Nucleoid structure and partition in Methanococcus jannaschii: an archaeon with multiple copies of the chromosome.

We measured different cellular parameters in the methanogenic archaeon Methanococcus jannaschii. In exponential growth phase, the cells contained multiple chromosomes and displayed a broad variation in size and DNA content. In most cells, the nucleoids were organized into a thread-like network, although less complex structures also were observed. During entry into stationary phase, chromosome replication continued to termination while no new rounds were initiated: the cells ended up with one to five chromosomes per cell with no apparent preference for any given DNA content. Most cells in stationary phase contained more than one genome equivalent. Asymmetric divisions were detected in stationary phase, and the nucleoids were found to be significantly more compact than in exponential phase.     

Cell cycle arrest in archaea by the hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane

Hypusination is an essential posttranslational modification unique to archaeal and eukaryotic protein synthesis initiation factor 5A (aIF5A and eIF5A, respectively). We have investigated the effect of the efficient hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane (GC(7)) on four archaeal and one bacterial species. We found that (i) archaea are sensitive to GC(7), whereas the bacterium Escherichia coli is not, (ii) GC(7) causes rapid and reversible arrest of growth of the archaeon Sulfolobus acidocaldarius, and (iii) the growth arrest is accompanied by a specific reversible arrest of the cell cycle prior to cell division. Our findings establish a link between hypusination and sustained growth of archaea and thereby provide the framework to study molecular details of archaeal cell cycle in connection with in vivo functions of hypusine and of aIF5A and eIF5A.     

Serological and molecular size characterization of flagellins of Pseudomonas syringae pathovars and related bacteria

Flagella from a total of 118 strains representing mostly pathovars of the phytopathogenic group Pseudomonas syringae, but also P. chlororaphis, P. cichorii, P. corrugata, P. fluorescens, P. fuscovaginae, P. stutzeri, P. viridiflava, as well as related phytopathogenic genera (Burkholderia cepacia and Ralstonia solanacearum) were characterized by immuno-fluorescent staining, SDS-PAGE, and immunoblotting. Eighty-six strains of the P. syringae group pathovars, P. cichorii and P. viridiflava were shown to possess flagella of serotypes H1 or H2, composed of a unique flagellin, whose molecular size varied between 31 and 31.5 kDa. Similarities between the P. syringae flagellin and a 31 kDa surface protein involved in pathogenicity are pointed out. The distribution of H1 and H2 antigens in the nine recently described genomospecies of P. syringae-P. viridiflava group suggested that flagellin would represent a phylogenetic marker within the arginin-dihydrolase-negative fluorescent pseudomonads. The characterization of flagellin was proposed as an identification tool at a level situated between genus and species.     

Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii

We analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus(Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea.     

Development of a microtitre-based spectrophotometric method to monitor Babesia divergens growth in vitro

Babesia divergens multiplication cycle involves erythrocyte invasion, intracellular division, and erythrocyte lysis with the simultaneous liberation of hemoglobin. We have decided to set up a spectrophotometric protocol based on hemoglobin concentration in the culture supernatants to monitor B. divergens in vitro growth. After the selection of 405 nm as the most appropriate endpoint hemoglobin wavelength in our conditions (hemoglobin concentration in the supernatant), cultures were standardized [1 x 10(9) red blood cell (RBC)/ml, 1-2.5 x 10(5) infected red blood cell (iRBC)/ml] to allow their monitoring over 3 days. The protocol was then compared to the most commonly used growth measurement methods: parasitemia counting and [(3)H]hypoxanthine incorporation. An excellent correlation was demonstrated between A(405) of the culture supernatant and parasitemia of the iRBC, whatever the RBC concentration used in the medium. This correlation was also evidenced between A(405) and [(3)H]hypoxanthine incorporation for [(3)H]hypoxanthine concentrations lower than 4 microCi/ml. Our assays also highlighted the inhibitory effect of [(3)H]hypoxanthine on B. divergens growth even when used at low concentrations (0.8 microCi/ml) and for a short incorporation duration (24 h). This effect was confirmed by both A(405) and parasitemia counting. In conclusion, A(405) measurement of B. divergens culture supernatant represents a simple, rapid, safe, and reliable way to measure the in vitro growth of this parasite. Generation times of three different B. divergens strains were then determined by the protocol described here and varied between 8 h 36 min and 13 h 8 min.     

Distribution of Pseudomonas syringae pathovars into twenty-three O serogroups.

Serological reactions of Pseudomonas syringae and Pseudomonas viridiflava were studied by Ouchterlony double diffusion. A total of 55 polyclonal antisera, containing anti-lipopolysaccharide (anti-LPS) precipitating antibodies, were cross-tested against antigenic suspensions of 51 strains. Twenty-three O serogroups were defined, primarily on the reaction of the type strains. Two families of O serogroups showed antigenic crossreactivities (PHA, MOP1, MOP2, MOP3, HEL1, HEL2, and SYR1; PERSAVTOM1, PERSAVTOM2, DEL, POR, and SYR2). Ten O serogroups showed a clearcut specificity: APTPIS, TAB, VIR1, VIR2, VIR3, SYR3, SYR4, SYR5, HUS, and LAC. The last serogroup (RIB) contained strains with rough colony morphology and side chain-deficient LPSs, as evidenced by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The LPS basis of the O serogroups was demonstrated by immunoblotting. Serological reference strains were designated for all of the O serogroups and correspondence was established between the O serogroups studied and seven previous serogroups (L. T. Pastushenko and I.D. Simonovich, Mikrobiol, Zh. 41:222-229 and 330-339, 1979). A total of 355 strains of P. syringae (sensu lato) belonging to 15 pathovars, not including pathovar syringae, were typed into the 23 described O serogroups. O serogroups were assigned after double-diffusion reactions, with each strain compared with serological references. The utility of O serogrouping to study P. syringae pathovar structure and diversity is discussed.     

Antibody prevalence and molecular identification of Babesia spp. In roe deer in France

In a region-wide serologic study carried out in 2004 on free-ranging hunted roe deer in various landscapes, we found that 58% of the animals (237 out of 406) were antibody positive for Babesia divergens antigen. Serologic and infection status was also analyzed for 327 roe deer live-trapped in two fenced forest areas over 5 yr (2004-08). For two consecutive years during this period, 92 and 94% of the deer in these closed populations were antibody-positive for B. divergens. Babesia spp. were isolated in autologous red blood cell culture for 131 of the trapped animals (40%). Molecular typing was done on 76 isolates with polymerase chain reaction (PCR)-restriction fragment length polymorphism methods targeted at the 18S ribosomal subunit gene (18 isolates) and the Bd37 gene coding for a merozo?te surface antigen implicated in a protective response (60 isolates). Results indicated continuous cocirculation of B. capreoli and B. venatorum in both forests and possible coinfection of animals with both species. No infection with B. divergens was detected. Fifteen isolates were confirmed to be B. capreoli by sequencing part of the 18S rRNA gene. Using PCR detection of the Bd37 gene, all nine isolates of B. venatorum in this study were negative, whereas the 15 confirmed and 50 putative B. capreoli isolates showed very variable restriction profiles, distinct from those known for Bd37 in B. divergens. Two isolates showed conflicting results, suggestive of mixed infection.