FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for degradation of C-24 branched sterol side chains

The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32ΔfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.     

Peptide nanotube aligning side chains onto one side

A novel pseudo cyclic penta‐β‐peptide composed of a β‐naphthylalanine, two β‐alanines, and a sequence of ethylenediamine‐succinic acid (CP5ES) is synthesized and investigated on peptide nanotube (PNT) formation. When the PNT is formed with the maximum number of intermolecular hydrogen bonds between the cyclic peptides, the sequence enables the alignment of the side chains, naphthyl groups, on one side of the PNT. Microscopic and spectroscopic observations of CP5ES crystals reveal that CP5ES forms rod‐ or needle‐shaped molecular assemblies showing exciton coupling of the Cotton effect and predominant monomer emission, which are different from a reference cyclic tri‐β‐peptide composed of a β‐naphthylalanine and two β‐alanines. Insertion of a sequence of ethylenediamine‐succinic acid into β‐amino acids in the cyclic skeleton is therefore suggested to be effective to make the side chains aligning on one side of the PNT. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

木质纤维素的微生物降解

木质纤维素广泛存在于自然界中,因结构复杂,其高效降解需要多种微生物的协同互作,由于参与木质纤维素降解的微生物种类繁多,其协同降解机理尚不完全明确。随着微生物分子生物学和组学技术的快速发展,将为微生物协同降解木质纤维素机制的研究提供新的方法和思路。笔者前期研究发现,细菌复合菌系在50℃下表现出强大的木质纤维素降解能力,菌系由可分离培养和暂时不可分离培养细菌组成,但是可分离培养细菌没有降解能力。通过宏基因组和宏转录组研究表明,与木质纤维素降解相关的某些基因表达量发生显著变化,通过组学方法有可能更加深入解释微生物协同降解木质纤维素的微生物学和酶学机理。文中从酶、纯培养菌株和复合菌群三个方面综述了木质纤维素微生物降解研究进展,着重介绍了组学技术在解析复合菌群作用机理方面的现状和应用前景,以期为探索微生物群落协同降解木质纤维素的机理提供借鉴。     

Microbial degradation of alkenylbenzenes

Alkenylbenzenes are produced in large quantities by the petrochemical industry. The simplest of these alkenylbenzenes, styrene, is in widespread use in the polymer-processing industry and is thus found in many industrial effluents. Airborne gaseous emissions of styrene are particular problems due to the potential toxicity and carcinogenicity of the compound. The catabolic pathways involved in the degradation of styrene have been well characterised. With an increased knowledge of the adaptative response which microorganisms exhibit when exposed to higher styrene concentrations, together with an understanding of the genetic regulation of the catabolic pathways which operate in these microbial strains, it is likely that these organisms could be exploited in areas such as biotransformations, biocatalysis and bioremediation.The authors are with the Microbiology Department, University College, Cork, Ireland.     

Microbial degradation of pentachlorophenol

Pentachlorophenol (PCP) was the most prevalent wood preservative for many years worldwide. Its widespread use had led to contamination of various environments. Traditional methods of PCP clean-up include storage in land-fill sites, incineration and abiotic degradation processes such as photodecomposition. Some aerobic and anaerobic microorganisms can degrade PCP under a variety of conditions. Axenic bacterial cultures, Flavobacterium sp., Rhodococcus sp., Arthrobacter sp., Pseudomonas sp., Sphingomonas sp., and Mycobacterium sp., and fungal cultures, Phanerochaete sp. and Trametes sp. exhibit varying rates and extent of PCP degradation. This paper provides some general information on properties of PCP and reviews the influence of nutrient amendment, temperature and pH on PCP degradation by various aerobic and anaerobic microorganisms. Where information is available, proposed degradation pathways, intermediates and enzymes are reviewed.     

Microbial degradation of lignin

The transformations of lignin that occur during its biodegradation are complex and incompletely understood. Certain fungi of the white-rot group, and possibly other fungi and bacteria, completely decompose lignin to carbon dioxide and water. Other fungi and bacteria apparently degrade lignin incompletely. Differences in lignin-degrading abilities observed for different organisms may result from differences in the completeness of their ligninolytic enzyme systems. Not all lignin components may be attacked by a particular organism. Alternatively, different organisms may differ in their basic mechanisms of attack on lignin. The basic pathways of lignin degradation have been elucidated only for certain representatives of the white-and brown-rot fungi. Although it is known that each of the principal structural components of lignin is attacked by other fungi and bacteria, the biochemistry of that attack has not been elucidated. Work with low molecular weight lignin models has provided only limited information on possible pathways of lignin degradation by microorganisms. There is little evidence to suggest a correlation between abilities to degrade single-ring aromatic or lignin model compounds and the ability to degrade polymeric lignin. More evidence has come from analysis of spent culture media for lignin breakdown products and from comparative chemical analyses of sound lignins versus decayed lignin residues. Accumulated evidence with the most thoroughly studied white-rot fungi suggests that with these fungi lignin degradation proceeds by way of extracellular mixed-function oxygenases and dioxygenases, which catalyse demethylations, hydroxylations and ring-fission reactions within a largely intact polymer, concomitant with some release of low molecular weight lignin fragments. There are also apparent relationships between lignin, carbohydrate and nitrogen metabolism for some organisms, but the relationships may vary from one organism to another. Although research is now mostly at a basic level, industrial applications may result from lignin degradation research. Considerable potential exists for the development of bioconversions which might produce low molecular weight chemicals from waste lignins, and thereby reduce our dependence on petroleum as a source of these chemicals. Alternatively, such bioconversions might produce chemically altered forms of polymeric lignin that may be valuable industrially.     

聚乙烯塑料的微生物降解

聚乙烯(polyethylene,PE)是产量最大的通用塑料之一,通常被加工成一次性包装材料(包括塑料袋及容器)和农用薄膜等。PE塑料的广泛应用导致大量PE废弃物的累积,对生态环境造成严重的威胁。自20世纪70年代以来,一些研究陆续报道了PE塑料被微生物降解的现象,并从土壤、海洋、垃圾堆置点及昆虫肠道等生境中分离筛选到了若干种具有一定PE塑料降解能力的菌株,而且发现一些单加氧酶、过氧化物酶和漆酶等氧化还原酶对PE塑料具有氧化降解能力。这些研究为发展PE塑料废弃物生物降解处理技术提供了一定的依据。本文总结和分析了PE塑料降解微生物的分离和筛选方法,以及已报道的PE塑料降解微生物和降解酶的研究进展,以期为进一步研究PE塑料的微生物降解机理和处理技术提供参考。     

Microbial degradation of polyethers

This paper summarizes studies on microbial degradation of polyethers. Polyethers are aerobically metabolized through common mechanisms (oxidation of terminal alcohol groups followed by terminal ether cleavage), well-characterized examples being found with polyethylene glycol (PEG). First the polymer is oxidized to carboxylated PEG by alcohol and aldehyde dehydrogenases and then the terminal ether bond is cleaved to yield the depolymerized PEG by one glycol unit. Most probably PEG is anaerobically metabolized through one step which is catalyzed by PEG acetaldehyde lyase, analogous to diol dehydratase. Whether aerobically or anaerobically, the free OH group is necessary for metabolization of PEG. PEG with a molecular weight of up to 20,000 was metabolized either in the periplasmic space (Pseudomonas stutzeri and sphingomonads) or in the cytoplasm (anaerobic bacteria), which suggests the transport of large PEG through the outer and inner membranes of Gram-negative bacterial cells. Membrane-bound PEG dehydrogenase (PEG-DH) with high activity towards PEG 6,000 and 20,000 was purified from PEG-utilizing sphingomonads. Sequencing of PEG-DH revealed that the enzyme belongs to the group of GMC flavoproteins, FAD being the cofactor for the enzyme. On the other hand, alcohol dehydrogenases purified from other bacteria that cannot grow on PEG oxidized PEG. Cytoplasmic NAD-dependent alcohol dehydrogenases with high specificity towards ether-alcohol compound, either crude or purified, showed appreciable activity towards PEG 400 or 600. Liver alcohol dehydrogenase (equine) also oxidized PEG homologs, which might cause fatal toxic syndrome in vivo by carboxylating PEG together with aldehyde dehydrogenase when PEG was absorbed. An ether bond-cleaving enzyme was detected in PEG-utilizing bacteria and purified as diglycolic acid (DGA) dehydrogenase from a PEG-utilizing consortium. The enzyme oxidized glycolic acid, glyoxylic acid, as well as PEG-carboxylic acid and DGA. Similarly, dehydrogenation on polypropylene glycol (PPG) and polytetramethylene glycol (PTMG) was suggested with cell-free extracts of PPG and PTMG-utilizing bacteria, respectively. PPG commercially available is atactic and includes many structural (primary and secondary alcohol groups) and optical (derived from pendant methyl groups on the carbon backbone) isomers. Whether PPG dehydrogenase (PPG-DH) has wide stereo- and enantioselective substrate specificity towards PPG isomers or not must await further purification. Preliminary research on PPG-DH revealed that the enzyme was inducibly formed by PPG in the periplasmic, membrane and cytoplasm fractions of a PPG-utilizing bacterium Stenotrophomonas maltophilia. This finding indicated the intracellular metabolism of PPG is the same as that of PEG. Besides metabolization of polyethers, a biological Fenton mechanism was proposed for degradation of PEG, which was caused by extracellular oxidants produced by a brown-rot fungus in the presence of a reductant and Fe3+, although the metabolism of fragmented PEG has not yet been well elucidated.     

Microbial degradation of octamethylcyclotetrasiloxane

The microbial degradation of low-molecular-weight polydimethylsiloxanes was investigated through laboratory experiments. Octamethylcyclotetrasiloxane was found to be biodegraded under anaerobic conditions in composted sewage sludge, as monitored by the occurrence of the main polydimethylsiloxane degradation product, dimethylsilanediol, compared to that found in experiments with sterilized control samples.     

Sterols with modified side chains.

Radio immunoassays for aldosterone and deoxycorticosterone (DOC)are described in which a simple separation procedure using ammonium sulfate stabilization of bound steroid and extraction of free steroid into toluene scintillant allows an “in vial” assay without mechanical separation of the two phase system. Extraction and thin layer Chromatographic methods for purification of aldosterone and DOC are free of solvent and plate blank effects. Normal values are given for unconjugated aldosterone and DOC in urine, for aldosterone and DOC in plasma and for aldosterone 18-glucuronide in urine.     

Shorter side chains optimize helix-helix packing

A systematic study of helix-helix packing in a comprehensive database of protein structures revealed that the side chains inside helix-helix interfaces on average are shorter than those in the noninterface parts of the helices. The study follows our earlier study of this effect in transmembrane helices. The results obtained on the entire database of protein structures are consistent with those obtained on the transmembrane helices. The difference in the length of interface and noninterface side chains is small but statistically significant. It indicates that helices, if viewed along their main axis, statistically are not circular, but have a flattened interface. This effect brings the helices closer to each other and creates a tighter structural packing. The results provide an interesting insight into the aspects of protein structure and folding.     

Environment of tryptophan side chains in proteins

Although relatively rare, the tryptophan residue (Trp), with its large hydrophobic surface, has a unique role in the folded structure and the binding site of many proteins, and its fluorescence properties make it very useful in studying the structures and dynamics of protein molecules in solution. An analysis has been made of its environment and the geometry of its interaction with neighbors using 719 Trp residues in 180 different protein structures. The distribution of the number of partners interacting with the Trp aromatic ring shows a peak at 6 (considering protein residues only) and 8 (including water and substrate molecules also). The means of the solvent-accessible surface areas of the ring show an exponential decrease with the increase in the number of partners; this relationship can be used to assess the efficiency of packing of residues around Trp. Various residues exhibit different propensities of binding the Trp side chain. The aromatic residues, Met and Pro have high values, whereas the smaller and polar-chain residues have weaker propensities. Most of the interactions are with residues far away in sequence, indicating the importance of Trp in stabilizing the tertiary structure. Of all the ring atoms NE1 shows the highest number of interactions, both along the edge (hydrogen bonding) as well as along the face. Various weak but specific interactions, engendering stability to the protein structure, have been identified.     

Microbial food chains and food webs

Mathematical models for simple microbial food chains and food webs in continuous culture are developed and analyzed. A model for competition of two microbial species for a single scarce resource is also presented as a degenerate case of the food web model. Two models for food chains are developed. The first is based on a model of microbial growth (Monod's) that is widely mentioned and used at the present time. The second is based on a generalization of that model that recent experimental results on microbial food chains seem to require. Experimental data for microbial food webs are almost entirely lacking but a tentative model having what are felt to be the right properties is developed and analyzed. The results obtained from these models seem to be consistent in most circumstances with current ecological thinking on community dynamics.     

Microbial degradation of 1,3-dichlorobenzene

A gram-negative, peritrichously flagellated rod, tentatively identified as an Alcaligenes sp., was isolated from a mixture of soil and water samples by using 1,3-dichlorobenzene as the sole carbon and energy source. During growth on 1,3-dichlorobenzene, almost stoichiometric amounts of chloride were released. Simultaneous adaptation studies, as well as enzyme studies, indicated that 1,3-dichlorobenzene was metabolized via 3,5-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene to 3,5-dichlorocatechol. Subsequently, the latter product was cleaved, yielding 2,4-dichloromuconate. No initial hydrolytic step yielding 3-chlorophenol was detected in this species.     

石油基塑料的微生物降解

石油基塑料种类繁多、数量巨大、应用广泛,常见的有聚乙烯(PE)、聚丙烯(PP)、聚苯乙烯(PS)、聚氯乙烯(PVC)、聚对苯二甲酸乙二醇酯(PET)、聚氨酯(PUR)等。这些合成塑料因其高分子量、高疏水性及高化学键能的特点难以被微生物降解,从而在环境中长期存在和累积,"白色污染"已经成为一个全球性问题。因此安全经济的微生物降解合成塑料是人类面临的一个选择和难题。文中从微生物资源及相关酶学研究方面综述了聚苯乙烯、聚乙烯、聚丙烯、聚氨酯、聚对苯二甲酸乙二醇酯和聚氯乙烯这6种石油基塑料的生物降解的研究现状。目前关于上述6种石油基塑料的微生物降解研究依然大多停留在微生物资源的寻找中,已发现的具备相关能力的菌株种类较少,并且微生物降解效率均非常缓慢;对于其降解机理及关键基因和酶的研究比较少。文中为进一步开展塑料生物降解研究,寻找高效的塑料降解菌株资源以及进一步在遗传、分子和生化水平研究塑料生物降解机理研究,从而最终实现合成塑料的彻底降解和高值化利用提供了借鉴。