Evolution of Protein Molecules

Microbial diversity studies based on the cloning and sequencing of DNA from nature support the conclusion that only a fraction of the microbial diversity is currently represented in culture collections. Out of over 40 known prokaryotic phyla, only half have cultured representatives. In an effort to culture the uncultured phylotypes from oligotrophic marine ecosystems, we developed high-throughput culturing procedures that utilize the concept of extinction culturing to isolate cultures in small volumes of low-nutrient media. In these experiments, marine bacteria were isolated and cultivated at in situ substrate concentrations—typically 3 orders of magnitude less than common laboratory media. Microtiter plates and a newly developed procedure for making cell arrays were employed to raise the throughput rate and lower detection sensitivity, permitting cell enumeration from 200-μl aliquots of cultures with densities as low as 103 cells/ml. Approximately 2,500 extinction cultures from 11 separate samplings of marine bacterioplankton were screened over the course of 3 years. Up to 14% of the cells collected from coastal seawater were cultured by this method, which was 14- to 1,400-fold higher than the numbers obtained by traditional microbiological culturing techniques. Among the microorganisms cultured were four unique cell lineages that belong to previously uncultured or undescribed marine Proteobacteria clades known from environmental gene cloning studies. These cultures are related to the clades SAR11 (α subclass), OM43 (β subclass), SAR92 (γ subclass), and OM60/OM241 (γ subclass). This method proved successful for the cultivation of previously uncultured marine bacterioplankton that have consistently been found in marine clone libraries.

High-Throughput Methods for Culturing Microorganisms in Very-Low-Nutrient Media Yield Diverse New Marine Isolates

The epsilon-proteobacteria have recently been recognized as globally ubiquitous in modern marine and terrestrial ecosystems, and have had a significant role in biogeochemical and geological processes throughout Earth's history. To place this newly expanded group, which consists mainly of uncultured representatives, in an evolutionary context, we present an overview of the taxonomic classification for the class, review ecological and metabolic data in key sulphidic habitats and consider the ecological and geological potential of the epsilon-proteobacteria in modern and ancient systems. These integrated perspectives provide a framework for future culture- and genomic-based studies.

The versatile epsilon-proteobacteria: key players in sulphidic habitats.

Abundant, micrometer-scale, spherical aggregates of 2- to 5-nanometer-diameter sphalerite (ZnS) particles formed within natural biofilms dominated by relatively aerotolerant sulfate-reducing bacteria of the family Desulfobacteriaceae. The biofilm zinc concentration is about 106 times that of associated groundwater (0.09 to 1.1 parts per million zinc). Sphalerite also concentrates arsenic (0.01 weight %) and selenium (0.004 weight %). The almost monomineralic product results from buffering of sulfide concentrations at low values by sphalerite precipitation. These results show how microbes control metal concentrations in groundwater- and wetland-based remediation systems and suggest biological routes for formation of some low-temperature ZnS deposits.

https://science.sciencemag.org/content/sci/290/5497/1744.full.pdf

Formation of sphalerite (ZnS) deposits in natural biofilms of sulfate-reducing bacteria.

This review of anaerobic metabolism emphasizes aerobic oxidation, because the two processes cannot be separated in a complete treatment of the topic. It is process oriented and highlights the fascinating microorganisms that mediate anaerobic biogeochemistry. We begin this review with a brief discussion of CO 2 assimilation by autotrophs, the source of most of the reducing power on Earth, and then consider the biological processes that harness this potential energy. Energy liberation begins with the decomposition of organic macromolecules to relatively simple compounds, which are simplified further by fermentation. Methanogenesis is considered next because CH 4 is a product of acetate fermentation, and thus completes the catabolism of organic matter, particularly in the absence of inorganic electron acceptors. Finally, the organisms that use nitrogen, manganese, iron, and sulfur for terminal electron acceptors are considered in order of decreasing free-energy yield of the reactions.

Anaerobic Metabolism: Linkages to Trace Gases and Aerobic Processes

A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “Candidatus Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO2 fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO2 fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “Candidatus Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

/pdf/characterization-of-an-autotrophic-sulfide-oxidizing-marine-1r9z9cutei.pdf

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.

/pdf/family-and-genus-level-16s-rrna-targeted-oligonucleotide-4qf43a8uob.pdf

Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

Environmentally dominant members of the genus Beggiatoa and Thioploca spp. are united by unique morphological and physiological adaptations (S. C. McHatton, J. P. Barry, H. W. Jannasch, and D. C. Nelson, Appl. Environ. Microbiol. 62:954–958, 1996). These adaptations include the presence of very wide filaments (width, 12 to 160 μm), the presence of a central vacuole comprising roughly 80% of the cellular biovolume, and the capacity to internally concentrate nitrate at levels ranging from 150 to 500 mM. Until recently, the genera Beggiatoa and Thioploca were recognized and differentiated on the basis of morphology alone; they were distinguished by the fact that numerous Thioploca filaments are contained within a common polysaccharide sheath, while Beggiatoa filaments occur singly. Vacuolate Beggiatoa or Thioploca spp. can dominate a variety of marine sediments, seeps, and vents, and it has been proposed (H. Fossing, V. A. Gallardo, B. B. Jorgensen, M. Huttel, L. P. Nielsen, H. Schulz, D. E. Canfield, S. Forster, R. N. Glud, J. K. Gundersen, J. Kuver, N. B. Ramsing, A. Teske, B. Thamdrup, and O. Ulloa, Nature [London] 374:713–715, 1995) that members of the genus Thioploca are responsible for a significant portion of total marine denitrification. In order to investigate the phylogeny of an environmentally dominant Beggiatoa sp., we analyzed complete 16S rRNA gene sequence data obtained from a natural population found in Monterey Canyon cold seeps. Restriction fragment length polymorphism analysis of a clone library revealed a dominant clone, which gave rise to a putative Monterey Beggiatoa 16S rRNA sequence. Fluorescent in situ hybridization with a sequence-specific probe confirmed that this sequence originated from wide Beggiatoa filaments (width, 65 to 85 μm). A phylogenetic tree based on evolutionary distances indicated that the Monterey Beggiatoa sp. falls in the gamma subdivision of the class Proteobacteria and is most closely related to the genus Thioploca. This vacuolate Beggiatoa—Thioploca cluster and a more distantly related freshwater Beggiatoa species cluster form a distinct phylogenetic group.

https://aem.asm.org/content/aem/65/1/270.full.pdf

Phylogenetic Affinity of a Wide, Vacuolate, Nitrate-Accumulating Beggiatoa sp. from Monterey Canyon, California, with Thioploca spp.

Molecular characterization of methanotrophic communities in forest soils that consume atmospheric methane

Damping off of soybean and corn, caused by Pythium spp., is favored by cool temperatures and wet soil conditions and is primarily managed using fungicide seed treatments. The goal of this research was to determine the effect of temperature on aggressiveness and fungicide sensitivity of Pythium spp. recovered from soybean and corn in Iowa. A total of 21 isolates of four of the most prevalent Pythium spp. in Iowa were screened. Seed and seedling assays were used to quantify the aggressiveness of P. lutarium, P. oopapillum, P. sylvaticum, and P. torulosum on soybean and corn at 13, 18, and 23°C. Isolates recovered from soybean or corn were equally pathogenic on both hosts. P. torulosum was more aggressive at 13°C compared with 18 and 23°C. Conversely, P. sylvaticum was more aggressive at 18 and 23°C than at 13°C. A plate assay was used to assess fungicide sensitivity to seven fungicides that are commonly used as seed treatments, and EC50 values at each of the three temperatures were determined and compared. EC50 values for P. torulosum were higher for all fungicides tested at 13°C, compared with 18 or 23°C, whereas EC50 values for P. sylvaticum were higher for all fungicides at 18 and 23°C compared with 13°C. These data contribute to our understanding of the effect of soil temperature on the risk of soybean and corn damping off, which may aid in the development of more effective management practices.

https://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-04-15-0487-RE

Azeem Ahmad

Papers

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

Phylogenetic Affinity of a Wide, Vacuolate, Nitrate-Accumulating Beggiatoa sp. from Monterey Canyon, California, with Thioploca spp.

Molecular characterization of methanotrophic communities in forest soils that consume atmospheric methane

Temperature Affects Aggressiveness and Fungicide Sensitivity of Four Pythium spp. that Cause Soybean and Corn Damping Off in Iowa.