A genomic perspective on a new bacterial genus and species from the Alcaligenaceae family, Basilea psittacipulmonis
© Whiteson et al.; licensee BioMed Central Ltd. 2014
Received: 4 October 2013
Accepted: 6 February 2014
Published: 1 March 2014
A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads.
Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters.
Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063).
The study of parakeet respiratory infection has had important implications for biomedical research since December of 1929, when psittacosis caused by Chlamydophila psittaci created a health scare which eventually led to the formation of the National Institutes of Health . Here we describe a novel bacterium from the family Alcaligenaceae that was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) from a petshop in Basel, Switzerland. The bacterial family Alcaligenaceae includes genera that have been isolated from humans, animals and the environment. They are Gram-negative rods or coccobacilli that possess oxidase and catalase, growing well on complex media under aerobic or microaerobic conditions.
There are nearly 25000 prokaryote genome projects registered in the NCBI database as of early 2014 , many of them human-associated. Pathogens of animals that are not important for agriculture or zoonotic transmission of disease are poorly studied. Filling out the tree of life is important for improving genome sequence annotation and creating good phylogenetic landmarks to analyze metagenomic data [3, 4].
The genome of a bacterium isolated from the lungs of a parakeet (Melopsittacus undulatus) in captivity was sequenced using Illumina sequencing. Here we describe the success and limitation of a comparative genomics approach to studying this newly discovered bacterium. This bacterium is most closely related to Pelistega europaea according to a Ribosome Database Project (RDP) classifier assessment of the similarity of their 16S rDNA (rrs) gene [5, 6], a stable and frequently used phylogenetic marker . The closest fully sequenced relatives, from genus Taylorella and genus Bordetella[8–11], share a great number of putative genes and functions, but are too distant to make specific analyses through simple sequence comparisons.
Bacterial isolation, phenotypic and biochemical characterization
The carcass of a suddenly dead parakeet (M. undulatus) from a petshop without previous presentation of clinical signs was brought to the Institute of Animal Pathology, University of Bern, Switzerland for post mortem examination and histological analysis.
Lung and liver samples from the deceased parakeet were cultured on tryptone soy agar with 5% sheep blood (Oxoid, Basel, Switzerland) at 37°C in an atmosphere of air with 5% CO2 for 48 hours. Phenotypic and biochemical characterization were performed with a VITEK2 instrument (bioMérieux, Geneva, Switzerland) and the API ZYM, API NH and API 20 NE (bioMérieux) according to the manufacturer’s instructions. Analysis of respiratory quinones, polar lipids and fatty acids were carried out by the Identification Service of the DSMZ and DR. BJ Tindall, DSMZ, Braunschweig, Germany. Plates were stained with 5% molybdophosphoric acid to show all lipids.
Submission to international culture collections
The strain JF4266 was submitted to the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, deposited under the name Alcaligenaceae bacterium DSM 24701) and the Institute Pasteur (number CIP 110308 T) with the name B. psittacipulmonis. Both repositories have made the strain publicly available under the name B. psittacipulmonis in addition to the strain number assigned by each repository, in accordance with the Rules of Bacteriological Code (1990 revision) as revised by the International Committee on Systematics of Prokaryotes (ICSP) at the plenary sessions in Sydney and Paris .
The material from the bottom of three cages (with live parakeets) and cage water were obtained from three petshops in Switzerland and France. Cage water was concentrated 50-fold in a vacuum concentrator. The cage samples were mixed with the lysis buffer [final concentration Tris 10 mM, EDTA 1 mM (pH 8), Tween 0.5%, proteinase K (Fermentas, Burlington, Canada) 200 μg/ml] and incubated for 2.5 hours at 55°C . Proteinase K was inactivated by a 10 min incubation at 95°C and the samples were frozen at -20°C. The PCR contained 6 μl of lysate and 0.5 μM of both forward and reverse primers in 50 μl of PrimeStar HS Premix (Takara, Otsu, Shiga, Japan). The PCR mix was amplified for 36 cycles (for three putative protein coding regions) or 30 cycles (for the 16S rDNA gene) of 98°C for 10 seconds, 56°C for 15 seconds, and 72°C for 1 min. One μl of the amplified reaction mix was run on the Agilent Bioanalyzer using a DNA1000 lab chip to determine if the product was generated. The Per-1 F/R, Per-2 F/R and Per-3 F/R primer pairs amplify 730, 522 and 533 bp regions of the DSM 24701 genomic DNA. The primers were designed to amplify RAST predicted genes of unknown function that are unique to the parakeet genome (there are no Blast hits to the nr/nt database). Primer pair Per-11 F/R specifically amplifies a unique298 bp region of the DSM 24701 16S rDNA, from position 202 to 499. Primer sequences were as follows: Per-1 F 5′ TCTGGGTGATTTTGGAGAGG 3′, Per-1R 5′ ATTCTCGCGTTCTTGCTGTT 3′, Per-2 F 5′ TTCGTATCTGGCAGAGGCTT 3′, Per-2R 5′ AACAATTGGGTTCCCACAAA 3′, Per-3 F 5′ AGATGATGGAGCAAGCTCGT 3′, Per-3R 5′ CAATTGGTCTACCGTTGCCT 3′, Per-11 F 5′ AAAGCAGGGGACCGCAAGGC 3′, Per-11R 5′ TCAGGTACCGTCATCACTCAATGGT 3′.
Controls to ensure that the parakeet cage samples did not inhibit PCR reactions were performed in two ways: 1) The parakeet cage material and water lysate were spiked with genomic DNA from DSM 24701, in which case all 3 pairs of DSM 24701 specific primers successfully amplified the expected product. 2) A PCR targeting the first three variable regions of the 16S rDNA gene (V123) was also performed on the parakeet cage samples using broad range bacterial 16S primers (8 F 5 GAGTTTGATCMTGGCTCAG 3 and 534R 5 CCGCGRCTGCTGGCAC 3). These primers amplified the expected segment of the bacterial 16S rDNA gene from all three parakeet cage material and water samples, suggesting that there are bacteria in the sample, as we would expect, but not DSM 24701.
Genomic DNA was prepared using the procedure in Hernandez et al.  using the DNEasy kit (Qiagen, Venlo, Netherlands) and sequenced with the Solexa Illumina Genome Analyzer. The 454 sequencing was conducted by Microsynth in Balgach, Switzerland. Optical mapping was carried out by digestion of genomic DNA by NheI with OpGen in Madison, Wisconsin, USA.
Assembly and annotation
The paired Illumina reads were assembled with the Edena assembler . The assembly of 454 sequencing data was performed with the dedicated GS De Novo Assembler available from Roche (Roche Applied Science, Indianapolis, IN, USA). The final 195 contigs were submitted to the RAST server (Chicago, IL, USA) for annotation .
Top BLASTn hits for DSM 24701 16S rDNA gene sequence
Advenella kashmirensis WT001
Bordetella sp. p23 (2011)
Uncultured compost bacterium clone ASC718
Taylorella equigenitalis 14/56
Taylorella equigenitalis ATCC 35865
Taylorella equigenitalis MCE9
Bordetella sp. d16
Achromobacter sp. CH1
Achromobacter sp. MT-E3
An array was constructed containing rows of putative genes and columns of fully sequenced bacterial genomes, following the strategy of Wu and Eisen . The absence and presence of a gene in the species is indicated by 0 or 1, as determined by BLASTp of the predicted genes from DSM 24701 against the SEED database of proteins from fully sequenced genomes with an E-value cut-off of 10E-05. Clusters were made using CLUSTER 3.0 with a complete linkage hierarchical analysis and weighting of the species in an attempt to remove phylogenetic bias, and visualized with JavaTreeview (both available at http://rana.lbl.gov/EisenSoftware.htm).
BLASTp of the predicted protein sequences from DSM 24701 was performed against a database of the same set of sequences, to find duplicates inside the genome (paralogs). Reciprocal hits and self-hits were excluded, and BLAST results with an E-value cut-off of 10E-05, >150aa long, and >30% sequence identity were counted as duplicates, largely following the strategy of Gevers et al. . We excluded all 57 sequences <150aa long in order to avoid overestimating the duplication rate by only including short sequences that do not have a paralog.
Results and discussion
Differential taxonomic characteristics between DSM 24701, T . equigenitalis (DSM 10668 T), T . asinigenitalis (CIP 79.7 T) and P . europaea (LMG 10982 T)
P. europaeaLMG 10982 T
T. asinigenitalisCIP 79.7 T
T. equigenitalisDSM 10668 T
API ZYM resultsa
API NH results b
Cellular fatty acid composition of DSM 24701
Fatty acid composition
14:1 w5c, 14:1 w5t or both
Summed feature 1
Summed feature 2
Summed feature 3
Summed feature 5
Description of Basilea gen. nov.
Basilea (Ba.si.le’a L. fem. N. referring to the Swiss town Basel, where the type strain was isolated)
Cells are small, Gram-negative, non-motile rods. Oxidase-positive and grows in aerobic or capnophillic conditions. Visible colonies appear after 2 days growth on blood agar plates at 30-42°C with 5% CO2. The major respiratory quinone is Q8 and the major polar lipids are phosphatidylethanolamine, phosphatidylglycerol, two unknown phosphoaminolipids, two unknown phospholipids and two unknown aminolipids. The major fatty acids were C16:0 and C18:1ω7c; C12:0was only detected in trace amounts. The type species is Basilea psittacipulmonis. The DNA G + C content of the type strain of this type species is 40%.
Description of psittacipulmonis sp. nov.
B. psittacipulmonis (psitt.a.ci.pul.mon’is named because the type and only known strain was isolated from the lung of a parakeet). The description is the same as for the genus, with the following additions. Grows at 30°C, 37°C and 42°C with 5% CO2, and in aerobic conditions at 30°C, and 42°C. Does not grow in LB broth or enriched Mycoplasma broth medium. Enzyme tests did not indicate a reaction forindol, trypsin, chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase, urease and prolin arylamidase, alkaline phosphatase, lipase, cystin arylamidase or penicillinase. However, the species exhibits strong enzyme activity of esterase, leucine arylamidase, naphtol-AS-BI-phosphohydrolase and γ-glutamyl transferase, and intermediate activity of esterase lipase, valine arylamidase, acid phosphatase and ornithine decarboxylase. The chemotaxonomic characteristics listed in the type strain genus apply to this strain.
The type strain is B. psittacipulmonis DSM 24701, isolated from the lungs of a parakeet from Basel, Switzerland (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063).
Distribution in the cages and homes of pet owners
We explored whether this microorganism is common in the environment of pet parakeets by conducting PCRs on environmental templates with PCR primers that are unique to the B. psittacipulmonis. Primers were designed to specifically amplify the B. psittacipulmonis 16S rDNA gene and several protein-coding genes that were considered unidentified on RAST, and did not yield any hits on BLAST in the nr/nt database. PCR amplification of sample templates from the drinking water and bottom of cages housing healthy parakeets from various pet stores and private homes using these primers were all negative, while positive samples obtained by artificial contamination of the same material with 1 ng of DSM 24701 genomic DNA were positive. This suggests that the DSM 24701 is not commonly found in the cages of healthy parakeets.
Comparison of DSM 24701 with other betaproteobacteria including many members of the family Alcaligenaceae
Genome size (Mb)
Observed growth rate (hours)
Taylorella equigenitalis MCE9
Taylorella asinigenitalis 14/45
Acidovorax avenae subsp. citrulli
Burkholderia ambifaria AMMD
P. europaea has been found in the lungs, trachea, liver and spleen of acutely diseased pigeons; clinical observations have led microbiologists to conclude that it is a pathogenic organism . Low GC content and small genome size, features which are shared by P. europaea, Taylorella spp., and this novel bacterium DSM 24701 , are different from the closely related, fully sequenced members of the Alcaligenaceae family such as the Bordetella with higher GC content (62-68%) and genome size (3.7-5.3 Mb) (Table 4).
Illumina sequencing data and assembly statistics of the draft genome
Number of reads
Average pairing distance (standard deviation)
Number of contigs
Average contig size
Max contig size
RAST predicted coding sequences
Contigs included in annotation
Genome size as determined by contig assembly and optical mapping is near 2 Mb
The size of the DSM 24701 genome is estimated to be near 2 Mb by both Solexa-Illumina and 454 sequencing in addition to the results of an optical map generated by electrophoresis of fragments generated by an NheI digest of the genomic DNA (results not presented). The large effort which would be required to complete the genome was not undertaken. The 195 contigs were submitted for Rapid Annotation using Subsystem Technology  (http://rast.nmpdr.org/). The annotation process found 1664 coding sequences on 88 contigs. The remaining contigs were shorter than the average gene length, suggesting that any gene which may occur on those contigs could be truncated and would be harder for gene-calling algorithms to identify. RAST describes each of the coding sequences as a protein expression gene (peg) numbered 1-1664 as they appear on the contigs which are ordered largest to smallest, i.e. peg.1 is the first gene on the largest contig.
Common protein coding marker genes and dinucleotide frequency recapitulate relationships found in 16S rDNA gene tree
Amino acid sequence homology shows that about a third of the predicted genes from DSM 24701 are shared with related genomes
GC Content analysis of concatenated DSM 24701 contigs suggests more recent genetic exchange with organisms that have low GC content
Phylogenetic profiling yields a unique profile of gene clusters, some shared with Bordetella, phage or other respiratory pathogens
There are also examples of gene clusters formed in the phylogenetic profile that are shared almost exclusively with the Bordetella species. Thirty-four genes in a cluster which is present consistently only in the Bordetella species are mostly described as hypothetical, but include genes predicted to be integral membrane proteins, TolA and a RecB-family exonuclease. Another intriguing cluster of 11 predicted genes that are all present in both genome sequences of B. avium encodes putative phage proteins, including the small terminase subunit involved in DNA packaging. Ten of the eleven genes in this cluster are located together on a contig of the DSM 24701 genome with the same gene order as the Bordetella species. We were surprised to find that the GC content of the DSM 24701 genes in this cluster ranged from 42-48%, while the orthologs from Bordetella species and several sequenced Bordetella phages have GC contents similar to that of their genomes just under 70%. The predicted phage terminase from DSM 24701 has 48% GC, which is high compared to the rest of the genome (Figure 4). It is interesting that this putative prophage cassette has such different GC content in DSM 24701 and Bordetella species; the difference may derive from a different phage or quick adaptation of the cassette sequence to a lower GC content in DSM 24701.
DSM 24701 shares some gene loss events with obligate intracellular bacteria
Of the 100 COGs lost by all obligate intracellular bacteria in a study of 317 genomes , only ~30 of them had equivalent representatives in the genome of strain DSM 24701 using the RAST annotation of predicted gene function. Strain DSM 24701 is not dependent on host cells; it is able to grow on blood agar plates. However, small genome size, high GC content and lack of ~70 genes also missing in obligate intracellular bacteria may indicate that DSM 24701 has taken steps on the one-way road toward gene loss like that which led other bacteria to become host dependent. Merkej et al  found that free living bacteria with larger genomes often have more genes that are described as virulence factors than pathogenic bacteria, challenging many early hypotheses that the presence of particular virulence factors was predictive of the pathogenicity of an organism . In addition, HGT is more difficult for intracellular bacteria, which are isolated from encounters with genetically diverse microorganisms and phage. Mutations that affect gene regulation may also drive virulence in bacteria that can otherwise inhabit humans as harmless commensals, such as Streptococcus pyogenes[42, 43], a bacterial species with similar genome size. Future annotation methods may become better at capturing these aspects of pathogenicity and bacterial lifestyle from genomic data.
Distribution of gene function annotation is similar to Taylorella genomes, and reflects the diverse repertoire of metabolic genes in DSM 24701
DSM 24701 shares low duplication rate with bacteria of similar genome size
Distribution of gene paralogs within the DSM 24701genome
Genes in 2 pairs
Genes in 3 pairs
Genes in 4 pairs
Genes in 7 pairs
Genes with paralogs (all pairs)
Total genes (>150 bp)
Excluded genes (<150 bp)
Shared gene homology varies widely inside bacterial families
Several recent genome comparison studies have drawn intriguing conclusions about genome evolution and organization. For example, the Mycoplasma agalactiae genome, long assumed to have undergone genome reduction in order to become one of the simplest free-living organisms with a minimal genome, was unexpectedly found to have a large fraction of predicted genes – 18% - likely acquired by HGT from species in distinct phylogenetic groups . Sequencing of 16 Mycoplasma genomes allowed for detailed comparison between closely related species, revealing that the genomes are not very similar. For example, in a comparison of M. agalactiae strain PG2 with four other Mycoplasma genomes, no predicted genes with a blastp identity >90% were found, and only few (16%) with >50%. The genome of DSM 24701 is actually more similar to Bordetella species than this – about a third of the DSM 24701 genome has >50% identity with the sequenced Taylorella and Bordetella genomes (Figure 2).
In an attempt to better understand the biology of the newly discovered DSM 24701, and to intimate whether it is a pathogen, we also examined the putative genes that are unique to DSM 24701 in comparison to B. avium, T. equigenitalis and T. asinigenitalis (Additional file 1: Table S6). The unique genes include potential antibiotic resistance genes, CRISPR-related proteins, and members of the Tad (tight adherence gene) macromolecular transport system that may indicate that the secretion systems used by DSM 24701 are different (Additional file 5: Figure S4). This ancient secretion system is found in a long list of pathogenic genera, such as species belonging to the genera of Haemophilus and Yersinia. The tad genes found in many bacteria, including DSM 24701, are known to be involved in biofilm formation and colonization , which are essential in the first steps of infection by many bacterial pathogens.
The organism described in our study (internal strain nr. JF4266, and referred to in this paper as DSM 24701) is different from the other genera belonging to the family Alcaligenaceae, according to phylogenetic, phenotypic and chemotaxonomic data. A new bacterial genus and species are proposed in order to place it taxonomically, with the name Basilea psittacipulmonis gen. nov., sp. nov. (originating from Basel, Switzerland and found in the lungs of Psittacidae). The presence of this easily cultured and yet unassigned bacterial strain, isolated from a common parakeet in a Basel petshop suggests that there may still be large parts of the bacterial kingdom which remain underexplored, even in the midst of the metagenomic revolution that has already yielded many Proteobacteria genome sequences.
The genomic sequence of a newly detected bacterium DSM 24701 will contribute to available sequence knowledge, with many genes that are not similar to any found in current databases. Sequence homology with related genomes, biochemical comparisons, dinucleotide usage, Crispr-detection and phylogenetic profiling allowed us to highlight several interesting features of this genome. However, as the passing of the 10 year anniversary of the human genome and our still vague understanding of its contents remind us, sequence information provides only limited biological knowledge of a live species. Additional sequence information from more closely related organisms would enable improved phylogenetic placement and, to some extent, functional characterization. Sequencing novel organisms – even an under-represented branch of a well-studied phyla - adds more unique information to the sequence databases, as recently shown by Jonathan Eisen and colleagues from the Genomic Encyclopedia of Bacteria and Archaea (GEBA) . Although it is more difficult to analyze novel genomic sequence in comparative studies, the novel sequences may become starting material for unforeseen biotechnology projects or discoveries in microbial evolution.
The assembled and annotated genome is publically on the RAST server with a guest account under the ID 666666.4954, and the 16S sequence has the Genbank accession number JX412111 and GI 406042063. 16S rDNA and rpoB gene alignments for phylogenetic tree construction can be found in the Dryad database: http://doi.org/10.5061/dryad.b341k.
We greatly appreciate the contribution of Myriam Girard, who cultured this bacterium and extracted genomic DNA multiple times, enabling these experiments. We would like to acknowledge Manuel Stark and Christian von Mering, Zürich, for running ML Tree before the standalone program was available. Michael Richter and Ramon Rosello-Mora from Esporles, Spain ran J species in a generous attempt to make an ANI calculation. Manon and Loane Croset, H. Dussolier, along with Maud and Alix François made critical contributions in the collection of parakeet cage and water samples. Jonathan Eisen made several helpful suggestions, especially with regard to phylogenetic profiling. COST (C05.0103) provided funding for common projects to Jacques Schrenzel and Joachim Frey. Katrine Whiteson was supported by an NIH Fellowship (R01GM095384 – 01S1). Finally, we would like to thank a certain parakeet who encountered this bacterium, enabling our study.
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