Effects of inserting eight amino acid residues into the major lipoprotein on its assembly in the outer membrane of Escherichia coli.

A DNA sequence consisting of 24 base pairs was inserted into the structural gene (lpp) coding for the major lipoprotein of the Escherichia coli outer membrane which was carried on a high-copy-number plasmid in which expression was regulated through a lac promoter-operator region. This modification resulted in the insertion of eight amino acid residues, Glu-Glu-Phe-Leu-Glu-Glu-Phe-Leu, between the glutamine residue at position 9 and the leucine residue at position 10 of the wild-type lipoprotein sequence. When production of the mutant lipoprotein was induced by a lac inducer, the cells became swollen, showed unusual morphology, and eventually lysed. When the membrane fraction was analyzed after the induction, the mutant lipoprotein was found to have been normally secreted across the cytoplasmic membrane and assembled in the outer membrane. This lipoprotein was modified with glycerol and palmitic acid and even formed the bound form, which was linked covalently to peptidoglycan. The major difference between the membrane-associated mutant lipoprotein and the wild-type lipoprotein was that the mutant lipoprotein became sensitive to trypsin treatment. These results indicate that the substantial alteration in mutant lipoprotein structure near the amino-terminal end does not interfere with modification of the amino-terminal cysteine residue or cleavage of the signal peptide by the prolipoprotein-specific signal peptidase. However, this mutant lipoprotein assembled in the outer membrane appears to have deleterious effects with respect to envelope structure and cellular morphology and viability.     

Novel mutation that causes a structural change in a lipoprotein in the outer membrane of Escherichia coli.

A novel mutation which caused a structural change in a lipoprotein in the outer-membrane has been found in Escherichia coli K-12. The lipoprotein of the wild-type strain is known to have a peculiar amino terminal structure: glycerylcysteine with two fatty acids attached by ester linkages and one fatty acid by an amide linkage. In contrast to the wild-type lipoprotein, the mutant lipoproteins is isolated from the E. coli envelope as a dimer of molecular weight of about 15,000. The dimer can be reduced by mercaptoethanol to the lipoprotein monomer of molecular weight of about 7,500. The monomer has a free thiol group which is susceptible to monoiodacetie mutant lipoprotein is extremely low in comparison with that into the wild-type lipoprotein. These results suggest that the mutant is defective in transferring a glycerol group to the thiol group of the amino terminal cysteine residue of the lipoprotein. The gene responsible for this modification reaction has been located at 36.5 min on the E. coli chromosome.     

Preferential selection of deletion mutations of the outer membrane lipoprotein gene of Escherichia coli by globomycin.

Globomycin is an antibiotic which inhibits the processing of the prolipoprotein. Eighty globomycin-resistant mutants were independently isolated from Escherichia coli K-12 which had a deletion mutation in chromosomal lipoprotein gene (lpp), but contained a plasmid carrying the wild-type lpp gene. Twenty-six of the mutants did not have the lipoprotein in the membrane fractions. From the analysis of the plasmids of these mutants, all of the lipoprotein-deficient mutations were found to be due to deletion mutations around the lpp gene.     

A new form of structural lipoprotein of outer membrane of Escherichia coli.

Among the membrane proteins synthesized in toluene-treated cells of Escherichia coli were two distinct membrane proteins of different molecular weights, which were cross-reactive with antiserum against a structural lipoprotein of the outer membrane. One was thought to be the known membrane lipoprotein since it migrated to the same position as that of the lipoprotein (Mr = 7,200) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the other protein migrated slower than the lipoprotein. No protein corresponding to the slower-migrating species was detected in the membrane proteins synthesized in vivo. The apparent molecular weight of the protein at the new peak was estimated to be between 10,000 and 15,000. Both the new protein and the lipoprotein were found to be synthesized from stable mRNA(s) in the toluene-treated cells. The synthesis of the new protein as well as the lipoprotein was sensitive to chloramphenicol, indicating that both proteins were synthesized on ribosomes. Peptides mapping of the new protein revealed the same COOH-terminal sequence as in the lipoprotein. This indicates that the new protein has an extra sequence at the NH2-terminal end. This hypothesis is supported by the finding that the NH2 terminus of the new lipoprotein is methionine, while that of the lipoprotein is a substituted cysteine. From double label experiments with each of 17 different amino acids and arginine, the amino acid composition of the extra region was deduced. The new protein was found to contain at least 18 to 19 extra amino acid residues over the lipoprotein, if it is assumed that the new protein has no extra arginine residues. It was found that 4 out of the 5 amino acids which were deficient in the lipoprotein (phenylalanine, tryptophan, proline, and histidine) were also deficient in the new protein, but the fifth one, glycine, was present in the new protein. From these results, it seems possible that this new form of the lipoprotine is a precursor of the lipoprotein (prolipoprotein) in the process of biosynthesis and assembly of the lipoprotein in the outer membrane.     

Restriction enzyme cleavage sites surrounding the structural gene for the lipoprotein of the Escherichia coli outer membrane.

The purified messenger ribonucleic acid (mRNA) for the lipoprotein of the Escherichia coli outer membrane was hybridized with fragments obtained by digestion of E. coli chromosomal deoxyribonucleic acid (DNA) with eight different restriction enzymes. For each restriction enzyme digestion, one specific fragment separated by agarose gel electrophoresis was found to hybridize with the lipoprotein mRNA. From the analysis of restriction fragments generated by double digestions with various combinations of restriction enzymes, cleavage sites for the restriction enzymes near the locus of the lipoprotein structural gene (lpp) were mapped. No restriction fragments of DNA from the E. coli lpp-2 mutant hybridized with the lipoprotein mRNA, confirming that the mutant has a deletion mutation in the vicinity of the lpp gene.     

Neither lipid modification nor processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in Escherichia coli.

The relationship between the modification and processing of prolipoprotein and the formation of murein-bound lipoprotein has been investigated using Escherichia coli mutants altered in the signal sequence of prolipoprotein and an E. coli strain producing OmpF-Lpp hybrid protein. The glyceride-modified prolipoprotein in mutant lppT20 and in globomycin-treated wild-type strain were covalently attached to the peptidoglycan. Likewise, the unmodified prolipoproteins in mutants lppL20, lppV20, and lppG21 were attached to the peptidoglycan. The OmpF-Lpp hybrid protein that is processed but not modified with lipid due to the absence of the cysteine-containing modification site in the hybrid protein was also covalently linked to the peptidoglycan. These results indicate that neither lipid modification nor the processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in E. coli. In contrast, introduction of a charged amino acid residue such as Asp or Arg at the 14th position of prolipoprotein affected not only the lipid modification and processing of the mutant prolipoprotein but also the formation of murein-bound lipoprotein. Replacement of the Gly14 with Glu or Lys partially affected the lipid modification and processing of prolipoprotein; the peptidoglycan of the lppE14 and lppK14 mutants contained a reduced amount of mature lipoprotein but no mutant prolipoprotein. In addition, lpp mutants A20I23I24 and A20I23K24 were found to be defective in both lipid modification/processing of prolipoprotein and the formation of murein-bound lipoprotein. The defective formation of murein-bound lipoprotein in the latter mutants may be related to an alteration in the secondary structure at the modification/processing site of the mutant prolipoproteins.     

Inhibition of secretion of a mutant lipoprotein across the cytoplasmic membrane by the wild-type lipoprotein of the Escherichia coli outer membrane.

A globomycin-resistant mutant of Escherichia coli was found to produce a precursor of the major outer membrane lipoprotein (prolipoprotein), in which the glycine residue at position 14 within the signal peptide was replaced by an aspartic acid residue. The same mutation has been reported by Lin et al. (Proc. Natl. Acad. Sci. U.S.A. 175:4891-4895, 1978). The structural gene of the mutant prolipoprotein was inserted into an inducible expression cloning vehicle. When the mutant prolipoprotein was produced in lipoprotein-minus host cells, 82% of the unprocessed protein was found in the membrane fraction, with the remaining 18% localized in the soluble fraction. However, when the production of the mutant prolipoprotein was induced in the wild-type lpp+ host cells, only 31% of the mutant prolipoprotein was found in the membrane fraction, leaving the remaining 69% in the soluble, cytoplasmic fraction. In addition, the assembly of the wild-type lipoprotein in these cells was not affected, whether the mutant prolipoprotein was produced or not. These results suggest that secretions of both mutant and wild-type prolipoproteins utilize the same component(s) responsible for the initial stages of secretion across the cytoplasmic membrane. However, it appears that the wild-type lipoprotein has a higher affinity for these components than does the mutant lipoprotein.     

Alterations of the carboxyl-terminal amino acid residues of Escherichia coli lipoprotein affect the formation of murein-bound lipoprotein.

Mutations in the Escherichia coli lpp gene resulting in the alterations of the COOH-terminal region of the lipoprotein have been isolated by oligonucleotide-directed mutagenesis. As might be expected, substitution of Lys78 with Arg78 completely abolished the formation of murein-bound lipoprotein. Each of the following single amino acid substitutions did not significantly affect the formation of bound-form lipoprotein: Asp70 to Glu70 or Gly70; Lys75 to Thr75; and Tyr76 to His76, Ile76, or Leu76. In contrast, mutational alterations of Tyr76 to Cys76, Gly76, Asn76, Pro76, or Ser76 resulted in a reduction of the bound-form lipoprotein to levels of 14-32% of that in the wild-type strain. A common feature of these lpp COOH-terminal mutations affecting the formation of bound-form lipoprotein is the presence of a beta-turn secondary structure at the COOH-terminal region of all these mutant lipoproteins. In addition, substitution of Tyr76 to Asp76 or Glu76, and Arg77 to Asp77 or Leu77 also resulted in a reduced formation of the bound-form lipoprotein. These results suggest that the formation of murein-bound lipoprotein requires a COOH-terminal Lys residue and a positively charged COOH-terminal region. Furthermore, a beta-turn secondary structure in the COOH-terminal random coil region interferes with the attachment of the lipoprotein to the peptidoglycan.     

Mutation and Mutagenesis of thiol peroxidase of Escherichia coli and a new type of thiol peroxidase family.

A novel thioredoxin-linked thiol peroxidase (Px) from Escherichia coli has been reported previously (M. K. Cha, H. K. Kim, and I. H. Kim, J. Biol. Chem. 270:28635-28641, 1995). In an attempt to perform physiological and biochemical characterizations of the thiol Px, a thiol Px null (tpx) mutant and a functional-residue mutant of thiol Px were produced. The tpx mutant was viable in aerobic culture but grew more slowly than the wild-type cells. The difference in growth rate became more pronounced when oxidative-stress-inducing reagents, such as peroxides and paraquat, were added to the cultures. The viability of the individual tpx mutant under oxidative stress was much lower than that of wild-type cells. tpx mutants growing aerobically respond to paraquat with a sixfold greater induction of Mn-superoxide dismutase than that of the wild-type cells. The deduced amino acid sequence of the thiol Px was found to be from 42 to 72% identical to the sequences of proteins from Haemophilus influenzae (ToxR regulon), Vibrio cholerae (ToxR regulon), and three kinds of streptococci (coaggregation-mediating adhesins), suggesting that they all belong to a new thiol Px family. Alignment of the amino acid sequences of the thiol Px family members showed that one cysteine, which corresponds to Cys-94 in E. coli thiol Px, is perfectly conserved. The substitution of serine for this cysteine residue resulted in complete loss of Px activity. These results suggest that the members of the thiol Px family, including E. coli thiol Px, have a functional cysteine residue and function in vivo as peroxidases.     

Deletion of internal twenty-one amino acid residues of Escherichia coli prolipoprotein does not affect the formation of the murein-bound lipoprotein.

Mutation pgsA affecting the phosphatidylglycerol phosphate synthesis is lethal for all but certain E. coli strains such as strains deleted for the lpp gene or strains containing unmodifiable prolipoprotein like lppD14. Strain SD312 pgsA3 is tolerant to pgsA mutation, which suggests the lpp alleles in strain SD312 pgsA3 and its parental strain SD12 may be defective. DNA sequence analysis of the lpp genes in Escherichia coli strains SD12 and SD312 pgsA using asymmetric polymerase chain reaction showed that the lpp alleles in these two strains contained a 63 base pair deletion corresponding to the 37th to 57th codons of the wild-type lpp gene. [3H]Palmitate labeling of strains SD12 and SDS312 showed that the mutant lipoprotein in SD12 strain was modified with lipid, while the prolipoprotein in SD312 was not modified. The shortened mature lipoprotein in SD12 and the lipid-modified prolipoprotein in globomycin-treated SD12 were found to be covalently attached to the peptidoglycan, while the unmodified prolipoprotein in SD312 did not form significant amounts of murein-bound lipoprotein.     

Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli

A DNA sequence of 532 base pairs encompassing the entire Morganella morganii lipoprotein gene (lpp) was determined. Sequence comparisons of the M. morganii lpp gene with the lpp genes from Escherichia coli, Serratia marcescens, and Erwinia amylovora reveal that the M. morganii lpp gene is more distantly related to the E. coli lpp gene than any of the other lpp genes examined. Between the E. coli and M. morganii lpp genes, the following homologies were found: 44% in the promoter region (bases, -45 to -1), 88% in the 5'-end untranslated region of the mRNA, 58% in the signal sequence coding region, 75% in the coding region for the first 51 and 43% for the last 7 amino acid residues. Upstream of the promoter region and downstream of the termination codon, there are extensive insertions, deletions, and base substitutions. In spite of the differences in the DNA sequences, the lipoprotein structure was found to be highly conserved except for the carboxyl-terminal sequence of 7 amino residues. The coding region of the M. morganii lpp gene including the signal sequence was inserted into an expression cloning vector so that the production of the M. morganii lipoprotein could be induced in E. coli by a lac inducer, isopropyl-beta-D-thioglactoside. It was found that when induced, the M. morganii prolipoprotein was apparently secreted normally across the E. coli cytoplasmic membrane, modified with glycerol and palmitic acid, processed to the mature lipoprotein, and assembled in the E. coli outer membrane. The bound form covalently linked to the peptidoglycan was also found.     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Primary sequence analysis of ATP2 encoding the F1-ATPase beta-subunit precursor

The yeast nuclear gene ATP2 encodes a F1-ATPase beta-subunit protein of 509 amino acids with a predicted mass of 54,575 daltons. In contrast to the ATPase beta-subunit proteins determined previously from Escherichia coli and various plant sources, the yeast mitochondrial precursor peptide contains a unique cysteine residue within its immediate amino terminus. Expression of an in-frame deletion in ATP2 between residues 28 and 34 to eliminate this single cysteine residue located near the processing site of the matrix protease does not prevent the in vivo delivery of the subunit to mitochondria or its assembly into a functional ATPase complex. Thus, the import F1 beta-subunit into mitochondria does not require a covalent modification of the type utilized for the secretion of the major lipoprotein from E. coli. In addition, analysis of the level of the major F1-ATPase subunits in mitochondria prepared from an atp2- disruption mutant demonstrates that the in vivo import of these catalytic subunits is not dependent on each other. These data and additional studies, therefore, suggest that the determinants for mitochondrial delivery reside within the amino terminus of the individual precursors.     

lpp deletion as a permeabilization method

Our earlier studies with outer membrane permeability in E. coli showed that an insertion mutation in lpp gene (encoding Braun's lipoprotein) drastically changed the outer membrane permeability, resulting in significant acceleration of whole-cell catalyzed reactions. In order to gain a mechanistic understanding of the nature of permeability change, the lpp region was sequenced. The results revealed that Lpp was not expressed in the insertion mutant, suggesting that the absence, rather than the alteration, of Lpp is responsible for the observed permeability change. This surprising result prompts us to investigate the possibility of establishing lpp deletion as a general permeabilization method. Two lpp deletion mutants were generated from strains with different genetic background and the effect of lpp deletion on cell physiology was investigated. While lpp deletion had no significant effect on cell growth, carbon metabolism, and fatty acid compositions, it enhanced permeability of various small molecules, consistent with the results with the insertion mutant. This phenotype is useful in a wide range of biotechnological applications. We illustrate here the use of the mutant with organophosphate hydrolysis and L-carnitine synthesis, where permeability is known to be a limiting factor. Both processes were significantly improved with the mutant because of enhanced permeability through the outer membrane. Therefore, this study has established an easy yet generally applicable method for permeabilizing E. coli cells without significant adverse effects. Further, as lpp homolog is known to exist in gram-negative bacteria, we expect that this method will be applicable to other gram-negative bacteria.     

Site-directed mutants of a soluble form of penicillin-binding protein 5 from Escherichia coli and their catalytic properties

Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated. Alkylation of cysteine 115 with sulfhydryl reagents has previously been shown to inhibit severely the D-alanine carboxypeptidase activity of PBP 5. Alkylation also inhibits the hydrolysis of bound penicillin G, with only a slight effect on its binding. Cysteine 115 in sPBP 5 was changed to either a serine (sPBP 5C-S) or an alanine (sPBP 5C-A) residue. The wild-type and mutant sPBPs were purified in milligram amounts from induced cultures by ampicillin affinity chromatography. The mutant PBPs showed only a 2-fold increase in the half-life of the penicilloyl-PBP complex, and had a binding affinity for penicillin G identical to wild-type PBP 5. The Km for the release of D-alanine from the peptide L-Ala-D-gamma-Glu-L-Lys-D-Ala-D-Ala was 5.0, 3.5, and 7.8 mM for PBP 5, PBP 5C-S, and PBP 5C-A, respectively, while the values for Vmax were 2.5, 3.3, and 5.1 mumol/min/mg. From these data it was concluded that the cysteine residue does not directly participate in the enzymatic mechanism.     

Prolipoprotein signal peptidase of Escherichia coli requires a cysteine residue at the cleavage site

A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected. These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.     

New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation.

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.     

Staphylococcal lactose phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis on the lacE gene gives evidence that a cysteine residue is responsible for phosphorylation

The lactose-specific phosphocarrier protein enzyme II of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacE gene in order to replace the histidine residues 245, 274 and 510 and the cysteine residue 476 of the amino acid sequence with a serine residue. The wild-type and mutant genes were expressed in Escherichia coli and the gene products were characterized in different in vitro test systems. In vitro phosphorylation studies on mutant derivatives of the lactose-specific enzyme II led to the conclusion that cysteine residue 476 is the active-site for phosphorylation of this enzyme II by a phospho-enzyme III of the same sugar specificity. A cysteine residue phosphorylated intermediate was first postulated for the mannitol-specific enzyme II of E. coli and studies performed independently concerning the lactose-specific enzyme II of Lactobacillus casei are in agreement with the above results.     

Site-directed lipid modification of IgG-binding protein by intracellular bacterial lipoprotein process.

IgG-binding protein was genetically expressed and lipid-modified in a site-directed manner in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E. coli (lpp) was fused to the sequence of B-domain which was one of the IgG binding domains of Staphylococcal Protein A (SpA). The N-terminal cysteine residue of the resulting protein was enzymatically linked with lipids in the bacterial membrane. The lipid-modified protein was translocated at the bacterial membrane in a manner similar to native bacterial lipoprotein, and it was purified with IgG-Sepharose by affinity chromatography. The lipid modified proteins (lppB1 and lppB5) showed a similar IgG binding activity to unmodified proteins, which was estimated by competitive ELISA. Proteoliposomes of lipid modified proteins were prepared in an elegant fashion so that the IgG binding site should be properly oriented on the surface of an individual liposome by anchoring the lipid-tail into the hydrophobic layer of the liposome membrane. As compared with the unmodified one, the lipid modified protein incorporated into the proteoliposome exhibited higher IgG binding activity.     

Studies on the modification and processing of prolipoprotein in Escherichia coli. Effects of structural alterations in prolipoprotein on its maturation in wild type and lpp mutants

We have previously shown that an Escherichia coli mutant ( mlpA allele) containing a structurally altered murein prolipoprotein due to substitution of Gly14 by Asp14 , is globomycin resistant. In addition, the mutant prolipoprotein is not modified with glyceride and consequently remains uncleaved. Spontaneous revertants possessing a mature lipoprotein of apparent normal structure can be isolated by EDTA selection. Three revertants were chosen in the present study which included the analysis of kinetics of lipoprotein maturation and the determination of globomycin sensitivity. These pseudorevertants in the lpp gene which could be recognized by the anomalous prolipoprotein mobility in sodium dodecyl sulfate gels, exhibited altered globomycin sensitivity in vivo. Our results indicate that alterations in prolipoprotein structure affect the kinetics of prolipoprotein modification and processing reactions, both in vivo and in vitro. Pulse-chase experiments revealed the transient existence of unmodified prolipoprotein and modified prolipoprotein as biosynthetic intermediates of mature lipoprotein. The rate of prolipoprotein modification appeared to be slightly faster than that of processing in the wild type cell. In contrast, modification of prolipoprotein was rate limiting in a pseudorevertant strain 14R21 , and the processing of 14R21 modified prolipoprotein appeared to proceed more rapidly than that of wild type prolipoprotein, both in vitro and in vivo.     

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K_m value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K_m values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V_max of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.