Bacillus subtilis research notes

[eyesmo]

Creating this thread to post potentially useful info about Bacillus subtilis that people come across.

Quick background: Bacillus subtilis is a bacterium that is widely used in industrial biotechnology for enzyme manufacturing. B. subtilis has a number of useful traits: it is Generally Regarded As Safe by the US Food and Drug Administration (it's actually present in some human food, being the active microbe in the Japanese fermented soybean dish called natto); it is gram positive, having only one cell wall (unlike E. coli's two) and active protein secretion machinery, enabling B. subtilis to secrete grams per liter of native or recombinant proteins in bioreactors; it can be naturally competent, able to take up, genomically integrate and express genes on DNA from outside the cell; it can maintain replicating plasmids as well as efficiently integrate plasmids into its genome; and it can sporulate, forming extremely hardy spores that make it easy to transport and store B. subtilis cell lines at room temperature, without a cold chain.

[eyesmo]

Notes from Wong et al's 1995 review of B. subtilis as a platform organism for recombinant protein expression
Structural requirements of a signal sequence

1. Bsub secTags have +3 avg charge
2. Have hydrophobic stretch 15 residues long, on average
   E. coli secTags have 12 residue hydrophobic stretch
3. Ala-X-Ala or Val-X-Ala signal peptide cleavage sequence

Secretion apparatus

1. E. coli Sec pathway key components: SecA (PrlD), SecD, SecE (PrlG), SecF, SecY (PrlA), and signal peptidases

Molecular chaperones and accessory factors

1. Increasing expression of intracellular molecular chaperones like GroES, GroEL, DnaK, DnaJ and GrpE may increase production yield, proper folding
   2.5-fold increase single chain antibody production with co-expression of groES and groEL
2. Overexpressing different combinations of these can help with secretion in E. coli
3. Co-expression of E. coli SecB can help increase secretion of a protein in B. subtilis
   Though the Bsub Apr secTag works even better, at least for secreting MBP.
4. Overexpressing Bsub membrane lipoprotein (chaperone?) PrsA can increase production of O-amylase 6-fold
5. Structural gene bdb from Bacillus braves might facilitate proper disulfide formation

Protease-deficient strains

1. Proteases are major impediments to high yields in Bsub
2. Knocking out 6 and 7 proteases greatly increases recombinant Bsub secreted protein yields

Protein engineering

1. Even some proteins secreted from 6 or 7 protease null Bsub can have parts clipped by proteases, e.g. streptokinase
2. But mutating a stretch of hydrophobic amino acids near the C terminus of the protein to hydrophilic residues can prevent this
3. Some partially folded proteins can induce cell lysis (e.g. a certain scFv). Mutating framework residues solved this problem
4. Increasing the hydrophilicity of the surface of a protein, as well as adding a 3-lysine solubilization motif, can increase its secretion yield in Bsub

Other interesting examples

1. Bsub can produce functional secreted streptavidin, while the protein forms inclusion bodies in E. coli
2. Bsub can express foreign genes with high GC content
3. Bsub is good at manufacturing recombinant secreted proteases
   Yields up to 0.25g/L to 0.g g/L as of 1995.

[eyesmo]

Harwood et al 1992 review of Bacillus biology and biotechnological applications
Intro

1. Marburg strain is the type strain of B. subtilis?
2. B. subtilis variant natto is responsible for fermenting soybeans into the food Natto
   -- 100,000,000 kg consumed annually!
3. GC content of different Bacillus strains varies from 33%-66%
   -- B. subtilis has 43% GC content
4. B. subtilis has a well-developed natural transformation system
   -- First non-pathogenic microbe to be transformed
5. Strain 168: a tryptophan auxotroph
   -- Equivalent to E. coli K-12, model strain

Transformation, fine structure mapping

1. Transformation: cells take up exogenous DNA
   -- Competence: cellular state that enables transformation
2. B. subtilis become naturally competent at the end of exponential growth
3. Congression: when a single cell takes up multiple molecules of DNA
   -- B. subtilis can do congression at high DNA concentrations (>1 ug/mL)
   -- 1-4% of transformants with a selected marker will have a second, unselected marker

Cloning vectors

1. pUB110 is the B. subtilis equivalent of pBR322 (a widely used workhorse plasmid in E. coli)
   -- Kanamycin resistant
2. Original E. coli/B. subtilis shuttle vectors were chimeras of E. coli and Staphylococcus plasmids
3. Consensus vegetative promoter sequences are very similar for E. coli and B. subtilis
   -- TTGACA—16-19 bp—TAATAT
4. But B. subtilis requires an AT-rich region upstream of the -35 hexamer
5. Higher complementarity between ribosome and RBS in B. subtilis than E. coli (∆G -17.6 kcal/mol vs -11.0 kcal/mol)
6. ‘Phage-like’ single stranded replication of plasmids leads to genetic instability and recombination
7. Theta replicating plasmids are more stable
   -- e.g. pHT43, which Friendzymes' B. subtilis wetware toolkit used as a starting point.

B. subtilis reporter genes

1. amyF / alpha-amylase, zones of clearing around colonies on starch plates
2. cat-86, chloramphenicol acetyltransferase, resistance to chloramphenicol
3. Beta galactosidase
4. luxAB, bacterial luciferase
5. xylE, Catechol 2,3-dioxygenase, yellow colonies when sprayed with catechol; catechol 2,3 deoxygenase assay

Promoters

1. SPO1 bacteriophage constitutive vegetative promoter (Ref 34)
2. Alpha-amylase promoter from B. amyloliquefasciens (stationary phase induced)
3. Thermo-inducible promoter from Bacillus phage phi-105
4. PN25/O promoter (lac/IPTG)
5. Hybrid spac-1 / lacO promoter (lac/IPTG)

Transformation techniques

1. Efficient uptake of linear fragments of homologous DNA
   -- But, circular plasmid DNA uptake is very inefficient
2. Multimeric plasmid DNA
3. Plasmids containing internal repeats
4. ‘Rescue’ of monomeric plasmid DNA with a related resident plasmid
5. Efficient protoplast transformation
   -- 10% of regenerated protoplasts contain the plasmid, even monomers
   -- Drawbacks: laborious method, difficult to reproduce; complex media, long (36-48h) time required to regenerate cells

Bacillus and Biotechnology

1. Secrete grams/liter of enzymes into the growth medium
2. Easy to grow
3. Well-proven safety

Industrial enzymes

1. Annual sales of industrial enzymes was 75000 tons in 1992!
   --Unclear if that's including carriers/bulking agents, or just the polypeptides
2. In 1992, 75% of industrial enzymes are hydrolytic enzymes (cleave molecules apart using water)
   -- Mainly proteases for detergent, dairy and leather industries
   -- Also carbohydrases for baking, brewing etc
3. Most species of Bacillus secrete proteases and alpha-amylases
4. Properties vary from strain to strain

Heterologous Proteins

1. ‘Conventional’ purification approaches:
   -- Size exclusion chromatography
   -- Ion exchange chromatography
   -- Need more research to find if there's a way to do these frugally
2. S-layer proteins on Bacillus brevis observed at up to 12 g/L in culture
   -- Any progress on expression and signal sequences from S-layer proteins? Don't know.
3. No native protein glycosylation systems in B. subtilis

Protein Export: B. subtilis vs E. coli

1. B. subtilis cell envelope lacks outer membrane, lipopolysaccharides, or membrane-enclosed periplasm
2. Lack of lipid A from LPS reduces endotoxic shock from co-purified endotoxins
3. Steps of protein export:
   A. Ribosome begins translating exoprotein, with signal peptide at its N-terminus
   B. Export chaperone SecB binds to the signal peptide during and/or after translation, prevents folding into secretion-incompetent forms
   C. SecA, peripheral membrane protein associated with SecY-E-1 translocate complex, interacts with SecB, the signal peptide and the partially folded protein.
   D. Protein translocates through SecE-1-Y complex, powered by ATP hydrolysis from SecA
   E. Signal peptide is cleaved off by a signal peptidase on the outer side of the cell membrane
   F. Many Bacillus exoproteins also have a ‘pro-‘ sequence between the signal sequence and the mature polypeptide, also proteolytically cleaved after translocation; may function in secretion and folding of mature exoprotein.
4. Secretion components of B. subtilis
   -- Cytosolic: GroES, GroEL, DnaK
   -- Internal, peripheral to membrane: SecA
   -- Membrane: SecY, SPasel
   -- External, peripheral to membrane: PrsA
   -- External: Fe(III) (Iron III)??

Limitations and advantages of B. subtilis

1. Seven extracellular proteases
   -- Neutral protease A
   -- Subtilisin (alkaline protease
   -- A minor extracellular protease
   -- A metalloprotease
   -- Bacillopeptidase F
   -- Neutral protease B
   --A minor serine protease
2. Proteins exiting the cell surface must pass the cell wall to be fully secreted into the extracellular medium
   -- Membrane is mostly peptidoglycan and anionic polymers
   -- Large folded proteins, and cationic proteins, may struggle to escape the cell wall
   -- Can’t secrete human serum albumin from intact B. subtilis, but can secrete it from B. subtilis protoplasts.
   -- Other poorly secreted proteins: lysozyme, E. coli OmpA, some alpha-interferons
3. There are examples of highly secreted homologous proteins (as of 1992):
   -- Alpha-interferon (15 mg/L)
   -- Growth hormone (200 mg/L)
   -- Epidermal growth factor (240 mg/L)
   -- Porcine pepsinogen (>500 mg/L)
   -- However, none of these are secreted at the levels of homologs of natively secreted enzymes, like alpha-amylases.
4. Bsub is also useful for intracellular production of outer membrane proteins from virulent gram-negative bacteria
   -- Used in vaccines
   -- Normally form tight associations with endotoxins in gram-negative bacteria; not a problem in gram-positive B. subtilis

Other products produced in B. subtilis

1. Fine biochemicals
   -- Hypoxanthine
   -- Inosine
   -- Guanine
   -- Xanthanylic acid
   -- Guanilic acid
   -- Inosinic acid
2. As of 1992, B. subtilis strains were still not producing Tryptophan, Phenylalanine or Histidine at titers competitive with industrial strains of Brevibacterium, Corynebacterium or Serratia.
3. Antibiotics Bacillus species produce:
   -- Bacitracin (cell wall synthesis inhibitor)
   -- Gramicidin’s, polymyxin (membrane-active)
   -- Edeines (basics peptides)
   -- Aminoglyoside ribosome inhibitors
   -- Bacilysin and subtilin from B. subtilis strains
4. Several B. subtilis strains produce surfactin, an ‘exceptional’ biosurfactant
5. Insecticidal toxins produced by:
   -- B. thuringinesis (BT toxin, lepidopteran larvae)
   -- B. larvae (kills honey bee larvae)
   -- B. lentimorbus, B. popilliae (kills scarabaeid beetle larvae)
   -- B. sphaericus (lepidopteran larvae)
   -- Toxins are produced during sporulation
   -- Toxins are highly active (hundreds to tens of thousands of times more potent than synthetic insecticides)
   -- Toxins are proteolytically cleaved to their active form in the alkaline midgut
   -- Toxins bind to specific receptors on the gut wall, paralyzing gut and mouth parts and altering K+ permeability
   -- Toxins are highly specific

Sporulation

1. B. subtilis is the best studied model of bacterial differentiation
2. As of 1992, nine sigma factors had been identified in Bsub
   -- 43 kDa sigma_v, vegetative sigma factor
   -- Sigma E, F, G and K are specific for sporulation genes
   -- Sigma B, D, and H function in stationary phase
   -- Sigma L functions during nitrogen starvation
3. Sporulation is induced by a cascading phosphorelay system
   -- Variant of two-component signaling systems

[danwchan]

I copied these notes into references for these 2 resources in the Zotero library:

1. Wong SL. Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Curr Opin Biotechnol. 1995;6: 517–522. doi:10.1016/0958-1669(95)80085-9
2. Harwood CR. Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends in Biotechnology. 1992;10: 247–256. doi:10.1016/0167-7799(92)90233-L