Targeted hypermutation of putative antigen sensors in multicellular bacteria.

Diversity-generating retroelements (DGRs) are used by bacteria, archaea, and viruses as a targeted mutagenesis tool. Through error-prone reverse transcription, DGRs introduce random mutations at specific genomic loci, enabling rapid evolution of these targeted genes. However, the function and benefits of DGR-diversified proteins in cellular hosts remain elusive. We find that 82% of DGRs from one of the major monophyletic lineages of DGR reverse transcriptases are encoded by multicellular bacteria, which often have two or more DGR loci in their genomes. Using the multicellular purple sulfur bacterium Thiohalocapsa sp. PB-PSB1 as an example, we characterized nine distinct DGR loci capable of generating 10282 different combinations of target proteins. With environmental metagenomes from individual Thiohalocapsa aggregates, we show that most of PB-PSB1's DGR target genes are diversified across its biogeographic range, with spatial heterogeneity in the diversity of each locus. In Thiohalocapsa PB-PSB1 and other bacteria hosting this lineage of cellular DGRs, the diversified target genes are associated with NACHT-domain anti-phage defenses and putative ternary conflict systems previously shown to be enriched in multicellular bacteria. We propose that these DGR-diversified targets act as antigen sensors that confer a form of adaptive immunity to their multicellular consortia, though this remains to be experimentally tested. These findings could have implications for understanding the evolution of multicellularity, as the NACHT-domain anti-phage systems and ternary systems share both domain homology and conceptual similarities with the innate immune and programmed cell death pathways of plants and metazoans.

Environmental structure impacts microbial composition and secondary metabolism.

Determining the drivers of microbial community assembly is a central theme of microbial ecology, and chemical ecologists seek to characterize how secondary metabolites mediate these assembly patterns. Environmental structure affects how communities assemble and what metabolic pathways aid in that assembly. Here, we bridged these two perspectives by addressing the chemical drivers of community assembly within a spatially structured landscape with varying oxygen availability. We hypothesized that structured environments would favor higher microbial diversity and metabolite diversity. We anticipated that the production of a compound would be more advantageous in a structured environment (less mixing) compared to an unstructured environment (more mixing), where the molecule would have a diminished local effect. We observed this to be partially true in our experiments: structured environments had similar microbial diversity compared to unstructured environments but differed significantly in the metabolites produced. We also found that structured environments selected for communities with higher evenness, rather than communities with higher richness. This supports the idea that when characterizing the drivers of community assembly, it matters less about who is there and more about what they are doing. Overall, these data contribute to a growing effort to approach microbial community assembly with interdisciplinary tools and perspectives.

Using Plate-Wash PCR and High-Throughput Sequencing to Measure Cultivated Diversity for Natural Product Discovery Efforts.

Molecular techniques continue to reveal a growing disparity between the immense diversity of microbial life and the small proportion that is in pure culture. The disparity, originally dubbed "the great plate count anomaly" by Staley and Konopka, has become even more vexing given our increased understanding of the importance of microbiomes to a host and the role of microorganisms in the vital biogeochemical functions of our biosphere. Searching for novel antimicrobial drug targets often focuses on screening a broad diversity of microorganisms. If diverse microorganisms are to be screened, they need to be cultivated. Recent innovative research has used molecular techniques to assess the efficacy of cultivation efforts, providing invaluable feedback to cultivation strategies for isolating targeted and/or novel microorganisms. Here, we aimed to determine the efficiency of cultivating representative microorganisms from a non-human, mammalian microbiome, identify those microorganisms, and determine the bioactivity of isolates. Sequence-based data indicated that around 57% of the ASVs detected in the original inoculum were cultivated in our experiments, but nearly 53% of the total ASVs that were present in our cultivation experiments were not detected in the original inoculum. In light of our controls, our data suggests that when molecular tools were used to characterize our cultivation efforts, they provided a more complete and more complex, understanding of which organisms were present compared to what was eventually detected during cultivation. Lastly, about 3% of the isolates collected from our cultivation experiments showed inhibitory bioactivity against an already multidrug-resistant pathogen panel, further highlighting the importance of informing and directing future cultivation efforts with molecular tools.

Builders, tenants, and squatters: the origins of genetic material in modern stromatolites.

Micro-organisms have long been implicated in the construction of stromatolites. Yet, establishing a microbial role in modern stromatolite growth via molecular analysis is not always straightforward because DNA in stromatolites can have multiple origins. For example, the genomic material could represent the microbes responsible for the construction of the stromatolite (i.e., "builders"), microbes that inhabited the structure after it was built (i.e., "tenants"), or microbes/organic matter that were passively incorporated after construction from the water column or later diagenetic fluids (i.e., "squatters"). Disentangling the role of micro-organisms in stromatolite construction, already difficult in modern systems, becomes more difficult as organic signatures degrade, and their context is obscured. To evaluate our ability to accurately decipher the role of micro-organisms in stromatolite formation in geologically recent settings, 16/18S SSU rRNA gene sequences were analyzed from three systems where the context of growth was well understood: (a) an actively growing stromatolite from a silicic hot spring in Yellowstone National Park, Wyoming, where the construction of the structure is controlled by cyanobacteria; (b) a mixed carbonate and silica precipitate from Little Hot Creek, a hot spring in the Long Valley Caldera of California that has both abiogenic and biogenic components to accretion; and (c) a near-modern lacustrine carbonate stromatolite from Walker Lake, Nevada that is likely abiogenic. In all cases, the largest percentage of recovered DNA sequences, especially when focused on the deeper portions of the structures, belonged to either the tenant or squatter communities, not the actual builders. Once removed from their environmental context, correct interpretation of biology's role in stromatolite morphogenesis was difficult. Because high-throughput genomic analysis may easily lead to incorrect assumptions even in these modern and near-modern structures, caution must be exercised when interpreting micro-organismal involvement in the construction of accretionary structures throughout the rock record.

The microbiology, pH, and oxidation reduction potential of larval masses in decomposing carcasses on Oahu, Hawaii.

Previous studies have begun to characterize the microbial community dynamics of the skin, soil, gut, and oral cavities of decomposing remains. One area that has yet to be explored in great detail is the microbiome of the fly larval mass, the community of immature flies that plays a significant role in decomposition. The current study aimed to characterize the microbiology and chemistry of larval masses established on pig (Sus scrofa domesticus) carcasses and to determine if these characteristics have potential as temporal evidence. Carcasses (n = 3) were decomposed on the soil surface of a tropical habitat on Oahu, Hawaii, USA and sampled over three days at 74 h, 80 h, 98 h, 104 h, 122 h, and 128 h (∼85-142 Accumulated Degree Days) postmortem. Larval masses were analyzed via high-throughput 16S rRNA sequencing and in situ chemical measurements (pH, temperature, oxidation-reduction potential). A trend was observed that resulted in three distinct microbial communities (pre-98 h, 98 h, and post-98 h). The oxidation-reduction potential (Eh) of larval masses apparently regulated microbial community structure with the most negative Eh being associated with the least rich and diverse microbial communities. Overall, a significant interaction between time and taxa was observed, particularly with bacterial phyla Firmicutes and Proteobacteria. The current results provide new insight into the microbial community and chemical parameters of larval masses and indicate a temporal shift that could be further studied as a PMI estimator.

Draft Genome Sequence of Picocystis sp. Strain ML, Cultivated from Mono Lake, California.

The microscopic alga Picocystis sp. strain ML is responsible for recurrent algal blooms in Mono Lake, CA. This organism was characterized by only very little molecular data, despite its prominence as a primary producer in saline environments. Here, we report the draft genome sequence for Picocystis sp. strain ML based on long-read sequencing.

Characterizing forensically important insect and microbial community colonization patterns in buried remains.

During violent criminal actions in which the perpetrator disposes of the victim's remains by burial, the analysis of insects and bacterial colonization patterns could be necessary for postmortem interval (PMI) estimation. Our research aimed to assess the decomposition process of buried rat carcasses from shallow graves (40 cm), the diversity and dynamics of insects and bacteria throughout the decomposition stages, and the environmental parameters' influence on these variations. The results provide further insight on decomposition in soil and contribute to a broader understanding of the factors involved in decomposition by qualitatively and quantitatively analysing the decomposer community (bacteria and insects). Additionally, two bacterial taxa, Enterococcus faecalis and Clostridium paraputrificum that were investigated for the first time as PMI indicators using quantitative polymerase chain reaction (qPCR) showed differential abundance over time, promising data for PMI estimation. The current study on the decomposition of buried rat carcasses in a natural environment will strengthen the current knowledge on decomposed remains from shallow graves and represents an effort to quantify insect and bacterial taxa as PMI estimators.

Characterization and microbial analysis of first recorded observation of Conicera similis Haliday (Diptera: Phoridae) in forensic decomposition study in Romania.

The identification of necrophagous insect diversity and dynamics has forensic significance for postmortem interval estimation specific to burial. Few studies regarding the necrophagous entomofauna from buried remains have been performed to date. In contrast to the exposed carcasses, the accessibility of soil to insects is limited due to burial depth and is dependent on soil type. This study highlights the colonization behavior of Conicera similis (Haliday 1833) (Diptera: Phoridae) during carcass decomposition, a previously unobserved taxon in Romania. Adult and larvae specimens were collected from rat (Rattus norvegicus) carcasses buried at 40 cm depth in an urban park environment in June 2016 during active decomposition and their presence and activity period was correlated with the environmental parameters variation. Bacterial diversity from C. similis female adult and larvae specimens was determined via 16S rRNA gene Illumina sequencing to further characterize these commonly encountered and forensically important necrophagous insects. This report signals the easternmost geographical location in Europe (Bucharest, Romania) of C. similis to date.

An initial investigation into the ecology of culturable aerobic postmortem bacteria.

Postmortem microorganisms are increasingly recognized for their potential to serve as physical evidence. Yet, we still understand little about the ecology of postmortem microbes, particularly those associated with the skin and larval masses. We conducted an experiment to characterize microbiological and chemical properties of decomposing swine (Sus scrofa domesticus) carcasses on the island of Oahu, Hawaii, USA, during June 2013. Bacteria were collected from the head, limb, and larval mass during the initial 145h of decomposition. We also measured the pH, temperature, and oxidation-reduction potential of larval masses in situ. Bacteria were cultured aerobically on Standard Nutrient Agar at 22°C and identified using protein or genetic signals. Carcass decomposition followed a typical sigmoidal pattern and associated bacterial communities differed by sampling location and time since death, although all communities were dominated by phyla Actinobacteria, Firmicutes, and Proteobacteria. Larval masses were reducing environments (~-200mV) of neutral pH (6.5-7.5) and high temperature (35°C-40°C). We recommend that culturable postmortem and larval mass microbiology and chemistry be investigated in more detail, as it has potential to complement culture-independent studies and serve as a rapid estimate of PMI.

Using bacterial and necrophagous insect dynamics for post-mortem interval estimation during cold season: Novel case study in Romania.

Considering the biogeographical characteristics of forensic entomology, and the recent development of forensic microbiology as a complementary approach for post-mortem interval estimation, the current study focused on characterizing the succession of necrophagous insect species and bacterial communities inhabiting the rectum and mouth cavities of swine (Sus scrofa domesticus) carcasses during a cold season outdoor experiment in an urban natural environment of Bucharest, Romania. We monitored the decomposition process of three swine carcasses during a 7 month period (November 2012-May 2013) corresponding to winter and spring periods of a temperate climate region. The carcasses, protected by wire cages, were placed on the ground in a park type environment, while the meteorological parameters were constantly recorded. The succession of necrophagous Diptera and Coleoptera taxa was monitored weekly, both the adult and larval stages, and the species were identified both by morphological and genetic characterization. The structure of bacterial communities from swine rectum and mouth tissues was characterized during the same time intervals by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene fragments. We observed a shift in the structure of both insect and bacterial communities, primarily due to seasonal effects and the depletion of the carcass. A total of 14 Diptera and 6 Coleoptera species were recorded on the swine carcasses, from which Calliphora vomitoria and C. vicina (Diptera: Calliphoridae), Necrobia violacea (Coleoptera: Cleridae) and Thanatophilus rugosus (Coleoptera: Silphidae) were observed as predominant species. The first colonizing wave, primarily Calliphoridae, was observed after 15 weeks when the temperature increased to 13°C. This was followed by Muscidae, Fanniidae, Anthomyiidae, Sepsidae and Piophilidae. Families belonging to Coleoptera Order were observed at week 18 when temperatures raised above 18°C, starting with Cleridae, Silphidae, and followed by Histeridae, Staphylinidae and Dermestidae. For bacteria, 53 taxa belonging to phyla Proteobacteria (Gammaproteobacteria, Betaproteobacteria), Firmicutes and Bacteroidetes were identified from the mouth cavity (36 OTUs) and rectum cavity (17 OTUs). These shifts in insect and bacterial communities indicated their complementary role in the carcass decomposition process. These results represent the first forensic entomological and microbiological survey in Romania. This study shows the value of forensic entomology as a tool for forensic investigations in Romania and neighboring areas with similar biogeographical characteristics. Moreover, this study represents a novel approach for understanding taphonomy and estimating post-mortem interval during cold season by combining entomological and microbiological methods.