Analysis of DNA Regulatory Elements Required for Expression of theLegionella pneumophilaicmanddotVirulence Genes

Abstract

To investigate the regulation of the Legionella pneumophila icm and dot genes required for intracellular growth, a series of nine icm::lacZ fusions were constructed. These icm::lacZ fusions were found to have different levels of expression in L. pneumophila, and five of them were more highly expressed at stationary phase than at exponential phase. When the expression of these fusions in Escherichia coli was tested, all of them were found to be expressed but three of them had dramatic changes in their levels of expression in comparison to those in L. pneumophila. Site-directed and PCR random mutagenesis with these icm::lacZ fusions was used to identify DNA regulatory elements of icm genes. Four icm genes (icmT, icmP, icmQ, and icmM) that had low levels of expression in L. pneumophila were found to contain a 6-bp sequence (TATACT) essential for their expression. This sequence was shown by primer extension to serve as their ؊10 promoter elements. A similar sequence, which constitutes the ؊10 promoter elements of the icmV, icmW, and icmR genes which had high levels of expression in L. pneumophila, was also identified. In addition, regulatory elements that probably serve as binding sites for transcription regulators were found in these genes. Altogether, 12 regulatory elements, 7 of which constitute the ؊10 promoter elements of the icm genes, were found. Even though all the icm and dot genes are part of one system required for L. pneumophila intracellular growth and even though their promoters are probably recognized by the vegetative sigma factor, it seems that they are subjected to different regulation mediated by several regulatory factors.Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen. L. pneumophila is able to infect, multiply within, and kill human macrophages, as well as free-living amoebae (28, 38). The bacteria are taken up by regular phagocytosis or by a special mechanism termed "coiling" phagocytosis (9, 26). They are then found within a specialized phagosome that does not fuse with lysosomes or acidify (9,24,27). The specialized phagosome undergoes several recruitment events, which include association with smooth vesicles, mitochondria, and the rough endoplasmic reticulum (1,25,49); the bacteria multiply within the specialized phagosome until the cell eventually lyses, releasing bacteria that can start new rounds of infection (28,38).Two regions of genes required for human macrophage killing and intracellular multiplication have been discovered in L. pneumophila (reviewed in references 44 and 51). Region I contains 7 genes (icmV, -W, and -X and dotA, -B, -C, and -D) (8, 10, 32, 50), and region II contains 17 genes (icmT, -S , -R, -Q, -P, -O, -N, -M, -L, -K, -E, -G, -C, -D, -J, -B, and -F) (3,35,41,43,50). Complementation analysis of these genes indicated that they are probably organized as nine transcriptional units (icmTS, icmR, icmQ, icmPO, icmMLKEGCD, icmJB, icmFtphA, icmWX, and icmV-dotA) (8,10,35,41,43,50). Most of these genes were also...

Characterization of theicmHandicmFGenes Required forLegionella pneumophilaIntracellular Growth, Genes That Are Present in Many Bacteria Associated with Eukaryotic Cells

“…7A). This result indicates that the Ϫ10 promoter element of this transcriptional unit is unique among the icm genes, as it does not contain the conserved TATACT consensus sequence that was identified in the other icm gene regulatory regions (24).…”

“…This result indicates that the upstream regulatory element (probably located between the F6 and F12 icmH::lacZ fusions) is probably independent of the site mutated (located between the F10 and F11 icmH::lacZ fusions), since the level of expression of the F5 icmH::lacZ fusion was reduced only by the portion contributed by the downstream regulatory element. Comparison of the level of expression of the F5 icmH::lacZ fusion to the level of expression of the other icm::lacZ fusion (24) clearly shows that the icmH-icmF-tphA transcriptional unit has the highest level of expression of all the icm genes and operons examined.…”

“…In a previous study, a regulatory element (CTATAGAAT) located between the F10 and F11 truncations was identified, and one nucleotide substitution in this site (constructed on the F4 icmH::lacZ fusion) completely abolished the expression of the fusion (24). To determine the relationship between this site and the potential regulatory element located between the F6 and F12 FIG.…”

Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein

“…L. pneumophila contains homologs of at least six sigma factors (RpoD, RpoH, RpoF, RpoE, RpoS, and RpoN) (14). In contrast, E. chaffeensis encodes only two sigma factor homologs: a constitutive 70 factor and a single alternative factor, 32 (RpoH) (17).…”

“…In Legionella, the icm/dot T4S system is organized into at least 11 transcriptional units that contain monocistronic as well as polycistronic transcripts, several of which contain a conserved sequence (TATAYT) that serves as their putative RpoD ( 70 ) recognition element and is essential for their expression (14). L. pneumophila contains homologs of at least six sigma factors (RpoD, RpoH, RpoF, RpoE, RpoS, and RpoN) (14).…”