Genetic Analysis of Phenoxyalkanoic Acid Degradation in Sphingomonas herbicidovorans MH

Phenoxyalkanoic acid degradation is well studied in Beta- and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for α-ketoglutarate-dependent dioxygenases, sdpA_MH and rdpA_MH, were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA_I/II), two for dienelactone hydrolases (dccD_I/II), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA_MH was 99% identical to rdpA_MC1, an (R)-dichlorprop/α-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha- and Betaproteobacteria. Conversely, DccA_I and DccA_II did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta- and Gammaproteobacteria.     

Genetic analysis of phenoxyalkanoic acid degradation in Sphingomonas herbicidovorans MH

Phenoxyalkanoic acid degradation is well studied in Beta- and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for alpha-ketoglutarate-dependent dioxygenases, sdpA(MH) and rdpA(MH), were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA(I/II)), two for dienelactone hydrolases (dccD(I/II)), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA(MH) was 99% identical to rdpA(MC1), an (R)-dichlorprop/alpha-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha- and Betaproteobacteria. Conversely, DccA(I) and DccA(II) did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta- and Gammaproteobacteria.     

Partial purification and properties of a β-N-acetylglucosaminidase from the fungus Sclerotinia fructigena

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a beta-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN'-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The K(m) value for hydrolysis of p-nitrophenyl beta-2-acetamido-2-deoxy-d-glucopyranoside at 37 degrees C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.     

Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1

A genomic library was prepared in Escherichia coli from DNA of wild-type Xanthomonas campestris pv. campestris (aetiological agent of crucifer black rot), partially digested with endonuclease EcoRI, using the mobilisable broad host range cosmid vector pLAFR1. Recombinant plasmids contained inserts ranging in size from 19.1 to 32.3 kb (mean 26.6). Certain of the clones complemented E. coli auxotrophic markers. Using the narrow host range plasmid pRK2013 as a helper the pooled recombinant plasmids were transferred conjugally to X. c. campestris mutants, and clones were identified which restored yellow pigmentation to white mutants, prototrophy to amino acid auxotrophs and pathogenicity towards turnip plants to two non-pathogenic mutants. The lesion in one mutant (8288, complemented by the plasmid pIJ3000) is unknown. However mutant 8237 is defective in production of extracellular protease and polygalacturonate lyase and restoration of pathogenicity by complementation with the plasmid pIJ3020 concomitantly restored both enzyme levels to wild-type values.