Heterologous gene expression of bovine plasmin in Lactococcus lactis

Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin produced was shown to be biologically active. Received: 2 December 1996 / Received revision: 17 March 1997 / Accepted: 27 April 1997     

Cloning and expression of a novel lactococcal aggregation factor from Lactococcus lactis subsp. lactis BGKP1

Background

Lateral Gene Transfer (LGT) is a major contributor to bacterial evolution and up to 25% of a bacterium's genome may have been acquired by this process over evolutionary periods of time. Successful LGT requires both the physical transfer of DNA and its successful incorporation into the host cell. One system that contributes to this latter step by site-specific recombination is the integron. Integrons are found in many diverse bacterial Genera and is a genetic system ubiquitous in vibrios that captures mobile DNA at a dedicated site. The presence of integron-associated genes, contained within units of mobile DNA called gene cassettes makes up a substantial component of the vibrio genome (1-3%). Little is known about the role of this system since the vast majority of genes in vibrio arrays are highly novel and functions cannot be ascribed. It is generally regarded that strain-specific mobile genes cannot be readily integrated into the cellular machinery since any perturbation of core metabolism is likely to result in a loss of fitness.

Results

In this study, at least one mobile gene contained within the Vibrio rotiferianus strain DAT722, but lacking close relatives elsewhere, is shown to greatly reduce host fitness when deleted and tested in growth assays. The precise role of the mobile gene product is unknown but impacts on the regulation of outermembrane porins. This demonstrates that strain specific laterally acquired mobile DNA can be integrated rapidly into bacterial networks such that it becomes advantageous for survival and adaptation in changing environments.

Conclusions

Mobile genes that are highly strain specific are generally believed to act in isolation. This is because perturbation of existing cell machinery by the acquisition of a new gene by LGT is highly likely to lower fitness. In contrast, we show here that at least one mobile gene, apparently unique to a strain, encodes a product that has integrated into central cellular metabolic processes such that it greatly lowers fitness when lost under those conditions likely to be commonly encountered for the free living cell. This has ramifications for our understanding of the role mobile gene encoded products play in the cell from a systems biology perspective.     

Lactococcus lactis and stress

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis.Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase.To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37°C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.     

Expression of a beta-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis

A beta-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high beta-galactosidase activity but utilized lactose only slightly faster than the recipient. beta-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the beta-galactosidase gene. Growth rates of Lac+ H1 and 7962 derivatives were not affected after introduction of the clostridial beta-galactosidase, even though beta-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-beta-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-beta-galactosidase activity. We suggest that beta-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-beta-galactosidase genes.     

大肠埃希菌Mn-SOD在乳酸乳球菌中的表达

本文根据GenBank中报道的大肠埃希菌MG1655全基因组DNA序列中SOD的编码基因序列设计引物,PCR扩增大肠埃希菌锰超氧化物歧化酶(Mn-SOD)基因,并将其克隆入原核高效表达质粒载体pBV220中构建重组质粒pBV220-sod,并将其电转入乳酸乳球菌MG1363中获得了成功表达,为SOD发酵奶的研制奠定了基础。     

Divergence of Genomic Sequences between Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris

Relatedness between Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis was assessed by Southern hybridization analysis, with cloned chromosomal genes as probes. The results indicate that strains of the two subspecies form two distinct groups and that the DNA sequence divergence between L. lactis subsp. lactis and L. lactis subsp. cremoris is estimated to be between 20 and 30%. The previously used phenotypic criteria do not fully discriminate between the groups; therefore, we propose a new classification which is based on DNA homology. In agreement with this revised classification, the L. lactis subsp. lactis and L. lactis subsp. cremoris strains from our collection have distinct phage sensitivities.     

Lactococcus lactis gene yjgB encodes a gamma-D-glutaminyl-L-lysyl-endopeptidase which hydrolyzes peptidoglycan

YjgB is one of five peptidoglycan hydrolases previously identified in Lactococcus lactis. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithiothreitol. Peptidoglycan-hydrolyzing activity of YjgB was detected in growing cells of an L. lactis strain overexpressing YjgB, as revealed by the presence of disaccharide (DS)-dipeptide in the muropeptide composition of the overexpressing strain. YjgB hydrolyzes the peptide chains of L. lactis muropeptides between gamma-D-Gln and L-Lys residues. Its hydrolytic activity was detected on DSs with tetra- and pentapeptide chains, whereas hydrolytic activity was very low on DS-tripeptides. Thus, we demonstrated that YjgB is an endopeptidase which cleaves gamma-D-Gln-L-Lys bonds in peptide chains of L. lactis peptidoglycan.     

ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression

The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.     

The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis

The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis. Received: 8 February 1996 / Accepted: 11 March 1996     

Two tandem promoters to increase gene expression in Lactococcus lactis

Two plasmids, pPAH and pAH, containing a staphylokinase variant gene (sakXH) under the control of two tandem promoters (P₃₂-P_lacA) or promoter P_lacA alone were constructed and introduced into Lactococcus lactis MG5267. The expression of sakXH in the strain MG5267(pPAH) was approximately twice as high as that in the strain MG5267(pAH), according to the formation of fibrinolytic halos on fibrinolytic plates detected at the same conditions, indicating that the two tandem promoters were stronger than one alone. Difference between the expressions of sakXH under the inducible and non-inducible conditions suggested that P_lacA retained its feature as an inducible promoter when fused to promoter P₃₂.     

Lysozyme expression in Lactococcus lactis

Summary Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.     

Genome plasticity in Lactococcus lactis

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.     

Gene expression in Lactococcus lactis

Abstract Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis . A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis . This feature allowed the expression of a number of L. lactis -derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.     

Oxidative stress in Lactococcus lactis

Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses.     

Citrate permease gene expression in Lactococcus lactis subsp. lactis strains IL1403 and MG1363

Abstract Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis . Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.