The methanotrophic bacterium Methylococcus capsulatus (Bath) grows on pure methane. However, in a single cell protein production process using natural gas as methane source, a bacterial consortium is necessary to support growth over longer periods in continuous cultures. In different bioreactors of Norferm Danmark A/S, three bacteria consistently invaded M. capsulatus cultures growing under semi-sterile conditions in continuous culture. These bacteria have now been identified as a not yet described member of the Aneurinibacillus group, a Brevibacillus agri strain, and an acetate-oxidiser of the genus Ralstonia. The physiological roles of these bacteria in the bioreactor culture growing on natural, non-pure methane gas are discussed. The heterotrophic bacteria do not have the genetic capability to produce either the haemolytic enterotoxin complex HBL or non-haemolytic enterotoxin.

The use of the Gram-positive bacterium Lactococcus lactis in recombinant protein production has several advantages, including the organism's long history of safe use in food production and the fact that it does not produce endotoxins. Furthermore the current non-dairy L. lactis production strains contain few proteases and can secrete stable recombinant protein to the growth medium. The P170 expression system used for recombinant protein production in L. lactis utilizes an inducible promoter, P170, which is up-regulated as lactate accumulates in the growth medium. We have optimised the components of the expression system, including improved promoter strength, signal peptides and isolation of production strains with increased productivity. Recombinant proteins are produced in a growth medium with no animal-derived components as a simple batch fermentation requiring minimal process control. The accumulation of lactate in the growth medium does, however, inhibit growth and limits the yield from batch and fed-batch processes. We therefore combined the P170 expression system with the REED™ technology, which allows control of lactate concentration by electro-dialysis during fermentation. Using this combination, production of the Staphylococcus aureus nuclease reached 2.5 g L(-1).

CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which ␤-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein syntheses are inhibited. Expression of pyrG responds only to the cellular level of CTP, since expression of pyrG has no correlation to alterations in UTP, GTP, and ATP pool sizes. In the untranslated pyrG leader sequence a potential terminator structure can be identified, and this structure is required for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing an antiterminator to form and transcription to proceed. This model therefore does not include any trans-acting protein for sensing the CTP concentration as previously proposed for Bacillus subtilis.

The pyrG gene from Lactococcus lactis encodes CTP synthase (EC 6.4.3.2), an enzyme converting UTP to CTP. A series of strains were constructed with different levels of pyrG expression by insertion of synthetic constitutive promoters with different strengths in front of pyrG. These strains expressed pyrG levels in a range from 3 to 665% relative to the wild-type expression level. Decreasing the level of CTP synthase to 43% had no effect on the growth rate, showing that the capacity of CTP synthase in the cell is in excess in a wild-type strain. We then studied how pyrG expression affected the intracellular pool sizes of nucleotides and the correlation between pyrG expression and nucleotide pool sizes was quantified using metabolic control analysis in terms of inherent control coefficients. At the wild-type expression level, CTP synthase had full control of the CTP concentration with a concentration control coefficient close to one and a negative concentration control coefficient of )0.28 for the UTP concentration. Additionally, a concentration control coefficient of 0.49 was calculated for the dCTP concentration. Implications for the homeostasis of nucleotide pools are discussed.Keywords: pyrG; CTP synthase; metabolic control analysis; metabolism; EC 6.4.3.2.1 Synthesis of ribonucleotides and deoxyribonucleotides is an essential part of cellular metabolism, as synthesis of RNA requires ribonucleotides and DNA replication is dependent on deoxyribonucleotides. The involvement of nucleotides in these central cellular pathways suggests that it is important for the cell to control the synthesis of nucleotides and to be able to maintain a steady supply of these essential precursors either by de novo biosynthesis or by uptake of precursors from the growth medium. In addition, the involvement of nucleotides in regulatory processes such as regulation of gene expression and modulation of kinetic properties of enzymes emphasizes the need for tight regulation of the level of nucleotides in the cell. Indeed, expression of genes responsible for the de novo biosynthesis of ribonucleotides in Gram-positive bacteria such as Lactococcus lactis and Bacillus subtilis are regulated by the availability of purines and pyrimidines. The pyrimidine biosynthetic genes are regulated by the RNA-binding regulatory protein PyrR that regulates gene expression by an attenuation mechanism through sensing of the UMP concentration in the cell [1][2][3][4][5]. However, PyrR is not involved in the regulation of expression of the pyrG gene encoding CTP synthase (EC 6.4.3.2) in L. lactis and B. subtilis, as pyrG expression is probably regulated by an attenuation mechanism responding to the CTP concentration in the cell [6,7]. The reaction catalyzed by CTP synthase (UTP + glutamine + ATP fi CTP + glutamate + ADP + P i ) involves all four ribonucleotides; UTP and CTP are substrate and product, respectively, ATP is used as an energy source and GTP is an allosteric activator of the reaction [8]. CTP synthase has a central role in pyrimidine metabolism, as ...

The PyrR protein regulates expression of the genes of de novo pyrimidine nucleotide biosynthesis (pyr genes) in nearly all Gram-positive and many other bacteria by a transcription attenuation mechanism [1]. PyrR acts by binding to a segment of pyr mRNA with conserved sequence and secondary structure [1,2]. When PyrR is bound, a downstream antiterminator stemloop structure is prevented from forming, and formation of a transcription terminator is permitted. The affinity of PyrR for pyr mRNA is increased by uridine nucleotides [2,3], so an elevated pyrimidine level in the cells results in greater termination of transcription at sites upstream of the ORF of the pyr genes. Three sites of PyrR binding and transcription attenuation have been identified in the pyr operons of Bacillus subtilis and related Bacillus species [1]. These are located in the 5¢ untranslated leader of the operon (binding loop 1 or BL1), between the first cistron of the operon, pyrR, and the second cistron pyrP (BL2), and between pyrP and the third cistron pyrB (BL3) (Fig. 1A).All of the initial genetic [4][5][6][7] and biochemical [2,3,8,9] studies of the regulation of pyr genes by PyrR in our laboratory were conducted with B. subtilis strains and PyrR purified from B. subtilis. However, The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5¢ leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. The apparent dissociation constants at 0°C were in the range 0.13-0.87 nm in the absence of effectors; dissociation constants were decreased by three-to 12-fold by uridine nucleotides and increased by 40-to 200-fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied.Abbreviation ATCase, aspartate transcarbamylase.