Microbial Diversity MB 451
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Date Created: 10/15/15
solid lines 1 and every 50th nucleotide Is numbered Every 10th nucleotide is marked with a tick mark uou Noncanonical base pair GOA GA base pair c c o c c c c o Acoco c c o c o c c c A A cu GU base pair Canonical base pair AU cic GC IIIIII oocgtoco 0 an Symbols Used In This Diagram 0 c 0 Man Coo Cco ooooooo ooccgto Nov 1999 4 Enterobacteriaceae and related symbionts 5 Enterobacteriaceae 6 Escherichia 1 Bacteria 2 Proteobacteria 3 gamma subdivision c Coogtco gtgto OI 0 o x t a 4gtCcoocoogtocgt cuuccccuccn G WA quotti 6 J01695 Escherichia coli a a gtgtgtgtgtgtooccooocogtooocgtgtoogtgtogtoco0 90 UUUAACCGUAGG mic CCCOC0gtCCOgtC ocooooco 0C0 0 c C c c NEIEI oogtoc c4 0 0 0 x o gtococoocgt IIII IIIID Co 00 ooooooc cocooc0gt Secondary Structure small subunit ribosomal RNA SAR11 Pelagibacter ubique et al A genome sequence paper What is SAR11 S Cultivating the uncultivatable What does the genome tell us w v SAR11 Pelagibacter ubique et al A genome sequence paper What is SAR11 S Cultivating the uncultivatable What does the genome tell us w v clones sequenced 4 cyanobacteria SAR7 cltr fer 8 k SAR11 is a group of quotuncultivatablequot Oiproteobacteria KG Field Giovannoni 1997 AEM 6363 TABLE 3 Phylogenetic subgroups among SAR11 cluster 16S rRNA genes Name Accession my Depth Position reherence noc m E C021 numbering SARI 16 X52280 Atlantic Hydrostation S Surface 2071191 SAR95 16 M63812 Atlantic Hydrostation S Surface 4971406 SAR407 16 U75253 Atlantic BATS 80 871541 252 U75261 Atla 39 80 116171540 BDA1725 15 L11942 Atlantic near Bermuda 10 37415 OM242b U70689 Atlantic Cape Hatteras 10 56467 OM188 U70687 Atlantic Cape Hatteras 10 56464 0CS12 U75252 Paci c Oregon Coast 10 971005 FLll 10 L10935 Paci c Santa Barbara Channel 10 1471448 FL1 10 L10934 Paci c Santa Barbara Channel 10 871114 ALO21 41 M64525 Paci c ALOHA S a 39on Surface 307499 ALO38 41 M64532 Paci c ALOHA Station Surface 307499 ALO39 41 M64533 Paci c ALOHA Station Surface 307499 NH1671 15 L11949 Northeast Paci c 100 5377761 NH25710 15 L11967 Northeast Paci c 100 537317 SAR11 16 X52172 Atlantic Hydrostation S Surface 2271191 SAR193 U75649 Atlantic BATS 250 54413 BDA171 15 L11934 Atlantic near Bermuda 250 537415 BDA1720 15 L11941 Atlantic near Bermuda 10 5377765 BDA1715 15 L11939 Atlantic near Bermuda 10 5377752 NH16V2A 15 L11961 Northeast Paci c 10 537317 NH16711 15 L11951 Northeast Paci c 100 5377763 NH25 15 L11974 Northeast Paci c 100 537357 SARleb U75256 Atlantic BATS 250 871542 AR46 U75254 Atlantic BATS 80 871541 SAR466b U75263 Atlantic BATS 80 87357 SAR440 U75262 Atlantic BATS 80 87357 SAR414b U75259 Atlantic BATS 80 87355 116171540 SAR490b U75264 Atlantic BATS 80 87357 116171540 SAR418b U75260 Atlantic BATS 80 87350 118071540 AR492b U75265 Atlantic BATS 80 r 6 BDA1727 15 L11943 tlantic near Ber 10 5377741 OM239 U70688 Atlantic Cape Hatteras 10 497319 OM136b U70684 Atlantic Cape Hatteras 10 49494 OM258b U70691 Atla c Cape Hatteras 10 607551 OCS143b U75266 Paci c Oregon Coast 10 106411 NH4971 15 L11987 Northeast Paci c 500 5377756 SARZZOZ U75257 Atlantic BATS 250 871541 SARZA lb U75258 Atlantic BATS 250 871542 BDA1717 15 L11940 Atlantic near Bermuda 10 5377790 SARZOSZ U75255 Atlantic BATS 250 5 377772 NH2973 15 L11982 Northeast Paci c 100 5377756 Genes in boldface type are al c GenBank so shown in the tree in Fig 1 Line spaces separate phylogenetic suhgroups b This gene was rst reported in this paper VOL 63 1997 DEPTHeSP SAR407 SAR11 SAR193 Agrobacterium tumefaciens 1 0 FIG 1 Phylogenetic relationships among SAR11 cluster los rRNA genes interred hy neighhor joining 38 from E C021 positions 9 through 1005 the nrs12 internal segments are the percentages of boomtrap replicates which supported the branching order tor the neighborrjoining tree hoootrap values tor the h P are not shown SARI 711 and 795 surface SAR193 7203 7211 7220 and 7241 250 m SAR407 and 7464 80 m SAR Sargasso Sea ocslz Oregon coast FL11 California coast 10 neighhor joining 38 We interred parsimony trees with the heuristic search option of PAU39P 44 The hootstrap 12 with 100 replicates was used to esti CDLCILLC uUIu UULll LllC Lay PIUUC dllu nidino 2 phenylindole dihydrochlor scence in situ hybridization FISH is nining 1 that lOI CSCCI lCC signals from cells are high Because some microbial cells such as lankton are dif cult to detect by this ve been used to increase signal inten Some of these studies have focused whereas others have explored strat 1t signal per cell Our strategy was It different regions of the 16S rRNA to on signal intensity coupled with a coupled device camera for detecting ybridization image composite Dual image with DAPI blue and the Cy3 probe red Cells Cy3 probe are both blue and red and cells ii probes are blue The identical fields of view how the characteristic size and curved rod white box Scale bar 1 pm C k heSVhR lh1lemiek e gimme t abundance of c lls mf un mlbwvannom n a P1 r WCIC I Wllll I used standard DAPI staining procedures for counting cells Agree oieQ im ml ib gf r hybridization procedure 2002 Nature 4202806 I P1 cyan a LlUllb LlldL A c 50 5 1 100 2 D 150 200 ORR 39 IIquot39 Iquotlquotquotquotquotquotquotquotquot Iquotquotquotquotquotquotquot 2 4 6 2 4 6 a 10 4 6 E Cells per x105 o d 1e 500 g 10 E 1000 V1500 1003 2000 239500 1000 E 3000 5mg 4 6 z 20 40 60 3010 Cells per x103 DAPI Figure 3 SARll probe counts bacterial probe counts and direct cell counts DAPI staining particles in the northwestern Sargasso Sea a d SARll clade squares Bacteria circles and DAPI diamonds counts at 32 N 64 W CDOM Oi BATS site a 30 N 64 W CDOM 03 b 28 N 64 W CDOM 05 c and 26 N 64 W CDOM 07 d e A transect composite shows the mean abundance values by depth for SARH clade and bacterial cell counts as percentages of direct cell counts DAPI staining particles n 4 except for depths below 250 m where n 1 Standard deviations are given for depths of l 250 m One sample point was obtained for depths ioplankton below 250m at 26 N 64 W CDOM O7 k SAR11 are mostly very small lt1pm long curved 200 RM Morris et al Giovannoni 2002 Nature 420806 0 Figure 2 SAR11 fluorescence in situ hybridization image composite Dual image overlay of DNA containing cells stained with DAPI blue and the CyS probe red Cells emitting a signal for both DAPI and the CyS probe are both blue and red and cells that did not hybridize to the set of SAR11 probes are blue The identical fields of view in the DAPI and CyS stained images show the characteristic size and curved rod morphology of a magnified SAR11 cell white box Scale bar 1 pm SAR11 Pelagibacter ubique et al A genome sequence paper What is SAR11A S Cultivating the uncultivatable What does the genome tell us w v 5 Standard methods grow weeds SA Connon amp SJ Giovannoni 2002 AEM 6823878 Problems with standard methods and the solutions o Slow growers are overwhelmed by weeds Inoculate small cultures with very dilute samples o Typical media is far too rich for oligotrophs Use buffered sterilized seawater for media o Many organisms grow only to very low densities Concentrate cultures by filtration for examination Direct count ofthe inoculum by uorescence microscopy Dilute inoculum into prepared medium at 15 cells per ml and fill 48well microtiter plate with ml per well Incubate under the desired time and conditions Array ZOOuI aliquots onto a 48 sector lter manifold stain and transfer to a microscope slide Screen for positive growth by uorescence microscopy I l Identify cultures Transfer to fresh Store cultures by PCR RF LP medium with DMSO and sequencing and01 glycerol in liquid N2 FIG 1 Flow chart of HTC procedures DMSO dimethyl sulfoxr ide is Up to 15 of microbes can be grown using this method SA Connon amp SJ Giovannoni 2002 AEM 6823878 TABLE 1 Extinction culturability statistics compared to traditional culturability oounts Date modayyr Culturability on and location of lnoculum sample Avg no of Total no of No of Culture Culmrabihtyc nutrient rich agar inoculation cellsml cellswell wells inoculated positive wellsb designations sample 110R2A RZA MA2216 52198 J 11 X 106 11 144 7 HTCC1 7 45 18 93 6598 J 15 X 106 15 192 37 HTCC8 44 143 100 197 7698 8 km 37 X 106 37 192 62 HTCC45 106 105 80 135 7698 25 km 15 X 106 15 192 37 HTCC107 143 143 100 197 61799 J 56 X 106 30 192 21 HTCC144 164 39 24 59 102999 J 19 X 106 30 192 10 HTCC165 174 18 09 33 122199 J 81 X 105 50 384 10 HTCC175 184 05 03 10 12600 J 11 X 106 50 192 11 HTCC185 191 12 06 21 001 001 002 193 196 4500 J 90 X 105 50 192 20 HTCC197 216 22 13 34 015 012 71200 J 19 X 106 30 228 33 HTCC217 233 52 36 73 098 015 012 236 251 10900 8 km 13 X 106 30 384 5 HTCC252 256 04 01 10 029 009 002 1 Samples were collected on the date indicated from the jetty J or 8 or 25 km out from the mouth of Yaquina Bay Oreg 5 Wells were scored for growth after 3 weeks of incubation at 16 C Ninety ve percent con dence inteivals are shown in parentheses 391 lnoculum was the same as that used for the microtiter plates 7 not determined 8 Most grow slowly and only to low density SA Connon amp SJ Giovannoni 2002 AEM 6823878 TABLE 2 Cell densities and inferred doublings attained after 3 weeks of incubation No of No of inferred F1nal no of cellsml cultures doublingsb 10 X 103 99 X 103 66 100 133 10 X 104 99 X 104 120 133 166 10 X 105 99 X 105 62 166 199 10 X 106 99 X 106 5 199 233 1 Out of 253 cultures 5 This inference is based on the assumption that only one inoculated cell in each well grew S SAR11 isolates are small curved rods SA Connon amp SJ Giovannoni 2002 AEM 683878 DAPI images of HTCC isolates HTCCISO HTCCISO HTCCISO SAR11 HTCC148 HTCCISl HTCC154 SAR92 HTCC163 HTCC163 HTCCI75 OM43 HTCC160 HTCC160 HTCC160 OM60 OM241 FIG 2 Fluorescence microscopy images of several of the novel isolates The cells were stained with DAPI Size bars 1 pm 16 Oehler D Z amp Smi1l1W Isotopic composition ofreduced and oxidized carbon in Early Archaean Thmepmrmbuetem b i m mp om 2002 Nature and H20 Variabilin in 39 uscopic techniques Geochim Com Acknowledgements We thank Wahlen and B L Deck for providin ies carbon an su sequent isotopic measurement D l H39lton fo stepped combustion extraction of reduced car carbonate 3ses J Finarelli for39determination n composition 39 e1 for roviding coordinatio n acilities for eldWo fl search Project Support by the Marianne and Marcu Wa gnberg d NASA Exobiology is gratefully acknowledged We L P Knauth S Moorbath and I M Hayes for their comments on this manuscript Competing interests statement The authors declare that they have no competing nancial interests Correspondence and requests for materials should be addressed to MiAiviZi e mail quot ed m Bw 7 into microtitre dish wells by dilution such that on avera 3 each well r 530 EWWEEBWQNQIIEMeagf i ih ng t l e coast s3aWP f Y q 9lilg st acW Qtl lEPt39 llB E RLt e Y thWBBeand thf l j i gg tile W8 1 I i 995 mr nl il c g39 i ea amnergttnp Ucca 9 aW dmrwetecnnimnmmstgn cgl39fs WHi fi thl c l g z l a l ijweeW FWSHP Qah l U a H q gmylfl ng PEQJ oSEt P eltTWEWMmg ngf39 HEB ampW t 0 um Z kl SAR11 clade E 3 m aProteobacteria Caedibacter caryophila Mmbacterium tumefaciens Roseobacter denitn39 cans Shingamonas alaskensis Brevundimonas bacteroides yProteobacteria B Proteobacteria gural L L quotT 01009 and representatives oftne SAR11 ciade and ochroteobacteria inferred from 168 rRNA gene sequence comparisons The Gramrpositive bacteria Bacillus subtiis and Marinococcus naOpnius were used as outgroups Bootstrap proportions over 70 tnat supported tne brancning order are snown Scaie bar correspondsto 005 substitutions per nucieotide position Aiso inciuded in tne anaiysis were tne erroteobacteria Ateromonas maceodii and Marinobacter hydrocarbonociasticus and tne BrProteobacteria Metnyopnius metnyotropnus and Polynuceobacter necessarius Delagibacter ubique is a robust SAR11 S Rapp 2002 Cultivation of the ubiquitous SAR11 marine bacterioplankton clade Nature 418630 2 Cells per ml 3i 20 25 30 35 40 45 Time 1 50 Figure 3 Growth of strain HTCC1062 in Oregon coast seawater media Mediaconsisted of steriie sea water suppiemented with 10 MM NH4Ci and 01 MM KHEPO4 fiiied circies 10 MM NH4Ci 01 MM KH2P04 and mixed carbon open circies 10 MM NH4Ci 01 MM KH2P04 and Va vitamins fiiied triangies 10 MM NH4Ci 01 MM KH2P04 mixed carbon and Va vitamins open triangies 10 MM NHACi 01 MM KH2P04 mixed carbon Va vitamins and 0001 wv proteose peptone fiiied sduares For aii cuitures ceii counts attempted on days 7 and 12 were beiowtne iimit of detection dotted iine 3000 ceiis per mi as were counts on day 31 and after day 33 for tne cuiture containing proteose peptone The point at day 0 is tne inocuium density Pelagibacter ubique is really really small MS Rapp 2002 Cultivation of the ubiquitous SAR11 marine bacterioplankton clade Nature 418630 E coli 13um X 4um average considered small for a typical bacterium 13 2 x 4 676umA3 iv lume 213 2413X4 24um 2 surface area 24676 36um 2um 3 surfacevolume ratio 20000 ribosomes make up 30 of the cell mass cell wall amp membranes make up 20 of cell mass DNA makes up 2 of cell mass P ubique 015um X 06um average 015 2 X 06 00135umA3 1 500th the volume of E coli 2015A24015X06 04um 2 1100th the SA of Ecoli 0013504 30um 239Um 3 8X the SAN ratio of Ecoli DNA makes up 30 of the cell volume SAR11 Pelagibacter ubique et al A genome sequence paper What are SAR11 amp Pubique S Cultivating the uncultivatable What does the genome tell us w v P ubique has the smallest genome with the fewest genes of any freeliving organism Steve Giovannoni et al 2005 Genome streamlining in a cosmopolitan oceanic bacterium Science 309 1242 100 Fig 1 Number of pre 39 39 39 39 dicted proteinencoding O 9 genes versus genome 0 Q i 50 size for 244 complete 0 0 gggp w m a published genomes 39om bacteria and archaea P ubique has the smallest SIIIctbacterpomeroyI 0 number 0f genes 1354 Coxiella burnetii 3 open reading frames for BananaIa henselaex 6quot Synechococcus SpWH8102 any freeUVng organism Th ermop asma ddoph umu 0 Prochlorococcus marinus MIT9313 a E artonella qumtana R Q Ehrlichia rIvminantium Prochlorococcus marinus 88120 E 39 39 Prochlorococcus marinus MED4 I 39 5 M Pelagibacter ubique 392 I 1 0 Rickettsia conorii 1 308 759bp m J J a K 39 E Mesoplasma florum g I Wigglesworthia glossinidia 8 05 390 Mycoplasma genitalium I anoarchaeum equitans I i O Freeliving 0 Hostassociated 0 Obligate 39 39 I quot I Pelagibacter ubique 01 u 100 500 1000 5000 10000 Nunger of protein encoding ger es K The Rubique genome is highly streamlined but hasn t discarded any basic metabolism Steve Giovannoni et al 2005 Genome Y pesrr39s 151 Pt profundum 1 37 Table 1 Metabolic pathways in Pelagibacter Pathway Prediction EV ooh 85 Glycolysis Uncertain V39 chmerae 63 TCA cycle Present sa la ymxhgluconeogenesis Glyoxylate shunt Present E organics no C fixation Respiration Present m respiration Pentose phosphate cycle Present 395 Fatty acid biosynthesis Present lateral Cell wall biosynthesis Present a enes T h m Biosynthesis of all 20 amino acids Present g If ormOp ius Heme biosynthesis Present reclaimean Ubiquinone Present mOStly A Nicotinate and nicotinamide Present faCtorS 39 V 95 e1 atfiquoti l r Folate Present networ Riboflavin Present DO not respond to Pantothenate Absent operon Ba Absent genes why it top gPOWing at Thiamine Absent Biotin Absent 12 Absent 100 200 300 Retinal Present Nucleotides The streamlined genome is the result of opposing evolutionary forces the demand to retain the ability to make what it needs to independently live is a sparse environment and the need to minimize the genome size to lower it s resource cost and physical size Rubique has the smallest intergenic spacers of any organism known only 3bp on average Steve Giovannoni et al 2005 Genome streamlining in a cosmopolitan oceanic bacterium Science 309 1242 Fig 2 Median size of a V 9951393057 intergenic spacers for P profundum S bacterial and ardwaeal I genomes Inset shows 5 30 35 expanded view of range Vchoieraef83 far orglimisms with t e sma est inter enic S pomeroyi 7539 Spacers g P abyssi C jejuni P harfkoshi T fhermophia us T ihermoph us A sections C jejuni M geniiaiium T maritime P ubique Organism C 1 00 20 D Nucleotides Rubique has an RNase P RNA gene ran in one of it s largest quotintergenic spacers not annotated of course U A Pelaglbacler ubique gg 3 If RNase P RNA 140 33 Agrobaclermm lumefactens A U AU RNase P RNA C G 06 26 06 Izu icA GUA E GA 7n C GA A G AU GU AAC GGU 160 AU 3 A G A c A A A 6 AA A A A GUA A A G G 666 G 99 A A WIquot 3 A I 6 cc A c ccACGCGAG A col 6 A G c G 130 c GU 6 A cec c I U u c 0 06 G GcA CA 0 GGUGICUG A U AG UUG 120 G FUG I GU A CccuEGAG CAAAAGG UAIG AAGAA GAA 200 c AGGIIIGC AC U c A AUG GGG c 2mm 8 AA 22m UG A A cccA G C c I Ac AGUA I GUAA 3 9H I G c 220 AAUA 240 gm A G 66 C I a co U A A e c A A G G I GA IGII I ICAAE I W clcF f F I em F E I H E I A C I G Ac G A CGCUC UUGA cuA c39quot i 0 0 GO ccu UG CCUACUG If U UA G G UG GA UGA A on U A G 300 GU 6 cc 6 AA 6n GAAAC c i 6 25m U G c G GUA AAAGGC G G is c U G c U c u G 06 c m GU U Uc A A 60 GCCUUUG 2 AGICIUIH I TGU 6 S F Gc G A A Uc SAA A A nuecceUAAA m H A L G A 40 393 G G I A A A AGACGGUAA GUG U AAG A IIII U NH A QCUGCCAUUCCGC c c U U A G U A 360 Gc7 A 20 c 340 G A 2n U A c CA A U G c U A A 036 UOG m 1 c the cic I I G UG cu AUGAACGA UUUAAAUG CCAGUUGGCCGG ACGG IIIIlOlII A IIIIIIIIIII CCAUA GAUCUGUUUGU c GGUCAACCGGC c c SADA A A A A A UUCGGCccAAG 400 UUCGGtFCCAAG A A 330 SAR11 Pelagibacter ubique et al Pelagibacter ubique is a member of the SAR11 quotunculturable group of alphaproteobacteria that predominate the oceanic pelagic ecosystem This organism like most SAR11 species is a freeliving planktonic oligotrophic facultative photochemotroph It is very small 015gtlt 06um 1500th the volume of Ecoli providing a large surfacevolume ratio for absorbing trace nutrients and light The 13Mbp genome of Pelagibacter ubique is extremely streamlined with no repeated sequences prophage ampc and has the smallest known intergenic spacers However the genome retains all of the usual metabolic capabilities of alpha proteobacteria and is specialized for slow growth egtlttracting trace dissolved organics nitrogen and phosphorous from the open ocean water Environmental Microbiology 2003 58 631 640 Minireview The hidden lifestyles of Bacillus cereus and relatives G B Jensen B M Hansen2 J Eilenberg3 and J Mahillon National Institute of Occupational Health Lersa Parkalle 105 2100 Copenhagen Denmark 2National Environmental Research Institute Frederiksborgve 399 4000 Hoskilde Denmark 3The Royal Veterinary and Agricultural University Thorvaldsensve 40 1871 Frederiksberg C Denmark 4Laboratory of Food and Environmental Microbiology Universit Catholique de Louvain Place CroiX du Sud 212 31348 LouvainlaNeuve Belgium Summary Bacillus cereus sensu lato the species group com prising Bacillus anfhracis Bacillus thuringiensis and B cereus sensu stricto has previously been scruti nized regarding interspecies genetic correlation and pathogenic characteristics So far little attention has been paid to analysing the biological and ecological properties of the three species in their natural envi ronments In this review we describe the B cereus sensu lato living in a world on its own all B cereus sensu lato can grow saprophytically under nutrient rich conditions which are only occasionally found in the environment except where nutrients are actively collected As such members of the B cereus group have recently been discovered as common inhabit ants of the invertebrate gut We speculate that all members disclose symbiotic relationships with appropriate invertebrate hosts and only occasionally enter a pathogenic life cycle in which the individual species infects suitable hosts and multiplies almost unrestrained Introduction The Bacillus cereus group a very homogeneous cluster within the Bacillus genus comprises six recognized spe cies B cereus B thuringiensis B anthracis B mycoides B pseudomycoides and B weihenstephanensis These Received 29 November 2002 accepted 17 March 2003 For correspondence Email gbjamidkTel 45 3916 5244 Fax 45 3916 5201 2003 Society for Applied Microbiology and Blackwell Publishing Ltd species are closely related but their precise phylogenetic and taxonomic relationships are still debated Recent data based on multilocus enzyme electrophoresis MEE Helgason etal 2000 and DNA sequence variations of the 168238 internal transcribed spacers Daffonchio etal 2000 suggested that B anthracis B thuringiensis and B cereus sensu stricto are members of a single species B cereus sensu lato Whereas intensive work has been performed to decipher their genetic relationship Harrell etal 1995 Helgason etal 2000 Hansen etal 2001 Chen and Tsen 2002 less attention has been paid to comparing the biological and ecological properties of the three species in their natural environments The main purpose of this review is to elucidate the ecological and biological properties of B cereus ie the three species B anthracis B thuringiensis and B cereus with special focus on interactions with other organisms Furthermore to the extent of the limited information available the species B mycoides B pseudomycoides and B weihen stephanensis are also included in this analysis Properties of Bacillus anthracis Bacillus anthracis is the causative agent of anthrax which is primarily a disease in mammals including man for recent reviews see Mock and Fouet 2001 Apart from being one of the oldest known diseases described as one of the Egyptian plagues in the time of Moses many of the ecological and epidemiological questions about anthrax are still unanswered Anthrax has been linked with endemic soil environments long before B anthracis was identified as the causative agent Flayer 1850 Davaine 1863 The virulence of B anthracis is based on the presence of two virulence plasmids pXO1 1817 kbp and pXO2 948 kbp The plasmid pXO1 encodes three toxicfactors the protective antigen PA the lethal factor LF and the oedema factor EF Bhatnagar and Batra 2001 These components associate into two bipartite exotoxins PALF and PAEF The plasmid pXO2 encodes a poly D glutamic acid capsule enabling the bacterium to withstand phago cytosis The loss of pXO2 renders the cells incapable of establishing an infection ie the bacterium becomes attenuated a trait that is the basis of the Sterne vaccine 632 G B Jensen B M Hansen J Eilenberg and J ManiIon B anthracis M ingested sporuiation numbers into blood in nal hours of life B thuringiensis h a Activation of dissolved toxin bindin to epithelium cells followed b lysis Germination and proliferation in hemocoei ea ive orms released in massive shed aiaeam in final hours of life B cereus ingested F maestet u a K SPCRESL L L 1 sporuiation sporlulatlon VEGETATivE FORMS shed at death 39 Fig 1 An illustration of the known pathogenic life cycles of B anthracis and B thuringiensis Although a human pathogen B cereus has not been shown to enter a pathogenic life cycle similar to those of B anthracis and B thuringiensis strain Both plasmids have been sequenced recently Okinaka etal 1999ab Current models of B anthracis ecology rely on its patho genicity ie the spores are ingested by herbivores the animals becomes infected and the bacteria proliferate in the lymphoid glands concomitantly expressing the exotox ins which ultimately leadsto the death of the animal see Fig 1 Once the animal is dead the vegetative cells of B anthracis having reached a serum concentration of gt107 cells mlquot will be outcompeted by anaerobic bacteria from the gastrointestinal tract through antagonistic inter actions Dragon and Rennie 1995 The environmental fate of the spore is not known in detail The spores will survive indefinitely in dry and protected environments However exposure to sunlight for 4 h has a significant negative effect on the survival of the spores Lindeque and Turnbull 1994 Furthermore photoinduced repair of UV damage is absent in B anthracis spores of the Sterne vaccine strain Knudson 1986 A few reports stated that spores could actually germinate in nature when conditions are favourable Van Ness 1971 reported that soil pH above 60 and temperatures above 155 C favour out breaks of anthrax The term incubator areas has been introduced to describe puddles in which decaying grass and other organic matter constitute the nutrients neces sary for the germination of B anthracis spores However the study by Van Ness 1971 did not show actual growth of B anthracis in these incubator areas and other studies indicated that B anthracis has very specific growth requirements making it very unlikely for the spores to germinate outside a host Minett and Dhanda 1941 It is also noteworthy that growth of B anthracis outside a host often leads to loss of virulence caused by loss of the plasmid carrying the capsule gene pXOZ an argument that further reduces the incubator area theory As a con sequence Dragon and Rennie 1995 renamed the areas storage areas Tabaniid flies horse and deer flies from for instance the genera Tabanus and Chrysops have been reported to disseminate anthrax and to excrete B anthracis in their faeces up to 13 days the average lifetime of adult tabaniid flies after initial feeding on animals infected with anthrax Khrisna Flao and Mohiyudeen 1958 These flies have also shown ability to transmit anthrax even after subse quent feeding on uninfected hosts Krinsky 1976 In an experiment with radioactivelabelled blood from an impala carcass Braack and De Vos 1990 were able to show that the faeces of carrionfeeding blowflies Diptera Fam ily Calliphoridae were deposited in the vicinity of the carcass on leaves and twigs The kudu antelopes in South Africa normally eat leaves and twigs and could therefore be more at risk of acquiring anthrax disseminated this way Moreover these browsers are normally severely affected in anthrax epizootic episodes ln laboratory experiments stable flies and mosquitoes have been shown to transmit B anthracis after feeding on infected animals Turell and Knudson 1987 Also faeces samples collected from scavengers in the Etosha National Park in Namibia revealed B anthracis spores in more than half the samples Lindeque and Turnbull 1994 indicating a possible route of dissemination Furthermore the same study showed a rapid decline in shed vegetative bacilli and failed to demonstrate multiplication of B anthracis in the environment Properties of Bacillus lhuringiensis Bacillus thuringiensis is generally regarded as an insect 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Environmental Microbiology 5 631 640 pathogen because of its ability to produce large crystal protein inclusions 6endotoxins during sporulation the only feature that can distinguish B thuringiensis from B cereus Baumann etal 1984 These inclusions which constitute up to 25 of the dry weight of the sporulated cells Agaisse and Lereclus 1995 are responsiblefor the biopesticide activity of the bacterium and its target spec ificity van File et al 1990 see Fig 1 The genes encod ing the insecticidal proteins are generally located on large transferable plasmids Kronstad etal 1983 Gonzalez and Carlton 1984 The B thuringiensis denomination actually comprises a considerable number of isolates cov ering a broad range of toxins active against larvae from different insect orders especially Lepidoptera Diptera and Coleoptera At present more than 235 deltaendot oxin gene sequences have been described Crickmore etal 2002 and 82 different serotypes have been reported Lecadet etal 1999 The numbers of delta endotoxin genes are thought to grow steadily as most B thuringiensis strains carry more than one deltaendotoxin gene Furthermore several B thuringiensis strains are known to produce vegetative insecticidal proteins VIPs Unlike the 6endotoxins the expression of which is restricted to sporulation VIPs are expressed in the vege tative stage of growth starting at midlog phase as well as during sporulation Although B thuringiensis is an insect pathogen the ecology of the bacteria is still somewhat of an enigma According to Martin and Travers 1989 B thuringiensis is a ubiquitous soil microorganism but it is also found in environmental niches including phylloplane and insects Descriptions of natural epizootic episodes are very rare but were reported in thefirst observation of B thuringiensis by lshiwata Milner 1994 in water mills Vankova and Purrini 1979 in a corn crop Porcar and Caballero 2000 and in mosquito breeding habitats Damgaard 2000 In addition to being organized into a structured parasporal crystal the 6endotoxins can also be embedded in the spore wall Du and Nickerson 1996 found that germina tion of spores of B thuringiensis ssp kurstaki HD73 with Cry1Ac embedded in the spore coat could be activated by alkaline conditions whereas selected Crynegative B thuringiensis ssp kurstaki HD73 could not Furthermore cry spores could bind to toxin receptors in brush border membrane preparations a binding that also stimulated spore germination Du and Nickerson 1996 This phe nomenon may in part explain the evolutionary advantage of possessing 6endotoxins namely the ability for B thu ringiensis to germinate faster than B cereus and thus have a greater chance to proliferate and dominate in an insect gut even in the absence of the crystalline 6endotoxins It is important to note however that 6endotoxins have per se no apparent antimicrobial effect for enhancing colonization efficacy Koskella and Stotzky 2002 The hidden lifestyles of B cereus and relatives 633 Several facts andor premises on the ecological niche occupied by B thuringiensis have been reported i B thuringiensis does not grow in soil but is deposited there by insects Glare and O Callaghan 2000 ii B thuring iensis may grow in soil when nutrient conditions are favourable Saleh etal 1970 and iii it occupies the same niche as B cereus iv vegetative B thuringiensis proliferates in the gut of earthworms leather jacket larvae and in plant rhizospheres Hendriksen and Hansen 2002 v multiplication of B thuringiensis occurs in insects weakened by the presence of other pathogens Eilenberg etal 2000 and vi germinating B thuring iensis ssp israelensis were found in excreted food vacu oles of protozoa Manasherob etal 1998 These different possibilities are not mutually exclusive It is conceivable that B thuringiensis is a natural inhabit ant of the intestinal systems of certain insects with or without provoking disease and eventually death Thus the bacterium is able to be released in soil and can subse quently proliferate when conditions are propitious Hansen and Salamitou 2000 hypothesized that B thuringiensis is a natural inhabitant of the digestion system of many invertebrates As such if the animal is diseased the B thuringiensis present in the digestion system can start to grow in the dyingdead carcass As nutrients become lim ited sporulation occurs along with the production of 6 endotoxins These spores and toxins can then contribute to a local epizootic in dense populations of target organ isms The presence of B thuringiensis in the intestine of mammals is transient indicating that the food of these animals has varying contents of B thuringiensis Swiecicka etal 2002 Along the same lines longterm sheep feeding with B thuringiensis based biopesticide preparations 10 2 spores daily for 5months did not harm the animals Hadley etal 1987 Furthermore recent studies of faecal samples from greenhouse work ers did not show adverse effects after exposure to B thuringiensis Jensen etal 2002 Rhizoidgrowing and psychrotolerant bacteria Rhizoid growth is characterized by the production of col onies with filaments or rootlike structures that may extend several centimetres from the site of inoculation Relatively few data are available on the rhizoidgrowing bacteria B mycoides and B pseudomycoides and more specifically on their ecology As for the other members of the B cereus group they have been isolated from various environmental niches including manured soils Klimanek and Greilich 1976 activated sludge arthropod guts C Vannieuwen burgh and J Mahillon unpublished results or plant rhizo sphere where they are thought to have antagonistic activity against fungal species Pandey et ai 2001 Sim ilarly inhibition of the pathogen Listeria monocytogenes 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Environmental Microbiology 5 631 640 634 G B Jensen B M Hansen J Ellenberg and J Mahlllon by putative B mycoldes has also been reported in silage Irvin 1969 Although the rhizoid growth is characteristic of B mycoldes nonrhizoid variants have been described in a study of environmental isolates of B mycoldes by von Wintzingerode etal 1997 it was found that fatty acid analysis identified the majority of the isolates as B mycoldes even though they lacked the characteristic rhiz oid growth Even less information has been gathered on B welhenstephanensls which regroups part of the psy chrotolerant B cereus isolates Lechner et al 1998 Sten fors and Granum 2001 except for their wide distribution in natural habitats von Stetten etal 1999 The hidden life cycle of B cereus One major consequence of the lack of knowledge on the ecology of B cereus is that pleomorphism of B cereus has not been given much attention This could result partly from the general notion of modern microbiologists that bacteria only occasionally show slight morphological vari ation In the very early days of microbiology the study of microorganisms was almost exclusively restricted to microscopical observations and hence surprisingly detailed observations were made then Bacillus cereus is a wellknown food poisoning bacte rium B cereus causes two distinct types of food poison ing characterized either by diarrhoea and abdominal pain diarrhoeal syndrome or by nausea and vomiting emetic syndrome The latter has often been associated with fried rice Apart from food poisoning cases there are only a few reports on intestinal carriage of B cereus Turnbull and Kramer 1985 reported seasonal changes in the isolation of B cereus ranging from 243 in the winter to 43 in the summer from faecal samples from 120 school children Ghosh 1978 reported the presence of B cereus in 100 samples from 711 adults 14 Both papers stated that because of the omnipresence of B cereus in many food products the bacteria are inevitably ingested in small numbers and thus contribute to the transitory intestinal flora A place to look for this bacterium in its natural niche is the gut microflora of invertebrates In certain arthropods the intestinal stage of B cereus has been shown to be filamentous the socalled Arthromltus stage In fact this filamentous stage of the bacterium was discovered in different soildwelling arthropods as early as 1849 Leidy 1849 The filamentous forms of B cereus have been studied in continuous cultures Wahren etal 1967 and have lately been proposed as the normal intestinal stage of B cereus sensu lato in soildwelling insects Margulis et al 1998 Furthermore colonization of mosquito larvae and various soildwelling pests by B cereus has been observed Feinberg etal 1999 Luxananil etal 2001 Wenzel etal 2002 see Fig 1 Other circumstantial evidence supports the data on B cereus colonization of insect gut systems In aphids Dasch etal 1984 reported that the introduction of pen icillin had little effect on growth and as evident from Table 1 the majority of the members of the B cereus group are known to produce Blactamases In one case the symbiont of Cletus slgnatus a hemipteran insect is identified as B cereus var slgnatus Singh 1974 A high frequency of vegetative B cereus and B mycoldes has been found in the gut of the earthworm Lumbrlcus terres trls B M Hansen and N B Hendriksen unpublished results Gene transfer in the environment Interestingly earthworms are known to contribute to gene transfer activity with gut passage being a prerequisite for DNA transfer Daane etal 1996 Thimm etal 2001 Other insects have been shown to promote gene transfer and transfer of B thurlnglensls plasmids has been observed in lepidopteran larvae Jarrett and Stephenson 1990 Tho mas et al 2000 2001 It is therefore tempting to envisage the continuous exchange of B thurlnglensls plasmids as by transduction mobilization or conjugation Reddy etal 1987 Green etal 1989 Stepanov etal 1989 Jensen etal 1996 The actual exchange of DNA is further cor roborated bythe data presented in Table 1eg the replicon of the virulence plasmid pXO2 is almost identical to the replicon found on the conjugative plasmid pAW63 from B thurlnglensls ssp kurstakl HD73 Wilcks etal 1999 Other genotypical features such as the presence of genetic markers for phospholipase C and nonhaemolytic entero toxin genes characteristic of B cereusfurther substantiate the close relationship among these species Furthermore serotyping of B thurlnglensls has revealed that the number of crossreacting Hantigens among B cereus strains is increasing Lecadet etal 1999 However the case of B anthracls has its own particu larities It now seems likely that B anthracls does not simply stem from the superposition of the virulence genes borne by the pXO1 and pXO2 plasmids on the chromo somic background of an opportunistic bacteria B cereus Recent studies have indeed indicated that the emer gence of B anthracls as a specialized animal and human pathogen has most probably proceeded through a step wise reciprocal adaptation between its chromosomal and extrachromosomal genomes Mignot 2002 This has resulted in a finely tuned gene regulation of different oper ons and regulons such as those involved in sporulation germination haemolytic activity capsule formation or exo toxin expression These complex genomic crosstalks are thought to be mediated by an arsenal of gene regulators among which are the plasmidencoded PagFi and AtxA 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Environmental Microbiology 5 631 640 Table 1 Selected phenotypic genotypic and ecological features of B anthracis B thuringiensis and B cereus The hidden lifestyles of B cereus and relatives 635 B anthracis B thuringiensis B cereus Phenotype Penicillin resistant Blactamase production Haemolytic activitya on sheep erythrocytes Motility Crystalline parasporal inclusions Mucoid colon capsule synthesis Gamma phage sensitivity Chitinase activity Genotype pXO1 pXO2 Phospholipase C nheA gene accession no Y19005 Ecology Host ran e toxin specific Distribution Prevalence in hosts 11 of tested B anthracis strains showed resistance to penicillin G Cavallo et al 2002 Weak haemolysis by some Isolated monoflagellar anthracis have been described Liang and Yu 1999 Occasional motile strains Brown and Cherry 1955 No Yes Several rare B anthracis are refractory Abshire et al 2001 Activity was not found in B anthracis Guttmann and Ellar 2000 Yes Growth of B anthracis outside a host often leads to loss of pXO2 Mignot etal 2001 G B Jensen unpublished results or Mignot etal 2001 G B Jensen unpublished results Ve rteb rates Worldwide but many areas not yet studie Endemic in AfricaAsia Yes Yes Spontaneous flagellaminus mutants of B thuringiensis can be readily isolated 6 of tested strains showed no inclusions Logan and Berkeley 1984 No No Yes Sequence homology to thoxis of Bt ssp israelensis Berry et al 2002 found In pX Okinaka et al 1999b B thuringiensis ssp kurstaki ATCC 33679 shows high homology to an unknown ORF in pXO1 base numbers 121815 122327 Okinaka at al 1999b Pannucci etal 2002 The replicons of pAW63 from Bt ssp kurstaki are almost identical to that of pXO2 Wilcks etal 1999 S231 from Bt ssp ni mus O1 Invertebrates Specific toxins are only active Worldwide but lack of success in isolating in Antarctica Wasano et al 1999 Generally low natural levels of infection occasional epidemics among mosquitoes and insects in stored product environment Milner 1994 mutants have been isolated 4 of tested strains showed no motility Logan and Berkeley 1984 Occasional isolation of nonmotile variants Brown and Cherry 1955 o No B cereus ATCC4342 susceptible Abshire et al 2001 Yes B cereus ATCC 43881 shows high homology to an unknown ORF of pXO1 coordinates 121815 122327 Okinaka etal 1999b Pannucci et al 2002 Not known Worldwide Present in invertebrates gut system but not regarded as a disease of invertebrates a Note that at least four distinct haemolytic protein complexes can participate in the haemolytic activity 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Environmental Microbiology 5 631 640 636 G B Jensen B M Hansen J Eilenberg and J ManiIon Guignot etai 1997 Mignot etal 2001 Mignot 2002 For instance the pleiotrophic regulator Plththat regulates several virulence functions in B cereus Gohar etal 2002 is inactive in B anthracis because of a nonsense mutation The introduction of a functional Plth in B anthracis activates several B cereus like virulence func tions which are not normally expressed in B anthracis Mignot etai 2001 This is in agreement with the data of Bonventre 1965 who found that in contrast to B cereus filtrates from liquid cultures of B anthracis were not toxic to animal tissue culture cells Table 1 lists the textbook characteristics of each mem ber of the B cereus group together with exceptions found in the literature These data are intended to display both the close relationship among the species and subse quently the possible pitfalls of data misinterpretation Thus B anthracis seems to constitute a narrow group of highly similar strains which have only recently been dis tinguished genetically Jackson etal 1999 Ticknor et al 2001 Consequently and as the most significant differ ences are plasmid encoded it seems appropriate to preserve the name B anthracis for B cereus strains possessing the pXO1 and pXO2 plasmids Likewise L C a U spores c L 397 L be 7 H spores J L L Q endosymbiotic cycle emetic B cereus strains constitute a narrow group of bacteria most of which belong to the B cereus H1 sero type Furthermore strains that produce the emetic toxin do not show expression of enterotoxins and starch hydro lytic activity Agata etal 1996 Pirttijarvi etai 2000 Conclusion The presence of B anthracis in both vultures and various biting insects reveals multiple routes of recycling of B anthracis Whether there is de facto colonization of the intestinal systems of both the vultures and the insects or the observations cited here resulted from transient expo sures resulting from feeding habits is still debatable How ever the carnivorous nature of the Tabanus larvae may equip the adult fly with an intestinal flora comprising any members of the B cereus group and although much of the data on anthrax transmission by tabaniid flies is exper imental the importance of tabaniid flies in natural out breaks is conceivable According to previously presented data B cereus can enter a filamentous stage in which it colonizes a variety of insects In this context it is sug gested as illustrated in Fig 2 that members of the B Fig 2 A supposed model in which the mem bers of the B cereus group experience two life cycles one type in which the bacteria live in 4 a a symbiotic relation with their invertebrate hosts and another more infrequent life cycle in which the bacteria can multiply rapidly in another infected insect host or a mammal infective cycle K 1 u g L spores e i L LL invertebrate host for symbiotic interaction infective cycle invertebrate or vertebrate host for pathogenic interaction 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Environmental Microbiology 5 631 640 cereus group experience two types of life cycles one in which the bacteria live in a symbiotic relation with their invertebrate hosts and another more infrequent life cycle in which the bacteria can multiply rapidly in another and infected host invertebrate or vertebrate The rela tionship between the two types of life cycle has not yet been documented experimentally but some indications exist In the case of a pathogenic relationship the inver tebrate host from the symbiotic relationship becomes the vector of the disease For example a recent study showed that female mos quitoes are attracted to culture filtrates of B thurlnglensis for ovipositioning Poonam etal 2002 It is possible that these and other insects could have a preference for ovi positioning in areas where B thurlnglensis is frequently located ie soil Martin and Travers 1989 activated sludge Mizuki etal 2001 water lchimatsu etal 2000 Maeda etal 2000 and the storage areas mentioned earlier subsequently giving the larvae a possibility of being fitted with an intestinal flora consisting of members of the B cereus group These bacteria can then provide their host with enhanced capabilities for instance degrad ing cellulose Wenzel etal 2002 Further studies on the ecology of B anthracis B cereus and B thurlnglensis will hopefully not only shed light on the working models proposed here They will also enable us to set up better controlling programmes that could cope with different objectives One objective is to avoid B anthracis outbreaks especially in risk areas Other objectives are to improve the biotechnological use of B thurlnglensis and consequently obtain better control of insect pests Although experimental evidence is still missing it is likely that the rhizoidgrowing bacteria share part of the horizontal gene pool of the B cereus senso lato group using plasmid conjugation phage transduction or DNA transformation Consequently it remains to be seen whether and how these still cryptic bacteria participate directly or indirectly in the various life cycles of the other members of the B cereus group Acknowledgements We are indebted to Tam Mignot for his inspiring PhD thesis We are thankful to Lars Andrup for fruitful discussions and critical reading of the manuscript JM is a research associ ate at the National Fund for 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