Reconstruction of the complete genome of a WWE1 bacterium, “Candidatus Cloacamonas acidaminovorans.”
WWE1 is a recently characterized candidate division deeply branching from the Spirochaetes
. Dot blot hybridization and fluorescence in situ hybridization analysis have shown that WWE1 bacteria are one of the subdominant groups in several anaerobic digesters (8
). As part of a metagenomic project, we investigated the presence and diversity of the WWE1 bacteria. Sludge samples were obtained from a full-scale anaerobic mesophilic digester located in the municipal wastewater treatment plant in Evry, France. Hybridization screening of a subset of a fosmid library constructed from this sludge revealed that 10% of the 16S rRNA gene-containing fosmids (64/599 clones) were affiliated with the WWE1 division. The 16S rRNA genes of these fosmids were classified into four phylotypes (based on a 97% identity threshold). In order to evaluate their genomic diversity, we fully shotgun sequenced 33 of the fosmids. In addition, we systematically determined the FES from the library and identified overlapping fosmids under the above-mentioned stringent matching criteria. The sequence of a phylotype 1 fully shotgun-sequenced fosmid (clone TCI) was found densely covered with FES (10- to 12-fold in terms of sequence coverage) and prompted us to reconstruct the complete tentative genome sequence of a representative bacterium of division WWE1.
An iterative assembly process was set up (see Materials and Methods) and applied starting from the phylotype 1 TCI fosmid insert sequence. After 31 iterations, the process stopped gathering new FES, and a scaffold was obtained. Completion and circularization of the genome sequence of phylotype 1 (“Candidatus Cloacamonas acidaminovorans”) was achieved by standard finishing techniques using fosmid clones as a matrix for PCR amplification and/or direct sequencing.
When applied to fosmids not affiliated with phylotype 1, the iterative assembly process failed to significantly extend the seed contig. Since “Candidatus Cloacamonas acidaminovorans” was not obtained in pure culture, it is difficult to check whether the reconstructed genome is a composite one. However, its reconstruction was probably made possible because of the extremely low sequence polymorphism (1.7 × 10−4) of the “Candidatus Cloacamonas acidaminovorans” dominant phylotype within the WWE1 population, as computed from the occurrence frequency of high-quality individual-read bases of “Candidatus Cloacamonas acidaminovorans” assigned FES differing from the consensus genome sequence.
Genome assembly robustness assessment.
Genome assembly robustness was assessed using fosmid coverage coherence (the relative orientation and spacing of the two FES of each clone localized). From the 1,741,439 available FES, 38,709 reads (2.2%, representing a cumulative 24,998,287 bp, that is, a mean genome coverage of 10.9) coming from 21,621 distinct fosmid clones were localized on the “Candidatus Cloacamonas acidaminovorans” genome using the above-mentioned criteria. Among those clones, 17,088 had their two FES localized. Only a few fosmids seemed to have incoherent features in regard to the “Candidatus Cloacamonas acidaminovorans” genome (172 of 199 rejected clones had one of the extremities localized more than one time [repeated regions], 21 had their end sequences in an incorrect orientation, and 6 were inconsistent in size). The 16,889 correctly localized fosmids allowed us to compute a clone coverage (with a mean value of 302.6 covering clones per base) showing no gap, with a minimum value of 4 over a 507-bp region. The 27 clones displaying incoherent pair end localization on the genome (either incorrect orientation or an insert size out of the normal fosmid range) were neither colocalized nor overlapping and matched in regions otherwise covered by several coherently positioned inserts. On the other hand, the 172 clones with multiply positioned ends were clustered, with all the ambiguous ends corresponding to a repeated region, and therefore were validated during the finishing of the genome. As, after reconstruction, the genome sequence of “Candidatus Cloacamonas acidaminovorans” was subjected to standard finishing, the impact of these reads on the structure of the sequence was carefully examined and ruled out.
Two percent of the (complete) FES library was assigned to the “Candidatus Cloacamonas acidaminovorans” genome, suggesting that the prevalence of the genome in this environment is quite low. It should, however, be noted that FES prevalence as defined gives only an approximation, as the number is dependent on (i) the complete genome size and (ii) the possible cloning biases.
“Candidatus Cloacamonas acidaminovorans” genome properties.
The genome of “Candidatus
Cloacamonas acidaminovorans” is represented by a single circular chromosome consisting of 2,246,820 nucleotides with a GC content of 37.9% (Fig. 2
). We do not know if “Candidatus
Cloacamonas acidaminovorans” harbors any plasmid. Two rRNA operons (5S, 16S, and 23S) were identified, together with two small noncoding RNA genes (ssrS
, encoding a 6S RNA, and rnpB
, encoding the RNA moiety of RNase P). A total of 47 tRNA genes representing all amino acids were annotated. Although only four insertion sequences were found, the fraction of repeated sequences was quite high (8.4%). One of these corresponded to a prophagic region (34.7 kb in length; Cloam0195-0237 and Cloam0474-0509) and displayed a strong GC percent deviation (Fig. 2
) and an atypical codon usage compared to the rest of the genome (see Fig. S1 in the supplemental material). Moreover, two large clustered regularly interspaced short palindromic repeats (CRISPR) (12.4 and 6 kb) (17
) have been identified. CRISPR, together with associated cas
genes, have been found in the majority of bacterial genera and are ubiquitous in Archaea
. They provide resistance against phages (3
). These regions are free of coding sequences and could be partly responsible for the atypical “Candidatus
Cloacamonas acidaminovorans” GC skew (not shown). “Candidatus
Cloacamonas acidaminovorans” has a rather low density of protein coding sequences (81%). Among the 1,820 predicted genes, 926 (51%) were assigned a probable biological function. Some 163 (9%) were similar to proteins with unknown functions, and 731 (40%) were unique to “Candidatus
Cloacamonas acidaminovorans” (Table 1
) . The percentage of genes found in groups of colocalized orthologs shared with other bacteria was generally very small (the maximum value was 15.6%, obtained with Pelobacter carbinolicus
) (see Table S2 in the supplemental material), showing that the “Candidatus
Cloacamonas acidaminovorans” genome is remote from known microbial genomes. The highest proportion of orthologs was found with anaerobic bacteria, such as Kuenenia stuttgartiensis
, Geobacter metallireducens
, and Thermoanaerobacter tengcongensis
(see Table S2 in the supplemental material). The genetic information necessary for self-maintenance and reproduction in the presence of a full complement of essential nutrients (13
) seemed to be present in “Candidatus
Cloacamonas acidaminovorans” (see Table S3 in the supplemental material for a detailed analysis) and supported the tentative reconstruction of the present genome assembly. Some interesting features of the “Candidatus
Cloacamonas acidaminovorans” genome were noted. First, it harbors proteins with sequences that are more related to their eukaryotic than their prokaryotic counterparts. For example, Cloam0177 is highly similar to the ftdD
gene of rat, mouse, and human (identity of 40% on the total length of the protein), a bifunctional enzyme involved in histidine degradation (glutamate formimidoyltransferase fused with formimidoyltetrahydrofolate cyclodeaminase). Secondly, it uses pyrophosphate-dependent enzymes: fructose-6-phosphate 1-phosphotransferase (EC 18.104.22.168) and pyruvate-phosphate dikinase (EC 22.214.171.124). Finally, the presence of the enzymes involved in the biosynthesis of lipopolysaccharides indicates that “Candidatus
Cloacamonas acidaminovorans” is a gram-negative bacterium. This is further confirmed by the detection of the typical signature of gram-negative bacteria found in the dnaK
“Candidatus Cloacamonas acidaminovorans” does not possess the enzymes necessary for the synthesis of 12 amino acids: leucine, isoleucine, valine, threonine, methionine, proline, arginine, histidine, phenylalanine, tyrosine, tryptophan, and cysteine. Moreover, it cannot produce polyamines and several cofactors: thiamine, biotin, lipoic acid, pyrroloquinolinequinone, coenzyme B12, folic acid, pyridoxine, and heme. Thus, all these compounds must be obtained from the environment. The in silico proteome analysis suggests that “Candidatus Cloacamonas acidaminovorans” could grow on a medium that includes glucose, alanine, asparagine, aspartate, glutamate, histidine, lysine, proline, serine, and certain aliphatic carboxylic acids (succinate, lactate, and acetate).
Cloacamonas acidaminovorans” possesses several proteins typical of anaerobic bacteria (the oxygen-sensitive class III ribonucleoside triphosphate reductase, several ferredoxin oxidoreductases, and radical S
-adenosylmethionine-dependent proteins), it seems to be adapted to an anaerobic lifestyle (Table 2
; see Table S4 in the supplemental material). However, the presence of the aerotolerant class II ribonucleotide reductase and some enzymes involved in protection against oxidative stress (i.e., superoxide reductase, ruberythrin, and thioredoxin reductase) may enable the bacterium to survive under minimal oxygen concentrations (Table 2
No electron transfer chain necessary for respiration was found in “Candidatus
Cloacamonas acidaminovorans,” and none of the classical terminal electron acceptors of anaerobic bacteria appeared to be used. Therefore, the bacterium seems to get its energy through the glycolytic Embden-Meyerhof pathway and the fermentation of amino acids, sugars, and carboxylic acids, as well as by the excretion of protons and sodium ions coupled with the reaction of the hydrogenase, the pyrophosphate-energized proton pump, and the methylmalonyl-coenzyme A (CoA) decarboxylase, respectively (Table 2
). This protein catalyzes a reaction characteristic of anaerobic prokaryotes. ATP synthesis is performed by a membrane-bound ATP synthase. More than 30 proteases and peptidases were identified. “Candidatus
Cloacamonas acidaminovorans” derives most of its carbon and nitrogen sources from the degradation of proteins. In addition, the bacterium possesses five different ferredoxin oxidoreductases, which are principally involved in the fermentation process of amino acids (Table 2
). Among these processes, the best identified was the lysine fermentation pathway. In fact, all the enzymes of this pathway except one have been detected. In contrast, the exact outcome of the fermentation of the other amino acids is unknown. These catabolic-degradation pathways are generally coupled with substrate level phosphorylation (ATP formation) and synthesis of reductants, mainly ferredoxins. Most of these ferredoxins are shared with anaerobic bacteria, known or not, to develop syntrophic interactions (see Table S4 in the supplemental material).
Besides these reducing agents, “Candidatus
Cloacamonas acidaminovorans” possesses a remarkable variety of redoxins (thioredoxin, rubredoxin, glutaredoxin, peroxiredoxin, and polyferredoxin). They principally act as hydrogen donors for the reduction of metabolites and of disulfide bonds in proteins and also function as electron carriers for the hydrogenase to form hydrogen. “Candidatus
Cloacamonas acidaminovorans” possesses one Fe-only hydrogenase and one putative Fe-only hydrogenase. In addition, an association of another putative hydrogenase with a putative formate dehydrogenase was detected. This complex might function as a simple type of formate hydrogen lyase, generating CO2
from formate (14
). Figure 3
highlights the metabolic specificities likely to be used by “Candidatus
A characteristic metabolic pathway in obligate syntrophic bacteria is the oxidation of propionate into acetate and carbon dioxide via methylmalonyl-CoA, succinate, fumarate, malate, oxaloacetate, pyruvate, and acetyl-CoA as intermediates (26
). This pathway is thermodynamically favorable only when hydrogen pressure is low. Thus, propionate-oxidizing bacteria form syntrophic consortia with hydrogen-scavenging bacteria, such as methanogenic, sulfate-reducing, and acetogenic bacteria (1
). All the genes involved in this pathway have been identified in “Candidatus
Cloacamonas acidaminovorans.” One step, the carboxylation of propionyl-CoA, is coupled with the decarboxylation of oxaloacetate by means of a transcarboxylase. This protein consists of three subunits: 12S, 5S, and 1.3S. Interestingly, the two genes encoding the 5S and the 1.3S subunit were fused. The presence of the oxidative propionate degradation pathway in “Candidatus
Cloacamonas acidaminovorans,” as well as the ability of the bacterium to produce hydrogen via fermentation and its Fe-only hydrogenases, makes it likely that “Candidatus
Cloacamonas acidaminovorans” is a syntrophic bacterium. Furthermore, compared to the other sequenced microbial genomes, the largest groups of colocalized orthologous genes with “Candidatus
Cloacamonas acidaminovorans” were found in P. carbinolicus
, Syntrophobacter fumaroxidans
, and Syntrophus aciditrophicus
(see Table S2 in the supplementary material), which are known to develop syntrophic interactions (10
Detection and culture enrichment.
Trials of enrichment cultures of “Candidatus Cloacamonas acidaminovorans” from activated sludge obtained from the anaerobic digester of Evry were carried out using a synthetic growth medium in which lysine served as a carbon and energy source (see Materials and Methods). A number of experiments were set up in order to obtain enrichment cultures of “Candidatus Cloacamonas acidaminovorans.” Although “Candidatus Cloacamonas acidaminovorans” was still detectable in some cases after 1 year of cultivation, quantitative PCR assays showed no detectable increase in the “Candidatus Cloacamonas acidaminovorans” cell numbers.
Moreover, “Candidatus Cloacamonas acidaminovorans” had disappeared or at least had become undetectable by PCR in the digester of Evry at the time (July 2005) we started enrichment cultures. The presence of these bacteria was then checked for monthly. One year later, “Candidatus Cloacamonas acidaminovorans” reappeared. This phenomenon was also observed in another digester (in Creil, France).
The presence of WWE1 and “Candidatus
Cloacamonas acidaminovorans” bacteria was checked for by PCR on DNAs extracted from 43 anaerobic digesters (19
; see Table S5 in the supplemental material). In addition to a few pilot or laboratory scale digesters, most of them were industrial installations processing wastewater with different amounts of industrial wastes. While WWE1 bacteria were detected in 32 anaerobic digesters, “Candidatus
Cloacamonas acidaminovorans” was found in 13 of them. WWE1 and “Candidatus
Cloacamonas acidaminovorans” bacteria thus seem to be widespread in anaerobic digesters.