生长抑制剂所诱导的乳酸乳球菌MG1363的自溶表型及其生长阶段特异的性质

【目的】自溶是细菌在压力环境下通过自身裂解而获得的一种生理适应现象,研究的目的是全面探讨乳酸菌株在生长抑制剂条件下的自溶表型及机理。【方法】对多种来源的乳酸菌株的自溶能力进行检测,通过在不同生长条件和抑制剂压力条件下乳酸乳球菌MG1363的生长检测对其自溶表型进行分析。【结果】在葡萄糖严格受限的培养基中,氨苄青霉素的加入能够显著诱导MG1363的自溶,而且该自溶现象只发生在葡萄糖耗尽的时间点,展现出一种狭窄的生长时期依赖的特征。与此同时,因为氨苄青霉素的加入,4种主要的自溶酶的表达都发生了不同程度的显著改变。此外,所有受试的抑制剂都削弱了MG1363在非营养条件下的自溶,表明该菌株可能具有一种涉及细胞壁合成和降解酶的共同调控的模式。【结论】乳酸菌株在不同生长抑制剂条件下的自溶表型存在很大差异,且该自溶体现出营养条件和生长时期严格依赖的特征。     

Changes in Glycolytic Activity of Lactococcus lactis Induced by Low Temperature

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30°C increased approximately 2.5-fold following a shift from 30 to 10°C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.     

Time dependent responses of glycolytic intermediates in a detailed glycolytic model of Lactococcus lactis during glucose run-out experiments

Glucose addition and subsequent run-out experiments were compared to simulations with a detailed glycolytic model of Lactococcus lactis. The model was constructed largely on bases of enzyme kinetic data taken from literature and not adjusted for the specific simulations shown here. Upon glucose depletion a rapid increase in PEP, inorganic phosphate and a gradual decrease in fructose 1,6-bisphosphate (FBP) were measured and predicted by simulation. The dynamic changes in these and other intermediate concentrations as measured in the experiments were well predicted by the kinetic model.     

In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis.

The metabolism of glucose by nongrowing cells of L. lactis strain MG5267 was studied under controlled conditions of pH, temperature, and gas atmosphere (anaerobic and aerobic) using a circulating system coupled to nuclear magnetic resonance (NMR) detection that allowed a noninvasive determination of intracellular pools of intermediate metabolites by 13C-NMR with a time resolution of 30 seconds. In addition, intracellular parameters, such as pH, NTP levels, and concentration of inorganic phosphate in the cytoplasm, could be monitored on-line by 31P-NMR with a time resolution of approx. 3 min. The time course for the concentrations of intracellular fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP), together with kinetic measurements of substrate consumption and endproducts formation, were used as a basis for the construction of a mechanistic model for glycolysis. In vivo measurements were complemented with determinations of phosphorylated metabolites in perchloric acid extracts. A top-down model was developed by simplifying the metabolism to the resolution allowed by the experimental data collected by in vivo NMR (grouped in seven metabolic steps). This simplified mechanistic model was adjusted to the metabolite concentrations determined by in vivo NMR. The results obtained led to the rationalization of the dynamics of glucose metabolism as being driven largely by ATP surplus. This excess causes accumulation of FBP due to NAD+ limitation, whose regeneration is dependent on downstream pyruvate reduction. The model was capable of predicting qualitative shifts in the metabolism of glucose when changing from anaerobic to aerobic conditions.     

Twofold reduction of phosphofructokinase activity in Lactococcus lactis results in strong decreases in growth rate and in glycolytic flux

Two mutant strains of Lactococcus lactis in which the promoter of the las operon, harboring pfk, pyk, and ldh, were replaced by synthetic promoters were constructed. These las mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions. One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two- to fourfold in the las mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.     

Purification and characterization of the lytic activity induced by the prolate-headed bacteriophage P001 in Lactococcus lactis

The lytic activity induced by the lactococcal bacteriophage P001 was isolated from phage lysates of Lactococcus lactis by a four-step purification procedure. Two proteins lytic for L. lactis were identified with molecular weights of 28 kDA and 8 kDa, respectively. The N-terminal amino acid sequences of the two proteins were determined and degenerated oligonucleotide probes corresponding to these sequences were synthesized. DNA hybridization experiments with phage P001-DNA and lactococcal DNA revealed that both proteins were apparently encoded by a single lysin gene located on the phage P001 genome. This was confirmed by alignment of the determined N-terminal amino acid sequences with nucleotide sequences which were deduced from cloned Lactococcus bacteriophage lysin genes.     

Energy-based dynamic model for variable temperature batch fermentation by Lactococcus lactis

We developed a mechanistic mathematical model for predicting the progression of batch fermentation of cucumber juice by Lactococcus lactis under variable environmental conditions. In order to overcome the deficiencies of presently available models, we use a dynamic energy budget approach to model the dependence of growth on present as well as past environmental conditions. When parameter estimates from independent experimental data are used, our model is able to predict the outcomes of three different temperature shift scenarios. Sensitivity analyses elucidate how temperature affects the metabolism and growth of cells through all four stages of fermentation and reveal that there is a qualitative reversal in the factors limiting growth between low and high temperatures. Our model has an applied use as a predictive tool in batch culture growth. It has the added advantage of being able to suggest plausible and testable mechanistic assumptions about the interplay between cellular energetics and the modes of inhibition by temperature and end product accumulation.     

Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in Lactococcus lactis (B. Poolman, B. Bosman, J. Kiers, and W. N. Konings, J. Bacteriol. 169:5887-5890, 1987). Those studies were based on inhibitor titrations with iodoacetate, which specifically inhibits GAPDH, and the data suggested that it should be possible to increase the glycolytic flux by overproducing GAPDH activity. To test this hypothesis, we constructed a series of mutants with GAPDH activities from 14 to 210% of that of the reference strain MG1363. We found that the glycolytic flux was unchanged in the mutants overproducing GAPDH. Also, a decrease in the GAPDH activity had very little effect on the growth rate and the glycolytic flux until 25% activity was reached. Below this activity level, the glycolytic flux decreased proportionally with decreasing GAPDH activity. These data show that GAPDH activity has no control over the glycolytic flux (flux control coefficient = 0.0) at the wild-type enzyme level and that the enzyme is present in excess capacity by a factor of 3 to 4. The early experiments by Poolman and coworkers were performed with cells resuspended in buffer, i.e., nongrowing cells, and we therefore analyzed the control by GAPDH under similar conditions. We found that the glycolytic flux in resting cells was even more insensitive to changes in the GAPDH activity; in this case GAPDH was also present in a large excess and had no control over the glycolytic flux.     

Increasing acidification of nonreplicating Lactococcus lactis deltathyA mutants by incorporating ATPase activity

Lactococcus lactis MBP71 deltathyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.     

Antimicrobial activity of divercin RV41 produced and secreted by Lactococcus lactis

Divercin V41 is a class IIa bacteriocin produced by Carnobacterium divergens V41 with a strong anti-Listeria activity. We have previously produced a recombinant form of divercin V41 (DvnRV41) in Escherichia coli strain Origami, by cloning a synthetic gene that codes for a mature divercin RV41 peptide. In this work we describe the inducible expression and secretion of DvnRV41 in the food-grade lactic acid bacterium, Lactococcus lactis. The production of DvnRV41 by recombinant L. lactis was confirmed and quantified by Western blot and ELISA assays. In addition, anti-Listeria activity of DvnRV41 was determined using an agar diffusion test. Although the levels of DvnRV41 produced by recombinant L. lactis were similar to those produced by the natural host, C. divergens V41, the specific activities were lower. In conclusion, our data show that the bacteriocin DvnRV41 is produced and secreted in an active form by L. lactis and that this approach may have important applications in the preservation of foods.     

A new protein of alpha-amylase activity from Lactococcus lactis

An extracellular alpha-amylase from Lactococcus lactis IBB500 was purified and characterized. The optimum conditions for the enzyme activity were pH 4.5, temperature of 35 degrees C, enzyme molecular mass of 121 kDa. The genome analysis and a plasmid curing experiment indicated that amy+ genes were located in a plasmid of 30 kb. An analysis of phylogenetic relationships strongly supported a hypothesis of horizontal gene transfer. A strong homology was found for the peptides with the sequence of alpha-amylases from Ralstonia pikettii and Ralstonia solanacearum. The protein of alpha-amylase activity purified in this study is the first one described for the Lactococcus lactis species, and this paper is the first report on Lactococcus lactis strain as a microorganism belonging to amylolytic lactic acid bacteria (ALAB).     

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。     

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.     

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.     

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.     

Diacetyl and alpha-acetolactate overproduction by Lactococcus lactis subsp. lactis biovar diacetylactis mutants that are deficient in alpha-acetolactate decarboxylase and have a low lactate dehydrogenase activity

Lactococcus lactis subsp. lactis biovar diacetylactis strains are utilized in several industrial processes for producing the flavoring compound diacetyl or its precursor alpha-acetolactate. Using random mutagenesis with nitrosoguanidine, we selected mutants that were deficient in alpha-acetolactate decarboxylase and had low lactate dehydrogenase activity. The mutants produced large amounts of alpha-acetolactate in anaerobic milk cultures but not in aerobic cultures, except when the medium was supplemented with catalase, yeast extract, or hemoglobin.     

Towards Enhanced Galactose Utilization by Lactococcus lactis

Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.Lactococcus lactis is a lactic acid bacterium widely used in the dairy industry for the production of fermented milk products. Because of its economic importance, L. lactis has been studied extensively in the last 40 years. A small genome, a large set of genetic tools, a wealth of physiological knowledge, and a relatively simple metabolic potential render L. lactis an attractive model with which to implement metabolic engineering strategies (reviewed in references 21 and 57).In the process of milk fermentation by L. lactis, lactose is taken up and concomitantly phosphorylated at the galactose moiety (C-6) by the lactose-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS^Lac), after which it is hydrolyzed to glucose and galactose 6-phosphate (Gal6P) (64). The glucose moiety enters the glycolytic pathway upon phosphorylation via glucokinase to glucose 6-phosphate (G6P), whereas Gal6P is metabolized to triose phosphates via the d-tagatose 6-phosphate (Tag6P) pathway, encompassing the steps catalyzed by galactose 6-phosphate isomerase (LacAB), Tag6P kinase (LacC), and tagatose 1,6-bisphosphate aldolase (LacD) (Fig. ​(Fig.1).1). Curiously, during the metabolism of lactose by L. lactis, part of the Gal6P is dephosphorylated and excreted into the growth medium, while the glucose moiety is readily used (2, 7, 51, 56, 60).Open in a separate windowFIG. 1.Schematic overview of the alternative routes for galactose uptake and further catabolism in L. lactis. Galactose can be imported by the non-PTS permease GalP and metabolized via the Leloir pathway (galMKTE) to α-G1P, which is converted to the glycolytic intermediate G6P by α-phosphoglucomutase (pgmH). Alternatively, galactose can be imported by PTS^Lac (lacFE) and further metabolized to triose phosphates by the Tag6P pathway (lacABCD). Here, we propose a new uptake route consisting of galactose translocation via the galactose PTS, followed by dephosphorylation of the internalized Gal6P to galactose, which is further metabolized via the Leloir pathway (highlighted in the gray box). galP, galactose permease; galM, galactose mutarotase; galK, galactokinase; galT, galactose 1-phosphate uridylyltransferase; galE, UDP-galactose-4-epimerase; pgmH, α-phosphoglucomutase; lacAB, galactose 6-phosphate isomerase; lacC, Tag6P kinase; lacD, tagatose 1,6-bisphosphate aldolase; lacFE, PTS^Lac; PTS^Gal, unidentified galactose PTS; Phosphatase; unidentified Gal6P-phosphatase; pgi, phosphoglucose isomerase; pfk, 6-phosphofructo-1-kinase; fba, fructose 1,6-bisphosphate aldolase; tpi, triose phosphate isomerase; α-Gal1P, α-galactose 1-phosphate; α-G1P, α-glucose 1-phosphate; UDP-gal, UDP-galactose; UDP-glc, UDP-glucose; G6P, glucose 6-phosphate; Gal6P, galactose 6-phosphate; Tag6P, tagatose 6-phosphate; TBP, tagatose 1,6-bisphosphate; FBP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate. The dotted arrow represents the conversions of GAP to pyruvate via the glycolytic pathway. Steps essential to improve galactose consumption are shown in black boxes.As a result of incomplete lactose utilization, some fermented dairy products contain significant residual amounts of galactose. The presence of galactose has been associated with shoddier qualities of the fermented product (6, 27, 43). In particular, galactose is a major contributor to the browning that occurs when dairy products (e.g., yogurt and mozzarella, Swiss, and cheddar cheese) are cooked or heated in the manufacture of pizzas, sauce preparation, or processed cheese. In addition, availability of residual galactose may result in production of CO₂ by heterofermentative starters and, consequently, in textural defects such as the development of slits and fractures in cheeses. Therefore, the availability of starter strains with improved galactose utilization capacity is desirable to develop higher-quality dairy products. Additionally, strains with increased galactose metabolism could provide galactose-free foods for individuals and, in particular, children suffering from the rare disease galactosemia (36). To this end, a comprehensive understanding of galactose catabolism is essential.Galactose metabolism in L. lactis was thoroughly studied in the past and has been and still is the subject of some controversy. Indeed, conflicting results regarding the type of PTS involved in galactose uptake have been published. Some authors advocated that galactose is exclusively transported via the plasmid-encoded PTS^Lac, whereas others proposed transport via a galactose-specific PTS (PTS^Gal) to the extreme of questioning the contribution of the PTS^Lac (17, 20, 50, 59). However, a gene encoding PTS^Gal has never been identified in L. lactis. Independently of the nature of the PTS, it is generally accepted that the resulting Gal6P is metabolized via the Tag6P pathway (lac operon) (Fig. ​(Fig.1).1). On the other hand, galactose translocated via the highly specific galactose permease (GalP) is metabolized via the Leloir pathway to α-glucose 1-phosphate (α-G1P) through the sequential action of galactose mutarotase (GalM), galactokinase (GalK), and galactose 1-phosphate uridylyltransferase (GalT)/UDP-galactose-4-epimerase (GalE) (gal operon). Entry in glycolysis is preceded by the α-phosphoglucomutase (α-PGM)-catalyzed isomerization of α-G1P to G6P. The use of the Leloir and/or the Tag6P pathway for galactose utilization is currently viewed as being strain dependent (9, 16, 25, 32, 33, 58), but the relative efficacy in the degradation of the sugar has not been established.The ultimate aim of this study was to engineer L. lactis for improved galactose-fermenting capacity as a means to minimize the galactose content in dairy products. To gain insight into galactose catabolism via the Leloir (gal genes) and the Tag6P (lac genes) pathways, a series of L. lactis subsp. cremoris NZ9000 isogenic gal and lac mutants were constructed. Carbon 13 labeling experiments coupled with nuclear magnetic resonance (NMR) spectroscopy were used to investigate galactose metabolism in the gal and lac strains. The data obtained revealed a novel route for galactose dissimilation and provided clues to further enhance galactose utilization.     

Permeabilization and lysis induced by bacteriocins and its effect on aldehyde formation by Lactococcus lactis

Permeabilization induced by lacticin 3147, lactococcins A, B and M, enterocin AS-48 and nisin, bacteriocins described as cell membrane-pore forming and lytic agents, enhanced in all cases aldehyde formation by Lactococcus lactis IFPL730. Nevertheless, the conversion of isoleucine into 2-methylbutyraldehyde depended not only on the degree of permeabilization but also on the bacteriocin that caused the cell membrane damage. The highest values of 2-methylbutyraldehyde corresponded to cell suspensions containing lacticin 3147 and lactococcins, treatments that provoked further lysis in addition to induced permeabilization.     

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.