Identification and functional characterisation of genes and corresponding enzymes involved in carnitine metabolism of Proteus sp.

Enzymes involved in carnitine metabolism of Proteus sp. are encoded by the cai genes organised as the caiTABCDEF operon. The complete operon could be sequenced from the genomic DNA of Proteus sp. Amino acid sequence similarities and/or enzymatic analysis confirmed the function assigned to each protein involved in carnitine metabolism. CaiT was suggested to be an integral membrane protein responsible for the transport of betaines. The caiA gene product was shown to be a crotonobetainyl-CoA reductase catalysing the irreversible reduction of crotonobetainyl-CoA to gamma-butyrobetainyl-CoA. CaiB and CaiD were identified to be the two components of the crotonobetaine hydrating system, already described. CaiB and caiD were cloned and expressed in Escherichia coli. After purification of both proteins, their individual enzymatic functions were solved. CaiB acts as betainyl-CoA transferase specific for carnitine, crotonobetaine, gamma-butyrobetaine and its CoA derivatives. Transferase reaction proceeds, following a sequential bisubstrate mechanism. CaiD was identified to be a crotonobetainyl-CoA hydratase belonging to the crotononase superfamily. Because of amino acid sequence similarities, CaiC was suggested to be a betainyl-CoA ligase. Taken together, these results show that the metabolism of carnitine and crotonobetaine in Proteus sp. proceeds at the CoA level.

Effect of salt stress on crotonobetaine and D(+)-carnitine biotransformation into L(-)-carnitine by resting cells of Escherichia coli.

The biotransformation of crotonobetaine and D(+)-carnitine into L(-)-carnitine is affected by salt stress in the resting cells of E. coli O44 K74 and the transformed E. coli K38 pT7-5KE32. A yield of 65 and 80% of L(-)-carnitine, respectively, were obtained with 0.5 M NaCl with the wild and transformed strain compared with the 40% obtained with the control. Higher salt levels reduced the conversion. In L(-)-carnitine transport studies using both strains, the transformed strain presented slightly lower apparent K(m) and V values. Arsenate reduced both the transport and biotransformation of crotono-betaine in the presence or absence of 0.5 M NaCl, whereas vanadate only inhibited these processes under salt stress conditions. Hg(II) inhibited both the transport and biotransformation and Pb(II) reduced the biotransformation only under salt stress conditions. Cu(II) produced a significantly higher decrease than Pb(II) in the biotransformation with both substrates in the absence of salt stress conditions, but only affected transport in the presence of such conditions. Furthermore, salt stress affected the CaiT transporter for L(-)-carnitine and crotonobetaine and induced ProU and ProP in the absence of the inducer of the L(-)-carnitine metabolism. It is highly likely that the increase in L(-)-carnitine production was not only due to improved transport but also to the permeabilization effect caused by NaCl, as transport and 1-N-phenylnaphthylamine uptake studies revealed.

Screening for soluble methane monooxygenase in methanotrophic bacteria using combined molecular and biochemical methods for hydroxylase detection.

Three well known methanotrophic bacteria (Methylosinus trichosporium OB3b, Methylocystis sp. WI 14, and Methylocystis sp. GB 25) and three newly isolated methanotrophic bacteria (Methylocystis sp. WI 11, Methylocystis sp. X, and FI-9) were screened for sMMO considering the existence of hydroxylase (component A) genes as well as its gene expression. For these purposes monoclonal antibodies that specifically recognize each subunit of the hydroxylase of Methylocystis sp. WI 14 (alpha-subunit [9E5/F2], beta-subunit [4E2/G11], gamma-subunit [10G3/D7]) were produced. PCR amplification using well known primers showed that the hydroxylase encoding genes appear to be only present in M. trichosporium OB3b, Methylocystis sp. WI 11 and WI 14, and in the isolate FI-9. Western and ELISA analysis using the monoclonal antibodies revealed that all subunits of hydroxylase were present. However, in FI-9, only the alpha-subunit of the hydroxylase might be expressed. Surprisingly, in Methylocystis sp. GB 25, where no sMMO activity and no amplification with sMMO specific primers was obtained, the antibody 4E2/G11 recognized a protein band with exactly the same molecular mass as the beta-subunit of the hydroxylase. Methylocystis sp. X showed no positive reaction in any of the tests. In combination with the detection methods currently used, the described antibodies provide a powerful tool for detecting even partially expressed hydroxylase genes.