Biology subjects

Matthew I Hutchings

Publications and source records attributed to Matthew I Hutchings.

2 recordsLinked to original sources

bioRxiv2016

Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP- and dRNA-seq with in vitro biochemistry to characterize a putative NsrR homologue in Streptomyces venezuelae. ChIP-seq analysis revealed that rather than regulating the nitrosative stress response like Streptomyces coelicolor NsrR, Sven6563 binds to a conserved motif at a different, much larger set of genes with a diverse range of functions, including a number of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the putative target genes are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator.

Molecular Biology↗

bioRxiv2016

Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene

Bacterial lipoproteins are a class of extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing the lsp. We report resequencing of the genomes of the wild-type M145 and the cis-complemented {Delta}lsp mutant (BJT1004), mapping and identifying secondary mutations, including an insertion into a novel putative small RNA, scr6809. Disruption of scr6809 led to a range of developmental phenotypes. However, these secondary mutations do not increase the efficiency of disrupting lsp suggesting they are not lsp specific suppressors. Instead we suggest that these were induced by introducing the cosmid St4A10{Delta}lsp as part of the Redirect mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using no gene duplication resulted in the previously observed phenotype. We conclude that lsp is not essential in S. coelicolor but loss of lsp does lead to developmental defects due to the loss of lipoproteins from the cell. Significantly, our results indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to unexpected phenotypes not necessarily linked to the gene or pathway of interest.

Genetics↗