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Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria. Theo H. M. Smits, 2002.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 pGEc47  B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C6 to C12), 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 C12 to C16 alkanes . All alkane hydroxylase homologs, except the Acinetobacter sp . ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes .
Gene Expression Profile of Campylobacter jejuni in Response to Growth Temperature Variation. Alain Stintzi, 2003.The foodborne pathogen Campylobacter jejuni is the primary causative agent of gastroenteritis in humans . In the present study a whole genome microarray of C . jejuni was constructed and validated . These DNA microarrays were used to measure changes in transcription levels over time, as C . jejuni cells responded to a temperature increase from 37 to 42°C . Approximately 20% of the C . jejuni genes were significantly up- or downregulated over a 50-min period after the temperature increase . The global change in C . jejuni transcriptome was found to be essentially transient, with only a small subset of genes still differentially expressed after 50 min . A substantial number of genes with a downregulated coexpression pattern were found to encode for ribosomal proteins . This suggests a short growth arrest upon temperature stress, allowing the bacteria to reshuffle their energy toward survival and adaptation to the new growth temperature . Genes encoding chaperones, chaperonins, and heat shock proteins displayed the most dramatic and rapid upregulation immediately after the temperature change . Interestingly, genes encoding proteins involved in membrane structure modification were differentially expressed, either up- or downregulated, suggesting a different protein membrane makeup at the two different growth temperatures . Overall, these data provide new insights into the primary response of C . jejuni to surmount a sudden temperature upshift, allowing the bacterium to survive and adapt its transcriptome to a new steady state . Synergistic Degradation of Linuron by a Bacterial Consortium and Isolation of a Single Linuron-Degrading Variovorax Strain. Winnie Dejonghe, 2003.The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays . Three strains, Variovorax sp . strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp . strain WDL5, were isolated directly from the linuron-degrading culture . In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7 . Of these five strains, only Variovorax sp . strain WDL1 was able to use linuron as the sole source of C, N, and energy . WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded . To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates . Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains . D . acidovorans WDL34 and C . testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H . sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine . The role of Pseudomonas sp . strain WDL5 needs to be further elucidated . The degradation of linuron can thus be performed by a single isolate, Variovorax sp . strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture .
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