Insertion mutation of the form I cbbL gene encoding ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in Thiobacillus neapolitanus results in expression of form II RuBisCO, loss of carboxysomes, and an increased CO2 requirement for growth

Abstract

It has been previously established that Thiobacillus neapolitanus fixes CO2 by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme, cbbL and cbbS, are part of a putative carboxysome operon. In the present study, cbbL and cbbS were cloned and sequenced. Analysis of RNA showed that cbbL and cbbS are cotranscribed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into cbbL resulted in a premature termination of transcription; a polar mutant was generated. The mutant is able to fix CO2, but requires a CO2 supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO2 by using a form II RuBisCO. This was confirmed by immunoblot analysis using antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant.

FIG. 1

Restriction map of T. neapolitanus putative carboxysome operon (30). The enlargement depicts the inactivation of the cbbL gene. The kanamycin resistance (Km^r) cartridge was inserted into the EcoRV site in the coding region of cbbL. The mutation was introduced into the genome by homologous recombination. E, EcoRI; H, HindIII; P, PstI; R, EcoRV; S, SalI. Arrows indicate direction of transcription.

FIG. 2

Confirmation of correct replacement of the wild-type cbbL gene with the insertionally inactivated gene by hybridization with the cbbL gene (A) and the kanamycin resistance gene (B). (A) Lane 1, T. neapolitanus chromosomal DNA digested with EcoRI; lane 2, T. neapolitanus cbbL::Km chromosomal DNA digested with EcoRI. (B) Lane 1, T. neapolitanus cbbL::Km chromosomal DNA digested with PstI; lane 2, T. neapolitanus cbbL::Km chromosomal DNA digested with EcoRI. Sizes of the restriction fragments are indicated on the sides.

FIG. 3

Northern blot analysis of cbbL and cbbS. RNA (20 μg) was resolved on a denaturing 1.5% agarose gel and transferred to a nylon membrane. Lane 1, wild-type RNA probed with the cbbL gene; lane 2, T. neapolitanus cbbL::Km RNA probed with the cbbL gene; lane 3, wild-type RNA probed with the cbbS gene; lane 4, T. neapolitanus cbbL::Km RNA probed with the cbbS gene. Sizes (in kilobases) of RNA markers (Life Technologies, Grand Island, N.Y.) are indicated to the left.

FIG. 4

Growth curves of wild-type (WT) T. neapolitanus and T. neapolitanus cbbL::Km grown in air supplemented with 5% CO₂ (A) and in air (B). Growth was monitored at A₆₀₀.

FIG. 5

RuBisCO activity profiles of sucrose gradients from wild-type (WT) T. neapolitanus grown in air and wild-type and cbbL::Km T. neapolitanus grown in air supplemented with 5% CO₂. The left peak represents the form II enzyme; the right peaks represent the form I enzyme.

FIG. 6

Immunoblot analysis of RuBisCO form I- and form II-enriched fractions from sucrose gradients. (A) Immunoblot of proteins transferred from an SDS–10% polyacrylamide gel and probed with form I antibodies. Lane 1, purified Synechococcus sp. strain 6301 RuBisCO; lane 2, purified R. sphaeroides form I RuBisCO; lane 3, purified R. sphaeroides form II RuBisCO; lane 4, form I peak fraction (fraction 26 in Fig. 5) from wild-type T. neapolitanus grown in air; lane 5, form I peak fraction (fraction 26) from wild-type T. neapolitanus grown in air supplemented with 5% CO₂; lane 6, corresponding fraction (fraction 26) from T. neapolitanus cbbL::Km. (B) Immunoblot of proteins from an SDS–10% polyacrylamide gel and probed with form II antibodies. Lane 1, purified Synechococcus sp. strain 6301 RuBisCO; lane 2, purified R. sphaeroides form I RuBisCO; lane 3, purified R. sphaeroides form II RuBisCO; lane 4, corresponding fraction (fraction 21 in Fig. 5) from wild-type T. neapolitanus grown in air; lane 5, corresponding fraction (fraction 21) from wild-type T. neapolitanus grown in air supplemented with 5% CO₂; lane 6, form II peak fraction (fraction 21) from T. neapolitanus cbbL::Km. Sizes (in kilodaltons) of Kaleidoscope prestained standards (Bio-Rad) are indicated to the left.

FIG. 7

Electron micrographs of wild-type and cbbL::Km T. neapolitanus. (A) Wild-type T. neapolitanus grown in batch culture in air. (B) Wild-type T. neapolitanus grown in batch culture supplemented with 5% CO₂. (C) T. neapolitanus cbbL::Km grown in batch culture supplemented with 5% CO₂. Arrowheads mark empty polyhedral inclusions. (D) Enlargement of the empty inclusions (arrowheads) observed in T. neapolitanus cbbL::Km. The scale bar represents 100 nm.

FIG. 8

Detection of downstream gene products of the T. neapolitanus carboxysome operon by Northern blotting with the carboxysome gene csoS1A as a probe. Lane 1, wild-type T. neapolitanus RNA (20 μg); lane 2, T. neapolitanus cbbL::Km RNA (20 μg). Sizes (in kilobases) of RNA markers are indicated to the left.