In order to determine the role of ferrous iron transport in Legionella pathogenesis, we identified and mutated the feoB gene in virulent Legionella pneumophila strain 130b. As it is in Escherichia coli, the L. pneumophila feoB gene was contained within a putative feoAB operon. L. pneumophila feoB insertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss of L. pneumophila FeoB. However, the L. pneumophila feoB mutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition of feoB in trans. In unsupplemented buffered yeast extract broth, the feoB mutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2-dipyridyl. The feoB mutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of the feoB mutant was noted within iron-depleted Hartmannella vermiformis amoebae and human U937 cell macrophages. The reduced intracellular infectivity of the feoB mutant was complemented by the introduction of a plasmid containing feoAB. The L. pneumophila feoB gene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate that L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.

In the course of characterizing a locus involved in heme utilization, we identified a Legionella pneumophila gene predicted to encode a protein with homology to the product of the Salmonella enterica serovar Typhimurium pagP gene. In Salmonella, pagP increases resistance to the bactericidal effects of cationic antimicrobial peptides (CAMPs). Mutants with insertions in the L. pneumophila pagP-like gene were generated and showed decreased resistance to different structural classes of CAMPs compared to the wild type; hence, this gene was designated rcp for resistance to cationic antimicrobial peptides. Furthermore, Legionella CAMP resistance was induced by growth in low-magnesium medium. To determine whether rcp had any role in intracellular survival, mutants were tested in the two most relevant host cells for Legionnaires' disease, i.e., amoebae and macrophages. These mutants exhibited a 1,000-fold-decreased recovery during a Hartmannella vermiformis coculture. Complementation of the infectivity defect could be achieved by introduction of a plasmid containing the intact rcp gene. Mutations in rcp consistently reduced both the numbers of bacteria recovered during intracellular infection and their cytopathic capacity for U937 macrophages. The rcp mutant was also more defective for lung colonization of A/J mice. Growth of rcp mutants in buffered yeast extract broth was identical to that of the wild type, indicating that the observed differences in numbers of bacteria recovered from host cells were not due to a generalized growth defect. However, in low-Mg 2؉ medium, the rcp mutant was impaired in stationary-phase survival. This is the first demonstration of a pagP-like gene, involved in resistance to CAMPs, being required for intracellular infection and virulence.

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phasedependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.In recent years there has been growing interest in the use of high hydrostatic pressure (HHP) as a means of food preservation. The attraction of HHP lies in the production of microbiologically safe foodstuffs with minimal use of chemical additives and without adversely affecting the organoleptic qualities of the food (11). HHP processing could replace traditional thermal pasteurization or be used in conjunction with existing techniques (3,10,14,22). However, exploitation of this potential requires a better understanding of the effects of HHP on microorganisms. HHP resistance varies among genera and species and is dependent on the physiological state of the organisms at the time of pressurization (6, 18). It is of concern that certain E. coli O157:H7 strains are among the most pressureresistant vegetative bacteria known (2, 18). It is therefore critically important to characterize the innate HHP resistance in these strains.We demonstrated recently that the pressure resistance of certain natural isolates of E. coli O157:H7 varied greatly (2, 17). Strains C9490 and 30-2C4 were the most pressure resistant and were able to withstand 500 MPa for 5 min with little viability loss; strains NCTC 12079 and W2-2 were of intermediate pressure resistance (ca. 3 to 4 log units decrease under the same conditions), whereas H1071 and an O124 strain, NCTC 8003, were the least pressure resistant (5 to 6 log decrease). However, this variation in pressure resistance was stationary-phase dependent, with the strains exhibiting similar pressure resistance in exponential phase (2). This led us to speculate that the differences in pressure resistance among the isolates were related to differences in RpoS. This sigma factor changes the specificity of RNA polymerase, allowing it to activate more than 30 genes, some of which are involved in stationary-phase stress survival (8). Stationary-phase bacteria are generally more resistant to other stresses, such as oxidative and osmotic stress (21). It is thus possible that resistance to inactivation by HHP is also controlled by mechanisms used to survive stationary-phase stress. These studies attempted to identify the genetic basis, and specifically the role of RpoS, in the wide variation observed in HHP resistance of natural isolates of E. coli O157:H7 stra...

We have constructed a DNA library from a virulent Salmonella typhimurium strain, in an avirulent strain. The process of selecting the components of interest from the library involved iterative growth in liquid culture. This resulted, after four cycles, in the culture becoming homogeneous for a single plasmid, which was much smaller than the average size for the library. We have identified the larger precursor of this plasmid which has two regions of sufficient homology to allow recombination resulting in the formation of the small plasmid. S. typhimurium carrying the small plasmid have a smaller genetic burden than other members of the library and survive better in spent culture medium, facilitating selection on repeated subculture. Such rapid adventitious selection has important implications for isolation of clones of interest from genomic libraries.

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