Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

Friedman, Alan M.; Long, Sharon R.; Brown, Susan E.; Buikema, William J.; Ausubel, Frederick M.

doi:10.1016/0378-1119(82)90167-6

Cited by 810 publications

(272 citation statements)

References 13 publications

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“…Chromosomal DNA of F. nucleatum 12230 was purified, partially digested with Tsp509I, and cloned into the EcoRI site of cosmid pLAFR2 (19). The ligation mixture was incubated with Gigapack III XL packaging extract (Stratagene), and the cosmid phage lysate was prepared according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

Han¹,

Ikegami²,

Rajanna³

et al. 2005

J Bacteriol

201

204

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Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.

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Section: Methodsmentioning

confidence: 99%

Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

Han¹,

Ikegami²,

Rajanna³

et al. 2005

J Bacteriol

201

204

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“…In addition, the bacterial strains E. coli JM 107 (Yanisch-Perron et al, 1985) and E. coli TG1 (Sambrook et al, 1989) were used in this study. Plasmids used included the broad host-range cosmid pLAFR3 (Friedman et al, 1982) for the construction of the genomic library and high-copy-number plasmid pUC18 as a cloning vector (Yanisch-Perron et al, 1985). MI3 vectors mp18 and mp19 were used for DNA sequencing 198.5).…”

Section: Methodsmentioning

confidence: 99%

4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3

Seibold

Matthes

Eppink

et al. 1996

European Journal of Biochemistry

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Technical University Hamburg-Harburg, Hamburg, Germany 4-Hydroxybenzoate hydroxylase from Pseudomonus sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50-fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44-kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4-hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45°C. Chloride ions were inhibitory, and competitive with respect to NADH.4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-tluoro-4-hydroxybenzoate, 2-cliloro-4-hydroxybenzoate, and 2,4-dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed.The gene encoding 4-hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 11 82 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promotor was identified, which led to constitutive expression of the cloned gene in E. coli TG1 . The deduced amino acid sequence of Pseudomanus sp. CBS3 4-hydroxybenzoate hydroxylase revealed 53 % identity with that of the pobA enzyme from Pseudomonasfluorescens for which a three-dimensional structure is known. The active-site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three-dimensional fold. Based on an isolated region of sequence divergence and site-directed mutagenesis data of 4-hydroxybenzoate hydroxylase from f? ,fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.

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“…This library was constructed by the ligation of ϳ20 -25-kb fragments of genomic DNA (24), generated by partial EcoRI digestion, into the cosmid pLAFR-1 (25). E. coli 803 was employed as the host strain (26).…”

Section: Quantitative Assay For Measuring the Conversion Of [ 14 C]b mentioning

confidence: 99%

Origin of the 2-Amino-2-deoxy-gluconate Unit inRhizobium leguminosarum Lipid A

Que-Gewirth

Karbarz

Kalb

et al. 2003

Journal of Biological Chemistry

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An unusual feature of the lipid A from the plant endosymbionts Rhizobium etli and Rhizobium leguminosarum is the presence of a proximal sugar unit consisting of a 2-amino-2-deoxy-gluconate moiety in place of glucosamine. An outer membrane oxidase that generates the 2-amino-2-deoxy-gluconate unit from a glucosaminecontaining precursor is present in membranes of R. leguminosarum and R. etli but not in S. meliloti or Escherichia coli. We now report the identification of a hybrid cosmid that directs the overexpression of this activity by screening 1800 lysates of individual colonies of a R. leguminosarum 3841 genomic DNA library in the host strain R. etli CE3. Two cosmids (p1S11D and p1U12G) were identified in this manner and transferred into S. meliloti, in which they also directed the expression of oxidase activity in the absence of any chromosomal background. Subcloning and sequencing of the oxidase gene on a 6.5-kb fragment derived from the ϳ20-kb insert in p1S11D revealed that the enzyme is encoded by a gene (lpxQ) that specifies a protein of 224 amino acid residues with a putative signal sequence cleavage site at position 28. Heterologous expression of lpxQ using the T7lac promoter system in E. coli resulted in the production of catalytically active oxidase that was localized in the outer membrane. A new outer membrane protein of the size expected for LpxQ was present in this construct and was subjected to microsequencing to confirm its identity and the site of signal peptide cleavage. LpxQ expressed in E. coli generates the same products as seen in R. leguminosarum membranes. LpxQ is dependent on O 2 for activity, as demonstrated by inhibition of the reaction under strictly anaerobic conditions. An ortholog of LpxQ is present in the genome of Agrobacterium tumefaciens, as shown by heterologous expression of oxidase activity in E. coli.As demonstrated in the accompanying article (1), the outer membranes of Rhizobium leguminosarum and Rhizobium etli contain an unusual oxidase that converts the proximal glucosamine unit of 1-dephosphorylated lipid A (or related molecules) to a novel 2-aminogluconate moiety. The membranes of Sinorhizobium meliloti and Escherichia coli do not normally contain such an oxidase activity. Although the function of this unusual covalent modification of lipid A is unknown (2-4), the existence of an oxidative enzyme in the outer membrane of a Gram-negative bacterium is without precedent. The few outer membrane enzymes described to date are all phospholipases (5, 6), acyltransferases (7, 8), or proteases (9). Other characterized outer membrane proteins function either as porins or specialized transporters (10 -12). The presence of the oxidase in outer membranes of certain strains of Rhizobium suggests that lipid A oxidation (1), when it occurs, is a late event in lipopolysaccharide assembly.Given the considerable progress that has recently been made with the structural biology of outer membrane proteins by x-ray crystallography (12) and NMR spectroscopy (8, 13) and the great interest in li...

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Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

Cited by 810 publications

References 13 publications

Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3

Origin of the 2-Amino-2-deoxy-gluconate Unit inRhizobium leguminosarum Lipid A

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