Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the ␥-proteobacterium Legionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells. L. pneumophila translocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of the L. pneumophila Philadelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G؉C biases compared to other L. pneumophila genes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that in L. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.The ␥-proteobacterium Legionella pneumophila is an opportunistic human pathogen that multiplies within alveolar macrophages and causes the nosocomial and community-acquired pneumonia known as Legionnaires' disease (18,25,48). Human disease occurs when aerosolized L. pneumophila is inhaled from man-made or natural freshwater reservoirs harboring the bacteria. L. pneumophila poses a significant worldwide public health problem, particularly for individuals with compromised immune systems (19,38,40). Eradication of the pathogen from freshwater, industrial settings has proven difficult, since L. pneumophila thrives in environments that exclude antibacterial agents, such as biofilms and the intracellular compartments of protozoa (8,46,47).In order to create a replicative niche inside eukaryotic cells suitable for replication, L. pneumophila is believed to modulate host cell functions by the delivery of effector proteins through a type IVB secretion system known as the Icm-Dot complex (52, 59). Effector proteins presumably regulate several pathways in the host, including up-regulation of phagocytosis (23), delay in phagosome-lysosome fusion (24), recruitment of ARF1 to the phagosome (43), acquisition of endoplasmic reticulum-derived vesicles (29), and nonlytic egress from the host cell (11).Several laboratories found Icm-Dot substrates through genetic screens and bioinformatic approaches (3,7,11,13,36,43,44,57). Most of the known effector proteins are not individually required for intracellular multiplication, since knocking out th...

The kil-kor regulon of the self-transmissible, broad-host-range plasmid RK2 is a unique network with eight coregulated operons. Among the genes encoded by the kil-kor regulon are trfA, which encodes the replication initiator, and several kil loci (kiL4, kilB, WidC, and WilE), each of which is lethal to the host cell in the absence of appropriate negative regulatory elements encoded by the korA, korB, korC, and korE determinants. We have proposed that the functions of the kil loci are related to RK2 maintenance or host range. Here, we report the nucleotide sequence of a 2.44-kb region that includes the lethal kilB determinant. We identified the first three genes of the kilB operon (designated klbA, k1bB, and klbC), and we determined by deletion analysis that the host-lethal phenotype requires klbB. The predicted amino acid sequence of the 34,995-Da klbA product reveals a potential ATP-binding fold. The klbB product is predicted to be a membrane protein with a molecular mass of 15,012 Da with homology to the RK2 KlaC membrane protein encoded by the kiL4 operon. The amino acid sequence of the 12,085-Da IdbC product contains a perfect match to the leucine zipper motif common to eukaryotic regulatory proteins. Primer extension analysis revealed unambiguously that transcription of the kilB operon begins 46 nucleotides upstream of kibA. No transcription was initiated from the sequence previously presumed by other investigators to be the kilB promoter. The abundance of kilB transcripts is reduced in the presence of KorB, consistent with the prediction that KorB acts at the level of transcription. A degenerate KorB-binding site that contains a perfect half-palindrome overlaps the kilB promoter, but this site is insufficient for regulation by KorB. The region containing a KorB-binding site located 183 bp upstream of the transcriptional start is required for regulation by KorB, indicating that KorB acts at a distance to regulate transcription of kilB. Our studies with the mutant plasmid pRP101, a transfer-defective derivative of the RK2-like plasmid RP4, demonstrated that the kiB operon includes the conjugal transfer and surface exclusion genes of the Tra2 region. Nucleotide sequence analysis revealed that the transposon Tn7 insertion in pRP101 is located in the kdbC gene, and complementation analysis showed that this mutation has a strong polar effect on the expression of genes for conjugal transfer and surface exclusion located several kilobases downstream. A idbA mutant was constructed and found to be both transfer defective and complementable, thus demonstrating a requirement for the kibA product in plasmid transmissibility. These results have demonstrated a role for the kilB operon in conjugal transfer. The kil-kor regulon of RK2 is the only known example of plasmid-mediated coregulation of replication and transfer.The self-transmissible plasmids of incompatibility group P (IncP) are remarkable for autonomous replication in a wide variety of gram-negative bacterial hosts (17,67,98). In addition, the range of organisms...

The amino acid sequence of the 13-kDa polypeptide (P116) encoded by the first gene of the trfA operon of IncP plasmid RK2 shows significant similarity to several known single-stranded DNA-binding proteins. We found that unregulated expression of this gene from its natural promoter (tifAp) or induced expression from a strong heterologous promoter (trcp) was sufficient to complement the temperature-sensitive growth phenotype of an Escherichia coli ssb-1 mutant. The RK2 ssb gene is the first example of a plasmid singlestranded DNA-binding protein-encoding gene that is coregulated with replication functions, indicating a possible role in plasmid replication.Plasmids of incompatibility group P (IncP) are known for their broad host range among gram-negative bacteria (10, 48). One well-studied IncP plasmid is the 56.4-kb selftransmissible plasmid RK2 (22,28,45 In addition, the last 40 residues are highly conserved and may be involved in protein-protein interactions (6,8). P116 is homologous to the first 105 residues of the known SSBs (Fig. 2). Three of the four residues thought to be involved in DNA binding are identical, and the fourth is similar (Phe versus Trp-54).* Corresponding author.E. coli ssb is an essential gene required for DNA replication (29). E. coli strains with the ssb-1 mutation are temperature sensitive (Ts) for growth; i.e., they grow normally at 33°C and poorly at 42°C (16, 29, 30). Complementation of ssb-1 has been used to identify SSB-encoding genes on several plasmids (18), including F (25) and ColIb-P9 (21). However, RK2 and derivatives of RK2 were previously found to be unable to complement the ssb-1 mutation (18,47). Because the trfA promoter is negatively controlled, we considered the possibility that expression of P116 from the regulated trfA operon was inadequate for complementation.We therefore tested the ability of the RK2 P116 gene to complement the E. coli ssb-1 allele under conditions in which the trfA promoter was not repressed.Complementation of the E. coli ssb-1 mutation by the trfA region of RK2 in the absence of kor functions. The ssb-1 mutant KLC436 [thyA36 rha-8 ssb-1 melA7 amp-S0 deoC2 IN(rmD-rrnE)1] and the isogenic ssb+ strain KLC438 (49) were used in the complementation assay. Strains carrying the plasmids to be tested for complementation of ssb-1 were grown in LB broth at 33°C to a density of -5 x 108 cells per ml, washed with M9 CAA medium (M9 containing 0.5% Casamino Acids [27]) containing thymine (50 ,ug/ml), and plated on M9 CAA-thymine agar plates at 33 and 42°C. The ssb+ strain grew normally at 33 and 42°C (see Table 2). The ssb-1 strain grew normally at 33°C but gave pinpoint colonies at 42°C, as expected. KLC436(F) yielded normal, healthy colonies at 33 and 42°C because the F plasmid encodes an ssb gene known to complement ssb-1 (25). In agreement with the results of previous work (18), RK2 alone did not suppress the Ts growth phenotype of the ssb-1 strain. The pinpoint colonies of KLC436(RK2) at 42°C were indistinguishable from those of KLC436. P116-encoding plasm...

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