Genome comparison provides molecular insights into the phylogeny of the reassigned new genus Lysinibacillus
© Xu et al.; licensee BioMed Central. 2015
Received: 16 October 2014
Accepted: 19 February 2015
Published: 27 February 2015
Lysinibacillus sphaericus (formerly named Bacillus sphaericus) is incapable of polysaccharide utilization and some isolates produce active insecticidal proteins against mosquito larvae. Its taxonomic status was changed to the genus Lysinibacillus in 2007 with some other organisms previously regarded as members of Bacillus. However, this classification is mainly based on physiology and phenotype and there is limited genomic information to support it.
In this study, four genomes of L. sphaericus were sequenced and compared with those of 24 representative strains belonging to Lysinibacillus and Bacillus. The results show that Lysinibacillus strains are phylogenetically related based on the genome sequences and composition of core genes. Comparison of gene function indicates the major difference between Lysinibacillus and the two Bacillus species is related to metabolism and cell wall/membrane biogenesis. Although L. sphaericus mosquitocidal isolates are highly conserved, other Lysinibacillus strains display a large heterogeneity. It was observed that mosquitocidal toxin genes in L. sphaericus were in close proximity to genome islands (GIs) and mobile genetic elements (MGEs). Furthermore, different copies and varying genomic location of the GIs containing binA/binB was observed amongst the different isolates. In addition, a plasmid highly similar to pBsph, but lacking the GI containing binA/binB, was found in L. sphaericus SSII-1.
Our results confirm the taxonomy of the new genus Lysinibacillus at the genome level and suggest a new species for mosquito-toxic L. sphaericus. Based on our findings, we hypothesize that (1) Lysinibacillus strains evolved from a common ancestor and the mosquitocidal L. sphaericus toxin genes were acquired by horizontal gene transfer (HGT), and (2) capture and loss of plasmids occurs in the population, which plays an important role in the transmission of binA/binB.
Lysinibacillus sphaericus (formerly named Bacillus sphaericus) is a Gram-positive, aerobic, mesophilic, and spore-forming bacterium that is commonly isolated from soil. It is also an archaic organism whose spores have even been found in 25–40-million-year-old amber . L. sphaericus has very distinctive phenotypic properties, including an inability to utilize polysaccharide pathways and employment of exclusive metabolic pathways for synthesis of a wide variety of organic compounds and amino acids . Some strains produce active insecticidal proteins against mosquito larvae, and thus have been widely used as biocontrol agents for disease-transmitting mosquitoes . The mosquitocidal properties are associated with the sporulation-specific binary toxin (Bin proteins) and vegetative-specific Mtx toxins , as well as a novel two-component toxin (Cry48 and Cry49 proteins) produced during sporulation . Compared with another mosquito pathogen, Bacillus thuringiensis subsp. israelensis, L. sphaericus demonstrates a higher efficiency for killing mosquito larvae and a better persistence in the field .
The evolutionary model and systematic classification of L. sphaericus continues to be debated. On the basis of flagellar agglutination, L. sphaericus isolates can be grouped into 49 serotypes . According to DNA homology between strains, five major groups (I to V) are indicated, each probably corresponding to a separate species because of the relatively low level of homology between groups . However, relatively few biochemical and morphological tests are available to distinguish L. sphaericus as a different species. Recently, a multi-locus sequence typing (MLST) study has indicated that the mosquitocidal strains are highly conserved and appear near-clonal . This is consistent with a previous report which observed that toxic L. sphaericus strains are all found within DNA subgroup IIA, although in association with nine serotypes (H1, H2, H3, H5, H6, H9, H25, H26, and H48).
In 2007, Bacillus sphaericus was formally renamed L. sphaericus and, together with Lysinibacillus boronitolerans and Lysinibacillus fusiformis (formerly named Bacillus fusiformis), was proposed to belong to a novel genus named Lysinibacillus gen. nov. Since then, more and more novel isolates have been assigned to Lysinibacillus. The species classification was mainly based on common features in physiology and phenotype, e.g. Gram-positive, spore-forming, rod-shaped, motile, presence of the Lys–Asp type of peptidoglycan in the cell wall, the main fatty acids as iso-C15: 0, and the predominant menaquinones as MK-7 , but there is little evidence to support this classification on a genomic basis. Thus, there is a need to analyze the relationship between Lysinibacillus and Bacillus on the genomic level, and to understand the evolution of mosquitocidal L. sphaericus.
Although a broad spectrum of data has been collected for L. sphaericus, there is limited genome sequence available. One complete genome sequence is available for mosquitocidal strain C3-41 (accession numbers CP000817 and CP000818) , and two gapped genome sequences from reference strains KCTC 3346 (or ATCC14577) and OT4b.31(both non-toxic) have also been published [12,13]. In this study we report genome sequences of four L. sphaericus strains, comprising three toxic strains (2297, LP1-G, SSII-1) and one non-toxic strain (NRS1693). We also investigate their phylogenetic relationship with genome sequences for Lysinibacillus and Bacillus strains. Our results provide the first support for the taxonomy of the reassigned new genus Lysinibacillus at the genome level and suggest a new species for mosquitocidal L. sphaericus, providing new insight into the evolution of Lysinibacillus.
Strains and genome information used in this study
Genome size (bp)
GC content (%)
No. of contigs
No. of proteins
Genbank accession No.
biovar anthracis str. CI
Bacillus sp. NRRL B-14905
The total genome sizes vary from 4.0 to 6.7 Mb across species and strains. All Lysinibacillus strains have larger chromosome sizes (4.5 ~ 4.8 M) compared to B. subtilis (4.0 ~ 4.2 M) but smaller sizes compared to B. cereus group strains (5.2 ~ 6.7 M). Conversely, their G + C content (~37%) is higher than that of B. cereus group strains (~35%) but lower than that of B. subtilis (~43%).
The numbers of predicted genes in L. sphaericus genomes varied from 4,470 to 4,701, but is likely a factor of incomplete genome assemblies as well as individual strain differences. With C3-41 as a reference, the predicted gene numbers of other L. sphaericus strains varied from 2,791 to 4,202, corresponding to 62.8 to 90.4% of the total gene numbers of the individual genome. The novel strains presented in this study (2297, LP1-G, SSII-1 and NRS1693) harbor over 80% genes predicted to be homologous to genes in C3-41, whereas the corresponding numbers in the two L. sphaericus reference strains KCTC3346 and OT4b.31 were only 62.8% and 65.4% respectively.
Based on the distance matrix Nexus file exported from Gegenees, a dendrogram was produced using SplitsTree 4 (using the neighbor joining method) (Figure 1 left). The tree classifies all Lysinibacillus genomes into two main clusters. The L. sphaericus toxic isolates and the marine Bacillus spp. NRRL B-14905 are clustered and closer to B. cereus group strains, whereas the three non-toxic L. sphaericus strains are clustered with L. fusiformis and L. boronitolerans. Thus the L. sphaericus strains are diverse and scattered at the genomic level.
In addition, the genomes of Solibacillus silvestris [GenBank: NC_018065], Sporosarcina pasteurii [GenBank: AYOX00000000], and Ureibacillus thermosphaericus [GenBank: AJIK00000000], which are thought to be sphaericus-like organisms close to L. sphaericus based on 16 s rDNA and phenotypic analysis  were investigated. The results showed that these sphaericus-like organisms were quite divergent at the genome level and there is no obvious relationship with Lysnibacillus and Bacillus (data not shown).
Core conserved genes consensus tree
Gene content of pan- and core genomes
Function features of the pan- and core genomes
Differences in the distribution of the COG categories involved in cellular process and signaling were also observed. For instance, Lysinibacillus displays larger proportions for category T (Signal transduction mechanisms) and N (Cell motility) both in the pan- and in the core genome compared to the B. cereus group and B. subtilis. Also, Lysinibacillus harbors the smallest distribution of category M (Cell wall/membrane biogenesis) features, which is almost identical in its pan- and core genome, whereas a shift was observed in B. cereus group and in B. subtilis, with a slight overrepresentation in the pan genome compared to the core genome.
A subset of COG proteins that were unique in both the pan and core genome of Lysinibacillus were also identified (Additional file 2: Table S2) which is probably related to species-specific characteristics. For instance, six proteins were related to ethanolamine utilization, two proteins were associated with the carbon dioxide concentrating mechanism, six were involved in cobalamin (vitamin B12) biosynthesis, one was related with the cell mobility and one with chromosome segregation.
Characterization of gene contents of Lysinibacillus strains
Pairwise comparison of the genomes of all the Lysinibacillus strains indicate a strong syntenic relationship with L. sphaericus C3-41 (Additional file 3: Figure S1), indicating that Lysinibacillus strains may have shared a common “chromosome backbone” in a very ancient stage.
The unique genes in the 11 Lysinibacillus strains, varying from 34 to 711, were COG categorized (data not shown), and appear to reflect observed functional diversity for each strain. For instance, OT4b.31 displayed a large number of unique genes encoding proteins which may be related to its tolerance for heavy-metals, e.g. Co/Zn/Cd/Mg/Ni cation transporters (6 genes), metal-dependent hydrolases (3 genes), membrane proteins related to metalloendopeptidases (3 genes), Zinc metalloprotease, Mn-containing catalase, Fe-S cluster formation, and other related Oxidoreductases. KTCC 3346 contained 19 unique genes related to cell wall/membrane/envelope biogenesis, which may be associated with its ability to produce specific surface layer proteins [18-20]. It was also interesting that a gene homologous to the virion core protein of lumpy skin disease virus was identified in the marine strain Bacillus sp. B14905. In addition, genes encoding unique bacteriophage related proteins were identified in L. sphaericus 2297 (4), C3-41 (3), SSII-1 (4), OT4b.31 (11), and KCTC 3346 (7), indicating the presence of different bacteriophage(s) or prophage remnants. However, it should be noted that these data are not exact since, with the exception of C3-41, the genomes are not completely sequenced.
A previous study showed that many strains of L. sphaericus produce restriction endonucleases which could form a barrier to genetic manipulation . The restriction enzymes and DNA methyltransferases (R-M systems) of the 11 Lysnibacillus strains were predicted by REBASE (http://rebase.neb.com). The result showed that the R-M systems in the L. sphaericus strains all belong to type II. C3-41 has the most abundant genes encoding DNA methyltransferases, with three on the chromosome and three on the plasmid pBsph, whereas 2297 and OT4b.31 only have one.
Evolution of mosquitocidal L. sphaericus
Genome Islands (GIs) predicted in L. sphaericus C3-41
Bsph1038 ~ Bsph1073
Cell wall/membrane/envelope biogenesis, Mtx2
Bsph1085 ~ Bsph1110
cell division or chromosome partitioning
Bsph 1936 ~ Bsph 1953
Bsph2575 ~ Bsph 2615
Multiple classes, major in information storage and processing
Bsph2815 ~ Bsph 2824
Bsph2913 ~ Bsph 2922
Lipid transport and metabolism
Bsph3179 ~ Bsph 3195
Bsph3265 ~ Bsph 3275
Bsph3521 ~ Bsph 3538
Replication, recombination and repair
Bsph4022 ~ Bsph 4035
In addition, a large contig in the genome of strain SSII-1 has a high overlap (>70%) and similarity (>95%) with pBsph, indicating that SSII-1 harbors a pBsph-like plasmid (named pBsph-2). Gene function analysis revealed that this contains some genes involved in replication, recombination and repair, but no GI7 was observed in pBSph-2. It is interesting that the large plasmid pBSph and pBSph-2 contain five genes which are predicted to encode proteins homologous to the type IV secretion system (e.g. VirD4, VirB4, and VirB6) and one gene encoding pilus assembly ATPase, all which may be involved in conjugal transfer. However, the function of the pBsph-2 is still to be characterized.
Lysinibacillus belongs to the family Bacillaceae. Organisms in this genus were previously regarded as members of Bacillus, but their taxonomic status was changed to the genus Lysinibacillus in 2007  and it remains for the classification to be confirmed on a genomic level. Moreover, as an important model bacterium for metabolism and mosquito control, the evolutionary model and systematic classification of L. sphaericus is a continual source of interest and debate. Therefore, exploring the phylogenetic relationship amongst members of the L. sphaericus genus in order to confirm the taxonomy of the reassigned new genus Lysinibacillus at the genomic level is of major importance. In this study, several novel genome sequences of L. sphaericus are reported, and their phylogenetic relationship with other genome sequences of Lysinibacillus and Bacillus strains are investigated.
The results showed that the genomes of all the studied Lysinibacillus strains and the marine strain Bacillus sp. B14905 show a high syntenic relationship with that of L. sphaericus C3-41, indicating these strains may have a common ancestor. Furthermore, the consensus trees based on the core genes and the genomic content indicated all the tested 10 Lysinibacillus organisms and B14905 are phylogenetically related and fall into a distinct and well defined cluster, confirming the taxonomy of the new Lysinibacillus genus. A previous study showed that one subspecies of B. subtilis is closely related with L. sphaericus based on 16 s rDNA analysis [22,23]. However, at the genome level, Lysinibacillus and B. subtilis are clustered separately. Moreover, it is interesting that despite being intergenus of Bacillus, the B. cereus group is not closely related to B. subtilis.
We also observed that a major difference between L. sphaericus and the two Bacillus species is the proportion of proteins encoded by the genome related to metabolism. This is in accordance with the observed species-specific metabolic characteristics; Lysinibacillus cannot utilize polysaccharides but alternatively metabolizes a wide variety of organic compounds and amino acids as an energy source . This may explain our observation that, compared to B. cereus group and B. subtilis, Lysinibacillus has an abundance of genes for amino acid transport and metabolism but fewer and less variable genes related to carbohydrate transport and metabolism (probably due to functional degradation). It is interesting to note that all the Lysinibacillus strains have an ethanolamine utilization gene cluster. This could be a complementary pathway for an insect pathogen unable to use polysaccharide for surviving in the insect gut . In addition, a difference was observed in the proportion of proteins with a COG classification of cell wall biosynthesis-related proteins, with members of the B. cereus group displaying a larger proportion in the pan genome than that in the core genome. This is probably because some B. cereus group strains, e. g. Bacillus mycoids and Bacillus pseudomycoids, have a different cell wall/membrane phenotype . In contrast, the cell wall biosynthesis-related proteins in the pan-genome of Lysinibacillus gen. strains are almost completely complimentary to the set identified in the core genome, suggesting the strains within this genus have specific and common features in their cell wall/membrane composition .
Amongst L. sphaericus, the genomes of toxic isolates are highly conserved, whereas those of the non-toxic strains are clearly variant. This confirms a recent MLST study which indicated that there is considerably more heterogeneity amongst non-toxic strains than amongst toxic ones, with the toxic strains tested appearing near-clonal . This is also consistent with a previous study which showed that recombination among L. sphaericus strains was relatively rare compared to the rates for most species, such as the B. cereus group, Campylobacter coli, and Listeria monocytogenes, and suggested that mutations were largely responsible for the generation of sequence diversity in L. sphaericus . Due to the large heterogeneity, it is supposed that the evolutionary distance and timescale of divergence between toxic and non-toxic strains of L. sphaericus should be large. In contrast to the lesser variation within a single species in other Bacillus spp., the toxic L. sphaericus strains may be separated from non-toxic strains and we propose a new species should be introduced.
This raises the question of how L. sphaericus strains obtained mosquitocidal toxin genes and evolved into a separate population. The proximity of mosquitocidal toxin genes with the GIs and the MGEs indicates a HGT origin and the structure of GI7, a pathogenicity island containing the major mosquitocidal toxin gene binA/binB and MGEs, provides a possible clue. GI7 possesses multiple genomic locations across the various genomes: it is present in both the chromosome and plasmid of C3-41, but is only found in the chromosome of 2297 and LP1-G, and is absent in SSII-1; furthermore, it is present in pBSph but absent in the highly similar plasmid pBSph-2. In order to assess the basic transfer potential of pBSph and pBSph-2, homologs of the T4SS genes virB4, virB6, and virD4 that were identified to be in the transfer region of the conjugative plasmids, e.g. the Ti-plasmid from Agrobacterium tumefaciens, plasmid pIP501 from Enterococcus faecalis, and plasmid pAW63 from B. thuringiensis [26,27], were investigated. The result showed that each harbor five T4SS genes displaying low levels of homology to known T4SS genes, making it doubtful that they could function as the concerted secretion machinery required for conjugation. The conjugative and transfer promoting capacities of pBsph and pBsph-2 were assessed by tri-parental matings as previously described . None were indicative of self-conjugative or mobilizable activities, at least under the conditions used in the assay (detection limit of 10–7 T/R) (data not shown). One interpretation of these results is that the ancestral form of the plasmid was conjugative and genetic drifts in subsequent lineages lead to the loss of transfer capability.
A previous study surveyed the presence of toxin genes and the associated mosquitocidal activities of L. sphaericus isolates. It showed that non-toxic strains contain only mtx2 or no toxin gene at all; low toxicity strains possess mtx1, mtx2 and mtx3; and moderately or highly toxic strains contain mtx3, binA/binB and/or cry48Aa/cry49Aa, in which some isolates also contains mtx1 and mtx2 . In addition, mtx2 and mtx3 are homologous and have close orthologs in Bacillus sp. strain NRRL B-14905 . It is also interesting that Mtx2 and Mtx3 are members of Clostridium epsilon toxin ETX/MTX2 family (pfam 03318) of pore forming toxins defined in the NCBI Conserved Domain Database . Combining the results of our analysis with these other findings, we propose the following hypothesis for the evolution of mosquitocidal L. sphaericus: 1) Lysnibacillus strains share a common ancestor; 2) A mtx2 or mtx3 ortholog was initially acquired by HGT; 3) The acquisition of mtx2/mtx3 was followed by acquisition of binA/binB, cry48a/cry49a and mtx1 also by HGT at a later time; 4) The GI containing binA/binB was obtained by phage integration into the chromosome and/or plasmid; 5) The ancestral form of pBsph and pBsph-2 was conjugative, whose capture and loss probably occurred in the population, probably playing an important role for the transmission of binA/binB. However, while the data collected to date supports this hypothesis, additional L. sphaericus genomes are needed together with complementary experimental and bioinformatics analysis.
We present the genome sequences of four Lysinibacillus strains and investigate their phylogenetic relationship to other available Lysinibacillus strains based on analysis of genome structure and identified core genes. Our results provide the first support at the genome level for the classification of these strains into a separate genus. Our analysis also indicates that mosquitocidal L. sphaericus isolates appear distinct from other Lysinibacillus organisms at the genome level, suggesting they should be classified into a separate species. Based on our findings, we hypothesis that Lysnibacillus strains evolved from a common ancestor, and the mosquitocidal toxin genes were acquired by horizontal gene transfer (HGT) resulting in the evolution of the mosquitocidal L. sphaericus.
Genome sequencing of L. sphaericus 2297, LP1-G, SSII-1 and NRS1693 was carried out using an Illumina HiSeq 2000 system by Encode Genomics Bio-Technology Co. (Suzhou, China). Paired-end reads with average length 72 and minimum read quality of 35 were used for assembly using the Velvet-1.0.14 software package . Using the genome sequence of L. sphaericus C3-41 [GenBank: CP000817 and CP000818] as reference, strains 2297, LP1-G and SSII-1 showed ~91% coverage, and their assembly produced 278, 143 and 138 contigs respectively. Strain NRS1693 showed ~74% coverage, and the assembly produced 546 contigs (Table 1).
Selection of genomes used in this study
All the 10 Lysinibacillus genomes available at the time of analysis and one Lysinibacillus-related strain Bacillus sp. NRRL B-14905  were included. The selection of 20 genomes from two representative species of Bacillus, B. subtilis and B. cereus group, was based on a previous study , which showed that B. subtilis is classified into two subspecies and one is closely related with L. sphaericus, and that B. cereus group is located on a clade neighboring L. sphaericus/B. subtilis. Thus, the five selected genomes of B. subtilis were well representative of the two subspecies. Since the seven members (i.e. B. cereus, B. thuringiensis, B. anthracis, B. weihenstephanensis, B. mycoides, B. pseudomycoides and B. cytotoxicus) of B. cereus group share close genetic and biochemical relatedness, only 15 genomes of the three major members (i.e. B. cereus, B. thuringiensis, B. anthracis) were selected as representative of the strains and species, other closely related or derivative strains were not included. In summary, a total of 17 complete and 11 gapped genomes from Lysinibacillus, B. cereus group, and B. subtilis strains were selected for analysis in this study (Table 1).
In addition, the genomes of Solibacillus silvestris [GenBank: NC_018065], Sporosarcina pasteurii [GenBank: AYOX00000000], and Ureibacillus thermosphaericus [GenBank: AJIK00000000], which are thought to be sphaericus-like organisms close to L. sphaericus based on 16 s rDNA and phenotypic analysis and previously thought belong to Bacillus  were also selected to compare with Lysinibacillus strains.
Genome annotation was performed using the xBASE web service ( http://www.xbase.ac.uk/annotation/ ), which comprises the following steps: (i) Glimmer is used for gene prediction; (ii) tRNA genes are predicted using tRNAScan-SE ; (iii) ribosomal RNA genes are searched for with RNAmmer ; (iv) protein BLAST is run using the translated coding sequences as a query against the reference sequence; (v) the best result for each BLAST search is imported as the gene annotation (if under the user-supplied E-value cutoff) [33,34]. Primary parameters were set as default, which sets the minimum length of a gene to be 90 bp, while the permitted maximum overlap of two genes is 50 bp, and the BLAST e-value cutoff is1e-10.
Each annotated protein was then compared to the COG database using BLASTP to identify its member functional groups.
Fragmented alignment of multiple genomes and phylogenomic relationship
A all-against-all fragment comparison analysis was performed using Gegenees (version 1.1.5) software by fragmenting genomes and comparing all pieces with all genomes . The heat-plot was based on a fragmented alignment using BLASTN with settings 500/500. The cutoff threshold for non-conserved material was 30%. A dendrogram was produced in SplitsTree version 4.12.8 (using the neighbor-joining method) made from a Nexus file exported from Gegenees .
Pan- and core genome analysis
The respective pan- and core genomes of 12 B. cereus group strains, 5 B. subtilis strains and 11 Lysinibacillus strains were calculated using the PanGP software package (http://pangp.big.ac.cn) [16,38], and a BLAST Matrix was constructed using a cutoff of 1e−10, and 50% identity and coverage. An R-script was used to analyze the COG protein composition in the pan- and core genomes, and the results were visualized in a bar chart .
Gene Islands (GIs) prediction
The GIs in the chromosome of L. sphaericus C3-41 were predicted using IslandViewer ( http://www.pathogenomics.sfu.ca/islandviewer/query.php ) . Using the C3-41 chromosome as the reference, the draft genome sequences of 2297, LP1-G, SSII-1, NRS1693 and Bacillus sp. NRRL B-14905 were compared and mapped with BRIG (version 0.95)  and GBrowse (version 2.49) [42,43], with the complete genomes of B. anthracis strain Ames, B. cereus strain AH187, B. thuringiensis strain BMB171, and B. subtilis strain 168 as outgroups. Some distinct special sites, including the predicted GIs, prophages and the mosquitocidal toxin genes were presented graphically outside the circle map.
Nucleotide sequence accession numbers
All four draft L. sphaericus genomes have been deposited at GenBank. Accession numbers are listed in Table 1.
Availability of supporting data
The data sets supporting the results of this article are included within the article and the additional files.
This project was supported by NFSC grants (30800002 and 31272384) and a 973 grant (2009CB118902), China.
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