Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Functions of S-layers.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding.

Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.

Transient expression in mammalian cells of the bacterial reporter gene encoding mercuric reductase: effects of various regulatory elements.

The effect of several transcriptional regulatory elements on gene expression in mammalian cells was investigated. As a reporter gene we have used the bacterial gene merA coding for the enzyme mercuric reductase. Several plasmids were constructed with different promoter/enhancer sequences (pSV/E, pSV/L, pMT, pRSV or pAd) at the 5' end and different splicing (small intron of the T antigen of SV40 or the second intron of the rabbit beta-globin gene) and/or polyadenylation signals (AEn, ALn or AR beta Gn) at the 3' end of the merA gene. Expression was measured in five different mammalian cell lines. In COS cells the highest level of expression is obtained with pSV/L and the lowest level with pSV/E. In HeLa, CV-1, Ltk-, and CHO cells merA expression is relatively high, under control of pRSV and pMT and relatively low under control of pSV/L and pAd. The introns studied have a negative effect on the expression of merA. The presence of a polyadenylation signal downstream from the gene is essential for its expression. The three different polyadenylation signals studied give a similar stimulatory effect on the level of expression of the merA gene.

Construction of new vectors for cloning of promoters.

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.

A system for the analysis of expression signals in Aspergillus.

To analyse gene expression signals in Aspergillus we have constructed a set of integration vectors each of which contains in front of the Escherichia coli 'lacZ gene a unique BamHI site in one of the three possible translational reading frames and the A. nidulans argB gene as a selection marker. The vectors allow in-phase translational fusion of any gene to 'lacZ. After transformation of an A. nidulans argB strain, the vectors integrate with a high percentage at the argB locus of the genome, as a single copy. The insertion of the fusion genes at the argB locus assures the constancy of influences of the chromosomal environment on gene expression.

Construction of expression plasmids for Saccharomyces cerevisiae: application for synthesis of poliovirus protein VP2.

A series of expression plasmids was constructed to compare the usefulness of various promoters for the synthesis of a given protein in the Saccharomyces cerevisiae. The plasmids pMBL212, -213, -214, -215 and -216 can be used to synthesize the protein of interest directly as a non-fused protein or, if the protein is difficult to detect, indirectly as an enzymatically active beta-galactosidase fusion protein. The plasmids were employed to identify which yeast promoter and strain are suitable for the synthesis of poliovirus protein VP2. It was concluded that the GAL7 and PGK promoters in combination with strain X904 can be used for efficient synthesis of a VP2 in the form of a N-terminally fused VP2-beta-galactosidase protein.

An upstream activating sequence from the Aspergillus nidulans gpdA gene.

Introduction of a previously identified promoter element of the Aspergillus nidulans gpdA gene (encoding glyceraldehyde-3-phosphate dehydrogenase), the so-called gpd box, into the upstream region of the highly regulated A. nidulans amdS gene (encoding acetamidase), significantly increased (up to 30-fold) the expression of the lacZ reporter gene fused to these expression signals. This increase was dependent on the orientation of the gpd box and on the site of introduction into the amdS upstream region. The presence of additional gpdA sequences which flank the gpd box reduced or even extinguished positive effects of the gpd box. omega-Amino acid and carbon catabolite regulation of the amdS promoter were retained after introduction of the gpd box, indicating that the gpd box does not abolish interactions of the regulatory proteins, AmdR and CreA, with the amdS transcription control sequences. Based on the results, it is suggested that the gpd box comprises at least two separate activities: one being orientation dependent, but relatively independent of position of the gpd box in the upstream region, and the other is only functional near other sites of transcriptional control. Most likely, both activities are not involved in regulation of the amdS promoter.

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers.

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.

Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans.

The isolation and characterization of the highly expressed glyceraldehyde-3-phosphate dehydrogenase (GPD)-coding gene (gpdA) of Aspergillus nidulans is described. The gene was isolated from an A. nidulans lambda gene library with a Saccharomyces cerevisiae GPD-coding gene as a probe. Unlike many other eukaryotes, A. nidulans contains only one GPD-coding gene. At the amino acid level, homology with other GPD enzymes is extensive. The A. nidulans gene contains seven introns, one of which is positioned in the 5'-untranslated part of the gene. The major transcription start point is found at 172 bp upstream from the start codon. Polyadenylation occurs at several sites about 200 bp downstream from the stop codon. Comparison of 5' and 3' flanking sequences with flanking sequences of other highly expressed (glycolytic) genes shows several regions of similar sequence.

Isolation and characterization of the Aspergillus niger trpC gene.

The Aspergillus niger trpC gene was isolated by complementation experiments with an Escherichia coli trpC mutant. Plasmid DNA containing the A. niger trpC gene transforms an Aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpC gene, to Trp+, indicating the presence of a complete and functional trpC gene. Southern blot analysis of DNA from these Trp+ transformants showed that plasmid DNA was present but that this DNA was not integrated at the site of the chromosomal trpC locus. The A. niger trpC gene was localized on the cloned fragment by heterologous hybridization experiments and sequence analysis. These experiments suggest that the organization of the A. niger trpC gene is identical to that of the analogous A. nidulans trpC and the Neurospora crassa trp-1 genes.

Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli.

A new, heterologous, dominant marker for selection of Aspergillus transformants is described. This marker is based on the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph). Expression of the hph gene is controlled by A. nidulans gpd and trpC expression signals. An Aspergillus transformation vector was constructed which contains this marker and confers HmB resistance to Aspergillus species. With both A. niger and A. nidulans, transformation frequencies of 5-20 transformants per micrograms vector DNA were obtained. Cotransformation with other vectors was shown to be very efficient in both species, when selection for HmB resistance was applied.

A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi.

To analyze the promoter region(s) of divergently transcribed fungal genes, a twin-reporter vector was constructed. This vector contains two divergently oriented reported genes, encoding Escherichia coli beta-glucuronidase (uidA) and E. coli beta-galactosidase (lacZ). Terminator regions of the Aspergillus nidulans nitrate and nitrite reductase-encoding genes, niaD and niiA, respectively, have been cloned 3' to the reporter genes to ensure proficient transcription termination of the reporter genes. The reporter genes have been separated by a unique NotI restriction site, which can be used for the insertion of expression signals. A mutant argB selection marker has been introduced in order to obtain A. nidulans transformants with a single copy of the vector integrated at the argB locus. The use of the vector was demonstrated by insertion of the A. nidulans niaD-niiA intergenic region and analysis of A. nidulans transformants obtained with this construct. Control of expression of both reporter genes was found to be in accordance with previously published data on control of nitrate assimilation [Cove, Biol. Rev. 54 (1979) 291-327].

Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus.

A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed. The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1. All four fusion proteins were efficiently produced in E. coli host bacteria. Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats. With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions.

Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase.

Analysis of the promoter region of the highly expressed Aspergillus nidulans gpdA gene is described. The nucleotide (nt) sequence of a 1.3-kb region upstream from the ATG was determined. Comparison with promoter regions of other Aspergillus and Neurospora genes revealed several regions of similar sequence. Both random and site-specific mutations were introduced into the promoter region of the gpdA gene, and the resulting mutant promoters were fused to the Escherichia coli lacZ gene. The constructed fusions were introduced into A. nidulans and transformants that contained one copy of these fusions at the argB locus were analysed. beta-Galactosidase assays and primer extension experiments were used to identify sequence elements involved in transcription activation and transcription initiation. Two elements, located around 650 and 250 nt upstream from the major transcription start point (tsp), were identified as transcription activation elements. These elements coincide with regions of putative secondary structure (direct or inverted repeats). A third element, a C + T-rich region directly upstream from the major tsp, was shown to be involved in correct initiation of transcription.

Expression of an Escherichia coli beta-galactosidase fusion gene in Aspergillus nidulans.

We inserted in frame the Escherichia coli lacZ gene into the protein-coding region of the Aspergillus nidulans trpC gene and introduced the resultant fused gene into the A. nidulans genome. A functional beta Gal fusion protein was produced. Removal of the trpC transcription and translation initiation sequences from the fusion gene abolished production of the fusion protein, showing that expression is dependent on these sequences. Thus, lacZ fusions should be of use for estimating gene activity in a. nidulans.

Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors.

Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 10 to 10 transformants per mug of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far.

Live bacterial delivery systems for development of mucosal vaccines.

By expression of foreign antigens in attenuated strains derived from bacterial pathogens and in non-pathogenic commensal bacteria, recombinant vaccines are being developed that aim to stimulate mucosal immunity. Recent advances in the pathogenesis and molecular biology of these bacteria have allowed rational development of new and improved bacterial carriers and more effective gene expression systems. These advances have improved the performance and versatility of these delivery systems to induce mucosal immunity to recombinant antigens in animal models. Application of these (improved) technologies for development of human vaccines is still limited and awaits further exploration.

The potential of Lactobacillus as a carrier for oral immunization: development and preliminary characterization of vector systems for targeted delivery of antigens.

Oral administration of lactobacilli evokes mucosal and systemic immune responses against epitopes associated with these organisms (Gerritse et al., 1990, 1991). The adjuvant function of different Lactobacillus species was investigated under the conditions of intraperitoneal (i.p.) injection or oral administration. After i.p. injection of trinitrophenylated chicken gamma-globulin, high DTH responses were observed with Lactobacillus casei and Lactobacillus plantarum, but low responses with Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus. In different experimental model systems L. casei and L. plantarum consistently showed significant adjuvanticity. A series of expression and expression-secretion vectors containing the strong constitutive promoter of the L. casei L-ldh gene or the regulatable promoter of the Lactobacillus amylovorus amy gene (Pouwels and Leer, 1995) was used for the intracellular, extracellular and surface-bound expression of an influenza virus antigenic determinant fused to Escherichia coli beta-glucuronidase. Intracellular expression of the fusion protein amounted to 1-2% of total soluble protein. Lactobacilli synthesizing the fusion protein intracellularly evoked an oral immune response after subcutaneous priming.

Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene.

The expression in Aspergillus is described of genes, coding for intracellular and extracellular proteins controlled by the promoter region of the constitutively and efficiently expressed glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) of Aspergillus nidulans. Both the homologous gpdA and the heterologous Escherichia coli beta-galactosidase (lacZ) and beta-glucuronidase (uidA) genes could be expressed intracellularly at levels as high as 10-25% of total soluble protein. Efficient extracellular production of A. niger glucoamylase could be achieved with a fusion-gene containing the region of the glucoamylase gene coding for the mature protein preceded by a synthetic fungal signal sequence. Extracellular production of a heterologous protein, E. coli beta-glucuronidase, with such a fusion was much less efficient. Only very low levels of beta-glucuronidase were detected in the culture fluid, whereas considerable enzyme activity was detected in the mycelium.