Production and secretion stress caused by overexpression of heterologous alpha-amylase leads to inhibition of sporulation and a prolonged motile phase 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 alpha-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 (sigmaD-dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the alpha-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 sigmaD-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 alpha-amylase overproduction.

FIG. 1.

FIVA analysis of interaction and GO (gene ontology) categories of the transcriptome data. Genes from the DNA macroarray data set were divided into up- and downregulated clusters [comparison of gene expression ratios between B. subtilis 168(pKTH10)/B. subtilis 168]. “Interaction” (A) and GO (B) categories are shown. The size of each cluster is presented in blue underneath the cluster name. Numbers in rectangles represent how many genes were up- or downregulated per cluster in each category. The colors of square boxes depict the significance of gene enrichment per category as defined in the key. Detailed information on significance analyses is available in Blom et al. (4).

FIG. 2.

Binding of CssR-His₆ to the indicated promoter regions. Gel retardation reactions were performed with radiolabeled DNA fragments prepared by PCR and end labeled with ³²P. Promoter regions were incubated with increasing concentrations of purified CssR-His₆ (see Materials and Methods), ranging from 0.125 μM to 1 μM of the protein. In each panel lane X corresponds to the reaction with no protein added.

FIG. 3.

Single-cell analysis of sporulation- and motility-specific gene expression. Strains were grown in TY medium at 37°C with shaking. Samples for flow-cytometric analysis were taken every hour during growth. Two time points are represented, T2 and T3, which correspond to 2 and 3 hours after the entry into stationary phase, respectively. The numbers of cells measured are indicated on the y axis, and their relative fluorescence levels are indicated on the x axis. (A and B) Strains IIA/E (P_spoIIA-gfp), IIA/Q (AmyQ overproduction, dark gray line), and IIA/QAla (AmyQ-Ala overproduction). (C and D) Strains hag/E (P_hag-gfp), hag/Q (AmyQ overproduction), and hag/QAla (AmyQ-Ala overproduction).

FIG. 4.

Activity of secreted amylase from B. subtilis 168 wild-type (wt) and sporulation- and motility-deficient strains. Amylase activity was quantified in the culture medium of the wild-type strain, the spo0A mutant, and the sigD mutant. All strains were transformed with the pKTHM10 plasmid (AmyQ overproduction), and levels of activity of secreted AmyQ were determined at different time points of growth by using the EnzCheck Ultra amylase assay kit as described in Materials and Methods. T0 corresponds to the transition phase of growth. Error bars represent standard errors over three independent biological replicates.