Morio Ishizuka

Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3

The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps.

Inactivation of Ribosomal Protein Genes in Bacillus subtilis Reveals Importance of Each Ribosomal Protein for Cell Proliferation and Cell Differentiation

Among the 57 genes that encode ribosomal proteins in the genome of Bacillus subtilis, a Gram-positive bacterium, 50 genes were targeted by systematic inactivation. Individual deletion mutants of 16 ribosomal proteins (L1, L9, L15, L22, L23, L28, L29, L32, L33.1, L33.2, L34, L35, L36, S6, S20, and S21) were obtained successfully. In conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in B. subtilis, at least for cell proliferation. Although several mutants that harbored a deletion of a ribosomal protein gene did not show any significant differences in any of the phenotypes that were tested, various mutants showed a reduced growth rate and reduced levels of 70S ribosomes compared with the wild type. In addition, severe defects in the sporulation frequency of the ΔrplA (L1) mutant and the motility of the ΔrpsU (S21) mutant were observed. These data provide the first evidence in B. subtilis that L1 and S21 are required for the progression of cellular differentiation.

An Escherichia coli cyoE gene homologue in thermophilic Bacillus PS3 encodes a thermotolerant heme O synthase.

The cyoE gene of the Escherichia coli bo‐type quinol oxidase operon (cyo ABCDE) has been previously shown to encode heme O synthase. To demonstrate a catalytic role of a cyoE homologue (the caaE gene) in the gene cluster for caa 3‐type cytochrome c oxidase of thermophilic Bacillus PS3, we have carried out genetic complementation analysis using the chimeric operon cyo ABCD‐caaE and heme O synthase assay using the CaaE‐overproduced E. coli membranes. We found that the caaE gene encodes a thermotolerant heme O synthase which provides an intermediate for heme A biosynthesis.

Defect in the Formation of 70S Ribosomes Caused by Lack of Ribosomal Protein L34 Can Be Suppressed by Magnesium

To elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the ΔrpmH mutant exhibited a severe slow-growth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the ΔrpmH strain. Either the disruption of yhdP, which is thought to be involved in the efflux of Mg(2+), or overexpression of mgtE, which plays a major role in the import of Mg(2+), could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg(2+) content was lower in the ΔrpmH cells than in the wild type, and the Mg(2+) content in the ΔrpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg(2+). These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg(2+). In addition, the Mg(2+) content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the ΔrplA (L1) and ΔrplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg(2+) content is influenced by the amount of 70S ribosomes.

Cloning and characterization of flagellin genes and identification of flagellin glycosylation from thermophilic Bacillus species.

Flagellin glycosylation was identified in Bacillus sp. PS3 and Geobacillus stearothermophilus. In vivo complementation showed that these flagellin genes did not restore the motility of a Bacillus subtilis flagellin mutant, whereas the genes encoding non-glycosylated flagellin from Geobacillus kaustophilus and Bacillus sp. Kps3 restored motility. Moreover, four types of flagellins expressed in B. subtilis were not glycosylated. We speculate that glycosylation is required for flagellar filament assembly of these bacilli.

Control of aerial mycelium formation by the BldK oligopeptide ABC transporter in Streptomyces griseus

An oligopeptide permease family ATP-binding cassette (ABC) transporter encoded by SGR2418-SGR2414 was shown to be essential for aerial mycelium formation on glucose-containing media in Streptomyces griseus. In spite of only weak sequence similarity, the operon was equivalent to the bldK operon of Streptomyces coelicolor A3(2) in terms of chromosomal location and function. Transcription of the operon appeared not to be directly regulated by AdpA, a global regulator of morphological and physiological development in S. griseus, although it was affected by adpA inactivation. This study revealed that an ABC transporter was essential for aerial mycelium formation not only in S. coelicolor A3(2) but also in S. griseus, indicating that extracellular signaling by certain peptides should be conserved among different Streptomyces species.

Superinducers for induction of thermostable lipase production by Pseudomonas species NT-163 and other Pseudomonas-like bacteria

Among various additives that affected production of a new thermostable extracellular lipase from Pseudomonas sp. NT-163 , stearyl alcohol was the most effective. Addition of stearyl alcohol (0.5%) brought about ca. 500 fold enhancement of the lipase activity (200 U/ml by p-nitorophenyllaurate method) compared to the case with no additive (≤ 0.4 U/ml), while olive oil attained only 12–15 U/ml. Palmityl and oleyl alcohols also were highly effective as lipase inducers (150–160 U/ml could be attained). Furthermore, stearyl alcohol induced lipase formation in several other Bacteriol strains 10-times more than olive oil.

The cytochrome C oxidase genes in blue-green algae and characteristics of the deduced protein sequence for subunit II of the thermophilic cyanobacterium Synechococcus vulcanus

Blue-green algae (cyanobacteria) contain both primitive photosynthetic and respiratory systems in their membranes. The controversial genes coding for an alpha alpha 3-type cytochrome oxidase in cyanobacteria were examined. The DNA probe coding for the most conserved part of subunit I hybridized with DNA fragments from four cyanobacterial species. We have cloned the genes coding for subunits I and II from the genomic library of the thermophilic cyanobacterium Synechococcus vulcanus and determined the nucleotide sequence of the subunit II gene. The deduced protein sequence (327 amino acid residues) indicates that there are two hydrophobic segments near the N-terminus and a hydrophilic intermembrane domain containing ligands for CuA (the ESR-active Copper) similar to other subunit IIs. The S. vulcanus subunit II does not contain the cytochrome c moiety that is present in bacilli and thermophiles.

Flagellar Glycosylation: Current Advances

In this chapter, we present the current advances in flagellar glycosylation. Glycosylation is well-known as one of the most frequent posttranslational protein modification. Glycosylation is well studied in eukaryotes as the superficial and secretory proteins are mostly glycosylated in the eukaryotic cell. Protein glycosylation was considered to be a eukaryotic organism specific modification for many years. However, reports of bacterial glycosylation have increased since the discovery of surface layer glycosylation on the cell envelope in archaea and hyperthermophiles in the mid-1970’s (Mescher & Strominger, 1976; Sleytr, 1975; Sleytr & Thorne, 1976).

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic Bacterium Geobacillus stearothermophilus

A group I intron that can be spliced in vivo and in vitro was identified in the flagellin gene of the thermophilic bacterium Geobacillus stearothermophilus. We also found one or two intervening sequences (IVS) of flagellin genes in five additional bacterial species. Furthermore, we report the presence of these sequences in two sites of a highly conserved region in the flagellin gene.