CaCO 3 , eventually favoring precipitation. Moreover, carbonate precipitation will
alter the extracellular environment according to 5.14 and Fig. 5.3, Sect. C (decrease
soluble Ca
2+ and increase acidity), so that it becomes more favorable for bacterial
proliferation.
In this view, microbial calcium metabolism is an inevitable event under typical
precipitation conditions, active calcium metabolism potentially creates unique
precipitation conditions, and carbonate precipitation chemically favors bacterial
survival and proliferation.
An active calcium metabolism controlling precipitation leads to the question if
BCCP is under genetic control, i.e., specific molecules or mechanisms could be
involved in controlling precipitation. In broad terms, all the metabolic pathways
described in Sect. 5.3.1, which affect environmental conditions favoring precipitation, are under genetic control and manipulation of one of those pathways could
enhance precipitation, for instance a higher urease activity increases calcite precipitation in Sporosarcina pasteurii (Achal et al. 2009).
To find genes which might specifically control calcite precipitation, the laboratory bacterium Bacillus subtilis, which can produce calcite precipitates, was used.
The starting point was to look for mutant strains impaired in precipitation. Six
mutants, impaired in calcite crystal formation, were isolated. In most cases, the
putative function of the mutated genes was linked to fatty acid metabolism (Perito
et al. 2000); moreover, mutations were found in ysiB and in the adjacent gene
ysiA. Genes ysiB and ysiA, together with lcfA, etfB, and etfA, belong to a five-gene
cluster, called lcfA operon. Further analysis of this cluster strongly suggested that
the last gene, etfA, is essential for the precipitation phenotype (Barabesi et al. 2007).
Putative functions assigned to the products of the lcfA operon genes are all involved
in fatty acid metabolism.
The physiology behind the lack of precipitation in mutant FBC5 (etfA
inactivated) was investigated, and compared to that of the wild-type strain
B. subtilis 168, by Marvasi et al. (2010). Two main factors were considered as
possibly affected by the etfA inactivation: EPS production (calcium-binding sites)
and pH. EPS extracted from both strains revealed similar FT-IR patterns, with
presence of bands typical of proteins and carbohydrates.
The pH decrease in FBC5 biofilm grown on solid medium was, on the contrary,
the main process responsible for the absence of calcite formation. The phenomenon
was reversible and crystal formation was restored when cells were incubated under
alkaline conditions, suggesting that no specific inhibitors of precipitation were
produced. The decrease of pH during FBC5 development resulted from an excess
of H
+ production in the mutant; specifically, the H
+ extruded by the mutant was
estimated to be 0.7 H
+ mole/L higher than in the wild-type strain. The excess of
protons could result from a miss-regulation in some part of the EtfA pathway. Even
though the function of the two EtfA-B proteins in B. subtilis is only putative, in
Clostridium acetobutylicum etfA and etfB co-expression is essential for the butyrylCoA dehydrogenase (BCD) activity, and etfA-B genes are involved in the oxidation
of NADH in the butyryl-CoA synthesis pathway (Inui et al. 2008). If the reduction
of b-hydroxybutyryl-CoA takes place in B. subtilis in the same way as in
5 Molecular Basis of Bacterial Calcium Carbonate Precipitation
131
alter the extracellular environment according to 5.14 and Fig. 5.3, Sect. C (decrease
soluble Ca
2+ and increase acidity), so that it becomes more favorable for bacterial
proliferation.
In this view, microbial calcium metabolism is an inevitable event under typical
precipitation conditions, active calcium metabolism potentially creates unique
precipitation conditions, and carbonate precipitation chemically favors bacterial
survival and proliferation.
An active calcium metabolism controlling precipitation leads to the question if
BCCP is under genetic control, i.e., specific molecules or mechanisms could be
involved in controlling precipitation. In broad terms, all the metabolic pathways
described in Sect. 5.3.1, which affect environmental conditions favoring precipitation, are under genetic control and manipulation of one of those pathways could
enhance precipitation, for instance a higher urease activity increases calcite precipitation in Sporosarcina pasteurii (Achal et al. 2009).
To find genes which might specifically control calcite precipitation, the laboratory bacterium Bacillus subtilis, which can produce calcite precipitates, was used.
The starting point was to look for mutant strains impaired in precipitation. Six
mutants, impaired in calcite crystal formation, were isolated. In most cases, the
putative function of the mutated genes was linked to fatty acid metabolism (Perito
et al. 2000); moreover, mutations were found in ysiB and in the adjacent gene
ysiA. Genes ysiB and ysiA, together with lcfA, etfB, and etfA, belong to a five-gene
cluster, called lcfA operon. Further analysis of this cluster strongly suggested that
the last gene, etfA, is essential for the precipitation phenotype (Barabesi et al. 2007).
Putative functions assigned to the products of the lcfA operon genes are all involved
in fatty acid metabolism.
The physiology behind the lack of precipitation in mutant FBC5 (etfA
inactivated) was investigated, and compared to that of the wild-type strain
B. subtilis 168, by Marvasi et al. (2010). Two main factors were considered as
possibly affected by the etfA inactivation: EPS production (calcium-binding sites)
and pH. EPS extracted from both strains revealed similar FT-IR patterns, with
presence of bands typical of proteins and carbohydrates.
The pH decrease in FBC5 biofilm grown on solid medium was, on the contrary,
the main process responsible for the absence of calcite formation. The phenomenon
was reversible and crystal formation was restored when cells were incubated under
alkaline conditions, suggesting that no specific inhibitors of precipitation were
produced. The decrease of pH during FBC5 development resulted from an excess
of H
+ production in the mutant; specifically, the H
+ extruded by the mutant was
estimated to be 0.7 H
+ mole/L higher than in the wild-type strain. The excess of
protons could result from a miss-regulation in some part of the EtfA pathway. Even
though the function of the two EtfA-B proteins in B. subtilis is only putative, in
Clostridium acetobutylicum etfA and etfB co-expression is essential for the butyrylCoA dehydrogenase (BCD) activity, and etfA-B genes are involved in the oxidation
of NADH in the butyryl-CoA synthesis pathway (Inui et al. 2008). If the reduction
of b-hydroxybutyryl-CoA takes place in B. subtilis in the same way as in
5 Molecular Basis of Bacterial Calcium Carbonate Precipitation
131