MAMP-elicited changes in amino acid transport activity contribute to restricting bacterial growth.

Plants live under the constant challenge of microbes that probe the environment in search of potential hosts. Plant cells perceive microbe-associated molecular patterns (MAMPs) from incoming microbes and activate defense responses that suppress attempted infections. Despite the substantial progress made in understanding MAMP-triggered signaling pathways, the downstream mechanisms that suppress bacterial growth and disease remain poorly understood. Here, we uncover how MAMP perception in Arabidopsis (Arabidopsis thaliana) elicits dynamic changes in extracellular concentrations of free L-amino acids (AA). Within the first 3 h of MAMP perception, a fast and transient inhibition of AA uptake produces a transient increase in extracellular AA concentrations. Within 4 and 12 h of MAMP perception, a sustained enhanced uptake activity decreases the extracellular concentrations of AA. Gene expression analysis showed that salicylic acid-mediated signaling contributes to inducing the expression of AA/H+ symporters responsible for the MAMP-induced enhanced uptake. A screening of loss-of-function mutants identified the AA/H+ symporter lysin/histidine transporter-1 as an important contributor to MAMP-induced enhanced uptake of AA. Infection assays in lht1-1 seedlings revealed that high concentrations of extracellular AA promote bacterial growth in the absence of induced defense elicitation but contribute to suppressing bacterial growth upon MAMP perception. Overall, the data presented in this study reveal a mechanistic connection between MAMP-induced plant defense and suppression of bacterial growth through the modulation of AA transport activity.

Environmental Dependence of Competitive Fitness in Rifampin-Resistant rpoB Mutants of Bacillus subtilis.

RNA polymerase (RNAP) is a highly conserved macromolecular machine that contributes to the flow of genetic information from genotype to phenotype. In Bacillus subtilis, mutations in the rpoB gene encoding the ß-subunit of RNAP have been shown to alter a number of global phenotypes, including growth, utilization of unusual nutrient sources, sporulation, germination, and production of secondary metabolites. In addition, the spectrum of mutations in rpoB leading to rifampin resistance (Rifr) can change dramatically depending upon the environment to which B. subtilis cells or spores are exposed. RifrrpoB mutations have historically been associated with slower growth and reduced fitness; however, these assessments of fitness were conducted on limited collections of mutants in rich laboratory media that poorly reflect natural environments typically inhabited by B. subtilis. Using a novel deep-sequencing approach in addition to traditional measurements of growth rate, lag time, and pairwise competitions, we demonstrated that the competitive advantages of specific rpoB alleles differ depending on the growth environment in which they are determined. IMPORTANCE Microbial resistance to antibiotics is a growing threat to public health across the world. Historically, resistance to antibiotics has been associated with reduced fitness. A growing body of evidence indicates that resistance to rifampin, a frontline antibiotic used to treat mycobacterial and biofilm-associated infections, may increase fitness given an appropriate environment even in the absence of the selective antibiotic. Here, we experimentally confirm this phenomenon by directly comparing the fitness of multiple rifampin-resistant mutants of Bacillus subtilis in rich LB medium and an asparagine minimal medium. Our research demonstrates that the fitness cost of rifampin resistance can vary greatly depending upon the environment. This has important implications for understanding how microbes develop antimicrobial resistance in the absence of antibiotic selection.

The Spectrum of Spontaneous Rifampin Resistance Mutations in the Bacillus subtilis rpoB Gene Depends on the Growth Environment.

Results from previous investigations into spontaneous rifampin resistance (Rifr) mutations in the Bacillus subtilis rpoB gene suggested that the spectrum of mutations depends on the growth environment. However, these studies were limited by low sample numbers, allowing for the potential distortion of the data by the presence of "jackpot" mutations that may have arisen early in the growth of a population. Here, we addressed this issue by performing fluctuation analyses to assess both the rate and spectrum of Rifr mutations in two distinct media: LB, a complete laboratory medium, and SMMAsn, a minimal medium utilizing l-asparagine as the sole carbon source. We cultivated 60 separate populations under each growth condition and determined the mutation rate to Rifr to be slightly but significantly higher in LB cultures. We then sequenced the relevant regions of rpoB to map the spectrum of Rifr mutations under each growth condition. We found a distinct spectrum of mutations in each medium; LB cultures were dominated by the H482Y mutation (27/53 or 51%), whereas SMMAsn cultures were dominated by the S487L mutation (24/51 or 47%). Furthermore, we found through competition experiments that the relative fitness of the S487L mutant was significantly higher in SMMAsn than in LB medium. We therefore conclude that both the spectrum of Rifr mutations in the B. subtilis rpoB gene and the fitness of resulting mutants are influenced by the growth environment. IMPORTANCE The rpoB gene encodes the beta subunit of RNA polymerase, and mutations in rpoB are key determinants of resistance to the clinically important antibiotic rifampin. We show here that the spectrum of mutations in Bacillus subtilis rpoB depends on the medium in which the cells are cultivated. The results show that the growth environment not only plays a role in natural selection and fitness but also influences the probability of mutation at particular bases within the target gene.