We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms . Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediatelyafter their synthesis, since no accumulation of either of themwas detected in the mother cell compartment, where their synthesisoccurs . The other two components, 12.5 and 30 kDa, were generated2 h later and were probably the products of posttranslationalmodifications of the two early forms occurring directly on thecoat surface during spore maturation . None of the CotC formswas found either on the spore coat or in the mother cell compartmentof a cotH mutant . This indicates that CotH serves a dual roleof stabilizing the early forms of CotC and promoting the assemblyof both early and late forms on the spore surface.

 
The Bacillus subtilis spore is encased within a complex multilayered protein structure known as the coat, whose role is to protect the spore against bactericidal enzymes and chemicals, such as lysozyme and chloroform, and to influence the spore's abilityto germinate in response to appropriate germinants . However,the recent finding that a component of the B . subtilis coathas laccase activity [20] suggests that the coat may have other, so far unexplored, roles . The coat is composed of a heterogeneous group of over 25 polypeptides arranged into three main structural layers: a diffuse undercoat, a laminated lightly staining inner layer, and a thick electron-dense outer coat . Several of these polypeptides have been studied, and their structural genes [cot genes] have been identified . Expression of all cot genes is governed by a cascade of four transcription factors, acting specifically in the mother cell compartment of the sporangiumin the sequence sigma E-SpoIIID-sigma K-GerE, with sigma E andsigma K being RNA polymerase sigma factors and SpoIIID and GerEbeing DNA-binding proteins acting in conjunction with sigmaE- and sigma K-driven RNA polymerase [5, 11].

In addition to the transcriptional control, a variety of posttranslational modifications have been shown to occur during coat formation. At least two coat-associated polypeptides [of about 8 and 9kDa] appear to be glycosylated [11], while others are derived from proteolytic processing of larger precursors [1, 3, 27].Cross-linking of structural proteins is also believed to occurand result in the insolubilization of specific components . Sinceseveral coat proteins are tyrosine rich and since dityrosinebonds are present in purified coat material, it is believedthat this type of modification may contribute to the assemblyand function of the coat [13] . Also [-glutamyl]lysine cross-linksare found in purified spores, and a coat-associated transglutaminasehas been identified [17] . The occurrence of transglutaminase-dependentcross-linking of the outermost coat layer has been suggested[12].

The initial stages in coat assembly occur early after the onsetof sporulation and involve functional interactions among atleast two morphogenetic proteins, both made under sigma E control.First, the SpoIVA protein localizes at the outer forespore membrane.Second, SpoIVA directs the assembly of CotE in a ring-like structurethat surrounds the forespore at a distance of about 75 nm fromit [6] . The gap generated by the localization of SpoIVA andCotE is thought to be the site of assembly of the inner coatcomponents . Within this region, the inner coat may correspondto the more internal sector, adjacent to the SpoIVA protein.In contrast, the outer coat proteins are assembled on the outsideof the CotE structure [5, 11] . Additional proteins with morphogenetic functions are needed for coat formation . SpoVID and SafA aremade under sigma E control; SpoVID interacts with SafA and directsit to the forming spore and is also required to maintain theCotE ring around the forespore [5, 11, 23] . In contrast CotHis a morphogenetic protein produced under sigma K control thatplays a role in outer coat assembly and the lysozyme resistanceof the spore and that, in conjunction with CotE, is also responsiblefor efficient spore germination [21, 33].

Most coat components are produced at late stages of sporulation, with some proteins, such as CotD, CotT, and CotS, targeted tothe inner coat, and others, such as CotB, CotC, and CotG, directedto the outer coat [11] . Of these outer coat components, CotB has been recently identified as exposed on the spore surface[7, 15].

This study focuses on the incorporation of CotC into the coat structure and on how this event is controlled by the morphogenetic protein CotH . CotC is a coat component initially identifiedby a reverse genetic approach [4] and later associated with the outer coat layer [32] . Together with CotD and CotG, CotCrepresents about 50% of the total solubilized coat proteins and, being alkali soluble, can be selectively extracted from purified spores by an NaOH treatment [11] . CotC is highly similarto the protein encoded by ynzH, an open reading frame identifiedduring the analysis of the B . subtilis genome [18], and hasrecently been proposed as a new coat component and renamed CotU[19] . CotC and CotU have almost identical N-terminal regions,diverging in only 1 out of 24 amino acid residues . In addition,CotC is also relatively similar to CotG, and, intriguingly,assembly of both CotC and CotG proteins is under the controlof the morphogenetic protein CotH [21].

 
Bacterial strains and transformation. B . subtilis strains utilized are listed in Table 1 . Plasmid amplification for nucleotide sequencing, subcloning experiments,and transformation of Escherichia coli competent cells were performed with E . coli strain DH5 [26] . Bacterial strains weretransformed by previously described procedures for CaCl₂-mediated transformation of E . coli competent cells [26] and two-steptransformation of B . subtilis [2].

 
Genetic and molecular procedures. Isolation of plasmids, restriction digestion, and ligation ofDNA were carried out by standard methods [26] . Chromosomal DNAfrom B . subtilis was isolated as described elsewhere [2] . Fragmentsof cotC and cotU DNA were amplified by PCR from the B . subtilischromosome, and the amplification was primed with the syntheticoligonucleotides listed in Table 2 . The PCR products were visualizedon ethidium bromide-stained agarose gels and gel purified bythe QIAquick gel extraction kit [Qiagen] as specified by themanufacturer.

 
A cotU null mutant was obtained by transforming competent cells of the B . subtilis strain PY79 with plasmid pRH41, carrying a neomycin resistance [neo] cassette in the cotU coding region.A purified 274-bp DNA fragment originating from the amplificationof B . subtilis chromosomal DNA with Ys2 and Ya2 oligonucleotides[Table 2] was digested with NotI and EcoRI and cloned into plasmidpBEST501 [16] . The plasmid obtained was cleaved with SphI andSalI and used to clone, to the 5' end of the neo cassette, asecond PCR fragment of 242 bp, originating from amplificationof the B . subtilis chromosome with Ys1 and Ya1 oligonucleotides[Table 2] . Neo^r clones were the result of double-crossover recombination,resulting in the interruption of the cotU gene on the B . subtilischromosome . Several Neo^r clones were analyzed by PCR, and oneof them, RH202, was used for further studies . The cotU nullmutation was then moved by chromosomal DNA-mediated transformationto the following isogenic strains [Table 1]: BD063 [cotA], generatingRH214; BD071 [cotC], generating RH203; ER203, [cotG], generatingRH217; ER209 [cotH], generating RH216; RH211, [cotE] generatingRH218; and RH201 [cotB], generating RH213.

Strain RH211 was obtained by transforming strain BZ213 [cotE::cat] with a linearized form of plasmid pJL62 [cat::spc] [a gift fromA . Grossman] to inactivate cat and introduce a spectinomycinresistance gene . Several clones resistant to spectinomycin butsensitive to chloramphenicol were isolated, and one of them,RH211, was used for further studies.

cotC expression in E . coli. The cotC coding region was amplified by PCR from B . subtilis chromosomal DNA with primers CotCcoding and CotCSTOP [Table 2] . The 210-bp PCR product was cleaved with XhoI and SalI andligated into XhoI-digested expression vector pRSETA [Invitrogen].The recombinant plasmid carrying an in-frame fusion of the 5'end of the cotC coding region to six histidine codons underthe transcriptional control of a T7 promoter was used to transformcompetent cells of E . coli BL21[DE3] [Invitrogen], yieldingstrain RH52 . This strain was grown in ampicillin-supplemented[50 µg/ml] TY medium [26] to an optical density of 0.7at 600 nm . The T7 promoter was then induced by adding isopropyl-ß-D-thiogalactopyranoside [IPTG; final concentration, 0.5 mM] to the culture, which was incubated for 2 h at 37°C . The six-His-tagged CotC proteinwas purified under denaturing conditions via Ni-nitrilotriaceticacid affinity chromatography as recommended by the manufacturer[Qiagen, Inc.].

Western blotting. Sporulation of wild-type and recombinant strains was inducedby the exhaustion method [2, 22] . After a 30-h incubation at37°C, spores were collected, washed four times, and purifiedby lysozyme treatment as previously described [2, 22] . The number of purified spores obtained was measured by direct counting with a Bürker chamber under an optical microscope [Olympus;BH-2 with 40x lenses] . Aliquots of 10¹⁰ spores suspended in0.3 ml of distilled water were used to extract coat proteinsby 0.1 N NaOH treatment at 4°C as previously reported [2]. The concentration of the extracted coat proteins was determined by the Bio-Rad DC [detergent-compatible] protein assay to avoid potential interference by the NaOH present [final concentration,0.2 to 0.6 mN] in the extraction buffer and 15 µg of totalproteins fractionated on 18% denaturing poly-acrylamide gels.Proteins were then electrotransferred to nitrocellulose filters[Bio-Rad] and used for Western blot analysis by standard procedures.For the analysis of sporulating cells samples were harvestedat various times during sporulation and disrupted by sonicationin 25 mM Tris [pH 7.5]-0.1 M NaCl-1 mM EDTA-15% [vol/vol] glycerol-0.1mg of phenylmethylsulfonyl fluoride/ml . Sonicated material wasthen fractionated by centrifugation at 12,000 x g for 20 min.The pellet, containing the forming spores resistant to the sonicationtreatment, was solubilized by 0.1 N NaOH treatment at 4°C, and the total protein concentration was determined as described above . Fifty [mother cell extract] or 15 µg [foresporeextract] of total proteins was fractionated on 18% denaturingpolyacrylamide gels . Western blot filters were visualized bythe SuperSignal West Pico chemiluminescence [Pierce] methodas specified by the manufacturer.

CotC-specific antibodies were raised in rabbits immunized witha 14-amino-acid synthetic peptide [NH₂-YDYVVEYKKHKKHY-COOH] designed on the base of the C-terminal region of CotC [IGtech, Salerno, Italy].

Yeast two-hybrid system. The Matchmaker two-hybrid system [Clontech] was used as describedby Ozin et al . [23], with only minor modifications . The cotCcoding region was amplified by PCR using primer pair C/5/Ncoand C2 [Table 2] . The PCR product was digested with NcoI and EcoRI and inserted between the same sites of plasmids pAS2-1 and pACT2 [Clontech] to create fusions to the Gal4 DNA binding or activation domains, yielding plasmids pRZ99 and pRZ98, respectively. Saccharomyces cerevisiae strains Y187 [MAT ura3-52 his3-200ade2-101 trp1-901 leu2-3,112 gal4 met^- gal80 URA3::GAL1_UAS-GAL1_TATA-HIS3] and Y190 [MATa ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3,112gal4 gal80 cyh^r2 LYS2::URA::GAL1_UAS-HIS3_TATA-HIS3 URA3::GAL1_UAS-GAL1_TATA-HIS3] [Clontech] were independently transformed with the pAS2-1 or pACT-2 vector and/or each of the cotC constructs according to the protocols suggested by the manufacturer . The resulting clones were used in pairwise matings selecting for Leu and Trp . Colonylift assays for detection of ß-galactosidase activitywere as described by the manufacturer [Clontech].

 
Multiple cotC-dependent polypeptides are present in the spore coat. The cotC gene of B . subtilis encodes a 66-amino-acid polypeptide[CotC] having a deduced molecular mass of 8.8 kDa but migratingon sodium dodecyl sulfate-polyacrylamide gel electrophoresis[SDS-PAGE] gel as a 12-kDa polypeptide [4] . CotC has been identifiedas a component of the outer layer of the coat [32], and itsassembly requires the action of the morphogenetic protein CotH[21] . To study the mechanism of CotC assembly in more detail,we raised specific antibodies against a 14-amino-acid syntheticpeptide designed on the basis of the CotC C-terminal sequenceand performed a Western blot analysis on the coat protein fractionextracted from purified spores of wild-type and isogenic cotCmutant strains [4] . Anti-CotC antibodies recognized six polypeptidesin the coat proteins of wild-type spores, five of which wereabsent in the coat protein fraction of cotC null mutant spores[Fig . 1, lanes 1 and 2] . The remaining 17-kDa polypeptide, presentalso in cotC mutant spores, is evidently encoded by a differentgene and is, therefore, not directly dependent on cotC expression[Fig. 1, lanes 1 and 2] . Only two of the five cotC-dependent polypeptides [12 and 21 kDa] were extracted from wild-type spores in amounts large enough to be observed by Coomassie blue staining of the SDS-PAGE gel [data not shown].

 
Due to the high level of sequence similarity between CotC andthe putative product of ynzH, an open reading frame identifiedin the analysis of the B . subtilis genome [18], recently identifiedas the coat component CotU [19], it was possible that at leastsome of the polypeptides recognized by our anti-CotC antibodieswere the product[s] of cotU expression . To test this possibility,we constructed a cotU null mutant [see Materials and Methods]and, by chromosomal-DNA-mediated transformation, a double cotCcotU null mutant . Coat proteins were extracted from purifiedspores and analyzed by Western blotting with anti-CotC antibodies.In addition to the 17-kDa cotC-independent polypeptide [seeabove], another polypeptide [23 kDa] was dependent on the expressionof both cotC and cotU [Fig. 1] . We decided to focus our studyon the four polypeptides exclusively dependent on cotC expression,and, to optimize definition of these four bands, we used thecotU null mutant in most of the Western blot experiments ofthis study.

Expression of cotC in E . coli produces two polypeptides. The cotC gene was fused to codons for a six-His tag at its 5'end, placed under the control of the T7lac promoter, and introducedinto cells of the E . coli host BL21[DE3] [Novagen] . Cells ofthe recombinant strain obtained, RH52, were induced with IPTGand lysed as described in Materials and Methods, and total proteinswere purified by affinity chromatography on Ni²⁺ columns . Asshown in Fig . 2, two polypeptides of 16 and 32 kDa were purifiedand both were recognized by CotC-specific and six-His tag-specificantibodies . We identify the 16-kDa polypeptide as the six-His-CotCfusion product [4.5 and 8.8 kDa, respectively, with the lattermigrating with an apparent mass of 12 kDa; Fig . 1], whose expectedapparent mass is 16.5 kDa . The slower-migrating CotC polypeptide,with an apparent mass of 32 kDa [Fig . 2], is most easily explained if two CotC molecules are bound together . Based on this, wesuggest that the faster-migrating protein corresponds to themonomeric form of CotC, whereas the slower one results fromthe assembly of CotC monomers into a homodimer . Cases of dimersand also oligomers resistant to detergent treatment and strongreducing conditions have been reported previously and are ratherfrequent [25, 28, 29].

 
CotC-CotC interaction in a yeast two-hybrid system. The results shown in Fig . 2 suggested that the 12-kDa polypeptide is most likely a CotC monomer able to self-interact to originate a homodimer in E . coli without the action of specific B . subtilisfactors . We used a yeast two-hybrid system [8, 9] to confirmthat CotC monomers can spontaneously self-interact . The cotCcoding sequence was fused to either the activation domain orthe DNA-binding domain of the yeast transcriptional activatorGAL4, and the gene fusions were introduced into yeast reporterstrains Y187 and Y190 [see Materials and Methods] . Interactionof the fusion proteins within yeast cells results in the expressionof a lacZ reporter gene [10] . No ß-galactosidase activitywas detected when the individual fusion proteins were expressedwith either control vector [data not shown] . In contrast, aninteraction between CotC and itself was detected, thus confirmingthe results of Fig . 2 and indicating that CotC molecules havethe potential to self-interact.

Assembly of all CotC-dependent polypeptides depends on cotH and cotE expression. Spores of strains carrying a cotU null mutation along with anull mutation in one other cot gene were solubilized by NaOHtreatment, and the released proteins were compared with thosereleased by a strain with mutations only in cotU [Fig . 3] . Thisanalysis showed that CotC-dependent polypeptides of 30, 21,12.5, and 12 kDa were all present in the cotU single mutantas well as in the cotU cotA, cotU cotB, and cotU cotG doublemutants . All four cotC-dependent polypeptides were absent instrains with double-null mutation cotU cotH or cotU cotE [Fig. 3, lanes 5 and 7, respectively] . Moreover, the amount of the 30-kDa polypeptide extracted from cotU cotB mutant spores [Fig. 3, lane 6] was repeatedly less than that extracted from sporeswith cotU mutations only [Fig . 3, lane 2] . Identical resultswere obtained in the single null mutant strains with mutationsin cotA, cotB, cotG, cotH, or cotE [not shown], thus suggestingthat assembly of all four cotC-dependent polypeptides in thespore coat strictly requires cotE and cotH expression and that,in addition, assembly of the 30-kDa polypeptide is partiallydependent on cotB expression.

 
CotC 12.5- and 30-kDa polypeptides appear late during spore formation. Sporulating cells of a cotU null mutant were harvested at various times during sporulation and lysed by sonication as described in Materials and Methods, and the forming spores surviving the treatment were separated by centrifugation . The forming sporeswere then extracted by alkali treatment, and the released proteinswere compared with those present in the mother cell cytoplasm.For each time point both protein fractions were analyzed byWestern blotting with CotC-specific antibodies . At all timepoints analyzed we detected no CotC-specific polypeptides inthe mother cell fraction [Fig . 4] . In the forespore fraction,CotC monomeric [12-kDa] and homodimeric [21-kDa] forms wereobserved starting 8 h after the onset of sporulation [T8], whilethe other two cotC-dependent polypeptides of 12.5 and 30 kDaappeared 2 h later [Fig. 4] . Appearance of CotC-specific polypeptides starting from T8 was in perfect agreement with the previously described cotC expression pattern [14, 31] . Identical resultswere obtained with a wild-type strain [not shown] . While 15µg of total proteins was used to detect CotC from purifiedspores and forespore fractions, 50 µg of total proteins was analyzed in the case of mother cell samples . This amountof total protein allowed visualization of CotB [15] and CotA [see below] in the mother cell fraction of sporulating cells. Therefore, absence of CotC-specific polypeptides in the mothercell fraction together with the appearance of the 12.5- and30-kDa polypeptides 2 h later than the 12- and 21-kDa formsof CotC [Fig. 4] suggests that [i] CotC does not accumulatein the mother cell, probably as a consequence of its immediateassembly on the forming spore, and [ii] the 12.5- and 30-kDapolypeptides are most likely generated on the forming sporecoat by specific posttranslational modifications of the previouslyassembled forms of 12 and 21 kDa.

 
CotH stabilizes CotC in the mother cell compartment of sporulating cells. As a consequence of our hypothesis that CotC assembles on the forming spore immediately after its synthesis in the mother cell compartment and that the 12.5- and 30-kDa polypeptidesform only on the forespore, we expected to find only the 12-and 21-kDa CotC polypeptides in the mother cells of mutantsthat fail to assemble CotC . To test this prediction, we performeda Western blot analysis of CotC polypeptides from sporulatingcells of cotU cotE and cotU cotH null mutant strains that, asshown in Fig. 3, do not assemble CotC . Cells were collectedat various times during sporulation and lysed by sonication,and forespore and mother cell fractions were separated as described above . The forming spores were then extracted by alkali treatment, and the released proteins were compared with those present inthe mother cell cytoplasm by using CotC-specific antibodies.In agreement with our prediction, accumulation of only two CotC-specific polypeptides, of 12 and 21 kDa, could be observed in the mothercell fraction of a cotU cotE null mutant [Fig . 5A] . The intensitiesof the signals observed for the 12- and 21-kDa polypeptides,compared to those obtained for the same polypeptides from wild-typespores, make it extremely unlikely that the absence of the othertwo forms of CotC could be due to their low concentrations. To our surprise, we repeatedly failed to detect any CotC-specific polypeptide in the mother cell protein fraction of a cotH mutant at all time points analyzed [Fig . 5B], as well as in the foresporefraction [not shown] . The same mother cell extracts were reactedwith CotA-specific antibodies [L . Martins and A . O . Henriques,unpublished results]; this revealed the presence of CotA-specificpolypeptides of the expected sizes [Fig . 5C], thus suggestingthat the phenomenon observed in the cotH mutant is restrictedto CotC forms and is not due to aspecific protein degradation.

 
In the present work we report evidence that the CotC componentof the B . subtilis spore coat is assembled into at least fourforms, all dependent on the expression of the cotH gene . Expression of the CotC structural gene in E . coli produced two polypeptides, most likely corresponding to a CotC monomer and a homodimer resistant to the detergent treatment and the reducing conditions used . Since it is unlikely that an E . coli enzyme forms specific covalent bonds between CotC molecules and since CotC has the potential to self-interact, as shown by our experiment withyeast cells [data not shown], we believe that the CotC homodimeris most probably formed by spontaneous, noncovalent assemblyof monomers . Additional biochemical experiments, out of thescope of this study, are needed to understand the nature ofthis interaction.

CotC assembly depends on cotE and cotH expression . Dependency on cotE was expected, since it has been previously shown that cotE null mutant spores do not assemble the outer coat [31]. Dependency on cotH expression was previously reported for the most abundant 12-kDa CotC form [21] . Assembly of two other coatcomponents, CotB and CotG, also depends on cotH expression.Since assembly of CotB, in turn, depends on cotG expression[24], hierarchical control CotH-CotG-CotB was proposed [21].Here we show [Fig . 3] that all four CotC polypeptides strictly require cotE and cotH expression and do not depend on the expressionof the cotA, cotB, or cotG gene and that only the 30-kDa formhas a partial requirement for cotB expression.

Western blotting performed at various times during sporulation with the wild type and cotH and cotE mutants allowed three conclusions.[i] The 12- and 21-kDa CotC forms are assembled on the formingspore immediately after their synthesis in the mother cell compartment.This is based on the observation that the 12- and 21-kDa formsaccumulate in the mother cell of a cotE mutant [unable to assemblethem] but not in the mother cell of wild-type spores . [ii] The12.5- and 30-kDa forms of CotC are generated on the forming spore coat, since they are never found in the mother cell of wild-type or cotE mutant cells . Their formation is most likely due to specific posttranslational modifications of the previously assembled forms of 12 and 21 kDa . The nature of such modifications has not been clarified . However, since CotC contains several tyrosines [30.3% of total residues] and since dityrosine bond formation may be involved in coat assembly [reference 13 and references therein], it is possible that this type of cross-link is, at least in part, responsible for the formation of the 12.5- and 30-kDa forms of CotC . [iii] CotH or a cotH-controlled factor allows assembly of the 12- and 21-kDa forms of CotC on the spore surface . This is based on the observation that the 12- and 21-kDa forms do not accumulate in the mother cell of a cotH mutant. Since CotC is present in the cytoplasm of a cotE mutant [Fig. 5A] as well as in E . coli [Fig . 2] but is not found in the cytoplasmof a cotH mutant [Fig. 5B], its absence cannot be due to thelow stability of the protein . We believe it more likely thata specific factor [possibly a protease] degrades CotC in theabsence of CotH [or a cotH-dependent protein] . According tothis model, in a wild-type strain CotH [or a cotH-dependentprotein] would prevent CotC degradation either by interactingin a chaperone-like manner with CotC or its specific proteasein the mother cell or by immediately recruiting CotC into thecoat of the forming spore . Either way, CotC is not present inthe mother cell or on the forespore of a cotH mutant . Our datado not allow us to establish whether CotC is degraded or isonly cleaved near its C-terminal end, making it undetectablefor our antibodies . However, such hypothetical cleavage doesnot occur in a wild-type strain and would in any case lead to a nonphysiological situation [i.e., assembly of a shorter form of CotC].

 
This work was supported by the European Union grant no . QLK5-CT-2001-01729 to E.R . and A.O.H . and by MIUR [Cofin 2002; FIRB 2002] grants to E.R.

 
* Corresponding author . Mailing address: Dipartimento di Fisiologia Generale ed Ambientale, Università Federico II, via Mezzocannone 16, 80134 Naples, Italy . Phone: 39-081-2534636 . Fax: 39-081-5514437 . E-mail: ericca@unina.it .

Present address: Gulbenkian Institute of Science, Oeiras, Portugal.

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What Is Water Purification?, What Is Molecular Biology?, What Is Yeast?, What Is Botulism?, What Is Biotechnology?, c, Microorganisms, o, Microbe, a, Microbes, r, Microorganism, i, Microbiology, a, Antibiotics, a, Escherichia coli, n, Escherichia coli, s, Escherichia coli, r, Pasteurella, o, Lactobacillus, i, S. cerevisiae, r, Cell suspensions, r, S. cerevisiae, e, Clostridia, o, Microbial, i, S. cerevisiae, r, Escherichia coli, r, Gram positive, a, Thermophiles, c, Pseudomonas aeruginosa, i, Enterobacters, s, Cell suspensions, e, Salmonella typhimurium, n, Microorganisms, e, Antibiotics




 

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