The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussis adenylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-twohybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila.Chaperonins constitute a family of highly conserved proteins found in all prokaryotic and eukaryotic organisms (34). Their primary role is to facilitate the folding of nascent and stressdenatured proteins into their functional native states in an ATP-dependent manner (54). Group I chaperonins, referred to as Hsp60, Cpn60, or GroEL, are prokaryotic proteins found in bacteria and in eukaryotic organelles such as mitochondria and chloroplasts (34). Group II chaperonins, also known as CCT or TCP-1, are found in the eukaryotic cytosol and in the archaea (34). Structural and functional studies of Escherichia coli GroEL have established the role of group I chaperonins as intracellular mediators of protein folding (7, 94). GroEL is an essential protein in E. coli (23) whose intracellular level increases substantially in response to defined stressful stimuli (55, 85). The protein-folding paradigm of group I chaperonins has changed with accumulating reports of surface-and membrane-associated chaperonins that perform other diverse functions. For instance, the extracytoplasmically localized chaperonins of Haemophilus ducreyi (25), Helicobacter pylori (9, 92), Borrelia burgdorferi (77), and Clostridium difficile (37) have been implica...

Volume 193, no. 17, pages 4346 -4360, 2011. Page 4355: We erroneously stated, "The only genes encoding polyamine biosynthetic enzymes in L. pneumophila were metK (methionine adenosyltransferase) and speA (arginine decarboxylase). 1-14, 1998). The other two enzymes encoded in the arginine decarboxylase (speA) locus in L. pneumophila genomes are agmatine deiminase and N-carbamoyl putrescine amidohydrolase. We did not account for these three enzymes, mainly because in our bioinformatics analysis we looked only for the conventional polyamine biosynthetic enzymes present in Escherichia coli and Vibrio cholerae. We thus acknowledge here the presence of a complete putrescine-homospermidine alternative biosynthetic pathway in Legionella. Our suggestion that "L. pneumophila cannot synthesize all polyamines" (end of p. 4355) still stands, and our finding that L. pneumophila grows better in the presence of putrescine, spermidine, and/or spermine is not affected by this correction.

BackgroundInfectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae, genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts.ResultsTwo different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' and 5' NCR sequences of the 8 RNA genome segments of both genotypes of ISAV. The 3' sequences of the mRNA transcripts of ADL-ISAV-07 terminated 13-18 nucleotides from the full 3' terminus of cRNA, continuing as a poly(A) tail, which corresponded with the location of the polyadenylation signal. The lengths of the 3' and 5' NCRs of the vRNA were variable in the different genome segments, but the terminal 7 and 11 nucleotides of the 3' and 5' ends, respectively, were highly conserved among the eight genomic segments of ISAV. The first three nucleotides at the 3' end are GCU-3' (except in segment 5 with ACU-3'), whereas at the 5' end are 5'-AGU with the polyadenylation signal of 3-5 uridines 13-15 nucleotides downstream of the 5' end terminus of the vRNA. Exactly the same features were found in the respective complementary 5' and 3' end NCR sequences of the cRNA transcripts of ADL-ISAV-07, indicating that the terminal sequences of the 8 RNA genome segments are highly conserved among the two ISAV genotypes. The 5' NCR sequence...