[Bilateral panuveitis in Whipple's disease: Case report]. / Panuvéite bilatérale dans la maladie de Whipple : à propos d'un cas.

Whipple's disease is a rare disease linked to chronic infection with the intracellular gram-positive bacterium, Tropheryma whipplei. The clinical signs suggestive of this disease are the association of unexplained fever, lymphadenopathy, gastroenterological disorders (malabsorption) and inflammatory joint disorders (arthritis). However, isolated cardiological, neurological or ophthalmological forms have been described. We report the rare case of a 56-year-old patient complaining of floaters and recent visual loss, who presented with bilateral panuveitis in the absence of any systemic disorder. Clinical examination showed inflammation of the anterior segment, vitritis, inflammatory optic disc edema, focal retinitis, and venous vasculitis in both eyes. We describe the clinical characteristics and ancillary findings of the disease (fundus photos, visual field, auto-fluorescence, macular OCT, fluorescein and indocyanine green angiography). The diagnosis was made with the blood (T. whipplei) PCR test and with the help of accessory salivary gland biopsies. We describe the work-up leading to the diagnosis of Whipple's disease, the laboratory tests, and the recommended extended work-up. The patient's course was marked by complete resolution of the symptoms and clinical signs within a few months following corticosteroid therapy (1mg/kg/day) combined with hydroxychloroquine (600mg per day for 1 year) and life-long doxycycline therapy (200mg per day). In conclusion, this is a rare disease which should be discussed when dealing with steroid-resistant and/or steroid-dependent chronic uveitis with a negative work-up (especially in the presence of joint and/or digestive involvement).

Characterization of IS1201, an insertion sequence isolated from Lactobacillus helveticus.

IS1201, a 1387-bp insertion sequence isolated from Lactobacillus helveticus, was identified by its nucleotide (nt) sequence. It carries a single open reading frame encoding a 369-amino-acid protein, which shares homology with transposases found in a class of related IS, including ISRm3 from Rhizobium meliloti, IS256 from Staphylococcus aureus, IS6120 from Mycobacterium smegmatis, IS1081 from M. bovis, IST2 from Thiobacillus ferroxidans and IS406 from Pseudomonas cepacia. IS1201 has terminal inverted repeats of 24 bp in length and a target site duplication of 8 bp. Its copy number ranges from 3 to about 16 per L. helveticus genome. No homology was found between the nt sequence of IS1201 and those of the other bacterial IS from the same class. These results, together with previous observations [de los Reyes-Gavilán et al., Appl. Environ. Microbiol., 58 (1992) 3429-3432], confirm that IS1201 can be used as a specific DNA probe for the identification of L. helveticus strains.

Resistance of 17 mesophilic lactic Streptococcus bacteriophages to pasteurization and spray-drying.

For 17 phages active against Streptococcus cremoris, Str. lactis and Str. lactis subsp. diacetylactis, the killing efficiency of pasteurization (log No/N) at 72 degrees C for 15 s in skim-milk showed large variations from greater than 6 to 0; the efficienty of killing during spray-drying ranged from 3.7 to 0.2 and phages survived well storage of milk powder at room temperature. Destruction in a heat exchanger was found to be greater than that calculated from biphasic curves obtained by heating phages in sealed ampoules. No relationship was established between lytic classification of phages and their heat resistance.

Plasmid-Determined Systems for Restriction and Modification Activity and Abortive Infection in Streptococcus cremoris.

Streptococcus cremoris strain IL964 possessed a restriction and modification (R/M) activity which resulted in a bacteriophage efficiency of plating of 5 x 10. Phage sensitivity of protoplast-induced plasmid-cured derivatives indicated that two plasmids called pIL103 (5.7 kilobases) and pIL107 (15.2 kilobases) were each coding for one R/M system. Plasmid pIL103-encoded R/M was ascertained by transfer into the plasmid-free, R/M strain IL1403 of S. lactis, using protoplast cotransformation. This procedure failed for pIL107 because of some degree of incompatibility between pIL107 and the indicator plasmid pHV1301 used in cotransformation experiments. We also observed that plasmid pIL105 (8.7 kilobases) which showed no incidence on phage sensitivity in the parental strain IL964, mediated abortive infection in strain IL1403. In 97% of the infected cells, the phage infection was abortive, while in the remaining 3% phages were produced with a decreased burst size (50 instead of 180).

Tubular heads in bacteriophages from lactic streptococci.

Tubular bacteriophage heads were observed in the lysate of two phages from Streptococcus lactis obtained from single plaques without mutagenesis. The frequency of appearance of the tubular heads was 2.5 and 16%.

Definition of bacteriophage groups according to their lytic action on mesophilic lactic streptococci.

The lytic activity of 132 phages isolated during slow acid production in cheese factories situated in all the dairying regions of France during the past 16 years has been determined on 291 strains of mesophilic lactic streptococci. The results have been treated according to a method of analysis of data so as to establish a classification. Six groups of phages have thus been formed. Sixty-six percent of the phages studied, which are very similar and for the most part nonspecific to one species, have been gathered together in one group. On the other hand, a classification of the bacterial strains has been made on their sensitivity to the phages. Six groups, each corresponding to one of these groups of phages, have thus been defined. One of them accounts for 40% of the strains studied, of which certain ones are sensitive to a large number of phages.

Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability.

Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.

High-efficiency transformation of Streptococcus lactis protoplasts by plasmid DNA.

Streptococcus lactis IL1403 protoplasts were transformed by plasmid pIL204 (5.5 kilobases), which conferred erythromycin resistance with an average efficiency of 5 X 10(6) transformants per microgram of supercoiled DNA. The procedure used and transformation efficiencies obtained were close to those described for Bacillus subtilis (G. Chang and S. N. Cohen, Mol. Gen. Genet. 168:111-115, 1979).

Sequence and organization of the lactococcal prolate-headed bIL67 phage genome.

bIL67 is a broad-host-range prolate-headed phage that is active against Lactococcus cells. The complete phage genome sequence of 22195 bp was established. Thirty-seven open reading frames (ORFs) organized in two clusters were identified. Functions were assigned to the putative products of six of the ORFs on the basis of comparison of the deduced amino acid sequences to known proteins, analysis of structural features of the proteins and search for conserved motifs. These were a DNA polymerase, a protein involved in recombination, a lysin, a terminase subunit, a structural protein and a holin.

Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome.

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.

Branched-chain amino acid biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes for biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine in Lactococcus lactis subsp. lactis NCDO2118 were characterized by cloning, complementation in Escherichia coli and Bacillus subtilis, and nucleotide sequence analysis. Nine structural genes are clustered on a 12-kb DNA fragment in the order leuABCD ilvDBNCA. Upstream of these genes, the nucleotide sequence suggests the existence of regulation by transcriptional attenuation. Between the leuD and ilvD genes is an unexpected gene, encoding a protein which belongs to the ATP-binding cassette protein superfamily.

Dramatic decay of phage transcripts in lactococcal cells carrying the abortive infection determinant AbiB.

The abortive infection determinant AbiB prevents growth of the sensitive phage bIL170, but not of the resistant phage bIL41, on Lactococcus lactis strain IL1403. Here we show that AbiB promotes a dramatic degradation of sensitive phage transcripts, starting 10-15 min after infection. The decay of the transcripts is the probable cause of the arrest of the sensitive phage development. Mapping of the 5' end of degradation products established that they result from endonucleolytic cleavage preferentially at U/U, A/U and U/A sites. We propose that an early product of the sensitive phage either induces the synthesis or stimulates the activity of an RNase in an AbiB+ cell.

Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1.

Phage bIL66 is unable to grow on Lactococcus lactis cells harboring the abortive infection gene abiD1. Spontaneous phage mutants able to grow on AbiD1 cells were used to study phage-Abi interaction. A 1.33-kb DNA segment of a mutant phage allowed growth of AbiD1s phages in AbiD1 cells when present in trans. Sequence analysis of this segment revealed an operon composed of four open reading frames, designated orf1 to orf4. The operon is transcribed 10 min after infection from a promoter presenting an extended -10 consensus sequence but no -35 sequence. Analysis of four independent AbiD1r mutants revealed a different point mutation localized in orf1, implying that this open reading frame is needed for sensitivity to AbiD1. However, the sensitivity is partly suppressed when orf3 is expressed in trans on a high-copy-number plasmid, suggesting that AbiD1 acts by decreasing the concentration of an available orf3 product.

Lactococcus lactis phage operon coding for an endonuclease homologous to RuvC.

The function of the Lactococcus lactis bacteriophage bIL66 middle time-expressed operon (M-operon), involved in sensitivity to the abortive infection mechanism AbiD1, was examined. Expression of the M-operon is detrimental to Escherichia coli cells, induces the SOS response and is lethal to recA and recBC E. coli mutants, which are both deficient in recombinational repair of chromosomal double-stranded breaks (DSBs). The use of an inducible expression system allowed us to demonstrate that the M-operon-encoded proteins generate a limited number of randomly distributed chromosomal DSBs that are substrates for ExoV-mediated DNA degradation. DSBs were also shown to occur upstream of the replication initiation point of unidirectionally theta-replicating plasmids. The characteristics of the DSBs lead us to propose that the endonucleolytic activity of the M-operon is not specific to DNA sequence, but rather to branched DNA structures. Genetic and physical analysis performed with different derivatives of the M-operon indicated that two orfs (orf2 and orf3) are needed for nucleolytic activity. The orf3 product has amino acid homology with the E. coli RuvC Holliday junction resolvase. By site-specific mutagenesis, we have shown that one of the amino acid residues constituting the active centre of RuvC enzyme (Glu-66) and conserved in ORF3 (Glu-67) is essential for the nucleolytic activity of the M-operon gene product(s). We therefore propose that orf2 and orf3 of the M-operon code for a structure-specific endonuclease (M-nuclease), which might be essential for phage multiplication.

Lysogenic strains of lactic Acid streptococci and lytic spectra of their temperate bacteriophages.

A total of 113 strains of mesophilic strains lactic streptococci of the species Streptococcus lactis, S. lactis subsp. diacetilactis, and S. cremoris, chosen from 291 strains that had been previously classified into six groups on the basis of their sensitivity to 132 virulent phages, were subjected to induction with mitomycin C. Among these strains, 43% produced phages capable of forming plaques of lysis on an indicator strain either spontaneously or after induction. There was a close correlation between the lytic spectra of temperate and virulent phages. Among the strains studied, 25% were shown to be indicator strains. These results emphasized the high probability of development of temperate phages in a starter culture containing mesophilic lactic streptococci and therefore their importance as a cause of accidents in cheese making.

Cloning and DNA sequence analysis of an X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763.

Lactococcus lactis subsp. lactis NCDO 763 (also designated ML3) possesses an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). X-PDAP mutants were selected by an enzymatic plate assay on the basis of their inability to hydrolyze an L-phenylalanyl-L-proline-beta-naphthylamide substrate. A DNA bank from L. lactis subsp. lactis NCDO 763 was constructed in one of these X-PDAP mutants, and one clone in which the original X-PDAP phenotype was restored was detected by the enzymatic plate assay. The X-PDAP gene, designated pepXP, was further subcloned and sequenced. It codes for a protein containing 763 residues. Comparison of the amino-terminal sequence of the X-PDAP enzyme with the amino acid sequence deduced from the pepXP gene indicated that the enzyme is not subjected to posttranslational modification or exported via processing of a signal peptide. The pepXP gene from L. lactis subsp. lactis NCDO 763 in more than 99% homologous to the pepXP gene from L. lactis subsp. cremoris P8-2-47 described elsewhere (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and is also conserved in other lactococcal strains.

Cloning and sequencing of pepC, a cysteine aminopeptidase gene from Lactococcus lactis subsp. cremoris AM2.

A gene coding for an aminopeptidase (PepC) from Lactococcus lactis subsp. cremoris AM2 was cloned by complementation of an Escherichia coli mutant lacking aminopeptidase activity. The nucleotide sequence was determined. A portion of the predicted amino acid sequence of PepC (436 amino acids) showed strong homology to the active site of cysteine proteases. No signal sequence was found, indicating an intracellular location of the enzyme.

Biochemical and genetic characterization of PepF, an oligopeptidase from Lactococcus lactis.

Lactococcus lactis possesses a complex proteolytic system which is essential for its growth in milk. We characterized one of the peptidases of this system, oligopeptidase PepF, together with its structural gene. PepF hydrolyzed peptides containing between 7 and 17 amino acids with a rather wide specificity. It was purified to homogeneity. The N-terminal sequences of PepF and of peptides resulting from tryptic digestion of PepF were determined and used to design degenerate oligonucleotides which served to amplify a DNA fragment internal to pepF. This fragment was used as a probe to screen a lactococcal genomic library in Escherichia coli and to clone the entire gene pepF. The gene coded for a 70 kDa protein and was located on a 55-kilobase lactose-protease plasmid. A motif His-Glu-X-X-His, characteristic of metallopeptidases was evidenced. Two regions of PepF were found similar, first to a stretch of 43 amino acids around the zinc-binding site of several other peptidases, second to a stretch of 33 amino acids well conserved among creatine and arginine kinases. Preliminary results suggest the presence of a second copy of pepF.

Phage abortive infection mechanism from Lactococcus lactis subsp. lactis, expression of which is mediated by an Iso-ISS1 element.

A 5-kb DNA fragment conferring a phage abortive infection phenotype (Abi+) has been cloned from Lactococcus lactis subsp. lactis IL416. The Abi+ determinant was subcloned on a 2-kb fragment which carried an Iso-ISS1 element and an open reading frame of 753 bp designated ORFX. Deletion within ORFX entailed the loss of the Abi+ phenotype, establishing that ORFX is the structural abi-416 gene. The expression of abi-416 was shown to be mediated by the Iso-ISS1 element, which contains a sequence fitting the consensus sequence for gram-positive promoters.