scholarly journals Functional Roles of the Conserved Glu304 Loop of Bacillus subtilis Glutamine Synthetase

2010 ◽  
Vol 192 (19) ◽  
pp. 5018-5025 ◽  
Author(s):  
Lewis V. Wray ◽  
Susan H. Fisher
Keyword(s):  
Bacillus Subtilis ◽  
Glutamine Synthetase ◽  
Enzymatic Activity ◽  
Feedback Inhibition ◽  
Constitutive Expression ◽  
Methionine Sulfoximine ◽  
Wild Type ◽  
Functional Roles ◽  
Glutamate Binding ◽  
High Level

ABSTRACT The enzymatic activity of Bacillus subtilis glutamine synthetase (GS), which catalyzes the synthesis of glutamine from ammonium and glutamate, is regulated by glutamine feedback inhibition. The feedback-inhibited form of B. subtilis GS regulates the DNA-binding activities of the TnrA and GlnR nitrogen transcriptional factors. Bacterial GS proteins contain a flexible seven-residue loop, the Glu304 flap, that closes over the glutamate entrance to the active site. Amino acid substitutions in Glu304 flap residues were examined for their effects on gene regulation, enzymatic activity, and feedback inhibition. Substitutions in five of the Glu304 loop residues resulted in constitutive expression of both TnrA- and GlnR-regulated genes, indicating that this flap is important for regulating the activity of these transcription factors. The residues in the highly conserved Glu304 flap appear to be optimized for glutamate binding because mutant enzymes with substitutions in five of the flap residues had increased glutamate Km values compared to that for wild-type GS. The E304A and E304D substitutions increased the ammonium Km values compared to that for wild-type GS and conferred high-level resistance to inhibition by glutamine, glycine, and methionine sulfoximine. A model for the role of the Glu304 residue in glutamine feedback inhibition is proposed.

scholarly journals Feedback-Resistant Mutations in Bacillus subtilis Glutamine Synthetase Are Clustered in the Active Site

2006 ◽  
Vol 188 (16) ◽  
pp. 5966-5974 ◽  
Author(s):  
Susan H. Fisher ◽  
Lewis V. Wray
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Glutamine Synthetase ◽  
Active Site ◽  
Feedback Inhibition ◽  
Constitutive Expression ◽  
Methionine Sulfoximine ◽  
Mobility Shift ◽  
Mutant Proteins

ABSTRACT The feedback-inhibited form of Bacillus subtilis glutamine synthetase regulates the activity of the TnrA transcription factor through a protein-protein interaction that prevents TnrA from binding to DNA. Five mutants containing feedback-resistant glutamine synthetases (E65G, S66P, M68I, H195Y, and P318S) were isolated by screening for colonies capable of cross-feeding Gln− cells. In vitro enzymatic assays revealed that the mutant enzymes had increased resistance to inhibition by glutamine, AMP, and methionine sulfoximine. The mutant proteins had a variety of enzymatic alterations that included changes in the levels of enzymatic activity and in substrate Km values. Constitutive expression of TnrA- and GlnR-regulated genes was seen in all five mutants. In gel mobility shift assays, the E65G and S66P enzymes were unable to inhibit TnrA DNA binding, while the other three mutant proteins (M68I, H195Y, and P318S) showed partial inhibition of TnrA DNA binding. A homology model of B. subtilis glutamine synthetase revealed that the five mutated amino acid residues are located in the enzyme active site. These observations are consistent with the hypothesis that glutamine and AMP bind at the active site to bring about feedback inhibition of glutamine synthetase.

scholarly journals Novel trans-Acting Bacillus subtilis glnA Mutations That Derepress glnRA Expression

2009 ◽  
Vol 191 (8) ◽  
pp. 2485-2492 ◽  
Author(s):  
Susan H. Fisher ◽  
Lewis V. Wray
Keyword(s):  
Bacillus Subtilis ◽  
Transcription Factors ◽  
Dna Binding ◽  
Glutamine Synthetase ◽  
Protein Interactions ◽  
Feedback Inhibition ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Enzymatic Assays

ABSTRACT Bacillus subtilis contains two nitrogen transcription factors, GlnR and TnrA. The activities of GlnR and TnrA are regulated by direct protein-protein interactions with the feedback-inhibited form of glutamine synthetase (GS). To look for other factors involved in regulating GlnR activity, we isolated mutants with constitutive glnRA expression (GlnC). The twenty-seven GlnC mutants isolated in this mutant screen all contained mutations tightly linked to the glnRA operon which encodes GlnR (glnR) and GS (glnA). Four GlnC mutants contained mutations in the glnR gene that most likely impair the ability of GlnR to bind DNA. Three other GlnC mutants contained novel glnA mutations (S55F, V173I, and L174F). GlnR regulation was completely relieved in the three glnA mutants, while only modest defects in TnrA regulation were observed. In vitro enzymatic assays showed that the purified S55F mutant enzyme was catalytically defective while the V173I and L174F enzymes were highly resistant to feedback inhibition. The V173I and L174F GS proteins were found to require higher glutamine concentrations than the wild-type GS to regulate the DNA-binding activities of GlnR and TnrA in vitro. These results are consistent with a model where feedback-inhibited GS is the only cellular factor involved in regulating the activity of GlnR in B. subtilis.

scholarly journals Production and Secretion Stress Caused by Overexpression of Heterologous α-Amylase Leads to Inhibition of Sporulation and a Prolonged Motile Phase in Bacillus subtilis

2007 ◽  
Vol 73 (16) ◽  
pp. 5354-5362 ◽  
Author(s):  
Andrzej T. Lulko ◽  
Jan-Willem Veening ◽  
Girbe Buist ◽  
Wiep Klaas Smits ◽  
Evert Jan Blom ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Secretion ◽  
Dependent Manner ◽  
Wild Type ◽  
Positive Bacterium ◽  
Expression Levels ◽  
High Affinity ◽  
Flow Cytometric ◽  
Secretion Stress ◽  
High Level

ABSTRACT Transcriptome analysis was used to investigate the global stress response of the gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon, which responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example, citM, ylxF, yloA, ykoJ, and several genes of the flgB operon. However, high-affinity CssR binding was observed only for htrA, htrB, and, possibly, citM. In addition, the DNA macroarray approach revealed that several genes of the sporulation pathway are downregulated by AmyQ overexpression and that a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the α-amylase, the process of sporulation is severely inhibited. Similar experiments were performed to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction.

scholarly journals Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis

2005 ◽  
Vol 71 (4) ◽  
pp. 1899-1908 ◽  
Author(s):  
Helga Westers ◽  
Peter G. Braun ◽  
Lidia Westers ◽  
Haike Antelmann ◽  
Michael Hecker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protease Inhibitor ◽  
Bactericidal Activity ◽  
Industrial Applications ◽  
High Potential ◽  
Proteolytic Degradation ◽  
Wild Type ◽  
Factor Production ◽  
Protease Deficient ◽  
High Level

ABSTRACT Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.

scholarly journals Identification of the RsmG Methyltransferase Target as 16S rRNA Nucleotide G527 and Characterization of Bacillus subtilis rsmG Mutants

2007 ◽  
Vol 189 (16) ◽  
pp. 6068-6073 ◽  
Author(s):  
Kenji Nishimura ◽  
Shanna K. Johansen ◽  
Takashi Inaoka ◽  
Takeshi Hosaka ◽  
Shinji Tokuyama ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
16S Rrna ◽  
Type Strain ◽  
Wild Type Strain ◽  
Culture Conditions ◽  
Wild Type ◽  
Mutation Pattern ◽  
Elevated Frequency ◽  
Ribosomal Protein S12 ◽  
High Level

ABSTRACT The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10−6. Importantly, in the rsmG mutant background, high-level-streptomycin-resistant rpsL (encoding ribosomal protein S12) mutants emerged at a frequency 200 times greater than that seen for the wild-type strain. This elevated frequency in the emergence of high-level streptomycin resistance was facilitated by a mutation pattern in rpsL more varied than that obtained by selection of the wild-type strain.

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

2002 ◽  
Vol 184 (10) ◽  
pp. 2780-2788 ◽  
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.

scholarly journals Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168

2009 ◽  
Vol 191 (15) ◽  
pp. 4835-4844 ◽  
Author(s):  
Jae-Hoon Shim ◽  
Jong-Tae Park ◽  
Jung-Sun Hong ◽  
Ki Woo Kim ◽  
Myo-Jeong Kim ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Glycogen Metabolism ◽  
Outer Side ◽  
Wild Type ◽  
Amylolytic Enzymes ◽  
In The Wild ◽  
Maltogenic Amylase ◽  
Molecular Masses ◽  
Average Molecular Masses ◽  
High Level

ABSTRACT The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and β-cyclodextrin (β-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and β-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched β-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the α-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.

scholarly journals Null Mutants of the Neurospora Actin-related Protein 1 Pointed-End Complex Show Distinct Phenotypes

2001 ◽  
Vol 12 (7) ◽  
pp. 2195-2206 ◽  
Author(s):  
In Hyung Lee ◽  
Santosh Kumar ◽  
Michael Plamann
Keyword(s):  
Atpase Activity ◽  
Cytoplasmic Dynein ◽  
Related Protein ◽  
Wild Type ◽  
Functional Roles ◽  
Distinct Phenotype ◽  
High Level ◽  
Pointed End

Dynactin is a multisubunit complex that regulates the activities of cytoplasmic dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end–binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Δro-2, Δro-7, and Δro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level dynein ATPase activity, and we find that all three mutants have low dynein ATPase activity. Surprisingly, Δro-12 differs from Δro-2 and Δro-7 and other previously characterized dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct dynein/dynactin localization pattern. All three mutants also show stronger dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.

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