Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides

Abstract

With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of "whole genome writing" in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF) of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in M. mycoides.

Keywords: complementation; minimal cell; nusG; synthetic cell; transposome.

Figure 1

Diagram depicting the genomic region, deletion and complementation fragments examined in this study. The genomic region contains nine genes indicated with arrows: (1) MMSYN1_0839, (2) nusG, and (3–9) MMSYN1_0841- MMSYN1_0847. The orientation of each gene is indicated (right-pointing arrow denotes a clock-wise direction in the genome). The deletion removes seven genes shown in green (genes 2–8), starting with the initiator codon of gene 2 and ending with the stop codon of gene 8. Complementation fragment 1 includes gene 1, gene 2 and their corresponding upstream and downstream intergenic regions. This complementation fragment was used in a marker-less approach (see Figure 2). Complementation fragment 2 includes only the ORF of gene 2. This fragment was combined with an expression vector and used in a “marker-driven” approach.

Figure 2

Designer transposons used in a marker-less approach (A) and a marker-driven approach (B). Asterisk denotes a 19-bp mosaic end recognized by Tn5 transposon. In (A), a query fragment can be generated using polymerase chain reaction or prepared as a synthetic DNA fragment. In (B), a query ORF is cloned downstream of the pac puromycin resistance marker. The start codon of the query ORF immediately follows the stop codon of the pac gene (see Supplementary Figure 2). This ORF can be amplified from a template or prepared as a synthetic DNA block. The tuf promoter that drives the expression of the pac gene also drives the expression of the query ORF. T denotes transcriptional terminator.

Figure 3

Flow charts of procedures for complementation of mycoplasma mutants using the marker-less approach and the marker-driven approach.

Figure 4

Complementation using the marker-less approach. (A) Transformed mycoplasma cells were grown without any antibiotic selection in liquid culture through three serial passages with the dilution levels and culture durations indicated. After a new culture was started, the previous culture was stored at 4°C. (B) After the fourth culture, cells from each of the four successive cultures were diluted and plated on agar medium (also not containing any antibiotic) with X-gal to visualize the colonies. The enrichment of fast-growing colonies in later cultures suggests that the transformed and complemented cells overtook the population during later cultures.

Figure 5

Restoration of the replication rate of JCVI-syn1.0 ΔL by inserting complementation fragment 1 (containing nusG) via the marker-less approach. The line for JCVI-syn1.0 (blue) and the line for rescued clone ML1 (green) overlap. Doubling times calculated from PicoGreen fluorescence (RFU) are 62 min for JCVI-syn1.0 (y = 54.732e^0.0112x, R² = 0.9951), 64 min for complemented clone ML1 (y = 58.374e^0.0108x, R² = 0.9951), and 116 min for JCVI-syn1.0 ΔL (y = 31.425e^0.006x, R² = 0.983).

Figure 6

Comparison of a rescued JCVI-syn1.0 ΔL clone with the positive- and negative-control strains for a clone (ML1) obtained using the marker-less approach (A,B) and for a clone (MD1) obtained using the marker-driven approach (C,D). (B) and (D) are magnified views of colonies in (A) and (C), respectively, showing rescued mutant (top) and wild-type JCVI-syn1.0 (bottom) populations.

Figure 7

Colony phenotypes associated with nusG deletion and rescue. (A–C) Individual colonies were photographed 72 h after plating on agar medium from sparse fields where colony growth would be unaffected by neighboring colonies. Colonies represent (A) JCVI-syn1.0, (B) JCVI-syn1.0 ΔL, and (C) complemented clone ML1. (D) represents a field of colonies from the second culture in Figure 4, showing the initial emergence of the faster growing colonies among the more prevalent smaller colonies within the population derived from transformation of JCVI-syn1.0 ΔL using the marker-less approach. Centers of colonies display the typical mycoplasma “fried-egg” characteristic. Blue color is from expression of lacZ in medium containing X-gal (smaller colonies had not yet developed color at the time of assay). Bars represent 500 μm.