Phosphate

Phosphorus is a major growth-limiting substrate in natural environments such as soil (67); consequently, under phosphate limitation, B. subtilis induces a programmed series of responses to reduce the cell's requirement for phosphorus, increase its affinity for phosphate uptake, and pro

Figure 3. The sporulation pathway in Bacillus subtilis and its relationship to the vegetative cell cycle. Sporulation is only initiated toward the end of the vegetative cell cycle when the cell has two complete copies of the chromosome.

duce extracellular hydrolytic enzymes to recover inorganic phosphate from organic sources (68,69).

B. subtilis responds to phosphate stress by the coordinated induction of approximately 40 genes (69). Prominent among these are genes of the Pho regulon (67,70-72), whose products include alkaline phosphatases (APase), alkaline phosphodiesterase (APDase), a high-affinity phosphate transporter, and enzymes required for teichuronic acid synthesis.

The Pho regulon of B. subtilis is controlled by two transacting regulators, PhoP and PhoR, that together form the components of a two-component, environment-sensing, signal transduction system (70,73). PhoR is a membrane-spanning sensor protein with histidine protein kinase (HPKase) activity that is activated at low phosphate concentrations to PhoR-P. This in turn acts as a substrate for the activation of PhoP, a response regulator required for the induction of the Pho regulon. B. subtilis genes activated by PhoP-P, which include the bicistronic operon encoding PhoP and PhoR, constitute the Pho regulon. The response regulators of at least two other signal transduc-tion systems, SpoOA-P and ResD, indirectly affect the expression of the Pho regulon by repressing the phoP/R op-eron (71,74,75).

The promoters of the Pho regulon genes/operons that have been studied to date have a conserved sequence, TTAACA (74), located around position — 22. This sequence may be equivalent to the Pho box observed in E. coli (76), which also tends to be located toward the 5' end of the promoter region of Pho regulon genes/operons.

Carbon

Bacillus species can grow on a wide range of simple carbon sources, including glucose, sucrose, and amino acids as well as a range of complex substrates such as proteins and polysaccharides, particularly plant-derived substrates such as starch and arabinans. In the case of more complex substrates, extracellular enzymes are required to reduce the size of these substrates before uptake and utilization. Carbon substrates are transported into the cell either in an unmodified form or via a PTS system (77).

In B. subtilis many genes/operons encoding enzymes involved in the catabolism of substrates as diverse as starch and histidine are repressed by readily metabolizable carbon sources such as fructose, glucose, glycerol, or manni-tol—so-called PTS sugars. Catabolite repression functions at the level of transcription, but differs substantially from the cyclic AMP-mediated system observed in E. coli. Three components of the B. subtilis system have been identified: a cis-acting catabolite-responsive element (CRE) and two trans-acting factors, CcpA and Hpr. The 14-bp palindomic CRE sequence (78) is essential for catabolite repression. CREs are found at diverse locations in relation to the tran-scriptional units of catabolite-sensitive genes, in some cases overlapping the promoter (amyE), in others being located within the structural gene itself (xylA). The latter positioning makes it extremely difficult to engineer catab-olite insensitivity in such genes.

CREs are likely to be binding sites for a catabolite re-pressor protein. Although it has not been directly shown to be the case, CcpA, which is related to E. coli Lacl-type repressors, is the most likely candidate for this protein. Mutants in the ccpA gene abolish catabolite repression, including glucose repression of sporulation, without affecting substrate-specific induction of the individual genes/ operons concerned. They also severely affect growth on substrates such as glucose, fructose, glucitol, and glycerol that are metabolized via glycolysis or the TCA cycle, but not on substrates such as xylose which are metabolized via the pentose phosphate pathway. In the former case, growth is restored if TCA cycle intermediates are added to the growth medium. Interestingly, a CRE appears to be responsible for glucose-mediated activation of the acetate kinase gene (ackA) and in genes encoding enzymes of central metabolism.

HPr is a component of the phosphotransferase system (PTS) which, when activated by phosphorylation to HPr(Ser-P), binds to CcpA. The key signaling component of the catabolite repression pathway appears to be the Embden-Meyerhof pathway (EMP) intermediate fructose-1,6-diphosphate (FDP). High concentrations of this compound activate the ATP-dependent HPr serine kinase and possibly stimulates an interaction between CcpA and HPr(Ser-P). This complex may then bind to the CREs of catabolite-responsive genes/operons (49).

Nitrogen

Bacillus spp. can use ammonium and a number of other nitrogen-containing compounds (notably certain amino acids) as sole sources of nitrogen (79). Ammonium is assimilated via glutamine synthetase (GS), glutamine:2-oxo-glutarate amidotransferase (GOGAT), and glutamate dehydrogenase (GDH) (80). Most members of the genus are able to use all three enzymes, while a smaller group, including many (but possibly not all) strains of B. subtilis, use GS and GOGAT, while nitrogen-fixing Bacillus species use GDH. In B. subtilis, the relative levels of GS and GOGAT are dependent on the available sources of nitrogen. The levels of GS synthesis are dependent on the need for glutamine synthesis; they are lowest in the presence of glutamine, a nitrogen source favored by this bacterium, and highest in the presence of nonfavored nitrogen sources such as ammonium. No global regulatory network analogous to that of Ntr of E. coli (81) has been detected in B. subtilis, although GS synthesis is autoregulated via the GlnR repressor and has also been implicated as a key signal for the nitrogen status of the cell modulating the activity of a variety of other metabolic pathways.

GOGAT levels are low in the presence of glutamate and amino acids (e.g., glutamine, arginine, and histidine) that can be catabolized to glutamate, and high in the presence of ammonium and amino acids that are not broken down to glutamate and other sources of nitrogen such as nitrate.

In addition to its use as a nitrogen source, nitrate (and nitrite) can be used by B. subtilis in place of oxygen as an electron acceptor, allowing growth under anaerobic conditions. Nitrate respiration requires the products of the narGHJIoperon (10,11).

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