Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates.

The selective transformation of ubiquitous but inert C–H bonds to other functional groups has far-reaching practical implications, ranging from more efficient strategies for fine chemical synthesis to the replacement of current petrochemical feedstocks by less expensive and more readily available alkanes. The past twenty years have seen many examples of C–H bond activation at transition-metal centres, often under remarkably mild conditions and with high selectivity. Although profitable practical applications have not yet been developed, our understanding of how these organometallic reactions occur, and what their inherent advantages and limitations for practical alkane conversion are, has progressed considerably. In fact, the recent development of promising catalytic systems highlights the potential of organometallic chemistry for useful C–H bond activation strategies that will ultimately allow us to exploit Earth's alkane resources more efficiently and cleanly.

Understanding and exploiting C–H bond activation

The use of biocatalysis for industrial synthetic chemistry is on the verge of significant growth. Biocatalytic processes can now be carried out in organic solvents as well as aqueous environments, so that apolar organic compounds as well as water-soluble compounds can be modified selectively and efficiently with enzymes and biocatalytically active cells. As the use of biocatalysis for industrial chemical synthesis becomes easier, several chemical companies have begun to increase significantly the number and sophistication of the biocatalytic processes used in their synthesis operations.

Industrial biocatalysis today and tomorrow

Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters

The growth of Pseudomonas oleovorans on n-octane was characterized by the formation of intracellular structures. These inclusions were isolated and characterized. Morphologically, they resembled the poly-beta-hydroxybutyrate granules found in Bacillus cereus, as shown by freeze-fracture electron microscopy. The elemental analysis of isolated granules showed, however, that they do not contain poly-beta-hydroxybutyric acid. Instead, the analysis was consistent with a C8 polyester, which interpretation was supported by the fatty acid analysis of hydrolyzed granules. From the evidence presented here, we conclude that P. oleovorans forms poly-beta-hydroxyoctanoate granules when grown on n-octane.

Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane.

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter 21 h 21 , corresponding to a mass transfer coefficient of 188 h 21 ) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter 21 during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.

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Methods for Intense Aeration, Growth, Storage, and Replication of Bacterial Strains in Microtiter Plates

A bacterium that is able to utilize a number of halogenated short-chain hydrocarbons and halogenated carboxylic acids as sole carbon source for growth was identified as a strain of Xanthobacter autotrophicus. The organism constitutively produces two different dehalogenases. One enzyme is specific for halogenated alkanes, whereas the other, which is more heat stable and has a higher pH optimum, is specific for halogenated carboxylic acids. Haloalkanes were hydrolyzed in cell extracts to produce alcohols and halide ions, and a route for the metabolism of 1,2-dichloroethane is proposed. Both dehalogenases show a broad substrate specificity, allowing the degradation of bromine- and chlorine-substituted organic compounds. The results show that X. autotrophicus may play a role in the degradation of organochlorine compounds and that hydrolytic dehalogenases may be involved in the microbial metabolism of short-chain halogenated hydrocarbons in microorganisms.

https://aem.asm.org/content/aem/49/3/673.full.pdf

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10.

The first step in the aerobic degradation of alkanes by bacteria, yeasts, and fungi is catalyzed by oxygenases. These enzymes, which introduce oxygen atoms derived from molecular oxygen into the alkane substrate, play an important role in oil bioremediation and in the cometabolic degradation of compounds such as trichloroethylene and fuel oxygenates. In addition, they are useful biocatalysts and important models for a difficult chemical reaction: the regio- and stereospecific activation of C-H bonds. Several unrelated enzyme classes catalyze the oxidation of alkanes. Alkane-degrading yeast strains contain multiple alkane hydroxylases belonging to the P450 superfamily, while many bacteria contain enzymes related to the Pseudomonas putida GPo1 membrane-bound alkane hydroxylase system. Short-chain alkanes are probably oxidized by alkane hydroxylases related to the soluble and particulate methane monooxygenases. Only the membrane-bound enzymes have been studied with respect to their prevalence in environments such as soils or aquifers.

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Diversity of Alkane Hydroxylase Systems in the Environment

Many Pseudomonads are able to use linear alkanes as sole carbon and energy source. The genetics and enzymology of alkane metabolism have been investigated in depth forPseudomonas oleovorans, which is able to oxidize C5-C12 n-alkanes by virtue of two gene regions, localized on the OCT-plasmid. The so-calledalk-genes have been cloned in pLAFR1, and were subsequent analyzed using minicell expression experiments, DNA sequencing and deletion analysis. This has led to the identification and characterization of thealkBFGHJKL andalkST genes which encode all proteins necessary to convert alkanes to the corresponding acyl-CoA derivatives. These then enter the β-oxidation-cycle, and can be utilized as carbon- and energy sources. Medium (C6-C12)- or long-chain (C13-C20) n-alkanes can be utilized by many strains, some of which have been partially characterized. The alkane-oxidizing enzymes used by some of these strains (e.g. twoP. aeruginosa strains, aP. denitrificans strain and a marinePseudomonas sp.) appear to be closely related to those encoded by the OCT-plasmid.

Bernard Witholt

Papers

Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane.

Methods for Intense Aeration, Growth, Storage, and Replication of Bacterial Strains in Microtiter Plates

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10.

Diversity of Alkane Hydroxylase Systems in the Environment

Genetics of alkane oxidation by Pseudomonas oleovorans