Genetic dissection of the Francisella novicida restriction barrier

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10(6)-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.     

Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.     

Live Attenuated Francisella novicida Vaccine Protects against Francisella tularensis Pulmonary Challenge in Rats and Non-human Primates

Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform.     

利用大肠杆菌克隆在原核生物中有活性的油菜基因启动子

利用本室构建的基因启动子探针型载体ｐＳＵＰＶ４直接在大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ（Ｍｉｇｕｌａ）ＣａｓｔｅｌａｎｉｅｔＣｈａｌｍｅｒｓ）中分离油菜（ＢｒａｓｉｃａｎａｐｕｓＬ．）基因启动子片段,获得卡那霉素抗性重组子３３个。对重组子ｐＲＰ１０做了进一步鉴定：Ｓｏｕｔｈｅｒｎ杂交表明ＲＰ１０片段来自油菜基因组,并与基因组中的另一些序列具有同源性;ＲＰ１０片段５′端区域的缺失可使卡那霉素抗性水平从１００ｍｇ／Ｌ降至２５ｍｇ／Ｌ,说明缺失的序列可能影响基因转录效率;序列分析发现ＲＰ１０片段内存在几个真核基因启动子的保守序列;用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ（ＳｍｉｔｈｅｔＴｏｗｎｓｅｎｄ）Ｃｏｎｎ）转化法将ＲＰ１０ＧＵＳ基因转入油菜,组织化学分析显示ＲＰ１０在愈伤组织中可以启动融合的ＧＵＳ基因表达     

Construction of targeted insertion mutations in Francisella tularensis subsp. novicida

Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species.     

Temperature-sensitive lesions in the Francisella novicida valA gene cloned into an Escherichia coli msbA lpxK mutant affecting deoxycholate resistance and lipopolysaccharide assembly at the restrictive temperature.

The valAB locus of Francisella novicida has previously been found to be highly similar at the deduced amino acid level to msbA lpxK of Escherichia coli. Both ValA and MsbA are members of the superfamily of ABC transporters, and they appear to have similar functions. In this study we describe the isolation of a temperature-sensitive valAB locus. DNA sequence analysis indicates that the only changes to the ValAB deduced amino acid sequence are changes of S453 to an F and T458 to an I in ValA. E. coli strains defective in msbA and expressing temperature-sensitive ValA rapidly ceased growth when shifted from a permissive temperature to a restrictive temperature. After 1 h at the restrictive temperature, cells were much more sensitive to deoxycholate treatment. To test the hypothesis that ValA is responsible for the transport or assembly of lipopolysaccharide, we introduced gseA, a Kdo (3-deoxy-D-manno-octulosonic acid) transferase from Chlamydia trachomatis, into a strain with a temperature-sensitive valA allele and a nonfunctional msbA locus. These recombinants were defective in cell surface expression of the chlamydial genus-specific epitope within 15 min of a shift to the nonpermissive temperature. Also, there was enhanced association of the epitope with the inner membrane after a shift to the nonpermissive temperature. Thus, we propose that ValA is involved in the transport of lipopolysaccharide to the outer membrane.     

Bile Salt Activation of Stress Response Promoters in Escherichia coli

Bile salts are prevalent in the mammalian intestine, a natural habitat of Escherichia coli. The bile salts deoxycholate, chenodeoxycholate, ursodeoxycholate, and glycocholate were tested for their effect on induction of 13 specific stress response genes. The most consistently activated E. coli promoters were those for genes micF, osmY, and dinD. MicF and osmY gene products are associated with membrane functions and are responsive to oxidative stress. DinD is induced by DNA damage as part of the SOS response. These results indicate that bile acids, to which E. coli are naturally exposed, induce expression of specific stress response genes, possibly in response to membrane perturbation, oxidative stress, and DNA damage. Altered expression of stress-response genes may also promote interaction of E. coli with cells of the colonic epithelium. Received: 5 March 1999 / Accepted: 2 April 1999     

MglA and MglB are required for the intramacrophage growth of Francisella novicida

Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated that are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for both the intracellular growth defect and the colony morphology on LB (X-p) media. The locus consists of an apparent operon of two genes, designated mglAB , for macrophage growth locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 on LB (X-p) media. Sequencing of mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild-type F. novicida . The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli , SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may affect the expression of genes whose products contribute to survival and growth within macrophages.     

Production of Outer Membrane Vesicles and Outer Membrane Tubes by Francisella novicida

Francisella spp. are highly infectious and virulent bacteria that cause the zoonotic disease tularemia. Knowledge is lacking for the virulence factors expressed by Francisella and how these factors are secreted and delivered to host cells. Gram-negative bacteria constitutively release outer membrane vesicles (OMV), which may function in the delivery of virulence factors to host cells. We identified growth conditions under which Francisella novicida produces abundant OMV. Purification of the vesicles revealed the presence of tube-shaped vesicles in addition to typical spherical OMV, and examination of whole bacteria revealed the presence of tubes extending out from the bacterial surface. Recently, both prokaryotic and eukaryotic cells have been shown to produce membrane-enclosed projections, termed nanotubes, which appear to function in cell-cell communication and the exchange of molecules. In contrast to these previously characterized structures, the F. novicida tubes are produced in liquid as well as on solid medium and are derived from the OM rather than the cytoplasmic membrane. The production of the OMV and tubes (OMV/T) by F. novicida was coordinately regulated and responsive to both growth medium and growth phase. Proteomic analysis of purified OMV/T identified known Francisella virulence factors among the constituent proteins, suggesting roles for the vesicles in pathogenesis. In support of this, production of OM tubes by F. novicida was stimulated during infection of macrophages and addition of purified OMV/T to macrophages elicited increased release of proinflammatory cytokines. Finally, vaccination with purified OMV/T protected mice from subsequent challenge with highly lethal doses of F. novicida.     

Distinct Promoters Affect Pyrroloquinoline Quinone Production in Recombinant Escherichia coli and Klebsiella pneumoniae

Pyrroloquinoline quinone (PQQ) is a versatile quinone cofactor participating in numerous biological processes. Klebsiella pneumoniae can naturally synthesize PQQ for harboring intact PQQ synthesis genes. Previous metabolic engineering of K. pneumoniae failed to overproduce PQQ due to the employment of strong promoter in expression vector. Here we report that a moderate rather than strong promoter is efficient for PQQ production. To screen an appropriate promoter, a total of four distinct promoters—lac promoter, pk promoter of glycerol dehydratase gene (dhaB1), promoter of kanamycin resistance gene, and T7 promoter (as the control)—were individually used for overexpressing the endogenous PQQ genes in K. pneumoniae along with heterologous expression in Escherichia coli. We found that all recombinant K. pneumoniae strains produced more PQQ than recombinant E. coli strains that carried corresponding vectors, indicating that K. pneumoniae is superior to E. coli for the production of PQQ. Particularly, the recombinant K. pneumoniae recruiting the promoter of kanamycin resistance gene produced the highest PQQ (1,700 nmol), revealing that a moderate rather than strong promoter is efficient for PQQ production. Furthermore, PQQ production was roughly proportional to glucose concentration increasing from 0.5 to 1.5 g/L, implying the synergism between PQQ biosynthesis and glucose utilization. This study not only provides a feasible strategy for production of PQQ in K. pneumoniae, but also reveals the exquisite synchronization among PQQ biosynthesis, glucose metabolism, and cell proliferation.