Biology subjects

Redfield, R. J.

Publications and source records attributed to Redfield, R. J..

4 recordsLinked to original sources

bioRxiv2019

Genome-wide analysis of DNA uptake by naturally competent Haemophilus influenzae

The genomes of naturally competent Pasteurellaceae and Neisseriaceae have many short uptake sequences (USS), which allow them to distinguish self-DNA from foreign DNA. To fully characterize this preference we developed genome-wide maps of DNA uptake using both a sequence-based computational model and genomic DNA that had been sequenced after uptake by and recovery from competent Haemophilus influenzae cells. When DNA fragments were shorter than the average USS spacing of ~1000 bp, sharp peaks of uptake were centered at USS and separated by valleys with 1000-fold lower uptake. Long DNA fragments (1.5-17 kb) gave much less variation, with 90% of positions having uptake within two-fold of the mean. All detectable uptake biases arose from sequences that fit the USS uptake motif. Simulated competition predicted that, in its respiratory tract environment, H. influenzae will efficiently take up its own DNA even when human DNA is present in 100-fold excess.

microbiology↗

bioRxiv2019

A competence-regulated toxin-antitoxin system in Haemophilus influenzae

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining both nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenzae, the genes needed for DNA uptake are induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that one of these genes, HI0659, encodes the antitoxin of a competence-regulated toxin-antitoxin operon ( toxTA), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the putative toxin (HI0660) restores uptake to the antitoxin mutant. The full toxTA operon was present in only 17 of the 181 strains we examined; complete deletion was seen in 22 strains and deletions removing parts of the toxin gene in 142 others. In addition to the expected Sxy-and CRP-dependent-competence promoter, HI0659/660 transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We propose that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture. Although the high frequency of toxT and toxTA deletions suggests that this competence-regulated toxin-antitoxin system may be mildly deleterious, it could also facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions. Although our analyses were focused on the effects of toxTA, the RNA-seq dataset will be a useful resource for further investigations into competence regulation.\n\nABBREVIATED SUMMARYThe competence regulon of Haemophilus influenzae includes an unprecedented toxin/antitoxin gene pair. When not opposed by antitoxin, the toxin completely prevents DNA uptake but causes only very minor decreases in cell growth and competence gene expression. The TA gene pair was acquired by horizontal gene transfer, and the toxin gene has undergone repeated deletions in other strains.

microbiology↗

bioRxiv2018

A competence-regulated toxin-antitoxin system in Haemophilus influenzae

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenae, the DNA uptake machinery is induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that HI0659--which is absolutely required for DNA uptake-- encodes the antitoxin of a competence-regulated toxin-antitoxin operon ( toxTA), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the toxin restores uptake to the antitoxin mutant. In addition to the expected Sxy-and CRP-dependent-competence promoter, transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We present evidence that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture, and we show that the toxin gene has been inactivated by deletion in many H. influenzae strains. We suggest that this competence-regulated toxin-antitoxin system may facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions, or alternatively be a simple genetic parasite.

microbiology↗

bioRxiv2018

Finding reliable phenotypes and detecting artefacts among in vivo and in vitro assays to characterize the refractory transcriptional activator Sxy (TfoX) in Escherichia coli

The Sxy (TfoX) protein is required for expression of a distinct subset of the genes regulated by the cAMP receptor protein (CRP) in the model organisms Escherichia coli, Haemophilus influenzae, and Vibrio cholerae. Genetic studies have established that CRP and Sxy co-activate transcription at gene promoters containing DNA binding sites called CRP-S sites. In contrast, CRP acts without Sxy at gene promoters containing canonical CRP-N sites, suggesting that Sxy makes physical contacts with CRP and/or DNA to assist in transcriptional activation at CRP-S promoters. Despite growing interest in Sxys activity as a transcription factor, Sxy remains poorly characterized due to a lack of reliable phenotypes in E. coli. Experiments are further hampered by growth inhibition and formation of inclusion bodies when Sxy is overexpressed. In this study we applied diverse phenotypic and molecular assays to test for postulated Sxy functions and interactions. Mutations in conserved regions of Sxy and truncations in the Sxy C-terminus abolish transcriptional activation of a CRP-S promoter, and a 37 amino acid truncation of the C-terminus relieves the growth inhibition normally caused by Sxy overexpression. Sxy was unable to augment weakened CRP interactions to restore carbon metabolism phenotypes. Bandshift analysis and chromatin pull-down assays of Sxy-CRP-DNA interactions yielded intriguing evidence of CRP-Sxy and Sxy-DNA physical interactions. However, despite the careful application of standard protein purification protocols and quality control steps for nickel affinity column purification, protein mass spectrometry revealed the enrichment of additional DNA-binding proteins in nickel column eluates, presenting a probable source of artefactual protein-protein and protein-DNA interaction results. These findings highlight the importance of extensive controls and phenotypic assays for the study of poorly characterized and recalcitrant proteins like Sxy.

microbiology↗