Human serum albumin: from bench to bedside.

One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major “cell factories” for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major “Sec” pathway for protein secretion. In contrast, the twin-arginine translocation “Tat” pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as “special-purpose” pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.

https://mmbr.asm.org/content/mmbr/64/3/515.full.pdf

Signal Peptide-Dependent Protein Transport in Bacillus subtilis : a Genome-Based Survey of the Secretome

We have used a modified CaMV 35S promoter to direct the expression of chimaeric genes encoding human serum albumin (HSA) in transgenic potato and tobacco plants. To secrete the protein, either the human prepro-sequence or the signal sequence from the extracellular tobacco protein PR-S was used. We demonstrate secretion of HSA with both types of signal sequences in transgenic leaf tissue and in suspension cultures. HSA produced in transgenic potato plants was purified to chromatographic homogeneity. N-terminal amino acid sequence analysis revealed that the processing of the precursor protein was dependent on the type of signal sequence. Expression of the human preproHSA gene lead to partial processing of the precursor and secretion of proHSA. Fusion of HSA to the plant PR-S presequence resulted in cleavage of the presequence at its natural site and secretion of correctly processed HSA that is indistinguishable from the authentic human protein.

Production of correctly processed human serum albumin in transgenic plants.

https://mmbr.asm.org/content/mmbr/57/1/109.full.pdf

Protein secretion in Bacillus species.

Comparative genomics is the cornerstone of identification of gene functions. The immense number of living organisms precludes experimental identification of functions except in a handful of model organisms. The bacterial domain is split into large branches, among which the Firmicutes occupy a considerable space. Bacillus subtilis has been the model of Firmicutes for decades and its genome has been a reference for more than 10 years. Sequencing the genome involved more than 30 laboratories, with different expertises, in a attempt to make the most of the experimental information that could be associated with the sequence. This had the expected drawback that the sequencing expertise was quite varied among the groups involved, especially at a time when sequencing genomes was extremely hard work. The recent development of very efficient, fast and accurate sequencing techniques, in parallel with the development of high-level annotation platforms, motivated the present resequencing work. The updated sequence has been reannotated in agreement with the UniProt protein knowledge base, keeping in perspective the split between the paleome (genes necessary for sustaining and perpetuating life) and the cenome (genes required for occupation of a niche, suggesting here that B. subtilis is an epiphyte). This should permit investigators to make reliable inferences to prepare validation experiments in a variety of domains of bacterial growth and development as well as build up accurate phylogenies.

/pdf/from-a-consortium-sequence-to-a-unified-sequence-the-58bg9m2mmq.pdf

From a consortium sequence to a unified sequence: The Bacillus subtilis 168 reference genome a decade later

We have fused the structural gene (hsa) for human serum albumin (HSA) to the expression elements and signal sequence coding region of each of two genes from Bacillus amyloliquefaciens P, an alpha-amylase gene (amyBamP) and a neutral protease gene (nprBamP). Bacillus subtilis strains harboring either of these gene fusions synthesized a protein with the antigenic characteristics and size (68 kilodaltons) of HSA. Results from pulse-labeling studies indicated that the bacterially produced HSA was secreted from cells which had been converted to protoplasts. Results from similar studies with intact cells suggested that the signal sequence was removed from the hybrid protein, providing further evidence that B. subtilis can translocate this foreign protein across the cell membrane. Signal sequence removal was efficient when the level of HSA synthesis was low. However, in strains which synthesized HSA at a high level, signal sequence removal was less efficient.

Secretion of human serum albumin from Bacillus subtilis.

With several different vectors, attempts were made to establish blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis. Stable establishment of blaZ in B. subtilis was achieved by use of a vector that allowed the integration of a single copy of the gene into the chromosome of that host. blaZ was expressed in the heterologous host since B. subtilis strains carrying integrated blaZ produced beta-lactamase and were more resistant to ampicillin than was wild-type B. subtilis. blaZ was stably inherited in such strains, as no loss of the ability to produce beta-lactamase was observed after growth in nonselective liquid medium or on solid medium. In contrast, a blaZ-containing restriction fragment could not be established in B. subtilis with either pUB110- or pC194-based vectors. Similarly, a pC194-based shuttle vector (pGX318) containing the 5' end of blaZ (including the promoter and the coding region for the signal sequence and the first few amino acids of the mature protein) was unable to transform B. subtilis. Two derivatives of pGX318 that could be stably established in B. subtilis were isolated. The structures of these derivatives suggested that inactivation of the blaZ promoter was associated with the acquisition of the ability to be established.

B J Schmidt

Papers

Secretion of human serum albumin from Bacillus subtilis.

Use of chromosomal integration in the establishment and expression of blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis.