GABA-modulating bacteria of the human gut microbiota.

The gut microbiota affects many important host functions, including the immune response and the nervous system1. However, while substantial progress has been made in growing diverse microorganisms of the microbiota2, 23-65% of species residing in the human gut remain uncultured3,4, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence in artificial media of key growth factors that are provided by neighbouring bacteria in situ5,6. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bioassay-driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter GABA (γ-aminobutyric acid). GABA was the only tested nutrient that supported the growth of KLE1738, and a genome analysis supported a GABA-dependent metabolism mechanism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome-based metabolic modelling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. A transcriptome analysis of human stool samples from healthy individuals showed that GABA-producing pathways are actively expressed by Bacteroides, Parabacteroides and Escherichia species. By coupling 16S ribosmal RNA sequencing with functional magentic resonance imaging in patients with major depressive disorder, a disease associated with an altered GABA-mediated response, we found that the relative abundance levels of faecal Bacteroides are negatively correlated with brain signatures associated with depression.

Quinones are growth factors for the human gut microbiota.

BACKGROUND: The human gut microbiome has been linked to numerous components of health and disease. However, approximately 25% of the bacterial species in the gut remain uncultured, which limits our ability to properly understand, and exploit, the human microbiome. Previously, we found that growing environmental bacteria in situ in a diffusion chamber enables growth of uncultured species, suggesting the existence of growth factors in the natural environment not found in traditional cultivation media. One source of growth factors proved to be neighboring bacteria, and by using co-culture, we isolated previously uncultured organisms from the marine environment and identified siderophores as a major class of bacterial growth factors. Here, we employ similar co-culture techniques to grow bacteria from the human gut microbiome and identify novel growth factors. RESULTS: By testing dependence of slow-growing colonies on faster-growing neighboring bacteria in a co-culture assay, eight taxonomically diverse pairs of bacteria were identified, in which an "induced" isolate formed a gradient of growth around a cultivatable "helper." This set included two novel species Faecalibacterium sp. KLE1255-belonging to the anti-inflammatory Faecalibacterium genus-and Sutterella sp. KLE1607. While multiple helper strains were identified, Escherichia coli was also capable of promoting growth of all induced isolates. Screening a knockout library of E. coli showed that a menaquinone biosynthesis pathway was required for growth induction of Faecalibacterium sp. KLE1255 and other induced isolates. Purified menaquinones induced growth of 7/8 of the isolated strains, quinone specificity profiles for individual bacteria were identified, and genome analysis suggests an incomplete menaquinone biosynthetic capability yet the presence of anaerobic terminal reductases in the induced strains, indicating an ability to respire anaerobically. CONCLUSIONS: Our data show that menaquinones are a major class of growth factors for bacteria from the human gut microbiome. These organisms are taxonomically diverse, including members of the genus Faecalibacterium, Bacteroides, Bilophila, Gordonibacter, and Sutterella. This suggests that loss of quinone biosynthesis happened independently in many lineages of the human microbiota. Quinones can be used to improve existing bacterial growth media or modulate the human gut microbiota by encouraging the growth of important symbionts, such as Faecalibacterium species.

Balanced transcription of cell division genes in Bacillus subtilis as revealed by single cell analysis.

Cell division in bacteria is carried out by a set of conserved proteins that all have to function at the correct place and time. A cell cycle-dependent transcriptional programme drives cell division in bacteria such as Caulobacter crescentus. Whether such a programme exists in the Gram-positive model organism Bacillus subtilis is unknown. Here, we investigate the role of gene transcription as a potential regulatory mechanism for control of division in B. subtilis. Transcriptional GFP fusions in combination with flow cytometry demonstrated a constitutive promoter activity, independent of growth rate, of nine tested cell division genes. These measurements were verified by quantitative real-time reverse-transcription PCR (qrtPCR). Time-lapse fluorescence microscopy was performed on a set of selected reporter strains to test transcriptional regulation during the cell cycle. Interestingly, although the average fluorescence remained constant during cell-cycle progression, individual cells demonstrated a roughly twofold higher promoter activity at the end of the cell cycle. This cell cycle-dependent increased promoter activity can be partly explained by the doubled promoter copy number after DNA replication. Our results indicate that the transcriptional activity of promoters for cell division genes remains constant regardless of growth rate and cell-cycle state, suggesting that regulation of cell division in B. subtilis predominantly takes place at the post-translational level.

Growing unculturable bacteria.

The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field.

Siderophores from neighboring organisms promote the growth of uncultured bacteria.

The majority of bacterial species do not grow on synthetic media. Many non-growers require growth factors from other bacteria, but the nature of these compounds is largely unknown. We show here that previously uncultured isolates from marine sediment biofilm grow on a Petri dish in the presence of cultured organisms from the same environment. The growth factors produced by one cultured helper strain were identified as new acyl-desferrioxamine siderophores. A panel of previously uncultured isolates exhibited a range of siderophore promiscuity for growth promotion. This siderophore-based approach has enabled the culturing of organisms only distantly related to previously cultured microbes. The lack of growth in the laboratory for many strains from this habitat stems from an inability to autonomously produce siderophores, and the resulting chemical dependence on other microorganisms regulates community establishment in the environment.

Direct visualization of horizontal gene transfer.

Conjugation allows bacteria to acquire genes for antibiotic resistance, novel virulence attributes, and alternative metabolic pathways. Using a fluorescent protein fusion, SeqA-YFP, we have visualized this process in real time and in single cells of Escherichia coli. We found that the F pilus mediates DNA transfer at considerable cell-to-cell distances. Integration of transferred DNA by recombination occurred in up to 96% of recipients; in the remaining cells, the transferred DNA was fully degraded by the RecBCD helicase/nuclease. The acquired integrated DNA was tracked through successive replication rounds and was found to occasionally split and segregate with different chromosomes, leading to the inheritance of different gene clusters within the cell lineage. The incidence of DNA splitting corresponds to about one crossover per cell generation.

Bet-hedging and epigenetic inheritance in bacterial cell development.

Upon nutritional limitation, the bacterium Bacillus subtilis has the capability to enter the irreversible process of sporulation. This developmental process is bistable, and only a subpopulation of cells actually differentiates into endospores. Why a cell decides to sporulate or not to do so is poorly understood. Here, through the use of time-lapse microscopy, we follow the growth, division, and differentiation of individual cells to identify elements of cell history and ancestry that could affect this decision process. These analyses show that during microcolony development, B. subtilis uses a bet-hedging strategy whereby some cells sporulate while others use alternative metabolites to continue growth, providing the latter subpopulation with a reproductive advantage. We demonstrate that B. subtilis is subject to aging. Nevertheless, the age of the cell plays no role in the decision of its fate. However, the physiological state of the cell's ancestor (more than two generations removed) does affect the outcome of cellular differentiation. We show that this epigenetic inheritance is based on positive feedback within the sporulation phosphorelay. The extended intergenerational "memory" caused by this autostimulatory network may be important for the development of multicellular structures such as fruiting bodies and biofilms.

Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation.

Aging, defined as a decrease in reproduction rate with age, is a fundamental characteristic of all living organisms down to bacteria. Yet we know little about the causal molecular mechanisms of aging within the in vivo context of a wild-type organism. One of the prominent markers of aging is protein aggregation, associated with cellular degeneracy in many age-related diseases, although its in vivo dynamics and effect are poorly understood. We followed the appearance and inheritance of spontaneous protein aggregation within lineages of Escherichia coli grown under nonstressed conditions using time-lapse microscopy and a fluorescently tagged chaperone (IbpA) involved in aggregate processing. The fluorescent marker is shown to faithfully identify in vivo the localization of aggregated proteins, revealing their accumulation upon cell division in cells with older poles. This accretion is associated with >30% of the loss of reproductive ability (aging) in these cells relative to the new-pole progeny, devoid of parental inclusion bodies, that exhibit rejuvenation. This suggests an asymmetric strategy whereby dividing cells segregate damage at the expense of aging individuals, resulting in the perpetuation of the population.

Mutations in two global regulators lower individual mortality in Escherichia coli.

There has been considerable investigation into the survival of bacterial cells under stress conditions, but little is known about the causes of mortality in the absence of exogenous stress. That there is a basal frequency of cell death in such populations may reflect that it is either impossible to avoid all lethal events, or alternatively, that it is too costly. Here, through a genetic screen in the model organism Escherichia coli, we identify two mutants with lower frequencies of mortality: rssB and fliA. Intriguingly, these two genes both affect the levels of different sigma factors within the cell. The rssB mutant displays enhanced resistance to multiple external stresses, possibly indicating that the cell gains its increased vitality through elevated resistance to spontaneous, endogenous stresses. The loss of fliA does not result in elevated stress resistance; rather, its survival is apparently due to a decreased physical stress linked to the insertion of the flagellum through the membrane and energy saved through the loss of the motor proteins. The identification of these two mutants implies that reducing mortality is not impossible; rather, due to its cost, it is subject to trade-offs with other traits that contribute to the competitive success of the organism.

Aging and death in an organism that reproduces by morphologically symmetric division.

In macroscopic organisms, aging is often obvious; in single-celled organisms, where there is the greatest potential to identify the molecular mechanisms involved, identifying and quantifying aging is harder. The primary results in this area have come from organisms that share the traits of a visibly asymmetric division and an identifiable juvenile phase. As reproductive aging must require a differential distribution of aged and young components between parent and offspring, it has been postulated that organisms without these traits do not age, thus exhibiting functional immortality. Through automated time-lapse microscopy, we followed repeated cycles of reproduction by individual cells of the model organism Escherichia coli, which reproduces without a juvenile phase and with an apparently symmetric division. We show that the cell that inherits the old pole exhibits a diminished growth rate, decreased offspring production, and an increased incidence of death. We conclude that the two supposedly identical cells produced during cell division are functionally asymmetric; the old pole cell should be considered an aging parent repeatedly producing rejuvenated offspring. These results suggest that no life strategy is immune to the effects of aging, and therefore immortality may be either too costly or mechanistically impossible in natural organisms.