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 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 UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity

2020 ◽  
Vol 21 (5) ◽  
pp. 1783
Author(s):  
Junyan Lin ◽  
Yongxing Ai ◽  
Hongda Zhou ◽  
Yan Lv ◽  
Menghan Wang ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Protease Inhibitor ◽  
High Throughput Screening ◽  
Marek's Disease ◽  
Marek’S Disease ◽  
Wild Type ◽  
Ubiquitin Chain ◽  
Protein Ubiquitination ◽  
High Level

(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the Marek’s disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480)H234A and UL36(480)D232A, but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.

scholarly journals Development and Characterization of a Subtilin-Regulated Expression System in Bacillus subtilis: Strict Control of Gene Expression by Addition of Subtilin

2005 ◽  
Vol 71 (12) ◽  
pp. 8818-8824 ◽  
Author(s):  
Roger S. Bongers ◽  
Jan-Willem Veening ◽  
Maarten Van Wieringen ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Industrial Applications ◽  
Wild Type ◽  
Control Of Gene Expression ◽  
Regulated Gene Expression

ABSTRACT A system for subtilin-regulated gene expression (SURE) in Bacillus subtilis that is based on the regulatory module involved in cell-density-dependent control of the production of subtilin is described. An integration vector for introduction of the essential sensor-regulator couple spaRK into the amyE locus of the B. subtilis chromosome and a B. subtilis 168-derived production host in which the spaRK genes were functionally introduced were constructed. Furthermore, several expression plasmids harboring the subtilin-inducible wild-type spaS promoter or a mutated derivative of this promoter were constructed, which facilitated both transcriptional and translational promoter-gene fusions. Functional characterization of both spaS promoters and the cognate expression host could be performed by controlled overproduction of the β-glucuronidase (GusA) and green fluorescent protein (GFP) reporters. Both spaS promoters exhibited very low levels of basal expression, while extremely high levels of expression were observed upon induction with subtilin. Moreover, the level of expression depended directly on the amount of inducer (subtilin) used. The wild-type spaS promoter appeared to be more strictly controlled by the addition of subtilin, while the highest levels of expression were obtained when the mutated spaS promoter was used. Induction by subtilin led to 110- and 80-fold increases in GusA activity for the spaS promoter and its mutant derivative, respectively. Since the SURE system has attractive functional characteristics, including promoter silence under noninducing conditions and a controlled and high level of expression upon induction, and since it is not subject to catabolite control, we anticipate that it can provide a suitable expression system for various scientific and industrial applications.

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 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 SsrA-Mediated Tagging in Bacillus subtilis

2001 ◽  
Vol 183 (13) ◽  
pp. 3885-3889 ◽  
Author(s):  
Thomas Wiegert ◽  
Wolfgang Schumann
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Detection System ◽  
General Mechanism ◽  
Transcriptional Terminator ◽  
Heat Stable ◽  
Gene Coding ◽  
Protease Deficient ◽  
High Level ◽  
Terminator Sequence

ABSTRACT A general mechanism in bacteria to rescue stalled ribosomes involves a stable RNA encoded by the ssrA gene. This RNA, termed tmRNA, encodes a proteolytic peptide tag which is cotranslationally added to truncated polypeptides, thereby targeting them for rapid proteolysis. To study this ssrA-mediated mechanism in Bacillus subtilis, a bipartite detection system was constructed that was composed of the HrcA transcriptional repressor and the bgaB reporter gene coding for a heat-stable β-galactosidase fused to an HrcA-controlled promoter. After the predicted proteolysis tag was fused to HrcA, the reporter β-galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA. Replacement of the two C-terminal alanine residues of the tag by aspartate rendered the repressor stable. Replacement of the hrcA stop codon by a transcriptional terminator sequence rendered the protein unstable; this was caused bytrans translational addition of the proteolytic tag. Inactivating the B. subtilis ssrA or smpB(yvaI) gene prevented the trans translational tagging reaction. Various protease-deficient strains of B. subtilis were tested for proteolysis of tagged HrcA. HrcA remained stable only in clpX or clpP knockouts, which suggests that this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in B. subtilis.

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 High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800

2005 ◽  
Vol 71 (7) ◽  
pp. 4101-4103 ◽  
Author(s):  
Xiao-Zhou Zhang ◽  
Zhong-Li Cui ◽  
Qing Hong ◽  
Shun-Peng Li
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Methyl Parathion ◽  
High Level Expression ◽  
Methyl Parathion Hydrolase ◽  
Parathion Hydrolase ◽  
Encoding Gene ◽  
Protease Deficient ◽  
High Level

ABSTRACT The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

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