Biology subjects

Price, M.

Publications and source records attributed to Price, M..

3 recordsLinked to original sources

bioRxiv2017

Filling Gaps in Bacterial Amino Acid Biosynthesis Pathways with High-throughput Genetics

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

microbiology↗

bioRxiv2017

Magic pools: parallel assessment of transposon delivery vectors in bacteria

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a parts-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term \"magic pools\". Within a magic pool, each transposon vector has a different combination of promoters and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. To identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use the best vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylum Bacteroidetes.

genomics↗

bioRxiv2017

A Cell Culture Platform For Cryptosporidium That Enables Long-Term Cultivation And New Tools For The Systematic Investigation Of Its Biology

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking. Currently, Cryptosporidium oocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains (Moredun, Iowa) produce sufficient infectious oocysts to infect subsequent cultures. Oocyst identity was confirmed by specific staining (Crypt-a-glo, Vicia Villosa lectin, Sporo-glo), PCR-based amplification of Cryptosporidium-specific genes, lipidomics fingerprinting, and atomic force microscopy (AFM). Antibody-stained oocysts produced unstained oocysts confirming production of novel oocysts. Infected cultures could be cryoconserved and continued to produce infectious oocysts after resuscitation. Transmission electron microscopy identified all key Cryptosporidium life cycle stages. Infected cultures produced thick-walled (primarily involved in Cryptosporidium transmission between organisms) and thin-walled oocysts (important for Cryptosporidium propagation within a host/tissue) as indicated by DAPI staining (only thin-walled oocysts are permeable to DAPI staining, thus allowing visualisation of sporozoites) and AFM. In conclusion, we present a novel, easy-to-handle cell culture system that enables the propagation, cryopreservation and detailed investigation of Cryptosporidium at a laboratory scale. Its availability will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.

microbiology↗