Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

Most studies of bacterial denitrification have used nitrate (NO₃⁻) as the first electron acceptor, whereas relatively less is understood about nitrite (NO₂⁻) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO₂⁻ (72 mM). Strain YD50.2, among several isolates, was taxonomically positioned within the α subclass of Proteobacteria and identified as Ochrobactrum anthropi YD50.2. This strain denitrified NO₂⁻, as well as NO₃⁻. The gene clusters for denitrification (nar, nir, nor, and nos) were cloned from O. anthropi YD50.2, in which the nir and nor operons were linked. We confirmed that nirK in the nir-nor operon produced a functional NO₂⁻ reductase containing copper that was involved in bacterial NO₂⁻ reduction. The strain denitrified up to 40 mM NO₂⁻ to dinitrogen under anaerobic conditions in which other denitrifiers or NO₃⁻ reducers such as Pseudomonas aeruginosa and Ralstonia eutropha and nitrate-respiring Escherichia coli neither proliferated nor reduced NO₂⁻. Under nondenitrifying aerobic conditions, O. anthropi YD50.2 and its type strain ATCC 49188^T proliferated even in the presence of higher levels of NO₂⁻ (100 mM), and both were considerably more resistant to acidic NO₂⁻ than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.Environmental bacteria maintain the global nitrogen cycle by metabolizing organic and inorganic nitrogen compounds. Denitrification is critical for maintenance of the global nitrogen cycle, through which nitrate (NO₃⁻) or nitrite (NO₂⁻) is reduced to gaseous nitrogen forms such as N₂ and nitrous oxide (N₂O) (19, 47). Decades of investigations into denitrifying bacteria have revealed their ecological impact (9), their molecular mechanisms of denitrification (13, 25, 47), and the industrial importance of removing nitrogenous contaminants from wastewater (31, 36). Bacterial denitrification is considered to comprise four successive reduction steps, each of which is catalyzed by NO₃⁻ reductase (Nar), NO₂⁻ reductase (Nir), nitric oxide (NO) reductase (Nor), and N₂O reductase (Nos). The reaction of each enzyme is linked to the electron transport chain on the cellular membrane and accompanies oxidative phosphorylation, implying that bacterial denitrification is of as much physiological significance as anaerobic respiration (25, 47). Most denitrifying bacteria are facultative anaerobes and respire with oxygen under aerobic conditions. Because denitrification is induced in the absence of oxygen, it is considered an alternative mechanism of energy conservation that has evolved as an adaptation to anaerobic circumstances (13, 47).Nitrite and NO are hazardous to bacteria, since they generate highly reactive nitrogen species (RNS) under physiological conditions and damage cellular DNA, lipid, and proteins (28, 37). Denitrifying bacteria are thought to be threatened by RNS since they reduce NO₃⁻ to generate NO₂⁻ and NO as denitrifying intermediates. Furthermore, denitrifying bacteria often inhabit environments where they are exposed to NO₂⁻ and NO and hence high levels of RNS. Recent reports suggest that pathogenic bacteria invading animal tissues are attacked by NO generated by macrophages (12). Such bacteria involve denitrifiers, and some of them, for example, Neisseria meningitidis (1) and Pseudomonas aeruginosa, acquire resistance to NO by producing Nor (44). The utilization (reduction) of NO by Brucella increases the survival of infected mice (2). These examples suggest that production of a denitrifying mechanism affects bacterial survival of threats from both endogenous and extracellular RNS. However, the mechanism of RNS tolerance induced by denitrifying bacteria is not fully understood.Ubiquitous gram-negative Ochrobactrum strains are widely distributed in soils and aqueous environments, where they biodegrade aromatic compounds (11), organophosphorus pesticides (45), and other hydrocarbons (38) and remove heavy metal ions such as chromium and cadmium (24). Having been isolated from clinical specimens, Ochrobactrum anthropi is currently recognized as an emerging opportunistic pathogen, although relatively little is known about its pathogenesis and factors contributing to its virulence (7, 30). Manipulation systems have been developed to investigate these issues at the molecular genetic level (33). Some O. anthropi strains have been identified as denitrifiers (21), although the denitrifying properties of these strains have not been investigated in detail. This study was undertaken to examine the denitrifying properties of O. anthropi in more detail. O. anthropi YD50.2 was selected for this study and was isolated herein. The strain denitrified high levels of NO₂⁻ (up to 40 mM) to dinitrogen under anaerobic conditions. The strain was highly resistant to acidified NO₂⁻ under nondenitrifying aerobic conditions. These results indicate that O. anthropi YD50.2 has mechanisms that produce tolerance to RNS.     

CD4 T-Cell Help Programs a Change in CD8 T-Cell Function Enabling Effective Long-Term Control of Murine Gammaherpesvirus 68: Role of PD-1-PD-L1 Interactions

We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II^−/− (CII^−/−) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII^−/− mice caused a significant reduction in lung viral titers, in contrast to those from control CII^−/− mice. Anti-CD40 treatment also greatly prolonged survival of infected CII^−/− mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII^−/−, CD40^−/−, or CD80/86^−/− mice, compared with that in wild-type or CD28/CTLA4^−/− mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen which is closely related to Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) (17, 64). Intranasal administration of MHV-68 to mice results in acute productive infection of lung epithelial cells and a latent infection in various cell types, including B lymphocytes, dendritic cells, epithelial cells, and macrophages (18, 19, 52, 53, 61, 65). The virus induces an inflammatory infiltrate in the lungs, lymph node enlargement, splenomegaly, and mononucleosis comprising increased numbers of activated CD8 T cells in the blood (53, 58). It has also been reported to induce lymphoproliferative disease/lymphoma in immunocompromised mice (30, 55, 60). Thus, the pathogenesis resembles that of EBV in humans, although structurally, the virus is more closely related to KSHV.Infectious MHV-68 is cleared from the lungs by a T-cell-dependent mechanism 10 to 15 days after infection (18, 53, 56). In wild-type mice, the lungs remain clear of replicating virus thereafter. Although CD4 T cells are not essential for primary clearance of replicating virus, they are required for effective long-term control (11). Thus, major histocompatibility complex (MHC) class II^−/− mice that lack CD4 T cells or mice rendered CD4 deficient by antibody treatment initially clear infectious virus from the lungs. However, infectious virus reactivates in the lungs 10 to 15 days later and gradually increases in titer (11, 43). The infected CD4-deficient mice eventually die, apparently from long-term lung damage due to continuing lytic viral replication (11). MHC class II^−/− mice do not produce antibody to T-dependent antigens (10). Cytotoxic T-lymphocyte (CTL) epitopes have been identified in open reading frame (ORF) 6 (p56, H-2D^b-restricted), and ORF 61 (p79, H-2K^b-restricted) gene products, which appear to encode early lytic-phase proteins (32, 49). The epitopes are presented during two distinct phases during MHV-68 infection, which changes the pattern of CTL dominance (32, 51). However, there is no significant difference in the numbers of CD8 T cells specific for each epitope in wild-type mice and CD4 T-cell-deficient mice (4, 50). In addition, CTL activity measured in vitro does not differ substantially in the lungs of wild-type mice or CD4 T-cell-deficient mice (4, 11, 50). Furthermore, postexposure vaccination with the p56 epitope failed to prevent viral reactivation in class II^−/− mice, despite dramatically expanding the number of CD8 T cells specific for the peptide (5). In contrast, vaccination of wild-type mice against these epitopes reduced lytic viral titers in the lung dramatically on subsequent challenge with MHV-68. B-cell-deficient mice clear MHV-68 with the kinetics of wild-type mice and do not show viral reactivation in the lungs (13, 61), suggesting that antibody is not essential for control of the virus. Depletion of CD4 T cells during the latent phase of infection in B-cell-deficient mice does not induce viral reactivation, whereas depletion of both CD4 and CD8 T-cell subsets provokes viral reactivation in the lungs (52). Short-term depletion of both CD4 and CD8 T-cell subsets during the latent phase of infection in wild-type mice does not lead to viral reactivation probably due to the presence of neutralizing antibody (11). Taken together, these results suggest that CD4 and CD8 T cells and B cells play overlapping roles in preventing or controlling reactivation of MHV-68 during the latent phase of infection. However, the B-cell- and CD8 T-cell-mediated control mechanisms do not develop in the absence of CD4 T cells.We, and others, have previously shown that the costimulatory molecule CD28 is not required for long-term control of MHV-68 (28, 29). However, interestingly, mice lacking both of the ligands for CD28, CD80 and CD86, show viral reactivation in the lung (21, 35). Our previously published data showed that agonistic antibodies to CD40 could substitute for CD4 T-cell function in the long-term control of MHV-68 (46). CD8 T-cell receptor-positive (TCR+) cells were required for this effect, while antibody production was not restored (45, 46). MHV-68-infected CD40L^−/− mice (7) and CD40^−/− mice (29) also showed viral reactivation in the lungs. However, no change in CD8 CTL activity was detected in in vitro assays following anti-CD40 treatment (46). A key question was whether anti-CD40 treatment (or CD4 T-cell help) caused a direct change in CD8 T-cell function or whether both CD8 T cells and an independent anti-CD40-sensitive step were required for viral control. To address this question, we used adoptive transfer of CD8 T cells from MHV-68-infected wild-type mice, anti-CD40-treated mice, or control MHC class II^−/− mice to MHV-68-infected class II^−/− recipients. We also investigated whether anti-CD40 treatment prolonged survival in addition to reducing lung viral titers. The heterodimeric molecule CD94/NKG2A has been implicated in negatively regulating the CD8 T-cell response to polyomavirus (38) and herpes simplex virus (HSV) (54), while the inhibitory receptor PD-1 (programmed death 1) has been implicated in T-cell exhaustion following infection with several other persistent viruses (2, 15, 20, 22, 26, 36, 39-41, 57, 67). In the present study, we investigated the effect of signaling via various costimulatory molecules on the expression of NKG2A and PD-1 and how these molecules influenced viral control.     

Requirement of JIP1-Mediated c-Jun N-Terminal Kinase Activation for Obesity-Induced Insulin Resistance

The c-Jun NH₂-terminal kinase (JNK) interacting protein 1 (JIP1) has been proposed to act as a scaffold protein that mediates JNK activation. However, recent studies have implicated JIP1 in multiple biochemical processes. Physiological roles of JIP1 that are related to the JNK scaffold function of JIP1 are therefore unclear. To test the role of JIP1 in JNK activation, we created mice with a germ line point mutation in the Jip1 gene (Thr¹⁰³ replaced with Ala) that selectively blocks JIP1-mediated JNK activation. These mutant mice exhibit a severe defect in JNK activation caused by feeding of a high-fat diet. The loss of JIP1-mediated JNK activation protected the mutant mice against obesity-induced insulin resistance. We conclude that JIP1-mediated JNK activation plays a critical role in metabolic stress regulation of the JNK signaling pathway.Diet-induced obesity causes insulin resistance and metabolic syndrome, which can lead to β-cell dysfunction and type 2 diabetes (15). It is established that feeding mice a high-fat diet (HFD) causes activation of c-Jun NH₂-terminal kinase 1 (JNK1) (10). Moreover, Jnk1^−/− mice are protected against the effects of HFD-induced insulin resistance (10). Together, these observations indicate that JNK1 plays a critical role in the metabolic stress response. However, the mechanism that accounts for HFD-induced JNK1 activation is unclear. Recent studies have implicated the JIP1 scaffold protein in JNK1 activation caused by metabolic stress (23, 39).JIP1 can assemble a functional JNK activation module composed of a mitogen-activated protein kinase (MAPK) kinase kinase (a member of the mixed-lineage protein kinase [MLK] group), the MAPK kinase MKK7, and JNK (40, 42). This complex may be relevant to JNK activation caused by metabolic stress (23, 39). Indeed, MLK-deficient mice (14) and JIP1-deficient mice (13) exhibit defects in HFD-induced JNK activation and insulin resistance.The protection of Jip1^−/− mice against the effects of being fed an HFD may be mediated by loss of the JNK scaffold function of JIP1. However, JIP1 has also been reported to mediate other biochemical processes that would also be disrupted in Jip1^−/− mice. For example, JIP1 interacts with AKT and has been implicated in the mechanism of AKT activation (8, 17, 18, 34). Moreover, JIP1 interacts with members of the Src and Abl tyrosine kinase families (4, 16, 24), the lipid phosphatase SHIP2 (44), the MAPK phosphatase MKP7 (43), β-amyloid precursor protein (20, 31), the small GTPase regulatory proteins Ras-GRF1, p190-RhoGEF, RalGDS, and Tiam1 (2, 8, 21), ankyrin G (35), molecular chaperones (35), and the low-density-lipoprotein-related receptors LRP1, LRP2, and LRP8 (7, 37). JIP1 also interacts with other scaffold proteins, including the insulin receptor substrate proteins IRS1 and IRS2 (35). Finally, JIP1 may act as an adapter protein for kinesin-mediated (11, 12, 16, 38, 42) and dynein-mediated (35) trafficking on microtubules. The JNK scaffold properties of JIP1 therefore represent only one of the possible biochemical functions of JIP1 that are disrupted in Jip1^−/− mice.The purpose of this study was to test the role of JIP1 as a JNK scaffold protein in the response of mice to being fed an HFD. Our approach was to examine the effect of a point mutation that selectively prevents JIP1-induced JNK activation. It is established that phosphorylation of JIP1 on Thr¹⁰³ is required for JIP1-mediated JNK activation by the MLK pathway (25). Consequently, the phosphorylation-defective Thr103Ala JIP1 protein does not activate JNK (25). Here we describe the analysis of mice with a point mutation in the Jip1 gene that replaces the JIP1 phosphorylation site Thr¹⁰³ with Ala. We show that this mutation suppresses HFD-induced JNK activation and insulin resistance. These data demonstrate that JNK activation mediated by the JIP1 scaffold complex contributes to the response of mice to an HFD.     

Identification of an aox System That Requires Cytochrome c in the Highly Arsenic-Resistant Bacterium Ochrobactrum tritici SCII24

Microbial biotransformations have a major impact on environments contaminated with toxic elements, including arsenic, resulting in an increasing interest in strategies responsible for how bacteria cope with arsenic. In the present work, we investigated the metabolism of this metalloid in the bacterium Ochrobactrum tritici SCII24. This heterotrophic organism contains two different ars operons and is able to oxidize arsenite to arsenate. The presence of arsenite oxidase genes in this organism was evaluated, and sequence analysis revealed structural genes for an As(III) oxidase (aoxAB), a c-type cytochrome (cytC), and molybdopterin biosynthesis (moeA). Two other genes coding for a two-component signal transduction pair (aoxRS) were also identified upstream from the previous gene cluster. The involvement of aox genes in As(III) oxidation was confirmed by functionally expressing them into O. tritici 5bvl1, a non-As(III) oxidizer. Experiments showed that the As(III) oxidation process in O. tritici requires not only the enzyme arsenite oxidase but also the cytochrome c encoded in the operon. The fundamental role of this cytochrome c, reduced in the presence of arsenite in strain SCII24 but not in an O. tritici ΔaoxB mutant, is surprising, since to date this feature has not been found in other organisms. In this strain the presence of an aox system does not seem to confer an additional arsenite resistance capability; however, it might act as part of an As(III)-detoxifying strategy. Such mechanisms may have played a crucial role in the development of early stages of life on Earth and may one day be exploited as part of a potential bioremediation strategy in toxic environments.Arsenic is naturally present in soil, water, and air, and arsenic contamination of drinking water constitutes an important public health problem in numerous countries throughout the world (33). Arsenic occurs in nature in the oxidation states +5 (arsenate), +3 (arsenite), 0 (elemental arsenic), and −3 (arsine). Although arsenic is most notorious as a poison threatening human health, recent studies suggest that arsenic species may have been involved in the ancestral taming of energy and played a crucial role in early stages in the development of life on Earth (reviewed in reference 34). The two soluble arsenic species, arsenate [As(V) as H₂AsO₄⁻ and HAsO₄²⁻] and arsenite [As(III) as H₃AsO₃⁰ and H₂AsO₃⁻] are the most common forms and exhibit different toxicities for living organisms. Several studies have documented the role of bacteria on speciation and mobilization of arsenic in the environment (23). Microorganisms are known to influence arsenic geochemistry by their metabolism, i.e., reduction, oxidation, and methylation (for reviews, see references 5, 19, and 22), affecting both the speciation and the toxicity of this element. Arsenate is less toxic than arsenite, but paradoxically, resistance to As(V) requires its reduction to As(III), which is then extruded by an active efflux pump.Another well-documented arsenic transformation is the microbiological oxidation of arsenite to arsenate. This redox reaction is generally carried out by microorganisms either for detoxification or for energy generation to support cellular growth (23). The oxidation of As(III) by heterotrophic microorganisms is generally considered to be a detoxification strategy, since the microbes do not gain energy from this reaction (32). These heterotrophic As-oxidizing organisms include the most-studied Alcaligenes faecalis (3), Herminiimonas arsenoxidans (21), Thermus species (13, 14), Hydrogenophaga sp. strain NT-14 (35), and Agrobacterium tumefaciens (17). In contrast, other organisms have been described as autotrophic As(III) oxidizers able to use the energy gained from the oxidation reaction for growth. Autotrophic As(III) oxidation has been best studied in strain NT-26 (27, 28) but has also been reported for Thiomonas sp. (10), strain MLHE1 (24), and other environmental isolates (7, 16, 25, 26).Of the arsenite-oxidizing bacteria, A. faecalis (3), NT-26 (27), and NT-14 (35) have been studied in detail and their arsenite oxidases purified and characterized. Moreover, a crystal structure of the A. faecalis arsenite oxidase has been elucidated (11). Genes encoding As(III) oxidases (aox) have also been identified and sequenced in several organisms, showing a common genetic organization, aoxA-aoxB, that encodes the small and large subunits, respectively. These aox operons usually contain additional genes, e.g., cytC, which encodes a cytochrome c, and moeA, which encodes an enzyme involved in molybdenum cofactor biosynthesis (32).The genome exploration of the alphaproteobacterium Ochrobactrum tritici revealed that it possesses heretofore-unsuspected mechanisms for coping with arsenic. This work reports the identification of a locus involved in arsenic oxidation in a heterotrophic bacterium previously characterized as carrying two operons involved in arsenic resistance. One operon confers resistance to arsenite and antimonite, while the second one is responsible for resistance to arsenate.     

c-Jun NH2-Terminal Kinase Is Required for Lineage-Specific Differentiation but Not Stem Cell Self-Renewal

The c-Jun NH₂-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1^−/− Jnk2^−/− ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.The c-Jun NH₂-terminal kinase (JNK) is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. JNK is encoded by two ubiquitously expressed genes (Jnk1 and Jnk2) and by a third gene (Jnk3) that is selectively expressed in neurons (14). Gene disruption studies demonstrate that mice without Jnk1 or Jnk2 are viable, but compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality (14). Murine embryonic fibroblasts (MEFs) isolated from Jnk1^−/− Jnk2^−/− mice exhibit a severe growth retardation phenotype (54). The markedly reduced growth of Jnk1^−/− Jnk2^−/− MEFs is consistent with the finding that JNK is critically required for the regulation of AP1-dependent gene expression (56) that is implicated in cellular proliferation (26). Thus, Jnk1^−/− Jnk2^−/− MEFs express low levels of AP1 proteins (e.g., c-Jun and JunD) and exhibit marked defects in AP1 target gene expression (34, 56). This loss of AP1 function is mediated, in part, by reduced phosphorylation of the activation domain of Jun family proteins and ATF2 (56).More recent studies using a conditional gene ablation strategy have demonstrated that compound JNK deficiency causes rapid senescence (12). This conclusion was confirmed by using chemical genetic analysis with MEFs isolated from mice with a germ line mutation that sensitizes JNK to inhibition by a predesigned small-molecule drug (12, 25). This form of senescence was found to be p53 dependent (12) and resembles the p53-dependent senescence of c-Jun^−/− MEFs (49). These data indicate that JNK plays a critical role in cellular proliferation. Indeed, it is possible that the p53-dependent senescence observed in JNK-deficient cells may contribute to aging. This is because altered p53 function is established to be an important determinant of early aging (36, 55). Importantly, this role of p53 in aging appears to be distinct from p53-mediated tumor suppression and DNA damage responses (21, 39, 43).One aspect of the aging process is a reduction in the regenerative capacity of stem cells (50). Indeed, it has been established that altered p53 activity associated with aging causes decreased stem cell function (8, 18, 42) and that disruption of the p53 pathway can increase stem cell function (1). Since JNK can influence p53-dependent senescence (12), these data indicate that JNK may be important for stem cell proliferation and self-renewal potential.Embryonic stem (ES) cells proliferate and are capable of both self-renewal and differentiation to multiple cell types. Indeed, murine ES cells can differentiate to create all tissues within a mouse. The profound growth retardation and rapid p53-dependent senescence of Jnk1^−/− Jnk2^−/− MEFs (12) suggests that JNK may play a critical role in the normal function of ES cells, including self-renewal and differentiation potential. The purpose of the present study was to test this hypothesis. Our approach was to isolate ES cells from wild-type and JNK-deficient mice. We demonstrate that JNK is not required for self-renewal or the proliferation of ES cells. However, JNK is required for ES cell differentiation.     

A Role for DNA Polymerase μ in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo

The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJ_H joints and compare them with those present in later life. The embryo DJ_H joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase μ (Polμ), which was widely expressed in the early ontogeny, as shown by analysis of Polμ^−/− embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polμ also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polμ participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development.The adaptive immune system is characterized by the great diversity of its antigen receptors, which result from the activities of enzymatic complexes that cut and paste the genomic DNA of antigen receptor loci. The nonhomologous end-joining (NHEJ) machinery is then recruited to repair the double-strand DNA breaks (DSBs) inflicted by the products of the recombination-activating genes (RAGs) (45, 65). Within B cells, each immunoglobulin (Ig) receptor represents a singular shuffling of two heavy (H) and two light (L) chains, which are derived from the recombination of V, D, and J gene segments of the IgH locus and of V and J for IgL (71). Besides these combinatorial possibilities, most Ig variability derives from extensive processing of the coding ends, including exonucleolytic trimming of DNA ends, together with the addition of palindromic (P) nucleotides templated by the adjacent germ line sequence and of nontemplated (N) nucleotides secondary to the activity of the terminal deoxynucleotidyl transferase (TdT), a lymphoid-specific member of family X of DNA polymerases (reviewed in reference 56). During B-lineage differentiation, IgH rearrangements occur before those of the IgL locus, and D-to-J_H rearrangements precede V-to-DJ_H rearrangements (62). DJ_H joints are formed in any of the three open reading frames (ORFs). ORF1 is predominantly used in mature Igs, ORF2 is transcribed as a Dμ protein that provides negative signals to the B-cell precursors, and ORF3 frequently leads to stop codons (32, 33, 37). Germ line V, D, and J gene segments display short stretches of mutually homologous nucleotides (SSH), which are frequently used in gene rearrangements during perinatal periods, when N additions are absent (27, 32, 55, 57). The actual Ig V-region repertoires represent both the results of the NHEJ process associated with genomic VDJ recombination and those of antigen-independent and -dependent selection events. Although the core NHEJ components (Ku-Artemis-DNA-PK and XLF-XRCC4-DNA ligase IV) are by themselves able to join RAG-induced, incompatible DNA ends, family X DNA polymerases can be recruited to fill gaps created by imprecise coding ends with 3′ overhangs (DNA polymerase μ [Polμ] and Polλ) and/or to promote diversity through the addition of N nucleotides (TdT) (34, 56).The lymphoid differentiation pathways and clonotypic repertoires are developmentally regulated and differ between the embryo-fetal and adult periods (2, 44, 68). The perinatal B cells result from a wave of B lymphopoiesis occurring during the last third of mouse gestation (13, 14, 21, 70). Perinatal V_H gene usage differs from that predominating in the adult (1, 69), and the former VDJ joints rarely display N additions, leading to V-region repertoires enriched in multi- and self-reactive specificities (36, 40). The program of B-cell differentiation starts at embryonic days 10 to 11 (E10 to E11) in embryo hematopoietic sites, after the emergence of multipotent progenitors (at E8.5 to E9.5) (18, 19, 23, 31, 51, 73). DJ_H rearrangements were detected in these early embryos, whereas full VDJ_H sequences were not observed before E14 (14, 18, 51, 66), when VJκ rearrangements were also found (63). The earliest mouse DJ_H/VDJ_H Ig sequences analyzed to date corresponded to late fetuses (E16) (14, 53). We reasoned that the true baseline of the Ig rearrangement process occurs in midgestation embryos, when the first DJ_Hs are not yet transcribed and, consequently, not subjected to selection and are conditioned only for the evolutionarily established and developmentally regulated usage of distinct NHEJ machineries.We report here the sequence profiles of the earliest embryo E10 to E12 DJ_H joints. Unexpected frequencies of embryonic DJ_H joints bearing N nucleotides, in the absence of detectable TdT expression, were found. Moreover, the embryo DJ_H joints lacking N nucleotides (N⁻) used fewer SSH to recombine than newborn DJ_Hs, and these SSH were widely dispersed along the embryo D sequences, in contrast to the most joint-proximal ones, which predominated in newborn DJ_Hs. Considering that Polμ is the closest relative of TdT (42% amino acid identity) (22), which is able to introduce N nucleotides in vitro (4, 22, 34, 39, 49) and to join DNA ends with minimal or even null complementarity (17, 58), and that it is expressed in early-embryo organs, we decided to investigate its putative contribution to the first embryo DJ_H joints. The DJ_H joints obtained from Polμ^−/− embryos (48) showed a significant reduction of N nucleotides compared to wild-type (WT) embryos. Moreover, highly preserved DJ_H joints (with <3 deleted nucleotides) were selectively depleted in the Polμ^−/− mouse embryos, while the remaining DJ_Hs preferentially relied upon longer stretches of homology for end ligation. These findings support the idea that Polμ is active during early-embryo DJ_H rearrangements and that both its template-dependent and -independent ambivalent functions may be used to fill in small nucleotide gaps generated after asymmetric hairpin nicking and also to extend coding ends via a limited TdT-like activity.     

Pseudomonas Exotoxin A-Mediated Apoptosis Is Bak Dependent and Preceded by the Degradation of Mcl-1

Pseudomonas exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax^−/−) MEFs but failed in Bak knockout (Bak^−/−) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax^−/− MEFs, but not in Bak^−/− or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-x_L, was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-x_L in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-x_L inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.Apoptosis is a mode of cell death utilized by multicellular organisms to remove unwanted cells. Also, many different cancer treatments, including chemotherapy and radiotherapy, induce apoptosis and result in the destruction of tumor cells. In some cases, apoptosis resistance can contribute to the failure of chemotherapy (14, 20, 24). Immunotoxins are a class of antitumor agents in which a powerful protein toxin is brought to the cancer cell by an antibody or an antibody fragment (for reviews, see references 28, 29, and 32). Several immunotoxins are currently in clinical trials, and one of these, BL22, targeting CD22, has shown excellent activity in drug-resistant hairy-cell leukemia (18, 19). Also, a fusion protein in which a fragment of diphtheria toxin is fused to the cytokine interleukin 2 (IL-2) (Ontak) is approved for the treatment of cutaneous T-cell lymphoma (26). Several studies carried out to determine how protein toxins and immunotoxins containing these toxins kill target cells have reported caspase activation (13, 16, 17, 30, 33). However, the steps leading up to caspase activation by these toxins that inhibit protein synthesis have not been elucidated.Bcl-2 family members are essential regulators of the mitochondrial (intrinsic) apoptosis pathway (1, 21). Proteins of this family have been divided into pro- and antiapoptotic proteins. Antiapoptotic proteins include the multi-Bcl-2 homology (BH) domain proteins Bcl-2, Bcl-x_L, Bcl-w, Mcl-1, Bcl-b, and Bcl2a1. Proapoptotic members can be further classified into two subfamilies, the multi-BH domain Bax homologues, including Bax, Bak, and Bok, and the BH3-only proteins, including Nbk/Bik, Noxa, Hrk, Bad, Bim, Puma, and Bmf. Bax and Bak are the most extensively studied central mediators in the mitochondrial apoptosis pathway (4, 6). Various stimuli, including pathogens, toxic drugs, irradiation, and starvation, induce a conformational change and activation of Bak/Bax, usually via BH3-only proapoptosis proteins. This results in the disruption of mitochondrial membranes and the release of apoptotic factors, such as cytochrome c, SMAC, and apoptosis-inducing factor, which lead to the activation of effector caspases (5, 37, 40, 42, 43).The roles of Bax and Bak can be redundant or nonredundant, depending on the apoptotic stimuli. Bak and Bax can compensate for each other in apoptosis induced by staurosporine, etoposide, UV irradiation, serum deprivation, tBid, Bim, Bad, or Noxa (37, 43). Bak plays an essential role for apoptosis induced by Semliki Forest virus, gliotoxin, Bcl-x_S, and vinblastine (22, 27, 34, 35), while Bax is favored for apoptosis induced by Nbk/Nik, a combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ionizing irradiation, or TRAIL and 5-fluorouracil (5-FU) (9, 10, 36, 38). Silencing of either Bak or Bax resulted in resistance to apoptosis induced by Neisseria gonorrhoeae and cisplatin (15). Sometimes the same stimulus may result in different outcomes in different cell types. NBK/Bik mediated Bax-dependent cell death in one study (9), while in another study, NBK/Bik activated BAK-mediated apoptosis (31).In the current study, we utilized mutant mouse embryo fibroblasts (MEFs) deficient in Bak, Bax, or both proteins and provided evidence for an essential role of Bak in apoptosis induced by Pseudomonas exotoxin A (PE) and other protein synthesis inhibitors. We found that Bak^−/− cells are resistant to killing by PE and that Mcl-1, which binds to Bak, controls apoptosis induced by PE.     

Microbial Iron Redox Cycling in a Circumneutral-pH Groundwater Seep

The potential for microbially mediated redox cycling of iron (Fe) in a circumneutral-pH groundwater seep in north central Alabama was studied. Incubation of freshly collected seep material under anoxic conditions with acetate-lactate or H₂ as an electron donor revealed the potential for rapid Fe(III) oxide reduction (ca. 700 to 2,000 μmol liter⁻¹ day⁻¹). Fe(III) reduction at lower but significant rates took place in unamended controls (ca. 300 μmol liter⁻¹ day⁻¹). Culture-based enumerations (most probable numbers [MPNs]) revealed significant numbers (10² to 10⁶ cells ml⁻¹) of organic carbon- and H₂-oxidizing dissimilatory Fe(III)-reducing microorganisms. Three isolates with the ability to reduce Fe(III) oxides by dissimilatory or fermentative metabolism were obtained (Geobacter sp. strain IST-3, Shewanella sp. strain IST-21, and Bacillus sp. strain IST-38). MPN analysis also revealed the presence of microaerophilic Fe(II)-oxidizing microorganisms (10³ to 10⁵ cells ml⁻¹). A 16S rRNA gene library from the iron seep was dominated by representatives of the Betaproteobacteria including Gallionella, Leptothrix, and Comamonas species. Aerobic Fe(II)-oxidizing Comamonas sp. strain IST-3 was isolated. The 16S rRNA gene sequence of this organism is 100% similar to the type strain of the betaproteobacterium Comamonas testosteroni ({"type":"entrez-nucleotide","attrs":{"text":"M11224","term_id":"151536","term_text":"M11224"}}M11224). Testing of the type strain showed no Fe(II) oxidation. Collectively our results suggest that active microbial Fe redox cycling occurred within this habitat and support previous conceptual models for how microbial Fe oxidation and reduction can be coupled in surface and subsurface sedimentary environments.Changes in iron (Fe) redox state are linked to carbon and energy flow as well as the behavior of various inorganic compounds in modern soils and sediments. Microorganisms play a pivotal role in the Fe redox cycle in such environments (29, 35, 39). A growing body of literature indicates that aerobic lithotrophic Fe(II)-oxidizing bacteria (FeOB) can contribute significantly to circumneutral-pH Fe(II) oxidation (4, 9, 15, 23, 25, 34) and that microbial catalysis can dominate Fe(II) oxidation in diffusion-limited reaction systems (32, 34). Microbial catalysis is strictly required for anaerobic nitrate-dependent Fe(II) oxidation (36), since an abiotic reaction between Fe(II) and nitrate does not take place under typical near-surface conditions (40).Circumneutral-pH Fe(II) oxidation produces Fe(III) oxide mineral phases which can function as electron acceptors for anaerobic respiration by dissimilatory Fe(III)-reducing bacteria (FeRB) (8, 37). This metabolism is widespread among prokaryotic taxa (19) and plays a key role in oxidation of natural organic compounds and in the bioremediation of organic and metal contaminants in the subsurface (18). The coupling of Fe(III) oxide reduction to oxidation of organic carbon or H₂ leads to release of Fe(II) into the aqueous phase. When the oxidative and reductive parts of the Fe redox cycle come together with ongoing input of energy, a self-sustaining microbial community based on Fe redox cycling may develop. Sustained microbial Fe redox cycling has been proposed in various redox interfacial environments like groundwater Fe seeps (8), plant roots (10), the sediment-water interface in circumneutral-pH (29, 33) and acidic (24) aquatic ecosystems, and hot springs and hydrothermal vents (16a, 24a).Here we present data that support the existence of a sustained microbial Fe redox cycle in a circumneutral-pH groundwater Fe seep in north central Alabama. Potential microbial involvement in Fe redox cycling was assessed by most probable number (MPN) enumerations, in vitro Fe(III) reduction experiments, and isolation of representative Fe(III)-reducing and Fe(II)-oxidizing microorganisms. A simple kinetic model was used to explore the impact that decay of dead chemolithotrophic biomass coupled to Fe(III) reduction could have on rates of Fe turnover.     

Evolution of the HIV-1 env Gene in the Rag2?/? γC?/? Humanized Mouse Model

Rag2^−/− γ_C^−/− mice transplanted with human hematopoietic stem cells (DKO-hu-HSC mice) mimic aspects of human infection with human immunodeficiency virus type 1 (HIV-1), including sustained viral replication and CD4⁺ T-cell decline. However, the extent of HIV-1 evolution during long-term infection in these humanized mice, a key feature of the natural infection, has not been assessed fully. In this study, we examined the types of genotypic and phenotypic changes in the viral env gene that occur in the viral populations of DKO-hu-HSC mice infected with the CCR5-tropic isolate HIV-1_JRCSF for up to 44 weeks. The mean rate of divergence of viral populations in mice was similar to that observed in a cohort of humans during a similar period of infection. Many amino acid substitutions were common across mice, including losses of N-linked glycosylation sites and substitutions in the CD4 binding site and in CD4-induced epitopes, indicating common selective pressures between mice. In addition, env variants evolved sensitivity to antibodies directed at V3, suggesting a more open conformation for Env. This phenotypic change was associated with increased CD4 binding efficiency and was attributed to specific amino acid substitutions. In one mouse, env variants emerged that exhibited a CXCR4-tropic phenotype. These sequences were compartmentalized in the mesenteric lymph node. In summary, viral populations in these mice exhibited dynamic behavior that included sequence evolution, compartmentalization, and the appearance of distinct phenotypic changes. Thus, humanized mice offer a useful model for studying evolutionary processes of HIV-1 in a complex host environment.Animal models of HIV-1 infection are important tools for studying transmission, replication, and pathogenesis, as well as therapeutic intervention, of HIV-1 infection. Nonhuman primates such as rhesus macaques, infected with simian or chimeric simian/human immunodeficiency viruses (SIV or SHIV, respectively), represent well-characterized and highly relevant models; however, key limitations include expense, genetic variability of the host animals, and the fact that SIV, while closely related, is distinct from HIV-1. Therefore, small animal models that support HIV-1 infection and recapitulate many aspects of the human infection have been sought using several approaches.Recent approaches have involved the use of genetically immunodeficient mice that have been reconstituted using human-derived hematopoietic stem cells (HSC) (known as humanized mice). Several models have been developed based on this approach, including Rag2^−/− γ_C^−/− (DKO) and NOD/SCID/γ_C^−/− (NOG or NSG) mice transplanted with human HSC (DKO-hu-HSC or NOG-hu-HSC mice) (40, 92) and the NOD/SCID mouse with transplanted human fetal thymus and liver tissue in addition to HSC (62). These models all support HIV-1 infection (1, 3, 6, 30, 87, 96, 102; for a review of these models, see the work of Denton and Garcia [22]). The DKO-hu-HSC mouse lacks both recombination activating gene 2 (Rag2) and the cytokine receptor common gamma chain (γ_C), and as a result, it does not generate murine T, B, and natural killer (NK) cells but supports engraftment of HSC and differentiation of human myeloid and lymphoid lineages. Immune reconstitution in this model likely involves education of human T cells in the mouse thymus and dissemination of differentiated human lymphoid subsets into the peripheral blood and to multiple lymphoid tissues, including lymph nodes, spleen, and bone marrow (92). The DKO-hu-HSC mouse, along with the other humanized mouse models, has been used in studies of transmission (5, 21), pathogenesis (43), and viral inhibition (16, 21, 53, 88, 94).One important feature of HIV-1 infection is the diversification and evolution of the viral genome over the course of infection. Diversification occurs most prominently in the envelope (env) gene, which encodes the viral surface glycoprotein (Env). Env mediates viral entry into cells through attachment to the primary receptor CD4, which primes Env for engagement with a coreceptor, either CCR5 or CXCR4, triggering virion fusion with the cellular plasma membrane (54). HIV-1 infection is typically established by one or a few CCR5-tropic (R5) variants that give rise to an initially homogenous viral population, which then diversifies over the course of chronic infection (45, 84). Diversification of Env results from immune selective pressures (27), isolation in or adaptation to different cellular and anatomical compartments (20, 28, 33, 46, 51), and selection for altered CD4 affinity (72, 90, 95) and coreceptor tropism (26, 39). In many cases, during late-stage infection, variants emerge from the R5 virus population that are CXCR4 tropic (X4), an event that is often associated with accelerated CD4 T-cell loss and progression to AIDS (9, 18, 89). In an effort to determine if any of these aspects of HIV-1 evolution are exhibited in the humanized mouse model, we examined the extent of HIV-1 diversification and the types of evolutionary changes that occur in env in mice infected with CCR5-tropic HIV-1 for up to 44 weeks.Sampling of viral env variants from the peripheral blood plasma over the course of the infection revealed increasing diversity and divergence of the viral population at rates similar to those observed in natural infection. Mutations were identified that affected Env conformation and sensitivity to neutralizing antibodies, CXCR4 coreceptor use, and potential N-linked glycosylation sites. Other mutations potentially affecting the Env phenotype were identified in CD4 binding sites and CD4-induced epitopes. The patterns of substitutions indicated that certain sites were under selection, particularly in cases where the same substitution was identified in multiple mice.This study demonstrates the potential for studying HIV-1 evolution in the DKO-hu-HSC mouse model and also gives insight into the types of selective pressures driving HIV-1 env evolution in this host environment. These findings, while highlighting some of the limitations of this model, will help to inform its appropriate use for studying different aspects of HIV-1 infection, such as the evolutionary constraints placed on HIV-1 during natural infection and in the face of pharmacological and immunological inhibition.