Purification and characterization of serine racemase from a hyperthermophilic archaeon, Pyrobaculum islandicum.

Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100 degrees C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95 degrees C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 micromol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 micromol/min/mg, respectively.

Lateral transfer of the lux gene cluster.

The lux operon is an uncommon gene cluster. To find the pathway through which the operon has been transferred, we sequenced the operon and both flanking regions in four typical luminous species. In Vibrio cholerae NCIMB 41, a five-gene cluster, most genes of which were highly similar to orthologues present in Gram-positive bacteria, along with the lux operon, is inserted between VC1560 and VC1563, on chromosome 1. Because this entire five-gene cluster is present in Photorhabdus luminescens TT01, about 1.5 Mbp upstream of the operon, we deduced that the operon and the gene cluster were transferred from V. cholerae to an ancestor of Pr. luminescens. Because in both V. fischeri and Shewanella hanedai, luxR and luxI were found just upstream of the operon, we concluded that the operon was transferred from either species to the other. Because most of the genes flanking the operon were highly similar to orthologues present on chromosome 2 of vibrios, we speculated that the operon of most species is located on this chromosome. The undigested genomic DNAs of five luminous species were analysed by pulsed-field gel electrophoresis and Southern hybridization. In all the species except V. cholerae, the operons are located on chromosome 2.

Freshwater bioluminescence in Vibrio albensis (Vibrio cholerae biovar albensis) NCIMB 41 is caused by a two-nucleotide deletion in luxO.

We previously proposed that the function of the lux operon is to produce a halotolerant flavodoxin, FP390 or P-flavin binding protein, and not to produce light. A crucial basis of this hypothesis is that almost all species of luminous bacteria emit light in culture media containing over 2% NaCl. However, Vibrio albensis (Vibrio cholerae biovar albensis) NCIMB 41 emits light in freshwater and this appears to be in direct conflict with our hypothesis. To determine why this exceptional freshwater bioluminescence is emitted, we studied the lux operon and the regulatory system of the operon in this strain, and found that expression of the operon is regulated by a system involving a derivative of 4,5-dihydroxy 2,3-pentanedione, DPD, as an inducer, and the repressor gene for the lux operon, luxO, is damaged by deletion of two nucleotides. Furthermore, to study the effect of damage to the luxO gene, pUC18 derivatives containing the damaged and repaired luxO sequences were prepared. Cells transfected with the damaged luxO sequence emitted light like the parental strain, whereas ones transfected with the repaired one did so only sparingly. Here we show that the light emission in freshwater by this strain is not in conflict with our hypothesis.

Stimulated biosynthesis of flavins in Photobacterium phosphoreum IFO 13896 and the presence of complete rib operons in two species of luminous bacteria.

Photobacterium phosphoreum IFO 13896 emits light strongly when cultured in medium containing 3% NaCl, but only weakly in medium containing 1% NaCl. It is known that dim or dark mutants appear frequently and spontaneously from this parent strain. To confirm that riboflavin biosynthesis is stimulated when the lux operon is active, the amount of light emitted and flavins synthesized under strongly or weakly light emitting conditions was determined. In comparison with the parent strain cultured in 3% NaCl, the same strain cultured in 1% NaCl emitted 1/36 the light and produced 1/4 the flavins, while three dim or dark mutants, M1, M2 and M3 cultured in 3% NaCl, emitted almost no light, 1/58 the light and 1/10 the light and produced 1/8, 1/5 and 1/3 the amount of flavins, respectively. From these results, we deduced that the genes for riboflavin synthesis, rib genes, are organized in an operon in this strain. In P. phosphoreum NCMB 844, it has been reported that a rib gene cluster is present just downstream of the lux operon. However, among rib genes, the gene for pyrimidine deaminase/pyrimidine reductase, ribD, was not found in this cluster. Because a complete rib operon seems to be necessary for efficient regulation at the transcriptional level, we expected ribD to be present downstream of this cluster and sequenced this region, using SUGDAT, Sequencing Using Genomic DNA As a Template. We could not find this gene but found a gene for hybrid-cluster protein (prismane protein). To find ribD in a different region, a partial ribD sequence was amplified and sequenced using a PCR-based method, and subsequently the genomic DNA was sequenced in both directions from this partial sequence using SUGDAT. Because ribC was found just downstream of ribD, we sequenced further downstream of ribC and confirmed that another complete set of rib genes, ribD, ribC, ribBA, and ribE, is present in P. phosphoreum. The presence of a complete rib operon in P. phosphoreum explains why this species can synthesize flavins at enhanced levels to sustain a strong light emission. Furthermore, we sequenced the rib operon in Vibrio fischeri, another representative luminous bacterium, in a manner similar to that described above, and confirmed that a complete operon is present also in this species. The organization of rib genes in an operon in the Proteobacteria gamma-subdivision is discussed.