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Secretion stress caused by overexpression of heterologous α-amylase in Bacillus subtilis

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 that 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, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented 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. Keywords: secretion stress response

ORGANISM(S): Bacillus subtilis Bacillus subtilis subsp. subtilis str. 168

PROVIDER: GSE8014 | GEO | 2007/10/01

SECONDARY ACCESSION(S): PRJNA100811

REPOSITORIES: GEO

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Project description: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 that 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, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented 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. Secretion stress was applied by overproducing the well-secreted AmyQ α-amylase (pKTH10 vector) from B. amyloliquefaciens. Besides examining secretion stress in wild-type cells, we compared transcriptome profiles of a cssS mutant strain under conditions of high-level AmyQ production. Samples for transcriptome analyses were collected at the late exponential growth stage (one hour before the transition point) and 3 hours upon entry in the stationary growth phase. Three independent cultures of each strain were used and cells were sampled for macroarray experiments. Duplicate spots were averaged in Array-Pro software (Media Cybernetics, Inc.) and the signal was normalized after background subtraction by calculation of the percentage of total signal per gene using Microsoft Excel.

Project description:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host. Samples for transcriptome analyses were induced at the exponential-growth phase (OD600 = 0.7) with 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). Cells were harvested 30 min after induction. Three or four independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.

Project description:Gram-positive bacteria are known to export many proteins to the cell wall and growth medium and, accordingly, many studies have addressed the respective protein export mechanisms. In contrast, very little is known about the subsequent fate of these proteins. The present studies were therefore aimed at determining the fate of native exported proteins in the model organism Bacillus subtilis. Specifically, we employed a GeLC-MS approach to distinguish the roles of the membrane-associated quality control proteases HtrA and HtrB from those of eight other proteases that are present in the cell wall and/or growth medium of B. subtilis. Notably, HtrA and HtrB were previously shown to counteract potentially detrimental 'protein export stresses' upon overproduction of membrane- or secreted proteins. Our results show that many secreted proteins, lipoproteins and membrane proteins of B. subtilis are potential substrates of extracytoplasmic proteases. Moreover, potentially important roles of HtrA and HtrB in the folding of native secreted proteins into a protease-resistant conformation, the liberation of lipoproteins from the membrane-cell wall interface, and the degradation of membrane proteins are uncovered. Altogether, our observations show that HtrA and HtrB are crucial for maintaining the integrity of the B. subtilis cell even under non-stress conditions. Bionformatics pipeline and data processing: MS and MS/MS data were acquired with the LTQ-Orbitrap mass spectrometer. After a survey scan in the Orbitrap (r= 30,000), MS/MS data were recorded for the five most intensive precursor ions in the linear ion trap. Singly charged ions were not taken into account for MS/MS analysis. The lock mass option was enabled throughout the analysis. The mass spectrometric data was then subjected to database searching via Sorcerer-Sequest. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, USA; version v.27, rev. 11). Sequest was set up to search a B. subtilis _target-decoy protein sequence database that included the complete proteome set of _B. subtilis _extracted from UniprotKB release 12.7, and a set of common laboratory contaminants compiled with Bioworks Browser (Thermo Fisher Scientific), assuming the digestion enzyme trypsin. Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a search tolerance of 10 ppm for the overview scans. Oxidation of methionine was specified in Sequest as a variable modification. Scaffold (version Scaffold_3_00_04, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they exceeded Xcorr values of 2.2, 3.5, 3.75 for doubly, triply and quadruply charged ions. A protein was regarded as significantly identified when at least two peptides per protein were identified in at least three of four biological replicates. These criteria resulted in no false positive identifications.

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Secretion stress caused by overexpression of heterologous α-amylase in Bacillus subtilis

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