Metabolic and evolutionary patterns in the extremely acidophilic archaeon Ferroplasma acidiphilum YT

Ferroplasmaceae represent ubiquitous iron-oxidising extreme acidophiles with a number of unique physiological traits. In a genome-based study of Ferroplasma acidiphilum YT, the only species of the genus Ferroplasma with a validly published name, we assessed its central metabolism and genome stability during a long-term cultivation experiment. Consistently with physiology, the genome analysis points to F. acidiphilum YT having an obligate peptidolytic oligotrophic lifestyle alongside with anaplerotic carbon assimilation. This narrow trophic specialisation abridges the sugar uptake, although all genes for glycolysis and gluconeogenesis, including bifunctional unidirectional fructose 1,6-bisphosphate aldolase/phosphatase, have been identified. Pyruvate and 2-oxoglutarate dehydrogenases are substituted by ‘ancient’ CoA-dependent pyruvate and alpha-ketoglutarate ferredoxin oxidoreductases. In the lab culture, after ~550 generations, the strain exhibited the mutation rate of ≥1.3 × 10−8 single nucleotide substitutions per site per generation, which is among the highest values recorded for unicellular organisms. All but one base substitutions were G:C to A:T, their distribution between coding and non-coding regions and synonymous-to-non-synonymous mutation ratios suggest the neutral drift being a prevalent mode in genome evolution in the lab culture. Mutations in nature seem to occur with lower frequencies, as suggested by a remarkable genomic conservation in F. acidiphilum YT variants from geographically distant populations

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical a/b-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal b-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. Copyright © 2023 Distaso et al.This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Program under grant agreement 101000327. M.F. and F.J.P. also acknowledge grants PID2020-112758RB-I00 (M.F.), PDC2021- 121534-I00 (M.F.), TED2021-130544B-I00 (M.F.), and PID2019-105838RB-C31 (F.J.P.) from MCIN/AEI/10.13039/501100011033 and the European Union (“NextGenerationEU/PRTR”). M.A.D., T.N.C., R.B., A.N.K., O.V.G., A.F.Y., and P.N.G. are thankful for support fromthe European Regional Development Fund (ERDF) through the Welsh Government to the Centre for Environmental Biotechnology, project number 81280. P.N.G. and A.F.Y. acknowledge the Natural Environment Research Council UK-funded Plastic Vectors project NE/S004548/1 and the Sêr Cymru program partly funded by the ERDF through the Welsh Government for support of the project BioPOL4Life.We are indebted to Connie Tulloch and GwionWilliams for their technical support.Supporting InformationPeer reviewe

Changes in managed care activity in outpatient substance abuse treatment organizations, 1995–2000

Using nationally representative data from 1995 and 2000, this study examines trends in managed care penetration and activity among outpatient drug treatment organizations in the United States. Further, it investigates how managed care activity varies across different types of treatment providers and for public and private managed care programs. Overall, managed care activity has increased, with a greater proportion of units having managed care arrangements and a larger percentage of clients covered by managed care. In general, public managed care activity has increased and private managed care activity has decreased. Treatment providers report that they have fewer managed care arrangements, which may reflect consolidation in the managed behavioral care sector. Finally, growth in managed care among outpatient substance abuse treatment units affiliated with hospitals and mental health centers may signal a preference for providers that can effectively link substance abuse treatment with medical and social service provision, or, alternatively, that linkages with such organizations may provide the size necessary to assume the risks associated with managed care contracts.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45778/1/11414_2005_Article_BF02287425.pd

NleG Type 3 Effectors from Enterohaemorrhagic Escherichia coli Are U-Box E3 Ubiquitin Ligases

NleG homologues constitute the largest family of type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9′ family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56±2 µM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes

Managed care and outpatient substance abuse treatment intensity

This study examines the extent to which managed care behavioral controls are associated with treatment intensity in outpatient substance abuse treatment facilities. Data are from the 1995 National Drug Abuse Treatment System Survey, a nationally representative survey that includes over 600 provider organizations with a response rate of 86%. Treatment intensity is measured in three ways: (1) the number of months clients spend in outpatient drug treatment, (2) the number of individual treatment sessions clients receive over the course of treatment, and (3) the number of group treatment sessions clients receive over the course of treatment. After accounting for selection bias and controlling for market, organization, and client characteristics, there is no significant relationship between the scope of managed care oversight and treatment intensity. However, the stringency of managed care oversight activities is negatively associated with the number of individual and group treatment sessions received over the course of treatment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45772/1/11414_2005_Article_BF02287231.pd

Managed care and access to substance abuse treatment services

Using nationally representative data from 1995 and 2000, this study examined how managed care penetration and other organizational characteristics were related to accessibility to outpatient substance abuse treatment. At an organizational level, access was measured as the percentage of clients unable to pay for services; the percentage of clients receiving a reduced fee; and the percentage of clients with shortened treatment because of their inability to pay. Treatment units with both relatively low and relatively high managed care penetration were more likely to support access to care; these units provided care to higher percentages of clients unable to pay and were less likely to shorten treatment because of client inability to pay. Treatment units with midrange managed care penetration were least likely to support access to care. The complexity of managing in an environment of conflicting incentives may reduce the organization's ability to serve those with limited financial means.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45776/1/11414_2005_Article_BF02289805.pd

Managed care and technical efficiency in outpatient substance abuse treatment units

This article examines (1) the extent to which managed care participation is associated with technical efficiency in outpatient substance abuse treatment (OSAT) organizations and (2) the contributions of specific managed care practices as well as other organizational, financial, and environmental attributes to technical efficiency in these organizations. Data are from a nationally representative sample survey of OSAT organizations conducted in 1995. Technical efficiency is modeled using data envelopment analysis. Overall, there were few significant associations between managed care dimensions and technical efficiency in outpatient treatment organizations. Only one managed care oversight procedure, the imposition of sanctions by managed care firms, was significantly associated with relative efficiency of these provider organizations. However, several organizational factors were associated with the relative level of efficiency including hospital affiliation, mental health center affiliation, JCAHO accreditation, receipt of lump sum revenues, methadone treatment modality, percentage clients unemployed, and percentage clients who abuse multiple drugs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45766/1/11414_2005_Article_BF02287509.pd