Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47

The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO₂. The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of α-, β-, and γ-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe₄S₄] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers “Aromatoleum aromaticum” EbN1, Azoarcus sp. strain T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetyl-CoA and CO₂. Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.Polycyclic aromatic hydrocarbons (PAHs) are constantly released into the environment by anthropogenic activities such as industrial use or by accidental contamination. Due to the low chemical reactivity caused by the resonance energy of the aromatic ring structure and the low bioavailability of PAHs, they are persistent in the environment (15). The understanding of microbial metabolic capabilities in terms of anaerobic PAH degradation is in its infancy. However, natural amelioration of contaminated sites relies on the degradation capacities of microorganisms, and therefore, it is an essential prerequisite to broaden knowledge about the microorganisms involved and their potentials concerning PAH breakdown.Numerous microorganisms that can degrade PAHs under aerobic conditions have already been identified, but only a small number of anaerobic cultures that degrade PAHs like naphthalene, 2-methylnaphthalene, and phenanthrene have been isolated so far (17, 20, 24, 31, 46-48, 50, 52, 66). It has been shown that these anaerobic degraders activate aromatic hydrocarbons by very unusual biochemical reactions which differ completely from those of aerobic degradation. The peripheral pathway of 2-methylnaphthalene degradation occurs in analogy to anaerobic toluene degradation by the addition of fumarate to the methyl group, catalyzed by the glycyl radical enzyme naphthyl-2-methyl-succinate synthase (Nms) (Fig. ​(Fig.1)1) (3). In subsequent reactions, naphthyl-2-methyl-succinate is activated to yield the coenzyme A (CoA) ester and oxidized to form naphthyl-2-methylene-succinyl-CoA. The following beta-oxidation of the side chain results in the formation of 2-naphthoyl-CoA and succinate (3, 53). The first three enzyme reactions of this pathway have been measured in vitro (3, 53). Recently, Musat et al. (48) identified the gene coding for the α-subunit of a putative naphthyl-2-methyl-succinate synthase (nmsA) in 2-methylnaphthalene-grown bacterial cultures. The molecular composition of the nmsA gene is analogous to that of the benzylsuccinate synthase α-subunit gene (bssA). The Bss enzyme is a well-investigated close homolog of Nms, catalyzing fumarate addition in the initial reaction of anaerobic toluene degradation (34, 40). Based on findings from comparative sequence studies, glycine radical-catalyzed fumarate addition has been shown to be a widely distributed initial reaction mechanism for anaerobic hydrocarbon degradation involving toluene and 2-methylnaphthalene, n-alkanes (12, 13, 25, 51), m-xylene (33), m- and p-cresols (9), and ethylbenzene (32).Open in a separate windowFIG. 1.Proposed pathway for anaerobic 2-methylnaphthalene degradation and reductive dearomatization of 2-naphthoyl-CoA (3, 4, 53). Genes found in the N47 genome encode the following enzymes (shown in gray boxes): NmsABC, naphthyl-2-methyl-succinate synthase; BnsEF, naphthyl-2-methyl-succinate CoA transferase; BnsG, naphthyl-2-methyl-succinyl-CoA dehydrogenase; BnsH, naphthyl-2-methylene-succinyl-CoA hydratase; BnsCD, naphthyl-2-hydroxymethyl-succinyl-CoA dehydrogenase; BnsAB, naphthyl-2-oxomethyl-succinyl-CoA thiolase; and NcrABCD, 2-naphthoyl-CoA reductase. The position of the double bond is not known for octahydro-2-naphthoyl-CoA. COSCoA, thioester of CoA and the respective carboxyl group.In a process analogous to the anaerobic benzoyl-CoA degradation pathway (7), 2-naphthoyl-CoA is subjected to aromatic ring reduction by a putative naphthoyl-CoA reductase, probably generating 5,6,7,8-tetrahydro-naphthoyl-CoA and further octahydro-2-naphthoic acid (4, 46). In the subsequent reactions, the ring system should be thiolytically cleaved and subjected to beta-oxidation, leading to the formation of acetyl-CoA and CO₂.In contrast to the first enzymatic reaction in the degradation of methylated aromatics, the first enzymatic reaction in anaerobic degradation of unsubstituted aromatic compounds such as naphthalene is still unresolved. In order to determine the initial activation reaction of anaerobic naphthalene degradation, studies based on the analysis of metabolites have been performed. Zhang and Young (66) observed the incorporation of ¹³C-labeled bicarbonate from the buffer into the carboxyl group of 2-naphthoic acid, hypothesizing that carboxylation is the initial activation reaction of anaerobic naphthalene degradation in the culture studied. Recently, Safinowski and Meckenstock (54) identified the deuterated metabolites naphthyl-2-methyl-succinate and naphthyl-2-methylene-succinate, which are exclusive intermediates of anaerobic 2-methylnaphthalene degradation, in the enrichment culture N47 when the culture was cultivated on fully deuterated naphthalene. Moreover, specific enzyme activities of the anaerobic 2-methylnaphtahlene degradation pathway have been detected in naphthalene-grown cells (54). Therefore, methylation of naphthalene to yield 2-methylnaphthalene as the initial activation reaction and subsequent degradation via the 2-methylnaphthalene pathway were proposed for this bacterial culture. The elucidation of 2-methylnaphthalene degradation may therefore reveal an important part of the naphthalene degradation pathway. However, Musat et al. (48) questioned methylation as the first reaction in naphthalene degradation for their marine naphthalene-degrading deltaproteobacterial NaphS strains.Whereas molecular components involved in anaerobic degradation of monoaromatic hydrocarbons are well known, knowledge about genes and enzymes involved in anaerobic PAH degradation is still missing (14). Here, we provide the first results of a whole-proteome- and whole-genome-based investigation of the sulfate-reducing enrichment culture N47 degrading naphthalene and 2-methylnaphtalene. We have identified some gene clusters encoding enzymes involved in 2-methylnaphthalene degradation, 2-naphthoyl-CoA dearomatization, and subsequent ring cleavage reactions in 2-methylnaphthalene-grown N47 cells.     

Patterns of Biofilm Succession on a Sheltered Rocky Shore in Hong Kong

Successional patterns are dependent on the nature of the substratum, water flow, concentrations of organics as well as the availability of bacteria, algal spores and invertebrate larvae in the coastal environment. Bacteria play an especially important role in biofilm formation as they are generally the earliest colonizers. In the present study, both winter and summer biofilm succession patterns were examined on glass coverslips inverted on experimental racks attached at two tidal levels on a sheltered shore in Hong Kong. In the succession, bacteria were followed by diatoms and cyanobacteria. Encrusting algae appeared in the late stages of the experiment (day 80 in summer and day 60 in winter). Colonization by bacteria was much slower in summer and their density remained low throughout the experimental period. The first appearance of diatoms and cyanobacteria, however, was more rapid in the summer. Bacteria and diatoms on the low-shore surfaces also had a faster succession rate than on the high-shore surfaces, suggesting that desiccation/aerial temperature are the causal factors for such differences.     

High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including enabling technologies such as the well-established multiwell/microwell plates and robotic spotting, and emerging technologies like microfluidics, micro-contact printing and lithography. We also discuss the studies that utilized high throughput screening platform to investigate stem cell response to extracellular matrix, topography, biomaterials and stiffness gradients in the stem cell niche. The combination of the aforementioned techniques could lay the foundation for new perspectives in further development of high throughput technology and stem cell research.     

RNA stable-isotope probing

At the heart of microbial ecology lies a true scientific dichotomy. On the one hand, we know microbes are responsible for processes on which all other life on Earth is dependent; their removal would mean the cessation of all known life. However, in opposition, the majority of extant microbial species in natural environments have never been cultured or studied in a laboratory as living organisms. Owing to these factors, the question of "who does what?" has been a major barrier to understanding how microbially mediated ecosystem level events occur. Recently, the use of stable isotopes (13C) to trace carbon from specific substrates into microbes that assimilate carbon from that substrate has significantly advanced our understanding of the relationship between environmental processes and microbial phylogeny.     

Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.     

Gestating for 22 months: luteal development and pregnancy maintenance in elephants

The corpus luteum, a temporally established endocrine gland, formed on the ovary from remaining cells of the ovulated follicle, plays a key role in maintaining the early mammalian pregnancy by secreting progesterone. Despite being a monovular species, 2-12 corpora lutea (CLs) were found on the elephant ovaries during their long pregnancy lasting on average 640 days. However, the function and the formation of the additional CLs and their meaning remain unexplained. Here, we show from the example of the elephant, the close relationship between the maternally determined luteal phase length, the formation of multiple luteal structures and their progestagen secretion, the timespan of early embryonic development until implantation and maternal recognition. Through three-dimensional and Colour Flow ultrasonography of the ovaries and the uterus, we conclude that pregnant elephants maintain active CL throughout gestation that appear as main source of progestagens. Two LH peaks during the follicular phase ensure the development of a set of 5.4 ± 2.7 CLs. Accessory CLs (acCLs) form prior to ovulation after the first luteinizing hormone (LH) peak, while the ovulatory CL (ovCL) forms after the second LH peak. After five to six weeks (the normal luteal phase lifespan), all existing CLs begin to regress. However, they resume growing as soon as an embryo becomes ultrasonographically apparent on day 49 ± 2. After this time, all pregnancy CLs grow significantly larger than in a non-conceptive luteal phase and are maintained until after parturition. The long luteal phase is congruent with a slow early embryonic development and luteal rescue only starts 'last minute', with presumed implantation of the embryo. Our findings demonstrate a highly successful reproductive solution, different from currently described mammalian models.     

Testing the limits of 454 pyrotag sequencing: reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes

The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ~4% read abundance across biological, and ~2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific 'in vivo' fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.