构建重组乳酸乳球菌生产谷胱甘肽

以大肠杆菌染色体DNA为模板,分别扩增得到编码γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶的基因gsbA和gshB。将gsbA和gshB基因克隆到质粒pNZSl48中,电转化乳酸乳球菌NZ9000,获得重组菌NZ9000(pNZ3203)。在添加10mmol／L谷氨酸、半胱氨酸和甘氨酸的M17培养基中培养该重组茵,当OD600达到0、4时用乳酸链球菌素诱导4h,胞内谷胱甘肽含量达到358mmol／mg蛋白(胞内浓度相当于140mmol／L),这是在革兰氏阳性茵中生产谷胱甘肽的首例报道。     

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     

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation

To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available. High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms. The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.). Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 microseconds to 1 s). Transformation efficiencies of 10(3) transformants per microgram of DNA were obtained when dense suspensions (final concentration, 5 x 10(10) CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30 degrees C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.     

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007 was examined. A plasmid of 88 MDa (pJS88) was identified in addition to the previously reported conjugally transferred plasmids of 32 (pKB32) and 4.8 MDa. Proteinase activity, reduced bacteriophage sensitivity, bacteriocin resistance, and conjugal transfer ability were encoded by pJS88. The ability to metabolize lactose (Lac+) was encoded by pKB32, and the 4.8-MDa plasmid was cryptic. When a strain containing both pKB32 and pJS88 was mated with a recipient deficient in host-mediated homologous recombination (Rec-), a plasmid of 40 MDa (pJS40) was observed in approximately 50% of the Lac+ transconjugants. DNA-DNA hybridization results indicated that pJS40 contained homology with both pKB32 and pJS88. These results indicated that pKB32 was conjugally transferred via conduction and suggested that pJS40 is a deletion derivative of a pKB32::pJS88 cointegrate. A Rec- strain containing pKB32 and pJS88 mediated Lac+ conjugal transfer, suggesting that the pKB32::pJS88 cointegrate could form via a rec-independent event. Resolution of the pKB32::pJS88 cointegrate was observed in both Rec- and Rec+ hosts. Cointegrate formation and resolution via rec-independent mechanisms suggest the involvement of a transposable element in the Tn3 family.     

Production of dehydroamino acid-containing peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.     

Host specificity of nisin production by Lactococcus lactis

Kinetics of nisin production have been investigated in terms of endogenous features of the producer organism, Lactococcus lactis. Nisin-producing transposons (Tn Nip) were transferred to different hosts by conjugation. Constructs were cultivated in batch cultures and nisin produced was measured. The proteinase function of C2Prt (Tn Nip)-1 was eliminated by plasmid curing, resulting in the construct C2Prt - (Tn Nip)-1. C2Prt - (Tn Nip)-1 produced nisin to a higher concentration compared to C2Prt (Tn Nip)-1 and was able to maintain the maximum concentration till the end of cultivation. The final concentration of nisin produced was host-specific, because when different constructs carrying the same Tn Nip were cultivated they produced nisin to different concentrations. However, when the same host carried Tn Nip transposons derived from different donors the concentration of nisin produced was similar, suggesting that the two Tn Nip transposons may be similar.     

Production of Dehydroamino Acid-Containing Peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.     

Autolysis of Lactococcus lactis Is Influenced by Proteolysis

The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in an L. lactis acmA deletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.     

Specificity of Milk Peptide Utilization by Lactococcus lactis

To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of α_s2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatography. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine.     

Conversion of methionine to methional by Lactococcus lactis

Lactic acid bacteria were screened for methional production from 4-methylthio-2-ketobutanoate. Only Lactococcus lactis IFPL730 produced high amounts of methional. It was demonstrated that production of this compound was an exclusively enzymatic reaction. The present work describes for the first time that L. lactis can convert enzymatically methionine to methional in a process mediated by aminotransferase and alpha-ketoacid decarboxylase activities. The activity seems to be strain dependent.     

The properties of citrate transport catalyzed by CitP of Lactococcus lactis ssp. lactis biovar diacetylactis

Abstract The SC3 hydrophobin gene of Schizophyllum commune was disrupted by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. The phenotype of the mutant was particularly clear in sealed plates in which formation of aerial hyphae was blocked. In non-sealed plates aerial hyphae did form but these were hydrophilic and not hydrophobic as in wild-type strains. Complementation with a genomic SC3 clone restored formation of hydrophobic aerial hyphae in sealed plates. In a dikaryon homozygous for the SC3 mutation normal sporulating fruiting bodies were produced but aerial hyphae were hydrophilic.     

Recognition of cyclonucleoside lesions by the Lactococcus lactis FPG protein

Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.     

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.     

Recognition of Cyclonucleoside Lesions by the Lactococcus lactis FPG Protein

Abstract

Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.     

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation.

To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available. High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms. The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.). Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 microseconds to 1 s). Transformation efficiencies of 10(3) transformants per microgram of DNA were obtained when dense suspensions (final concentration, 5 x 10(10) CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30 degrees C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.     

The MG1363 and IL1403 Laboratory Strains of Lactococcus lactis and Several Dairy Strains Are Diploid

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.In contrast to higher eukaryotes, bacteria are haploid (6, 19); i.e., they store their genetic information in a single chromosome, which is then duplicated during the cell cycle. If the growth rate is sufficiently low, bacteria are born with a single copy of the chromosome, which will then be duplicated before the bacterium divides.There are a few reports about bacteria that have more than one genome per cell, i.e., that are polyploid. Deinococcus radiodurans has been shown to have 4 to 10 copies of its genome (13, 14). The diplococcal bacterium Neisseria gonorrhoeae was found to be diploid per coccal unit (31). Azotobacter vinelandii bacteria amplify the genome during growth in rich medium more than 40 times (20, 24, 27). The giant bacterium Epulopiscium fishelsoni has been shown to amplify its genome into a polytene chromosome of 3,000-fold ploidy (2). In addition, noncomplementing diploid bacteria have been isolated from protoplast fusions in Bacillus subtilis (11) and, as a result of zygogenesis, in Escherichia coli (10). A few other bacteria with two to six different chromosomes have been reported (15, 30).The normal cell cycle is divided into three periods: (i) the B period from cell division until initiation of replication, (ii) the C period in which the cell replicates its DNA, and (iii) the D period from termination of productive replication until cell division. The D period thus includes processes such as proofreading and deconcatenation. The B period is found only in cells whose generation times exceed the length of the combined C and D periods. If the generation times become shorter than the combined lengths of the C and D periods, then the initiations of replication move into previous cell cycles (16). Fast-growing bacteria will therefore have more than one ongoing round of DNA replication at the same time; they might have 4, 8, or even 16 origins of replication (4). Normal haploid cells are born with one chromosome, either replicating or nonreplicating, and always with one terminus of replication. Not until the replication has ended do the cells have two termini. If the D period becomes longer than the generation time, which happens at high growth rates, the cells will be born with two termini as a result of the overlapping cell cycles. Long D periods are discussed further in the Discussion.We have examined the cell cycle of Lactococcus lactis subsp. cremoris MG1363 in order to determine the cell cycle periods. To our surprise, we found that slow-growing cultures of these bacteria were born with two complete chromosomes, which were replicated into four chromosomes during the C period. This strain thus fulfills the criterion of being diploid without overlapping chromosomal replication cycles. Comparison with other L. lactis strains showed that both of the subspecies, L. lactis subsp. cremoris and L. lactis subsp. lactis, had members that were either diploid, like MG1363, or haploid, like most bacteria.     

Sec-Mediated Secretion of Bacteriocin Enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

Sec-mediated secretion of bacteriocin enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.