Effective polyploidy causes phenotypic delay and influences bacterial evolvability.

Whether mutations in bacteria exhibit a noticeable delay before expressing their corresponding mutant phenotype was discussed intensively in the 1940s to 1950s, but the discussion eventually waned for lack of supportive evidence and perceived incompatibility with observed mutant distributions in fluctuation tests. Phenotypic delay in bacteria is widely assumed to be negligible, despite the lack of direct evidence. Here, we revisited the question using recombineering to introduce antibiotic resistance mutations into E. coli at defined time points and then tracking expression of the corresponding mutant phenotype over time. Contrary to previous assumptions, we found a substantial median phenotypic delay of three to four generations. We provided evidence that the primary source of this delay is multifork replication causing cells to be effectively polyploid, whereby wild-type gene copies transiently mask the phenotype of recessive mutant gene copies in the same cell. Using modeling and simulation methods, we explored the consequences of effective polyploidy for mutation rate estimation by fluctuation tests and sequencing-based methods. For recessive mutations, despite the substantial phenotypic delay, the per-copy or per-genome mutation rate is accurately estimated. However, the per-cell rate cannot be estimated by existing methods. Finally, with a mathematical model, we showed that effective polyploidy increases the frequency of costly recessive mutations in the standing genetic variation (SGV), and thus their potential contribution to evolutionary adaptation, while drastically reducing the chance that de novo recessive mutations can rescue populations facing a harsh environmental change such as antibiotic treatment. Overall, we have identified phenotypic delay and effective polyploidy as previously overlooked but essential components in bacterial evolvability, including antibiotic resistance evolution.

Antimicrobial nodule-specific cysteine-rich peptides disturb the integrity of bacterial outer and inner membranes and cause loss of membrane potential.

BACKGROUND: Certain legume plants produce a plethora of AMP-like peptides in their symbiotic cells. The cationic subgroup of the nodule-specific cysteine-rich (NCR) peptides has potent antimicrobial activity against gram-negative and gram-positive bacteria as well as unicellular and filamentous fungi. FINDINGS: It was shown by scanning and atomic force microscopies that the cationic peptides NCR335, NCR247 and Polymyxin B (PMB) affect differentially on the surfaces of Sinorhizobium meliloti bacteria. Similarly to PMB, both NCR peptides caused damages of the outer and inner membranes but at different extent and resulted in the loss of membrane potential that could be the primary reason of their antimicrobial activity. CONCLUSIONS: The primary reason for bacterial cell death upon treatment with cationic NCR peptides is the loss of membrane potential.

Perturbation of iron homeostasis promotes the evolution of antibiotic resistance.

Evolution of antibiotic resistance in microbes is frequently achieved by acquisition of spontaneous mutations during antimicrobial therapy. Here, we demonstrate that inactivation of a central transcriptional regulator of iron homeostasis (Fur) facilitates laboratory evolution of ciprofloxacin resistance in Escherichia coli. To decipher the underlying molecular mechanisms, we first performed a global transcriptome analysis and demonstrated that the set of genes regulated by Fur changes substantially in response to antibiotic treatment. We hypothesized that the impact of Fur on evolvability under antibiotic pressure is due to the elevated intracellular concentration of free iron and the consequent enhancement of oxidative damage-induced mutagenesis. In agreement with expectations, overexpression of iron storage proteins, inhibition of iron transport, or anaerobic conditions drastically suppressed the evolution of resistance, whereas inhibition of the SOS response-mediated mutagenesis had only a minor effect. Finally, we provide evidence that a cell permeable iron chelator inhibits the evolution of resistance. In sum, our work revealed the central role of iron metabolism in the de novo evolution of antibiotic resistance, a pattern that could influence the development of novel antimicrobial strategies.