Mechanism of 2'-fucosyllactose degradation by human-associated Akkermansia.

Among the first microorganisms to colonize the human gut of breastfed infants are bacteria capable of fermenting human milk oligosaccharides (HMOs). One of the most abundant HMOs, 2'-fucosyllactose (2'-FL), may specifically drive bacterial colonization of the intestine. Recently, differential growth has been observed across multiple species of Akkermansia on various HMOs including 2'-FL. In culture, we found growth of two species, A. muciniphila MucT and A. biwaensis CSUN-19,on HMOs corresponded to a decrease in the levels of 2'-FL and an increase in lactose, indicating that the first step in 2'-FL catabolism is the cleavage of fucose. Using phylogenetic analysis and transcriptional profiling, we found that the number and expression of fucosidase genes from two glycoside hydrolase (GH) families, GH29 and GH95, vary between these two species. During the mid-log phase of growth, the expression of several GH29 genes was increased by 2'-FL in both species, whereas the GH95 genes were induced only in A. muciniphila. We further show that one putative fucosidase and a ß-galactosidase from A. biwaensis are involved in the breakdown of 2'-FL. Our findings indicate that the plasticity of GHs of human-associated Akkermansia sp. enables access to additional growth substrates present in HMOs, including 2'-FL. Our work highlights the potential for Akkermansia to influence the development of the gut microbiota early in life and expands the known metabolic capabilities of this important human symbiont.IMPORTANCEAkkermansia are mucin-degrading specialists widely distributed in the human population. Akkermansia biwaensis has recently been observed to have enhanced growth relative to other human-associated Akkermansia on multiple human milk oligosaccharides (HMOs). However, the mechanisms for enhanced growth are not understood. Here, we characterized the phylogenetic diversity and function of select genes involved in the growth of A. biwaensis on 2'-fucosyllactose (2'-FL), a dominant HMO. Specifically, we demonstrate that two genes in a genomic locus, a putative ß-galactosidase and α-fucosidase, are likely responsible for the enhanced growth on 2'-FL. The functional characterization of A. biwaensis growth on 2'-FL delineates the significance of a single genomic locus that may facilitate enhanced colonization and functional activity of select Akkermansia early in life.

Increasing student interest with the Bacterial Unknown Identification Project: using mixed cultures to create real-world applications.

Many introductory-level classes teach fundamental concepts, as they are prerequisites for upper-division courses. Therefore, the student body in these classes has diverse interests. To address this breadth of career trajectory, introductory-level laboratory courses often include experiments that demonstrate a wide range of microbiological techniques and processes. One of the experiments that is a standard component of many microbiology classes, the Bacterial Unknown Identification Project (BUIP), is often limited to isolated organisms or a specific environment. Here, we describe an updated method for the BUIP that incorporates the projected student career diversity through the implementation of multiple mixed cultures of microorganisms associated with different environments. This update can be utilized in any microbiology laboratory classroom. We maintained the learning objectives, including applying appropriate microbiological methods to analyze and interpret results, and effectively communicate scientific findings, while modifying the sample composition. Assessment of the modification demonstrated that upon completion of the BUIP, students felt that the project applied to their career and it did not take too much of their free time to complete.

Mechanism of 2'-Fucosyllactose degradation by Human-Associated Akkermansia.

Among the first microorganisms to colonize the human gut of breastfed infants are bacteria capable of fermenting human milk oligosaccharides (HMOs). One of the most abundant HMOs, 2'-fucosyllactose (2'-FL), may specifically drive bacterial colonization of the intestine. Recently, differential growth has been observed across multiple species of Akkermansia on various HMOs including 2'FL. In culture, we found growth of two species, A. muciniphila Muc T and A. biwaensis CSUN-19, in HMOS corresponded to a decrease in the levels of 2'-FL and an increase in lactose, indicating that the first step in 2'-FL catabolism is the cleavage of fucose. Using phylogenetic analysis and transcriptional profiling, we found that the number and expression of fucosidase genes from two glycoside hydrolase (GH) families, GH29 and GH95, varies between these two species. During mid-log phase growth, the expression of several GH29 genes was increased by 2'-FL in both species, whereas the GH95 genes were induced only in A. muciniphila . We further show that one putative fucosidase and a ß-galactosidase from A. biwaensis are involved in the breakdown of 2'-FL. Our findings indicate that that plasticity of GHs of human associated Akkermansia sp. enable access to additional growth substrates present in HMOs, including 2'-FL. Our work highlights the potential for Akkermansia to influence the development of the gut microbiota early in life and expands the known metabolic capabilities of this important human symbiont. IMPORTANCE: Akkermansia are mucin degrading specialists widely distributed in the human population. Akkermansia biwaensis has recently been observed to have enhanced growth relative to other human associated Akkermansia on multiple human milk oligosaccharides (HMOs). However, the mechanisms for enhanced growth are not understood. Here, we characterized the phylogenetic diversity and function of select genes involved in growth of A. biwaensis on 2'-fucosyllactose (2'-FL), a dominant HMO. Specifically, we demonstrate that two genes in a genomic locus, a putative ß-galactosidase and α-fucosidase, are likely responsible for the enhanced growth on 2'-FL. The functional characterization of A. biwaensis growth on 2'-FL delineates the significance of a single genomic locus that may facilitate enhanced colonization and functional activity of select Akkermansia early in life.

Isolation of Uncultured Bacteria from Antarctica Using Long Incubation Periods and Low Nutritional Media.

Uncultured microorganisms comprise most of the microbial diversity existing on our planet. Despite advances in environmental sequencing and single-cell genomics, in-depth studies about bacterial metabolism and screening of novel bioproducts can only be assessed by culturing microbes in the laboratory. Here we report uncultured, or recalcitrant, microorganisms from an Antarctic soil sample, using relatively simple methods: oligotrophic media, extended incubation periods, observation under stereo microscopy, and selection of slow-growing bacteria. We managed to isolate several rare microorganisms belonging to infrequently isolated or recently described genera, for example Lapillicoccus, Flavitalea, Quadrisphaera, Motilibacter, and Polymorphobacter. Additionally, we obtained isolates presenting 16S rRNA sequence similarity ranging from 92.08 to 94.46% with any other known cultured species, including two distinct isolates from the class Thermoleophilia, that although common in Antarctic soils (as identified by metagenomics), was never reported to be isolated from such samples. Our data indicates that simple methods are still useful for cultivating recalcitrant microorganisms, even when dealing with samples from extreme environments.

Dehalococcoides mccartyi strain JNA dechlorinates multiple chlorinated phenols including pentachlorophenol and harbors at least 19 reductive dehalogenase homologous genes.

Pentachlorophenol and other chlorinated phenols are highly toxic ubiquitous environmental pollutants. Using gas chromatographic analysis we determined that Dehalococcoides mccartyi strain JNA in pure culture dechlorinated pentachlorophenol to 3,5-dichlorophenol (DCP) via removal of the ortho and para chlorines in all of the three possible pathways. In addition, JNA dechlorinated 2,3,4,6-tetrachlorophenol via 2,4,6-trichlorophenol (TCP) and 2,4,5-TCP to 2,4-DCP and 3,4-DCP, respectively, and dechlorinated 2,3,6-TCP to 3-chlorophenol (CP) via 2,5-DCP. JNA converted 2,3,4-TCP to 3,4-DCP and 2,4-DCP by ortho and meta dechlorination, respectively. 2,3-DCP was dechlorinated to 3-CP, and, because cultures using it could be transferred with a low inoculum (0.5 to 1.5% vol/vol), it may act as an electron acceptor to support growth. Using PCR amplification with targeted and degenerate primers followed by cloning and sequencing, we determined that JNA harbors at least 19 reductive dehalogenase homologous (rdh) genes including orthologs of pcbA4 and pcbA5, pceA, and mbrA, but not tceA or vcrA. Many of these genes are shared with D. mccartyi strains CBDB1, DCMB5, GT, and CG5. Strain JNA has previously been shown to extensively dechlorinate the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1260. Collectively the data suggest that strain JNA may be well adapted to survive in sites contaminated with chlorinated aromatics and may be useful for in situ bioremediation.

Vulnerabilities on the lagging-strand template: opportunities for mobile elements.

Mobile genetic elements have the ability to move between positions in a genome. Some of these elements are capable of targeting one of the template strands during DNA replication. Examples found in bacteria include (a) Red recombination mediated by bacteriophage λ, (b) integration of group II mobile introns that reverse splice and reverse transcribe into DNA, (c) HUH endonuclease elements that move as single-stranded DNA, and (d) Tn7, a DNA cut-and-paste transposon that uses a target-site-selecting protein to target transposition into certain forms of DNA replication. In all of these examples, the lagging-strand template appears to be targeted using a variety of features specific to this strand. These features appear especially available in certain situations, such as when replication forks stall or collapse. In this review, we address the idea that features specific to the lagging-strand template represent vulnerabilities that are capitalized on by mobile genetic elements.

Heteromeric transposase elements: generators of genomic islands across diverse bacteria.

Horizontally acquired genetic information in bacterial chromosomes accumulates in blocks termed genomic islands. Tn7-like transposons form genomic islands at a programmed insertion site in bacterial chromosomes, attTn7. Transposition involves five transposon-encoded genes (tnsABCDE) including an atypical heteromeric transposase. One transposase subunit, TnsB, is from the large family of bacterial transposases, the second, TnsA, is related to endonucleases. A regulator protein, TnsC, functions with different target site selecting proteins to recognize different targets. TnsD directs transposition into attTn7, while TnsE encourages horizontal transmission by targeting mobile plasmids. Recent work suggests that distantly related elements with heteromeric transposases exist with alternate targeting pathways that also facilitate the formation of genomic islands. Tn6230 and related elements can be found at a single position in a gene of unknown function (yhiN) in various bacteria as well as in mobile plasmids. Another group we term Tn6022-like elements form pathogenicity islands in the Acinetobacter baumannii comM gene. We find that Tn6022-like elements also appear to have an uncharacterized mechanism for provoking internal transposition and deletion events that serve as a conduit for evolving new elements. As a group, heteromeric transposase elements utilize diverse target site selection mechanisms adapted to the spread and rearrangement of genomic islands.

Dehalococcoides mccartyi strain JNA in pure culture extensively dechlorinates Aroclor 1260 according to polychlorinated biphenyl (PCB) dechlorination Process N.

We isolated Dehalococcoides mccartyi strain JNA from the JN mixed culture which was enriched and maintained using the highly chlorinated commercial PCB mixture Aroclor 1260 for organohalide respiration. For isolation we grew the culture in minimal liquid medium with 2,2',3,3',6,6'-hexachlorobiphenyl (236-236-CB)(20 µM) as respiratory electron acceptor. We repeatedly carried out serial dilutions to extinction and recovered dechlorination activity from transfers of 10(-7) and 10(-8) dilutions. Fluorescence microscopy, DGGE and RFLP analysis of PCR amplified16S rRNA genes, and multilocus sequence typing of three housekeeping genes confirmed culture purity. No growth occurred on complex media. JNA dechlorinated most hexa- and heptachlorobiphenyls in Aroclor 1260 (50 µg/mL) leading to losses of 51% and 20%, respectively. Dechlorination was predominantly from flanked meta positions of 34-, 234-, 235-, 236-, 245-, 2345-, 2346-, and 2356-chlorophenyl rings, as indicated by the underscores. The major products were 24-24-CB, 24-26-CB, 24-25-CB, and 25-26-CB. We identified 85 distinct PCB dechlorination reactions and 56 different PCB dechlorination pathways catalyzed by JNA. Dechlorination pathways were confirmed by mass balance of substrates and products. This dechlorination pattern matches PCB Dechlorination Process N. JNA is the first pure culture demonstrated to carry out this extensive and environmentally relevant PCB dechlorination pattern.

A Small-Group Activity Introducing the Use and Interpretation of BLAST.

As biological sequence data are generated at an ever increasing rate, the role of bioinformatics in biological research also grows. Students must be trained to complete and interpret bioinformatic searches to enable them to effectively utilize the trove of sequence data available. A key bioinformatic tool for sequence comparison and genome database searching is BLAST (Basic Local Alignment Search Tool). BLAST identifies sequences in a database that are similar to the entered query sequence, and ranks them based on the length and quality of the alignment. Our goal was to introduce sophomore and junior level undergraduate students to the basic functions and uses of BLAST with a small group activity lasting a single class period. The activity provides students an opportunity to perform a BLAST search, interpret the data output, and use the data to make inferences about bacterial cell envelope structure. The activity consists of two parts. Part 1 is a handout to be completed prior to class, complete with video tutorial, that reviews cell envelope structure, introduces key terms, and allows students to familiarize themselves with the mechanics of a BLAST search. Part 2 consists of a hands-on, web-based small group activity to be completed during the class period. Evaluation of the activity through student performance assessments suggests that students who complete the activity can better interpret the BLAST output parameters % query coverage and % max identity. While the topic of the activity is bacterial cell wall structure, it could be adapted to address other biological concepts.

Succession of microbial consortia in the developing infant gut microbiome.

The colonization process of the infant gut microbiome has been called chaotic, but this view could reflect insufficient documentation of the factors affecting the microbiome. We performed a 2.5-y case study of the assembly of the human infant gut microbiome, to relate life events to microbiome composition and function. Sixty fecal samples were collected from a healthy infant along with a diary of diet and health status. Analysis of >300,000 16S rRNA genes indicated that the phylogenetic diversity of the microbiome increased gradually over time and that changes in community composition conformed to a smooth temporal gradient. In contrast, major taxonomic groups showed abrupt shifts in abundance corresponding to changes in diet or health. Community assembly was nonrandom: we observed discrete steps of bacterial succession punctuated by life events. Furthermore, analysis of ≈ 500,000 DNA metagenomic reads from 12 fecal samples revealed that the earliest microbiome was enriched in genes facilitating lactate utilization, and that functional genes involved in plant polysaccharide metabolism were present before the introduction of solid food, priming the infant gut for an adult diet. However, ingestion of table foods caused a sustained increase in the abundance of Bacteroidetes, elevated fecal short chain fatty acid levels, enrichment of genes associated with carbohydrate utilization, vitamin biosynthesis, and xenobiotic degradation, and a more stable community composition, all of which are characteristic of the adult microbiome. This study revealed that seemingly chaotic shifts in the microbiome are associated with life events; however, additional experiments ought to be conducted to assess how different infants respond to similar life events.

Serotonin receptor activation leads to neurite outgrowth and neuronal survival.

Serotonin 5-HT1 receptors are implicated in anxiety and depression. These receptors belong to the family A of G-protein-coupled receptors and couple to inhibitory G-proteins. Recent studies show that chronic activation of 5-HT1A receptors leads to proliferation of hippocampal neurons suggesting that neurogenesis contributes to the effects of antidepressants. However, the molecular mechanisms and pathways involved are not understood. We used Neuro 2A cells transfected with 5-HT1A receptors and SK-N-SH cells endogenously expressing the receptor to examine the effect of receptor activation on neuronal survival and neurite outgrowth. We find that receptor activation leads to increased neurite outgrowth that can be blocked by the receptor selective antagonist and by treatment with pertussis toxin or lactacystin implicating inhibitory G-proteins and proteasomal degradation in this process. Interestingly, the small G-protein Rap and the transcription factor STAT-3 are also involved since reducing the levels of Rap1 (using small interfering RNA) or STAT-3 (using dominant negative STAT3) significantly blocks 5-HT1A-receptor-mediated neurite outgrowth. The observed increase in the phosphorylation of Src and STAT-3, at sites leading to their activation, further supports a crucial role for these proteins in neurite outgrowth. We also find that prolonged activation of endogenous 5-HT1A receptors leads to increased cell survival even under starving conditions; this is completely blocked by co-treatment with the antagonist. Taken together, these findings indicate that activation of the 5-HT1A receptor leads to a number of neurotropic events by activating a series of signal transduction molecules leading to long-term changes required for neurogenesis.

Mechanisms of action of antidepressants: from neurotransmitter systems to signaling pathways.

Antidepressants are commonly used in the treatment of anxiety and depression, medical conditions that affect approximately 17-20% of the population. The clinical effects of antidepressants take several weeks to manifest, suggesting that these drugs induce adaptive changes in brain structures affected by anxiety and depression. In order to develop shorter-acting and more effective drugs for the treatment of anxiety and depression, it is important to understand how antidepressants bring about their beneficial effects. Recent reports suggest that antidepressants can induce neurogenesis in the adult brain, although the mechanisms involved are not clearly understood. In this review, we describe the different neurotransmitter systems that are affected by anxiety and depression and how they are modulated by antidepressant treatment with a focus on signaling molecules and pathways that are activated during neurotransmitter receptor induced neurogenesis.