CFU. In addition, the mutant strain demonstrated increased susceptibility to antimicrobial peptide killing, but no differences were observed between the lipid A of the mutant and the parental strain, as has been observed with pmrA mutants of other microbes. The F. novicida pmrA mutant was 100% protective as a single-dose vaccine when challenge was with 10 6 CFU of F. novicida but did not protect against type A Schu S4 wild-type challenge. DNA microarray analysis identified 65 genes regulated by PmrA. The majority of these genes were located in the region surrounding pmrA or within the Francisella pathogenicity island (FPI). These FPI genes are also regulated by MglA, but MglA does not regulate pmrA, nor does PmrA regulate MglA. Thus, the orphan response regulator PmrA is an important factor in controlling virulence in F. novicida, and a pmrA mutant strain is an effective vaccine against homologous challenge.

Inhalational pneumonic tularemia, caused by Francisella tularensis, is lethal in humans. F. tularensis is phagocytosed by macrophages followed by escape from phagosomes into the cytoplasm. Little is known of the phagocytic mechanisms for Francisella, particularly as they relate to the lung and alveolar macrophages. Here we examined receptors on primary human monocytes and macrophages which mediate the phagocytosis and intracellular survival of F. novicida. F. novicida association with monocyte-derived macrophages (MDM) was greater than with monocytes. Bacteria were readily ingested, as shown by electron microscopy. Bacterial association was significantly increased in fresh serum and only partially decreased in heat-inactivated serum. A role for both complement receptor 3 (CR3) and Fc␥ receptors in uptake was supported by studies using a CR3-expressing cell line and by down-modulation of Fc␥ receptors on MDM, respectively. Consistent with Fc␥ receptor involvement, antibody in nonimmune human serum was detected on the surface of Francisella. In the absence of serum opsonins, competitive inhibition of mannose receptor (MR) activity on MDM with mannan decreased the association of F. novicida and opsonization of F. novicida with lung collectin surfactant protein A (SP-A) increased bacterial association and intracellular survival. This study demonstrates that human macrophages phagocytose more Francisella than monocytes with contributions from CR3, Fc␥ receptors, the MR, and SP-A present in lung alveoli.

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either A -or B -dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10 ؊2 ) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a "TA" dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo ؊ clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.Bacillus subtilis has long been a subject organism for bacteriological research, serving as a model for analyses of the physiology of gram-positive bacteria and microbial differentiation (sporulation). Genetic analyses have been key to unraveling the biology of microorganisms, with transposon mutagenesis as a powerful tool in these analyses. Transposons can generate insertion mutations that are readily mapped and, depending on the particular transposon used, can also create reporter gene fusions at the sites of their insertion (3). The Tn917 and Tn10 transposons have been modified for use in B. subtilis. Each was successfully applied in a number of studies, but both have properties that limit their usefulness. Tn917, a streptococcal Tn3-like transposon, was the first transposon developed for use in B. subtilis (39). Although Tn917 readily mobilizes into the B. subtilis chromosome, it does not insert randomly. Ninety-nine percent of all Tn917 insertions occur at several "hot-spot" regions of the B. subtilis chromosome (40). As a result, large numbers of transposants need to be screened if a desired mutant is to be found among the 1% of insertions that occur randomly. The second B. subtilis transposon system was derived from the Escherichia coli transposon Tn10 (22). Unlike Tn917, Tn10 does not appear to have preferred insertion sites in the B. subtilis chromosome; however, it requires a particular 6-bp sequence as its target element (12). Unique 6-bp sequences occur randomly in DNA at approximately 4-kbp intervals (i.e., once in 4 6 bases). This reduces the number of potential Tn10 insertion sites on the B. subtilis chromosome and, as a consequence, Tn10's effectiveness as a tool for random mutagenesis.Th...

AcpA of Francisella spp. is a respiratory-burst-inhibiting acid phosphatase that also exhibits phospholipase C activity. To better understand the molecular basis of AcpA in virulence, a deletion of acpA was constructed in Francisella novicida. The phosphatase and lipase activities were reduced 10-fold and 8-fold, respectively, in the acpA mutant compared to the wild type and were found mostly associated with the outer membrane. The acpA mutant was more susceptible to intracellular killing than the wild-type strain in the THP-1 human macrophage-like cell line. In addition, mice infected with the acpA mutant survived longer than the wild-type strain and were less fit than the wild-type strain in competition infection assays. Transmission electron microscopy showed that the acpA mutant was delayed in escape from macrophage phagosomes, as more than 75% of acpA mutant bacteria could still be found inside phagosomes after 12 h of infection in THP-1 cells and human monocyte-derived macrophages, whereas most of the wild-type bacteria had escaped from the phagosome by 6 h postinfection. Thus, AcpA affects intracellular trafficking and the fate of Francisella within host macrophages.