Our efforts had added 70 novel proteins to the Dot/Icm substrate inventory, which with previously proteins, have expanded experimentally confirmed Dot/Icm substrates to 275. Thus, judging by the number of proteins transferred, the L. pneumophila Dot/Icm system is arguably the most prolific bacterial translocator, whose substrates are more than five times of the effectors secreted by the Hrp type III secretion system of Pseudomonas syringae, a plant pathogen known to have a large repertoire of effectors. Many Dot/Icm substrates identified by an earlier genetic method are larger than 100 kDa. With a more complete list of its substrates, we analyzed the size distribution of these proteins. Surprisingly, only 39 genes are larger than 2 kbp of which 12 are more than 3 kbp. The majority of the genes are shorter than 2 kbp, with 113 sizing between 1 to 2 kbp and 91 shorter than 1 kbp. Thus, the length of most of the substrate genes is about 1 kbp, typical for proteins of bacterial origins. That many substrates genes identified in an earlier genetic screen are longer than 2 kbps may be a result of high probability of generating in-frame fusions of longer genes in the random library used for bacterial two-hybrid screenings. Type IV transporter-mediated protein Evofosfamide 918633-87-1 translocation is determined by at least three factors: Components of the transporter involved in recognizing the signals, signals encoded by amino acids embedded in the carboxyl portion of the substrates and chaperones responsible for proper folding of the substrate. In L. pneumophila, DotF, an important component of the Dot/Icm system interacts with a set of substrates, but whether this protein is involved in recognizing the substrates by interacting with patches of amino acids important for translocation is unknown. The roles played by the chaperones IcmS/IcmW in substrate translocation probably is by inducing conformational changes, which presumably will allow the translocation signals more properly exposed to the transporter. Accumulating evidence suggests that signals residing in the carboxyl end of Dot/Icm substrates are quite diverse. For examples, a hydrophobic residue located at -3 or -4 position is present in many Dot/Icm substrates and has been shown to be important for the translocation of RalF. More recently, Huang et al showed that an E-block motif is important for the translocation of a subset of substrates. Further, our results have revealed Lpg2844, a protein with a carboxyl amino acid composition highly different from most substrates can be translocated at high efficiencies. Thus, it appears that in L. pneumophila, successful translocation does not need the presence of all known signals. Residues important for protein translocation can contribute to substrate recognition by forming structural entities that directly bind to the transporter or by indirectly involved in this process by supporting the formation of such structures.