Exploring Vertical Transmission of Bifidobacteria from Mother to Child

Passage through the birth canal and consequent exposure to the mother''s microbiota is considered to represent the initiating event for microbial colonization of the gastrointestinal tract of the newborn. However, a precise evaluation of such suspected vertical microbiota transmission has yet to be performed. Here, we evaluated the microbiomes of four sample sets, each consisting of a mother''s fecal and milk samples and the corresponding infant''s fecal sample, by means of amplicon-based profiling supported by shotgun metagenomics data for two key samples. Notably, targeted genome reconstruction from microbiome data revealed vertical transmission of a Bifidobacterium breve strain and a Bifidobacterium longum subsp. longum strain from mother to infant, a notion confirmed by strain isolation and genome sequencing. Furthermore, PCR analyses targeting unique genes from these two strains highlighted their persistence in the infant gut at 6 months. Thus, this study demonstrates the existence of specific bifidobacterial strains that are common to mother and child and thus indicative of vertical transmission and that are maintained in the infant for at least relatively short time spans.     

The Translesion Polymerase ζ Has Roles Dependent on and Independent of the Nuclease MUS81 and the Helicase RECQ4A in DNA Damage Repair in Arabidopsis

DNA polymerase zeta catalytic subunit REV3 is known to play an important role in the repair of DNA damage induced by cross-linking and methylating agents. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the basic polymerase activity of REV3 is essential for resistance protection against these different types of damaging agents. Interestingly, its processivity is mainly required for resistance to interstrand and intrastrand cross-linking agents, but not alkylating agents. To better define the role of REV3 in relation to other key factors involved in DNA repair, we perform epistasis analysis and show that REV3-mediated resistance to DNA-damaging agents is independent of the replication damage checkpoint kinase ataxia telangiectasia-mutated and rad3-related homolog. REV3 cooperates with the endonuclease MMS and UV-sensitive protein81 in response to interstrand cross links and alkylated bases, whereas it acts independently of the ATP-dependent DNA helicase RECQ4A. Taken together, our data show that four DNA intrastrand cross-link subpathways exist in Arabidopsis, defined by ATP-dependent DNA Helicase RECQ4A, MMS and UV-sensitive protein81, REV3, and the ATPase Radiation Sensitive Protein 5A.The DNA of all living organisms is constantly exposed to damaging factors, and therefore a number of DNA damage repair and bypass mechanisms have evolved. DNA lesions that interfere with the replication machinery constitute a particular challenge for cells (Schröpfer et al., 2014a); if not repaired in a timely manner, such damage can result in the stalling or collapse of replication forks, which in turn can lead to cell death. Furthermore, one-sided double-strand breaks (DSBs) can occur when the replication fork encounters a single-strand break. Lesions within one DNA strand, such as alkylations or DNA intrastrand cross links, can be bypassed by postreplicative repair (PRR), a process that is best understood in yeast (Saccharomyces cerevisiae). This mechanism does not lead to repair of the lesion but prevents fatal long-lasting stalling of the replication fork. PRR can be divided into two branches: the error-prone pathway and the error-free pathway (for review, see Goodman and Woodgate, 2013; Haynes et al., 2015; Jansen et al., 2015). It is known from yeast that both branches of PRR are controlled by the Radiation sensitivity protein6 (Rad6) and Mms-Ubc13 E3 ubiquitin-conjugating enzyme complexes, which ubiquitinate the replicative processivity factor Proliferating Cellular Nuclear Antigen1. The monoubiquitination of PCNA at Lys-164 by Rad6-Rad18 initiates the error-prone pathway, whereas polyubiquitination additionally requires Mms2, Ubc13, and Rad5, and triggers the error-free PRR branch (Hoege et al., 2002; Moldovan et al., 2007; Lee and Myung, 2008). There are two possible competing models postulated for the error-free bypass of lesions at the replication fork, both of which depend on template-switch mechanisms; if the lesion concerns only one of the two sister strands, the undamaged strand can be used as the template for bypassing the lesion. One of the two models features the so-called overshoot synthesis, whereby the newly synthesized strand on the undamaged parental strand is elongated further than the strand blocked by the lesion. Regression of the replication fork then leads to the formation of a special type of four-way junction called a chicken-foot structure. This regression mechanism is thought to be accomplished by helicases, such as the RecQ helicase Bloom Syndrome Protein (BLM) in humans (Croteau et al., 2014). AtRECQ4A is the respective BLM homolog in Arabidopsis (Arabidopsis thaliana), and this enzyme has the ability to regress replication forks in vitro (Hartung et al., 2007, 2008; Schröpfer et al., 2014b). The second error-free subpathway entails invasion of the newly synthesized strand on the blocked sister chromatid into the complementary newly replicated strand on the other sister chromatid. Such a step forms a displacement loop-like structure in which synthesis over the damaged region can occur. In yeast, both error-free pathways are dependent on the multifunctional protein Rad5, which is known to recruit PRR factors and also exhibits helicase activity itself (Blastyák et al., 2007). We previously identified AtRAD5A as a functional Arabidopsis homolog of Rad5 (Chen et al., 2008). Interestingly, AtRAD5A is required for efficient repair by homologous recombination via the synthesis-dependent strand-annealing mechanism, a pathway that in some steps is related to the invasion model of PRR (Mannuss et al., 2010).The error-prone pathway is based on the function of translesion synthesis (TLS) polymerases, which promote replication through DNA lesions (Prakash et al., 2005). In a mechanism termed polymerase switch, the replicative polymerase is exchanged by such a TLS polymerase at a damaged site. After incorporation of a nucleotide opposite the damaged base by the TLS polymerase, a second polymerase switch exchanges the TLS polymerase for the replicative polymerase so that replication can proceed (Prakash and Prakash, 2002; Lehmann et al., 2007). TLS polymerases possess no 5′-3′-exonuclease activity, and therefore act in a potentially mutagenic manner. Nevertheless, depending on the damage incurred and the TLS polymerase used, damage bypass can be error free (Haracska et al., 2000; McCulloch et al., 2004).Polymerases can be divided into at least six families based on their amino acid sequences and crystal structures: A, B, C, D, X, and Y. All of them share the common structure analogous to a right hand grasping DNA with palm, finger, and thumb domains (Steitz, 1999). The amino acid sequences of the finger and thumb domains of different polymerase families are highly variable, whereas the palm domains share high similarity. The palm domain forms the largest part of the polymerase active site and contains highly conserved Asp residues that have been postulated to be involved in the catalytic activity of the enzyme (Joyce and Steitz, 1995; Steitz, 1999).Most TLS polymerases belong to the Y family of polymerases, a class of specially structured enzymes that catalyze replication over damaged templates (Ohmori et al., 2001; Sale et al., 2012). Although some polymerases of the A, B, or X family can also exhibit TLS activity, this is often not their primary function (Prakash et al., 2005). DNA Polymerase Zeta (POLζ) is a B family polymerase and consists of a DNA Polymerase Zeta subunit REV3-REV7 heterodimer, in which REV3 is the catalytic subunit with its accessory subunit and processivity factor REV7 (Nelson et al., 1996). Recent studies in yeast and human cells have shown that POLζ contains two additional subunits, Pol31 and Pol32 in yeast, orthologs to human POLD2 and POLD3, which are known to be accessory subunits of the replicative polymerase POLδ (Johnson et al., 2012; Lee et al., 2014). REV3 contains three regions that are highly conserved between organisms: an N-terminal region, a REV7 binding domain, and a B family-type polymerase domain. The polymerase domain carries the six common conserved regions, I to VI (IV-II-VI-III-I-V), of which I is the most and VI the least conserved region. The A (II), B (III), and C (I) motifs, located within regions I, II, and III (Wong et al., 1988), form the active site of the enzyme, and each harbors an essential Asp residue that coordinates two catalytic metal ions. Deficiency of REV3 in mice is embryo lethal (Bemark et al., 2000; Esposito et al., 2000), and vertebrate cells depleted in REV3 show hypersensitivity to various DNA-damaging agents, including UV and ionizing irradiation, cisplatin, MMS, and mitomycin C (MMC; Sonoda et al., 2003; Sharma and Canman, 2012). In Arabidopsis, rev3 mutants exhibit no obvious phenotype under standard growth conditions, but are hypersensitive to UV-B and gamma irradiation, MMC, MMS, and cisplatin (Sakamoto et al., 2003).Our previous work demonstrated the existence of several different pathways in Arabidopsis involved in repairing the DNA damage induced by cross-linking and methylating agents. These independent pathways are defined by the ATPase RAD5A, the helicase RECQ4A, and MMS and UV-sensitive protein81 (MUS81; Mannuss et al., 2010). The structure-specific endonuclease MUS81 together with its noncatalytic subunit (Mms4 in yeast, Eme1 in Schizosaccharomyces pombe, and MMS4 or Crossover Junction Endonuclease (EME1) in humans and plants) functions in the rescue of stalled replication forks. The enzyme is able to cleave the stalled fork at the lesion site, which leads to a one-sided DSB that is repaired by homologous recombination (HR) to restore the stalled fork (Hanada et al., 2006). We previously showed that mus81 transfer DNA (T-DNA) insertion lines in Arabidopsis are highly sensitive to treatment with MMS, cisplatin, hydroxyurea, ionizing irradiation, and MMC. We also found that AtMUS81 can form a complex with its heterologous binding partners AtEME1A or AtEME1B that is able to process intricate DNA structures, such as nicked Holliday junctions, which might also form at stalled replication forks (Hartung et al., 2006; Geuting et al., 2009).In the current study, we address whether fully functional polymerase activity is required for the repair of DNA damage induced by alkylating and cross-link-inducing agents. Moreover, we sought to clarify whether REV3 cooperates with other key factors identified in Arabidopsis in the repair of these different types of damage. Indeed, it has already been shown that AtREV3 and AtRAD5A do not cooperate in the repair of such DNA damage, confirming independent pathways of error-prone and error-free PRR in plants (Wang et al., 2011). However, as plants, animals, and yeast differ in their DNA cross-link repair machinery (e.g. Mannuss et al., 2010; Knoll et al., 2012; Dangel et al., 2014; Herrmann et al., 2015), it is of particular importance to define the role of REV3 in relation to MUS81 and RECQ4A.     

Regional Variations in Biomass Distribution in Brazilian Savanna Woodland

The Cerrado, the savanna biome in central Brazil, mostly comprised of woodland savanna, is experiencing intense and fast land use changes. To understand the changes in Cerrado carbon stocks, we present an overview of biomass distribution in different Cerrado vegetation types (i.e., grasslands, shrublands and forestlands). We surveyed 26 studies including 170 Cerrado sites. The grasslands presented mean total biomass of 24 Mg/ha, with 70 percent allocated in the belowground portion. In shrublands, the mean total biomass was 58 Mg/ha being 58 percent in the belowground portion. Finally, in forestlands the mean total biomass was 98 Mg/ha with 18 percent as belowground biomass. The surveyed studies presented 12 allometric equations for biomass estimate, most involving both diameter and height. Data on wood density for Cerrado shrubs and trees are not abundant and the average value was 0.66 g/cm³, similar to that found in the central portion of the Amazon Forest. We also examined the relationship between total precipitation and dry‐season intensity with biomass variation in the Cerrado shrubland using data from tropical rainfall measurement mission (TRMM) for the period 2000–2010. Dry‐season precipitation amount in cerrado areas in severe drought regions explained 29 percent of the variation in aboveground woody biomass. This finding is important in the face of the predictions of longer and more severe dry seasons in the region due to climate change.     

Dynamic and Thermodynamic Response of the Ras Protein Cdc42Hs upon Association with the Effector Domain of PAK3

Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately − 10 kcal mol^− 1 at 298 K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.     

A Type IV Translocated Legionella Cysteine Phytase Counteracts Intracellular Growth Restriction by Phytate

The causative agent of Legionnaires'' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique “Legionella-containing vacuole.” The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different “effector” proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys²³¹) and arginine (Arg²³⁷) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.     

Dynamic Balancing of Isoprene Carbon Sources Reflects Photosynthetic and Photorespiratory Responses to Temperature Stress

The volatile gas isoprene is emitted in teragrams per annum quantities from the terrestrial biosphere and exerts a large effect on atmospheric chemistry. Isoprene is made primarily from recently fixed photosynthate; however, alternate carbon sources play an important role, particularly when photosynthate is limiting. We examined the relative contribution of these alternate carbon sources under changes in light and temperature, the two environmental conditions that have the strongest influence over isoprene emission. Using a novel real-time analytical approach that allowed us to examine dynamic changes in carbon sources, we observed that relative contributions do not change as a function of light intensity. We found that the classical uncoupling of isoprene emission from net photosynthesis at elevated leaf temperatures is associated with an increased contribution of alternate carbon. We also observed a rapid compensatory response where alternate carbon sources compensated for transient decreases in recently fixed carbon during thermal ramping, thereby maintaining overall increases in isoprene production rates at high temperatures. Photorespiration is known to contribute to the decline in net photosynthesis at high leaf temperatures. A reduction in the temperature at which the contribution of alternate carbon sources increased was observed under photorespiratory conditions, while photosynthetic conditions increased this temperature. Feeding [2-¹³C]glycine (a photorespiratory intermediate) stimulated emissions of [¹³C_1–5]isoprene and ¹³CO₂, supporting the possibility that photorespiration can provide an alternate source of carbon for isoprene synthesis. Our observations have important implications for establishing improved mechanistic predictions of isoprene emissions and primary carbon metabolism, particularly under the predicted increases in future global temperatures.Many plant species emit isoprene (2-methyl-1,3-butadiene [C₅H₈]) into the atmosphere at high rates (Sharkey and Yeh, 2001). With an estimated emission rate of 500 to 750 teragram per year by terrestrial ecosystems (Guenther et al., 2006), isoprene exerts a strong control over the oxidizing capacity of the atmosphere. Due to its high reactivity to oxidants, it fuels an array of atmospheric chemical and physical processes affecting air quality and climate, including the production of ground-level ozone in environments with elevated concentrations of nitrogen oxides (Atkinson and Arey, 2003; Pacifico et al., 2009) and the formation/growth of organic aerosols (Nguyen et al., 2011). At the plant level, isoprene provides protection from stress, through stabilizing membrane processes (Sharkey and Singsaas, 1995; Velikova et al., 2011) and/or reducing the accumulation of damaging reactive oxygen species in plant tissues under stress (Loreto et al., 2001; Vickers et al., 2009b; Velikova et al., 2012). While the mechanism(s) are still under investigation, isoprene may directly or indirectly stabilize hydrophobic interactions in membranes (Singsaas et al., 1997), minimize lipid peroxidation (Loreto and Velikova, 2001), and directly react with reactive oxygen species (Kameel et al., 2014), yielding first-order oxidation products methyl vinyl ketone and methacrolein (Jardine et al., 2012, 2013). The two main environmental drivers for global changes in isoprene fluxes are light and temperature (Guenther et al., 2006). Isoprene production is closely linked to net photosynthesis, and both isoprene emissions and net photosynthesis are controlled by light intensity (Monson and Fall, 1989). There is also a positive correlation between net photosynthesis and isoprene emissions as leaf temperatures increase up to the optimum temperature for net photosynthesis (Monson et al., 1992).Despite the close correlation between photosynthesis and isoprene emissions, plant enclosure observations and leaf-level analyses have both shown that the fraction of net photosynthesis dedicated to isoprene emissions is not constant. During stress events that decrease net photosynthetic rates, isoprene emissions are often less affected or even stimulated; this results in an increase in relative isoprene production from 1% to 2% of net photosynthesis under normal conditions to 15% to 50% under extreme stress (Goldstein et al., 1998; Fuentes et al., 1999; Kesselmeier et al., 2002; Harley et al., 2004). In severe stress conditions such as drought, isoprene emissions can even continue in the complete absence of photosynthesis (Fortunati et al., 2008). An uncoupling of isoprene emissions from net photosynthesis has also been observed in a number of other studies where the optimum temperature for isoprene emissions was found to be substantially higher than that of net photosynthesis; under the high-temperature conditions, isoprene emissions can account for more than 50% of net photosynthesis (Sharkey and Loreto, 1993; Lerdau and Keller, 1997; Harley et al., 2004; Magel et al., 2006).Analyses of carbon sources using ¹³CO₂ leaf labeling have revealed that under standard conditions (i.e. leaf temperature of 30°C and photosynthetically active radiation [PAR] levels of 1,000 µmol m^–2 s^–1), isoprene is produced primarily (70%–90%) using carbon directly derived from the Calvin cycle (Delwiche and Sharkey, 1993; Affek and Yakir, 2002; Karl et al., 2002) via the chloroplastic methylerythritol phosphate (MEP) isoprenoid pathway (Zeidler et al., 1997). The relative contributions of photosynthetic and alternate carbon sources for isoprene are now recognized as being variable under different environmental conditions. Changes in net photosynthesis rates under drought stress (Funk et al., 2004; Brilli et al., 2007), salt stress (Loreto and Delfine, 2000), and changes in ambient O₂ and CO₂ concentrations (Jones and Rasmussen, 1975; Karl et al., 2002; Trowbridge et al., 2012) alter their relative contributions. Under heat stress-induced photosynthetic limitation in Populus deltoides (a temperate species), an increase in the relative contribution of alternate carbon sources was also observed (Funk et al., 2004). However, our current understanding of the responses of isoprene carbon sources to changes in temperature and light levels is poor, and the connection(s) of these responses to changes in leaf primary carbon metabolism (e.g. photosynthesis, photorespiration, and respiration) remains to be determined.Studies over the last decade have shown or suggested that potential alternate carbon sources include refixation of respired CO₂ (Loreto et al., 2004), intermediates from the cytosolic mevalonate (MVA) isoprenoid pathway (Flügge and Gao, 2005; Lichtenthaler, 2010), and intermediates from central carbon metabolism, including pyruvate (Jardine et al., 2010), phosphoenolpyruvate (Rosenstiel et al., 2003), and Glc (Schnitzler et al., 2004). Over 40 years ago, it was also proposed that photorespiratory carbon could directly contribute to isoprene production in plants (Jones and Rasmussen, 1975); however, subsequent studies (Monson and Fall, 1989; Hewitt et al., 1990; Karl et al., 2002) have concluded that photorespiration does not contribute to isoprenoid production.In this study, we examined the carbon composition of isoprene emitted from tropical tree species under changes in light and temperature, the two key environmental variables that affect isoprene emissions. Using a novel real-time analytical approach, we were able to observe compensatory changes in carbon source contribution to isoprene during thermal ramping at high temperatures, despite the overall isoprene emissions remaining relatively stable. By conducting leaf temperature curves under variable ¹³CO₂ concentrations and applying [2-¹³C]Gly leaf labeling, we also reopen the discussion on the role of photorespiration as an alternate source of carbon for isoprenoid formation.