William W. Mohn

The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse

Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1s 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O2-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.

Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma

Allergic asthma rates have increased steadily in developed countries, arguing for an environmental aetiology. To assess the influence of gut microbiota on experimental murine allergic asthma, we treated neonatal mice with clinical doses of two widely used antibiotics—streptomycin and vancomycin—and evaluated resulting shifts in resident flora and subsequent susceptibility to allergic asthma. Streptomycin treatment had little effect on the microbiota and on disease, whereas vancomycin reduced microbial diversity, shifted the composition of the bacterial population and enhanced disease severity. Neither antibiotic had a significant effect when administered to adult mice. Consistent with the ‘hygiene hypothesis’, our data support a neonatal, microbiota‐driven, specific increase in susceptibility to experimental murine allergic asthma.

A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages

Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis, degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted to proceed via 4-androstene-3,17-dione and 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). Inactivation of each of the hsaC, supAB, and mce4 genes in RHA1 substantiated their roles in cholesterol catabolism. Moreover, the hsaC− mutant accumulated 3,4-DHSA, indicating that HsaCRHA1, formerly annotated as a biphenyl-degrading dioxygenase, catalyzes the oxygenolytic cleavage of steroid ring A. Bioinformatic analyses revealed that 51 rhodococcal genes specifically expressed during growth on cholesterol, including all predicted to specify the catabolism of rings A and B, are conserved within an 82-gene cluster in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette–Guérin. M. bovis bacillus Calmette–Guérin grew on cholesterol, and hsaC and kshA were up-regulated under these conditions. Heterologously produced HsaCH37Rv and HsaDH37Rv transformed 3,4-DHSA and its ring-cleaved product, respectively, with apparent specificities ≈40-fold higher than for the corresponding biphenyl metabolites. Overall, we annotated 28 RHA1 genes and proposed physiological roles for a similar number of mycobacterial genes. During survival of M. tuberculosis in the macrophage, these genes are specifically expressed, and many appear to be essential. We have delineated a complete suite of genes necessary for microbial steroid degradation, and pathogenic mycobacteria have been shown to catabolize cholesterol. The results suggest that cholesterol metabolism is central to M. tuberculosiss unusual ability to survive in macrophages and provide insights into potential targets for novel therapeutics.

The Lung Tissue Microbiome in Chronic Obstructive Pulmonary Disease

RATIONALE Based on surface brushings and bronchoalveolar lavage fluid, Hilty and coworkers demonstrated microbiomes in the human lung characteristic of asthma and chronic obstructive pulmonary disease (COPD), which have now been confirmed by others. OBJECTIVES To extend these findings to human lung tissue samples. METHODS DNA from lung tissue samples was obtained from nonsmokers (n = 8); smokers without COPD (n = 8); patients with very severe COPD (Global Initiative for COPD [GOLD] 4) (n = 8); and patients with cystic fibrosis (CF) (n = 8). The latter served as a positive control, with sterile water as a negative control. All bacterial community analyses were based on polymerase chain reaction amplifying 16S rRNA gene fragments. Total bacterial populations were measured by quantitative polymerase chain reaction and bacterial community composition was assessed by terminal restriction fragment length polymorphism analysis and pyrotag sequencing. MEASUREMENT AND MAIN RESULTS Total bacterial populations within lung tissue were small (20-1,252 bacterial cells per 1,000 human cells) but greater in all four sample groups versus the negative control group (P < 0.001). Terminal restriction fragment length polymorphism analysis and sequencing distinguished three distinct bacterial community compositions: one common to the nonsmoker and smoker groups, a second to the GOLD 4 group, and the third to the CF-positive control group. Pyrotag sequencing identified greater than 1,400 unique bacterial sequences and showed an increase in the Firmicutes phylum in GOLD 4 patients versus all other groups (P < 0.003) attributable to an increase in the Lactobacillus genus (P < 0.0007). CONCLUSIONS There is a detectable bacterial community within human lung tissue that changes in patients with very severe COPD.

Degradation of polycyclic aromatic hydrocarbons at low temperature under aerobic and nitrate-reducing conditions in enrichment cultures from northern soils.

ABSTRACT Thepotential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not wellunderstood, but such biodegradation would be very useful forremediation of polluted sites. Biodegradation of a mixture of 11different PAHs with two to five aromatic rings, each at a concentrationof 10 μg/ml, was studied in enrichment cultures inoculated withsamples of four northern soils. Under aerobic conditions, lowtemperature severely limited PAH biodegradation. After 90 days, aerobiccultures at 20°C removed 52 to 88% of the PAHs. The mostextensive PAH degradation under aerobic conditions at 7°C,53% removal, occurred in a culture from creosote-contaminatedsoil. Low temperature did not substantially limit PAH biodegradationunder nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene weredegraded. The most extensive PAH degradation under nitrate-reducingconditions at 7°C, 39% removal, occurred in a culturefrom fuel-contaminated Arctic soil. In separate transfer cultures fromthe above Arctic soil, incubated anaerobically at 7°C, removalof 2-methylnaphthalene and fluorene was stoichiometrically coupled tonitrate removal. Ribosomal intergenic spacer analysis suggested thatenrichment resulted in a few predominant bacterial populations,including members of the genera Acidovorax,Bordetella, Pseudomonas, Sphingomonas, andVariovorax. Predominant populations from different soils oftenincluded phylotypes with nearly identical partial 16S rRNA genesequences (i.e., same genus) but never included phylotypes withidentical ribosomal intergenic spacers (i.e., different species orsubspecies). The composition of the enriched communities appeared to bemore affected by presence of oxygen, than by temperature or source oftheinoculum.

Limiting factors for hydrocarbon biodegradation at low temperature in Arctic soils

Hydrocarbon fuel spills are common in the Arctic. But, little is known about hydrocarbon-degrading microflora in Arctic tundra soils or the potential for bioremediation of these soils. We examined mineralization of radiolabeled hydrocarbons in microcosms containing soils collected from sites across the Canadian Arctic. The soils all contained psychrotolerant microorganisms which mineralized dodecane and substantially removed total petroleum hydrocarbons (TPH) at 7°C. Dodecane mineralization was severely limited by both N and P. Dodecane mineralization kinetics varied greatly among different soils. Multiple regression analysis showed that soil N and TPH concentrations together accounted for 73% of the variability of the lag time preceding dodecane mineralization. Soil characteristics were less effective as predictors of mineralization kinetic parameters other than lag time. High total C concentrations were associated with high mineralization rate constants, and high sand contents were associated with long times for half-maximal dodecane mineralization. Very high concentrations of TPH (100 mg g−1 of dry soil) and heavy metals (e.g., 1.4 mg Pb g−1 of dry soil) did not prevent dodecane mineralization. Inoculation of soils with indigenous or non-indigenous hydrocarbon-degrading microorganisms stimulated dodecane mineralization. Bioremediation of hydrocarbon-contaminated Arctic tundra soils appears to be feasible, and various engineering strategies, such as heating or inoculating the soil, can accelerate hydrocarbon biodegradation.

Microbiological removal of pentachlorophenol from soil using a Flavobacterium

Abstract Experiments reported here show that it is possible to remove pentachlorophenol (PCP) from a variety of contaminated soils, including actual waste-dump soils, by inoculating such soils with cells of a PCP-degrading Flavobacterium. However, soil conditions such as temperature, water content, PCP concentration and density of bacterial cells must be maintained within optimum ranges. Manipulation of the natural microflora so that it degrades PCP may be the preferred decontamination method in many instances. Very highly contaminated soils are so toxic that methods such as soil leaching followed by decontamination of the leachate may be required, as direct inoculation or self-purification are ineffective.

Microbial community dynamics in a seasonally anoxic fjord: Saanich Inlet, British Columbia.

Dissolved oxygen concentration plays a major role in shaping biotic interactions and nutrient flows within marine ecosystems. Throughout the global ocean, regions of low dissolved oxygen concentration (hypoxia) are a common and expanding feature of the water column, with major feedback on productivity and greenhouse gas cycling. To better understand microbial diversity underlying biogeochemical transformations within oxygen-deficient oceanic waters, we monitored and quantified bacterial and archaeal community dynamics in relation to dissolved gases and nutrients during a seasonal stratification and deep water renewal cycle in Saanich Inlet, British Columbia, a seasonally anoxic fjord. A number of microbial groups partitioned within oxygen-deficient waters including Nitrospina and SAR324 affiliated with the delta-proteobacteria, SAR406 and gamma-proteobacteria related to thiotrophic gill symbionts of deep-sea clams and mussels. Microbial diversity was highest within the hypoxic transition zone decreasing dramatically within anoxic basin waters and temporal patterns of niche partitioning were observed along defined gradients of oxygen and phosphate. These results provide a robust comparative phylogenetic framework for inferring systems metabolism of nitrogen, carbon and sulfur cycling within oxygen-deficient oceanic waters and establish Saanich Inlet as a tractable model for studying the response of microbial communities to changing levels of water column hypoxia.

The Actinobacterial mce4 Locus Encodes a Steroid Transporter

Bioinformatic analyses have suggested that Mce proteins in diverse actinobacteria are components of complex ATP-binding cassette transporter systems, comprising more than eight distinct proteins. In Mycobacterium tuberculosis, these proteins are implicated in interactions of this deadly pathogen with its human host. Here, we provide direct evidence that the Mce4 system of Rhodococcus jostii RHA1 is a steroid uptake system. Transcriptional analyses indicate that the system is encoded by an 11-gene operon, up-regulated 4.0-fold during growth on cholesterol versus on pyruvate. Growth of RHA1 on cholesterol and uptake of radiolabeled cholesterol both required expression of genes in the mce4 operon encoding two permeases plus eight additional proteins of unknown function. Cholesterol uptake was ATP-dependent and exhibited Michaelis-Menten kinetics with a Km of 0.6 ± 0.1 μm. This uptake system was also essential for growth of RHA1 on β-sitosterol, 5-α-cholestanol, and 5-α-cholestanone. Bioinformatic analysis revealed that all mce4 loci in sequenced genomes are linked to steroid metabolism genes. Thus, we predict that all Mce4 systems are steroid transporters. The transport function of the Mce4 system is consistent with proposed roles of cholesterol and its metabolism in the pathogenesis of M. tuberculosis.

Significant and persistent impact of timber harvesting on soil microbial communities in Northern coniferous forests

Forest ecosystems have integral roles in climate stability, biodiversity and economic development. Soil stewardship is essential for sustainable forest management. Organic matter (OM) removal and soil compaction are key disturbances associated with forest harvesting, but their impacts on forest ecosystems are not well understood. Because microbiological processes regulate soil ecology and biogeochemistry, microbial community structure might serve as indicator of forest ecosystem status, revealing changes in nutrient and energy flow patterns before they have irreversible effects on long-term soil productivity. We applied massively parallel pyrosequencing of over 4.6 million ribosomal marker sequences to assess the impact of OM removal and soil compaction on bacterial and fungal communities in a field experiment replicated at six forest sites in British Columbia, Canada. More than a decade after harvesting, diversity and structure of soil bacterial and fungal communities remained significantly altered by harvesting disturbances, with individual taxonomic groups responding differentially to varied levels of the disturbances. Plant symbionts, like ectomycorrhizal fungi, and saprobic taxa, such as ascomycetes and actinomycetes, were among the most sensitive to harvesting disturbances. Given their significant ecological roles in forest development, the fate of these taxa might be critical for sustainability of forest ecosystems. Although abundant bacterial populations were ubiquitous, abundant fungal populations often revealed a patchy distribution, consistent with their higher sensitivity to the examined soil disturbances. These results establish a comprehensive inventory of bacterial and fungal community composition in northern coniferous forests and demonstrate the long-term response of their structure to key disturbances associated with forest harvesting.