Mortality risk information and health-seeking behavior during an epidemic.

In a context where pessimistic survival perceptions have been widespread as a result of the HIV/AIDS epidemic (Fig. 1 A), we study vaccine uptake and other health behaviors during the recent COVID-19 pandemic. Leveraging a longitudinal cohort study in rural Malawi that has been followed for up to 25 y, we document that a 2017 mortality risk information intervention designed to reduce pessimistic mortality perceptions (Fig. 1 B) resulted in improved health behavior, including COVID-19 vaccine uptake (Fig. 1 C). We also report indirect effects for siblings and household members. This was likely the result of a reinforcing process where the intervention triggered engagement with the healthcare system and stronger beliefs in the efficacy of modern biomedical treatments, which led to the adoption of health risk reduction behavior, including vaccine uptake. Our findings suggest that health information interventions focused on survival perceptions can be useful in promoting health behavior and participation in the formal healthcare system, even during health crises-such as the COVID-19 pandemic-that are unanticipated at the time of the intervention. We also note the importance of the intervention design, where establishing rapport, tailoring the content to the local context, and spending time with respondents to convey the information contributed to the salience of the message.

The Growth Yield of Aminobacter niigataensis MSH1 on the Micropollutant 2,6-Dichlorobenzamide Decreases Substantially at Trace Substrate Concentrations.

2,6-Dichlorobenzamide (BAM) is an omnipresent micropollutant in European groundwaters. Aminobacter niigataensis MSH1 is a prime candidate for biologically treating BAM-contaminated groundwater since this organism is capable of utilizing BAM as a carbon and energy source. However, detailed information on the BAM degradation kinetics by MSH1 at trace concentrations is lacking, while this knowledge is required for predicting and optimizing the degradation process. Contaminating assimilable organic carbon (AOC) in media makes the biodegradation experiment a mixed-substrate assay and hampers exploration of pollutant degradation at trace concentrations. In this study, we examined how the BAM concentration affects MSH1 growth and BAM substrate utilization kinetics in a AOC-restricted background to avoid mixed-substrate conditions. Conventional Monod kinetic models were unable to predict kinetic parameters at low concentrations from kinetics determined at high concentrations. Growth yields on BAM were concentration-dependent and decreased substantially at trace concentrations; i.e., growth of MSH1 diminished until undetectable levels at BAM concentrations below 217 µg-C/L. Nevertheless, BAM degradation continued. Decreasing growth yields at lower BAM concentrations might relate to physiological adaptations to low substrate availability or decreased expression of downstream steps of the BAM catabolic pathway beyond 2,6-dichlorobenzoic acid (2,6-DCBA) that ultimately leads to Krebs cycle intermediates for growth and energy conservation.

Retirement age and disability status as pathways to later-life cognitive impairment: Evidence from the Norwegian HUNT Study linked with Norwegian population registers.

BACKGROUND: Research shows that retirement age is associated with later-life cognition but has not sufficiently distinguished between retirement pathways. We examined how retirement age was associated with later-life dementia and mild cognitive impairment (MCI) for people who retired via the disability pathway (received a disability pension prior to old-age pension eligibility) and those who retired via the standard pathway. METHODS: The study sample comprised 7210 participants from the Norwegian Trøndelag Health Study (HUNT4 70+, 2017-2019) who had worked for at least one year in 1967-2019, worked until age 55+, and retired before HUNT4. Dementia and MCI were clinically assessed in HUNT4 70+ when participants were aged 69-85 years. Historical data on participants' retirement age and pathway were retrieved from population registers. We used multinomial regression to assess the dementia/MCI risk for women and men retiring via the disability pathway, or early (<67 years), on-time (age 67, old-age pension eligibility) or late (age 68+) via the standard pathway. RESULTS: In our study sample, 9.5% had dementia, 35.3% had MCI, and 28.1% retired via the disability pathway. The disability retirement group had an elevated risk of dementia compared to the on-time standard retirement group (relative risk ratio [RRR]: 1.64, 95% CI 1.14-2.37 for women, 1.70, 95% CI 1.17-2.48 for men). MCI risk was lower among men who retired late versus on-time (RRR, 0.76, 95% CI 0.61-0.95). CONCLUSION: Disability retirees should be monitored more closely, and preventive policies should be considered to minimize the dementia risk observed among this group of retirees.

Barker's Hypothesis Among the Global Poor: Positive Long-Term Cardiovascular Effects of in Utero Famine Exposure.

An influential literature on the Developmental Origins of Health and Disease (DOHaD) has documented that poor conditions in utero lead to higher risk of cardiovascular disease at older ages. Evidence from low-income countries (LICs) has hitherto been missing, despite the fact that adverse in utero conditions are far more common in LICs. We find that Malawians exposed in utero to the 1949 Nyasaland famine have better cardiovascular health 70 years later. These findings highlight the potential context specificity of the DOHaD hypothesis, with in utero adversity having different health implications among aging LIC individuals who were exposed to persistent poverty.

Union formation, within-couple dynamics, and child well-being: A global macrolevel perspective.

Studies on global changes in families have greatly increased over the past decade, adopting both a country-specific and, more recently, a cross-national comparative perspective. While most studies are focused on the drivers of global changes in families, little comparative research has explored the implications of family processes for the health and well-being of children. This study aims to fill this gap and launch a new research agenda exploring the intergenerational implications of union-formation and within-couple dynamics for children's health and well-being across low- and middle-income countries (LMICs), both globally, regionally, and by the stage of fertility transition. We do so by adopting a macro-level perspective and a multi-axis conceptualization of children's outcomes - health at birth, health in later life, and schooling - and leveraging Demographic and Health Survey and World Bank data across 75 LMICs. Our results show that in societies where partnerships are characterized by more equal status between spouses - i.e., where the age range between spouses and differences in years of schooling between partners are narrower - children fare better on several outcomes. These associations are particularly strong in mid- and high-fertility settings. Despite a series of regularities, our results also highlight a set of findings whereby, at a macro-level, the prevalence of marriage and divorce/separation are not invariably associated with children's outcomes, especially in LMICs where fertility is comparatively lower. We document little cross-regional heterogeneity, primarily highlighting the centrality of demographic factors such as age vis-à-vis, for instance, region-specific characteristics that are more tied to the social fabric of specific societies.

Unequal Transitions to Adulthood: Widening Disparities in Age at First Union, Sex, and Birth in Many Low- and Middle-Income Countries.

Research on the timing of events during the transition to adulthood, such as first union, sex, and birth in low- and middle-income countries (LMICs), focused predominantly on measures of central tendency, notably median or mean ages. In this report, we adopt a different perspective on this topic by examining disparities in the timing of these events in 46 LMICs spanning four decades. Using Demographic and Health Surveys, we estimate ages at which 25 percent, 50 percent, and 75 percent of women have first union, birth, and sex. We compute interquartile ranges to measure within-country variation and disparities in the timing of sexual initiation and family formation. Variation in the timing of first union, birth, and sex generally increases as the median ages at these events increase. Disparities in the timing of first union and birth grew in West Africa and Latin America, and women who experience these events relatively early increasingly lag behind women who experience them relatively late. Documenting trends in measures of central tendency is insufficient to capture the complexity of ongoing changes because they mask growing disparities in the timing of family formation across many LMICs. These results are important for assessing progress toward the achievement of sustainable development goals related to the reduction of early marriages and pregnancies and highlight a need for more holistic approaches to measure the timing of family formation.

Viral suppression among pregnant adolescents and women living with HIV in rural KwaZulu-Natal, South Africa: a cross sectional study to assess progress towards UNAIDS indicators and Implications for HIV Epidemic Control.

BACKGROUND: South Africa has made significant progress in scaling up antiretroviral therapy (ART) to achieve the aspirational goal of HIV epidemic control. The aim of this study was to determine the prevalence of HIV, assess progress towards each of the Joint United Nations Programme on HIV/AIDS (UNAIDS) indicators and determine factors associated with achieving viral suppression among pregnant adolescents and women living with HIV in rural KwaZulu-Natal, South Africa. METHODS: Pregnant adolescents and women, 12 years and older seeking antenatal care at six primary health care clinics were enrolled in a cross-sectional study. Following written informed consent, structured questionnaires were administered, and finger-prick blood samples were collected for HIV antibody testing and viral load measurement. Viral suppression was defined as HIV viral load of < 400 copies per mL. RESULTS: Between Dec 2016 and March 2017, among the 546 enrolled participants, data for 545 were analysed. The overall HIV prevalence was 40.2% [95% Confidence Interval (CI) 36.1-44.3]. Age-stratified prevalence increased from 22.1% (95% CI, 15.9-30.0) in the 14-19 year age group to 63.9% (95% CI, 55.1-71.9) among women ≥ 30 years (Χ2 trend P < 0.0001). Of the HIV positive participants, 84.5% (95% CI, 79.0-88.8) knew their HIV positive status, 98.3% (95% CI 95.1-99.4) who knew their status were on ART, and of those on ART, 95.9% (95% CI 91.8-98.0) were virally suppressed. Among all HIV-positives 90.8% (95% CI, 86.3-94.0) had achieved viral suppression, whilst those in the 14-19 year age group were least likely to be virally suppressed at 82.8% (95% CI 65.5-92.4) compared to those in the older age groups. Married women compared to those unmarried were more likely to have achieved viral suppression (PRR) of 1.11 (95% CI 1.05-1.18), P < 0.001. CONCLUSIONS: The proportion of HIV positive pregnant women achieving viral suppression was encouraging though far short of the target towards achieving epidemic control. Importantly, adolescent pregnant women were less likely to know their HIV status and to achieve viral suppression, underscoring the public health implications of sustained risk of HIV transmission. Thus, greater effort and strong social support are essential to improve HIV knowledge of status and care continuum towards the goal to achieving HIV epidemic control. To "fast-track" the response to achieve HIV epidemic control and end the AIDS epidemic, the Joint United Nations Programme on HIV/AIDS (UNAIDS) set ambitious HIV testing and treatment targets for people living with HIV. Meeting these targets through scaling up testing for HIV, initiating and sustaining antiretroviral therapy (ART) to maintain viral suppression provides both therapeutic and preventive benefits with the potential to reduce HIV transmission. Viral suppression among pregnant adolescents and women living with HIV is crucial for the prevention of mother-to-child transmission of HIV including onward transmission to sexual partners. As a public health approach, in South Africa all pregnant women are offered routine HIV testing and immediate initiation of lifelong ART irrespective of CD4 cell count. It is, therefore, important to ascertain progress towards reaching the targets. The proportion of HIV positive pregnant adolescents and women achieving viral suppression was encouraging though far short of the target towards achieving epidemic control. Importantly, pregnant adolescents were less likely to know their HIV status and to achieve viral suppression, underscoring the public health implications of sustained risk of HIV transmission. Thus, greater effort and strong social support are essential to improve HIV knowledge of status and care continuum towards the goal to achieving HIV epidemic control.

Curtailing Covid-19 on a dollar-a-day in Malawi: Role of community leadership for shaping public health and economic responses to the pandemic.

Utilizing population-based data from the Covid-19 phone survey ( N = 2 , 262 ) of the Malawi Longitudinal Study of Families and Health (MLSFH) collected during June 2nd-August 17th, 2020, we focus on the crucial role that community leadership and trust in institutions played in shaping behavioral, economic and social responses to Covid-19 in this low-income sub-Saharan African context. We argue that the effective response of Malawi to limit the spread of the virus was facilitated by the engagement of local leadership to mobilize communities to adapt and adhere to Covid-19 prevention strategies. Using linear and ordered probit models and controlling for time fixed effects, we show that village heads (VHs) played pivotal role in shaping individuals' knowledge about the pandemic and the adoption of preventive health behaviors and were crucial for mitigating the negative economic and health consequences of the pandemic. We further show that trust in institutions is of particular importance in shaping individuals' behavior during the pandemic, and these findings highlight the pivotal role of community leadership in fostering better compliance and adoption of public health measures essential to contain the virus. Overall, our findings point to distinctive patterns of pandemic response in a low-income sub-Saharan African rural population that emphasized local leadership as mediators of public health messages and policies. These lessons from the first pandemic wave remain relevant as in many low-income countries behavioral responses to Covid-19 will remain the primary prevention strategy for a foreseeable future.

Family change and variation through the lens of family configurations in low- and middle-income countries.

Using 254 Demographic and Health Surveys from 75 low- and middle-income countries, this study shows how the joint examination of family characteristics across rural and urban areas provides new insights for understanding global family change. We operationalise this approach by building family configurations: a set of interrelated features that describe different patterns of family formation and structure. These features include partnership (marriage/unions) regimes and their stability, gender relations, household composition and reproduction. Factorial and clustering techniques allow us to summarise these family features into three factorial axes and six discrete family configurations. We provide an in-depth description of these configurations, their spatial distribution and their changes over time. Global family change is uneven because it emerges from complex interplays between the relative steadiness of longstanding arrangements for forming families and organising gender relations, and the rapidly changing dynamics observed in the realms of fertility, contraception, and timing of family formation.

Returning to school after COVID-19 closures: Who is missing in Malawi?

During the early stages of the COVID-19 pandemic, almost all countries implemented school closures to prevent disease transmission. However, prolonged closures can put children at risk of leaving school permanently, a decision that can reduce their long-term potential and income. This study investigated the extent to which the COVID-19 pandemic and associated school closures reduced school attendance in Malawi, a low-income African country. We used longitudinal data from a cohort of adolescents interviewed before (2017/18; at age 10-16) and after (2021; at age 13-20) the pandemic school closures. Of those students who had been attending school prior to school closures, we find that 86% returned when schools re-opened. Dropouts were more pronounced among older girls: over 30% of those aged 17-19 did not return to school. This resulted in further lowering the gender parity index to the greater disadvantage of girls. We also found that students already lagging behind in school were more likely to dropout. Thus, our data suggest that the COVID-19 pandemic has magnified gender inequalities in schooling, at least partially erasing recent progress towards inclusive education. Urgent investments are needed to find and re-enroll lost students now, and to create more resilient and adaptable educational systems before the next pandemic or other negative shock arrives.

Prevalence and Risk Factors for HIV Infection Among Heterosexual Men Recruited from Socializing Venues in Rural KwaZulu-Natal, South Africa.

Young heterosexual men have low uptake of HIV prevention and treatment services and represent an important key population that may require novel strategies. We recruited 1271 heterosexual men, 12 years and older from socializing venues such as "shebeens", transport hubs, "spaza" shops, and community centers in rural KwaZulu-Natal, South Africa. Participants completed a questionnaire and were tested for HIV serostatus. Generalized estimating equations (GEE) with exchangeable covariance structure estimated factors independently associated with prevalent HIV infection. Median age was 25 years [Interquartile range (IQR) 21-29]. HIV prevalence was 15.5% [95% confidence interval (CI) 11.0-21.9] and increased significantly by age. Factors associated with higher odds of HIV infection were being 25 years and older [adjusted odds ratio (aOR) 4.82, 95% CI 3.47-6.69; p < 0.001), not completing high school (aOR 1.60, 95% CI 1.39-1.85; p < 0.001), not using condoms at first sex (aOR 1.43, 95% CI 1.20-1.70; p < 0.001), consuming alcohol (aOR 1.63, 95% CI 1.15-2.31; p = 0.006) or substances (aOR 1.37, 95% CI 1.31-1.44; p < 0.001), and absence of medical circumcision (aOR 2.05, 95% CI 1.71-2.44; p < 0.001). Risk was lower among those testing for HIV in last 12 months (aOR 0.54, 95% CI 0.36-0.80; p = 0.002). Greater effort is needed to implement innovative programs within settings that are easily accessible and where heterosexual men are likely to be.

Genetic risks, adolescent health, and schooling attainment.

We provide new evidence on the effect of adolescent health behaviors/outcomes (obesity, depression, smoking, and attention deficit hyperactivity disorder [ADHD]) on schooling attainment using the National Longitudinal Study of Adolescent to Adult Health. We take two different approaches to deal with omitted variable bias and reverse causality. Our first approach attends to the issue of reverse causality by estimating the effect of health polygenic scores (PGSs) on schooling. Second, we estimate the effect of adolescent health using sibling fixed-effects models that control for unmeasured genetic and family factors shared by siblings. We use the PGSs as additional controls in the sibling fixed-effects models to reduce concerns about residual confounding from sibling-specific genetic differences. We find consistent evidence across both approaches that being genetically predisposed to smoking and smoking regularly in adolescence reduces schooling attainment. Estimates for depression are more imprecise, but also suggest that a high genetic risk of depression and adolescent depression reduce schooling attainment. We find mixed evidence for ADHD. Our estimates suggest that having a high genetic risk for ADHD reduces grades of schooling, but we do not find any statistically significant negative effects of ADHD. Finally, we find no consistent evidence for a detrimental effect of obesity on schooling attainment.

Marital dissolutions and changes in mental health: Evidence from rural Malawi.

BACKGROUND: Advancing efforts to unpack the complex relationship between marital dissolutions and health outcomes increasingly requires assessing the marital histories and health of individuals who have lived long enough to experience divorce or widowhood ‒ or even multiples of each ‒ and measurable changes in health. OBJECTIVE: To explore this line of inquiry, we chose a sample from rural Malawi where a high prevalence of marital dissolutions and remarrying exists, as an ideal theoretical foil to the predominant literature found in high-income countries (HICs). We examine if changes in having experienced a marital dissolution, one's total number of dissolutions, and the percentage of one's life spent outside of marriage since first becoming married are associated with changes in mental health. METHODS: Our analyses focus on 1,266 respondents aged 45 years and older who participated in the 2012 Mature Adults Cohort of the Malawi Longitudinal Study of Families and Health (MLSFH-MAC), linked back to cohort information from 2008 and 2010 available through the MLSFH. Fixed-effects regressions guide our inferences over the 2008, 2010, and 2012 waves of data. RESULTS: For men, spending more life outside of marriage is associated with worse mental health, while more marital dissolutions are surprisingly associated with better mental health for women. CONCLUSIONS: These results could suggest that larger portions of one's life spent unmarried are associated with a type of role strain for men or simply that men are burdened with taking up tasks that their spouses had previously done in order to survive. For women, many may have gotten out of 'bad' marriages that otherwise would have been detrimental to their mental health and/or those in good mental health are the ones able to remarry. CONTRIBUTIONS: Our research from rural Malawi provides a type of litmus test for many HICs where marriage, remarriage, and dissolution rates are lower but quite consequential for mental health outcomes. Measuring time outside of marriage should be more strongly considered in such settings. These results also inform increasingly important research on the relationship between marital dissolutions and mental health in other African nations as noncommunicable diseases play a continually more important role in people's lives.

Interest in HIV pre-exposure prophylaxis (PrEP) among adolescents and their caregivers in Malawi.

Over a third of new HIV infections occur in adolescents aged 10-19 globally. Pre-exposure prophylaxis (PrEP) could be a powerful tool for prevention. Understanding more about the drivers of PrEP interest could inform implementation strategies among this age group. Moreover, family dynamics may play a uniquely critical role for this younger age group, thus it is important to gauge whether caregivers would support their children's use of PrEP. We surveyed 2,089 adolescents (aged 10-16) and their caregivers in Malawi during 2017-2018. Data were collected on PrEP interest, factors that may facilitate PrEP use, and preferences for PrEP modality. We used multivariate logistic regression to estimate the association between the above characteristics and PrEP interest. We find that young adolescents are engaging in behaviors that would put them at substantial risk of acquiring HIV, would likely benefit from PrEP, are largely (82%) interested in using such, would prefer to get an injection over taking a daily pill, and are considerably discouraged by the prospect of side effects. Endorsement by caregivers was even greater (87%). Our findings demonstrate initial support for adolescent PrEP, and suggest parents may be a surprising advocate.

Quantification of Synthetic Polyesters from Biodegradable Mulch Films in Soils.

Soil biodegradable mulch films composed of the polyester polybutylene adipate-co-terephthalate (PBAT) are being increasingly used in agriculture. Analytical methods to quantify PBAT in field soils are needed to assess its soil occurrence and fate. Here, we report an analytical method for PBAT in soils that couples Soxhlet extraction or accelerated solvent extraction (ASE) with quantitative protonnuclear magnetic resonance (q-1H NMR) spectroscopy detection. The 1H NMR peak areas of aromatic PBAT protons increased linearly with PBAT concentrations dissolved in deuterated chloroform (CDCl3), demonstrating accurate quantitation of PBAT by q-1H NMR. Spike-recovery experiments involving PBAT addition to model sorbents and soils showed increased PBAT extraction efficiencies into chloroform (CHCl3) with methanol (MeOH) as cosolvent, consistent with MeOH competitively displacing PBAT from H-bond donating sites on mineral surfaces. Systematic variations in solvent composition and temperatures in ASE revealed quantitative PBAT extraction from soil with 90/10 volume % CHCl3/MeOH at 110-120 °C. Both Soxhlet extraction and ASE resulted in the complete recovery of PBAT added to a total of seven agricultural soils covering a range of physicochemical properties, independent of whether PBAT was added to soils dissolved in CHCl3, as film, or as particles. Recovery was also complete for PBAT added to soil in the form of a commercial soil biodegradable mulch film with coextractable polylactic acid (PLA). The presented analytical method enables accurate quantification and biodegradation monitoring of PBAT in agricultural field soils.

The Psychological Benefits of Marriage and Children in Rural Malawi.

Despite an extensive literature on the psychological rewards of marriage and children in high-income countries, research on these relationships in low-income countries remains limited. This paper draws on data from 4,133 adult women and men interviewed in the Malawi Longitudinal Study of Families and Health to examine how marital status, categorized as never, formerly, monogamously, and polygynously married, and number of children are associated with psychological well-being. With respect to marital status, we find that women in polygynous unions fare worse than monogamously married women and this detrimental effect is stronger for women than for men. Formerly married men and women of reproductive age experience the worst psychological outcomes, although this association wanes with age. In contrast, the benefits of having children is only evident among older Malawian women. These findings offer novel insights into the patterns of nearly universal marriage and high fertility that characterize Malawi and much of sub-Saharan Africa.

Enzyme Kinetics of Organic Contaminant Oxygenations.

Enzymatic oxygenations initiate biodegradation processes of many organic soil and water contaminants. Even though many biochemical aspects of oxygenation reactions are well-known, quantifying rates of oxidative contaminant removal as well as the extent of oxygenation remains a major challenge. Because enzymes use different strategies to activate O2, reactions leading to substrate oxygenation are not necessarily limiting the rate of contaminant removal. Moreover, oxygenases react along unproductive pathways without substrate metabolism leading to O2 uncoupling. Here, we identify the critical features of the catalytic cycles of selected oxygenases that determine rates and extents of biodegradation. We focus most specifically on Rieske dioxygenases, a subfamily of mononuclear non-heme ferrous iron oxygenases, because of their ability to hydroxylate unactivated aromatic structures and thus initiate the transformation of the most persistent organic contaminants. We illustrate that the rate-determining steps in their catalytic cycles range from O2 activation to substrate hydroxylation, depending on the extent of O-O cleavage that is required for generating the reactive Fe-oxygen species. The extent of O2 uncoupling, on the other hand, is highly substrate-specific and potentially modulated by adaptive responses to oxidative stress. Understanding the kinetic mechanisms of oxygenases will be key to assess organic contaminant biotransformation quantitatively.

Assessing Aerobic Biotransformation of Hexachlorocyclohexane Isomers by Compound-Specific Isotope Analysis.

Contamination of soils and sediments with the highly persistent hexachlorocyclohexanes (HCHs) continues to be a threat for humans and the environment. Despite the existence of bacteria capable of biodegradation and cometabolic transformation of HCH isomers, such processes occur over time scales of decades and are thus challenging to assess. Here, we explored the use of compound-specific isotope analysis (CSIA) to track the aerobic biodegradation and biotransformation pathways of the most prominent isomers, namely, (-)-α-, (+)-α-, ß-, γ-, and δ-HCH, through changes of their C and H isotope composition in assays of LinA2 and LinB enzymes. Dehydrochlorination of (+)-α-, γ-, and δ-HCH catalyzed by LinA2 was subject to substantial C and H isotope fraction with apparent 13C- and 2H-kinetic isotope effects (AKIEs) of up to 1.029 ± 0.001 and 6.7 ± 2.9, respectively, which are indicative of bimolecular eliminations. Hydrolytic dechlorination of δ-HCH by LinB exhibited even larger C but substantially smaller H isotope fractionation with 13C- and 2H-AKIEs of 1.073 ± 0.006 and 1.41 ± 0.04, respectively, which are typical for nucleophilic substitutions. The systematic evaluation of isomer-specific phenomena showed that, in addition to contaminant uptake limitations, diffusion-limited turnover ((-)-α-HCH), substrate dissolution (ß-HCH), and potentially competing reactions catalyzed by constitutively expressed enzymes might bias the assessment of HCH biodegradation by CSIA at contaminated sites.

Kinetic Isotope Effects of the Enzymatic Transformation of γ-Hexachlorocyclohexane by the Lindane Dehydrochlorinase Variants LinA1 and LinA2.

Compound-specific isotope analysis (CSIA) can provide insights into the natural attenuation processes of hexachlorocyclohexanes (HCHs), an important class of persistent organic pollutants. However, the interpretation of HCH stable isotope fractionation is conceptually challenging. HCHs exist as different conformers that can be converted into each other, and the enzymes responsible for their transformation discriminate among those HCH conformers. Here, we investigated the enzyme specificity of apparent 13C- and 2H-kinetic isotope effects (AKIEs) associated with the dehydrochlorination of γ-HCH (lindane) by two variants of the lindane dehydrochlorinases LinA1 and LinA2. While LinA1 and LinA2 attack γ-HCH at different trans-1,2-diaxial H-C-C-Cl moieties, the observed C and H isotope fractionation was large, typical for bimolecular eliminations, and was not affected by conformational mobility. 13C-AKIEs for transformation by LinA1 and LinA2 were the same (1.024 ± 0.001 and 1.025 ± 0.001, respectively), whereas 2H-AKIEs showed minor differences (2.4 ± 0.1 and 2.6 ± 0.1). Variations of isotope effects between LinA1 and LinA2 are small and in the range reported for different degrees of C-H bond cleavage in transition states of dehydrochlorination reactions. The large C and H isotope fractionation reported here for experiments with pure enzymes contrasts with previous observations from whole cell experiments and suggests that specific uptake processes by HCH-degrading microorganisms might modulate the observable HCH isotope fractionation at contaminated sites.

Aminobacter sp. MSH1 Mineralizes the Groundwater Micropollutant 2,6-Dichlorobenzamide through a Unique Chlorobenzoate Catabolic Pathway.

2,6-Dichlorobenzamide (BAM) is a major groundwater micropollutant posing problems for drinking water treatment plants (DWTPs) that depend on groundwater intake. Aminobacter sp. MSH1 uses BAM as the sole source of carbon, nitrogen, and energy and is considered a prime biocatalyst for groundwater bioremediation in DWTPs. Its use in bioremediation requires knowledge of its BAM-catabolic pathway, which is currently restricted to the amidase BbdA converting BAM into 2,6-dichlorobenzoic acid (2,6-DCBA) and the monooxygenase BbdD transforming 2,6-DCBA into 2,6-dichloro-3-hydroxybenzoic acid. Here, we show that the 2,6-DCBA catabolic pathway is unique and differs substantially from catabolism of other chlorobenzoates. BbdD catalyzes a second hydroxylation, forming 2,6-dichloro-3,5-dihydroxybenzoic acid. Subsequently, glutathione-dependent dehalogenases (BbdI and BbdE) catalyze the thiolytic removal of the first chlorine. The remaining chlorine is then removed hydrolytically by a dehalogenase of the α/ß hydrolase superfamily (BbdC). BbdC is the first enzyme in that superfamily associated with dehalogenation of chlorinated aromatics and appears to represent a new subtype within the α/ß hydrolase dehalogenases. The activity of BbdC yields a unique trihydroxylated aromatic intermediate for ring cleavage that is performed by an extradiol dioxygenase (BbdF) producing 2,4,6-trioxoheptanedioic acid, which is likely converted to Krebs cycle intermediates by BbdG.