1. Question 1:
Tat system, e.g., folded (TAT) or
T1SS: unfolded (Sec), twin arg (TAT) or a hydrophobic N-
terminal leader sequence (Sec). Most proteins secreted
Mechanism are toxins and degradative enzymes.
There are three components of which the T1SS is
made, i.e. ABC(ATP-binding cassette) transporter on
the inner membrane (IM), membrane fusion protein T3SS:
(MFP) in the periplasmic space, and the outer
Mechanism
membrane protein (OMP, could be alternatively called
outer membrane factor as well) on the outer membrane A discussion involving the mechanism by which T3SS
(OM). As could be inferred from the presence of a ABC translocates its substrates should begin with the
transporter at the cytoplasmic membrane, the introduction of its structure. T3SS is a protein complex
mechanism of substrate transportation is initiated by the spanning across the IM, periplasmic space and OM like
energy provided by ATP binding to the ABC transporter that of T1SS; however, T3SS has a needle-shaped
and the subsequent conformational change that send structure termed "injectisome" that enables the bacteria
the substrate through the inner membrane and into the to translocates their substrates directly into target cell's
MFP. With the example of the well-studied #alpha- plasma membrane. The injectisome protrudes outward
hemolysin secretion in uropathogenic Escherichia coli, into the extracellular surface, functioning like a needle.
the opening of the OMP is believed to be triggered by For the translocation to occur, ATP hydrolysis via
MFP, creating a pore of about 16-20 A that allows the ATPase components at the cytoplasmic surface
protein to be secreted extracellularly. Therefore, the energizes the translocation. Therefore, T3SS is an one-
T1SS is an one-step secretion system and is Sec/ Tat- step secretion system and is Sec/ Tat- independent.
independent.
Substrate specificity Substrate specificity
The T1SS is compatible with a wide range of Substrates of T3SS includes the extracellular needle
substrates, ranging from both proteinaceous and non- components of T3SS (during the construction of a
proteinaceous substrates. For proteinaceous functional T3SS) and, as usual, the effector proteins.
substrates, most of the proteins to be secreted via T3SS may use a protein-based signal rather than a
T1SS possess a C-terminal signal peptides. The nucleotide-based signal to regulate substrate specificity.
secreted proteins are mostly toxins and degradative As an example, if there are four or five serine residues
enzymes. between amino acid residue 2 to 8 of the protein YopE,
this peptide has the greatest likelihood of promoting
secretion. [ref.1]
T2SS
Mechanism T4SS
Transportation through T2SS is mediated by Sec/Tat
Mechanism
although T2SS does possesses IM components, i.e.,
substrates are sent across IM via Sec or Tat system T4SS is similar to T3SS in that they both possess
first, then exported by T2SS from the periplasmic space injectisomes yet unique to all other SS since T4SS
into the extracellular space. In the model system of translocates ssDNA as well as proteins. The most well-
Vibrio cholerae, the IM component (EspE) of this known example being the Agrobacterium tumefaciens
bacterium's T2SS has an ATP-binding motif and C58 causing crown gall. In A. tumefaciens,the T4SS is
autokinase activity. It is assumed that the type A T4SS. There is type B T4SS which is best
conformational change driven by ATP hydrolysis of exemplified by Legionnella pneumophilia. The T4SS is
EspE would eventually be transmitted all the way to the an one-step secretion system and is Sec/ Tat-
periplasmic domain and OM domain, translocating the independent. During translocation, ATP is required as
substrates from periplasm to the extracellular space. an energy source. T4SS is a one-step secretion system
Therefore, the T2SS is an two-step secretion system and Sec/Tat-independent.
and is Sec/Tat-dependent.
Substrate specificity
For the reason that T2SS is Sec/Tat-dependent, the
substrates must comply with the same rule of the Sec or

2. Substrate specificity As implied by the fact that it is Sec-dependent, proteins
to be secreted via T5SS must have the characteristic N-
Substrates of T4SS includes the extracellular needle terminal signal sequence.
components of T4SS (during the construction of a
functional T4SS) and, as usual, the effector proteins
along with the unique ssDNA. T6SS
Mechanism
T5SS
T6SS has an injectisome structure analogous to that of
Mechanism T3SS and T4SS except for the injectisome in T6SS is
T4-phage-like. A cytoplasmic chaperone with ATPase
Except for the presence of the injectisome, T5SS is may provide it with energy necessary to translocation of
more similar to T2SS in that it is Sec/Tat-dependent and substrates. The secretion is one step and is Sec/Tat-
is thus a two step translocator. Besides these, T5SS are independent.
more complicated in its translocation system as there
are three known subclasses of T5SS, each of which Substrate specificity
possessing different translocation mechanisms. In
T5aSS (auto-transporter), the secreted proteins T6SS is only recently discovered in 2006, it translocates
consists of an N-terminal passenger domain and a substrates such as Hcp and VgrG which contains C-
conserved C-terminal domain which will form a β-barrel. terminal actin crosslinking domain that is the effector
Once the peptide is translocated into periplasm via Sec/ protein responsible for the virulence of V. cholerae.
Tat, the β-barrel of the C-terminal is inserted into the VgrG is also the component of the T6SS.
OM and the passenger domain at the N-terminal is
inserted into the β-barrel. There are now two options for
this protein. In one option, the passenger domain is References:
cleaved and is thus “secreted” into the extracellular
space. In the other option, the passenger domain
1. Gerlach, R.G. and Hensel, M. (2007) Protein secretion system and
remains anchored in the barrel, such as adherin. In adhesins: The molecular armory of Gram-negative pathogens. Int.
T5bSS, the substrate to be secreted is a trimeric protein J. Med. Microbiol. 297: 401-15
complex in which each of the three peptides contributes
to the formation of a single β-barrel. Lastly, in T5cSS, 2. Tseng, T.T., Tyler, B.M. and Setubal, J.C. (2009) Protein secretion
which is termed two partner secretion (TPS), one in a system in bacterial-host associations, and their description in the
pair of proteins forms the β-barrel and the other is gene ontology. BMC Microbiol. 9 (Suppl 1):S2
secreted.
3. Ghosh, P., (2004) Process of Protein Transport by the Type III
Secretion System. Microbiol. Mol. Biol. R. 68: 771-95
Substrate specificity
To confirm, we could delete the identified gene in the
Question 2: wild-type Xaa to see if it lose the ability to cause spot
disease or to show its original phenotype. If it does,
Method 1 send the gene back again to see if regains the function
or phenotype, in this case, the ability to cause spot
The pathogenic Xaa bacteria are first treated with
disease.
transposon mutagenesis with random insertion of the
transposons. Then the mutated bacteria are subjected Method 2
to screening for a lose of function. For example, the
absence of a clear zone of an otherwise digestible With the assumption that there is a protein secreted by
substance on the petri-dish often indicates that an Xaa which causes the spot disease, search the Xaa
essential gene encoding a digestive enzyme is genome to see if there is any homologous proteins that
disrupted. In order to confirm that the Xaa bacterium causes similar disease in other pathogenic bacterium.
indeed carries the transposon, they are selected on A double-stranded siRNA about the size of 20
agar containging antibiotics such as streptomycin and/ nucleotides long is synthesized to target the
or kanamycin. Once a loss-of-function Xaa mutant is homologous proteinsʼ mRNA. Run an experiment to
selected, it will be sequenced to determine where the confirm if Xaa bacterium lose its ability to cause spot
transposon is inserted, i.e., which gene is disrupted. disease.
When the gene is identified, a bioinformatics survey of
homologous gene would usually give hints of other
functions of this gene.

3. References:
1. Pukatzki, S., Ma, A.T., Sturtevant, D., Krastins, B., Sarracino, D.,
Nelson, W.C., Heidelberg, J.F. and. Mekalanos, J.J. (2006)
Identification of a conserved bacterial protein secretion system in
Vibrio cholerae using the Dictyostelium host model system. PNAS.
103: 1528-33