A molecular method to study population and activity dynamics in anaerobic digestors.

The applicability of a new molecular fingerprinting method (Single Strand Conformation Polymorphism) to study the microbial populations of anaerobic digestors was investigated. After extraction of total nucleic acids, the 16S rDNA and 16S rRNA molecules were amplified and the amplicons were separated by SSCP electrophoresis. Characteristic and complex peak patterns were obtained, where each peak could be correlated with the 16S rDNA sequence of one micro-organism. The rDNA peak patterns should consist of the most abundant sequences and thus would reflect the diversity of prominent species of different digestors. Ribosomal DNA patterns were compared to rRNA patterns and revealed the bacteria that were the most active metabolically. The SSCP method also revealed dynamic changes in the presence and activity of populations, following perturbations such as an acidic shock which caused an increase in activity of two species. After cloning the 16S rDNA, the species corresponding to the peaks of interest, such as the archaeal species, could be identified by screening the clones according to their SSCP patterns and sequencing the 16S rDNA.

Aerobic Denitrifiers Isolated from Diverse Natural and Managed Ecosystems.

Twenty-eight bacterial strains were isolated from an ecosystem adapted to fluctuating oxic-anoxic conditions. This ecosystem comprised a mixture of different natural and wastewater treatment environments. Among the 28 strains isolated, 10 exhibited aerobic denitrifying activity, i.e., co-respiration of oxygen and nitrate and simultaneous production of nitrite by 4 of them and of nitrogen gas by the remaining 6. Comparisons between the 16S rDNA sequences of the 10 strains showed that 3 of them were identical to M. aerodenitrificans, whereas RAPD profiles showed that the 3 strains were identical to each other but that they were different from M. aerodenitrificans. This implies that alternating aerobic-anoxic conditions allowed the isolation of a new strain of this aerobic denitrifier. Moreover, other denitrifying bacteria belonging to the genera Paracoccus, Thiobacillus, Enterobacter, Comamonas, and Sphingomonas were isolated in this way. These data imply that a wide variety of bacteria are able to carry out this type of metabolism. M. aerodenitrificans was also detected in methanogenic, denitrifying, nitrifying, phosphate removal, and activated sludge ecosystems by two-step PCR amplification. After 4 months of acclimation to oxic-anoxic phases, the strain was also detected in a canal and in a pond. This suggests that there is no specific natural ecological niche for aerobic denitrifiers but, as soon as selective pressure such as alternating aeration conditions is applied, this metabolism is amplified.

Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction (PCR) single-strand conformation polymorphism analysis.

The structures of the bacterial and archaeal communities in an anaerobic digester were monitored over a 2 year period. The study was performed on a fluidized bed reactor fed with vinasse. The objective was to characterize the population dynamics over a long time period under constant environmental parameters. Total bacterial and archaeal populations were measured independently by fluorescence-based polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) analysis using an automated DNA sequencer. With the current level of accuracy, the technique was able to monitor 45 bacterial and seven archaeal 16S rDNA molecules. The community dynamics were compared with molecular inventories of the microbial community based on 16S rDNA sequences done at the beginning of the study. The six archaeal and the 22 most frequent bacterial operational taxonomic units (OTUs) identified were associated with their SSCP peak counterparts. Overall, the data indicated that, throughout the period of the study, rapid significant shifts in the species composition of the bacterial community occurred, whereas the archaeal community remained relatively stable.

Degradation of morpholine, piperidine, and pyrrolidine by mycobacteria: evidences for the involvement of a cytochrome P450.

Nine bacterial strains that grew on morpholine and pyrrolidine as sole carbon, nitrogen, and energy sources were isolated from three different environments with no known morpholine contamination. One of these strains could also degrade piperidine. These bacteria were identified as Mycobacterium strains. A phylogenetic analysis based on the partial 16S rDNA sequences indicated that the isolated strains clustered within the fast growing group of mycobacteria. When the above-mentioned cyclic amines were used as growth substrates, the synthesis of a soluble cytochrome P450 was induced in all these bacteria. Other laboratory strains, Mycobacterium fortuitum and Mycobacterium smegmatis mc(2)155, were tested for their abilities to degrade morpholine. Neither of them degraded morpholine but could use pyrrolidine and piperidine. The growth of M. fortuitum and M. smegmatis mc(2)155 on these compounds involved a soluble cytochrome P450, suggesting that mycobacterial strains are naturally able to use pyrrolidine and have developed a similar enzymatic pathway to metabolize this amine.

Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

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.

Sequence analysis of the Bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome.

The standard strategies of genome sequencing based on lambda-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli. One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis, established by using yeast artificial chromosomes (YACs). This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it. The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs. The complete 104,109 bp insert sequence of this YAC clone was thus established. The strategy used allowed us to avoid resequencing the two largest, previously sequenced, contigs (13,695 and 20,303 bp) of the YAC insert. We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning, as well as for larger inserts of eukaryotic origin cloned in YACs. Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg. The organization of the newly determined region is presented. Of the 138 ORFs identified in the spollA-kdg region, 57 have no clear putative function from their homology to proteins in the databases.

A 29.425 kb segment on the left arm of yeast chromosome XV contains more than twice as many unknown as known open reading frames.

The nucleotide sequence of a 29.425 kb fragment localized on the left arm of chromosome XV from Saccharomyces cerevisiae has been determined. The sequence contains 13 open reading frames (ORFs) of which four encode the known genes ADH1, COQ3, MSH2 and RCF4. Predictions are made concerning the functions of the unknown ORFs. Some of the ORFs contain sequences similar to expressed sequence tags (EST) found in the database made available by TIGR. In particular, the highly expressed ADH1 gene is represented in this database by no less than 20 EST sequences. Two ARS sequences and a putative functional GCN4 motif have also been detected. One ORF (O0953) containing nine putative transmembrane segments is similar to a hypothetical membrane protein of Arabidopsis thaliana. Characteristic features of the other ORFs include ATP/GTP binding sites, a fungal Zn(2)-Cys(6) binuclear centre, an endoplasmic reticulum targeting sequence, a beta-transducin repeat signature and in two instances, good similarity to the prokaryotic lipoprotein signal peptide motif.

Sequence of a 10.27 kb segment on the left arm of chromosome XV from Saccharomyces cerevisiae includes part of the IRA2 gene and a putative new gene.

A 10,270 bp fragment from the left arm of chromosome XV of Saccharomyces cerevisiae was sequenced and analysed. The sequence reveals the presence of two open reading frames (ORFs), one of them is the larger part of the previously sequenced gene IRA2 (YOL0951). The other ORF, YOL0950, has a length of 1245 nucleotides and exhibits no significant homology with any known gene, although there is some similarity of its upstream region to the corresponding region of the Schizosaccharomyces pombe cdr1/nim1 gene which is involved in the control of mitotic cell size.

The organization of the Bacillus subtilis 168 chromosome region between the spoVA and serA genetic loci, based on sequence data.

Three different lambda phage clones with overlapping inserts of Bacillus subtilis DNA, which cover the region from spoIIAA to serA, have been isolated. The nucleotide sequence of their inserts, starting after spoVAF and ending at serA, has been determined. A contiguous sequence of 35,354 bp was established, including previously analysed overlapping adjacent regions. Within the newly determined sequence 31 open reading frames (ORFs) with putative ribosome-binding sites have been found. Nine of them correspond to previously sequenced and characterized genes: spo-VAF, lysA, sipS, ribG, ribB, ribA, ribH, ribTD and dacB. Comparison of the amino acid sequences of the products encoded by the other ORFs to known proteins allowed putative functions to be assigned to seven of these ORFs. Among these are the following: (i) the ppiB gene, encoding a cytoplasmic peptidylprolyl isomerase; (ii) two pairs of signal-transducers, one homologous to phoR-phoP of B. subtilis, encoding regulators of phosphatase biosynthesis, and the second to the fecI-fecR of Escherichia coli, which is responsible for the regulation of the citrate-dependent iron (III) transport system; (iii) aroC and serA genes, involved in the biosynthesis of aromatic amino acids and serine, respectively, the function of which has been confirmed by constructing corresponding mutants with disrupted ORFs. The organization of putative operons has been postulated on the basis of the sequences of their transcription terminators, promoters and regulatory elements.

An ordered collection of Bacillus subtilis DNA segments cloned in yeast artificial chromosomes.

A collection of 772 Bacillus subtilis DNA segments was obtained by cloning in yeast artificial chromosomes. The B. subtilis inserts of 288 clones were mapped by hybridization using as probes 65 cloned genes and 188 isolated insert ends. In this way, 59 inserts were ordered in four contigs that cover > 98% of the B. subtilis chromosome. This ordered collection is now available for further genetic and physical analysis of the B. subtilis genome.