The new bacteriology

The conventional fossil record of bones, shells, tracks and trails traces an evolutionary history of animals some 600 million years in duration.  Phylogenies, however, indicate that animals are evolutionary late-comers, implying a deeper, largely microbial history of life.  Geochronology, in turn, demonstrates the conventional fossil record documents only the last 15 percent of recorded earth history.  Microfossils, stromatolites, preserved lipids, and biologically informative isotopic ratios quadruple the known history of life, providing a substantial record of bacterial diversity and biogeochemical cycles in Proterozoic (2500-541 Ma) oceans that can be interpreted, at least broadly, in terms present day organisms and processes.  Archean (>2500 Ma) sedimentary rocks add an additional billion years to this account, but phylogenetic and functional details are limited.  Geochemistry provides a major constraint on early evolution, indicating that early bacteria were shaped by anoxic environments, with distinct patterns of major and micro-nutrient availability different.  The Archean biosphere may document Earth’s first iron age, as reduced Fe was the principal electron donor for photosynthesis, oxidized Fe the most abundant terminal electron acceptor for respiration and Fe a key cofactor in proteins.  With the permanent oxygenation of the atmosphere and surface ocean ca. 2400 Ma, photic zone O2 limited the access of photosynthetic bacteria to electron donors other than water, while expanding the inventory of oxidants available for respiration and chemoautotrophy.  Thus, nearly halfway through Earth history, the microbial underpinnings of functioning ecosystems began to resemble their present form.  

The peptidoglycan cell wall is a defining structure of the bacteria. It is the target for our best antibiotics and fragments of the wall trigger powerful innate immune responses against infection. Surprisingly, many bacteria can switch almost effortlessly into a cell wall deficient “L-form” state. These cells become completely resistant to many antibiotics and may be able to pass under the radar screen of our immune systems. Studies of L-forms have provided surprising insights into various aspects of bacterial cell physiology and biochemistry, as well as providing a model illuminating how the earliest true cells on the planet might have proliferated. Recent studies have revealed that rapid growth of L-forms, as well as accurate chromosome segregation and assembly of components of the division machinery, can occur in the absence of a cell wall, provided that the L-forms are artificially constrained into a cylindrical shape of appropriate dimensions.

Key references

Leaver et al., 2009, Nature 457, 849-853. Mercier et al., 2013, Cell 152, 997-1007. Errington, 2013, Open Biology 3, 120143. Mercier et al., 2014, eLife 04629. Kawai et al., 2015, Current Biology 25, 1613-1618.

Genomes represent not only the set of instructions for an organism, but also a record of how that set of instructions has evolved and adapted over short and long timescales, a record that is enhanced by the ability to compare related genomes. New technologies have allowed us to sequence very large numbers of closely-related bacterial genomes, enabling the construction of phylogenetic relationships within recently-evolved clones of human and animal pathogens. We can now see bacteria as measurably-evolving populations, amenable to analysis with Bayesian tools developed for the analysis of fast-evolving viruses. The resulting data can allow us to answer specific hypotheses, but perhaps more interestingly, these vast data sets allow us to explore without preconceptions, answering questions we didn’t know that we should be asking. Sequence gazing is hypothesis generating, and I will discuss some examples of novelty that has come from such analyses.

The RNA-programmable CRISPR-Cas9 system has recently emerged as a transformative technology in biological sciences, allowing rapid and efficient targeted genome engineering, chromosomal marking and gene regulation in a large variety of cells and organisms. In this system, the endonuclease Cas9 or catalytically inactive Cas9 variants are programmed with single guide RNAs (sgRNAs) to target site-specifically any DNA sequence of interest given the presence of a short sequence (Protospacer Adjacent Motif, PAM) juxtaposed to the complementary region between the sgRNA and target DNA.

Originally, CRISPR-Cas is an RNA-mediated adaptive immune system that protects bacteria and archaea from invading mobile genetic elements. Short crRNA (CRISPR RNA) molecules containing unique genome-targeting spacers guide Cas protein(s) to invading cognate nucleic acids to affect their maintenance. CRISPR-Cas has been classified into three main types and further subtypes. CRISPR-Cas9 originates from the type II system that has evolved unique molecular mechanisms for maturation of crRNAs and targeting of invading DNA, which my laboratory has identified in the human pathogen Streptococcus pyogenes. During the step of crRNA biogenesis, a unique CRISPR-associated RNA, tracrRNA, base pairs with the repeats of precursor-crRNA to form anti-repeat-repeat dual-RNAs that are cleaved by RNase III in the presence of Cas9, generating mature tracrRNA and intermediate forms of crRNAs. Following a second maturation event, the mature dual-tracrRNA-crRNAs guide Cas9 to cleave cognate target DNA and thereby affect the maintenance of invading genomes. We have shown that Cas9 can be programmed with sgRNAs mimicking the natural dual-tracrRNA-crRNAs to target site-specifically any DNA sequence of interest. I will discuss the biological roles of CRISPR-Cas9, the mechanisms involved, the evolution of type II CRISPR-Cas components in bacteria and the applications of CRISPR-Cas9 as a novel genome engineering technology.

Bacterial biofilms are architecturally and morphologically highly structured multicellular communities with essentially tissue-like properties. Structure and functions of a biofilm depend on an elaborate spatiotemporal control of the synthesis of an extracellular matrix that usually contains adhesins, amyloid fibres and exopolysaccharides (e.g. cellulose) (1). In E. coli the production of amyloid curli fibres and cellulose is activated by the biofilm regulator CsgD, whose expression requires the stationary phase sigma factor RpoS and the nucleotide second messenger c-di-GMP. The latter is synthesized by 12 diguanylate cyclases (DGC, with GGDEF domains) and degraded by 13 phosphodiesterases (PDE, with EAL domains). Some of these DGCs and PDEs show specialized functions in matrix control that are based on highly specific macromolecular interactions, i.e. 'local c-di-GMP signaling'.
The key player in the switch that turns on CsgD expression in cells that begin to starve, is the PDE PdeR (formerly YciR). It acts as a 'trigger PDE'  that – in response to the rising c-di-GMP level generated by RpoS-driven induction of the DGC DgcE – allows the equally RpoS-controlled DGC DgcM and the transcription factor MlrA to co-activate csgD transcription by an intriguing mechanism: PdeR initially inhibits both DgcM and MlrA by direct specific interactions, which are relieved when c-di-GMP levels get high enough for PdeR to efficiently bind and degrade c-di-GMP. As the prototype of a 'trigger PDE', PdeR thus combines three activities, i.e. it is (i) a direct and therefore locally acting antagonist for DgcM and MlrA, (ii) a sensor and effector for the cellular c-di-GMP level (driven up by DgcE), and (iii) a PDE (2). 
A second mode of local c-di-GMP signaling in E. coli is exemplified by DgcC and PdeK that directly interact with each other and with the cellulose synthase complex BcsA/BcsB. DgcC and PdeK thus dynamically determine the local c-di-GMP concentration in close vicinity to the regulatory c-di-GMP-binding PilZ domain of BcsA. However, PdeK does not act as a 'trigger PDE', i.e. its direct interaction with cellulose synthase does not have a direct regulatory impact, but rather allows it to act as a local sink for c-di-GMP and thus to specifically counteract local c-di-GMP accumulation driven by DgcC.

(1) Serra and Hengge (2014) Environ. Microbiol. 16, 1455-1471.
(2) Lindenberg et al. (2013) EMBO J. 32, 2001-2014.

The bacterial Type VI secretion system (T6SS) is a large dynamic organelle that is functionally analogous to contractile tails of bacteriophages. T6SS is used by Gram-negative bacteria to inhibit adjacent cells via translocation of toxic effector proteins and thus plays an important role in bacterial pathogenesis and ecology. Time-lapse fluorescence light microscopy revealed that T6SS sheath, which powers the secretion, cycles between assembly, quick contraction, disassembly and re-assembly. Single cell analysis of subcellular localization of T6SS assembly revealed that T6SS organelle encoded by Pseudomonas aeruginosa H1-T6SS cluster is assembled and aimed to specifically retaliate against attack by other bacteria. I will present latest update on the structure, function and dynamics of T6SS as well on mechanisms of effector delivery and function in various T6SS+ organisms. I will also focus on the structure of the T6SS sheath solved by cryo-electron microscopy and the implications for T6SS dynamics and assembly. I will show examples of how various imaging techniques helped us to understand many aspects of T6SS.

Our immune system has likely evolved under the double constraint to tolerate the commensal microbiota, particularly in the gut, where bacteria can reach astronomic numbers, and to quickly recognize and eliminate the few pathogenic bacteria that reach and colonize this same surface. The cross talks that allow the host to discriminate between the « good » microbes and the « bad » microbes are starting to be fully recognized.

We have focused on the intestinal crypt that accounts for epithelial regeneration, due to the presence of stem cells, their environmental niche and the transit amplifying epithelial compartment. We have identified a « Crypt-specific core microbiota (CSCM) » corresponding to a limited group of strictly aerobic bacteria that populate the cecal and colonic crypts of mammals. We can now decipher the molecular crosstalks between CSCM members and the crypt regenerative apparatus. For instance we have shown that, thanks to activation of a Nod-2 dependent signaling process by muramyl dipeptide from the microbiota, stem cells under stress express increased surviving capacities, thus securing better epithelial restitution. This illustrates the depth of our symbiosis with gut commensals and illuminates the function of Nod-2 beyond innate immune regulation. Alternatively, we have identified the intestinal crypt as the site of invasion by Shigella, a bona fide enteric pathogen. We are currently deciphiring the cross-talks occuring in these various situations.

The intestinal tract is inhabited by a large and diverse community of microbes collectively referred to as the gut microbiota. While the gut microbiota provides important benefits to its host, disturbance of the microbiota–host relationship is associated with numerous chronic inflammatory diseases, including inflammatory bowel disease and metabolic syndrome. A primary means by which the intestine is protected from its microbiota is via multi-layered mucus structures that cover the intestinal surface, allowing the vast majority of gut bacteria to be kept at a safe distance from epithelial cells that line the intestine. Thus, agents that disrupt mucus–bacterial interactions might have the potential to promote diseases associated with gut inflammation. We recently reported that, in mice, relatively low concentrations of two commonly used emulsifiers, detergent-like molecules and ubiquitous component of processed foods, induced low-grade inflammation and obesity/metabolic syndrome in wild-type hosts and promoted robust colitis in mice predisposed to this disorder. Emulsifier-induced metabolic syndrome was associated with microbiota encroachment, altered species composition and increased pro-inflammatory potential. Use of germ-free mice and faecal transplants indicated that such changes in microbiota were necessary and sufficient for both low-grade inflammation and metabolic syndrome. These results support the emerging concept that perturbed host–microbiota interactions resulting in low-grade inflammation can promote adiposity and its associated metabolic effects. Moreover, they suggest that the broad use of emulsifying agents might be contributing to an increased societal incidence of obesity/metabolic syndrome and other chronic inflammatory diseases.