Chromosomal constraints in Gram-positive bacteria revealed by artificial inversions.

We used artificial chromosome inversions to investigate the chromosomal constraints that preserve genome organization in the Gram-positive bacterium Lactococcus lactis. Large inversions, 80-1260 kb in length, disturbing the symmetry of the origin and terminus of the replication axis to various extents, were constructed using the site-specific Cre-loxP recombination system. These inversions were all mechanistically feasible and fell into various classes according to stability and effect on cell fitness. The L. lactis chromosome supports only to some extent unbalance in length of its replication arms. The location of detrimental inversions allowed identification of two constrained chromosomal regions: a large domain covering one fifth of the genome that encompasses the origin of replication (Ori domain), and a smaller domain located at the opposite of the chromosome (Ter domain).

[Intimal muscular fibrodysplasia responsible for an ischemic gastric ulcer in a patient with a von Recklinghausen's disease: a case report]. / Fibrodysplasie musculaire artérielle intimale responsable d'un ulcère gastrique ischémique chez une patiente ayant une maladie de von Recklinghausen: un cas.

A 78 year-old woman, suffering from a von Recklinghausen's disease sought medical assistance for hematemesis with anemia. This patient had previously experienced an amputation of the right arm for gangrene. Gastric fibroscopy unveiled a deep chronic ulcus developed in the antrum, highly suspect of malignancy. Multiple biopsies of the ulcer showed mainly interstitial gastritis. The persistence of the hematemesis imposed a subtotal gastrectomy. Pathological examination of the operative specimen evidenced an ischemic ulcer caused by arterial intimal muscular fibrodysplasia with associated neurofibromatosis in the neighboring sub-mucosal layer. This case report highlights the frequent association of phacomatosis especially von Recklinghausen's disease, with vascular lesions whose clinical expression mainly depends on the involved vascular area.

Genome plasticity among related ++Lactococcus strains: identification of genetic events associated with macrorestriction polymorphisms.

The genomic diversity of nine strains of the Lactococcus lactis subsp. cremoris (NCDO712, NCDO505, NCDO2031, NCDO763, MMS36, C2, LM0230, LM2301, and MG1363) was studied by macrorestriction enzyme analysis using pulsed-field gel electrophoresis. These strains were considered adequate for the investigation of genomic plasticity because they have been described as belonging to the same genetic lineage. Comparison of ApaI and SmaI genome fingerprints of each strain revealed the presence of several macrorestriction fragment length polymorphisms (RFLPs), despite a high degree of similarity of the generated restriction patterns. The physical map of the MG1363 chromosome was used to establish a genome map of the other strains and allocate the RFLPs to five regions. Southern hybridization analysis correlated the polymorphic regions with genetic events such as chromosomal inversion, integration of prophage DNA, and location of the transposon-like structures carrying conjugative factor or oligopeptide transport system.

A natural large chromosomal inversion in Lactococcus lactis is mediated by homologous recombination between two insertion sequences.

Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related Lactococcus lactis subsp. cremoris strains MG1363 and NCDO763 revealed the presence of a large inversion covering half of the genome. To determine what kind of genetic element could be implicated in this rearrangement, the two inversion junctions of MG1363 and NCDO763 chromosomes were cloned and characterized. Nucleotide sequence analysis showed the presence of one copy of the lactococcal IS905 element in each junction. Each copy of this element contained the same nucleotide mutation that inactivates the putative transposase. Comparison of the sequences surrounding the insertion sequence demonstrated that the large inversion arose from a single-step homologous recombination event between the two defective copies of the IS905 element. The large inversion presumably conferred no selective disadvantage on strain NCDO763 because this rearrangement did not alter the oriC-terC symmetry of the chromosome and the local genetic environment.

Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion.

A physical and genetic map of the chromosome of the Lactococcus lactis subsp. cremoris reference strain MG1363 was established. The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments. The MG1363 chromosome appeared to be circular and 2,560 kb long. Seventy-seven chromosomal markers were located on the physical map by hybridization experiments. Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome. The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction. Comparison of the L. lactis subsp. cremoris MG1363 physical map with those of the two L. lactis subsp. lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism. At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.

Chromosome mapping in lactic acid bacteria.

The chromosome structure of lactic acid bacteria has been investigated only recently. The development of pulsed-field gel electrophoresis (PFGE) combined with other DNA-based techniques enables whole-genome analysis of any bacterium, and has allowed rapid progress to be made in the knowledge of the lactic acid bacteria genome. Lactic acid bacteria possess one of the smallest eubacterial chromosomes. Depending on the species, the genome sizes range from 1.1 to 2.6 Mb. Combined physical and genetic maps of several species are already available or close to being achieved. Knowledge of the genomic structure of these organisms will serve as a basis for future genetic studies. Macrorestriction fingerprinting by PFGE is already one of the major tools for strain differentiation, identification of individual strains, and the detection of strain lineages. The genome data resulting from these studies will be of general application strain improvement.

Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403.

A combined physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403 was determined. We constructed a restriction map for the NotI, ApaI, and SmaI enzymes. The order of the restriction fragments was determined by using the randomly integrative plasmid pRL1 and by performing indirect end-labeling experiments. The strain IL1403 chromosome was found to be circular and 2,420 kb in size. A total of 24 chromosomal markers were mapped on the chromosome by performing hybridization experiments with gene probes for L. lactis and various other bacteria. Integration of pRC1-derived plasmids via homologous recombination allowed more precise location of some lactococcal genes and allowed us to determine the orientation of these genes on the chromosome. Recurrent sequences, such as insertion elements and rRNA gene (rrn) clusters, were also mapped. At least seven copies of IS1076 were present and were located on 50% of the chromosome. In contrast, no copy of ISS1RS was detected. Six ribosomal operons were found on the strain IL1403 chromosome; five were located on 16% of the chromosome and were transcribed in the same direction. A comparison of the physical maps of L. lactis subsp. lactis IL1403 and DL11 showed that these two strains are closely related and that the variable regions are located mainly near the rrn gene clusters. In contrast, despite major restriction pattern dissimilarities between L. lactis IL1403 and MG1363, the overall genetic organization of the genome seems to be conserved between these two strains.

New tools for the physical and genetic mapping of Lactococcus strains.

Tools for the genetic and physical analysis of the Lactococcus lactis subsp. lactis genome were developed. Plasmid pRC1 does not replicate in Gram+ bacteria; it contains unique ApaI, NotI and SmaI restriction sites and an erythromycin-resistance (ErR) encoding gene, ermAM, functional in L. lactis subsp. lactis. When a chromosomal L. lactis subsp. lactis DNA fragment was cloned into this vector, the resulting plasmid became integrated, after transformation, into the bacterial chromosome by homologous recombination in a Campbell-like manner. The integration lead to the generation of new rare restriction sites near to the host fragment. This procedure allows precise mapping of cloned genes onto the chromosomal restriction map. The mapping of the his operon of L. lactis subsp. lactis provides an illustration. The cloning into pRC1 of an IS element able to transpose into the chromosome of the target cell, gave rise to an integration plasmid able to insert randomly rare restriction sites onto the bacterial chromosome. The L. lactis IS element, ISS1RS, was cloned into pRC1, yielding pRL1. Pulsed-field gel electrophoresis analysis of ErR clones obtained after transformation with pRL1, showed that this plasmid was stably integrated at a number of different sites in the L. lactis subsp. lactis chromosome, via transposition. Plasmids pRC1 and pRL1 can greatly facilitate the construction of the physical and genetic map of the chromosome of lactococcal strains.

Genome comparison of Lactococcus strains by pulsed-field gel electrophoresis.

The two restriction enzymes SmaI and ApaI were found to produce distributions of DNA fragments useful for genome analysis of some lactic acid bacteria (Lactococcus lactis and Streptococcus salivarius subsp. thermophilus) by pulsed-field gel electrophoresis. The genome size was estimated to be 1750 to 2500 kb depending on the species. Each strain displayed unique restriction patterns; nevertheless, the percentage of the comigrating fragments of two isogenic or closely related strains was about 80% and fell to 20-40% when the patterns of two non-related strains of the same species or two strains belonging to different species were compared.

[Peri-anal ulceration. Tuberculosis should not be overlooked]. / Ulcération péri-anale. Ne pas oublier la tuberculose.

Two cases of peri-anal ulceration of tuberculous origin are reported: one revealed an active pulmonary and ileo-caecal tuberculosis, the other complicated a chronic pulmonary tuberculosis of several years' duration. Cutaneous manifestations of tuberculosis are exceptional. In patients with protracted peri-anal ulceration, a biopsy should be performed that will show a typical tuberculoid granuloma. The most frequently encountered anorectal tuberculous lesions are suppurations and fistulae. The main differential diagnosis is Crohn's disease with anorectal manifestations.

[Diffuse recto-colic malacoplakia: association with cryptogenic colitis and remission following long-term antibiotic therapy]. / Malakoplakie recto-colique diffuse: association à une colite cryptogénétique et rémission après antibiothérapie prolongée.

Diffuse digestive malakoplakia appears exceptional. A case of rectocolic malakoplakia with multiple localisations is reported in a 22 year old man presenting with inflammatory bowel disease. The most prominent clinical features were deterioration of his general condition, fever, and rectal bleeding, with fistula. Endoscopy revealed pseudotumoral masses and multiple colorectal ulcerations. Diagnosis was based on histological examination of colorectal biopsies. Clinical and histological remission was obtained after colonic diversion associated with broad spectrum antibiotherapy. These findings raise the problem of the possible association between inflammatory bowel disease and malakoplakia. They also confirm that, as previously reported, favourable outcome in digestive malakoplakia is possible.