Characterization of the sporulation control protein SsgA by use of an efficient method to create and screen random mutant libraries in streptomycetes

Abstract

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function.

FIG. 1.

A schematic overview of cloning and propagation in E. coli of the plasmid library of ssgA variants.

FIG. 2.

(A) Typical example of an SFM plate containing a replicate of transformants from deep-frozen 96-deep-well MTPs. The colony at position A1 is the control (GSA3 containing pHJL401 without the insert), displaying the white, nonsporulating phenotype characteristic of the ssgA null mutant. All other colonies represent different GSA3 transformants of the library. Several phenotypes are observed: nonsporulating phenotypes (no complementation) such as those in colonies in positions A4 and E1, sporulating (full complementation) phenotypes such as those in colonies in positions A5 and B2, and intermediate phenotypes (partial complementation) such as those in colonies in positions B6 and H2. (B) PCR products for DNA sequencing. An initial PCR was done using a few microliters of deep-frozen suspensions of individual mutants as the template. The amounts of product formed in different reactions were highly variable (upper panel). A second PCR was performed using products of the first PCR as the DNA template, resulting in saturated amounts for all samples (lower panel).

FIG. 3.

Amino acid sequence of SsgA. The theoretical secondary structure is shown; barrels indicate predicted α-helices, and arrows indicate predicted β-sheets. Residues not subjected to mutagenesis in this study (and hence invariably wild type) are shown in lower case. Residues 100% conserved (identical or similar) among all 24 known Streptomyces SALPs (see the text for further explanation) are underlined. Identical amino acids are further highlighted in bold. Bullets above the sequence indicate single amino acid substitutions causing loss of function (Table 3). Asterisks above the sequence indicate amino acids which obtained an importance score of 80% or higher from this study (see Results and Fig. 4). Amino acid numbering is shown below the primary sequence (numbers correspond to the amino acid above the first digit in each number).

FIG. 4.

Graph showing the importance scores (gray bars) and specificity scores (black bars) in percentages on the y axis. The amino acid sequence of SsgA is shown along the x axis. The SsgA importance score represents the frequency at which a certain mutation occurs in a nonfunctional ssgA clone, where 100% would indicate that an amino acid is essential for SsgA function. By dividing the importance score by the conservation value, amino acids are identified as primarily important for the function of SsgA and less important for the other SALPs. A score of 100% would indicate that an amino acid is essential for SsgA function and unique for the SsgA protein. Data sets below the statistical threshold (specific residues mutated in fewer than four clones) were not included in the analysis.