The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.

A nitrifying sequencing batch reactor was inoculated twice with the aerobic denitrifying bacterium Microvirgula aerodenitrificans and fed with acetate. No improvement was obtained on nitrogen removal. The second more massive inoculation was even followed by a nitrification breakdown, while at the same time, nitrification remained stable in a second reactor operated under the same conditions without bioaugmentation. Fluorescent in situ hybridization with rRNA-targeted probes revealed that the added bacteria almost disappeared from the reactor within 2 days, and that digestive vacuoles of protozoa gave strong hybridization signals with the M. aerodenitrificans-specific probe. An overgrowth of protozoa, coincident with the disappearance of free-living bacteria, was monitored by radioactive dot-blot hybridization only in the bioaugmented reactor. Population dynamics were analysed with a newly developed in situ quantification procedure of the probe-targeted bacteria. The nitrifying groups of bacteria decreased in a similar way in the bioaugmented and non-bioaugmented reactors. Other bacterial groups evolved differently. The involvement of different ecological parameters are discussed separately for each reactor. These results underline the importance of predator-prey interaction and illustrate the undesirable effects of massive bioaugmentation.

The microbial community structure of pig manure slurry (PMS) was determined with comparative analysis of 202 bacterial, 44 archaeal and 33 eukaryotic small subunit (SSU) rDNA partial sequences. Based on a criterion of 97% of sequence similarity, the phylogenetic analyses revealed a total of 108, eight and five phylotypes for the Bacteria, Archaea and Eukarya lineages, respectively. Only 36% of the bacterial phylotypes were closely related (>or=97% similarity) to any previously known sequence in databases. The bacterial groups most often represented in terms of phylotype and clone abundance were the Eubacterium (22% of total sequences), the Clostridium (15% of sequences), the Bacillus-Lactobacillus-Streptococcus subdivision (20% of sequences), theMycoplasma and relatives (10% of sequences) and the Flexibacter-Cytophaga-Bacteroides (20% of sequences). The global microbial community structure and phylotype diversity show a close relationship to the pig gastrointestinal tract ecosystem whereas phylotypes from the Acholeplasma-Anaeroplasma and the Clostridium purinolyticum groups appear to be better represented in manure. Archaeal diversity was dominated by three phylotypes clustering with a group of uncultured microorganisms of unknown activity and only distantly related to the Thermoplasmales and relatives. Other Archaea were methanogenic H2/CO2 utilisers. No known acetoclastic Archaea methanogen was found. Eukaryotic diversity was represented by a pluricellular nematode, two Alveolata, a Blastocystis and an Entamoebidae. Manure slurry physico-chemical characteristics were analysed. Possible inhibitory effects of acetate, sulphide and ammonia concentrations on the microbial anaerobic ecosystem are discussed.

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RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented X sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a X site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a X site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with X. Results show that (1i) MW accumulation is X orientation dependent, and (ii) a succession of X sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate X activity in plasmids. The rolling circle mechanism produces a a structure during plasmid replication; we propose that the double-stranded DNA tail of this ar form allows RecBCD entry; the tail is degraded unless it is protected by a X site. By analogy, a principal role of X in the survival of A redgam-mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD.The RecBCD enzyme has principal roles in both DNA degradation and homologous recombination in Escherichia coli (1, 2). The enzyme is multifunctional; it acts as a single-stranded (ss) and double-stranded (ds) DNA exonuclease, helicase, ATPase (see refs. 3 and 4 for review), and site-specific endonuclease which recognizes the 8-base-pair (bp) sequence, 5'-GCTGGTGG-3', known as X (5). RecBCD recognizes and nicks 3' ofX if it enters a dsDNA end from the right of the X site as written here (6). It is proposed that a 3' tail formed after X nicking by RecBCD stimulates homologous recombination to the left of X (4, 6-8).The in vivo interaction of RecBCD with X and its effect on recombination were elucidated by using bacteriophage A as a model system (9-12). In this paper, another system is described to study RecBCD-X interaction. We are studying broad host range plasmids that utilize a unidirectional rolling circle replication (rcr) mechanism (13, 14), resembling that of bacteriophage 4X174 (15): the replication protein (Rep) encoded by the rcr plasmid creates a site-specific nick in the plus strand to initiate replication and attaches to the 5' end of the displaced strand (16). The 3'-OH end produced serves as a primer for DNA synthesis. Replication intermediates are a-shaped molecules (unpublished data; D. Halpern and L. Janniere, personal communication) with less than monomerlength tails (15). After one round of replication, Rep nicks a second time at the origin to terminate replication. If termination does not occur-due, for example, to a break in the displaced strand or to a nonattached Rep protein-rcr would continue indefinitely, giving rise to a or molecule with a long tail. We have identified high molecular weight plasmid species that may correspond to nonterminated replication intermediates (17,18). In E. coli as well as in...