Genome editing in ubiquitous freshwater Actinobacteria.

Development of genome-editing tools in diverse microbial species is an important step both in understanding the roles of those microbes in different environments, and in engineering microbes for a variety of applications. Freshwater-specific clades of Actinobacteria are ubiquitous and abundant in surface freshwaters worldwide. Here, we show that Rhodoluna lacicola and Aurantimicrobium photophilum, which represent widespread clades of freshwater Actinobacteria, are naturally transformable. We also show that gene inactivation via double homologous recombination and replacement of the target gene with antibiotic selection markers can be used in both strains, making them convenient and broadly accessible model organisms for freshwater systems. We further show that in both strains, the predicted phytoene synthase is the only phytoene synthase, and its inactivation prevents the synthesis of all pigments. The tools developed here enable targeted modification of the genomes of some of the most abundant microbes in freshwater communities. These genome-editing tools will enable hypothesis testing about the genetics and (eco)physiology of freshwater Actinobacteria and broaden the available model systems for engineering freshwater microbial communities. IMPORTANCE: To advance bioproduction or bioremediation in large, unsupervised environmental systems such as ponds, wastewater lagoons, or groundwater systems, it will be necessary to develop diverse genetically amenable microbial model organisms. Although we already genetically modify a few key species, tools for engineering more microbial taxa, with different natural phenotypes, will enable us to genetically engineer multispecies consortia or even complex communities. Developing genetic tools for modifying freshwater bacteria is particularly important, as wastewater, production ponds or raceways, and contaminated surface water are all freshwater systems where microbial communities are already deployed to do work, and the outputs could potentially be enhanced by genetic modifications. Here, we demonstrate that common tools for genome editing can be used to inactivate specific genes in two representatives of a very widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are found in almost all tested surface freshwater environments, where they co-occur with primary producers, and genome-editing tools in these species are thus a step on the way to engineering microbial consortia in freshwater environments.

Genome sequences of four bacterial strains isolated from organofluorine enrichment cultures.

Because fluorinated organic compounds are broadly used and highly persistent, microbes isolated from wastewater may be able to degrade these contaminants. Here, we report the genome sequences of Flavobacterium sp. str. WV_118_3, Nocardioides sp. str. WV_118_6, Ochrobactrum anthropi str. WV_118_8, and Sphingomonas sp. str. VL_57B, isolated from wastewater.

Genome sequence of Methylobacterium fujisawaense strain C14, isolated from concrete.

Methylobacterium fujisawaense strain C14 was isolated from a weathered concrete cylinder. Using PacBio sequencing, we generated a complete genome for strain C14, which includes one circular chromosome (6,656,731 bp) and six putative plasmids (35,452 to 85,428 bp).

A Bacillus velezensis strain shows antimicrobial activity against soilborne and foliar fungi and oomycetes.

Biological control uses naturally occurring antagonists such as bacteria or fungi for environmentally friendly control of plant pathogens. Bacillus spp. have been used for biocontrol of numerous plant and insect pests and are well-known to synthesize a variety of bioactive secondary metabolites. We hypothesized that bacteria isolated from agricultural soil would be effective antagonists of soilborne fungal pathogens. Here, we show that the Delaware soil isolate Bacillus velezensis strain S4 has in vitro activity against soilborne and foliar plant pathogenic fungi, including two with a large host range, and one oomycete. Further, this strain shows putative protease and cellulase activity, consistent with our prior finding that the genome of this organism is highly enriched in antifungal and antimicrobial biosynthetic gene clusters. We demonstrate that this bacterium causes changes to the fungal and oomycete hyphae at the inhibition zone, with some of the hyphae forming bubble-like structures and irregular branching. We tested strain S4 against Magnaporthe oryzae spores, which typically form germ tubes and penetration structures called appressoria, on the surface of the leaf. Our results suggest that after 12 hours of incubation with the bacterium, fungal spores form germ tubes, but instead of producing appressoria, they appear to form rounded, bubble-like structures. Future work will investigate whether a single antifungal molecule induces all these effects, or if they are the result of a combination of bacterially produced antimicrobials.

Metagenomic Analysis of a Concrete Bridge Reveals a Microbial Community Dominated by Halophilic Bacteria and Archaea.

Concrete hosts a small but diverse microbiome that changes over time. Shotgun metagenomic sequencing would enable assessment of both the diversity and function of the microbial community in concrete, but a number of unique challenges make this difficult for concrete samples. The high concentration of divalent cations in concrete interferes with nucleic acid extraction, and the extremely low biomass in concrete means that DNA from laboratory contamination may be a large fraction of the sequence data. Here, we develop an improved method for DNA extraction from concrete, with higher yield and lower laboratory contamination. To show that this method provides DNA of sufficient quality and quantity to do shotgun metagenomic sequencing, DNA was extracted from a sample of concrete obtained from a road bridge and sequenced with an Illumina MiSeq system. This microbial community was dominated by halophilic Bacteria and Archaea, with enriched functional pathways related to osmotic stress responses. Although this was a pilot-scale effort, we demonstrate that metagenomic sequencing can be used to characterize microbial communities in concrete and that older concrete structures may host different microbes than recently poured concrete. IMPORTANCE Prior work on the microbial communities of concrete focused on the surfaces of concrete structures such as sewage pipes or bridge pilings, where thick biofilms were easy to observe and sample. Because the biomass inside concrete is so low, more recent analyses of the microbial communities inside concrete used amplicon sequencing methods to describe those communities. However, to understand the activity and physiology of microbes in concrete, or to develop living infrastructure, we must develop more direct methods of community analysis. The method developed here for DNA extraction and metagenomic sequencing can be used for analysis of microbial communities inside concrete and can likely be adapted for other cementitious materials.

RNA-Seq Reveals that Light and Darkness Are Different Stimuli in Freshwater Heterotrophic Actinobacteria.

Light is a ubiquitous source of both energy and information in surface environments, and regulates gene expression not only in photosynthetic microorganisms, but in a broad range of photoheterotrophic and heterotrophic microbes as well. Actinobacteria are keystone species in surface freshwater environments, where the ability to sense light could allow them to coordinate periods of nutrient uptake and metabolic activity with primary production. The model freshwater Actinobacteria Rhodoluna (R.) lacicola strain MWH-Ta8 and Aurantimicrobium (A.) photophilum strain MWH-Mo1 grow faster in the light than in the dark, but do not use light energy to support growth. Here, we characterize transcription throughout a light-dark cycle in R. lacicola and A. photophilum. In both species, some genes encoding carbohydrate metabolism and storage are upregulated in the light. However, expression of genes of the TCA cycle is only coordinated with light availability in R. lacicola. In fact, the majority of genes that respond to light and darkness in these two species are different, even though their light-responsive phenotypes are similar. The ability to respond to light and darkness may be widespread in freshwater Actinobacteria, but the genetic networks controlled by these two stimuli may vary significantly.

Aurantimicrobium photophilum sp. nov., a non-photosynthetic bacterium adjusting its metabolism to the diurnal light cycle and reclassification of Cryobacterium mesophilum as Terrimesophilobacter mesophilus gen. nov., comb. nov.

The aerobic primarily chemoorganotrophic actinobacterial strain MWH-Mo1T was isolated from a freshwater lake and is characterized by small cell lengths of less than 1 µm, small cell volumes of 0.05-0.06 µm3 (ultramicrobacterium), a small genome size of 1.75 Mbp and, at least for an actinobacterium, a low DNA G+C content of 54.6 mol%. Phylogenetic analyses based on concatenated amino acid sequences of 116 housekeeping genes suggested the type strain of Aurantimicrobium minutum affiliated with the family Microbacteriaceae as its closest described relative. Strain MWH-Mo1T shares with the type strain of that species a 16S rRNA gene sequence similarity of 99.6 % but the genomes of the two strains share an average nucleotide identity of only 79.3 %. Strain MWH-Mo1T is in many genomic, phenotypic and chemotaxonomic characteristics quite similar to the type strain of A. minutum. Previous intensive investigations revealed two unusual traits of strain MWH-Mo1T. Although the strain is not known to be phototrophic, the metabolism is adjusted to the diurnal light cycle by up- and down-regulation of genes in light and darkness. This results in faster growth in the presence of light. Additionally, a cell size-independent protection against predation by bacterivorous flagellates, most likely mediated by a proteinaceous cell surface structure, was demonstrated. For the previously intensively investigated aerobic chemoorganotrophic actinobacterial strain MWH-Mo1T (=CCUG 56426T=DSM 107758T), the establishment of the new species Aurantimicrobium photophilum sp. nov. is proposed.

Bacterial Communities in Concrete Reflect Its Composite Nature and Change with Weathering.

Concrete is an extreme but common environment and is home to microbial communities adapted to alkaline, saline, and oligotrophic conditions. Microbes inside the concrete that makes up buildings or roads have received little attention despite their ubiquity and capacity to interact with the concrete. Because concrete is a composite of materials which have their own microbial communities, we hypothesized that the microbial communities of concrete reflect those of the concrete components and that these communities change as the concrete ages. Here, we used a 16S amplicon study to show how microbial communities change over 2 years of outdoor weathering in two sets of concrete cylinders, one prone to the concrete-degrading alkali-silica reaction (ASR) and the other having the risk of the ASR mitigated. After identifying and removing taxa that were likely laboratory or reagent contaminants, we found that precursor materials, particularly the large aggregate (gravel), were the probable source of ∼50 to 60% of the bacteria observed in the first cylinders from each series. Overall, community diversity decreased over 2 years, with temporarily increased diversity in warmer summer months. We found that most of the concrete microbiome was composed of Proteobacteria, Firmicutes, and Actinobacteria, although community composition changed seasonally and over multiyear time scales and was likely influenced by environmental deposition. Although the community composition between the two series was not significantly different overall, several taxa, including Arcobacter, Modestobacter, Salinicoccus, Rheinheimera, Lawsonella, and Bryobacter, appear to be associated with ASR.IMPORTANCE Concrete is the most-used building material in the world and a biologically extreme environment, with a microbiome composed of bacteria that likely come from concrete precursor materials, aerosols, and environmental deposition. These microbes, though seeded from a variety of materials, are all subject to desiccation, heating, starvation, high salinity, and very high pH. Microbes that survive and even thrive under these conditions can potentially either degrade concrete or contribute to its repair. Thus, understanding which microbes survive in concrete, under what conditions, and for how long has potential implications for biorepair of concrete. Further, methodological pipelines for analyzing concrete microbial communities can be applied to concrete from a variety of structures or with different types of damage to identify bioindicator species that can be used for structural health monitoring and service life prediction.

Complete Genome Sequence of Rhodococcus qingshengii Strain CL-05, Isolated from Concrete.

Here, we report the complete genome sequence of Rhodococcus qingshengii strain CL-05, which was isolated from pavement concrete in Newark, Delaware. The genome consists of a 6.29-Mbp chromosome and one plasmid (123,183 bp), encodes a total of 5,859 predicted proteins, and has a GC content of 62.5%.

Best Practices for Successfully Writing and Publishing a Genome Announcement in Microbiology Resource Announcements.

Microbiology Resource Announcements (MRA) provides peer-reviewed announcements of scientific resources for the microbial research community. We describe the best practices for writing an announcement that ensures that these publications are truly useful resources. Adhering to these best practices can lead to successful publication without the need for extensive revisions.

Complete Genome Sequence of Bacillus velezensis Strain S4, Isolated from Biochar-Treated Soil.

Here, we report the complete genome sequence of Bacillus velezensis strain S4, which was isolated from biochar-amended agricultural soil collected in Smyrna, Delaware. The genome is 4.07 Mbp, encodes 3,918 predicted proteins, and has a GC content of 46.4%.

Future-Proofing Your Microbiology Resource Announcements Genome Assembly for Reproducibility and Clarity.

Descriptions of resources, like the genome assemblies reported in Microbiology Resource Announcements, are often frozen at their time of publication, yet they will need to be interpreted in the midst of continually evolving technologies. It is therefore important to ensure that researchers accessing published resources have access to all of the information required to repeat, interpret, and extend these original analyses. Here, we provide a set of suggestions to help make certain that published resources remain useful and repeatable for the foreseeable future.

Complete Genome Sequence of Microbacterium sp. Strain 10M-3C3, Isolated from an Extremely Phosphorus-Poor Lake.

Here, we report the complete genome sequence of Microbacterium sp. strain 10M-3C3, which was isolated from Lake Matano, Indonesia. The genome is 3,387,846 bp long, encodes 3,351 predicted proteins, and has a G+C content of 71.6%.

Light Modulates the Physiology of Nonphototrophic Actinobacteria.

Light is a source of energy and an environmental cue that is available in excess in most surface environments. In prokaryotic systems, conversion of light to energy by photoautotrophs and photoheterotrophs is well understood, but the conversion of light to information and the cellular response to that information have been characterized in only a few species. Our goal was to explore the response of freshwater Actinobacteria, which are ubiquitous in illuminated aquatic environments, to light. We found that Actinobacteria without functional photosystems grow faster in the light, likely because sugar transport and metabolism are upregulated in the light. Based on the action spectrum of the growth effect and comparisons of the genomes of three Actinobacteria with this growth rate phenotype, we propose that the photosensor in these strains is a putative CryB-type cryptochrome. The ability to sense light and upregulate carbohydrate transport during the day could allow these cells to coordinate their time of maximum organic carbon uptake with the time of maximum organic carbon release by primary producers.IMPORTANCE Sunlight provides information about both place and time. In sunlit aquatic environments, primary producers release organic carbon and nitrogen along with other growth factors during the day. The ability of Actinobacteria to coordinate organic carbon uptake and utilization with production of photosynthate enables them to grow more efficiently in the daytime, and it potentially gives them a competitive advantage over heterotrophs that constitutively produce carbohydrate transporters, which is energetically costly, or produce transporters only after detection of the substrate(s), which delays their response. Understanding how light cues the transport of organic carbon and its conversion to biomass is key to understanding biochemical mechanisms within the carbon cycle, the fluxes through it, and the variety of mechanisms by which light enhances growth.

Distribution and Diversity of Rhodopsin-Producing Microbes in the Chesapeake Bay.

Although sunlight is an abundant source of energy in surface environments, less than 0.5% of the available photons are captured by (bacterio)chlorophyll-dependent photosynthesis in plants and bacteria. Metagenomic data indicate that 30 to 60% of the bacterial genomes in some environments encode rhodopsins, retinal-based photosystems found in heterotrophs, suggesting that sunlight may provide energy for more life than previously suspected. However, quantitative data on the number of cells that produce rhodopsins in environmental systems are limited. Here, we use total internal reflection fluorescence microscopy to show that the number of free-living microbes that produce rhodopsins increases along the salinity gradient in the Chesapeake Bay. We correlate this functional data with environmental data to show that rhodopsin abundance is positively correlated with salinity and with indicators of active heterotrophy during the day. Metagenomic and metatranscriptomic data suggest that the microbial rhodopsins in the low-salinity samples are primarily found in Actinobacteria and Bacteroidetes, while those in the high-salinity samples are associated with SAR-11 type AlphaproteobacteriaIMPORTANCE Microbial rhodopsins are common light-activated ion pumps in heterotrophs, and previous work has proposed that heterotrophic microbes use them to conserve energy when organic carbon is limiting. If this hypothesis is correct, rhodopsin-producing cells should be most abundant where nutrients are most limited. Our results indicate that in the Chesapeake Bay, rhodopsin gene abundance is correlated with salinity, and functional rhodopsin production is correlated with nitrate, bacterial production, and chlorophyll a We propose that in this environment, where carbon and nitrogen are likely not limiting, heterotrophs do not need to use rhodopsins to supplement ATP synthesis. Rather, the light-generated proton motive force in nutrient-rich environments could be used to power energy-dependent membrane-associated processes, such as active transport of organic carbon and cofactors, enabling these organisms to more efficiently utilize exudates from primary producers.

Freshwater bacteria release methane as a byproduct of phosphorus acquisition.

Freshwater lakes emit large amounts of methane, some of which is produced in oxic surface waters. Two potential pathways for aerobic methane production exist: methanogenesis in oxygenated water, which has been observed in some lakes, or demethylation of small organic molecules. Although methane is produced via demethylation in oxic marine environments, this mechanism of methane release has not yet been demonstrated in freshwater systems. Genes related to the C-P lyase pathway, which cleaves C-P bonds in phosphonate compounds, were found in a metagenomic survey of the surface water of Lake Matano, which is chronically P-starved and methane-rich. We demonstrate that four bacterial isolates from Lake Matano obtain P from methylphosphonate and release methane, and that this activity is repressed by phosphate. We further demonstrate that expression of phnJ, which encodes the enzyme that releases methane, is higher in the presence of methylphosphonate and lower when both methylphosphonate and phosphate are added. This gene is also found in most of the metagenomic data sets from freshwater environments. These experiments link methylphosphonate degradation and methane production with gene expression and phosphate availability in freshwater organisms, and suggest that some of the excess methane in the Lake Matano surface water, and in other methane-rich lakes, may be produced by P-starved bacteria. IMPORTANCE: Methane is an important greenhouse gas, and contributes substantially to global warming. Although freshwater environments are known to release methane into the atmosphere, estimates of the amount of methane emitted by freshwater lakes vary from 8 to 73 Tg per year. Methane emissions are difficult to predict in part because the source of the methane can vary: it is the end product of the energy-conserving pathway in methanogenic archaea, which predominantly live in anoxic sediments or waters, but have also been identified in some oxic freshwater environments. More recently, methane release from small organic molecules has been observed in oxic marine environments. Here we show that demethylation of methylphosphonate may also contribute to methane release from lakes, and that phosphate can repress this activity. Since lakes are typically phosphorus-limited, some methane release in these environments may be a byproduct of phosphorus metabolism, rather than carbon or energy metabolism. Methane emissions from lakes are currently predicted using primary production, eutrophication status, extent of anoxia, and the shape and size of the lake; to improve prediction of methane emissions, phosphorus availability and sources may also need to be included in these models.

Biochemical Analysis of Microbial Rhodopsins.

Ion-pumping rhodopsins transfer ions across the microbial cell membrane in a light-dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodopsin identification in environmental and genomic sequence data sets, in vitro analysis of their light-absorbing properties and in vivo analysis of ion pumping will remain critical to characterizing these proteins. As we learn more about the variety of physiological roles performed by microbial rhodopsins in different cell types and environments, observing the localization patterns of the rhodopsins and/or quantifying the number of rhodopsin-bearing cells in natural environments will become more important. Here, we provide protocols for purification of rhodopsin-containing membranes, detection of ion pumping, and observation of functional rhodopsins in laboratory and environmental samples using total internal reflection fluorescence microscopy. © 2016 by John Wiley & Sons, Inc.

Nutrient release and ammonium sorption by poultry litter and wood biochars in stormwater treatment.

The feasibility of using biochar as a filter medium in stormwater treatment facilities was evaluated with a focus on ammonium retention. Successive batch extractions and batch ammonium sorption experiments were conducted in both deionized (DI) water and artificial stormwater using poultry litter (PL) and hardwood (HW) biochars pyrolyzed at 400°C and 500°C. No measureable nitrogen leached from HW biochars except 0.07 µmol/g of org-N from 400°C HW biochar. PL biochar pyrolyzed at 400°C leached 120-127 µmol/g of nitrogen but only 7.1-8.6 µmol/g of nitrogen when pyrolyzed at 500°C. Ammonium sorption was significant for all biochars. At a typical ammonium concentration of 2mg/L in stormwater, the maximum sorption was 150 mg/kg for PL biochar pryolyzed at 400°C. In stormwater, ion competition (e.g. Ca(2+)) suppressed ammonium sorption compared to DI water. Surprisingly, ammonium sorption was negatively correlated to the BET surface area of the tested biochars, but increased linearly with cation exchange capacity. Cation exchange capacity was the primary mechanism controlling ammonium sorption and was enhanced by pyrolysis at 400°C, while BET surface area was enhanced by pyrolysis at 500°C. The optimal properties (BET surface area, CEC, etc.) of biochar as a sorbent are not fixed but depend on the target pollutant. Stormwater infiltration column experiments in sand with 10% biochar removed over 90% of ammonium with influent ammonium concentration of 2mg/L, compared to only 1.7% removal in a sand-only column, indicating that kinetic limitations on sorption were minor for the storm conditions studied. Hardwood and poultry litter biochar pyrolyzed at 500°C and presumably higher temperature may be viable filter media for stormwater treatment facilities, as they showed limited release of organic and inorganic nutrients and acceptable ammonium sorption.

Heterotrophic bacteria from an extremely phosphate-poor lake have conditionally reduced phosphorus demand and utilize diverse sources of phosphorus.

Heterotrophic Proteobacteria and Actinobacteria were isolated from Lake Matano, Indonesia, a stratified, ferruginous (iron-rich), ultra-oligotrophic lake with phosphate concentrations below 50 nM. Here, we describe the growth of eight strains of heterotrophic bacteria on a variety of soluble and insoluble sources of phosphorus. When transferred to medium without added phosphorus (P), the isolates grow slowly, their RNA content falls to as low as 1% of cellular dry weight, and 86-100% of the membrane lipids are replaced with amino- or glycolipids. Similar changes in lipid composition have been observed in marine photoautotrophs and soil heterotrophs, and similar flexibility in phosphorus sources has been demonstrated in marine and soil-dwelling heterotrophs. Our results demonstrate that heterotrophs isolated from this unusual environment alter their macromolecular composition, which allows the organisms to grow efficiently even in their extremely phosphorus-limited environment.

Complete genome sequence of the filamentous gliding predatory bacterium Herpetosiphon aurantiacus type strain (114-95(T)).

Herpetosiphon aurantiacus Holt and Lewin 1968 is the type species of the genus Herpetosiphon, which in turn is the type genus of the family Herpetosiphonaceae, type family of the order Herpetosiphonales in the phylum Chloroflexi. H. aurantiacus cells are organized in filaments which can rapidly glide. The species is of interest not only because of its rather isolated position in the tree of life, but also because Herpetosiphon ssp. were identified as predators capable of facultative predation by a wolf pack strategy and of degrading the prey organisms by excreted hydrolytic enzymes. The genome of H. aurantiacus strain 114-95(T) is the first completely sequenced genome of a member of the family Herpetosiphonaceae. The 6,346,587 bp long chromosome and the two 339,639 bp and 99,204 bp long plasmids with a total of 5,577 protein-coding and 77 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2005.