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Journal of Bacteriology, January 2007, p. 228-235, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01450-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Bacillus subtilis Gene Cluster Involved in Calcium Carbonate Biomineralization

Chiara Barabesi,¹ Alessandro Galizzi,² Giorgio Mastromei,^1* Mila Rossi,¹ Elena Tamburini,^1, and Brunella Perito¹

Dipartimento di Biologia Animale e Genetica Leo Pardi, Università degli Studi di Firenze, via Romana 17, 50125 Firenze, Italy,¹ Dipartimento di Genetica e Microbiologia A. Buzzati-Traverso, and Centro di Eccellenza in Biologia Applicata, Università degli Studi di Pavia, via Ferrata 1, 27100 Pavia, Italy²

Received 13 September 2006/ Accepted 27 October 2006

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Calcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Nevertheless, little is known of the molecular mechanisms by which bacteria foster calcium carbonate mineralization. In our laboratory, we are studying calcite formation by Bacillus subtilis, in order to identify genes involved in the biomineralization process. A previous screening of UV mutants and of more than one thousand mutants obtained from the European B. subtilis Functional Analysis project allowed us to isolate strains altered in the precipitation phenotype. Starting from these results, we focused our attention on a cluster of five genes (lcfA, ysiA, ysiB, etfB, and etfA) called the lcfA operon. By insertional mutagenesis, mutant strains carrying each of the five genes were produced. All of them, with the exception of the strain carrying the mutated lcfA operon, were unable to form calcite crystals. By placing transcription under IPTG (isopropyl-ß-D-thiogalactopyranoside) control, the last gene, etfA, was identified as essential for the precipitation process. To verify cotranscription in the lcfA operon, reverse transcription-PCR experiments were performed and overlapping retrocotranscripts were found comprising three adjacent genes. The genes have putative functions linked to fatty acid metabolism. A link between calcium precipitation and fatty acid metabolism is suggested.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Calcium carbonate biomineralization is a widespread process among organisms from bacteria to Chordata, but it is generally accepted that the mineralization capacities of prokaryotes and eukaryotes are different (59). Like other biomineralization processes, calcium carbonate biomineralization can occur by two different mechanisms: biologically controlled mineralization and biologically induced mineralization (32, 34).

In biologically controlled mineralization, the organism controls the process (nucleation and growth of the mineral particles) to a high degree. The mineral particles formed are synthesized or deposited on or within organic matrices or vesicles in a specific location with regard to the cell, and usually intracellularly (9). Biologically controlled calcium mineralization is predominantly carried out by tissue-forming multicellular eukaryotes and leads to the production of complex and specialized structures such as shells, teeth, and skeletons. Every organism synthesizes biogenic minerals in a form that is unique to that species, independently of environmental conditions. Because of these features, both the synthesis and the form of every specific biogenic mineral are thought to be under specific metabolic and genetic control ( 7, 8, 9). The best known examples of molecular and genetic control of mineralization processes by microorganisms are magnetite mineralization in magnetotactic bacteria and CaCO₃ mineralization and silica deposition in the unicellular algae coccolithophores and diatoms, respectively. They all represent situations of biologically controlled mineralization and are reviewed by Bäuerlein (7, 8).

In contrast, biologically induced mineralization is usually carried out in an open environment, and no specialized cell structure or specific molecular mechanism is thought to be involved. Calcium carbonate deposition by bacteria is generally regarded as induced, and the type of mineral produced is largely dependent on environmental conditions (10, 15, 42). It is a very diffuse phenomenon (13) and represents a fundamental part of the calcium biogeochemical cycle (59), contributing to the formation of calcium carbonate sediments, deposits, and rocks (19, 22, 25, 37, 53, 59).

Different mechanisms of bacterial involvement in calcification have been proposed (23, 24), and they have been a matter of controversy throughout the last century (54). It is generally accepted that this microbial activity can be influenced by environmental physical-chemical parameters, and it is correlated to both metabolic activities and cell surface structures (11, 21, 26). Metabolic activities of heterotrophic bacteria are considered, by some authors, to be the most relevant mechanisms in calcium carbonate precipitation (18). In general, metabolic pathways able to increase the environmental pH toward alkalinity can, in the presence of calcium ions, foster calcium carbonate precipitation (26). Bacterial surfaces also play an important role in calcium precipitation (26). Due to the presence of several negatively charged groups, at a neutral pH, positively charged metal ions could be bound on bacterial surfaces, favoring heterogeneous nucleation (7, 21). Commonly, carbonate precipitates develop on the external surface of bacterial cells by successive stratification (17, 38), and bacteria can be embedded in growing carbonate crystals (17, 43). However, the actual role played by bacteria in calcium mineralization is still debated (18, 54, 59).

Proposed applications of CaCO₃ mineralization by bacteria include the fields of biomimetic processes and materials (33, 50, 10, 30, 47) and bioremediation as leaching (27), solid-phase capture of inorganic contaminants (57), and plugging-cementation in rock and other porous media fissures (49, 4). Bacterial calcium carbonate mineralization has also been proposed as a new tool in the conservation of monumental calcareous stones (5, 18, 36, 40, 44, 51).

We are studying calcium carbonate formation by Bacillus subtilis in order to identify genes and cell structures involved in the biomineralization process (5). B. subtilis is a model laboratory bacterium which can produce calcite precipitates on suitable media supplemented with a calcium source (41, 51) (Fig. 1). To study genes involved in CaCO₃ crystal formation, a B. subtilis mutant unable to form precipitates was obtained by UV mutagenesis (40). We also screened 1,190 mutants of B. subtilis 168 obtained from the European B. subtilis Functional Analysis (BFA) consortium. These strains were constructed by insertional mutagenesis using the pMUTIN plasmid to study the functions of the uncharacterized open reading frames identified in the sequenced B. subtilis genome (52). In this way, six mutants impaired in calcite crystal formation were isolated. A preliminary sequence analysis of the mutated genes revealed that, in many cases, their putative function was linked to fatty acid metabolism (40).

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FIG. 1. Calcite crystals produced by B. subtilis strains on B4 solid medium.

In this paper we report the identification of a B. subtilis gene cluster involved in calcium carbonate biomineralization.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains and growth conditions. B. subtilis strains used are listed in Table 1. Escherichia coli strains XL1-Blue, DH5, and JM109 (45) were used as hosts for the construction of recombinant plasmids.

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TABLE 1. B. subtilis strains used in this study

Media used were nutrient broth and nutrient agar (Oxoid) for B. subtilis growth and liquid or agar Luria-Bertani medium (45) for E. coli growth. Calcium carbonate precipitation growth medium included B4 medium (0.4% yeast extract, 0.5% dextrose, 0.25% calcium acetate, 1.5% agar if solid) (13).

Media were supplemented with ampicillin (100 µg/ml), chloramphenicol (5 µg/ml), IPTG (isopropyl-ß-D-thiogalactopyranoside) 0.5 mM and 1 mM, and X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) 40 µg/ml, when appropriate.

Crystal observation and analysis. To produce crystals, strains were streaked on precipitation solid medium, incubated at 37°C for 21 days, and observed with a stereomicroscope each day. Crystals were visible after a few days of incubation.

Crystals produced on solid media were collected as described by Tiano et al. (51). Precipitates from liquid media were collected by filtration through a Millipore filter (0.45 µm). Filters were dried at 60°C overnight. Precipitates were analyzed by a Fourier transform infrared (FTIR) spectrophotometer and X-ray diffraction.

Recombinant DNA techniques. B. subtilis genomic DNA was extracted as described by Marmur (35). B. subtilis was transformed with chromosomal or plasmid DNA by the procedure described by Anagnostopoulos and Spizizen (2).

E. coli transformation, cloning, and other molecular techniques were carried out according to standard protocols (45).

PCR amplification was performed using a GeneAmp PCR System 9600 and the DNA polymerase Taq-Platinum (Invitrogen). Amplified fragments were purified from the PCR mixture with the High Pure PCR product purification kit (Roche).

B. subtilis mutagenesis. For random and directed B. subtilis mutagenesis, the integrative pJM103 plasmid was used (39). To modulate the transcription of lcfA operon genes, the integrative pDH88 vector carrying the IPTG-controlled Pspac promoter (28, 39) was used.

DNA sequencing and analysis. Sequencing was performed in a Perkin-Elmer ABI 310 sequence analyzer and analyzed with OMIGA software (OMIGA 1.1; Oxford Molecular). BLAST (1) was used at the web sites http://genolist.pasteur.fr/SubtiList/ and http://www.ncbi.nlm.nih.gov/.

RNA preparation and analysis. B. subtilis 168 cells were grown at 37°C in B4 liquid medium and collected during the exponential growth phase by centrifugation (6,000 rpm at 4°C for 15 min).

Total RNA was extracted with a Fast RNA kit Blue (Qbiogene). The pellet was dissolved in diethyl pyrocarbonate-treated water and treated with DNase (2 U DNase I RNase-free; Ambion) at 37°C for 1 h in the presence of 20 U RNaseOUT (Invitrogen). After incubation, the sample was precipitated with lithium chloride and ethanol. The yield was determined by UV spectrometry.

The primers used in reverse transcription (RT)-PCR are listed in Table 2. The absence of DNA in the RNA preparation was tested by PCR experiments with the same primers used for RT-PCR. To amplify the intragenic and short intergenic retrotranscripts of the five genes, 50 ng of total B. subtilis RNA and an RT-PCR one-step RT-Platinum Taq kit (Invitrogen) were used. To amplify retrotranscripts comprising three or more genes, a two-step RT-PCR with the reverse transcriptase Superscript II (Invitrogen), 1 µg of total B. subtilis RNA, and the DNA polymerase Platinum Taq High Fidelity (Invitrogen) were used. When needed, the band of the expected size was eluted from the gel by QIAquick gel extraction (QIAGEN) and sequenced with the same primers used for amplification.

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TABLE 2. Oligonucleotide primers matching the lcfA operon used in this study

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Isolation of B. subtilis mutant strains impaired in calcite precipitation. Mutants impaired in precipitation were searched by random insertional mutagenesis of B. subtilis PB19, a prototrophic derivative of strain 168. B. subtilis PB19 genomic fragments of 200 to 400 bp obtained by digestion with Sau3A restriction enzyme were cloned into the BamHI restriction site of the pJM103 integrative vector (39). Recombinant plasmids amplified in E. coli XL1-Blue were tested for the presence of the insert by PCR, using the universal M13 forward and reverse primers flanking the pJM103 polylinker. Recombinant plasmid pools derived from 50 white colonies each were used to transform B. subtilis PB19. Chloramphenicol-resistant transformants were tested for calcite crystal production on solid B4 medium. By this approach, one mutant unable to produce calcite was isolated. To verify that the precipitation phenotype was linked to chloramphenicol resistance and therefore to pJM103 insertion, B. subtilis PB19 was transformed with genomic DNA extracted from the mutant. All transformants resistant to chloramphenicol were impaired in crystal production. One of these strains was called B. subtilis CadA (Table 1); its precipitation phenotype is shown in Table 3. The same phenotype was observed upon transfer of the mutation to strain 168 (strain FB CadA, Table 1). To isolate the PB19 genomic fragment containing the mutation, the integrated plasmid was rescued from the genome together with adjacent chromosomal sequences and recloned in E. coli as described by Perego (39). Sequence analysis revealed that the insert corresponds to the 3' region of the ysiB gene located at 2917 kb on the chromosome. An independent screen of a large collection of insertion mutants constructed by a consortium of European laboratories (BFA), identified a mutant impaired in crystal formation with an insertion in the middle of the ysiB gene (strain BFS2427). An additional mutant with a similar phenotype had the insertion in the adjacent ysiA gene (strain BFS2426) (40). This prompted us to focus our attention on this region of the chromosome. The ysiB gene is preceded by ysiA and lcfA and followed by etfB and etfA. This group of genes is flanked by two putative intrinsic transcription terminators (http://www.pasteur.fr/Bio/SubtiList). We called it the lcfA operon. The organization of the region is shown in Fig. 2. To assess which gene was involved in crystal formation, isogenic insertional mutants were produced. For each gene, an amplified product of approximately 200 bp, corresponding to the central part of the putative coding region, was obtained by PCR from genomic DNA using couples of primers with BamHI or PstI restriction sites at their 5' ends (Tables 1 and 2). Each fragment was cloned into plasmid pJM103, and the recombinant plasmid was used to transform B. subtilis strain 168. Transformants resistant to chloramphenicol were tested for calcium carbonate precipitation on B4 solid medium. With the exception of the FBC1 mutant (lcfA inactivated), all other mutant strains (FBC2 to FBC5) were impaired in crystal production (Table 3; Fig. 1). To verify that the mutation was associated with the chloramphenicol resistance, genomic DNA was extracted from each mutant and used to transform back strain 168: chloramphenicol resistance and crystal formation were found to be inseparable.

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TABLE 3. Results of CaCO3 precipitation on B4 solid medium by B. subtilis wild-type and mutant strains

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FIG. 2. Map of the B. subtilis 168 lcfA operon and results of RT-PCR. Arrows show the direction of transcription. Two putative intrinsic transcription terminators are indicated at both ends of the region. Chromosomes are enumerated according to the system found at www.pasteur.fr/Bio/Subtilist. Bars indicate major cotranscripts obtained by RT-PCR; their sizes in base pairs (bp) are at the left of the bars. Primers used flank each bar.

These data exclude the involvement of the lcfA gene in calcium carbonate deposition. The effects of the insertions in genes ysiA, ysiB, and etfB could be due to inactivation of a function required for crystal formation or to a polar effect on the etfA gene, which appears to be the last gene of an operon.

To verify whether mutations could affect precipitation through changes in medium pH level, strains 168 and FBC1 to FBC5 were grown in B4 liquid medium with calcium acetate replaced by sodium acetate to avoid calcium carbonate precipitation and incubated statically at 37°C for 10 days. The pH was measured each day. No significant differences were detected in the pH values among cultures (data not shown).

Cell cultures grown on B4 solid medium were observed with a phase-contrast optical microscope: all the strains (wild type and mutants) sporulated on this medium as well as they did on other media (data not shown). This observation suggests that sporulation is not affected by calcite precipitation.

Specificity of biomineralization. To exclude the possibility that the mutant phenotype might be influenced by the presence of acetate as a carbon source, parental and mutant strains were tested on B4 solid medium supplemented with 0.25% CaCl₂ instead of calcium acetate. The precipitation behaviors of all strains were identical to those reported in Table 3, indicating that precipitation was independent from the anion present.

To test the specificity of calcium carbonate precipitation, B. subtilis 168 and mutants were grown on B4 solid medium with calcium acetate replaced by acetate salts of ammonium, barium, or strontium at a 0.25% concentration (46). B. subtilis 168 and FBC1 formed precipitates both on calcium (control) and on strontium acetate, while FBC2 to FBC5 did not. Under all other experimental conditions, no precipitate was observed after 21 days of incubation. Precipitates formed in the presence of strontium acetate were analyzed by FTIR and X-ray diffraction and found to be composed of strontium carbonate (strontianite) (data not shown).

Modulation of the expression of the lcfA operon genes. The insertional mutagenesis data showed that all genes of the lcfA operon, with the exception of lcfA, could be involved in the precipitation process. A polar effect on the expression of genes downstream of the interrupted gene could not be excluded. To evaluate this possibility, B. subtilis strains were constructed with an IPTG-inducible promoter upstream of each one of the four genes (ysiA, ysiB, etfA, and etfB). In these strains, the transcription of regions downstream of the inserted promoter is under IPTG control. The experimental procedure was based on the use of the pDH88 integrative vector carrying the IPTG-controlled Pspac promoter (28, 39). For each gene, a 5' region of about 200 bp containing the putative ribosome binding site and start codon was amplified by PCR from the B. subtilis 168 genome and cloned into the pDH88 ClaI and XbaI restriction sites. Recombinant plasmids were amplified in E. coli cells. B. subtilis chloramphenicol-resistant transformants were called strains FBC6 to FBC9. The target regions and primers used are described in Table 1 and 2. The correctness of the recombinant pDH88 insertion was verified by amplifying a region of about 400 to 500 bp using primer pDH88-fw (5'-CCAGACTATTCGGCAC-3') internal to pDH88, coupled to primers B1, C1, D1, and E1 (Table 2) for strains FBC6, FBC7, FBC8, and FBC9, respectively. The amplified products were sequenced.

Strains FBC6 to FBC9 were tested on B4 solid medium with 0.5 mM and 1 mM IPTG and without IPTG. Precipitation results are shown in Table 3. In the absence of IPTG, all strains were unable to produce crystals. The insertion into the chromosome of the plasmid with the spac promoter does not alter the resident promoter and should not affect the expression of genes upstream of the insertion. Thus, the absence of activity observed for all four constructs in the absence of an inducer strongly suggests that the products of ysiA, ysiB, and etfB are not sufficient to sustain the precipitation of calcite. On the other hand, induction of the promoter inserted upstream in all the genes resulted in the formation of calcite crystals (Table 3). This result strongly suggests that the last gene, etfA, is essential for the precipitation phenotype.

The mutant strains were also tested on solid medium supplemented with strontium acetate in the presence and absence of IPTG. The results were the same as those obtained with calcium acetate.

Transcription analysis of the lcfA operon genes. We used RT-PCR to study the transcription of the lcfA operon. Primers used are described in Table 2 and results are given in Table 4.

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TABLE 4. Results of RT-PCR of intragenic and intergenic regions of the lcfA operon

The presence of transcripts corresponding to the single open reading frames was tested by one-step RT-PCR. Single bands of the expected size were obtained for genes lcfA, ysiA, ysiB, and etfA (Table 4; Fig. 3A). In the case of the etfB gene, no band was obtained (Table 4; Fig. 3A). Single bands of the expected size were obtained when the presence of retrotranscripts of intergenic regions comprising two genes was tested. Testing for the presence of the amplified product by using D6 and E1 primers showed that the etfB gene as well was transcribed (at least cotranscribed with etfA) (Table 4).

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FIG. 3. Ethidium bromide-stained 1% agarose gels of RT-PCR products of the lcfA operon. RT reactions were performed with total RNA obtained from strain 168. (A) Retrotranscripts of single genes. Lane 1, retrotranscript of the intragenic lcfA region obtained with primers A3 and A2 (forward and reverse, respectively). Lane 2, retrotranscript of the intragenic ysiA region obtained with primers B3 and B2. Lane 3, retrotranscript of the intragenic ysiB region obtained with primers C1 and C2. Lane 4, RT-PCR of the intragenic etfB region with primers D3 and D2. Lane 5, retrotranscript of the intragenic etfA region obtained with primers E3 and E2. Lane 6, molecular weight marker 1 kb plus DNA ladder (Invitrogen). Lane 7, PCR of the intragenic lcfA region with primers A3 and A2. (B) Retrotranscripts comprising three adjacent genes. Lane 1, retrotranscript of the intergenic ysiB-etfA region obtained with primers E3 and C6. Lane 2, retrotranscript of the intergenic lcfA-ysiB region obtained with primers C1 and A4. Lane 3, retrotranscript of the intergenic ysiA-etfB region obtained with primers D1 and B6. Lane 4, molecular weight marker 1 kb plus DNA ladder (Invitrogen). Lane 5, PCR of the intergenic ysiB-etfA region with primers E3 and C6.

Since cotranscription was demonstrated for each couple of adjacent genes, the presence of cotranscripts of greater size, including all genes in the cluster, was tested. A two-step RT-PCR system with a DNA polymerase particularly indicated for long amplification products was used.

To test for the presence of transcripts including three adjacent genes, the cDNA obtained with primer E3 was used in a PCR with primer C6. The amplified product of the expected size (1,765 bp) was detected, including genes from ysiB to etfA (Table 4; Fig. 3B). The cDNA obtained with primer C1 was used in a PCR with primer A4. The amplified product of the expected size (1,688 bp) was detected, including genes from lcfA to ysiB (Table 4; Fig. 3B). The cDNA obtained with primer D1 was used in a PCR with primer B6. The amplified product of the expected size (1,155 bp) was detected, including genes from ysiA to etfB (Table 4; Fig. 3B). These three retrotranscripts overlapped each other at ysiB (Fig. 2). They were the longest retrotranscripts obtained by RT-PCR. Reactions directed to test for the presence of longer transcripts including four or five genes gave no products (Table 4).

From these data, we infer that the region from the lcfA gene to the etfA gene is an operon expressed as a single transcriptional unit, with the possible presence of additional internal promoters.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This paper describes the isolation of B. subtilis mutants impaired in calcite precipitation and the identification of a gene cluster involved in the process. In a previous work (40), we reported the identification of six B. subtilis 168 mutant strains, obtained from the B. subtilis European consortium, impaired in calcium precipitation. Two of the mutants, strains BFS2426 and BFS2427, carried mutations in genes ysiA and ysiB, respectively. By random insertional mutagenesis described in this paper, a B. subtilis PB19 mutant, called the CadA mutant and impaired in precipitation, was isolated. The target region of the mutational insertion was identified in the ysiB gene. The position of the ysiB mutation carried by the CadA mutant is different from that of BFS2427 (http://www.pasteur.fr/Bio/SubtiList). These preliminary data focused our attention on a group of genes (Fig. 2) called the lcfA operon, with functions possibly involved in fatty acid metabolism (58).

By insertional mutagenesis, five B. subtilis 168 isogenic strains were produced, each carrying a mutation in a single gene of the cluster (FBC1 to FBC5). A sixth isogenic mutant (FB CadA) was obtained by the transformation of B. subtilis 168 with genomic DNA from the CadA mutant.

With the sole exception of strain FBC1 (lcfA), the lcfA operon mutants do not form calcium carbonate precipitates under growth conditions in the way the parental strain does. These data point to an involvement of the whole cluster in the precipitation phenotype, but these results could also be due to polar effects on the downstream genes. For this reason, four additional mutants were constructed (FBC6 to FBC9) in which transcription was under the control of an IPTG-inducible promoter. All strains formed crystals only when IPTG was added. This phenotype suggests that the last gene (etfA) is essential for precipitation.

The expression at the transcriptional level of the cluster in cells grown on B4 medium was investigated by RT-PCR. Single retrotranscripts for all the genes were obtained, with the one exception of the etfB gene. Single retrotranscripts for all five genes were reported in B. subtilis cells grown in a different medium (29). In that study, the primers used for etfB were different from those used in the present study. On the other hand, in our experiments, the etfB gene was cotranscribed in retrotranscripts that included two or three genes, indicating that the failure to detect it as a single transcript could depend on the chosen primers. The longest retrocotranscripts included no more than three genes. All long retrotranscripts overlapped at ysiB, two at ysiA and two at etfB. This suggests that a unique cotranscript for the whole cluster could exist, but the instability of the RNAs does not allow it to be detected.

The putative transcriptional unit from lcfA to etfA was originally described by Wipat et al. (58) as a group of genes apparently encoding proteins involved in fatty acid metabolism. The functions assigned to their products at the SubtiList web site (http://genolist.pasteur.fr/SubtiList/) and at the NCBI web-site (http://www.ncbi.nlm.nih.gov/) are all putative. The product of the lcfA gene is homologous to prokaryotic and eukaryotic long-chain acyl coenzyme A (acyl-CoA) synthetases. The product of the ysiA gene is similar to prokaryotic transcriptional regulators of the TetR/AcrR family. The product of the ysiB gene is homologous to prokaryotic and eukaryotic proteins belonging to the enoyl-CoA hydratase/isomerase family. Finally, the products of genes etfA and etfB are similar to the and ß subunits of prokaryotic heterodimeric flavoproteins involved in electron transport, respectively.

None of these genes was essential (31; also see Micado [http://locus.jouy.inra.fr/cgi-bin/genmic/madbase_home.pl] and JAFAN [http://bacillus.genome.jp/]): ysiB gave a positive response to phenotype tests for the effects of glucose, N, or C sources and starvation (12), but no additional information is available. In general, no phenotype was associated with mutants carrying the five genes.

Many putative functions assigned to genes of the lcfA operon can be found in bacteria, involved in pathways linked to fatty acid metabolism, with different functional significance, such as the E. coli fad regulon (20), the BCS operon of Clostridium acetobutylicum (14), and the rpf cluster of Xanthomonas campestris (6, 56).

In E. coli, fatty acid degradation by the ß-oxidation cycle is controlled by the fad regulon (20). E. coli fadD encodes a fatty acyl-CoA synthetase (56% homologous to B. subtilis LcfA) which is postulated to activate fatty acids via an AMP-mediated CoA binding. According to DiRusso et al. (20), in the first step of the ß-oxidation cycle, an electron transferring flavoprotein, ETF, encoded by the fadE locus, should be required for the acyl-CoA dehydrogenases (ACDHs). In this case, an ETF activity level similar to those of the putative products of B. subtilis genes etfA and etfB could be found. The presence of an ETF homologue in the E. coli fad regulon is, however, controversial (16). The E. coli FadB1 (the FadB N-terminal domain encoded by fadB) is 53% homologous to B. subtilis YsiB and has an enoyl-CoA hydratase activity.

Wipat et al. (58) already noted similarities between the organization of the lcfA operon and the BCS cluster of Clostridium acetobutylicum involved in the conversion of aceto-acetyl-CoA to butyryl-CoA in the butanol-butyrate synthesis pathway of clostridia (14). The BCS operon includes the crt gene encoding crotonase (butyryl-CoA hydratase, which has 60% similarity to YsiB) and two etf genes encoding the and ß subunits of an electron transfer flavoprotein (64% similarity to B. subtilis ETFA and 57% similarity to B. subtilis ETFB, respectively).

Finally, two genes, rpfF and rpfB, of Xanthomonas campestris, involved in the regulation of phytopathogenicity, encode as putative products an enoyl-CoA hydratase (47% homologous to YsiB) and an acyl-CoA synthetase (57% homologous to LcfA), respectively. The two genes regulate pathogenic factor production via a diffusible signal factor identified as cis-11-methyl-2-dodecenoic acid (6, 56).

Therefore, our data suggest the possibility of a link between calcite precipitation and fatty acid metabolism. This link could be due to possible pleiotropic effects that mutations in fatty acid pathways may have on cellular metabolism, or the lcfA operon might be involved in the synthesis of a lipid intermediate (e.g., an acyl-CoA intermediate) directly involved in biomineralization. We can only speculate on the possible role of the putative intermediate in precipitation: a structural role (i.e., it could participate in or contribute to a surface structure responsible for nucleation and precipitation) or a regulatory role (as for the X. campestris rpfF and rpfB products [56]). It should be noted that a cDNA encoding a putative long-chain fatty acid CoA ligase was found among the cDNAs expressed under conditions promoting coccolithogenesis in the unicellular coccolithophorid Emiliania huxleyi (55). It is worth noting also that in our screen of BFS strains, we found four additional mutant strains impaired in calcium precipitation (40). Three of these strains (BFS1345, BFS1346, and BFS1347) were inactivated in genes yusJ, yusK, and yusL, respectively, with putative functions again linked to fatty acid metabolism.

Despite the lack of knowledge about the mechanism through which these genes are involved in the biomineralization process, the mechanism seems specific. In fact, inactivation of B. subtilis 168 genes yngI, yngF, and yngJ, whose deduced products are similar to those of lcfA, ysiB, and yusJ, respectively, did not affect calcite precipitation (data not shown). In addition, the mineralization is limited to calcium and strontium and is not affected by the anion source.

Intracellular calcium concentration must be maintained at low levels in the eukaryotic as well as in the prokaryotic cell because of the key role of calcium in the regulation of many fundamental processes and its potential harm to cell structures (48). Considering that it is a very diffuse phenomenon, calcium carbonate precipitation could be a detoxification mechanism to immobilize the excess of dangerous extracellular calcium in bacteria (3) as well as in eukaryotes (15). Considering the importance of the biomineralization process for cell life, it seems unlikely that it would be relied on only for aspecific mechanisms in bacteria. It seems more likely to hypothesize more than one bioprecipitation mechanism (23, 24) with a different involvement of bacterial cells.

To our knowledge, this is the first report of the isolation of bacterial mutants impaired in calcite biomineralization and the suggestion that the process is genetically controlled.

 
We thank Susanna Bracci (CNR, Florence, Italy) for spectrophotometer FTIR and X-ray diffraction crystal analysis and C. Indorato for technical assistance.

This work was supported by EC grant EVK4-CT2000-00037 BIOREINFORCE.

 
* Corresponding author. Mailing address: Dipartimento di Biologia Animale e Genetica Leo Pardi, Via Romana 17, 50125 Firenze, Italy. Phone: (39) 055 2288240. Fax: (39) 055 2288250. E-mail: giorgio.mastromei{at}unifi.it.

Published ahead of print on 3 November 2006.

Present address: Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e Biotecnologie Microbiche, Università degli Studi di Cagliari, Cagliari, Italy.

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Journal of Bacteriology, January 2007, p. 228-235, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01450-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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