Biology subjects

Kunka, K. S.

Publications and source records attributed to Kunka, K. S..

4 recordsLinked to original sources

bioRxiv2020

Manganese depletion leads to multisystem changes in the transcriptome of the opportunistic pathogen Streptococcus sanguinis

1Streptococcus sanguinis is a primary tooth colonizer and is typically considered beneficial due to its antagonistic relationship with the cariogenic pathogen Streptococcus mutans. However, S. sanguinis can also act as an opportunistic pathogen should it enter the bloodstream and colonize a damaged heart valve, leading to infective endocarditis. Studies have implicated manganese acquisition as an important virulence determinant in streptococcal endocarditis. A knockout mutant lacking the primary manganese import system in S. sanguinis, SsaACB, is severely attenuated for virulence in an in vivo rabbit model. Manganese is a known cofactor for several important enzymes in S. sanguinis, including superoxide dismutase, SodA, and the aerobic ribonucleotide reductase, NrdEF. To determine the effect of manganese depletion on S. sanguinis, we performed transcriptomic analysis on a {Delta}ssaACB mutant grown in aerobic fermentor conditions after the addition of the metal chelator EDTA. Despite the broad specificity of EDTA, analysis of cellular metal content revealed a decrease in manganese, but not in other metals, that coincided with a drop in growth rate. Subsequent supplementation with manganese, but not iron, zinc, or magnesium, restored growth in the fermentor post-EDTA. Reduced activity of Mn-dependent SodA and NrdEF likely contributed to the decreased growth rate post-EDTA, but did not appear entirely responsible. With the exception of the Dps-like peroxide resistance gene, dpr, manganese depletion did not induce stress response systems. By comparing the transcriptome of {Delta}ssaACB cells pre- and post-EDTA, we determined that manganese deprivation led to altered expression of diverse systems, including ethanolamine utilization, CRISPR/Cas, and a type IV pilus. Manganese depletion also led to an apparent induction of carbon catabolite repression in a glucose-independent manner. The combined results suggest that manganese limitation produces effects in S. sanguinis that are diverse and complex, with no single protein or system appearing entirely responsible for the observed growth rate decrease. This study provides further evidence for the importance of this trace element in streptococcal biology. Future studies will focus on determining mechanisms for regulation, as the multitude of changes observed in this study indicate that multiple regulators may respond to manganese levels.

microbiology↗

bioRxiv2019

Acid and Iron Experimental Evolution of Halobacterium sp. NRC-1

BackgroundHalobacterium sp. NRC-1 (NRC-1) is an extremely halophilic archaeon that is adapted to multiple stressors such as UV, ionizing radiation and arsenic exposure. We conducted experimental evolution of NRC-1 under acid stress. NRC-1 was serially cultured in CM+ medium modified by four conditions: optimal pH (pH 7.5), acid stress (pH 6.3), iron amendment (600 M ferrous sulfate, pH 7.5), and acid plus iron (pH 6.3, with 600 M ferrous sulfate). For each condition, four independent lineages of evolving populations were propagated. After 500 generations, 16 clones were isolated for phenotypic characterization and genomic sequencing. ResultsGenome sequences of all 16 clones revealed 378 mutations, of which 90% were haloarchaeal insertion sequences (ISH) and ISH-mediated large deletions. This proportion of ISH events in NRC-1 was five-fold greater than that reported for comparable evolution of E. coli. One acid-evolved clone had increased fitness compared to the ancestral strain when cultured at low pH. Seven of eight acid-evolved clones had a mutation within or upstream of arcD, which encodes an arginine-ornithine antiporter; no non-acid adapted strains had arcD mutations. Mutations also affected the arcR regulator of arginine catabolism, which protects bacteria from acid stress by release of ammonia. Two acid-adapted strains shared a common mutation in bop, which encodes the bacteriorhodopsin light-driven proton pump. Unrelated to pH, one NRC-1 minichromosome (megaplasmid) pNRC100 had increased copy number, and we observed several mutations that eliminate gas vesicles and arsenic resistance. Thus, in the haloarchaeon NRC-1, as in bacteria, pH adaptation was associated with genes involved in arginine catabolism and proton transport. ConclusionsOur study is among the first to report experimental evolution with multiple resequenced genomes of an archaeon. Haloarchaea are polyextremophiles capable of growth under environmental conditions such as concentrated NaCl and desiccation, but little is known about pH stress. Halobacterium sp. NRC-1 (NRC-1) is considered a model organism for the feasibility of microbial life in iron-rich brine on Mars. Interesting parallels appear between the molecular basis of pH adaptation in NRC-1 and in bacteria, particularly the acid-responsive arginine-ornithine system found in oral streptococci.

genetics↗

bioRxiv2019

Benzoate-Tolerant Escherichia coli Strains from Experimental Evolution Lose the Gad Regulon, Multi-Drug Efflux Pumps, and Hydrogenases

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. IMPORTANCEBenzoate 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.

microbiology↗

bioRxiv2018

CCCP experimental evolution of Escherichia coli K-12 selects for mutations that increase EmrA activity or that downregulate other PMF-driven drug efflux pumps

ABSTRACTExperimental evolution was conducted with Escherichia coli K-12 W3110 in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the proton motive force (PMF). Cultures were serially diluted daily 1:100 in broth medium containing 20-150 M CCCP at pH 6.5 or at pH 8.0. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Sequenced isolates showed mutations in emrAB or in its negative repressor mprA; the EmrAB-TolC multidrug efflux pump confers resistance to CCCP and nalidixic acid. Deletion of emrA abolished the CCCP resistance of these strains. One CCCP-evolved isolate lacked emrA or mprA mutations; this strain (C-B11-1) showed mutations in drug efflux regulators cecR (ybiH) (upregulates drug pumps YbhG and YbhFSR) and gadE (upregulates drug pump mdtEF). A cecR::kanR deletion conferred partial resistance to CCCP. A later evolved descendant of the C-B11 population showed mutations in ybhR (MDR efflux). Another isolate showed acrB (MDR efflux pump). The acrB isolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Other mutant genes in CCCP-evolved strains include adhE (alcohol dehydrogenase), rng (ribonuclease G), and cyaA (adenylate cyclase). Overall, experimental evolution revealed a CCCP fitness advantage for mutations increasing its own efflux via EmrA; and for mutations that may decrease proton-driven pumps that efflux other drugs not present (cecR, gadE, acrB, ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate).\n\nIMPORTANCEThe genetic responses of bacteria to depletion of proton motive force, and their effects on drug resistance, are poorly understood. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for and against various multidrug efflux pumps. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF. Organic acid uncouplers may select against multidrug resistance in evolving populations of enteric bacteria.

microbiology↗