Binding properties of a monoclonal antibody directed toward lead-chelate complexes.

A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution.

Deduced amino acid sequence, functional expression, and unique enzymatic properties of the form I and form II ribulose bisphosphate carboxylase/oxygenase from the chemoautotrophic bacterium Thiobacillus denitrificans.

The cbbL cbbS and cbbM genes of Thiobacillus denitrificans, encoding form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), respectively, were found to complement a RubisCO-negative mutant of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. denitrificans promoters were shown to function in R. sphaeroides, resulting in high levels of cbbL cbbS and cbbM expression in the R. sphaeroides host. This expression system provided high levels of both T. denitrificans enzymes, each of which was highly purified. The deduced amino acid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes from sulfur-oxidizing bacteria. The form I T. denitrificans enzyme possessed a very low substrate specificity factor and did not exhibit fallover, and yet this enzyme showed a poor ability to recover from incubation with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II RubisCO enzymes. The substrate specificity factor was characteristically low, and the lack of fallover and the inhibition by ribulose 1,5-bisphosphate were similar to those of form II RubisCO obtained from nonsulfur purple bacteria. Both form I and form II RubisCO from T. denitrificans possessed high KCO2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO2. These studies present the initial description of the kinetic properties of form I and form II RubisCO from a chemoautotrophic bacterium that synthesizes both types of enzyme.

Isolation and characterization of the replicon of a Thiobacillus intermedius plasmid.

The replicon of a cryptic Thiobacillus intermedius plasmid (pTiK12) has been isolated and sequenced. Functional analysis of deletion subclones in Escherichia coli localized the replicon to a 3.5-kb region of DNA. Sequencing of this region identified a 30-bp A-T-rich potential stem-loop structure. In addition, an 11-bp direct repeat, an 11-bp inverted repeat, and a 16-bp inverted repeat were observed at the stem-loop structure. Also found in the replicon was a series of four tandem direct repeats consisting of a perfectly conserved 8-bp core. A region near the stem-loop structure is involved in the regulation of plasmid copy number. Deletion subclones lacking this region have increased copy numbers, indicating a negative regulatory role. An open reading frame capable of encoding a 320-amino-acid protein was found near the stem-loop structure. The putative amino acid sequence shares significant similarity with the two Rep proteins from the ColE2 and ColE3 replicons. Replication of the T. intermedius replicon is dependent upon DNA polymerase I. The isolation and examination of the T. intermedius plasmid replicon are initial steps toward the establishment of a genetic system in T. intermedius.

Isolation and characterization of a carboxysome shell gene from Thiobacillus neapolitanus.

The gene coding for the major carboxysome shell peptide (csoS1) from Thiobacillus neapolitanus has been isolated and sequenced. Oligonucleotide primers for polymerase chain reaction (PCR) amplification of the 5' end of the gene were made possible by amino acid sequencing of the N-terminal residues of the shell peptide. A 41 bp PCR product was used as a probe to isolate the gene. The deduced amino acid composition of the 216 bp gene shows a high degree of hydrophobicity. The gene is located within a series of three repeated regions of DNA and appears to have arisen via gene duplication. The transcript of csoS1 is approximately 400 bases in length. The shell peptide shares significant homology with Synechococcus open reading frames implicated in carboxysome structure/assembly. These open reading frames and csoS1 are related and are probably members of a carboxysome gene family.

Two forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Thiobacillus denitrificans.

The autotrophic, sulfur-oxidizing bacterium Thiobacillus denitrificans possesses two forms of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The form I and form II genes were isolated from a cosmid library using heterologous DNA probes. Restriction enzyme analysis indicated that the genes are within 17 kbp of each other. Other Calvin cycle enzyme genes are not present. Analysis of T. denitrificans RNA indicated that the form I genes for the large and small subunits are co-transcribed with a length of 2800 nucleotides. The transcript for the form II gene is 1900 nucleotides in length.