Patterns of recovery in extant and extirpated seabirds after the world's largest multipredator eradication.

Eradicating invasive predators from islands can result in substantial recovery of seabirds, but the mechanisms that drive population changes remain poorly understood. Meta-analyses have recently revealed that immigration is surprisingly important to the recovery of philopatric seabirds, but it is not known whether dispersal and philopatry interact predictably to determine rates of population growth and changes of distribution. We used whole-island surveys and long-term monitoring plots to study the abundance, distribution, and trends of 4 burrowing seabird species on Macquarie Island, Australia, to examine the legacy impacts of invasive species and ongoing responses to the world's largest eradication of multiple species of vertebrates. Wekas (Gallirallus australis) were eradicated in 1988; cats (Felis catus) in 2001; and rabbits (Oryctolagus cuniculus), black rats (Rattus rattus), and mice (Mus mus) in 2011-2014. We compared surveys from 1976-1979 and 2017-2018 and monitoring from the 1990s and 2000s onward. Antarctic prions (Pachyptila desolata) and white-headed petrels (Pterodroma lessonii) increased ∼1% per year. Blue petrels (Halobaena caerulea) and gray petrels (Procellaria cinerea) recolonized following extirpation from the main island in the 1900s but remained spatially and numerically rare in 2018. However, they increased rapidly at 14% and 10% per year, respectively, since cat eradication in 2001. Blue and gray petrel recolonization occurred on steep, dry, west-facing slopes close to ridgelines at low elevation (i.e., high-quality petrel habitat). They overlapped <5% with the distribution of Antarctic prion and white-headed petrels which occurred in suboptimal shallow, wet, east-facing slopes at high elevation. We inferred that the speed of population growth of recolonizing species was related to their numerically smaller starting size compared with the established species and was driven by immigration and selection of ideal habitat.

Patrones de recuperación en aves marinas existentes y extirpadas después de la mayor erradicación mundial de multidepredadores Resumen La erradicación de depredadores invasores en las islas puede derivar en la recuperación sustancial de aves marinas, aunque entendemos muy poco los mecanismos que causan los cambios poblacionales. Los metaanálisis recientes han revelado que la inmigración es de gran importancia para la recuperación de aves marinas filopátricas, aunque no sabemos si la dispersión y la filopatría interactúan de forma predecible para poder determinar las tasas de crecimiento poblacional y los cambios en la distribución. Aplicamos censos de isla completa y parcelas de monitoreo a largo plazo para estudiar la abundancia, distribución y tendencias de cuatro especies de aves marinas cavadoras en la Isla Macquarie, Australia, para analizar los impactos heredados de las especies invasoras y la respuesta continua a la mayor erradicación mundial de varias especies de vertebrados. El rascón weka (Gallirallus australis) se erradicó en 1988; los gatos (Felis catus) en 2001; y los conejos (Oryctolagus cuniculus), ratas (Rattus rattus) y ratones (Mus mus) entre 2011 y 2014. Comparamos los censos de 1976-1979 y 2017-2018 y el monitoreo realizado en los 90s y del año 2000 en adelante. El pato petrel antártico (Pachyptila desolata) y el petrel cabeciblanco (Pterodroma lessonii) incrementaron ∼1% por año. El petrel azulado (Halobaena caerulea) y la pardela gris (Procellaria cinerea) recolonizaron la isla después de su extirpación en la década de 1900, pero todavía eran especies raras espacial y numéricamente en 2018. Sin embargo, esta especie incrementó rápidamente en un 14% y 10% por año respectivamente desde que se erradicaron los gatos en 2001. La recolonización ocurrió desde las laderas empinadas, secas y con orientación al oeste en los sistemas montañosos de baja elevación (es decir, hábitats de gran calidad para los petreles). La distribución del petrel azulado y la pardela gris ocurrió en laderas someras subóptimas y húmedas con orientación al este a altas elevaciones. Esta distribución se traslapó menos del 5% con la del pato petrel antártico y la del petrel cabeciblanco. Inferimos que la velocidad del crecimiento poblacional de las especies que recolonizaron estuvo relacionada con el menor tamaño inicial en comparación con las especies establecidas y fue causada por la inmigración y la selección del hábitat ideal.

A gel electrophoresis-based assay for measuring enzymatic RNA decapping activity.

RNA 5' ends are remarkably heterogeneous. In addition to the eukaryotic 5' methyl-7-Guanosine (m7G) cap, a number of primarily metabolite-based cap structures have been identified both in prokaryotic and eukaryotic systems. These metabolite caps include Nicotinamide Adenine Dinucleotide (NAD+/NADH), dephosphoCoenzyme A (dpCoA), Flavin Adenine Dinucleotide (FAD), dinucleotide polyphosphates and Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) (Chen et al., 2009; Kowtoniuk et al., 2009; Wang et al., 2019). The most highly studied of these new cap structures, 5' NAD, has significant effects on RNA stability (Bird et al., 2016; Jiao et al., 2017). Both prokaryotes and eukaryotes have decapping enzymes specific to these metabolite caps and decapping is an integral step in the control of RNA stability (Cahová et al., 2015; Jiao et al., 2017; Sharma et al., 2020; Zhang et al., 2020). To better study how these 5' metabolite RNAs are decapped, we present a method to (1) generate radiolabeled dinucleotide and "full length" 5' capped RNA substrates for use in decapping assays, (2) a simple decapping assay to test the activity of various enzymes on different 5' capped transcripts and (3) a gel electrophoresis-based method for the visualization and differentiation of 5' capped transcripts.

Guidelines for selecting an appropriate currency in biodiversity offset transactions.

When designing biodiversity offset transactions, selecting the appropriate currency for measuring losses and gains to biodiversity is crucial. Poorly designed currencies reduce the likelihood that the proposed offset will sufficiently compensate for the development impact on the affected biota. We present a framework for identifying appropriate offset currencies for terrestrial biodiversity features, either vegetation communities or particular species. The guidelines were developed based on a review of issues and solutions presented in the existing literature, including government policies and guidance. We assert that while benchmark-based condition scores provide a suitable offset transaction currency for vegetation communities, this approach is also commonly applied to individual species based on the often-unproven assumption that vegetation quality is a proxy for the value of a site to that species. We argue that species are better served by species-specific currencies based on either species abundance, or the suitability and amount of the habitat available. For species where it is practical and meaningful to measure the abundance on site, an abundance-based currency using either directly observable or proxy indicators is the most representative measure of the net impact on the species. In other instances, such as when species are difficult to locate, or not reliably present on site, a currency based on the quality and amount of habitat is preferable. The habitat-quality component should be measured relative to its value for the species, with the most important attributes weighted accordingly. Ensuring the currency used in biodiversity offset transactions is practical to measure, and relevant to the species or vegetation community is an important step in minimising the net biodiversity losses from unavoidable impacts.

In transcription antitermination by Qλ, NusA induces refolding of Qλ to form a nozzle that extends the RNA polymerase RNA-exit channel.

Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and they translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein of bacteriophage 21 (Q21) functions by forming a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein of bacteriophage λ (Qλ), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Qλ, the Qλ-QBE complex, the NusA-free pre-engaged Qλ-loading complex, and the NusA-containing engaged Qλ-loading complex. The results show that Qλ, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Qλ has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Qλ to form a pre-engaged loading complex, followed by NusA-facilitated refolding of Qλ to form an engaged loading complex. The results establish that Qλ and Q21 are not structural homologs and are solely functional analogs.

Generation lengths of the world's birds and their implications for extinction risk.

Birds have been comprehensively assessed on the International Union for Conservation of Nature (IUCN) Red List more times than any other taxonomic group. However, to date, generation lengths have not been systematically estimated to scale population trends when undertaking assessments, as required by the criteria of the IUCN Red List. We compiled information from major databases of published life-history and trait data for all birds and imputed missing life-history data as a function of species traits with generalized linear mixed models. Generation lengths were derived for all species, based on our modeled values of age at first breeding, maximum longevity, and annual adult survival. The resulting generation lengths varied from 1.42 to 27.87 years (median 2.99). Most species (61%) had generation lengths <3.33 years, meaning that the period of 3 generations-over which population declines are assessed under criterion A-was <10 years, which is the value used for IUCN Red List assessments of species with short generation times. For these species, our trait-informed estimates of generation length suggested that 10 years is a robust precautionary value for threat assessment. In other cases, however, for whole families, genera, or individual species, generation length had a substantial impact on their estimated extinction risk, resulting in higher extinction risk in long-lived species than in short-lived species. Although our approach effectively addressed data gaps, generation lengths for some species may have been underestimated due to a paucity of life-history data. Overall, our results will strengthen future extinction-risk assessments and augment key databases of avian life-history and trait data.

Duraciones Generacionales de las Aves del Mundo y sus Implicaciones para el Riesgo de Extinción Resumen Las aves han sido valoradas integralmente en la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza (UICN) más veces que cualquier otro grupo taxonómico. Sin embargo, a la fecha, las duraciones generacionales no han sido estimadas sistemáticamente para escalar las tendencias poblacionales cuando se realizan las valoraciones, como lo requieren los criterios de la Lista Roja de la UICN. Compilamos información a partir de las principales bases de datos de historias de vida y datos de características publicadas para todas las aves e imputamos los datos faltantes de historias de vida como una función de las características de especies con modelos lineales mixtos generalizados. La duración por generación estuvo derivada para todas las especies con base en nuestros valores modelados de edad durante la primera reproducción, la longevidad máxima y la supervivencia anual de adultos. La duración por generación resultante varió de 1.42 a 27.87 años (mediana: 2.99). La mayoría de las especies (61%) tuvo una duración generacional <3.33 años, lo que significa que el periodo de tres generaciones - durante el cual se valoran las declinaciones poblacionales bajo el Criterio A - es <10 años, el cual es el valor usado por la Lista Roja de la UICN para la valoración de especies con tiempos generacionales cortos. Para estas especies, nuestras estimaciones de duración por generación informados por características sugieren que diez años es un valor preventivo sólido para la valoración de amenazas. Para otros casos, sin embargo, como familias o géneros enteros o especies individuales, la duración generacional tuvo un impacto sustancial sobre su riesgo de extinción estimado, resultando así en un riesgo de extinción más elevado para las especies con mayor longevidad que aquellas especies con una menor longevidad. Aunque nuestra estrategia lidió efectivamente con los vacíos en los datos, la duración generacional para algunas especies podría estar subestimada debido a la escasez de datos de historia de vida. En general, nuestros resultados fortalecerán las futuras valoraciones de extinción de riesgo y aumentarán las bases de datos importantes de la historia de vida de las aves y los datos de características.

Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase.

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

"NAD-capQ" detection and quantitation of NAD caps.

RNA 5' cap structures comprising the metabolic effector nicotinamide adenine dinucleotide (NAD) have been identified in diverse organisms. Here we report a simple, two-step procedure to detect and quantitate NAD-capped RNA, termed "NAD-capQ." By use of NAD-capQ we quantitate NAD-capped RNA levels in Escherichia coli, Saccharomyces cerevisiae, and human cells, and we measure increases in NAD-capped RNA levels in cells from all three organisms harboring disruptions in their respective "deNADding" enzymes. We further show that NAD-capped RNA levels in human cells respond to changes in cellular NAD concentrations, indicating that NAD capping provides a mechanism for human cells to directly sense and respond to alterations in NAD metabolism. Our findings establish NAD-capQ as a versatile, rapid, and accessible methodology to detect and quantitate 5'-NAD caps on endogenous RNA in any organism.

CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD.

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs.

It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3'-desphospho-coenzyme A (dpCoA), can serve as 'non-canonical initiating nucleotides' (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA polymerases (RNAPs) and that the efficiency of the reaction is determined by promoter sequence (Bird et al., 2016). Here we describe a protocol to quantify the relative efficiencies of transcription initiation using an NCIN vs. transcription initiation using a nucleoside triphosphate (NTP) for a given promoter sequence.

5' End Nicotinamide Adenine Dinucleotide Cap in Human Cells Promotes RNA Decay through DXO-Mediated deNADding.

Eukaryotic mRNAs generally possess a 5' end N7 methyl guanosine (m7G) cap that promotes their translation and stability. However, mammalian mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD+) cap that, in contrast to the m7G cap, does not support translation but instead promotes mRNA decay. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD+ caps, and cocrystal structures of DXO/Rai1 with 3'-NADP+ illuminate the molecular mechanism for how the "deNADding" reaction produces NAD+ and 5' phosphate RNA. Removal of DXO from cells increases NAD+-capped mRNA levels and enables detection of NAD+-capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD+ caps can be added to 5'-processed termini. Our findings establish NAD+ as an alternative mammalian RNA cap and DXO as a deNADding enzyme modulating cellular levels of NAD+-capped RNAs. Collectively, these data reveal that mammalian RNAs can harbor a 5' end modification distinct from the classical m7G cap that promotes rather than inhibits RNA decay.

Sustainable intensification of agriculture for human prosperity and global sustainability.

There is an ongoing debate on what constitutes sustainable intensification of agriculture (SIA). In this paper, we propose that a paradigm for sustainable intensification can be defined and translated into an operational framework for agricultural development. We argue that this paradigm must now be defined-at all scales-in the context of rapidly rising global environmental changes in the Anthropocene, while focusing on eradicating poverty and hunger and contributing to human wellbeing. The criteria and approach we propose, for a paradigm shift towards sustainable intensification of agriculture, integrates the dual and interdependent goals of using sustainable practices to meet rising human needs while contributing to resilience and sustainability of landscapes, the biosphere, and the Earth system. Both of these, in turn, are required to sustain the future viability of agriculture. This paradigm shift aims at repositioning world agriculture from its current role as the world's single largest driver of global environmental change, to becoming a key contributor of a global transition to a sustainable world within a safe operating space on Earth.

The mechanism of RNA 5' capping with NAD+, NADH and desphospho-CoA.

The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency, and has been proposed to provide a layer of 'epitranscriptomic' gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate 'cap' in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated 'ab initio capping' may occur in all organisms.

A universal transcription pause sequence is an element of initiation factor σ70-dependent pausing.

The Escherichia coli σ70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, σ70 acts at other sites during transcription elongation as well. The pause is induced by σ70 binding to a repeat of the promoter -10 sequence. After σ70 binding, further RNA synthesis occurs as DNA is drawn (or 'scrunched') into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when σ70 first engages the -10 sequence repeat. We show that the actual pause site in the stabilized, scrunched complex is the 'elemental pause sequence' recognized from its frequent occurrence in the E. coli genome. σ70 binding and the elemental pause sequence together, but neither alone, produce a substantial transcription pause.

Multiplexed protein-DNA cross-linking: Scrunching in transcription start site selection.

In bacterial transcription initiation, RNA polymerase (RNAP) selects a transcription start site (TSS) at variable distances downstream of core promoter elements. Using next-generation sequencing and unnatural amino acid-mediated protein-DNA cross-linking, we have determined, for a library of 4(10) promoter sequences, the TSS, the RNAP leading-edge position, and the RNAP trailing-edge position. We find that a promoter element upstream of the TSS, the "discriminator," participates in TSS selection, and that, as the TSS changes, the RNAP leading-edge position changes, but the RNAP trailing-edge position does not change. Changes in the RNAP leading-edge position, but not the RNAP trailing-edge position, are a defining hallmark of the "DNA scrunching" that occurs concurrent with RNA synthesis in initial transcription. We propose that TSS selection involves DNA scrunching prior to RNA synthesis.

The lysis cassette of DLP12 defective prophage is regulated by RpoE.

Expression of the lysis cassette (essD, ybcT, rzpD/rzoD) from the defective lambdoid prophage at the 12th minute of Escherichia coli's genome (DLP12) is required in some strains for proper curli expression and biofilm formation. Regulating production of the lytic enzymes encoded by these genes is critical for maintaining cell wall integrity. In lambdoid phages, late-gene regulation is mediated by the vegetative sigma factor RpoD and the lambda antiterminator Qλ. We previously demonstrated that DLP12 contains a Q-like protein (QDLP12) that positively regulates transcription of the lysis cassette, but the sigma factor responsible for this transcription initiation remained to be elucidated. In silico analysis of essDp revealed the presence of a putative - 35 and - 10 sigma site recognized by the extracytoplasmic stress response sigma factor, RpoE. In this work, we report that RpoE overexpression promoted transcription from essDp in vivo, and in vitro using purified RNAP. We demonstrate that the - 35 region is important for RpoE binding in vitro and that this region is also important for QDLP12-mediated transcription of essDp in vivo. A bacterial two-hybrid assay indicated that QDLP12 and RpoE physically interact in vivo, consistent with what is seen for Qλ and RpoD. We propose that RpoE regulates transcription of the DLP12 lysis genes through interaction with QDLP12 and that proper expression is dependent on an intact - 35 sigma region in essDp. This work provides evidence that the unique Q-dependent regulatory mechanism of lambdoid phages has been co-opted by E. coli harbouring defective DLP12 and has been integrated into the tightly controlled RpoE regulon.

Prioritizing islands for the eradication of invasive vertebrates in the United Kingdom overseas territories.

Invasive alien species are one of the primary threats to native biodiversity on islands worldwide. Consequently, eradicating invasive species from islands has become a mainstream conservation practice. Deciding which islands have the highest priority for eradication is of strategic importance to allocate limited resources to achieve maximum conservation benefit. Previous island prioritizations focused either on a narrow set of native species or on a small geographic area. We devised a prioritization approach that incorporates all threatened native terrestrial vertebrates and all invasive terrestrial vertebrates occurring on 11 U.K. overseas territories, which comprise over 2000 islands ranging from the sub-Antarctic to the tropics. Our approach includes eradication feasibility and distinguishes between the potential and realistic conservation value of an eradication, which reflects the benefit that would accrue following eradication of either all invasive species or only those species for which eradication techniques currently exist. We identified the top 25 priority islands for invasive species eradication that together would benefit extant populations of 155 native species including 45 globally threatened species. The 5 most valuable islands included the 2 World Heritage islands Gough (South Atlantic) and Henderson (South Pacific) that feature unique seabird colonies, and Anegada, Little Cayman, and Guana Island in the Caribbean that feature a unique reptile fauna. This prioritization can be rapidly repeated if new information or techniques become available, and the approach could be replicated elsewhere in the world.

Interactions between RNA polymerase and the "core recognition element" counteract pausing.

Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G₋10Y₋1G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.

The impact of conservation on the status of the world's vertebrates.

Using data for 25,780 species categorized on the International Union for Conservation of Nature Red List, we present an assessment of the status of the world's vertebrates. One-fifth of species are classified as Threatened, and we show that this figure is increasing: On average, 52 species of mammals, birds, and amphibians move one category closer to extinction each year. However, this overall pattern conceals the impact of conservation successes, and we show that the rate of deterioration would have been at least one-fifth again as much in the absence of these. Nonetheless, current conservation efforts remain insufficient to offset the main drivers of biodiversity loss in these groups: agricultural expansion, logging, overexploitation, and invasive alien species.

Functional analysis of CbpA, a DnaJ homolog and nucleoid-associated DNA-binding protein.

DnaK/Hsp70 proteins are universally conserved ATP-dependent molecular chaperones that help proteins adopt and maintain their native conformations. DnaJ/Hsp40 and GrpE are co-chaperones that assist DnaK. CbpA is an Escherichia coli DnaJ homolog. It acts as a multicopy suppressor for dnaJ mutations and functions in vitro in combination with DnaK and GrpE in protein remodeling reactions. CbpA binds nonspecifically to DNA with preference for curved DNA and is a nucleoid-associated protein. The DNA binding and co-chaperone activities of CbpA are modulated by CbpM, a small protein that binds specifically to CbpA. To identify the regions of CbpA involved in the interaction of CbpA with CbpM and those involved in DNA binding, we constructed and characterized deletion and substitution mutants of CbpA. We discovered that CbpA interacted with CbpM through its N-terminal J-domain. We found that the region C-terminal to the J-domain was required for DNA binding. Moreover, we found that the CbpM interaction, DNA binding, and co-chaperone activities were separable; some mutants were proficient in some functions and defective in others.

Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators.

DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.