We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47ΔB, which encodes all proteins necessary for growth on medium-chain-length alkanes (C ₆ to C ₁₂ ), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C ₁₂ to C ₁₆ alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n -alkanes.

The alkane hydroxylase systems of twoRhodococcusstrains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1,alkB2,alkB3, andalkB4). In both strains, thealkB1andalkB2homologs were part ofalkgene clusters, each encoding two rubredoxins (rubA1andrubA2;rubA3andrubA4), a putative TetR transcriptional regulatory protein (alkU1;alkU2), and, in thealkB1cluster, a rubredoxin reductase (rubB). ThealkB3andalkB4homologs were found as separate genes which were not part ofalkgene clusters. Functional heterologous expression of some of the rhodococcalalkgenes (alkB2,rubA2, andrubA4[NRRL B-16531];alkB2andrubB[Q15]) was achieved inEscherichia coliandPseudomonasexpression systems.Pseudomonasrecombinants containing rhodococcalalkB2were able to mineralize and grow on C₁₂to C₁₆n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to thePseudomonas putidaGPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported inRhodococcus.

Fire blight, caused by the enterobacterium Erwinia amylovora, is a devastating disease of rosaceous plants that has global economic importance for apple and pear production and trade. The complete genome of E. amylovora CFBP 1430 was sequenced, annotated, and compared with the genomes of other Erwinia spp. Several singleton and shared features of the E. amylovora CFBP 1430 genome were identified that offer a first view into evolutionary aspects within the genus Erwinia. Comparative genomics identified or clarified virulence and fitness determinants and secretion systems. Novel insights revealed in the genome of E. amylovora CFBP 1430 hold potential for exploitation to improve the design of more effective fire blight control strategies.