Arvind A. Bhagwat

Benzoate, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline in Escherichia coli K-12. Genetic mechanisms of increased benzoate tolerance and decreased drug resistance were analyzed in strains isolated from experimental evolution with benzoate. Transcriptomes showed reversal of benzoate-dependent regulation, including the Gad regulon for acid resistance and multi-drug resistance (MDR). Benzoate-evolved strains knocked down the nitrate reductase narHJ and the acid-consuming hydrogenase Hyd-3, but upregulated OmpF and other porins that admit nutrients and antibiotics. Mutations were found affecting many genes of aromatic catabolism such as folD and add . Several candidate genes from benzoate-evolved strains had a role in benzoate tolerance. Growth with benzoate or salicylate was increased by deletion of the Gad activator ariR , or by deletion of the slp-gadX acid fitness island encoding Gad regulators and the MDR pump MdtEF. Benzoate growth was also increased by deletion of MDR component emrA , the RpoS post-transcriptional regulator cspC , or the adenosine deaminase add . Growth in chloramphenicol with benzoate was decreased by a point mutation in the RNA polymerase alpha-subunit rpoA , which appeared in one chloramphenicol-sensitive benzoate-evolved strain. Growth in chloramphenicol was also decreased by deletion of Mar activator rob , or of rfaY (lipopolysaccharide biosynthesis). Hydrogenase Hyd-3 deletion increased benzoate tolerance. Overall, long-term culture in the presence of benzoate or salicylate favors loss of MDR efflux pumps and of hydrogenases that generate a futile cycle of PMF; and favors upregulation of large-hole porins that increase uptake of fermentable nutrients and of antibiotics. IMPORTANCE: Benzoate is a common food preservative, and salicylate is the active form of aspirin. In E. coli , benzoate derivatives upregulate multiple regulons that export antibiotics and other toxic products, and downregulate large outer-membrane porins that allow antibiotic influx. But benzoate or salicylate exposure causes energy stress by depleting the proton motive force. In the absence of antibiotics, long-term benzoate exposure selects against energy-spending systems such as multi-drug efflux pumps and the proton-consuming hydrogenase. Selection favors upregulation of porins that admit fermentable substrates but also allow entry of antibiotics. Thus, evolution with benzoate requires a tradeoff for antibiotic sensitivity. Benzoate and salicylate are naturally occurring plant signal molecules that may influence the evolution of microbiomes in plants and in animal digestive tracts. Effects on hydrogenase activity may be relevant to the biotechnology of hydrogen production.

Movement of food-borne pathogens on moist surfaces enables them to migrate towards more favorable niches and facilitate their survival for extended periods of time. Salmonella enterica serovar Typhimurium mutants defective in Osmoregulated periplasmic glucans (OPG) synthesis are unable to exhibit motility on moist surfaces (swarming); however, their mobility in liquid (swim motility) remains unaffected. In order to understand the role of OPG in swarm motility, transcriptomic analysis was performed using cells growing on a moist agar surface. In opgGH deletion mutant, lack of OPG significantly altered transcription of 1039 genes out of total 4712 genes (22%). Introduction of a plasmid-borne copy of opgGH into opgGH deletion mutant restored normal expression of all but 30 genes, indicating a wide-range influence of OPG on gene expression under swarm motility condition. Major pathways that were differentially expressed in opgGH mutants were motility, virulence and invasion, and genes related to the secondary messenger molecule, cyclic di-GMP. These observations provide insights and help explain the pleiotropic nature of OPG mutants such as sub-optimal virulence and competitive organ colonization in mice, biofilm formation, and sensitivity towards detergent stress.

Background: Salmonella enterica serovar Typhimurium SL 1344 migrates on moist surfaces by swarming motility. S. enterica serovar Typhimurium synthesized osmoregulated periplasmic glucans (OPG) using

Controlling spread of human pathogens on fresh produce is a top priority for public health reasons. Isolation of compounds from agricultural waste that would control spread of human pathogens was explored using Salmonella enterica serovar Typhimurium as a model organism. In the environment, micro‐organisms migrate as a ‘community’ especially when they move on moist surfaces. This type of motility is characterized as swarming motility. We examined extracts from agricultural waste such as soya bean husk, peels of orange, pineapple, avocado and pomegranate for antiswarming activity. Avocado and pineapple peels showed moderate (~40%) inhibition of swarming motility while pomegranate peel extract had high antiswarming activity (~85% inhibition) and was examined in further detail. Although the pomegranate peel extract was acidic, swarm‐inhibitory activity was not due to low pH and the peel extract did not inhibit growth of Salmonella. Among the key swarm motility regulatory genes, class II (fliF, fliA, fliT and fliZ) and class III (fliC and fliM) regulators were downregulated upon exposure to pomegranate peel extract. Pomegranate peels offer great potential as a bioactive repellent for pathogenic micro‐organisms on moist surfaces. Significance and Impact of the Study Controlling the spread of food‐borne pathogens in moist environments is an important microbial food safety issue. Isolation of compounds from agricultural waste (such as fruit peels) that would control spread of human pathogens was explored using Salmonella enterica serovar Typhimurium as a model organism. Pomegranate peels offer great potential as a bioactive repellent for pathogenic micro‐organisms.

Enhanced virulence or infectivity after passage through a mammalian host has been reported for a number of enteric food-borne pathogens. Citrobacter rodentium is a mouse pathogen that mimics many aspects of enterohemorrhagic Escherichia coli infection of humans and serves as a useful model for studying virulence mechanisms. Emergence of a hyperinfectious state after passage through mouse gastrointestinal tract was reported for C. rodentium. We wanted to investigate if increased acid tolerance could explain hypervirulence status of C. rodentium. Although we were able to observe hyperinfectious state of C. rodentium upon host passage, the cells were extremely acid sensitive. Growth under mildly acidic conditions (LB-MES, pH 5.5) induced acid tolerance of C. rodentium, but did not improve the organism’s ability to establish infection. Growth under anaerobic environment on fecal components also did not induce hyperinfectious state. Thus, contrary to conventional anticipation, hypervirulent C. rodentium cells were found to be acid sensitive thereby revealing limitations of the role of mouse gastric acidity by itself in elucidating the hypervirulent phenotype.

Background: Using next-generation-sequencing technology to assess entire transcriptomes requires high quality starting RNA. Traditionally, the ratios of 23S to 16S ribosomal RNA bands from agarose gel

Osmoregulated periplasmic glucans (OPGs) are synthesized by the members of the family Enterobacteriaceae when grown under low osmotic growth conditions. Enteropathogens such as Shigella flexneri spend considerable time outside the host environment such as irrigation waters where low nutrient low osmolarity conditions normally may exist. We recently demonstrated that OPGs of S. flexneri are required for optimal growth under low osmolarity low nutrient conditions. Based on homology of the OPG biosynthesis genes to those of Escherichia coli, the presumptive function of opgC and opgB genes is to add succinate and phosphoglycerol residues respectively on OPGs, rendering them anionic. Using lambda-red recombination procedure, we constructed opgB, opgC, and opgBC mutants of S. flexneri. The mutant strain defective in opgC and opgB genes synthesized neutral OPGs. The OPGs without any anionic charges were beneficial for the organism's growth in hypo-osmotic media. However, with the loss of anionic charges from OPGs, mutants were compromised in their ability to combat stress caused by anionic detergents in hypo-osmotic growth conditions. Cloned wild-type genes opgB, opgC, and opgBC, when mobilized to respective opg mutants, simultaneously restored anionic charges to OPGs and tolerance to detergents. The data indicate that anionic charges on the OPGs contribute towards overcoming the stress caused by anionic detergents such as sodium dodecyl sulfate and sodium deoxycholate.

Salmonella enterica, a bacterial, food-borne pathogen of humans, can contaminate raw fruits and vegetables. Unfortunately for consumers, the bacteria can survive in water used to wash away contaminating bacteria. The ability to survive the low-osmotic conditions of the wash water is attributed to the OpgGH operon that leads to the production of osmotically regulated periplasmic glucans. Mutants lacking OpgGH grow slowly under low-osmotic conditions, but there are also unexpected traits such as abnormal flagellar motility and reduced virulence in mice. To get a broader understanding of these pleiotropic effects under low osmolarity, we examined the proteome of these mutants using high-throughput mass spectrometry. We identified approximately one-third of the proteins encoded by the genome and used label-free spectral counting to determine the relative amounts of proteins in wild-type cultures and mutants. Mutants had reduced amounts of proteins required for osmotic sensing, flagellar motility, purine and pyrimidine metabolism, oxidative energy production, and protein translation. By contrast, mutants had greater amounts of ABC transporters needed to balance cellular osmolarity. Hence, the effects of OpgGH reach across the proteome, and the data are consistent with the mutant phenotypes.