RNA was extracted from whole abdomens of the adult bees using a standard TRIzol (Invitrogen, Carlsbad, CA, USA) procedure while RNA was extracted from individual larvae using the RNAqueous kit (Ambion, Austin, TX). RNAs were pooled by sample duration for the eight larvae challenged with the bacterial pathogen P. larvae, and the eight controls prior to cDNA synthesis, giving six RNA pools. DNA was removed from all extracts, then first-strand cDNA was synthesized as described by Evans (2004) . Transcript abundances for these cDNAs were assayed by quantitative real-time PCR with an Icycler real-time PCR machine (Bio-Rad, Hercules, CA, USA). Primer pairs were designed to amplify 120-300 bp sections of 39 honey bee immune-related genes derived from Supplementary Material Table S1 and ribosomal protein S5 as a control gene (primers in Supplementary Material Table S2 ). Primer sequences were modified, where necessary, to run in duplicate on 96-well plates using a fixed thermal protocol consisting of 5 min at 95 °C, then 40 cycles of a four-step protocol consisting of 94 °C for 20 s, 60 °C for 30 s, 72 °C for 1 min, and 78 °C for 20 s was used (Evans, 2006) . Reactions were carried out on 0.5-2 µg cDNA along with 1 U Taq, the provided PCR buffer (Roche Applied Sciences, Indianapolis, IN, USA), 1 mM dNTP mix, 2 mM added MgCl 2 , 0.2 µM each primer, 1× concentration SYBR-Green I dye (Applied Biosystems, Foster City, CA, USA), and 10 nM fluorescein in a 25 µl reaction volume. Amplification was followed by a meltcurve dissociation program in order to confirm expected product size. Thresholds were calculated individually for each target gene on the 96-well plate. For adult bee samples, data were pooled for the three replicates in each single-bee injection treatment (or controls). Results were screened for the appropriate dissociation (melt-curve) values, and by 1% agarose gels, in order to ensure against primer artefacts and the presence of DNA contamination (which would have been evident for numerous primers spanning two exons). Immune-gene transcripts were normalized relative to expression levels for the gene encoding ribosomal protein S5, a gene with consistent expression across honey bee life stages and disease status (Evans & Wheeler, 2000; Evans, 2004) . For display purposes, transcript abundance values (CTcontrol-CTtarget) for each gene were median-normalized across each panel of genes and clustered by average linkage clustering (using Cluster 3.0, M. Eisen, www.rana.lbl.gov/EisenSoftware.htm) and presented as relative grey-scale values (using Treeview, M. Eisen). ElaD, a Deubiquitinating Protease Expressed by E. coli BACKGROUND: Ubiquitin and ubiquitin-like proteins (Ubl) are designed to modify polypeptides in eukaryotes. Covalent binding of ubiquitin or Ubls to substrate proteins can be reversed by specific hydrolases. One particular set of cysteine proteases, the CE clan, which targets ubiquitin and Ubls, has homologs in eukaryotes, prokaryotes, and viruses. FINDINGS: We have cloned and analyzed the E. coli protein elaD, which is distantly related to eukaryotic CE clan members of the ULP/SENP protease family that are specific for SUMO and Nedd8. Previously misannotated as a putative sulfatase/phosphatase, elaD is an efficient and specific deubiquitinating enzyme in vitro. Interestingly, elaD is present in all intestinal pathogenic E. coli strains, but conspicuously absent from extraintestinal pathogenic strains (ExPECs). Further homologs of this protease can be found in Acanthamoeba Polyphaga Mimivirus, and in Alpha-, Beta-and Gammaproteobacteria. CONCLUSION: The expression of ULP/SENP-related hydrolases in bacteria therefore extends to plant pathogens and medically relevant strains of Escherichia coli, Legionella pneumophila, Rickettsiae, Chlamydiae, and Salmonellae, in which the elaD ortholog sseL has recently been identified as a virulence factor with deubiquitinating activity. As a counterpoint, our phylogenetic and functional examination reveals that ancient eukaryotic ULP/SENP proteases also have the potential of ubiquitin-specific hydrolysis, suggesting an early common origin of this peptidase clan. Ubiquitin, as well as Ubls such as Nedd8 and SUMO, are proteins (almost) exclusively expressed by eukaryotes [1] . Ubiquitination controls many cellular processes, including degradation of proteins by the proteasome and intracellular trafficking. Nedd8 is a ubiquitin-like modifier that regulates the rate or extent of ubiquitination, whereas SUMO1 is involved mostly in regulation of transcription factors and in nuclear import. The attachment of ubiquitin or ubiquitin-like modifiers to substrate proteins is covalent, yet reversible [2] . A large family of eukaryotic cysteine proteases is involved not only in generation of ubiquitin(-like) proteins from their precursors, but also in their removal from modified substrates [3, 4] . Pathogens can tamper with the ubiquitin-proteasome system to cripple the cell's defenses [5] . For instance, ubiquitination and proteasomal degradation of p53, initiated by a Human Papillomavirus protein [6] , or stabilization of Ikb-a by Yersinia deubiquitinases [7] have been described. The continuing discovery of new deubiquitinating proteases in viruses broadly hints at how important it is for these pathogens to seize control of posttranslational modifications in host cells [8] [9] [10] . Here, we focus on cysteine proteases of the CE clan [11] , as defined by the MEROPS database [12] . CE peptidases are expressed by viruses, bacteria and eukaryotes. In eukaryotes, this clan represents the family of Ubl-specific proteases (ULP/SENP), which remove SUMO or Nedd8 from substrate proteins [13, 14] . Viral homologs of ULP/SENPs can act as deubiquitinases, but they also cleave unrelated proteins, as long as a glycine motif is present at the C-terminus of the substrate [15, 16] . Examples of bacterial deubiquitinases include YopJ and ChlaDUBs. First, YopJ is a protease that is secreted into host cells by Y. pestis [17] , and homologs to this peptidase can be found in other bacteria, too [18, 19] . Injection of YopJ eventually suppresses the inflammatory response in affected cells. The precise molecular mechanism of YopJ family peptidases has not yet been solved, as they lack a hallmark tryptophan following the active-site histidine [20] . Yet, in vitro and in vivo data strongly suggest that these proteins are indeed proteases with specificity for ubiquitin or SUMO [7, 18, 19] , although their effectiveness as virulence factors might depend on additional functions such as acetylation [21, 22] . A second example of bacterial CE peptidases can be found in pathogenic Chlamydiae. We have shown that pathogenic Chlamydiae, but not a nonpathogenic environmental strain, express proteases that specifically recognize ubiquitin and Nedd8. They do so presumably to remove both modifiers from target proteins of the host cell, as Chlamydiaelike most other bacteria-possess neither a ubiquitin nor a Nedd8 homolog [23] .