L-forms are "cell wall-deficient" bacteria which are able to grow as spheroplasts or protoplasts. They can be differentiated into four types depending on their ability to revert to the parental, cell-walled form and to the extent of their cell-wall modification. L-forms are significant in modern science because of their contributions to an improved understanding of principal questions and their interactions with eukaryotes. This review particularly focuses on research using stable protoplast-type L-forms which have contributed to a better understanding of the structural and functional organisation of the cytoplasmic membrane and of cell division. These L-forms, which have only a single surrounding bilayer membrane, also represent a unique expression system for production of recombinant proteins. A large proportion of L-form publications concern their putative role in human disease and its therapy, a topic which is discussed briefly. L-forms have also been used to form intracellular associations with plant cells and have been shown to elicit induced disease resistance offering a novel method for plant protection. The recent decline in active research on L-forms is a concern as knowledge and experience, as well as unique L-form strains which have been maintained for decades, are being lost.

Bacterial L-Forms

Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

https://aem.asm.org/content/aem/64/12/4862.full.pdf

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

The cells of an L-form strain of Streptomyces hygroscopicus have been grown for 20 years without a cell wall. Their cytoplasmic membranes have high stability and an unusual structural polymorphism. To clarify the importance of the lipid components for these membrane properties, a comparative analysis has been carried out with purified membranes of L-form cells, of parent vegetative hyphal cells (N-form cells), and of protoplasts derived from the latter. The phospholipid classes and fatty acids were determined by thin-layer chromatography (TLC), two-dimensional TLC, high-performance liquid chromatography, gas chromatography, and mass spectrometry. The qualitative compositions of cardiolipin (CL), lyso-cardiolipin (LCL), phosphatidylethanolamine (PE1 and PE2), lyso-phosphatidylethanolamine (LPE), phosphatidylinositolmannoside (PIM), phosphatidic acid (PA), dilyso-cardiolipin-phosphatidylinositol (DLCL-PI), and the 13 main fatty acids were the same in the three membrane types. However, significant quantitative differences were observed in the L-form membrane. They consist of a three- to fourfold-higher content of total, extractable lipids, 20% more phospholipids, an increased content of CL and PIM, and a reduced amount of the component DLCL-PI. Furthermore, the L-form membrane is characterized by a higher content of branched anteiso 15:0 and anteiso 17:0 fatty acids compared to that of the membranes of the walled vegetative cells. These fatty acids have lower melting points than their straight and iso-branched counterparts and make the membrane more fluid. The phospholipid composition of the protoplast membrane differs quantitatively from that of the N form and the L form. Whereas the phospholipid classes are mostly similar to that of the N form, the fatty acid pattern tends to be closer to that of the L-form membrane. The membranes of both the L-form cells and the protoplasts need to be more fluid because of their spherical cell shape and higher degree of curvature compared with N-form membranes.

Lipid and fatty acid composition of cytoplasmic membranes from Streptomyces hygroscopicus and its stable protoplast-type L form.

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one- or two-step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often-overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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Secretion of recombinant proteins from E. coli.

Biotechnological advances on penicillin G acylase: pharmaceutical implications, unique expression mechanism and production strategies.

L-form strains of Proteus mirabilis and Escherichia coli lacking the cell wall represent an alternative prokaryotic cell system for the production of recombinant proteins (KLESSEN et al. 1988, LAPLACE et al. 1988a, 1989b). We could demonstrate that they are also able to synthesize the enzyme penicillin G acylase (PAC)1). PAC was processed and secreted into the medium by recombinant L-form strains. The synthesis of PAC was growth-associated and stably regulated. Expression, secretion, and processing were not temperature-dependent and occurred at 26 degrees C, 32 degrees C and even 37 degrees C. The expression vector pHC1 carried the pac gene under the control of the lac UV promotor and a kanamycin resistance gene. It could be maintained in L-form cells, showing low structural as well as segregational instability. The secretion of the biologically active enzyme into the medium indicated that the postranslational processing of the PAC molecule, including the excision of a 54 amino acid spacer peptide between the alpha and beta subunit, is not carried out in the periplasmic space, but occurs at the cytoplasmic membrane or autocatalytically.

Heinrich Cron

Papers

Synthesis and secretion of recombinant penicillin G acylase in bacterial L‐forms