The carboxysomal polypeptides of Thiobacillus neapolitanus with apparent molecular masses of 85 and 130 kDa were isolated and subjected to N-terminal sequencing. The first 17 amino acids of the two peptides were identical. The sequence perfectly matched the deduced amino acid sequence of an open reading frame in the carboxysome operon. The gene was subsequently named csoS2. Expression of the gene in Escherichia coli resulted in the production of two peptides with apparent molecular masses of 85 and 130 kDa. Immunospecific antibodies generated against the smaller peptide recognized both peptides; the peptides were named CsoS2A and CsoS2B, respectively. A digoxigenin-hydrazide glycosylation assay revealed that both CsoS2A and CsoS2B are post-translationally modified by glycosylation. CsoS2 was localized to the edges of purified carboxysomes by immunogold electron microscopy using the monospecific CsoS2A antibodies. The molecular mass of CsoS2A calculated from the nucleotide sequence was 92.3 kDa.

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.

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.

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 K CO 2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO 2 . 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.Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) is one of the two unique enzymes of the reductive pentose phosphate pathway, or Calvin-Bassham-Benson cycle. In this pathway, RubisCO functions to catalyze the actual CO 2 assimilatory step to convert RuBP and CO 2 into two molecules of 3-phosphoglyceric acid via a six-carbon carboxylated intermediate. RubisCO also may act as an internal monooxygenase, resulting in the formation of one molecule each of 3-phosphoglycerate and 2-phosphoglycolate, the latter of which is converted to glycolate by phosphoglycolate phosphatase and further metabolized via the photorespiratory pathway. These two reactions follow a sequential and ordered mechanism in which enzyme-bound RuBP is converted to an enediolate which then reacts with CO 2 or O 2 (15, 38). In most cases, RubisCO is a hexadecamer with a native molecular weight of about 550,000 and is composed of two kinds of subunits, eight large catalytic and eight small subunits with molecular weights of about 56,000 and 15,000, respectively. This type of RubisCO is called form I (or type I) and is present in nearly all eukaryotic photosynthetic organisms and virtually all bacteria that use the Calvin cycle to fix CO 2 (50). A second type, form II (or type II), which is composed of only large (M r , ϳ56,000) subunits has also been described. The number of subunits in form II enzymes varies with the organis...