Accurately dating when the first bilaterally symmetrical animals arose is crucial to our understanding of early animal evolution. The earliest unequivocally bilaterian fossils are Ϸ555 million years old. In contrast, molecular-clock analyses calibrated by using the fossil record of vertebrates estimate that vertebrates split from dipterans (Drosophila) Ϸ900 million years ago (Ma). Nonetheless, comparative genomic analyses suggest that a significant rate difference exists between vertebrates and dipterans, because the percentage difference between the genomes of mosquito and fly is greater than between fish and mouse, even though the vertebrate divergence is almost twice that of the dipteran. Here we show that the dipteran rate of molecular evolution is similar to other invertebrate taxa (echinoderms and bivalve molluscs) but not to vertebrates, which significantly decreased their rate of molecular evolution with respect to invertebrates. Using a data set consisting of the concatenation of seven different amino acid sequences from 23 ingroup taxa (giving a total of 11 different invertebrate calibration points scattered throughout the bilaterian tree and across the Phanerozoic), we estimate that the last common ancestor of bilaterians arose somewhere between 573 and 656 Ma, depending on the value assigned to the parameter scaling molecular substitution rate heterogeneity. These results are in accord with the known fossil record and support the view that the Cambrian explosion reflects, in part, the diversification of bilaterian phyla.A lthough the Cambrian explosion is of singular importance to our understanding of the history of life, it continues to defy explanation (1). This defiance stems, in part, from our inability to distinguish between two competing hypotheses: whether the Cambrian explosion reflects the rapid appearance of fossils with animals having a deep but cryptic precambrian history, or whether it reflects the true sudden appearance and diversification of animals in the Cambrian (2). Because each hypothesis makes a specific prediction of when animals arose in time, one way to distinguish between these two hypotheses is to date animal diversifications by using a molecular clock (2). A number of previous clock studies (reviewed in refs. 3 and 4) have suggested that the last common ancestor of bilaterians (LCB) lived well over one billion years ago (5, 6), whereas others suggest that LCB arose Ϸ900 million years ago (Ma) (e.g., refs. 7-10), and still others are more consistent with an origination closer to the Cambrian (11-13). These deep estimates for the origin of LCB raise the question of how hundreds of millions of years of bilaterian evolution can escape detection, given that LCB and its near relatives should have had the capability of leaving both body and trace fossils (14-16).Because molecular clocks have several inherent problems, including how the clock is calibrated, how molecular substitution rates are estimated, and how heterogeneity in these rates is detected and corrected (3, 4), as well...

Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-13 C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and 13 C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAO1 and PA14 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa.A number of microbes, including Pseudomonas aeruginosa, can utilize glycine betaine (GB) as a sole carbon, nitrogen, and energy source (17,35,41). GB, an important osmoprotectant for many bacteria (6), is available to organisms in a variety of environments (5,14,34,41). Free GB can be released by roots (9), microbes (14, 15), or decaying animal (20) and plant (10) matter. Alternatively, GB can be derived from choline or carnitine (4,5,12,15,20,36). Choline and carnitine can be found in many eukaryote-associated environments, and bacteria, including P. aeruginosa, can use phospholipases and choline phosphatases to release choline from phosphatidylcholine (30, 38). In P. aeruginosa, choline is oxidized to GB by a two-step process catalyzed by BetA and BetB (29,36), while carnitine is predicted to be reduced and deacetylated by uncharacterized enzymes, ultimately yielding GB (16).The aerobic catabolism of GB in bacteria is best understood in Sinohizobium (35), Corynebacterium (8, 37), and Arthrobacter species (24). The data from these studies suggest that GB catabolism occurs via serial demethylation that forms dimethylglycine (DMG), then sarcosine (also called monomethylglycine), and finally glycine (Fig. 1). Thin-layer chromatographic analyses indicated that in P. aeruginosa DMG and sarcosine are also intermediates formed during GB catabolism (11). Furthermore, in the same study, a proteomics analysis of P. aeruginosa cul...

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Carnitine is a quaternary amine compound found at high concentration in animal tissues, particularly muscle, and is most well studied for its contribution to fatty acid transport into mitochondria. In bacteria, carnitine is an important osmoprotectant, and can also enhance thermotolerance, cryotolerance and barotolerance. Carnitine can be transported into the cell or acquired from metabolic precursors, where it can serve directly as a compatible solute for stress protection or be metabolized through one of a few distinct pathways as a nutrient source. In this review, we summarize what is known about carnitine physiology and metabolism in bacteria. In particular, recent advances in the aerobic and anaerobic metabolic pathways as well as the use of carnitine as an electron acceptor have addressed some long-standing questions in the field. Received 5 January 2015 Accepted 17 March 2015Introduction Carnitine (c-trimethylamino-b-hydroxybutyric acid) ( Fig. 1) is a quaternary amine compound that can be produced by all domains of life, and was discovered in muscle extract in 1905 by Gulewitsch & Krimberg (1905) and Kutscher (1905). It was shown to be essential for larval development of the mealworm Tenebrio molitor and was originally designated vitamin B T based on this requirement. Later, it was discovered that carnitine can be synthesized in mammals and is now considered to be a quasi-nutrient or conditionally essential nutrient (Flanagan et al., 2010), as neonates have reduced biosynthesis and rely on placental transfer of carnitine in utero and exogenous sources after birth (Combs, 2012). Fifty years after the discovery of carnitine, it was demonstrated that assorted Gram-positive and Gram-negative bacteria could use carnitine in either aerobic or anaerobic environments for a variety of cellular functions, including as an electron acceptor, as a compatible solute to survive environmental insults or as a sole carbon, nitrogen and energy source. Bacterial carnitine metabolism was most recently reviewed in 1998 (Bieber, 1988;Bremer, 1983;Kleber, 1997;Rebouche & Seim, 1998) and the field has seen important advances. This review summarizes what we knew at the time of the previous reviews and emphasizes what we have learned since, including: (i) how anaerobic bacteria synthesize and utilize crotonobetaine and carnitine as final electron acceptors, (ii) the impact of carnitine degradation by the intestinal microbiota and the genes responsible for this anaerobic conversion, (iii) the genes involved in aerobic degradation of carnitine, and (iv) how carnitine as a compatible solute impacts survival within and outside of the host. Carnitine in the environmentRecent work makes it clear that while animals represent the most readily accessible source of carnitine, carnitine is often present and sometimes abundant in soil and natural waters. Quaternary ammonium compounds are abundant in a number of soil ecosystems, including comprising a quarter of the most abundant organic nitrogen compounds in the soil water of a subalpine gras...

Regulation of ciliary and flagellar motility requires spatial control of dynein-driven microtubule sliding. However, the mechanism for regulating the location and symmetry of dynein activity is not understood. One hypothesis is that the asymmetrically organized central apparatus, through interactions with the radial spokes, transmits a signal to regulate dynein-driven microtubule sliding between subsets of doublet microtubules. Based on this model, we hypothesized that the orientation of the central apparatus defines positions of active microtubule sliding required to control bending in the axoneme. To test this, we induced microtubule sliding in axonemes isolated from wild-type and mutant Chlamydomonas cells, and then used electron microscopy to determine the orientation of the central apparatus. Transverse sections of wild-type axonemes revealed that the C1 microtubule is predominantly oriented toward the position of active microtubule sliding. In contrast, the central apparatus is randomly oriented in axonemes isolated from radial spoke deficient mutants. For outer arm dynein mutants, the C1 microtubule is oriented toward the position of active microtubule sliding in low calcium buffer, but is randomly oriented in high calcium buffer. These results provide evidence that the central apparatus defines the position of active microtubule sliding, and may regulate the size and shape of axonemal bends through interactions with the radial spokes. In addition, our results indicate that in high calcium conditions required to generate symmetric waveforms, the outer dynein arms are potential targets of the central pair-radial spoke control system.