The replication-recombination connection: insights from genomics.

One amazing characteristic of DNA processing enzymes is their breakneck speed. However, considering the metabolic traffic on DNA, it is not surprising that accidents that interrupt the replication process and require immediate repair occur. Recombination is an example of a repair mechanism, and as a result of accidents, the arrested or broken fork becomes the recombination substrate. Repair proteins are on hand to assist at accident sites, but responses may be sensitive to conditions and depend on the organism. Genomics has been used to identify genetic clues that indicate the occurrence of accidents and to detect sites of recombination. DNA and protein features that may affect replication and recombination are examined and discussed.

Improvement of bovine beta-lactoglobulin production and secretion by Lactococcus lactis.

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Characteristics of Chi distribution on different bacterial genomes.

The availability of full genome sequences provides the bases for analyzing global properties of the genetic text. For example, oligonucleotide sequences that are over- or underrepresented can be identified by taking into account the overall genome composition and organization. One of the most overrepresented oligonucleotides in Escherichia coli is the Chi site, an octanucleotide that stimulates DNA repair by homologous recombination. Here we analyze the genomic distribution of Chi in E. coli and in the three other bacteria where a Chi sequence has been identified; note that Chi is a different sequence in each organism. For each bacterial genome, Chi sequences are frequent, regularly distributed, and overrepresented. This suggests that selection for Chi may have occurred during evolution to favor efficient repair of a damaged chromosome. Other characteristics of Chi distribution are not conserved and might reflect specific features of DNA repair in each host. The different sequence and characteristics of Chi in each microorganism suggest that selection for Chi occurred independently in different bacteria.

Distinctive features of homologous recombination in an 'old' microorganism, Lactococcus lactis.

Homologous recombination is needed to assure faithful inheritance of DNA material, especially under stress conditions. The same enzymes that repair broken chromosomes via recombination also generate biodiversity. Their activities may result in intrachromosomal rearrangements, assimilation of foreign DNA, or a combination of these events. It is generally supposed that homologous recombination systems are conserved, and function the same way everywhere as they do in Escherichia coli, the accepted paradigm. Studies in an 'older' microorganism, the gram-positive bacterium of the low GC branch Lactococcus lactis, confirm that many enzymes are conserved across species lines. However, the main components of the double strand break (DSB) repair system, an exonuclease/helicase (Exo/hel) and a short DNA modulator sequence Chi, differ markedly between bacteria, especially when compared to the gram-negative analogues. Based on our studies, a model is proposed for the functioning of the two-subunit Exo/hel of L. lactis and other gram-positive bacteria, which differs from that of the three-subunit E. coli enzyme. The differences between bacterial DSB repair systems may underlie a selection for diversity when dealing with DSB. These and other features of homologous recombination in L. lactis are discussed.

Vaginal clear cell carcinoma in a young patient with ectopic termination of the left ureter in the vagina.

The association of clear cell adenocarcinoma of the vagina and vaginal adenosis with prenatal exposure to diethylstilbestrol (DES) is well-documented in the United States. In Europe, however, DES was never used in the therapy of threatened abortion and, therefore, clear cell adenocarcinoma and vaginal adenosis remained rare diseases. We report on the clinical and pathological features of a case of clear cell adenocarcinoma of the upper vagina in a 17-year-old German girl, who had a history of hypoplasia of the left kidney with an ectopic termination of the ureter in the upper vagina, removed surgically 2 years before. No previous report of a similar coincidence of vaginal clear cell carcinoma and a congenital disorder of the genitourinary tract exists. Congenital anomaly of the ureter interfering with the development and the differentiation of the distal Müllerian tract and its epithelium might have provided a similar histological basis for carcinogenesis in our patient to that in those provided exposed to DES.

Treatment results and long-term follow-up in stage I seminoma patients.

Between 1979 and 1992 seventy-nine patients with seminoma were treated at our institution, 62 of them with stage I. The mean follow-up time was 6.0 years (range: 36 months to 14 years). Preoperatively, serum beta human chorionic gonadotropin (beta-HCG) was elevated in 12 cases (19%) without prognostic significance. In addition to orchiectomy, 57 patients with stage I seminoma of the testis received adjuvant radiotherapy (mean dose: 33 Gy). Two patients were treated with primary retroperitoneal lymph node dissection (RPLND) and one patient with cisplatin-based chemotherapy. In 2 cases a surveillance strategy was used. Three patients (5%) had a relapse of the seminoma (2 in the retroperitoneum and one suprainguinally). The time interval between orchiectomy and relapse was 5 to 60 months. Salvage treatment consisted of chemotherapy and RPLND in 2 patients and chemotherapy and resection of the suprainguinal recurrent mass in one patient, and was successful in all 3 patients. A total of 60 patients evaluated (100%) are still alive with no evidence of disease. In conclusion, adjuvant radiotherapy is considered the routine treatment in seminomas stage I despite studies with a 'wait and watch' policy or a carboplatin monotherapy.

Lactococcus lactis, a bacterial model for stress responses and survival.

The dairy organism, Lactococcus lactis, is continuously exposed to stress conditions generated during industrial processes. To identify the mechanisms that confer resistance to the lethal effects of oxygen and thermal stress, we isolated resistant strains by insertional mutagenesis. Mutated genes were identified and mutations were shown to confer resistance to multiple stresses (including non-selected stresses such as carbon starvation). Our results revealed that metabolic flux plays an important role in L. lactis stress response, and suggested that phosphate and guanine pools may be intracellular stress sensors. As previously shown, we also observed an increase of stress resistance during the stationary phase. We have evidence that stationary phase actually initiates very early during growth. Taken together, these data show that the stationary phase is a very complex system with multiple participants interacting altogether. These results reinforce the idea of the interdependence of stress response and the intimate relation between metabolic flux and stress responses in L. lactis.

[Immunochemotherapy of metastatic renal cell carcinoma with interleukin 2, interferon-alpha and 5-fluorouracil]. / Immunchemotherapie des metastasierten Nierenzellkarzinoms mit Interleukin-2, Interferon-alpha und 5-Fluorouracil.

Interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) were both administered subcutaneously (SC) in combination with intravenously (IV) applied 5-fluorouracil (5-FU) for the treatment of patients with metastasized renal cell carcinoma (RCC). The therapy protocol consisted of a treatment cycle of 8 weeks, which could be carried out in an outpatient regimen. The IFN-alpha was given in each of the 8 weeks (6-9 MU/m2 once to three times weekly SC) combined sequentially with IL-2 (5-20 MU/m2 three times weekly SC for 4 weeks) and 5-FU (750 mg/m2 IV weekly for 4 weeks). Among the 30 consecutive patients treated, in 2 cases a complete, and in 9 cases a partial, remission was achieved in patients with mostly lung and skeletal metastases, with an overall objective response rate of 37%. Mean response duration was 8 months (range 3-18 months). A stable state of the disease lasting 3-18 months was observed in 10 cases. The side effects were only slight and corresponded to toxicity grade I (n = 2), grade II (n = 22) and grade III (n = 6), according to the WHO classification. In conclusion, this triple-drug biochemotherapy demonstrated significant clinical effectiveness comparable with that of an aggressive IL-2 treatment regimen (applied IV), but without its high toxicity.

Complementation of the Lactococcus lactis secretion machinery with Bacillus subtilis SecDF improves secretion of staphylococcal nuclease.

Unlike Bacillus subtilis and Escherichia coli, the gram-positive lactic acid bacterium Lactococcus lactis does not possess the SecDF protein, a component of the secretion (Sec) machinery involved in late secretion stages and required for the high-capacity protein secretion in B. subtilis. In this study, we complemented the L. lactis Sec machinery with SecDF from B. subtilis and evaluated the effect on the secretion of two forms of staphylococcal nuclease, NucB and NucT, which are efficiently and poorly secreted, respectively. The B. subtilis SecDF-encoding gene was tested in L. lactis at different levels. Increased quantities of the precursor and mature forms were observed only at low levels of SecDF and at high NucT production levels. This SecDF secretion enhancement was observed at the optimal growth temperature (30 degrees C) and was even greater at 15 degrees C. Furthermore, the introduction of B. subtilis SecDF into L. lactis was shown to have a positive effect on a secreted form of Brucella abortus L7/L12 antigen.

Plasmid instability in regenerating protoplasts of Staphylococcus aureus is caused by aberrant cell division.

Elimination of plasmids from regenerating S. aureus protoplasts occurred when the regeneration medium contained sucrose but not when it contained sodium succinate. This difference was caused by the occurrence of cell division prior to regeneration of the cell wall on sucrose but not on succinate. Coexisting compatible plasmids were cured independently; coexisting incompatible plasmids were cured jointly. These results support the hypothesis that plasmid pools exist as physically sequestered units in protoplasts and that curing is a consequence of the segregation of such units during abnormal division of wall-less organisms.

Insertion of foreign DNA into plasmids from gram-positive bacteria induces formation of high-molecular-weight plasmid multimers.

Plasmids pUB110, pC194, pE194, and pT181 are commonly used as cloning vectors in both Bacillus subtilis and Staphylococcus aureus. We report that insertion of foreign DNA into any of these plasmids results in the generation of high-molecular-weight plasmid multimers (HMW) of the recombinant, present as tandem head-to-tail copies. HMW was detected in wild-type B. subtilis and S. aureus strains. The production of HMW depended on the nature of the DNA insertion. Inserts of Escherichia coli DNA, e.g., pBR322 or pUC18, resulted in large amounts of HMW, whereas some inserts of S. aureus DNA of the same size had no effect on plasmid profile. The generation of HMW depended on the mode of plasmid replication; plasmids which replicate via a single-stranded DNA intermediate produced HMW upon foreign DNA insertion, whereas plasmid pAM beta 1, which does not generate single-stranded DNA, did not generate HMW. We propose that HMW is a product of imparied termination of rolling-circle replication and that the impairment is due to the nature of the DNA insertion.

The family of highly interrelated single-stranded deoxyribonucleic acid plasmids.

Many plasmids from gram-positive bacteria replicate via a single-stranded deoxyribonucleic acid (ssDNA) intermediate, most probably by a rolling-circle mechanism (these plasmids are referred to in this paper as ssDNA plasmids). Their plus and minus origins are physically separated, and replicative initiations are not simultaneous; it is this feature that allows visualization of ssDNA replication intermediates. The insertion of foreign DNA into an ssDNA plasmid may provoke a high frequency of deletions, changes of replicative products to high-molecular-weight forms, segregational loss, and decreased plasmid copy numbers. When an ssDNA plasmid is inserted into the chromosome, both deletions and amplifications may be induced. Both the mode of replication and the copy control mechanism affect the fate of inserted foreign material, usually selecting for its loss. Thus, after having tasted various morsels of DNA, the resulting plasmid stays trim. The features of the ssDNA plasmids seem to be beneficial for their viability and propagation, but not for their use as cloning vectors. However, plasmids replicating via ssDNA intermediates are being exploited to yield insights into the mechanisms of recombination and amplification.

Chemotherapy in advanced seminoma and the role of postcytostatic retroperitoneal lymph node dissection.

Between 1979 and 1992, 79 patients with seminoma were treated at our institution. Nineteen of these patients with advanced seminoma were treated with cisplatin-based chemotherapy (stage IIA, n = 2; stage IIB, n = 6; stage IIC, n = 2; stage III, n = 5 [2 with primary extragonadal tumor site]; relapse, n = 5 [1 with previously stage III seminoma]). One patient died of progressive disease 3 months after treatment with chemotherapy and retroperitoneal lymph node dissection (RPLND) which had led initially to a complete clinical response. Another patient (62 years old) died of an acute heart failure due to a sepsis caused by chemotherapy. Treatment of the other 17 patients was successful. Ten patients with a residual retroperitoneal mass after inductive chemotherapy underwent a RPLND. In 1 case viable seminoma (diameter of the residual mass 5 cm), in 4 cases necrotic tumor tissue, and in 5 cases fibrosis was diagnosed histopathologically. The 2 patients with extragonadal seminoma showed complete responses after surgery and chemotherapy. In conclusion, in patients with advanced seminoma, inductive cytostatic therapy seems to be the best treatment regimen. Residual retroperitoneal masses after chemotherapy with a diameter of > 3 cm should be treated with RPLND.

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 degrees 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.

Acid- and multistress-resistant mutants of Lactococcus lactis : identification of intracellular stress signals.

Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid-resistant insertional mutants of the L. lactis strain MG1363. Twenty-one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress-resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.

Evaluation of the T stage of carcinoma of the bladder by transurethral ultrasonography.

In order to evaluate the accuracy of transurethral ultrasonography in predicting the T stage of bladder tumors a prospective study was initiated. Transurethral ultrasonography was performed in 308 consecutive patients prior to cystoscopy, transurethral resection, biopsy or cystectomy. The findings of ultrasonography were compared with the histopathologic results. In 218 out of the 308 patients a carcinoma of the bladder was diagnosed histologically, whereas in 90 patients a tumor could not be detected on histological examination. In 78.2% of those patients, in whom a carcinoma of the bladder was present, it was possible to predict the correct T stage. It was not possible, however, to distinguish tumors of stage TA from those of stage T1. Further analysis of the results revealed an overstaging of the tumor in 12.0%, whereas in 8.0% of the cases the tumor was understaged or had not been recognized at all by ultrasonography.

Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.

Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp. lactis ML3. Using this fragment, we recovered and sequenced the entire lactococcal recA gene. The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair.