Genome Sequence of the Early 20th-Century Extreme Halophile Halobacterium sp. Strain NRC-34001.

Halobacterium sp. strain NRC-34001 is a red, extremely halophilic archaeon isolated in Canada in 1934. Single-molecule real-time sequencing revealed a 2.3-Mbp genome with a 2-Mbp chromosome and two plasmids (235 kb and 43 kb). The genome encodes all conserved core haloarchaeal groups (cHOGs) and a highly acidic proteome.

Androgen-Influenced Polarization of Activin A-Producing Macrophages Accompanies Post-pyelonephritic Renal Scarring.

Ascending bacterial pyelonephritis, a form of urinary tract infection (UTI) that can result in hospitalization, sepsis, and other complications, occurs in ~250,000 US patients annually; uropathogenic Escherichia coli (UPEC) cause a large majority of these infections. Although UTIs are primarily a disease of women, acute pyelonephritis in males is associated with increased mortality and morbidity, including renal scarring, and end-stage renal disease. Preclinical models of UTI have only recently allowed investigation of sex and sex-hormone effects on pathogenesis. We previously demonstrated that renal scarring after experimental UPEC pyelonephritis is augmented by androgen exposure; testosterone exposure increases both the severity of pyelonephritis and the degree of renal scarring in both male and female mice. Activin A is an important driver of scarring in non-infectious renal injury, as well as a mediator of macrophage polarization. In this work, we investigated how androgen exposure influences immune cell recruitment to the UPEC-infected kidney and how cell-specific activin A production affects post-pyelonephritic scar formation. Compared with vehicle-treated females, androgenized mice exhibited reduced bacterial clearance from the kidney, despite robust myeloid cell recruitment that continued to increase as infection progressed. Infected kidneys from androgenized mice harbored more alternatively activated (M2) macrophages than vehicle-treated mice, reflecting an earlier shift from a pro-inflammatory (M1) phenotype. Androgen exposure also led to a sharp increase in activin A-producing myeloid cells in the infected kidney, as well as decreased levels of follistatin (which normally antagonizes activin action). As a result, infection in androgenized mice featured prolonged polarization of macrophages toward a pro-fibrotic M2a phenotype, accompanied by an increase in M2a-associated cytokines. These data indicate that androgen enhancement of UTI severity and resulting scar formation is related to augmented local activin A production and corresponding promotion of M2a macrophage polarization.

Acid Experimental Evolution of the Haloarchaeon Halobacterium sp. NRC-1 Selects Mutations Affecting Arginine Transport and Catabolism.

Halobacterium sp. NRC-1 (NRC-1) is an extremely halophilic archaeon that is adapted to multiple stressors such as UV, ionizing radiation and arsenic exposure; it is considered a model organism for the feasibility of microbial life in iron-rich brine on Mars. We conducted experimental evolution of NRC-1 under acid and iron 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 µM ferrous sulfate, pH 7.5), and acid plus iron (pH 6.3, with 600 µM 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. Genome 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 Escherichia 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 bacterio-opsin, apoprotein for the bacteriorhodopsin light-driven proton pump. Thus, in the haloarchaeon NRC-1, as in bacteria, pH adaptation was associated with genes involved in arginine catabolism and proton transport. Our 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. 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.

Experimental Evolution of Escherichia coli K-12 in the Presence of Proton Motive Force (PMF) Uncoupler Carbonyl Cyanide m-Chlorophenylhydrazone Selects for Mutations Affecting PMF-Driven Drug Efflux Pumps.

Experimental evolution of Escherichia coli K-12 with benzoate, a partial uncoupler of the proton motive force (PMF), selects for mutations that decrease antibiotic resistance. We conducted experimental evolution in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a strong uncoupler. Cultures were serially diluted daily 1:100 in LBK medium containing 20 to 150 µM CCCP buffered at pH 6.5 or at pH 8.0. After 1,000 generations, the populations tolerated up to 150 µM CCCP. Sequenced isolates had mutations in mprA (emrR), which downregulates the EmrAB-TolC pump that exports CCCP. A mprA::kanR deletion conferred growth at 60 µM CCCP, though not at the higher levels resisted by evolved strains (150 µM). Some mprA mutant strains also had point mutations affecting emrA, but deletion of emrA abolished the CCCP resistance. Thus, CCCP-evolved isolates contained additional adaptations. One isolate lacked emrA or mprA mutations but had mutations in cecR (ybiH), whose product upregulates drug pumps YbhG and YbhFSR, and in gadE, which upregulates the multidrug pump MdtEF. A cecR::kanR deletion conferred partial resistance to CCCP. Other multidrug efflux genes that had mutations included ybhR and acrAB The acrB isolate was sensitive to the AcrAB substrates chloramphenicol and tetracycline. Other mutant genes in CCCP-evolved strains include rng (RNase G) and cyaA (adenylate cyclase). Overall, experimental evolution revealed a CCCP-dependent fitness advantage for mutations increasing CCCP efflux via EmrA and for mutations that may deactivate proton-driven pumps for drugs not present (cecR, gadE, acrAB, and ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved benzoate tolerance.IMPORTANCE The genetic responses of bacteria to depletion of proton motive force (PMF), and their effects on drug resistance, are poorly understood. PMF drives export of many antibiotics, but the energy cost may decrease fitness when antibiotics are absent. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for increased CCCP efflux but also against the expression of PMF-driven pumps for drugs not present. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF.

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism.

Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.