Microbial symbioses are diverse and central to the functions of life on Earth.

Symbiosis is defined as the close, long-term interaction between individuals of different species. Symbiotic relationships can be anything from parasitism to mutualism. The cost or benefits from the relationship can change over time, or depending on the environment, shifting the relationship along the spectrum. It can be difficult to empirically measure the costs or benefits to the host and symbiont. A symbiotic relationship may be vertically acquired from ancestors or horizontally acquired from the environment.

Bacteria engage in quorum sensing to regulate expression of genes involved in virulence/mutualism. Small diffusible autoinducer molecule interacts with a receptor at a certain threshold concentration leading to changes in gene expression. The autoinducer is species-specific but is generally N-acyl homoserine lactones for Gram negative bacteria and peptides for Gram positives. Quorum sensing allows coordinated gene expression by cells.

Eukaryotes generally have innate immune responses that sense the presence of microbes based on molecular patterns - essential and highly conserved molecules which the microbe would have trouble doing without or changing. These are known as microbe associated molecular patterns (MAMPs). There are different classes of pattern recognition receptors such as TLRs. The host must be able to tolerate beneficial microbes being present despite the fact they also contain MAMPs. In some cases, the microbe can mask, change or lose the MAMP to evade triggering the innate immune response eg Shigella have lost their flagellum, however this is rare.

Aliivibrio fischeri forms a symbiotic relationship with Euprymna scolopes. A. fischeri is a Gram negative, gammaproteobacteria related to Vibrio species. Its genome is made up of 2 circular chromosomes and A. fischeri is found in free living marine environments as well as in symbiosis with E. scolopes. Euprymna scolopes, aka Hawaiian bobtail squid, lives in shallow waters off islands in the central Pacific. Bioluminescence formed by A. fischeri prevents them casting shadow so they don’t alert their prey or predators. They are approximately 1.2 inches long and adults weigh up to 2.7g.

Immature E. scolopes are colonised by free-living A. fischerii present in the environment. They can be maintained aposymbiotically in the lab. The bacteria colonise a specialised light organ, establish an infection and divide using host-supplied nutrients. The squid controls the amount of bacteria in the light organ and the bacteria regulate bioluminescence by quorum sensing.

The squid need to acquire A. fischeri from the environment but this contains many other microbes. Only A. fischeri is able to colonise. Recruited A. ficheri bind host cilia and induce a suite of changes in gene expression. Host mucus contains chemoattractant chitobiose and immunity factors which may inhibit other bacteria. A. fischeri cells move into pores then into the crypts - approximately 1 cell per crypt. After around 12 hours, bioluminescence is produced. The squid are probably able to sanction dark mutants, meaning they can remove bacteria which aren’t benefitting them.

Colonisation of the crypts causes light organ morphogenesis. A. fischeri MAMPs signal cell death and destruction of the ciliated surface originally used for colonisation. Epithelial swelling and increase in microvilli density occurs. Crypt spaces contain V. fischeri in direct contact with epithelial cells. They are exposed to the same antimicrobials as during colonisation which may affect A. fischeri gene expression and/or population size. The host is able to take up molecules produced and released by A. fischeri.

Once the squid is mature, host behaviour switches from arrhythmic to completely nocturnal as the diel/circadian pattern of host and symbiont behaviour is established. Transcriptomes oscillate, the host crypt epithelium is restructured and bacterial metabolism changes dramatically. The bacteria probably use host membranes for growth during the day from anaerobic respiration and glycerol phosphate. At night the bacteria likely carry out fermentation of chitin provided by the host.

At dawn, the squid vent around 90% of the bacteria, seeding the environment with A. fischeri to colonise new hosts.

High cell density = high concentration of autoinducers. Bacteria sense quorum and turn on bioluminescence genes. Bioluminescence is energetically expensive therefore the genes only on when enough bacteria present.

Key regulators of bioluminescence are autoinducer synthase LuxI and regulator LuxR. LuxI produces specific AHL N-3-oxohexanoyl-homoserine lactone (3-oxo-C6-HSL). Regulator LuxR binds AHL at high concentration and directs transcription of target genes luxCDABEG.

Two quorum sensing systems regulate bioluminescence in A. fischeri. AinS produces C8 which is sensed by AinR → derepression of LitR → LuxR expression. LuxI produces 3OC6, which is sensed by LuxR → LuxR binding the lux box, activating expression of structural genes required for bioluminescence.

LuxAB luciferase: other lux gene products required to regenerate luciferase products.

LEGUMES AND RHIZOBIA:

Nitrogen is an essential element in biology eg in proteins and DNA. Nitrogen must be fixed from the relatively inert N2 to biologically accessible NH3. Industrial production of ammonia costs 1-2% of global energy production and produces 1% of manmade greenhouse gases (Haber Process).

Diazotrophy = ability to fix nitrogen to biologically relevant and useable compounds eg ammonia. Chemical methods are lightning and the Haber-Bosch process. Biological methods are bacteria associated with leguminous plants eg Rhizobium leguminosarum and free living diazotrophs eg Azotobacter vinelandii and P. stutzeri.

Biological N fixing feeds 4 billion and chemical N fixation feeds 3 billion. Nitrogen fixing is a very oxygen sensitive process catalysed by nitrogenase. All nitrogenase enzymes have Fe-S cofactor and either Fe/molybdenum or Fe/vanadium mixed reactive centres.

Rhizobial bacteria are Gram negative members of the proteobacteria - alpha and beta. They’re found free-living in soil or in association with the root nodules of legumes. They usually have very complex genome structures known as megasymbiotic plasmids and differentiate into bacteroids which are what fix nitrogen.

Formarion of the symbiotic relationship and partner selection involves flavonoids, nod factor and root nodule formation.

Rhizosphere = region of soil around plant roots, affected by plant exudates

Flavonoids are polyphenolic compounds released by plant roots when nitrogen is scarce. These flavenoids diffuse across rhizobial membranes and act as signals. Different plants produce different flavonoid profiles and there’s also variation amongst rhizobia in their ability to sense and respond to particular flavonoids.

Plant flavonoids in the bacterial cytoplasm bind transcription factor NodD. NodD then activates expression of the nod genes. The nod gene products synthesise nod factors aka lipochitooligosaccharides. The basic structures of nod factors are similar but there’s different chemical modifications amongst different rhizobial species due to species-specific genes. Nod factor helps determine species specificity.

Nod factors can induce plant responses at picomolar concentrations. They are perceived by LysM-RLKs. Perception of the signal causes calcium to spike, deformation of the root hair, formation of the infection thread and nodule organogenesis. There’s similarities in the early stages with steps involved in forming symbiosis with arbuscular mycorrhiza (all plants).

Critical steps in root nodule formation:

• rhicadhesin mediated recognition and attachment

• bacteria secretes Nod factors → root hair curling

• invasion - Rhizobia penetrate root hair and multiply within an infection thread

• bacteria in infection thread grow towards root cell

• formation of bacteroid state within plant root cells

• continued plant and bacterial cell division → nodules

Nodule development and bacteroid differentiation: invading bacteria are released into the plant cell cytoplasm in an endocytosis-like process, ending with the bacteria enveloped in the host cell membrane. Bacteria differentiate into bacteroids. There’s species-species variation in these processes eg determinate vs indeterminate nodules. Signals including EPS production by rhizobia ensure specificity of symbiosis.

Rhizobia differentiate, sometimes terminally, into bacteroids which are enveloped by a host peribacteroid membrane. This process is seemingly controlled by the plant via signals such as nodule-specific cysteine-rich peptides. Significant changes when compared to free-living cells include changes to cell morphology, DNA content, gene expression and metabolism. nif genes are expressed for nitrogen fixation leading to nitrogenase production and provision of plant with fixed N. Extensive cross-talk occurs between the symbiotic partners. The plant may be able to sanction non-nitrogen fixing bacteroids.

The legume-bacteria symbiosis is characterised by several metabolic reactions and nutrient exchange. Plant provides bacteroids with dicarboxylates, a carbon source. Some rhizobial bacteroids are auxotrophic for branched-chain amino acids provided by the plant. The rhizobia provide plant with fixed nitrogen in the form of ammonia and ammonium. The plant maintains a microoxic environment in the nodule using leghemoglobin as nitrogenase is very oxygen sensitive. The nodules eventually senesce but this is poorly understood.

Endosymbiotic theory:

The endosymbiont theory was proposed by Lynn Margulis in 1966. Chloroplasts and mitochondria have been suggested as descendants of ancient prokaryotic cells - primary endosymbiosis. Chloroplasts suggested to be descended from cyanobacteria and mitochondria descended from Rickettsiales.

Green and red algae are thought to have acquired chloroplasts later, by secondary endosymbiosis. Some organisms eg Giardia and Microsporidia lack mitochondria and are thought to have lost them at some point in evolution.

Evidence supporting endosymbiont theory:

• mitochondria and chloroplasts have own reduced chromosomal DNA

• mitochondria and chloroplasts have own 70S ribosomes

• antibiotics which affect prokaryote ribosome affect mitochondria and chloroplasts

• eukaryotic genomes often contain bacterial-deriven genes

• mitochondrial and chloroplast DNA phylogenetically related to bacteria

Mitochondria are semi-autonomous, meaning they can create some of their own proteins using their own ribosomes from their own DNA. However many proteins and functions are supplied by host. Mitochondria aren’t synthesised de novo but grow and divide like bacteria. In some bacteria such as slime mould Dictyostelium discoideum, mitochondrial fission uses a FtsZ homologue. FtsZ is a cell division protein used in free-living proteobacteria eg E. coli.

In animal mitochondria, the FtsZ homologue has been lost and proteins required for mt fission are supplied by the host.

Mitochondrial and chloroplast genomes are dsDNA, and usually circular. They have undergone extreme genome reduction eg animal mt DNA consists of 37 genes (2 rRNA, 22 tRNA, 13 proteins for oxidative phosphorylation). Many genes/functions have been transferred to the nucleus. The genomes are often present in multiple copies.

Genome reduction: free-living, extracellular → facultative intracellular → obligate intracellular → obligate intracellular mutualist → organelle.

Aphids and Buchnera aphidicola:

Insects often have primary symbionts which they require for reproduction, and may also have secondary symbionts. Buchnera synthesise amino acids for aphids which is necessary as the aphid diet of plant sap is low in essential amino acids, These symbionts exhibit extreme gene reduction of 160 to 800kbp compared with free-living bacteria genome of 2 to 10 Mbp. They retain only genes needed for host fitness and there is a potential gene transfer of some Buchnera genes to the aphid