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A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis

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Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis
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  RESEARCH LETTER A Bacillusthuringiensis   strainproducingapolyglutamatecapsuleresemblingthatof Bacillusanthracis  Elise Cachat 1 , Margaret Barker 1 , Timothy D. Read 2 & Fergus G. Priest 1 1 School of Life Sciences, Heriot Watt University, Edinburgh, UK; and  2 Biological Defense Research Directorate, Naval Medical Research Center, SilverSpring, MD, USA Correspondence:  Fergus G Priest, School ofLife Sciences, Heriot Watt University,Edinburgh EH14 4AS, UK. Tel.: 1 44 131 4513464; fax: 1 44 131 451 3009; e-mail:f.g.priest@hw.ac.uk Present address:  Elise Cachat, MoredunResearch Institute, Pentlands Science Park,Midlothian EH26 0PZ, UK.Received 11 February 2008; accepted15 May 2008.First published online 11 June 2008.DOI:10.1111/j.1574-6968.2008.01231.xEditor: Mark Enright Keywords Bacillus anthracis  ;  Bacillus cereus  ;  Bacillus thuringiensis  ;capsule;polyglutamicacid;MLST. Abstract Bacillus thuringiensis  serovar Monterrey strain BGSC 4AJ1 produced a micro-scopically visible capsule that reacted with a fluorescent antibody specific for thepoly- g - D -glutamic acid (PGA) capsule of   Bacillus anthracis . PGAcapsule biosynth-esis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the  B. anthracis capBCADE   cluster were present on a plasmid (pAJ1-1). StrainBGSC 4AJ1, together with five strains of   Bacillus cereus  that hybridized to a PGA cap  gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus  multilocus sequence typing scheme.  Bacillus thuringiensis  BGSC 4AJ1shared four identical alleles with  B. anthracis  and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typingdatabase. The other  cap 1 strains were distributed among various lineages of Clade 1 of the  B. cereus  group. Introduction Bacillus anthracis, Bacillus cereus  and  Bacillus thuringiensis share genomic content consistent with allocation to a singlespecies while possessing diverse virulence properties (Han et al  ., 2006).  Bacillus anthracis  is the causative agent of anthrax, primarily a disease of herbivorous animals (Mock & Fouet, 2001),  B. cereus  is a saprophyte and opportunisticpathogen responsible for endophthalmitis, pneumonia andsepticemia as well as food poisoning (Vassileva  et al  ., 2006),and  B. thuringiensis  is an insect pathogenwith application inbiological control (Crickmore, 2006). The principal viru-lence factors of   B. anthracis , the tripartite toxin and thecapsule composed of poly- g - D -glutamic acid (PGA), areencoded in plasmids pXO1 and pXO2, respectively (Mock & Fouet, 2001), the genes for emetic toxin biosynthesis in B. cereus  have been localized to a plasmid (Ehling-Schulz et al  ., 2006), and the insect-toxic crystal proteins of  B. thuringiensis  are also plasmid borne (Berry   et al  ., 2002).Examples of atypical combinations of host and toxin havebeen reported. In particular,  B. cereus  G9241 was associatedwith a case of severe, inhalation pneumonia, similar to thatcaused by   B. anthracis . The organism was confirmed to be astrain of   B. cereus  by multilocus sequence typing (MLST)but contained a plasmid that was 99.6% identical to pXO1and carried the lethal toxin genes of   B. anthracis  (Hoff-master  et al  ., 2004). Strain G9241 synthesizes a polysacchar-ide capsule rather than the PGA capsule of   B. anthracis , butthis apparently complements the absent PGA capsule anddefends the organism from the host immune system. Subse-quently, two more encapsulated strains of   B. cereus  fromfatal pneumonias were characterized (Hoffmaster  et al  .,2006). Both contained lethal toxin genes and, although B. anthracis cap  genes responsible for PGA capsule synthesiswere present in one strain, the capsules of both strains didnot react with antibody to the  B. anthracis  capsule and werepresumably composed of polysaccharide. Indeed, screeningof 47  B. cereus  isolates from various sources for capsuleproduction revealed only one additional encapsulatedstrain, again a polysaccharide capsule, from a case of pneumonia (Sue  et al  ., 2006). Finally, two, almost identical,encapsulated  B. cereus  strains were isolated from great apes FEMS Microbiol Lett  285  (2008) 220–226 c   2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved  inwest and central Africa that had died from an anthrax-likeinfection. The strains produced capsules and contained PGA cap  genes virtually identical to those of   B. anthracis  on apX02-like plasmid, but the composition of the capsule wasnot determined (Klee  et al  ., 2006). In summary, only six encapsulated strains of   B. cereus  have been described andalthough  cap  genes responsible for PGA synthesis have beendemonstrated in some of these bacteria, most producedpolysaccharide capsules.Non-encapsulated strains of   B. anthracis  are attenuated intheir virulence; indeed, the capsule is essential for dissemi-nation and lethality in a murine model of inhalation anthrax (Heninger  et al  ., 2006). Given the importance of thisvirulence determinant, we screened strains of the  B. cereus group for capsule and PGA  cap  genes and provide the firstconclusive demonstration of a PGA capsule in a strain of  B. thuringiensis . Materials and methods Strains and growth conditions Sixty strains of   B. cereus , of which seven were from invasiveinfections (Priest  et al  ., 2004; Barker  et al  ., 2005), and 18strains of   B. thuringiensis  (including strain BGSC 4AJ1 fromthe Bacillus Genetic Stock Center) were grown on tryptonesoy agar (TSA) for routine purposes and TSA supplementedwith 5% horse blood for enhancement of capsule produc-tion. Plates were incubated in a 5% CO 2  atmosphere for 16hat 37 1 C to promote capsule synthesis.  Bacillus cereus  G9241(Hoffmaster  et al  ., 2004) was used as a positive encapsulatedcontrol. Cells were dispersed in water and India ink on amicroscope slide and examined microscopically for thepresence of capsule. Cell suspensions were heated at 65 1 Cfor 30min and examined for capsule to determine covalentattachment of capsular material (Candela & Fouet, 2005). DNA hybridization Genomic DNA was prepared from cultures as describedpreviously (Priest  et al  ., 2004) and 1 m g was immobilized ona nylon membrane using a slot-blot apparatus. A probe wasPCR-amplified from  B. anthracis  Ames DNA using forwardprimer 5 0 -TCGTCATCGTCAATTTTTCG and reverse pri-mer 5 0 -CGAAGAACGCAGGCTTAGAT representing posi-tions 54934–54953 and 56012–56031 of the pXO2 plasmidof   B. anthracis  Ames (accession no. NC_007323). The probeof 1098bp partiallycovered the  capBCA genesof   B. anthracis .A probe of 1250bp was also prepared from the exopolysac-charide biosynthesis protein gene of pBC218 derived from B. cereus  G9241 using forward primer 5 0 -GACACAGTTAACATAGGGGG and reverse primer 5 0 -GGTGTTAGATCGATGAGGAGG representing positions 72287–72296 and73537–73557 of the plasmid (accession no. DQ889697).PCR reactions contained DIG-labelled dUTP (Roche) toprepare the probe and hybridizations were carried out at57 1 C for 16h, washed at 45 1 C, and developed according tothe manufacturer’s (Roche) instructions.Plasmid DNA was prepared from BGSC 4AJ1 (Jensen et al  ., 1995), separated on a 1% agarose gel and Southernblotted onto a nylon membrane. The membrane washybridized to the  cap  gene probe as described above.  Bacillussubtilis  168 was used as a negative control. Capsule analysis Capsule was extracted from cultures of strain BGSC 4AJ1grown as a lawn on plates of TSA, 5% horse blood andincubated as described above. After checking for the pre-sence of capsules microscopically, bacteria were collected in6mL phosphate-buffered saline (PBS) per plate, harvestedby centrifugation and washed in 5mL cold PBS. The cellpellet was weighed, resuspended in two volumes (v/w) waterand autoclaved at 121 1 C for 15min. After cooling, cellsuspensions were pelleted in microfuge tubes, the super-natants were pooled and precipitated on ice with fourvolumes of ethanol at   20 1 C overnight. Suspensions werecentrifuged at 4 1 C and the pellets were dissolved in theminimum amount of distilled water and dialyzed againstdistilled water overnight at 4 1 C. The extracts were electro-phoresed in polyacrylamide gel and stained with methyleneblue as described previously (Kambourova  et al  ., 2001).Direct fluorescent-antibody (DFA) detection of capsulein strain BGSC 4AJ1 was performed as described by De et al  .(2002) with cultures grown as described above andantibody kindly provided by J.W. Ezzell (United States Army Medical Research Institute for Infectious Diseases). Scanning electron microscopy (SEM) Strain BGSC 4AJ1 was grown in 100mL of nutrient brothsupplemented with MnCl 2  (50 m M), CaCl 2  (0.7mM) andMgCl 2  (1mM) at 30 1 C, with shaking until sporulation andlysisofmostofthecells(about3–4days).Culture(10mL)wascollectedbycentrifugation, washed twicein distilledwater andresuspended in 5mL of distilled water. Cells (50 m L) werepipetted onto an EM stub and allowed to dry overnight. Thesample of spores and crystals was coated with gold andobserved using a Philips XL30 scanning electron microscope. Nucleotide sequence and phylogenetic analyses Briefly, BGSC 4AJ1 genomic DNA was sequenced to highredundancy using bead-based pyrosequencing (Margulies et al  ., 2005). A total of 2015691 usable reads were producedwith an average size of 109 nucleotides for a total of 219.6Mbof raw sequence. Preliminary assembly using the 454 Inc NEWBLER   program produced 172 contigs  4 1kb for a total size FEMS Microbiol Lett  285  (2008) 220–226  c   2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved 221 Polyglutamate capsule of  B. thuringiensis  of 6.42Mb. The 7.9-kb contig containing the capsule clusterwas identified by alignment with the  B. anthracis  capsuleproteins using  TBLASTN  (Gertz  et al  ., 2006). The sequences of the genes were verified by PCR amplification and resequen-cing, and submitted to GenBank as accession nos EU444120( capBCA ) and EU669907 ( capDE  ). A full description of theBGSC4AJ1 genome and GenBank accession is currentlybeingprepared. Phylogenetic and molecular evolutionary analyseswere conducted using  MEGA version 3.1 (Kumar  et al  ., 2004). Results Screening for capsule Of more than 60 strains of   B. cereus  and  B. thuringiensis chosen to represent the genetic diversity within the group,only strain  B. thuringiensis  serovar Monterrey BGSC 4AJ1produced a convincing, microscopically visible capsule(Fig. 1a). Capsule biosynthesis in this bacterium was sporadicand we failed to find conditions that routinely promoted itsproduction. Of the various growth conditions, TSA supple-mented with 5% horse blood and incubated in a 5% CO 2 atmosphere generally enhanced capsule production, whichwas associated with postexponential growth. Not all cells insuch a culture would be capsulated. The capsule was notdispersed by heating cultures to 65 1 C, indicating that it wascovalently linked to the peptidoglycan (Candela & Fouet,2005). We supplemented visual screening with hybridizationof genomic DNA to a DIG-labelled probe derived from the capBCA  genes of   B. anthracis  Ames. This probe hybridizedstrongly to DNA from strain BGSC 4AJ1,  B. cereus  strains168287M and 191560K from a postoperative infection and aninfected insect bite, respectively (Barker  et al  ., 2005), and  B.cereus  S366 isolated from the North Sea. Weak hybridizationwas noted with  B. cereus  strains m1293 isolated from milk products and MM_4 isolated from a food processing plant(data not shown). Details of all these strains are available fromthe  B. cereus  MLSTwebsite (http://pubmlst.org/bcereus/). Wealso hybridized DNA from strains BGSC 4AJ1, 168287M and191560K to a probe prepared from the exopolysaccharidebiosynthesis protein gene of pBC218 derived from strainG9241. Only DNA from the control G9241 hybridized to thisprobe (Table 1).Concatenated sequences of six genes derived from theMLST profiles of strains showing hybridization to the  cap gene probe, together with reference sequence types (STs),were used to prepare a phylogenetic tree (Fig. 2). The  ilvD allele was excluded from the analysis because the sequencewas not available for  B. cereus  CA and CI, two encapsulatedstrains associated with anthrax in great apes (Klee  et al  ., Fig. 1.  Bacillus thuringiensis  serovar Monterrey strain BGSC 4AJ1 (a)stained for capsule using India ink and (b) following DFA stainingtargeted to the PGA capsule of  Bacillus anthracis . Table 1.  Summary of strains covered in this study and their characteristicsStrainSequencetypeCapsulepresentPGA  cap genes B. anthracis capsule (DFA)Polysaccharide cap  genes Source ReferenceBGSC 4AJ1 107  1 1  1   w Environmental This study; Kim  et al  . (2005a)CA, CI ND  1 1 z ND ND Ape anthrax Klee  et al  . (2006)G9241 78  1     1  Pneumonia Hoffmaster  et al  . (2004)G9898 78  1     1  Pneumonia/septicaemia Miller  et al  . (1997)03BB87 78  1     1  Pneumonia Hoffmaster  et al  . (2006);Sue  et al  . (2006)03BB102 11  1 1     Pneumonia Hoffmaster  et al  . (2006);Sue  et al  . (2006)03BB108 62    1    ND Environmental Hoffmaster  et al  . (2006)191560K 76    1    w Infected insect bite This study168287M 77    1    w Postoperative infection This studyPresence of PGA  cap  genes based on PCR detection of  capBCA  of  Bacilus anthracis  (Hoffmaster  et al  ., 2006).  Detection by hybridization. z Detection by sequence analysis.Presenceofpolysaccharide cap genesbasedonPCRdetection ofputative polysaccharidepolymeraseandtranslocaseofstrainG9241 (Sue et al  .,2006). w Detection by hybridization to G9241 gene probe.ND, not defined.[Correctionaddedon 24 June 2008, after first online publication: Strain 03BB102 possesses PGA  cap  genes contrary to srcinal entry in column 4 row6of Table 1.] FEMS Microbiol Lett  285  (2008) 220–226 c   2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved 222  E. Cachat  et al  .  2006). All  cap -hybridizing strains, including strains CA andCI, belonged to STs within Clade 1 of the  B. cereus  group,which includes  B. anthracis  and a predominance of   B. cereus strains (Fig. 2). Strain BGSC 4AJ1 was assigned to ST-107and shared four alleles with  B. anthracis  Ames (  gmk-1, pta-1, pur-1 and tpi-1 ). The other three alleles (  glpF-57, ilvD-52 and  pycA-52 ) differed in 2, 2 and 3 nucleotides from therespective  B. anthracis  alleles. This places strain BGSC 4AJ1on the periphery of the  B. anthracis  clonal complex and itremains one of the closest strains to  B. anthracis  of the 674strains currently in the MLST database. Strains that hybri-dized to the  cap  gene probe and strains CA and CI weredistributed throughout the phylogenetic tree and located inseveral lineages, but all were recovered in Clade 1 as definedby Priest  et al  . (2004). Demonstration of the polyglutamate capsule in B. thuringiensis   BGSC 4AJ1 The capsule material synthesized by   B. thuringiensis  BGSC4AJ1 was extracted, purified and separated by polyacryla-mide gel electrophoresis .  Bacillus cereus  G9241 was used as asource of polysaccharide capsule and  B. cereus  ATCC 10987was included because it has no discernible capsule. The ST-6ST-27ST-163ST-75ST-109CA, CIST-60ST-57ST-113 97-27ST-389 ST-112ST-130ST-134ST-2ST-1 AmesST-3ST-135ST-144 emetic  ST-47ST-26 emetic  ST-7ST-180ST-32ST-5ST-90ST-172ST-91ST-140ST-28ST-121ST-122ST-89 (from Clade 2)0.005 ST-11 03BB102 ST-76 191560.KST-62 03BB108ST-77 168287.MST-110ST-107 BGSC 4AJ1ST-102ST-31 S366ST-127 MM_4 ST-78 G9241, G9898, 03BB87 ST-45 m1293 Cereus IV Cereus V Cereus I Cereus II AnthracisCereus III Fig. 2.  Phylogenetic (neighbor-joining) tree ofstrains included in this study with representativeSTs from Clade 1 of the  Bacillus cereus group. The tree was constructed from theconcatenated sequences of six housekeepinggenes used for MLSTof the  B. cereus  group(Priest  et al  ., 2004).  Bacillus anthracis  STs areshown as stars,  B. cereus  STs as circles and Bacillus thuringiensis  STs as triangles. StrainsCA, CI (Klee  et al  ., 2006) and those thathybridized to the PGA capsule gene probe areincluded in a shaded box, and those with amicroscopically visible polysaccharide capsuleare given in an open box. Strains isolated fromcasesofpneumoniaareindicatedbyanasterisk.Lineage names are placed opposite clusters(Cereus I etc). FEMS Microbiol Lett  285  (2008) 220–226  c   2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved 223 Polyglutamate capsule of  B. thuringiensis  electrophoresed products were visualized with methyleneblue, which detects polyglutamic acid. High molecularweight PGA was obtained from strain BGSC 4AJ1 (supple-mentary Fig. S1) and was not evident in extracts of   B. cereus G9241. The presence of PGA was also demonstrated by immunoflourescence using a DFA assay targeted to the B. anthracis  capsule (Sue  et al  ., 2006). Cells exhibited greenfluorescence, demonstrating the presence of a PGA capsulesimilar to that of   B. anthracis  (Fig. 1b).The sequences of the  capBCADE   genes of strain BGSC4AJ1 were obtained froma preliminary genomic sequence of the bacterium and followed the same order as in pXO2 of  B. anthracis . Nucleotide sequence similarities to  B. anthracis Ames were  capB , 75%;  capC  , 81%;  capA , 72%;  capD , 65%;and  capE  , 63%. The relative coverage of the capsule genes interms of average reads per base was 53.4 compared with theaverage for the chromosome of about 25, suggesting thatthis locus was located on an extrachromosomal element.The plasmid location was confirmed by preparing plasmidfrom strain BGSC 4AJ1 and hybridizing a Southern blot tothe  cap  gene probe (supplementary Fig. S2).A tree was prepared from the  capB  gene of BGSC 4AJ1and homologs from other firmicutes (Fig. 3). Unfortunately,only   capB  sequences were available in GenBank for thisanalysis, but the tree shows that the gene (and presumably the associated genes) from BGSC 4AJ1 was more closely related to that of   B. anthracis  than the polyglutamatebiosynthesis genes of   B. subtilis  and its relatives. Bacillus cereus   or  B. thuringiensis  ? The close phylogenetic relationship between strain BGSC4AJ1 and  B. anthracis  led us to investigate its originalidentification as a strain of   B. thuringiensis . The only definitive feature distinguishing  B. cereus  from  B. thurin- giensis  is the presence of crystal proteins in the latter. SEMrevealed crystals in sporulating cultures of BGSC 4AJ1 thatformed bipyramidal shapes (Fig. 4). Discussion Capsule biosynthesis genes in  B. anthracis  are located onpXO2. PCR-based detection of this plasmid suggests that itis not commonly distributed in  B. cereus  and  B. thuringiensis strains (Ramisse  et al  ., 1996; Kim  et al  ., 2005b) and is absentfrom strain BGSC 4AJ1 (Kim  et al  ., 2005a,b). Moreover,screens for pXO2-located ORFs among  B. cereus  groupstrains by PCR similarly revealed their restricted distribu-tion and  cap  genes were not detected other than in B. anthracis  (Pannucci  et al  ., 2002). However, the latterstudy indicated that  B. thuringiensis  strains were morelikely than  B. cereus  strains to contain pXO2-derived genes.Such PCR-based detection systems rely on conservedprimer sequences and negative results may arise from smallchanges in primer sites. Sequence analysis of pAW63,a large plasmid from  B. thuringiensis  serovar Kurstaki,revealed a common backbone between pAW63, pXO2 andpBT9727, a plasmid from  B. thuringiensis  serovar Konku-kian (strain 97-27). The region of pXO2 encoding the cap  genes is a probable 37-kb pathogenicity island and wasnot evident in pAW63 and pBT9727 (Van der Auwera  et al  .,2005). These findings suggest that pXO2-like plasmids may be more commonly distributed in the  B. cereus  groupthan indicated by PCR-based screening. Indeed, pXO2plasmids complete with  cap  operons virtually identical insequence to those of   B. anthracis  have been detected in B. cereus  CA and CI from great apes (Klee  et al  ., 2006), Bacillus circulans  strain CBD 118 (Luna  et al  ., 2006) and Bacillus acidiciler   strain CBD 119 (Peak   et al  ., 2007) buttherewas no visible capsule in strain CBD 118 (although thisstrain reacted positively with the DFA assay on initialisolation) and the microscopically visible capsule in CBD B. licheniformis AM231532 BGSC 4AJ1 EU444120 B. subtilis AB016245 0.1 B. anthracis M24150 S. epidermidis  CP000029 B. pumilus  CP000813 Fig. 3.  Phylogenetic tree showing the relationships of the  capB  se-quence of  Bacillus thuringiensis  serovar Monterrey BGSC 4AJ1 withrelated sequences from GenBank (accession numbers indicated). Fig. 4.  SEM of a sporulated culture of  Bacillus thuringiensis  serovarMonterrey BGSC 4AJ1 showing spores and crystal proteins. FEMS Microbiol Lett  285  (2008) 220–226 c   2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved 224  E. Cachat  et al  .
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