Karin Hjort

Cell cycle regulation in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius

The regulation and co‐ordination of the cell cycle of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius was investigated with antibiotics. We provide evidence for a core regulation involving alternating rounds of chromosome replication and genome segregation. In contrast, multiple rounds of replication of the chromosome could occur in the absence of an intervening cell division event. Inhibition of the elongation stage of chromosome replication resulted in cell division arrest, indicating that pathways similar to checkpoint mechanisms in eukaryotes, and the SOS system of bacteria, also exist in archaea. Several antibiotics induced cell cycle arrest in the G2 stage. Analysis of the run‐out kinetics of chromosome replication during the treatments allowed estimation of the minimal rate of replication fork movement in vivo to 250 bp s−1. An efficient method for the production of synchronized Sulfolobus populations by transient daunomycin treatment is presented, providing opportunities for studies of cell cycle‐specific events. Possible targets for the antibiotics are discussed, including topoisomerases and protein glycosylation.

Hydrogenosomes in the diplomonad Spironucleus salmonicida

Acquisition of the mitochondrion is a key event in the evolution of the eukaryotic cell, but diversification of the organelle has occurred during eukaryotic evolution. One example of such mitochondria-related organelles (MROs) are hydrogenosomes, which produce ATP by substrate-level phosphorylation with hydrogen as a byproduct. The diplomonad parasite Giardia intestinalis harbours mitosomes, another type of MRO. Here we identify MROs in the salmon parasite Spironucleus salmonicida with similar protein import and Fe–S cluster assembly machineries as in Giardia mitosomes. We find that hydrogen production is prevalent in the diplomonad genus Spironucleus, and that S. salmonicida MROs contain enzymes characteristic of hydrogenosomes. Evolutionary analyses of known hydrogenosomal components indicate their presence in the diplomonad ancestor, and subsequent loss in Giardia. Our results suggest that hydrogenosomes are metabolic adaptations predating the split between parabasalids and diplomonads, which is deeper than the split between animals and fungi in the eukaryotic tree.

Antimicrobial peptide exposure selects for Staphylococcus aureus resistance to human defence peptides

Background The clinical development of antimicrobial peptides (AMPs) is currently under evaluation to combat the rapid increase in MDR bacterial pathogens. However, many AMPs closely resemble components of the human innate immune system and the ramifications of prolonged bacterial exposure to AMPs are not fully understood. Objectives We show that in vitro serial passage of a clinical USA300 MRSA strain in a host-mimicking environment containing host-derived AMPs results in the selection of stable AMP resistance. Methods Serial passage experiments were conducted using steadily increasing concentrations of LL-37, PR-39 or wheat germ histones. WGS and proteomic analysis by MS were used to identify the molecular mechanism associated with increased tolerance of AMPs. AMP-resistant mutants were characterized by measuring in vitro fitness, AMP and antibiotic susceptibility, and virulence in a mouse model of sepsis. Results AMP-resistant Staphylococcus aureus mutants often displayed little to no fitness cost and caused invasive disease in mice. Further, this phenotype coincided with diminished susceptibility to both clinically prescribed antibiotics and human defence peptides. Conclusions These findings suggest that therapeutic use of AMPs could select for virulent mutants with cross-resistance to human innate immunity as well as antibiotic therapy. Thus, therapeutic use of AMPs and the implications of cross-resistance need to be carefully monitored and evaluated.

Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica.

Heteroresistance, a phenomenon where subpopulations of a bacterial isolate exhibit different susceptibilities to an antibiotic, is a growing clinical problem where the underlying genetic mechanisms in most cases remain unknown. We isolated colistin resistant mutants in Escherichia coli and Salmonella enterica serovar Typhimurium at different concentrations of colistin. Genetic analysis showed that genetically stable pmrAB point mutations were responsible for colistin resistance during selection at high drug concentrations for both species and at low concentrations for E. coli. In contrast, for S. Typhimurium mutants selected at low colistin concentrations, amplification of different large chromosomal regions conferred a heteroresistant phenotype. All amplifications included the pmrD gene, which encodes a positive regulator that up‐regulates proteins that modify lipid A, and as a result increase colistin resistance. Inactivation and over‐expression of the pmrD gene prevented and conferred resistance, respectively, demonstrating that the PmrD protein is required and sufficient to confer resistance. The heteroresistance phenotype is explained by the variable gene dosage of pmrD in a population, where sub‐populations with different copy number of the pmrD gene show different levels of colistin resistance. We propose that variability in gene copy number of resistance genes can explain the heteroresistance observed in clinically isolated pathogenic bacteria.

Evolution of high-level resistance during low-level antibiotic exposure

It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.Mutational antibiotic resistance can emerge under either high or low antibiotic levels. Here, the authors show several small-effect resistance mutations are combined to confer high-level resistance in Salmonella enterica exposed to sub-MIC levels of streptomycin.