High concentrations of indole are known to be toxic to cells due to perturbations in membrane potential. Here, we report for the first time a transcriptome analysis of a soil model bacterium, Pseudomonas putida KT2440, under indole treatment. We demonstrated that 47 genes are differentially expressed, including 11 genes involved in the tricarboxylic acid cycle (TCA cycle) and 12 genes involved in chaperone and protease functions (hslV, hslU, htpG, grpE, dnaK, ibpA, groEL, groES, clpB, lon-1, lon-2, and hflk). Mutant analysis supported the observation that protease genes including hslU are essential for the indole resistance of Pseudomonas strains. Subsequent biochemical analyses have shown that indole increases the NADH/NAD(+) ratio and decreases the adenosine triphosphate (ATP) concentration inside cells, due to membrane perturbation and higher expression of TCA cycle genes in the presence of indole. This energy reduction leads to a reduction in cell size and an enhancement of biofilm formation in P. putida. The observed upregulation in many chaperones and proteases led us to speculate that protein folding might be inhibited by indole treatment. Interestingly, our in vitro protein-refolding assay using malate dehydrogenase with purified GroEL/GroES demonstrated that indole interferes with protein folding. Taken together, our data provide new evidence that indole causes toxicity to P. putida by inhibiting cellular energy production and protein folding.

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18Bactericidal antibiotics are powerful drugs due to their ability to not only inhibit essential 19 bacterial functions, but to convert them into toxic (and potentially lethal) processes. 20However, many important bacterial pathogens are remarkably tolerant against 21 bactericidal drugs, due to inducible stress responses that repair antibiotic-induced 22 damage. The mechanistic details of how stress responses promote whole population 23 tolerance in important human pathogens are unknown. The two-component system 24 VxrAB of the diarrheal pathogen Vibrio cholerae, a model system for high-level -lactam 25 2 tolerance, is induced by exposure to cell wall acting antibiotic and controls a gene 26 network encoding highly diverse functions, including cell wall synthesis functions and 27 iron uptake systems. Here, we show that positive control over cell wall synthesis 28 functions only partially explains high level -lactam tolerance. We find that in addition to 29 cell wall damage, -lactam antibiotics inappropriately induce the Fur-regulated iron 30 starvation response, causing an increase in intracellular free iron levels and colateral 31 oxidative damage. We propose that VxrAB reduces antibiotic-induced toxic influx of 32 Fe 2+ and concomitant metabolic perturbations by selectively downregulating iron uptake 33 transporters. Our results suggest that the ability to counteract diverse antibiotic-induced 34 stresses promotes high-level antibiotic tolerance and highlight the complex responses 35 elicited by antibiotics in addition to their primary mechanism of action. 36 37 by either developing the ability to grow in their presence (antibiotic resistance, ABR) or 49 to simply stay alive in their presence for extended time periods (antibiotic 50 tolerance/persistence) [3][4][5][6][7][8] . While the mechanisms and consequences of ABR are 51 relatively well-established, antibiotic tolerance remains poorly understood, limiting our 52 ability to develop antibiotic adjuvants that increase the efficacy of existing drugs. 53 54The -lactam antibiotics (penicillins, cephalosporins, carbapenems, cephamycins and 55 monobactams) are highly potent bactericidal agents. Their typically lethal action results 56 from their ability to simultaneously inhibit multiple targets (i.e, the transpeptidase 57 domain of multiple penicillin-binding proteins [PBPs]), which ultimately causes bacterial 58 cells to deplete essential cell wall precursors and self-destruct through the activity of 59 endogenous, cell wall lytic enzymes ('autolysins'; endopeptidases, amidases and lytic 60 transglycosylases) 9-12 . However, we and others have recently shown that many 61 clinically significant Gram-negative pathogens are remarkably -lactam tolerant. The 62 cholera pathogen Vibrio cholerae, the opportunistic pathogen Pseudomonas aeruginosa 63 and clinical isolates of Enterobacteriaceae all survive treatment with -lactam antibiotics 64 (including the "last resort" agent meropenem) by forming non-dividing, cell wall deficient 65 spheroplasts 11,13,14 . Upon...

Red clay is a type of soil, the red color of which results from the presence of iron oxide. It is considered an eco-friendly material, with many industrial, cosmetic, and architectural uses. A patented method was applied to red clay in order to change its chemical composition and mineral bioavailability. The resulting product was designated processed red clay. This study evaluates the novel use of red clay and processed red clay as biostimulation agents in diesel-contaminated soils. Diesel biodegradation was enhanced in the presence of red clay and processed red clay by 4.9- and 6.7-fold, respectively, and the number of culturable bacterial cells was correlated with the amount of diesel biodegradation. The growth of Acinetobacter oleivorans DR1, Pseudomonas putida KT2440, and Cupriavidus necator was promoted by both types of red clays. Culture-independent community analysis determined via barcoded pyrosequencing indicated that Nocardioidaceae, Xanthomonadaceae, Pseudomonadaceae, and Caulobacteraceae were enriched by diesel contamination. Bacterial strain isolation from naphthalene- and liquid paraffin-amended media was affiliated with enriched taxa based on 16S rRNA gene sequence identity. We suggest that the biostimulating mechanism of red clay and processed red clay is able to support bacterial growth without apparent selection for specific bacterial species.

We used culture-dependent and culture-independent methods to extract previously undescribed plasmids harboring tetracycline (TC) resistance genes from activated sludge. The extracted plasmids were transformed into naturally competent Acinetobacter oleivorans DR1 to recover a non-Escherichia coli-based plasmid. The transformed cells showed 80-100-fold higher TC resistance than the wild-type strain. Restriction length polymorphism performed using 30 transformed cells showed four different types of plasmids. Illumina-based whole sequencing of the four plasmids identified three previously unreported plasmids and one previously reported plasmid. All plasmids carried TC resistance-related genes (tetL, tetH), tetracycline transcriptional regulators (tetR), and mobilization-related genes. As per expression analysis, TC resistance genes were functional in the presence of TC. The recovered plasmids showed mosaic gene acquisition through horizontal gene transfer. Membrane fluidity, hydrophobicity, biofilm formation, motility, growth rate, sensitivity to stresses, and quorum sensing signals of the transformed cells were different from those of the wild-type cells. Plasmid-bearing cells seemed to have an energy burden for maintaining and expressing plasmid genes. Our data showed that acquisition of TC resistance through plasmid uptake is related to loss of biological fitness. Thus, cells acquiring antibiotic resistance plasmids can survive in the presence of antibiotics, but must pay ecological costs.