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The severe skin-destructive disease caused by Mycobacterium ulcerans, named Buruli ulcer, is the third most important mycobacterial disease in humans after tuberculosis and leprosy. Recently we demonstrated that M. ulcerans could colonize the salivary glands of the water bug, Naucoris cimicoides. In this study, we report that M. ulcerans may be delivered from the digested prey aspirate to the coelomic cavity via a unique headspace, the head capsule (HC). During the infected meal, we observed that M. ulcerans clusters adhered to the stylets that were retracted in the HC at the end of the meal. M. ulcerans was able to translocate from the HC to the coelomic cavity where it is phagocytosed by the plasmatocytes. These cells are subverted as shuttle cells and deliver M. ulcerans to the salivary glands. At this early stage of its parasitic life style, two other important features of M. ulcerans can be documented: first, mycolactone is not required for translocation of M. ulcerans into the HC, in contrast to the next step, colonization of the salivary glands; second, M. ulcerans clusters bind a member of the serpin protein family present in the salivary gland homogenate.

Buruli ulcer, a disease caused by Mycobacterium ulcerans, is emerging as an increasingly important cause of morbidity throughout the world, for which surgery is the only efficient treatment to date. The aim of this work was to identify potential vaccine candidates in an experimental model of mouse infection. In BALB/c mice infected with M. ulcerans subcutaneously, Hsp65 appeared to be an immunodominant antigen eliciting both humoral and cellular responses. However, vaccination of mice with a DNA vector encoding Mycobacterium leprae Hsp65 only poorly limited the progression of M. ulcerans infection. In contrast, a substantial degree of protection was conferred by subcutaneous vaccination with BCG, suggesting that BCG antigens that are conserved in M. ulcerans, such as TB10.4, the 19 kDa antigen, PstS3 and Hsp70, may be interesting to consider as subunit vaccines in future prospects.

Clostridium perfringensis a ubiquitous gram-positive pathogen that is present in the air, soil, animals, and humans. AlthoughC. perfringensis strictly anaerobic, vegetative and stationary cells can survive in a growth-arrested stage in the presence of oxygen and/or low concentrations of superoxide and hydroxyl radicals. Indeed, it possesses an adaptive response to oxidative stress, which can be activated in both aerobic and anaerobic conditions. To identify the genes involved in this oxidative stress response,C. perfringensstrain 13 mutants were generated by Tn916insertional mutagenesis and screened for resistance or sensitivity to various oxidative stresses. Three of the 12 sensitive mutants examined harbored an independently inserted single copy of the transposon in the same operon as two genes orthologous to theydaDandycdFgenes ofBacillus subtilis, which encode a putative NADPH dehydrogenase. Complementation experiments and knockout experiments demonstrated that these genes are both required for efficient resistance to oxidative stress inC. perfringensand are probably responsible for the production of NADPH, which is required for maintenance of the intracellular redox balance in growth-arrested cells. Other Tn916disrupted genes were also shown to play important roles in the oxidative stress response. This is the first time that some of these genes (e.g., a gene encoding an ATP-dependent RNA helicase, the β-glucuronidase gene, and the gene encoding the atypical iron sulfur prismane protein) have been shown to be involved in the oxidative response.

We report here an evaluation of the dissemination of nim genes, encoding 5-nitroimidazoles resistance, among Bacteroides clinical strains isolated in Morocco. This study was done using a PCR method. Among 60 strains studied, nine contain a copy of a nim gene. The sequence determination of these genes showed that they are homologous to three nim genes previously characterized in strains isolated in France: nimB (five genes), nimC (three genes), and nimA (one gene). Although the nimA and nimC genes were previously identified on plasmids pIP417 and pIP419, respectively, we found here that they have a chromosomal location. The MICs of three 5-nitroimidazole antibiotics (metronidazole, ornidazole, and tinidazole) of the nim gene-containing strains were very low (0.5-2 microg/ml), indicating that the nim genes were not efficiently expressed in these clinical isolates.

We investigated the metabolism of dimetridazole (1,2-dimethyl-5-nitroimidazole) (DMZ) by the resting cell method in a susceptible strain of Bacteroides fragilis and in the same strain containing the nimA gene, which conferred resistance to 5-nitroimidazole drugs. In both cases, under strict anaerobic conditions DMZ was metabolized without major ring cleavage or nitrate formation. However, one of two distinct metabolic pathways is involved, depending on the susceptibility of the strain. In the susceptible strain, the classical reduction pathway of nitroaromatic compounds is followed at least as far as the nitroso-radical anion, with further formation of the azo-dimer: 5,5'-azobis-(1,2-dimethylimidazole). In the resistant strain, DMZ is reduced to the amine derivative, namely, 5-amino-1,2-dimethylimidazole, preventing the formation of the toxic form of the drug. The specificity of the six-electron reduction of the nitro group, which is restricted to 4- and 5-nitroimidazole, suggests an enzymatic reaction. We thus conclude that nimA and related genes may encode a 5-nitroimidazole reductase.

The nucleotide sequence of the DNA mobilization region of the 5-nitroimidazole resistance plasmid pIP421, from strain BF-F239 of Bacteroides fragilis, was determined. It contains a putative origin of transfer (oriT) including three sets of inverted repeats and two sequences reminiscent of specific integration host factor binding sites. The product of the mobilization gene mob421 (42.2 kDa) is a member of the Bacteroides mobilization protein family, which includes the MobA of pBI143, NBUs, and Tn4555. Sequence similarity suggests that it has both oriT binding and nicking activities. The transfer frequency of pIP421 in a B. fragilis donor strain possessing a Tc(r) or Tc(r) Em(r)-like conjugative transposon was significantly enhanced by tetracycline. Moreover, the mobilization region of pIP421 confers the ability to be mobilized from Escherichia coli by an IncP plasmid.

The nucleotide sequence of the DNA replication origin region of a Bacteroides vulgatus plasmid, pIP417, encoding 5-nitroimidazole resistance has been determined. This region of 1934 bp presents some characteristics similar to those of other replication protein-dependent origins. It contains a large open reading frame which could encode a basic Rep protein (RepA) of 36.8 kDa. Upstream of this ORF exist an AT-rich region, three direct repeats (iterons) of 21 bp, multiple DnaA binding sites, and sites, and sites for the integration host factor (IHF). Moreover, the amino acid sequence of the pIP417 RepA protein shows similarities with those of other Rep proteins encoded by plasmids of gram-negative bacteria: pRO1600 from Pseudomonas aeruginosa; pPS10 from Pseudomonas syringae; pFA3 from Neisseria gonorrhoeae; and two cryptic plasmids from Campylobacter hyointestinalis and Butyrivibrio fibrisolvens. Although RepA can be expressed in an Escherichia coli in vitro transcription-translation assay, vectors containing the pIP417 replication origin did not replicate in E. coli. The homology of the pIP417 replication region with the corresponding regions of other Bacteroides spp, plasmids was also studied by Southern blot hybridization. The results indicated that the repA gene of plasmid pIP417 is homologous to that of plasmid pIP421, but not of plasmid pIP419. The replication region of plasmid pIP421 was sequenced and showed about 80% identity at the nucleotide level with that of pIP417. A small (3634-bp) cloning vector (pFK12) of entirely defined nucleotide sequence was constructed for Bacteroides spp.

The genetic organization of two different 5-nitroimidazole (5-Ni) resistance genes was investigated: nimC and nimD from Bacteroides plasmids plP419 and plP421, respectively. The nimC gene (492 bp) and the nimD gene (495 bp) directed the synthesis of polypeptides with deduced molecular masses of 18·37 kDa and 18·48 kDa, respectively. The predicted proteins showed 67-83% identity and 78-91% similarity with the products of two other nimA and nimB genes previously described and could be derived from a common ancestral gene. An insertion sequence element (IS1170) was identified upstream of the nimC gene. IS1170 is 1604 bp in length and is flanked by imperfect inverted repeats (15 bp). IS 1170 is similar to the Bacteroides insertion sequence element IS942 with an identity of 70% at the nucleotide level. The single copy of IS 1170 present on plasmid plP419 is integrated 24 bp upstream of the initiation codon of nimC. Similar genetic organization was found on plasmid plP421. One copy of another insertion sequence (IS1169) was found 4 bp upstream of the first ATG codon of the nimD gene. This element (1325 bp) shows a strong homology at the nucleotide level (70% identity) with IS 1186 and IS 1168 found to be associated with the Bacteroides carbapenem resistance gene cfiA, and the 5-Nir genes nifiA and nimB, respectively. There is strong evidence that, as in the case of the cfiA gene, the transcription of the four nim genes so far studied is directed by outward-oriented promoters, carried on the right ends of the different insertion sequence elements.

A 3.3-kb DNA fragment of Clostridium acetobutylicum conferred methyl methane sulfonate (MMS), mitomycin C (MC), and UV resistance to recA strains of E. coli when cloned on the pUC19 plasmid. Analysis of the nucleotide sequence of the total insert and results of in vitro transcription-translation experiments showed that the insert directed the synthesis of three polypeptides referred to as ORFa, ORFb, and ORFc of 23.6, 15.3, and 21 kDa, respectively. None of the polypeptides presented a relationship with the RecA protein of E. coli or products of genes involved in the SOS response. The deduced amino acid sequence of ORFb and ORFc are highly homologous to those deduced from two genes specifying resistance to tellurium salts present on plasmid pMER610 harbored by Alcaligenes sp.strains and to an AMP-binding protein (CABP1) found in Dictyostelium discoideum. The existence of these homologous proteins suggests that they may perform a similar key function in the three unrelated organisms.

This report describes a genetic and molecular analysis of two transferable Bacteroides plasmids, pIP417 and pIP419, which carry genetic determinants conferring low-level resistance to 5-nitroimidazoles. The restriction endonuclease cleavage sites for each plasmid were localized. The NiR genetic determinants of pIP417 and pIP419 plasmids have been cloned into the Bacteroides cloning vector pBI191 (C.J. Smith, J. Bacteriol. 164, 294-301, 1985) as PvuII and Sau3A fragments, respectively. Both inserts had different restriction sites and did not cross-hybridize by Southern blot analysis. Genetic data obtained by cloning into pBI191 clearly show that the PvuII-generated fragments A (Rep) and B (Mob) of pIP417 are involved in plasmid replication and transfer, respectively. Although encoding resistance to the same antibiotic, both plasmids appeared different with regard to the 5-nitroimidazole resistance and replication genetic determinants. However, they share a homology in a region involved, at least in one case, in plasmid transfer. Considering the spontaneous high level of resistance to 5-nitroimidazole in Escherichia coli, this work, based on direct gene cloning into Bacteroides, demonstrates the value of such an approach.

A new type II restriction endonuclease, named CacII was detected in Clostridium acetobutylicum strain ABKn8. CacII cleaved the hexanucleotide sequence [5'-GCN⇓ NGC-3'] and generated blunt ends. Up to now no isoschizomer of CacII has been described.

International audience; Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.

International audience; BACKGROUND: Buruli ulcer is a severe human skin disease caused by Mycobacterium ulcerans. This disease is primarily diagnosed in West Africa with increasing incidence. Antimycobacterial drug therapy is relatively effective during the preulcerative stage of the disease, but surgical excision of lesions with skin grafting is often the ultimate treatment. The mode of transmission of this Mycobacterium species remains a matter of debate, and relevant interventions to prevent this disease lack (i) the proper understanding of the M. ulcerans life history traits in its natural aquatic ecosystem and (ii) immune signatures that could be correlates of protection. We previously set up a laboratory ecosystem with predatory aquatic insects of the family Naucoridae and laboratory mice and showed that (i) M. ulcerans-carrying aquatic insects can transmit the mycobacterium through bites and (ii) that their salivary glands are the only tissues hosting replicative M. ulcerans. Further investigation in natural settings revealed that 5%-10% of these aquatic insects captured in endemic areas have M. ulcerans-loaded salivary glands. In search of novel epidemiological features we noticed that individuals working close to aquatic environments inhabited by insect predators were less prone to developing Buruli ulcers than their relatives. Thus we set out to investigate whether those individuals might display any immune signatures of exposure to M. ulcerans-free insect predator bites, and whether those could correlate with protection. METHODS AND FINDINGS: We took a two-pronged approach in this study, first investigating whether the insect bites are protective in a mouse model, and subsequently looking for possibly protective immune signatures in humans. We found that, in contrast to control BALB/c mice, BALB/c mice exposed to Naucoris aquatic insect bites or sensitized to Naucoris salivary gland homogenates (SGHs) displayed no lesion at the site of inoculation of M. ulcerans coated with Naucoris SGH components. Then using human serum samples collected in a Buruli ulcer-endemic area (in the Republic of Benin, West Africa), we assayed sera collected from either ulcer-free individuals or patients with Buruli ulcers for the titre of IgGs that bind to insect predator SGH, focusing on those molecules otherwise shown to be retained by M. ulcerans colonies. IgG titres were lower in the Buruli ulcer patient group than in the ulcer-free group. CONCLUSIONS: These data will help structure future investigations in Buruli ulcer-endemic areas, providing a rationale for research into human immune signatures of exposure to predatory aquatic insects, with special attention to those insect saliva molecules that bind to M. ulcerans.

Clostridium perfringens, a strictly anaerobic bacterium, is able to survive when exposed to oxygen for short periods of time and exhibits a complex adaptive response to reactive oxygen species, both under aerobic and anaerobic conditions. However, this adaptive response is not completely understood.C. perfringenspossesses specialized genes that might be involved in this adaptive process, such as those encoding superoxide dismutase (SOD), superoxide reductase and alkyl hydroperoxide reductase, but their contribution to the oxidative stress response and their control mechanisms are unknown. By a combination of functional complementation ofEscherichia colistrains impaired in either SOD, alkyl hydroperoxide reductase (AhpC) or catalase activity (Cat), transcription analysis and characterization of mutants impaired in regulatory genes, it was concluded that: (i) the product of thesodgene is certainly essential to scavenge superoxide radicals, (ii) theahpCgene, which is fully induced in all oxidative stress conditions, is probably involved in the scavenging of all intracellular peroxides, (iii) the three rubrerythrin (rbr) genes ofC. perfringensdo not encode proteins within vivoH2O2reductase activity, and (iv) the two rubredoxin (rub) genes do not contribute to the hypothetical superoxide reductase activity, but are likely to belong to an electron transfer chain involved in energy metabolism.

Resume Des resistances aux 5-nitroimidazoles, de niveau parfois faible, sont apparues chez les Bacteroides. Ces resistances sont souvent transferables; elles le sont alors de facon independante du transfert d'autres resistances. Les genes codant pour cette resistance ont une localisation soit plasmidique, soit chromosomique et appartiennent a au moins deux groupes d'hybridation dont on precise la repartition parmi des isolats cliniques.

International audience; The sensitivity of Clostridium perfringens strain 13 to oxygen and its toxic derivatives was investigated in a new, defined medium (MMP). Exponentially growing cells in MMP medium were very sensitive to exposure to air by vigorous shaking. When exposed to air, the cells survived only 1hour and then rapidly died. Addition of cysteine, ascorbic acid, or yeast extract to the medium significantly increased vegetative cell survival without inducing sporulation. The level of toxicity of peroxyl and hydroperoxyl radicals, generated by H2O2, t-butyl hydroperoxide or ethanol, was very similar with and without air exposure. By contrast, plumbagin or menadione, which generate superoxide radicals in the presence of oxygen, caused high levels of cell death only in aerobiosic culture. Growth-arrested cells were more resistant to H2O2and to redox-cycling agents than were exponentially growing cells, but the resistance required de novo synthesis of proteins. An adaptive response to oxidative stress was also suggested by the higher level of cell resistance to H2O2and to ethanol when cells were pretreated with sublethal doses of these oxidants.

The in vivo mutagenic potential of two 5-nitroimidazoles, metronidazole and dimetridazole, was evaluated in Bacteroides fragilis, a strictly anaerobic bacterium. Two antibiotic resistance genes, tetA(Q)3 and nimA, were used as DNA targets. The forward and back mutations were identified by nucleotide sequence analysis. Both drugs induced GC→CG transversion exclusively. The results suggest that the reactive molecules generated during the intracellular reduction of the 5-nitroimidazoles are responsible for both base pair substitutions and DNA strand breaks, although the mechanisms and targets may be different.

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.

DNA sequence analysis of regions from plasmid pIP417 and chromosome BF8 which encode 5-nitroimidazole resistance in Bacteroides strains allowed the identification of two open reading frames corresponding to new genes, nimA (528 bp) and nimB (492 bp). Either gene may confer 5-nitroimidazole resistance to susceptible strains of Bacteroides. The encoded polypeptides have deduced molecular masses of 20.1 and 18.6 kDa, respectively, and share about 73% identity and 85% similarity. A new insertion sequence (IS) element named IS1168 lies 14 bases upstream of the nimA gene. The complete sequence of IS1168 was determined. A similar IS exists 12 bp upstream of the nimB gene. About 60% of the BF8 IS element was also sequenced and shown to be almost identical to IS1168.

Nitroimidazoles are imidazoles that contain a nitro group at position 2, 4, or 5 of the imidazole ring. These compounds may have various substitutions on the nitrogen at position 1 of the imidazole ring. The 5-nitroimidazoles are synthetic compounds that appear to be very potent anaerobicidal agents. Three derivatives are commonly used in clinical therapy: metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitro-imidazole], ornidazole [1-(3-chloro-2-hydroxy-propyl) 2-methyl-5-nitroimidazole], and tinidα-zole (ethyl [2-(2-methyl-5-nitro-1-imidazolyl)-ethyl] sulfone). They are used extensively for treatment of infections from anaerobic eucaryotes (Entamoeba histolytica, Trichomonas vaginalis, Giardia) and anaerobic procaryotes, having a bactericidal effect against all anaerobic bacteria except the genera Bifidobacterium, Propionibacterium, and Actinomyces.

The introduction of DNA into bacteria by transformation remains an important step for the development of genetic and recombinant DNA technology. In addition to studying the structure and expression of exogenous genes either in Escherichia coli or Bacillus subtilis, it is important to reintroduce these genes in their original host. Genetic background, physiological, and environmental conditions are highly relevant to gene expression and regulation. This is particularly true for strict anaerobic bacteria such as Clostridia whose general physiology is very different than that of E. coli or B. subtilis. Further, doning foreign genes in strains of medical or economic significance with the purpose of modifying their genotype or improving their metabolic potentiality requires efficient gene transfer systems.

The role of biofilms in the pathogenesis of mycobacterial diseases remains largely unknown. Mycobacterium ulcerans, the etiological agent of Buruli ulcer, a disfiguring disease in humans, adopts a biofilm-like structure in vitro and in vivo, displaying an abundant extracellular matrix (ECM) that harbors vesicles. The composition and structure of the ECM differs from that of the classical matrix found in other bacterial biofilms. More than 80 proteins are present within this extracellular compartment and appear to be involved in stress responses, respiration, and intermediary metabolism. In addition to a large amount of carbohydrates and lipids, ECM is the reservoir of the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, and purified vesicles extracted from ECM are highly cytotoxic. ECM confers to the mycobacterium increased resistance to antimicrobial agents, and enhances colonization of insect vectors and mammalian hosts. The results of this study support a model whereby biofilm changes confer selective advantages to M. ulcerans in colonizing various ecological niches successfully, with repercussions for Buruli ulcer pathogenesis., Author Summary Mycobacterium ulcerans is the etiologic agent of Buruli ulcer, a necrotic skin disease affecting humans living close to wetlands in tropical countries. This mycobacteria resides in water where it could colonize many ecological niches such as aquatic plants, herbivorous animals, and water bugs. The latter were shown to be able to transmit the bacteria to mammalian hosts. Here, we described that the bacilli could be structured with a thick envelope called the extracellular matrix (ECM). This peculiar coat contains in small vesicles a toxin named mycolactone, the main virulence factor of M. ulcerans. The ECM confers to the mycobacterium increased resistance to antimicrobial agents and plays a role in virulence. Indeed, a bacteria with ECM is more potent for colonization of insect vectors and mammalian hosts compared to bacteria. Unraveling the regulation of the production of the ECM together with the export of mycolactone will be an important step in developing new pharmacological approaches for the treatment of Buruli ulcer, which has been greatly handicapped by the lack of effectiveness of the current antibiotics.

Several strains ofClostridium acetobutylicum, isolated from sugar beet pulps or Jerusalem artichokes, are able to utilize inulin, a β-polyfructosane polymer of fructose with glucose as the terminal residue. Inulin-degrading activity, which was detected in cultures of one such strain, ABKn8, grown in Basol-medium containing inulin, reached a maximum at the end of exponential phase. Most of the enzyme activity was detected in the supernatant. It was stably maintained in 0.1 M acetate buffer pH 5.0, and was optimal at pH 4.6. The enzyme, inulinase was induced by inulin, but not by xylose, fructose or sucrose and was repressed by glucose. Inulinase was active against inulin, sucrose and raffinose, but not melezitose. It had a higher affinity for inulin (Km: 1.2×10-2 mM) than all the other known inulinases.

Plasmids pIM13, pT127 and pBC16δ1, introduced by transformation into Clostridium acetobutylicum N1-4081, were shown to replicate in, and to confer antibiotic resistance upon this new host. Recombinant plasmids were contructed by inserting erythromycin-resistant plasmid pIM13 into the unique ClaI site of pBR322 or by ligating a tetracycline-resistant determinant of plasmid pT127 to HindIII-linearized pIM13. The hybrid plasmids replicated and expressed erythromycin resistance in C. acetobutylicum strain N1-4081 and in Escherichia coli or Bacillus subtilis, indicating that they might be useful as shuttle vectors for transferring genes between these strains. The efficiency and stability of different replicons in C. acetobutylicum were compared.

SUMMARY: A method for the production of protoplasts of Clostridium acetobutylicum strain N1-4080 utilizing lysozyme and penicillin G was optimized to yield about 100% protoplasts. Treatment of 109 cells left fewer than 102 that were viable and osmo-resistant. With a mutant strain deficient in autolytic activity, about 1% of the protoplasts regenerated walled cells, whereas most gave rise to stable L-form colonies. Addition of sodium polyanethole sulphonate to the medium improved the regeneration frequency by at least a factor of 10, probably by somehow inhibiting the residual autolytic activity of the strain.

A type II restriction endonuclease, named Cac I, was detected in Clostridium acetobutylicum strain N1-4081. Cac I cleaved the tetranucleotide sequence [5′↓-GATC-3′]. The modification system consisted of the methylation of the adenine present in this sequence. Cac I, an isoschizomer of Mbo I, is inactive on dam methylated substrates.

Summary A series of twelve glutamine-requiring mutants of B. megaterium was isolated by a procedure which did not include a sporulation step. Mutants belonged to different categories according to their residual level of glutamine synthetase activity, their glutamate synthase activity and their ability to produce cross-reacting material with an antiserum against glutamine synthetase. Nevertheless all of them were unable to sporulate. The comparison of the properties of these mutants with those of strain Gln − 26, a previously isolated sporogenous glutamine synthetase-deficient mutant, leads to the conclusion that glutamine synthetase is likely to play a regulatory role in the control of sporulation.

Summary: Addition of choline to growing Clostridium acetobutylicum led to abnormal cell septation, lack of cell separation and the consequent formation of chains. Similar results were obtained with the wild-type strain N1-4 and its autolysin-deficient mutant N1-4081. With strain N1-4, addition of choline at 1 to 2 mg ml−1 resulted in inhibition of autolysis assessed as autoplast formation in 0.6 M-sucrose, lysis by 0.3 M-NaCl/0.03 M-sodium citrate, lysis by 0.1% Triton X-100 and lysis by penicillin G. In vitro data confirmed the inhibition by choline of wall-degrading activity, using N1-4 cell walls as substrate. Choline was shown to be a component of the teichoic acid of C. acetobutylicum N1-4.

Mutants of B. subtilis 168 which exhibited an absolute requirement for glutamine have been isolated and characterized. Of the two mutants studied in detail, one had normal levels of glutamine synthetase and sporulated normally, the other had reduced glutamine synthetase and was asporogenic. Both mutants were mapped close to the thy A region of the chromosome by PBS1 transduction. A study of spontaneous revertants selected for glutamine prototrophy (or the sporulation character in the case of the asporogenic mutant) led to the conclusion that there is a relationship between the glutamine requirement and sporulation. However, the influence of glutamine could not be entirely explained by the catalytic properties of glutamine synthetase.

An improved technique of protoplast regeneration and a general procedure of transfection and plasmid transformation are described inClostridium acetobutylicum. Frequencies of 104–105 transfectants/transformants per μg of DNA were easily achieved.

An intracellular serylprotease has been characterized in B. , subtilis , Marburg strain. Like the extracellular serylprotease of that strain, it occurs only during sporulation, but otherwise differs in many characteristics, including its migration on electrophoresis, its absolute requirement for calcium and its narrower specificity toward esters. On the other hand, it is quite similar to the cytoplasmic serylprotease of B. , meqaterium also synthesized during sporulation. Since the latter enzyme can specifically modify the B. , subtilis vegetative RNA polymerase in vitro , it is conceivable that the intracellular B. subtilis serylprotease studied here carries out this modification in , vivo .

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.