So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.Many organisms from the domains Bacteria and Archaea possess a surface layer (S-layer) as the outermost structure of the cell envelope. S-layers are composed of regularly arranged proteinaceous subunits of a single protein or glycoprotein species with molecular masses ranging from 40 to 170 kDa (43, 48). S-layer proteins represent 10 to 15% of the total protein of the bacterial cell, and S-layer lattices cover the cell surface during all stages of growth, which indicates that efficient gene expression, S-layer protein synthesis, and secretion take place (4). A high content of hydrophobic and acidic amino acids and a low theoretical isoelectric point (pI) are typical features of S-layer proteins (48). In contrast, very high pI values have been described for the S-layer proteins from various lactobacilli (4, 54) and Methanothermus fervidus (6). In general, S-layers have been considered to function as cell shape determinants, protective coats, promoters for cell adhesion and surface recognition, and molecular and ion traps; however, no general function found in all S-layers has been recognized (47, 48).S-layers have been found from many species of the genus Lactobacillus (31, 56). However, the S-layer protein genes have been cloned and sequenced only from Lactobacillus brevis ATCC 8287 (54) and from the closely related species L. acidophilus (3), L. helveticus (8), and L. crispatus (46). Th...
Expression of D-(؊)-lactate dehydrogenase (D-LDH) and L-(؉)-LDH genes (ldhD andldhL, respectively) and production of D-(؊)-and L-(؉)-lactic acid were studied in Lactobacillus helveticus CNRZ32. In order to develop a host for production of pure L-(؉)-isomer of lactic acid, two ldhD-negative L. helveticus CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter. The two D-LDH-negative strains produced only L-(؉)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables for L-(؉)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41°C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.
Nisin Z, a post-translationally modified antimicrobial peptide of Lactococcus lactis, is positively autoregulated by extracellular nisin via the two-component regulatory proteins NisRK. A mutation in the nisin NisT transporter rendered L. lactis incapable of nisin secretion, and nisin accumulated inside the cells. Normally nisin is activated after secretion by the serine protease NisP in the cell wall. This study showed that when secretion of nisin was blocked, intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin. The isolated cytoplasm of a non-nisin producer could also cleave the leader from the nisin precursor, showing that the cytoplasm of L. lactis cells does contain proteolytic activity capable of cleaving the leader from fully modified nisin precursor. Nisin could not be detected in the growth supernatant of the NisT mutant strain with a nisin-sensing strain (sensitivity 10 pg ml "1 ), which has a green fluorescent protein gene connected to the nisin-inducible nisA promoter and a functional nisin signal transduction circuit. Northern analysis of the NisT mutant cells revealed that even though the cells could not secrete nisin, the nisin-inducible promoter P nisZ was active. In a nisB or nisC background, where nisin could not be fully modified due to the mutations in the nisin modification machinery, the unmodified or partly modified nisin precursor accumulated in the cytoplasm. This immature nisin could not induce the P nisZ promoter. The results suggest that when active nisin is accumulated in the cytoplasm, it can insert into the membrane and from there extrude parts of the molecule into the pseudoperiplasmic space to interact with the signal-recognition domain of the histidine kinase NisK. Potentially, signal presentation via the membrane represents a general pathway for amphiphilic signals to interact with their sensors for signal transduction. INTRODUCTIONIn bacteria as well as in some plants, fungi, protozoa and archaea, an adaptive response to environmental changes is regulated by two-component signal transduction (TCST) pathways (Koretke et al., 2000). These systems have in common a phosphoryl transfer between a sensor histidine protein kinase (HPK) and an effector response regulator (RR) (Kramer & Weiss, 1999;Fabret et al., 1999). A typical HPK is a transmembrane receptor with an amino-terminal extracellular sensing domain and a carboxy-terminal cytosolic signalling domain. The corresponding cytoplasmic RR mediates an adaptive response to this environmental signal, in many cases a change in gene expression (Dutta et al., 1999;Grebe & Stock 1999;Stock et al., 2000).The HPK-mediated TCST circuits may sense nutrients, chemoattractants or osmotic conditions, and be involved in stress-induced sporulation, host recognition for pathogen invasion, hyphal development and ethylene response (Loomis et al., 1997;Koretke et al., 2000;Wolanin et al., 2002). Recently, several chemical classes of microbially derived signalling molecules have ...
Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.
The cellulose-binding domain (CBD) of XylA was fused with PrtP, NisP and AcmA anchors derived from Lactococcus lactis under P45 promoter and Usp45 secretion signal. The fusion construct with the anchor PrtP (334 aa) was shown to exhibit the most efficient immobilization. The CBD-PrtP construct on the other hand was not efficiently attached to the cell wall and as such was found mainly in the supernatant. Results also showed that expression of the CBD-NisP anchor fusion led to a similar result. This raised the question if more efficient binding of the anchor to the cell wall by sortase could enhance the efficiency of cell immobilization to the cellulosic material. However, expressing sortase with the CBD-PrtP fusion did not improve the immobilization of the cells to cellulose.
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