Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo–electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

Domestication – the artificial selection of wild species to obtain variants with traits of human interest – was integral to the rise of complex societies. The oversupply of food was probably associated with the formalization of food preservation strategies through microbial fermentation. While considerable literature exists on the antiquity of fermented food, only few eukaryotic microbes have been studied so far for signs of domestication, less is known for bacteria. Here, we tested if cheese starter cultures harbour typical hallmarks of domestication by characterising over 100 community samples and over 100 individual strains isolated from historical and modern traditional Swiss cheese starter cultures. We find that cheese starter cultures have low genetic diversity both at the species and strain-level and maintained stable phenotypic traits. Molecular clock dating further suggests that the evolutionary origin of the bacteria approximately coincided with the first archaeological records of cheese making. Finally, we find evidence for ongoing genome decay and pseudogenization via transposon insertion related to a reduction of their niche breadth. Future work documenting the prevalence of these hallmarks across diverse fermented food systems and geographic regions will be key to unveiling the joint history of humanity with fermented food microbes.

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol–pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu¹³¹ in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.

Potassium channels from prokaryotes and eukaryotes are usually recognized by a typical amino acid sequence TXTGY(F)G representing the ionic selectivity filter. Using a screening approach with ion channel family profiles but without the above motif, we identified a gene in Trypanosoma brucei that exhibits homology to inward rectifying potassium channels. We report here cloning of this ion channel named TbIRK. The protein is localized to acidocalcisomes in procyclic and in bloodstream form parasites. Functional properties of this channel were established after expression in Xenopus oocytes. Currents recorded in potassium medium show inward rectification and little time dependence. Surprisingly, this channel retains selectivity for potassium ions over sodium ions >7, in spite of the lack of the classical selectivity filter. The sequence GGYVG was predicted in silico to replace this filter motif. Point mutations of the corresponding glycine residues confirmed this at the functional level. The channel is inhibited by caesium ions but remains unaffected by barium ions up to 10 mM. TbIRK is to our knowledge the first potassium channel in T. brucei that localizes to the acidocalcisomes, organelles involved in the storage of phosphates and the response to osmotic stress that occurs during the life cycle of trypanosomes.

As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis (LDL) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To this end, a phenotypic test, as well as a screening for antibiotic resistance genes (ARGs) in genome sequences, is commonly performed. Historically, microbiological cutoffs (MCs), which are used to classify strains as either ‘sensitive’ or ‘resistant’ based on the minimal inhibitory concentrations (MICs) of a range of clinically-relevant antibiotics, have been defined for the whole group of the obligate homofermentative lactobacilli, which includes LDL among many other species. This often leads to inaccuracies in the appreciation of the ABR status of tested LDL strains and to false positive results. To define more accurate MCs for LDL, we analyzed the MIC profiles of strains originating from various habitats by using the broth microdilution method. These strains’ genomes were sequenced and used to complement our analysis involving a search for ARGs, as well as to assess the phylogenetic proximity between strains. Of LDL strains, 52.1% displayed MICs that were higher than the defined MCs for kanamycin, 9.9% for chloramphenicol, and 5.6% for tetracycline, but no ARG was conclusively detected. On the other hand, all strains displayed MICs below the defined MCs for ampicillin, gentamycin, erythromycin, and clindamycin. Considering our results, we propose the adaptation of the MCs for six of the tested clinically-relevant antibiotics to improve the accuracy of phenotypic antibiotic testing.