 152 (3) ◽

pp. 605-616 ◽

Cited By ~ 85

Author(s):

Claudia S. López ◽

Alejandro F. Alice ◽

Horacio Heras ◽

Emilio A. Rivas ◽

Carmen Sánchez-Rivas

Keyword(s):

Bacillus Subtilis ◽

Stationary Phase ◽

Growth Curves ◽

Membrane Lipid ◽

Wild Type ◽

Lipid Packing ◽

Anionic Phospholipids ◽

Large Unilamellar Vesicles ◽

Mutant Strains ◽

Lag Period

The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1·2 M the growth curves were similar to those of its wild-type control, at 1·5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00089-10 ◽

2010 ◽

Vol 192 (12) ◽

pp. 3103-3113 ◽

Cited By ~ 3

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.

Download Full-text

The E1β and E2 Subunits of the Bacillus subtilis Pyruvate Dehydrogenase Complex Are Involved in Regulation of Sporulation

Journal of Bacteriology ◽

10.1128/jb.184.10.2780-2788.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2780-2788 ◽

Cited By ~ 30

Author(s):

Haichun Gao ◽

Xin Jiang ◽

Kit Pogliano ◽

Arthur I. Aronson

Keyword(s):

Bacillus Subtilis ◽

Enzymatic Activity ◽

Pyruvate Dehydrogenase ◽

Divalent Cations ◽

Pyruvate Dehydrogenase Complex ◽

Pyruvate Decarboxylase ◽

Stage Ii ◽

Wild Type ◽

Wild Type Level ◽

Late Stages

ABSTRACT The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1α and E1β), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH). There are two promoters: one for the entire operon and an internal one in front of the pdhC gene. The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1α and E1β. Disruptions of the pdhB, pdhC, and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful, suggesting that E1α is essential. The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium. The pdhB and pdhC mutants sporulated to only 5% of the wild-type level, whereas the pdhD mutant strain sporulated to 55% of the wild-type level. This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to a function(s) of these subunits independent of enzymatic activity. Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations. Results from the expression of various spo-lacZ fusions revealed that the pdhB mutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas the pdhC mutant was blocked after the completion of engulfment (stage III). This analysis was confirmed by fluorescent membrane staining. The E1β and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation.

Download Full-text

Properties of Spores of Bacillus subtilis Blocked at an Intermediate Stage in Spore Germination

Journal of Bacteriology ◽

10.1128/jb.183.16.4894-4899.2001 ◽

2001 ◽

Vol 183 (16) ◽

pp. 4894-4899 ◽

Cited By ~ 107

Author(s):

Barbara Setlow ◽

Elizabeth Melly ◽

Peter Setlow

Keyword(s):

Enzyme Activity ◽

Bacillus Subtilis ◽

Uv Irradiation ◽

Spore Germination ◽

Dipicolinic Acid ◽

Intermediate Stage ◽

Stage I ◽

Wild Type ◽

Resistance To Heat

ABSTRACT Germination of mutant spores of Bacillus subtilisunable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.

Download Full-text

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Journal of Bacteriology ◽

10.1128/jb.180.17.4555-4563.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4555-4563 ◽

Cited By ~ 45

Author(s):

Thomas Murray ◽

David L. Popham ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Cell Growth ◽

Divalent Cation ◽

Vegetative Growth ◽

Binding Protein ◽

Divalent Cations ◽

Penicillin Binding Protein ◽

Wild Type ◽

Deficient Medium ◽

Spore Outgrowth

ABSTRACT Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg2+ or Ca2+ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg2+ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg2+ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restoreponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg2+ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.

Download Full-text