Molecular cloning of the recA gene and construction of a recA strain of Francisella novicida

Berg, J M; Mdluli, Khisimuzi; Nano, Francis E.

doi:10.1128/iai.60.2.690-693.1992

Cited by 13 publications

(5 citation statements)

References 35 publications

(36 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The identification and characterization of virulence factors of Francisella has been hampered by a lack of tools for genetic manipulation of the bacterium. Transformation, allelic replacement, and in cis partial complementation of a mutation in Francisella have been described previously (Anthony et al, 1991b;Berg et al, 1992;Mdluli et al, 1994). Recently, cloning vectors have been developed based on a cryptic plasmid isolated from an F. novicidalike strain of Francisella (Norqvist et al, 1996;Pavlov et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

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

Baron

Nano

1998

Molecular Microbiology

181

200

View full text Add to dashboard Cite

SummaryFrancisella 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 wildtype 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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Baron

Nano

1998

Molecular Microbiology

181

200

View full text Add to dashboard Cite

show abstract

“…More than 10% of more than 1,000 colonies contained Lac ϩ papillae. The frequency of lac recombination was almost the same as that obtained with plasmid pJB4, which contains the Francisella novicida recA gene (2). No Lac ϩ papillae appeared when E. coli JC14604 was electroporated with pUC19 alone.…”

mentioning

confidence: 51%

Identification of the Chlamydia trachomatis RecA-encoding gene

et al. 1995

View full text Add to dashboard Cite

DNA sequencing of the major outer membrane protein (MOMP) gene (omp1) from Chlamydia trachomatis shows that some strains have a mosaic structure suggestive of homologous recombination between two distinct omp1 genes. On the basis of this conjecture, we attempted to clone by complementation and sequence the chlamydial recA homolog from C. trachomatis serovar L 2. Chlamydial genomic DNA was partially restricted with XbaI, and fragments of 2 to 4 kb were ligated into pUC19. The recombinant plasmid was electroporated into Escherichia coli HB101 (RecA ؊), and colonies were selected in the presence of methyl methanesulfonate (MMS). A 2.1-kb fragment of C. trachomatis DNA in pUC19 conferred relative MMS resistance to E. coli HB101. When this recombinant plasmid (pX203) was electroporated into E. coli JC14604 (RecA ؊ lacZ), lac ؉ recombinants were isolated. Rabbit polyclonal antibodies produced to purified E. coli RecA were immunoreactive in an immunoblot assay with a 35-kDa antigen in RecA ؊ strains of E. coli transformed with pX203. The 2.1-kb insert was cycle sequenced by the dideoxy chain termination method. An open reading frame of 1,056 bp encoding 352 amino acids that had 44% sequence identity with E. coli RecA was identified. The finding of a recA homolog in C. trachomatis suggests that homologous recombination may occur in this organism. The cloned C. trachomatis RecA-encoding gene will be useful for the construction of a recA mutant once a gene transfer system is developed for chlamydiae.

show abstract

“…Another possibility is that the Francisella cell wall loses its integrity as a result of abnormal septum formation induced by the MinD mutation, allowing bacteriocidal agents to penetrate the outer membrane. Although this work and other recent publications [17,24] demonstrate an expanded repertoire of genetic manipulations that can be done in F. tularensis we are still limited by the inability to complement mutations in trans; only one antibiotic marker has been shown to express in F. tularensis and efficient cloning vectors are not available. In this work we attempted to compensate for the inability to complement by classical in trans analysis: the presumptive mutagenized locus was molecularly cloned, and reintroduced to the WT strain to generate a mutant with the same virulence and MinD expression phenotype.…”

Section: Identification Of the Inactivated Locusmentioning

confidence: 90%

Isolation of a Francisella tularensis mutant that is sensitive to serum and oxidative killing and is avirulent in mice: Correlation with the loss of MinD homologue expression

Anthony

Cowley

Mdluli

et al. 1994

FEMS Microbiology Letters

View full text Add to dashboard Cite

We constructed mutant strains of Francisella tularensis biotype novicida by insertional mutagenesis with a kanamycm resistance (Kin R) cassette. One mutant, KEM7, was defective for survival in macrophages in comparison with the wild-type (WT) strain and a random insertion strain, KEM21. While all three strains exhibited intracellular growth, the number of viable KEM7 present after 24-48 h of infection was approximately 10 times less than that of WT or KEM21. This observation was apparently due to a reduced number of viable KEM7 associated with the macrophages one hour after phagocytosis. KEM7 was approximately 3 times more susceptible than WT or KEM21 to killing by the products of the xanthine-xanthine oxidase reaction or by hydrogen peroxide. KEM7 was also found to be susceptible to killing by serum, whereas WT and KEM21 were resistant. Upon intravenous inoculation of C57BL/6 mice, the number of KEM7 in the livers and spleens 48 h post-infection was found to be 1000-to 10000-times less than that of either KEM21 or WT. DNA sequence analysis at the Km R insertion site suggested that the F. tularensis homologue of minD had been interrupted. Western immunoblot analysis confirmed the presence of a MinD homologue in F. tularensis WT and KEM21, and demonstrated its absence in KEM7. tularensis multiplies intracellularly in macrophages, probably within a phagosome which has not fused with lysosomes [1]. Resolution of murine tularemia was found to be dependent upon the activity of class II-restricted T cells [2,3] as well as the macrophage activating lymphokine, gamma interferon [4,5] and the macrophage product, tumour necrosis factor [6]. Recently, it has been demonstrated that the mechanism of action of gamma interferon on the inhibition of intracellu-

show abstract

Molecular cloning of the recA gene and construction of a recA strain of Francisella novicida

Cited by 13 publications

References 35 publications

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

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

Identification of the Chlamydia trachomatis RecA-encoding gene

Isolation of a Francisella tularensis mutant that is sensitive to serum and oxidative killing and is avirulent in mice: Correlation with the loss of MinD homologue expression

Contact Info

Product

Resources

About