Identification and molecular analysis of lbpBA, which encodes the two-component meningococcal lactoferrin receptor.

We identified lbpB, encoding the lipoprotein component of the meningococcal lactoferrin receptor. An LbpB mutant was unable to acquire Fe from lactoferrin and exhibits decreased surface binding to lactoferrin. Primer extension and reverse transcription-PCR analysis indicate that lbpB and lbpA are cotranscribed on a polycistronic Fe-repressible mRNA.

Analysis of the alcABC operon encoding alcaligin biosynthesis enzymes in Bordetella bronchiseptica.

We previously cloned a B. bronchiseptica (Bb) genomic DNA fragment that complements a Bb alcaligin biosynthesis mutant, and reported the identification of a gene, alcA, with predicted protein sequence similarity to siderophore biosynthesis enzymes from other organisms. In the present study we show that further nt sequencing of this region revealed two open reading frames (ORFs) 3' to alcA that encode putative proteins AlcB and AlcC, with significant sequence similarity to the aerobactin biosynthesis enzymes IucB and IucC, respectively. RT-PCR analysis indicated that the three ORFs are encoded on a single transcript, and that this operon is repressed at the transcriptional level by Fe. Primer extension analysis placed the transcriptional start point (tsp) 35 nt from the 5' end of the Fur consensus sequence and 188 nt from the putative start of translation of AlcA.

Structures and characteristics of novel siderophores from plant deleterious Pseudomonas fluorescens A225 and Pseudomonas putida ATCC 39167.

When Pseudomonas putida ATCC 39167 and plant-deleterious Pseudomonas fluorescens A225 were grown in an iron-deficient culture medium, they each produced two different novel yellow-green fluorescent pseudobactins: P39167-I, II and PA225-I, II. Pseudobactin P39167-I has a molecular formula of C46H65O23N13 and is monoanionic at neutral pH. P39167-II has the molecular formula of C46H63O22N13 and no charge at neutral pH. Pseudobactin PA225-I has a molecular formula of C46H65O24N13 and is monoanionic at neutral pH whereas pseudobactin PA225-II has the molecular formula of C46H63O23N13 and no charge at neutral pH. All four of the pseudobactins contain a dihydroxyquinoline-based chromophore. The amino acid sequence for the octapeptide in case of pseudobactins from P. putida ATCC 39167 is Chr-Ser(1)-Ala(1)-AcOHOrn-Gly-Ala(2)-OHAsp-Ser(2)-Thr. In case of pseudobactins from P. fluorescens A225, the octapeptide has the sequence Chr-Ser(1)-Ala-AcOHOrn-Gly-Ser(2)-OHAsp-Ser(3)-Thr. For all four pseudobactins (P39167-I, II and PA225-I, II), the serine(1) residue of the octapeptide is attached to the carboxylic acid group on the C-11 of the fluorescent quinoline via an amide bond. Additionally, for pseudobactin P39167-II and PA225-II, the hydroxyl group of the serine(1) residue is also attached to the carboxyl group of threonine residue at the carboxy terminus of the peptide via an ester bond, resulting in a cyclic depsipeptide in contrast to the linear peptide chain of P39167-I and PA225-I. For all four pseudobactins, a malamide group is attached to the C-3 of the quinoline derived chromophore. The three bidentate iron(III) chelating groups in all four pseudobactins consist of a 1,2-dihydroxy aromatic group of the fluorescent chromophore, a hydroxy acid group of beta-hydroxy aspartic acid, and a hydroxamate group from the acylated Ndelta-hydroxyornithine. The amino acid constituents of the pseudobactins P39167 I, II are the same as those in pseudobactin A214, whereas those in A225 I, II are the same as in 7SR1, but in both cases the sequences are different. The uptake results indicate a single outer membrane receptor protein for ferric-pseudobactins in both organisms. The receptor proteins in the two species are similar but not identical.

Cloning and characterization of the Actinobacillus actinomycetemcomitans gene encoding a heat-shock protein 90 homologue.

In a search for clones from a lambda gt11 expression library of Actinobacillus actinomycetemcomitans (Aa) genomic DNA that expressed epitopes from a 70-kDa iron-repressible membrane protein, we inadvertently identified clones that encoded a member of the 90-kDa heat-shock protein (HSP 90) family. The gene appears to encode a homologue of HtpG, as the nucleotide sequence has approximately 70% identity with the Escherichia coli (Ec) and Vibrio fischeri htpG. Growth of an Aa htpG insertion mutant at 42 degrees C was reduced to 50% of the parent strain, similar to an Ec htpG deletion mutant. These data suggest that Aa HtpG performs a function similar to Ec HtpG.

Identification of alcA, a Bordetella bronchiseptica gene necessary for alcaligin production.

The alcA gene, essential for the production of the dihydroxamate siderophore, alcaligin, by Bordetella bronchiseptica, was cloned and sequenced. The alcA gene was identified on a 4.7-kb EcoRI genomic fragment adjacent to a Tn5lac transposon insertion that inactivated alcaligin production in strain MBORD846. Analysis of the alcA nucleotide sequence revealed a putative Fur-binding site, suggesting that expression of this gene is repressed by iron. The deduced amino-acid sequence of this open reading frame had significant homology with the Escherichia coli iucD gene product, an enzyme required for biosynthesis of the dihydroxamate siderophore aerobactin.

Two new cytotoxic peroxide-containing acids from a New Guinea sponge, Callyspongia sp.

Two new C14 cytotoxic, branched-chain acids, 1 and 2, containing a cyclic peroxide moiety were isolated from a sponge, Callyspongia sp., collected in New Guinea. The structures were elucidated through extensive spectral analysis.

Pantherinine, a cytotoxic aromatic alkaloid, and 7-deazainosine from the ascidian Aplidium pantherinum.

A new cytotoxic aromatic alkaloid, pantherinine [1], and a mixture of nucleosides including 7-deazainosine [3], have been isolated from the ascidian Aplidium pantherinum collected at Stenhouse Bay, South Australia. The structures were determined primarily from 1H- and 13C-nmr data, especially one-bond and multiple-bond proton-carbon correlations. Although 7-deazainosine [3] has been known as a synthetic compound for several decades, this appears to be the first report of its isolation as a natural product.