Metabolic Reconstruction for Metagenomic Data and Its Application to the Human Microbiome

Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis in the human microbiome. Shotgun sequencing of such communities' metagenomes provides information complementary to organismal abundances from taxonomic markers, but the resulting data typically comprise short reads from hundreds of different organisms and are at best challenging to assemble comparably to single-organism genomes. Here, we describe an alternative approach to infer the functional and metabolic potential of a microbial community metagenome. We determined the gene families and pathways present or absent within a community, as well as their relative abundances, directly from short sequence reads. We validated this methodology using a collection of synthetic metagenomes, recovering the presence and abundance both of large pathways and of small functional modules with high accuracy. We subsequently applied this method, HUMAnN, to the microbial communities of 649 metagenomes drawn from seven primary body sites on 102 individuals as part of the Human Microbiome Project (HMP). This provided a means to compare functional diversity and organismal ecology in the human microbiome, and we determined a core of 24 ubiquitously present modules. Core pathways were often implemented by different enzyme families within different body sites, and 168 functional modules and 196 metabolic pathways varied in metagenomic abundance specifically to one or more niches within the microbiome. These included glycosaminoglycan degradation in the gut, as well as phosphate and amino acid transport linked to host phenotype (vaginal pH) in the posterior fornix. An implementation of our methodology is available at http://huttenhower.sph.harvard.edu/human​n. This provides a means to accurately and efficiently characterize microbial metabolic pathways and functional modules directly from high-throughput sequencing reads, enabling the determination of community roles in the HMP cohort and in future metagenomic studies.National Institutes of Health (U.S.) (U54HG004968

The lactose operon from Lactobacillus casei is involved in the transport and metabolism of the human milk oligosaccharide core-2 N-acetyllactosamine

The lactose operon (lacTEGF) from Lactobacillus casei strain BL23 has been previously studied. The lacT gene codes for a transcriptional antiterminator, lacE and lacF for the lactose-specific phosphoenolpyruvate: phosphotransferase system (PTSLac) EIICB and EIIA domains, respectively, and lacG for the phospho-β-galactosidase. In this work, we have shown that L. casei is able to metabolize N-acetyllactosamine (LacNAc), a disaccharide present at human milk and intestinal mucosa. The mutant strains BL153 (lacE) and BL155 (lacF) were defective in LacNAc utilization, indicating that the EIICB and EIIA of the PTSLac are involved in the uptake of LacNAc in addition to lactose. Inactivation of lacG abolishes the growth of L. casei in both disaccharides and analysis of LacG activity showed a high selectivity toward phosphorylated compounds, suggesting that LacG is necessary for the hydrolysis of the intracellular phosphorylated lactose and LacNAc. L. casei (lacAB) strain deficient in galactose-6P isomerase showed a growth rate in lactose (0.0293 ± 0.0014 h-1) and in LacNAc (0.0307 ± 0.0009 h-1) significantly lower than the wild-type (0.1010 ± 0.0006 h-1 and 0.0522 ± 0.0005 h-1, respectively), indicating that their galactose moiety is catabolized through the tagatose-6P pathway. Transcriptional analysis showed induction levels of the lac genes ranged from 130 to 320-fold in LacNAc and from 100 to 200-fold in lactose, compared to cells growing in glucose

Spectroscopic and electrochemical studies of cocaine–opioid interactions

Abstract The drugs of abuse cocaine (C), heroin (H), and morphine (M) have been studied to enable understanding of the occurrence of cocaine–opioid interactions at a molecular level. Electrochemical, Raman, and NMR studies of the free drugs and their mixtures were used to study drug–drug interactions. The results were analyzed using data obtained from quantum-mechanical calculations. For the cocaine–morphine mixture (C–MH), formation of a binary complex was detected; this involved the 3-phenolic group and the heterocyclic oxygen of morphine and the carbonyl oxygen and the methyl protons of cocaine’s methyl ester group. NMR studies conducted simultaneously also revealed C–MH binding geometry consistent with theoretical predictions and with electrochemical and vibrational spectroscopy results. These results provide evidence for the occurrence of a cocaine–morphine interaction, both in the solid state and in solution, particularly for the hydrochloride form. A slight interaction, in solution, was also detected by NMR for the cocaine–heroin mixture. Figure "Schematic representation of the proposed model for cocaine:morphine salt interaction

Patterns of release of the secondary conidia of Claviceps africana, the sorghum ergot pathogen in Australia

Trials were conducted in southern Queensland, Australia between March and May 2003, 2004 and 2005 to study patterns of hourly and daily release of the secondary conidia of Claviceps africana and their relationships with weather parameters. Conidia were trapped for at least one hour on most (> 90%) days in 2003 and 2004, but only on 55% of days in 2005. Both the highest daily concentration of conidia, and the highest number of hours per day when conidia were trapped, were recorded 1-3 days after rainfall events. Although the pattern of conidial release was different every day, the highest hourly conidial concentrations occurred between 10.00 hours and 17.00 hours on 73% of all days in the three trials. Hours when conidia were trapped were characterized by higher median values of temperature, windspeed and vapour pressure deficit, lower relative humidity, and leaf wetness values of 0%, than hours when no conidia were recorded. The results indicate that fungicides need to be applied to the highly ergot-susceptible male sterile (A-) lines of sorghum in hybrid seed production blocks and breeders' nurseries as soon as possible after rainfall events to minimize ergot severity

Effects of stem canker (Leptosphaeria maculans) and light leaf spot (Pyrenopeziza brassicae) on yield of winter oilseed rape (Brassica napus) in southern England

The relationships between yield loss and incidence or severity of stem canker and light leaf spot in winter oilseed rape were analysed by correlation and regression analyses, using data from experiments at Rothamsted, England in 1992/93, 1994/95 and 1995/96. Growth stages (GS) 6,3/6,4 and 4,0/4,5 were identified as the critical points for relating percentage yield loss to stem canker and light leaf spot (on stems), respectively. Critical point (CP) and area under disease progress curve (AUDPC) models relating percentage yield loss to combined incidence or severity of stem canker and light leaf spot (stems) in each experiment were constructed by linear regression. There were no differences in the CP models for incidence between 1992/93, 1994/95 and 1995/96 experiments, or in the AUDPC models for incidence between 1992/93 and 1994/95 experiments. Therefore, a general CP model relating percentage yield loss (Delta Y) to combined incidence of stem canker (S-i) at GS 6,3/6,4 and light leaf spot (stems) (L-i) at GS 4,0/4,5 was constructed using data from the three experiments: Delta Y = 0.85 + 0.079S(i) + 0.065L(i) (R-2 = 43.7%, P < 0.001, 92 df). A general AUDPC model relating Delta Y to the AUDPC of combined incidence of stem canker (S-ia) from GS 5.7 to GS 6.5 and light leaf spot (stems) (L-ia) from GS 4.0 to GS 6.3 was constructed using data from the 1992/93 and 1994/95 experiments: Delta Y = 0.07 + 0.00096S(ia) + 0.0026L(ia) (R-2 = 43.6%, P < 0.001, 68 df). These two general yield-loss models were tested with data from Rothamsted in 1993/94 and Boxworth in 1992/93. The predictive accuracy of the CP model based on combined incidence of stem canker and light leaf spot (stems) was better than that of the AUDPC model. Yield losses predicted by summing the estimates from individual models for incidence of stem canker alone (GS 6,3/6,4) and light leaf spot alone (on leaves at GS 3,3) were greater than observed yield losses in experiments at Rothamsted in 1992/93, 1993/94, 1994/95 and 1995/96 and at Boxworth in 1992/93.Peer reviewe