proper length. several theories exist, among them the existence of a "ruler protein" that somehow connects the tip and the base of the needle. addition of new monomers to the tip of the needle should stretch the ruler protein and thereby signal the needle length to the base. energetics. the force that drives the passage of proteins inside the needle is not completely known. an atpase is associated with the base of the t3ss and participates in directing proteins into the needle; but whether it supplies the energy for transport is not clear. secretion signal. as mentioned above, the existence of a secretion signal in effector proteins is known. the signal allows the system to distinguish t3ss-transported proteins from any other protein. its nature, requirements and the mechanism of recognition are poorly understood, but methods for predicting which bacterial proteins can be transported by the type iii secretion system have recently been developed. activation of secretion. the bacterium must know when the time is right to secrete effectors. unnecessary secretion, when no host cell is in vicinity, is wasteful for the bacterium in terms of energy and resources. the bacterium is somehow able to recognize contact of the needle with the host cell. how this is done is still being researched, and the method may well be dependent on the pathogen. some theories postulate a delicate conformational change in the structure of the needle upon contact with the host cell; this change perhaps serves as a signal for the base to commence secretion. one method of recognition has been discovered in salmonella, which relies on sensing host cell cytosolic ph through the pathogenicity island 2-encoded t3ss in order to switch on secretion of effectors. binding of chaperones. it is not known when chaperones bind their effectors (whether during or after translation) and how they dissociate from their effectors before secretion. effector mechanisms. although much was revealed since the beginning of the 21st century about the ways in which t3ss effectors manipulate the host, the majority of effects and pathways remains unknown. evolution. as mentioned, the t3ss is closely related to the bacterial flagellum. there are three competing hypotheses: first, that the flagellum evolved first and the t3ss is derived from that structure, second, that the t3ss evolved first and the flagellum is derived from it, and third, that the two structures are derived from a common ancestor. there was some controversy about the different scenarios, since they all explain protein homology between the two structures, as well as their functional diversity. yet, recent phylogenomic evidence favours the hypothesis that the t3ss derived from the flagellum by a process involving initial gene loss and then gene acquisition. a key step of the latter process was the recruitment of secretins to the t3ss, an event that occurred at least three times from other membrane-associated systems. nomenclature of t3ss proteins flagellum of gram-negative bacteria. the rings of the base are very similar to needle-complex rings, although the existence of a c-ring in the needle complex has not been proven. the flagellar hook is homologous to the t3ss needle since the beginning of the 1990s new t3ss proteins are being found in different bacterial species at a steady rate. abbreviations have been given independently for each series of proteins in each organism, and the names usually do not reveal much about the protein's function. some proteins discovered independently in different bacteria have later been shown to be homologous; the historical names, however, have mostly been kept, a fact that might cause confusion. for example, the proteins sica, ipgc and sycd are homologs from salmonella, shigella and yersinia, respectively, but the last letter (the "serial number") in their name does not show that. below is a summary of the most common protein-series names in several t3ss-containing species. note that these names include proteins that form the t3ss machinery as well as the secreted effector proteins: yersinia yop: yersinia outer protein ysc: yersinia secretion (component) ypk: yersinia protein kinase salmonella spa: surface presentation of antigen sic: salmonella invasion chaperone sip: salmonella invasion protein prg: phop-repressed gene inv: invasion org: oxygen-regulated gene ssp: salmonella-secreted protein iag: invasion-associated gene shigella ipg: invasion plasmid gene ipa: invasion plasmid antigen mxi: membrane expression of ipa spa: surface presentation of antigen osp: outer shigella protein escherichia tir: translocated intimin receptor sep: secretion of e. coli proteins esc: escherichia secretion (component) esp: escherichia secretion protein ces: chaperone of e. coli secretion pseudomonas hrp: hypersensitive response and pathogenicity hrc: hypersensitive response conserved (or hrp conserved) rhizobium nop: nodulation protein rhc: rhizobium conserved in several species: vir: virulence "protochlamydia amoebophila" "sodalis glossinidius" following those abbreviations is a letter or a number. letters usually denote a "serial number", either the chronological order of discovery or the physical order of appearance of the gene in an operon. numbers, the rarer case, denote the molecular weight of the protein in kda. examples: ipaa, ipab, ipac; mxih, mxig, mxim; spa9, spa47. several key elements appear in all t3sss: the needle monomer, the inner rod of the needle, the ring proteins, the two translocators, the needle-tip protein, the ruler protein (which is thought to determine the needle's length; see above) and the atpase, which supplies energy for secretion. the following table shows some of these key proteins in four t3ss-containing bacteria: ↓ function / genus → shigella salmonella yersinia escherichia needle monomer mxih prgi yscf escf inner rod mxii prgj ysci esci needle-tip protein ipad sipd lcrv espa translocator ipab sipb yopb espd translocator ipac sipc yopd espb chaperone for the two translocators ipgc sica sycd cesd atpase spa47 invc yscn sepb (escn) ruler protein spa32 invj yscp orf16 switch spa40 spas yscu escu gatekeeper mxic inve yopn (tyea) sepl methods employed in t3ss research isolation of t3ss needle complexes the isolation of large, fragile, hydrophobic membrane structures from cells has constituted a challenge for many years. by the end of the 1990s, however, several approaches have been developed for the isolation of t3ss ncs. in 1998 the first ncs were isolated from salmonella typhimurium. for the isolation, bacteria are grown in a large volume of liquid growth medium until they reach log phase. they are then centrifuged; the supernatant (the medium) is discarded and the pellet (the bacteria) is resuspended in a lysis buffer typically containing lysozyme and sometimes a detergent such as ldao or triton x-100. this buffer disintegrates the cell wall. after several rounds of lysis and washing, the opened bacteria are subjected to a series of ultracentrifugations. this treatment enriches large macromolecular structures and discards smaller cell components. optionally, the final lysate is subjected to further purification by cscl density gradient. an additional approach for further purification uses affinity chromatography. recombinant t3ss proteins that carry a protein tag (a histidine tag, for instance) are produced by molecular cloning and then introduced (transformed) into the researched bacteria. after initial nc isolation, as described above, the lysate is passed through a column coated with particles with high affinity to the tag (in the case of histidine tags: nickel ions). the tagged protein is retained in the column, and with it the entire needle complex. high degrees of purity can be achieved using such methods. this purity is essential for many delicate assays that have been used for nc characterization. type iii effectors were known since the beginning of the 1990s, but the way in which they are delivered into host cells was a complete mystery. the homology between many flagellar and t3ss proteins led researchers to suspects the existence of an outer t3ss structure similar to flagella. the identification and subsequent isolation of the needle structure enabled researchers to: characterize the three-dimensional structure of the nc in detail, and through this to draw conclusions regarding the mechanism of secretion (for example, that the narrow width of the needle requires unfolding of effectors prior to secretion), analyze the protein components of the nc, this by subjecting isolated needles to proteomic analysis (see below), assign roles to various nc components, this by knocking out t3ss genes, isolating ncs from the mutated bacteria and examining the changes that the mutations caused. microscopy, crystallography and solid-state nmr as with almost all proteins, the visualization of t3ss ncs is only possible with electron microscopy. the first images of ncs (1998) showed needle structures protruding from the cell wall of live bacteria and flat, two-dimensional isolated ncs. in 2001 images of ncs from shigella flexneri were digitally analyzed and averaged to obtain a first semi-3d structure of the nc. the helical structure of ncs from shigella flexneri was resolved at a resolution of 16 å using x-ray fiber diffraction in 2003, and a year later a 17-å 3d structure of ncs from salmonella typhimurium was published. recent advances and approaches have allowed high-resolution 3d images of the nc, further clarifying the complex structure of the nc. numerous t3ss proteins have been crystallized over the years. these include structural proteins of the nc, effectors and chaperones. the first structure of a needle-complex monomer was nmr structure of bsal from "burkholderia pseudomallei" and later the crystal structure of mixh from shigella flexneri, which were both resolved in 2006. in 2012, a combination of recombinant wild-type needle production, solid-state nmr, electron microscopy and rosetta modeling revealed the supramolecular interfaces and ultimately the complete atomic structure of the salmonella typhimurium t3ss needle. it was shown that the 80-residue prgi subunits form a right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellum of salmonella typhimurium. the model also revealed an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen. proteomics several methods have been employed in order to identify the array of proteins that comprise the t3ss. isolated needle complexes can be separated with sds-page. the bands that appear after staining can be individually excised from the gel and analyzed using protein sequencing and mass spectrometry. the structural components of the nc can be separated from each other (the needle part from the base part, for instance), and by analyzing those fractions the proteins participating in each one can be deduced. alternatively, isolated ncs can be directly analyzed by mass spectrometry, without prior electrophoresis, in order to obtain a complete picture of the nc proteome. genetic and functional studies the t3ss in many bacteria has been manipulated by researchers. observing the influence of individual manipulations can be used to draw insights into the role of each component of the system. examples of manipulations are: deletion of one or more t3ss genes (gene knockout). overexpression of one or more t3ss genes (in other words: production in vivo of a t3ss protein in quantities larger than usual). point or regional changes in t3ss genes or proteins. this is done in order to define the function of specific amino acids or regions in a protein. the introduction of a gene or a protein from one species of bacteria into another (cross-complementation assay). this is done in order to check for differences and similarities between two t3sss. manipulation of t3ss components can have influence on several aspects of bacterial function and pathogenicity. examples of possible influences: the ability of the bacteria to invade host cells, in the case of intracellular pathogens. this can be measured using an invasion assay (gentamicin protection assay). the ability of intracellular bacteria to migrate between host cells. the ability of the bacteria to kill host cells. this can be measured by several methods, for instance by the ldh-release assay, in which the enzyme ldh, which leaks from dead cells, is identified by measuring its enzymatic activity. the ability of a t3ss to secrete a specific protein or to secrete at all. in order to assay this, secretion is induced in bacteria growing in liquid medium. the bacteria and medium are then separated by centrifugation, and the medium fraction (the supernatant) is then assayed for the presence of secreted proteins. in order to prevent a normally secreted protein from being secreted, a large molecule can be artificially attached to it. if the then non-secreted protein stays "stuck" at the bottom of the needle complex, the secretion is effectively blocked. the ability of the bacteria to assemble an intact needle complex. ncs can be isolated from manipulated