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

Engineering new functions and altering existing functions.

Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.

https://aem.asm.org/content/aem/63/9/3412.full.pdf

Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis

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.

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.

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

Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N′-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl-β-alanyl-l-proline (PhAc-βAla-Pro) phthalyl-l-leucine (Pht-Leu) or phthalylglycyl-l-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-gbAla-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The Km of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45° C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold.

Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity

Penicillin G acylase of Escherichia coli ATCC 11105 catalyzes hydrolysis as wellas synthesis of penicillin G. In this work a recombinant penicillin G acylase genewas mutagenized in vivo. A mutant with altered penicillin G acylase was selectedby its ability to grow with phthalyl-L-leucine as sole source of leucine. Themutant enzyme obtained was deficient in hydrolyzing penicillin G. A mutation ofGly359 to aspartic acid was mapped first by construction of chimeric pac genescomposed of wild type and mutant DNA, followed by nucleotide sequencing.

Helmut Niersbach

Papers

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

Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity

Isolation and mapping of a mutant penicillin G acylase with altered substrate specificity from Escherichia coli