Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum H2S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major H2S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H2S from l-cysteine (∼30 %) than Fn1220. The Fn0625 protein degraded a variety of substrates containing βC–S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from l-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of l-cysteine with Fn1220 produced H2S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except l-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H2S in distinct manners: Fn0625 carries out β-elimination of l-cysteine to produce H2S, pyruvate and ammonia, whereas Fn1220 catalyses the β-replacement of l-cysteine to produce H2S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum.

Indole produced via the b-elimination reaction of L-tryptophan by pyridoxal 59-phosphatedependent tryptophanase (EC 4.1.99.1) has recently been shown to be an extracellular and intercellular signalling molecule in bacteria, and controls bacterial biofilm formation and virulence factors. In the present study, we determined the molecular basis of indole production in the periodontopathogenic bacterium Porphyromonas gingivalis. A database search showed that the amino acid sequence deduced from pg1401 of P. gingivalis W83 is 45 % identical with that from tnaA of Escherichia coli K-12, which encodes tryptophanase. Replacement of the pg1401 gene in the chromosomal DNA with the chloramphenicol-resistance gene abolished indole production. The production of indole was restored by the introduction of pg1401, demonstrating that the gene is functionally equivalent to tnaA. However, RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends analyses showed that, unlike E. coli tnaA, pg1401 is expressed alone in P. gingivalis and that the nucleotide sequence of the transcription start site is different, suggesting that the expression of P. gingivalis tnaA is controlled by a unique mechanism. Purified recombinant P. gingivalis tryptophanase exhibited the Michaelis-Menten kinetics values K m 50.20±0.01 mM and k cat 51.37±0.06 s "1 in potassium phosphate buffer, but in sodium phosphate buffer, the enzyme showed lower activity. However, the cation in the buffer, K + or Na + , did not appear to affect the quaternary structure of the enzyme or the binding of pyridoxal 59-phosphate to the enzyme. The enzyme also degraded S-ethyl-L-cysteine and S-methyl-L-cysteine, but not L-alanine, L-serine or L-cysteine.

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Hydrogen sulfide (H 2 S) is a toxic gas that induces the modification and release of haemoglobin in erythrocytes; however, it also functions in methionine biosynthesis in bacteria. bC-S lyase, encoded by the lcd gene, is responsible for bacterial H 2 S production through the cleavage of Lcysteine. In this study, 26 of 29 crude extracts from reference and clinical strains of Streptococcus intermedius produced H 2 S from L-cysteine. The capacities in those strains were not higher than those in strains of the other anginosus group of streptococci, Streptococcus anginosus and Streptococcus constellatus, but were much greater than those in strains of Streptococcus gordonii, which is known to have an extremely low capacity for H 2 S production. Incubation of the remaining three extracts with L-cysteine did not result in H 2 S production. Sequence analysis revealed that the lcd genes from these three strains (S. intermedius strains ATCC 27335, IMU151 and IMU202) contained mutations or small deletions. H 2 S production in crude extracts prepared from S. intermedius ATCC 27335 was restored by repairing the lcd gene sequence in genomic DNA. The kinetic properties of the purified recombinant protein encoded by the repaired lcd gene were comparable to those of native proteins produced by H 2 S-producing strains, whereas the truncated protein produced by S. intermedius ATCC 27335 had no enzymic activity with L-cysteine or L-cystathionine. However, real-time PCR analysis indicated that the lcd gene in strains ATCC 27335, IMU151 and IMU202 is transcribed and regulated in a manner similar to that in the H 2 S-producing strain.

Background:Background: Long-term proteasome inhibitor (PI)-based therapy improves outcomes in multiple myeloma (MM).However, physical, social, and geographic access to health services and treatment toxicity may interfere with longterm PI therapy in clinical practice. Aims:Aims: To evaluate the efficacy and safety of treatment with the oral PI, ixazomib (IXA), lenalidomide (LEN) + dexamethasone (DEX) [IRd] in patients with relapsed/refractory MM (RRMM) initially treated with an injectable PIbased therapy.