Transfer and maintenance of small, mobilizable plasmids with ColE1 replication origins in Legionella pneumophila

With the mutagenesis of specific, virulence-associated genes of Legionella pneumophila as the eventual goal, methods for gene transfer to these bacteria were developed. Following the observations of others that conjugative, broad-host-range plasmids could be transferred from Escherichia coli to L. pneumophila at low frequency, we constructed a small mobilizable vector, pTLP1, which carries oriV from pBR322, oriT from pRK2, Kmr from Tn5, and an L. pneumophila-derived fragment to permit chromosomal integration. In triparental matings including an E. coli with a conjugative (Tra+) helper plasmid, kanamycin-resistance was transferred from E. coli to L. pneumophila. Southern hybridization of L. pneumophila transconjugants showed that pTLP1 was replicated autonomously. Additional matings of plasmids having deletions or substitutions of pTLP1 sequences confirmed that replication in L. pneumophila requires oriV only. pTLP1 was maintained in L. pneumophila with passage on medium containing kanamycin but was rapidly lost after passage on nonselective medium. This plasmid instability in L. pneumophila is most likely due to rapid generation of plasmid-free segregants because of plasmid multimerization and low plasmid copy number. We conclude that mobilizable pBR322-derived plasmids can be used as shuttle vectors to transfer cloned genes to L. pneumophila, a feature that can be exploited for the purposes of mutagenesis or genetic complementation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27233/1/0000240.pd

Site-specific mutagenesis in Legionella pneumophila by allelic exchange using counterselectable ColE1 vectors

To study the molecular pathogenesis of infection by Legionella pneumophila, a technique of site-specific mutagenesis by allelic exchange was evaluated. To develop this system, we optimized conjugal DNA transfer by isolating a mutant that functions 1000-fold more efficiently as a recipient than the wild type strain, identified two counter-selectable markers, rpsL and sacB, that function in L. pneumophila, and constructed a counterselectable Co1E1 vector. Allelic exchange of a L. pneumophila chrosomal gene was achieved at a frequency of 10-5 per transconjugant. The allelic exchange procedure itself did not alter the ability of L. pneumophila to infect macrophages, indicating that the system can be used to study this aspect of virulence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27030/1/0000018.pd

Cytochrome c4 is required for siderophore expression by Legionella pneumophila, whereas cytochromes c1 and c5 promote intracellular infection

A panel of cytochrome c maturation (ccm) mutants of Legionella pneumophila displayed a loss of siderophore (legiobactin) expression, as measured by both the chrome azurol S assay and a Legionella-specific bioassay. These data, coupled with the finding that ccm transcripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes siderophore expression by L. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individual c-type cytochromes. Whereas ubiquinol-cytochrome c reductase (petC) mutants specifically lacking cytochrome c1 and cycB mutants lacking cytochrome c5 had normal siderophore expression, cyc4 mutants defective for cytochrome c4 completely lacked legiobactin. These data, along with the expression pattern of cyc4 mRNA, indicate that cytochrome c4 in particular promotes siderophore expression. In intracellular infection assays, petC mutants and cycB mutants, but not cyc4 mutants, had a reduced ability to infect both amoebae and macrophage hosts. Like ccm mutants, the cycB mutants were completely unable to grow in amoebae, highlighting a major role for cytochrome c5 in intracellular infection. To our knowledge, these data represent both the first direct documentation of the importance of a c-type cytochrome in expression of a biologically active siderophore and the first insight into the relative importance of c-type cytochromes in intracellular infection events

In vivo structure of the Legionella type II secretion system by electron cryotomography

The type II secretion system (T2SS) is a multiprotein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane of many Gram-negative bacteria. Here, using electron cryotomography and subtomogram averaging methods, we reveal the in vivo structure of an intact T2SS imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily related type IV pilus (T4P) system, we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including, for example, that the T2SS–ATPase complex is usually present but disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our electron cryotomography map produced a complete architectural model of the intact T2SS that provides insights into the structure and function of its components, its position within the cell envelope and the interactions between its different subcomplexes

In vivo structure of the Legionella type II secretion system by electron cryotomography

The type II secretion system (T2SS) is a multiprotein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane of many Gram-negative bacteria. Here, using electron cryotomography and subtomogram averaging methods, we reveal the in vivo structure of an intact T2SS imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily related type IV pilus (T4P) system, we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including, for example, that the T2SS–ATPase complex is usually present but disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our electron cryotomography map produced a complete architectural model of the intact T2SS that provides insights into the structure and function of its components, its position within the cell envelope and the interactions between its different subcomplexes

The Legionella pneumophila tatB Gene Facilitates Secretion of Phospholipase C, Growth under Iron-Limiting Conditions, and Intracellular Infection

Our previous mutational analysis of Legionella pneumophila demonstrated a role for type II protein (Lsp) secretion and iron acquisition in intracellular infection and virulence. In gram-negative bacteria, the twin-arginine translocation (Tat) pathway is involved in secretion of proteins, including components of respiratory complexes, across the inner membrane to the periplasm. To assess the significance of Tat for L. pneumophila, tatB mutants were characterized. The mutants exhibited normal growth in standard media but grew slowly under low-iron conditions. They were also impaired in the Nadi assay, indicating that the function of cytochrome c oxidase is influenced by tatB. Consistent with this observation, a subunit of the cytochrome c reductase was shown to be a Tat substrate. Supernatants of the tatB mutants showed a 30% reduction in phospholipase C activity while maintaining normal levels of other Lsp secreted activities. When tested for infection of U937 macrophages, the tatB mutants showed a 10-fold reduction in growth. Double mutants lacking tatB and Lsp secretion were even more defective, suggesting tatB has an intracellular role that is independent of Lsp. tatB mutants were also impaired 20-fold in Hartmannella vermiformis amoebae cultured in the presence of an iron chelator. All mutant phenotypes were complemented by reintroduction of an intact tatB. Thus, L. pneumophila tatB plays a role in the formation of a respiratory complex, growth under low-iron conditions, the secretion of a phospholipase C activity, and intracellular infection

The Secreted Pyomelanin Pigment of Legionella pneumophila Confers Ferric Reductase Activity▿

The virulence of Legionella pneumophila is dependent upon its capacity to acquire iron. To identify genes involved in expression of its siderophore, we screened a mutagenized population of L. pneumophila for strains that were no longer able to rescue the growth of a ferrous transport mutant. However, an unusual mutant was obtained that displayed a strong inhibitory effect on the feoB mutant. Due to an insertion in hmgA that encodes homogentisate 1,2-dioxygenase, the mutant secreted increased levels of pyomelanin, the L. pneumophila pigment that is derived from secreted homogentisic acid (HGA). Thus, we hypothesized that L. pneumophila-secreted HGA-melanin has intrinsic ferric reductase activity, converting Fe3+ to Fe2+, but that hyperpigmentation results in excessive reduction of iron that can, in the case of the feoB mutant, be inhibitory to growth. In support of this hypothesis, we demonstrated, for the first time, that wild-type L. pneumophila secretes ferric reductase activity. Moreover, whereas the hyperpigmented mutant had increased secreted activity, an lly mutant specifically impaired for pigment production lacked the activity. Compatible with the nature of HGA-melanins, the secreted ferric reductase activity was positively influenced by the amount of tyrosine in the growth medium, resistant to protease, acid precipitable, and heterogeneous in size. Together, these data represent the first demonstration of pyomelanin-mediated ferric reduction by a pathogenic bacterium