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. 2024 Oct 3;25(1):922.
doi: 10.1186/s12864-024-10839-5.

Mobilome impacts on physiology in the widely used non-toxic mutant Microcystis aeruginosa PCC 7806 ΔmcyB and toxic wildtype

Gwendolyn F Stark  1 , Alexander R Truchon  1 , Steven W Wilhelm  2
Affiliations

Mobilome impacts on physiology in the widely used non-toxic mutant Microcystis aeruginosa PCC 7806 ΔmcyB and toxic wildtype

Gwendolyn F Stark et al. BMC Genomics. .

Abstract

The Microcystis mobilome is a well-known but understudied component of this bloom-forming cyanobacterium. Through genomic and transcriptomic comparisons, we found five families of transposases that altered the expression of genes in the well-studied toxigenic type-strain, Microcystis aeruginosa PCC 7086, and a non-toxigenic genetic mutant, Microcystis aeruginosa PCC 7806 ΔmcyB. Since its creation in 1997, the ΔmcyB strain has been used in comparative physiology studies against the wildtype strain by research labs throughout the world. Some differences in gene expression between what were thought to be otherwise genetically identical strains have appeared due to insertion events in both intra- and intergenic regions. In our ΔmcyB isolate, a sulfate transporter gene cluster (sbp-cysTWA) showed differential expression from the wildtype, which may have been caused by the insertion of a miniature inverted repeat transposable element (MITE) in the sulfate-binding protein gene (sbp). Differences in growth in sulfate-limited media also were also observed between the two isolates. This paper highlights how Microcystis strains continue to "evolve" in lab conditions and illustrates the importance of insertion sequences / transposable elements in shaping genomic and physiological differences between Microcystis strains thought otherwise identical. This study forces the necessity of knowing the complete genetic background of isolates in comparative physiological experiments, to facilitate the correct conclusions (and caveats) from experiments.

Keywords: Microcystis; Genomic rearrangement; Sulfur uptake; Transposable elements.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
A Chromosome map showing the inversion region, in blue of the Microcystis aeruginosa PCC 7806 genome from our lab relative to an alignment against the mutant Microcystis aeruginosa PCC7806 ΔmcyB mutant genome. Orange regions with arrows indicate the locations of a cluster of 14 identical genes, which occur in 3 locations in the genome.​ ​B Gene arrangement of the repetitive gene cluster that flanks the chromosome repeat regions in the genomes. Dark blue genes are all uncharacterized proteins, with no homology to known proteins. Genes not colored dark blue have homology to known genes/protein domains. Gene 5 has a Cro/C1 HTH-like protein domain. Gene 8 has phage site-specific integrase/tyrosine recombinase protein domains. ​ ​C Gene expression of chromosome repeat region 1 in control (26° C ) (left) and cold (19° C ) (right) growth conditions. ​For average TPM values, n = 2 for the wildtype, and n = 2 for the mutant. D Gene expression of chromosome repeat region 2 in control (26° C) (n = 4) and cold (19° C) (right) growth conditions. ​​For average TPM values, n = 2 for the wildtype, and n = 2 for the mutant. E Gene expression of chromosome repeat region 3 in control (26° C) (left) and cold (19° C) (right) growth conditions. ​For average TPM values, n = 2 for the wildtype, and n = 2 for the mutant
Fig. 2
Fig. 2
A Gene arrangement in ΔmcyB versus the PCC 7806 wildtype genome. In the wildtype (bottom arrangement), an IS1-family transposase is inserted before genes encoding an uncharacterized protein and a MBL-fold metallo-hydrolase protein. In ΔmcyB (top) the IS1-family transposase is absent from the intergenic region before the uncharacterized protein and MBL-fold metallohydrolase. The insertion of an IS1634 family transposase downstream of the MBL-fold metallo-hydrolase in ΔmcyB increases the length of the MBL-fold gene coding region by 80 nt residues. ​B Transcription of the genes in the order shown in Fig. 2A. Absence of the IS1-family transposase in the mutant leads to significantly increased expression (p < 0.001) of the uncharacterized protein and MBL-fold metallo-hydrolase compared to the expression of these genes in the wildtype, which has the IS1 transposase insertion
Fig. 3
Fig. 3
A Gene arrangement in ΔmcyB (top) versus the PCC 7806 wildtype (bottom) genome. In the PCC7806 wildtype, an IS1-family transposase is inserted in the region between genes encoding a tetratricopeptide repeat protein and an efflux RND transporter. B Transcription of the genes in the order shown in Fig. 3A. In the PCC 7806 wildtype, insertion of the IS1-family transposase in the region between genes encoding a tetratricopeptide repeat protein and an efflux RND transporter leads to significantly decreased expression (p < 0.001) of the gene encoding a tetratricopeptide repeat protein and a hypothetical protein. Expression of the efflux RND transporter is significantly different between ΔmcyB and PCC 7806 wildtype at control growth temperature (26° C) (p < 0.001) but not cold (19° C). Differences in expression for the RND transporter are probably not due to the IS1-family transposase, since the two strains have similar expression profiles in the cold. Gene expression data is not shown for the ORF at the end of the RND transporter (white arrow, transcribed on opposite strand) because TPM values were < 6 for both strains and did not appear impacted by the transposase IS1-family
Fig. 4
Fig. 4
A Gene arrangement in PCC 7806 wildtype genome (top) versus the ΔmcyB genome (bottom). In ΔmcyB, an IS1634 family transposase is inserted between genes encoding a diflavin flavoprotein and a putative lipoprotein. In the PCC 7806 wildtype, this region is absent of the transposase. B Transcription of the genes in the order shown in Fig. 4A. In ΔmcyB, insertion of the IS1634 family transposase significantly reduced (p < 0.001) the expression of four genes, which include a putative lipoprotein, an uncharacterized protein (white arrow), a DUF1995- domain containing protein, and a SAM-methyltransferase.
Fig. 5
Fig. 5
A Gene arrangement of the high-affinity sulfate transport gene cluster in Microcystis aeruginosa PCC 7806 wildtype (top) and ΔmcyB (bottom). Genes are labeled, homologous genes in the mutant are the same colors as those seen in the wildtype arrangement. In the mutant, the sulfate/substrate binding protein gene has a 187-nt long insertion, which is denoted by the striped box interrupting the gene. B Average normalized gene expression (normalized by TPM) of the sulfate transporter genes, in order of the gene arrangement seen in Fig. 5A. The open blue circles represent average TPM of ΔmcyB, and black circles are the average TPM of the PCC 7806 wildtype. The mutant has a marked differential expression of all five genes, which show fold changes > 2 at most time points. The wildtype shows very little transcriptional activity of any of these genes (all TPM < 20)
Fig. 6
Fig. 6
A In-silico determination of the substrate binding site conserved residues of the sulfate binding protein gene in the ΔmcyB strain, based on and adapted from the output of NCBI’s conserved domain search [23, 25]. The 187-nt insertion sequence (IS) is located downstream from the substrate binding domain. B Gene expression profile of the sulfate binding protein coding regions upstream and downstream of the MITE insertion sequence in the ΔmcyB strain. The 187-nt long MITE does not appear to affect the expression of the gene downstream of the insertion sequence in the ΔmcyB strain. C Growth assays of the PCC 7806 wildtype and ΔmcyB strain in sulfate replete, or sulfate limited media, grown at 19° C. The mutant ΔmcyB strain appears to have a growth advantage in sulfate limited media when grown at cold temperature compared to the PCC 7806 wildtype. The PCC 7806 wildtype barely grew above the initial inoculum of (avg. starting ~ 1.03 x106, avg. ending ~ 1.23 x 106), whereas the ΔmcyB starting inoculum was (avg. ~8.65 x 105), and it reached maximum cell concentrations of (avg. ~2.8 x 106)
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