Evaluation of E. coli biofilm as a protective barrier against microbiologically influenced deterioration of concrete (MICD) under mesophilic temperatures

In this study, Escherichia coli DH5α biofilm was evaluated for its potential to control and minimize microbiologically influenced concrete deterioration (MICD) under mesophilic temperatures (37 °C). Escherichia coli DH5α biofilm was first grown on Portland cement mortar disks for 8 days. Mortar disks were then exposed to two different types of sulfur oxidizing bacteria (SOB) (Thiobacillus neapolitanus and Thiobacillus thiooxidans), which use sulfur compounds as substrate and oxidize them to sulfate and sulfuric acid. The effectiveness of the biofilm against MICD was evaluated by measuring pH, sulfate, calcium concentrations in the reactors and surface analysis of the mortar samples using X-ray diffraction and visual inspection. Overall, the results indicate that the E. coli DH5α biofilm showed good protection against MICD induced by SOB at 37 °C.

Ultrastructural and Cytochemical Analyses of the Expression of the Thiobacillus Carboxysome Operon in Escherichia Coli

The cytoplasm of the bacterium Thiobacillus neapolitanus contains 117 nm diameter polyhedral inclusions, “carboxysomes” (Fig. 1) that contain ribulose-1,5- bisphosphate carboxylase/oxygenase (RuBisCO). Surrounding the polyhedron are nonmembranous proteinaceous plates devoid of lipid. The carboxysomes are composed of at least 8 major peptides, all coded within the same operon. Six (CsoSIA, CsoSIB, CsoSIC, CsoS2A, CsoS2B, and CsoS3) make up the shell, and two are the large (CbbL) and small subunits (CbbS) of RuBisCO. Using immunogold labeling on ultrathin sections, peptides CsoS2A, CsoS2B, and CsoS3 have been localized to the shell. Since the original characterization of the csoSl gene, we have also immunolocalized the CsoSl peptide to the shell.As part of our initial efforts to understand how these components are assembled into the symmetrical, functional entity, the carboxysome operon from T. neapolitanus was cloned into the pET-21a(+) plasmid, an expression vector that codes for resistance to ampicillin.

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 andcbbS were cloned and sequenced. Analysis of RNA showed thatcbbL 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 CO2supplement 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.

Sequence homologs of the carboxysomal polypeptide CsoS1 of the thiobacilli are present in cyanobacteria and enteric bacteria that form carboxysomes - polyhedral bodies

Carboxysomes containing the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been demonstrated in a variety of chemoautotrophic prokaryotes and cyanobacteria. The genes in the ccm and cso operon in Synechococcus sp. PCC7942 and Thiobacillus neapolitanus, respectively, code for several carboxysome polypeptides. The polypeptides CcmK and CsoS1 exhibit a high degree of conservation, and in turn show significant homology to the CchA and PduA polypeptides of the ethanolamine and propanediol operons of enteric bacteria. Probing Southern blots of Escherichia coli genomic DNA with csoS1A showed positive hybridization indicating the presence of a csoS1-like gene. Growing Salmonella enterica and Klebsiella oxytoca with propanediol, and E.coli with ethanolamine as the energy source under anaerobic conditions resulted in the formation of polyhedral bodies in these bacteria. The DNA - deduced amino acid sequence of three additional csoS1 genes in both Thiobacillus intermedius and Thiobacillus denitrificans was determined. The nine CsoS1 polypeptides, which includes the three previously determined for T.neapolitanus, exhibited greater than 67% sequence identity. Identity and similarity comparisons and phylogenetic analysis of known polyhedral body CsoS1-like polypeptides indicate a close structural relationship between polyhedral bodies of potentially very different function.Key words: polyhedral bodies, carboxysomes, ribulose-1,5-bisphosphate carboxylase-oxygenase, cyanobacteria, thiobacilli, enteric bacteria.