Molecular Switch Controlling Expression of the Mannose-Specific Adhesin, Msa, in Lactobacillus plantarum.

Some lactic acid bacteria, especially Lactobacillus spp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probiotic Lactobacillus plantarum strains, WCSF1 and 299v, display highly different mannose-specific adhesion, with L. plantarum 299v being superior to L. plantarum WCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of the msa gene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a stronger msa transcript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of the msa gene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate the msa transcript. In addition, the ribosome binding site upstream of the msa gene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of the msa transcript in the variants than in the wild type, correlating with a strong-Msa phenotype.IMPORTANCE Probiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. In Lactobacillus plantarum, interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of the msa gene in L. plantarum strain WCFS1. Strains with increased msa expression could be valuable in the development and manufacture of improved probiotic products.

Compliance with Guidelines on Thromboprophylaxis for Acutely Admitted Medical Patients.

INTRODUCTION: The risk of venous thromboembolism (VTE) is increased by more than 100-fold among hospitalised medical patients compared to subjects in the community. The Danish Council for the Use of Expensive Hospital Medicines has published national guidelines on thromboprophylaxis (TP) in which the risks of VTE and bleeding are balanced. We wanted to investigate the proportion of acutely admitted medical patients for whom thromboprophylaxis was indicated and to what extent the guidelines were followed. METHODS: Data from patients hospitalised at two medical wards were screened. We registered the proportion of patients for whom mechanical or pharmacologic TP (MTP and PTP, respectively) was indicated and whether national guidelines were followed. All data extraction and analyses were performed retrospectively. RESULTS: After exclusion criteria were applied, 340 cases remained. PTP was indicated in 26 patients (7.6%) but only 4 patients were treated besides 12 patients who were already in anticoagulant treatment at submission. Conversely, 8/306 patients, in whom TP was not indicated, were started on PTP. MTP was indicated in 8/340 patients (2.4%) but therapy was not initiated in any of them. The majority (320/340, 94.1%) of cases was managed in accordance with existing guidelines. However, this high proportion was mainly explained by the large number of untreated patients, where TP was not indicated. CONCLUSION: A large proportion of hospitalised medical patients was managed in conflict with national guidelines. A systematic approach to TP in patients with acute medical illness should be implemented. Plain language summary available for this article.

Expression, Purification and Characterization of GMZ2'.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite.

PURPOSE: Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. METHODS: GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS. RESULTS: CP-GMZ2'.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2'.10C. CP-GMZ2'.10C and IP-GMZ2'.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2'.10C and two reference fusion proteins encompassing similar parts of Pfs48/45. CONCLUSION: GMZ2'.10C, combining GMZ2' and correctly-folded Pfs48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.

Recombinant protein expression in Lactococcus lactis using the P170 expression system.

The use of the Gram-positive bacterium Lactococcus lactis in recombinant protein production has several advantages, including the organism's long history of safe use in food production and the fact that it does not produce endotoxins. Furthermore the current non-dairy L. lactis production strains contain few proteases and can secrete stable recombinant protein to the growth medium. The P170 expression system used for recombinant protein production in L. lactis utilizes an inducible promoter, P170, which is up-regulated as lactate accumulates in the growth medium. We have optimised the components of the expression system, including improved promoter strength, signal peptides and isolation of production strains with increased productivity. Recombinant proteins are produced in a growth medium with no animal-derived components as a simple batch fermentation requiring minimal process control. The accumulation of lactate in the growth medium does, however, inhibit growth and limits the yield from batch and fed-batch processes. We therefore combined the P170 expression system with the REED™ technology, which allows control of lactate concentration by electro-dialysis during fermentation. Using this combination, production of the Staphylococcus aureus nuclease reached 2.5 g L(-1).

Recombinant expression of Laceyella sacchari thermitase in Lactococcus lactis.

Thermitase (EC 3.4.21.66) is a thermostable endo-protease with the ability to convert various food relevant substrates into low-molecular weight peptides. A thermitase produced by Laceyella sacchari strain DSM43353 was found to have a mature amino acid sequence nearly identical to that of the original thermitase isolated from Thermoactinomyces vulgaris. The DSM43353 thermitase gene sequence contains a pro-peptide including parts of an I9 inhibitor motif. Expression of the thermitase gene in the Lactococcus lactis P170 expression system allowed secretion of stable thermitase in an auto-induced fermentation setup at 30°C. Thermitase accumulated in the culture supernatant during batch fermentations and was easily activated at 50°C or by prolonged dialysis. The activation step resulted in an almost complete degradation of endogenous L. lactis host proteins present in the supernatant. Mature activated product was stable at 50°C and functional at pH values between pH 6 and pH 11, suggesting that substrate hydrolysis can be performed over a broad range of pH values. The L. lactis based P170 expression system is a simple and safe system for obtaining food compatible thermitase in the range of 100 mg/L.

The capillary dysfunction hypothesis of Alzheimer's disease.

It is widely accepted that hypoperfusion and changes in capillary morphology are involved in the etiopathogenesis of Alzheimer's disease (AD). This is difficult to reconcile with the hyperperfusion observed in young high-risk subjects. Differences in the way cerebral blood flow (CBF) is coupled with the local metabolic needs during different phases of the disease can explain this apparent paradox. This review describes this coupling in terms of a model of cerebral oxygen availability that takes into consideration the heterogeneity of capillary blood flow patterns. The model predicts that moderate increases in heterogeneity requires elevated CBF in order to maintain adequate oxygenation. However, with progressive increases in heterogeneity, the resulting low tissue oxygen tension will require a suppression of CBF in order to maintain tissue metabolism. The observed biphasic nature of CBF responses in preclinical AD and AD is therefore consistent with progressive disturbances of capillary flow patterns. Salient features of the model are discussed in the context of AD pathology along with potential sources of increased capillary flow heterogeneity.

Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum.

In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Gel bands were excised and in-gel digested with trypsin. The resulting peptides were analysed by capillary-LC-ESI-MS/MS. The peptide sequences were used for a database search and allowed identification of a total of 29 proteins, many of which could potentially be involved in the action of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics.

Flavobacterium sp. strain 4221 and Pedobacter sp. strain 4236 beta-1,3-glucanases that are active at low temperatures.

Secretion of beta-1,3-glucanases by the arctic bacterial isolates 4221 and 4236, related to the genera Flavobacterium and Pedobacter, was discovered. Escherichia coli and Lactococcus lactis expression of beta-1,3-glucanases Glc4221-1 and Glc4236-1 from the respective isolates was achieved. The enzymes hydrolyzed fungal cell walls and retained activity at low temperatures.

Extracellular secretion of a maltogenic amylase from Lactobacillus gasseri ATCC33323 in Lactococcus lactis MG1363 and its application on the production of branched maltooligosaccharides.

A maltogenic amylase gene from Lactobacillus gasseri ATCC33323 (LGMA) was expressed in Lactococcus lactis MG1363 using the P170 expression system. The successful production of recombinant LGMA (rLGMA) was confirmed by the catalytic activity of the enzyme in liquid and solid media. The N-terminal amino acid sequencing analysis of the rLGMA showed that it was Met-Gln-Leu-Ala-Ala-Leu-, which was the same as that of genuine protein, meaning the signal peptide was efficiently cleaved during secretion to the extracellular milieu. The optimal reaction temperature and pH of rLGMA (55 degrees C and pH 5, respectively) and enzymatic hydrolysis patterns on various substrates (beta-cyclodextrin, starch, and pullulan) supported that rLGMA was not only efficiently secreted from the Lactococcus lactis MG1363 but was also functionally active. Finally, the branched maltooligosaccharides were effectively produced from liquefied corn starch, by using rLGMA secreted from Lactococcus lactis, with a yield of 53.1%.

Production of Recombinant Peanut Allergen Ara h 2 using Lactococcus lactis.

BACKGROUND: Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. RESULTS: A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. CONCLUSION: Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.

Immunological analysis of a Lactococcus lactis-based DNA vaccine expressing HIV gp120.

For reasons of efficiency Escherichia coli is used today as the microbial factory for production of plasmid DNA vaccines. To avoid hazardous antibiotic resistance genes and endotoxins from plasmid systems used nowadays, we have developed a system based on the food-grade Lactococcus lactis and a plasmid without antibiotic resistance genes. We compared the L. lactis system to a traditional one in E. coli using identical vaccine constructs encoding the gp120 of HIV-1. Transfection studies showed comparable gp120 expression levels using both vector systems. Intramuscular immunization of mice with L. lactis vectors developed comparable gp120 antibody titers as mice receiving E. coli vectors. In contrast, the induction of the cytolytic response was lower using the L. lactis vector. Inclusion of CpG motifs in the plasmids increased T-cell activation more when the E. coli rather than the L. lactis vector was used. This could be due to the different DNA content of the vector backbones. Interestingly, stimulation of splenocytes showed higher adjuvant effect of the L. lactis plasmid. The study suggests the developed L. lactis plasmid system as new alternative DNA vaccine system with improved safety features. The different immune inducing properties using similar gene expression units, but different vector backbones and production hosts give information of the adjuvant role of the silent plasmid backbone. The results also show that correlation between the in vitro adjuvanticity of plasmid DNA and its capacity to induce cellular and humoral immune responses in mice is not straight forward.

Safety of ESAT-6.

A recombinant dimer of the Mycobacterium tuberculosis (MTb) 6 kDa early secreted antigenic target (ESAT-6) was produced in Lactococcus lactis. Pharmacodynamic and safety studies were carried out in guinea pigs, rats, mice and dogs with intradermal (id), subcutaneous (sc) and intravenous (iv) administration of the antigen. In contrast to tuberculin purified protein derivative (PPD) the recombinant dimer (rdESAT-6) was able to discriminate MTb infection from BCG vaccination in vivo. In guinea pigs sensitized by infection with MTb, 1 microg rdESAT-6 gave a mean delayed-type hypersensitivity (DTH) response of 22 mm, a significantly stronger reaction than in animals sensitised by the environmental mycobacteria M. kansasii, M. szulgai and M. marinum. rdESAT-6 proved to be a safe tuberculin reagent in a dose range of 1-1000 microg with no or only minor local reactions.

Two acid-inducible promoters from Lactococcus lactis require the cis-acting ACiD-box and the transcription regulator RcfB.

We previously characterized three Lactococcus lactis promoters, P170, P1 and P3, which are induced by low pH. Here, we identified a novel 14 bp regulatory DNA region centred at around -41.5 and composed of three tetranucleotide sequences, boxes A, C and D. Boxes A and C contribute to P1 activity, whereas box D and the position of boxes ACD (renamed ACiD-box) are essential to P1 activity and acid response. We also identified a trans -acting protein, RcfB, which is involved in P170 and P1 basal activity and is essential for their pH induction. The regulator belongs to the Crp-Fnr family of transcription regulators. Overexpression of rcfB resulted in increased beta-galactosidase activities and lantibiotic lacticin 481 production from P170- and P1-controlled genes, respectively, in acid condition. RcfB is thus probably activated when cells encounter an acid environment. rcfB is co-transcribed with genes encoding an universal stress-like protein and a multidrug transporter. RcfB plays a role in acid adaptation, as the survival rate of an rcfB mutant after a lethal acid challenge was 130-fold lower than that of the wild-type strain, when the bacteria were first grown in acidic medium. The groESL promoter includes a sequence resembling an ACiD-box and the chaperone GroEL production is partly RcfB dependent in acid condition. Our results suggest that the ACiD-box could be the DNA target site of RcfB.

Expression of the Staphylococcus aureus surface proteins HtrA1 and HtrA2 in Lactococcus lactis.

Staphylococcus aureus encodes two HtrA-like serine surface proteases, called HtrA1 and HtrA2. The roles of these HtrA homologs were distinguished by expression studies in a heterologous host, Lactococcus lactis, whose single extracellular protease, HtrA(Ll), was absent. HtrA(Ll) is involved in stress resistance, and processing and/or degradation of extracellular proteins. Controlled expression of staphylococcal htrA1 and htrA2 was achieved in L. lactis strain NZ9000 DeltahtrA, as confirmed with anti-HtrA1 and anti-HtrA2 specific antibodies. HtrA1 fully restored thermo-resistance to the htrA-defective L. lactis strain, despite a poor capacity to degrade abnormal or truncated proteins. We therefore propose that activities of HtrA1 other than proteolysis may be sufficient for high-temperature growth complementation. HtrA2 is 36% identical to HtrA(Ll), and was highly expressed in L. lactis; nevertheless, it displayed nearly no detectable activities. The poor proteolytic activities of staphylococcal HtrA proteins in L. lactis may reflect a requirement for S. aureus-specific co-factors.

Research report: do general practitioners tell their patients about side effects to common treatments?

The principle of respect for patients' autonomy, or right to self-determination, has gained increasing importance in health care legislation during the last decade. To respect this principle the patients' informed consent to a proposed treatment is required. In relation to ordinary treatments in general practice, where several reasonable alternatives may be available and where non-treatment may be an acceptable alternative, this requirement is at least as strong as in other parts of the health care system. In this context, information about side effects may be crucial for the patient's decision to accept a proposed treatment or not. The aims of this study were to investigate the extent to which general practitioners in Denmark inform their patients about possible side effects without being asked when a common treatment is proposed. We also wished to examine the relation between physicians' estimation of the severity and frequency of these side effects, and their willingness to inform patients spontaneously as well as their preferred reasons for choosing to inform or not inform the patients. A questionnaire was sent to a random sample of 450 Danish general practitioners. The respondents differed considerably with regard to their willingness to inform patients about side effects but they were significantly more likely to give the information spontaneously if they considered the side effects frequent than when side effects were considered rare. In contrast, estimations of severity did not seem to be of any importance. The majority of the respondents informed their patients primarily to enable them to react appropriately to the side effects in question or to make sure that the patient would comply with the treatment. These findings indicate that the information given to patients about side effects by Danish general practitioners is not in accordance with the principle of respect for the patients' autonomy and not in accordance with the requirements of Danish legislation.

A Plasmodium falciparum GLURP-MSP3 chimeric protein; expression in Lactococcus lactis, immunogenicity and induction of biologically active antibodies.

Plasmodium falciparum malaria is a major cause of morbidity and mortality worldwide. To evaluate the efficacy of a possible vaccine antigen against P. falciparum infection, a fusion protein, derived from P. falciparum Glutamate-rich protein (GLURP) genetically coupled to P. falciparum Merozoite surface protein 3 (MSP3) was produced in Lactococcus lactis as a secreted recombinant GLURP-MSP3 fusion protein. The hybrid protein was purified to homogeneity by ion exchange and hydrophobic-interaction chromatography and its composition was verified by MALDI MS, SDS/PAGE and Western blotting with antibodies against antigenic components of GLURP and MSP3. Mice immunized with the hybrid protein produced higher levels of both GLURP- and MSP3-specific antibodies than mice immunized with either GLURP, MSP3 or a mix of both. The protective potential of the hybrid protein was also demonstrated by in vitro parasite-growth inhibition of mouse anti-GLURP-MSP3 IgG antibodies in a monocyte-dependent manner. These results indicate that the GLURP-MSP3 hybrid could be a valuable strategy for future P. falciparum vaccine development.

Melanoma-associated spongiform scleropathy: biochemical changes and possible relation to tumour extension.

PURPOSE: To investigate biochemical changes of the sclera in eyes with melanoma-associated spongiform scleropathy (MASS), and to analyse possible relationships between these changes and tumour extension. METHODS: Sections from 364 eyes, enucleated for choroidal and ciliary body melanoma, were examined for MASS and scleral tumour extension. Biochemical analysis was also performed on eight scleral specimens with MASS and eight specimens (controls) from morphologically normal sclera of the same eyes. The scleral thickness of each specimen was measured. Samples were delipidized, dried and weighed. The weight ratios of collagen-related amino acids were calculated based on quantitation by liquid chromatography. Amounts of glycosaminoglycans (GAGs) were determined by electrophoresis. RESULTS: Melanoma-associated spongiform scleropathy was seen in 140 eyes (38.5%). Tumour scleral extension was observed in 82 eyes. Of these 82 eyes, 75 (91.5%) had MASS (p<0.05). Biochemically, the majority of the main amino acids of the scleral collagen and total proteins were significantly lower in areas with MASS than in the control specimens. Specific GAGs and total GAGs were found in significantly higher concentrations in areas with MASS than in the control specimens. Scleral thickness was also significantly higher in areas with MASS than in the control specimens. CONCLUSIONS: The reduced content of collagen manifested by decreased amino acids and total proteins indicates collagen degradation in the vicinity of the tumour. The concomitant excessive deposition of GAGs accumulates water and may cause loosening of the already degraded collagen bundles, giving a histopathological picture of MASS. These changes could facilitate tumour cell migration and may explain the high incidence of MASS in eyes with scleral tumour extension.

Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis.

The authors have previously reported the identification of novel signal peptides (SPs) from Lactococcus lactis using transposon insertion. Of these, SP310 caused the highest level of secretion. However, the levels were lower than those obtained using the signal peptide from Usp45 (SPUSP), the major secreted lactococcal protein. In this study, site-directed mutagenesis of signal peptide SP310 was used to investigate the effect of amino acid alterations on lactococcal secretion and to improve secretion efficiency. Several mutated SPs caused higher secretion. This increase in secretion was due to modifications in the cleavage region. In fermenter experiments, the signal peptide SP310mut2 resulted in an extracellular Staphylococcus aureus nuclease (Nuc) yield which was 45 % higher than that with the natural SP310. Surprisingly, increasing the hydrophobicity of the hydrophobic core or increasing the number of positively charged amino acids in the N-terminal region of SP310 decreased secretion. High extracellular yields of Nuc resulted from more efficient secretion, as strains with less efficient SPs accumulated more intracellular SP-Nuc precursor.

Decrease of collagen deposition in wound repair in type 1 diabetes independent of glycemic control.

HYPOTHESIS: Type 1 and type 2 diabetes mellitus and glycemic control influence wound healing in humans. DESIGN: Experimental study using a human wound-healing model. SETTING: Collaboration among a multidisciplinary wound-healing department, department of medicine, and research laboratories. PATIENTS, CONTROL SUBJECTS, AND METHODS: In 34 patients with type 1 (insulin-dependent) and 25 with type 2 (non-insulin-dependent) diabetes and 5 nondiabetic control subjects matched with the type 2 diabetic patients, wound-healing capacity was determined as subcutaneous accumulation of collagen measured as hydroxyproline. Two expanded polytetrafluoroethylene tubes were implanted and removed 10 days later. The hydroxyproline level was determined by means of high-performance liquid chromatography; the collagenase activity, by using a radiolabeled collagen substrate. Proliferation of fibroblasts cultured from the wounds was studied in patient groups. RESULTS: The deposition of hydroxyproline decreased by 40% (P =.03) in type 1 compared with type 2 diabetes (median, 0.70 vs 1.16 nmol/mg; interquartile range, 0.48-1.04 vs 0.56-1.63 nmol/mg), which in turn did not differ significantly from that of controls (median, 1.35 nmol/mg; interquartile range, 0.72-1.88 nmol/mg). The decreased collagen deposition in type 1 diabetes was not caused by increased collagenase activity. The deposition of hydroxyproline did not correlate significantly (r(s) = 0.07; P =.63) with glycosylated hemoglobin levels in either diabetic group. Fibroblast growth was also decreased in type 1 compared with type 2 diabetic patients and controls. CONCLUSIONS: Collagen deposition in acute wounds is impaired in type 1 diabetes, possibly due to a decreased fibroblast proliferation. In type 2 diabetes, collagen deposition is normal. Glycemic control does not influence collagen deposition in acute wound repair in type 1 or in type 2 diabetes mellitus.

Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor.

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage phi31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage phi31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage phi31 was inhibited to an efficiency of plaquing (EOP) of 10(-6) in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the phi31 EOP to <10(-7). Phage phi31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the phi31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.