Characterization and Transcription of the Genes Involved in Butyrate Production in Butyrivibrio fibrisolvens TypeI and II Strains

The genes in Butyrivibrio fibrisolvens that encode the enzymes involved in butyrate production were sequenced. In a type I strain (ATCC 19171(T)), the genes coding for the enzymes that catalyze the conversion from acetyl-CoA to butyryl-CoA, thl (thiolase), crt (crotonase), hbd (beta-hydroxybutyryl-CoA dehydrogenase), bcd (butyryl-CoA dehydrogenase), etfB (electron transfer flavoprotein [ETF]-beta), and etfA (ETF-alpha), were found to be clustered and arranged in this order. A type II strain (ATCC 51255) had the same clustered genes with the same arrangement, except that crt was not present in the clustered genes. The deduced amino acid sequences of these enzymes did not greatly differ between the two strains, and even between B. fibrisolvens and clostridia. Amino acid identity appeared to be higher within the same type than between types I and II. The clustered genes were shown to be cotranscribed, and constitutively transcribed without being affected significantly by culture conditions.     

Genetic control of the histidine dissimilatory pathway in Pseudomonas putida

Summary In P. putida the first four enzymes involved in the dissimilation of histidine are induced by the first intermediate of the pathway, urocanic acid. The genes specifying these enzymes, hutH, hutU, hutF and (probably) hutI appear to be clustered on the chromosome between pcaE and pcaA (genes involved in p-hydroxybenzoic acid catabolism). Two mutants which produce the histidine-dissimilitory enzymes constitutively have been isolated. They appear to carry mutations in a regulatory locus, which maps in the same region as the structural genes of the pathway.     

Evolution of the clusters of genes for beta-lactam antibiotics: a model for evolutive combinatorial assembly of new beta-lactams.

beta-Lactam antibiotics are produced by prokaryotic and eukaryotic organisms. The genes for beta-lactam biosynthesis are organized in clusters but the location of the different genes is not identical. Biosynthesis genes are clustered with genes for resistance (bla, pbp) and for the efflux of the antibiotic (cmcT) in prokaryotes. Comparison of proteins reveals much larger differences for primary metabolism enzymes than for beta-lactam biosynthesis enzymes in producing organisms. This suggests a horizontal transfer of the beta-lactam antibiotic biosynthesis genes.     

The Evolution of Fungal Metabolic Pathways

Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters represent hotspots for the generation of fungal metabolic diversity.     

Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3

A 13.9-kb region, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 2-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight genes together were designated amnBACFEDHG. Each gene was similar to the corresponding gene operating in the meta-cleavage pathway, except for amnB, amnA, and amnD. The four 2-aminophenol-metabolizing enzymes, 2-aminomuconic 6-semialdehyde dehydrogenase, 2-aminomuconate deaminase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase, were purified and characterized. NH2-terminal amino acid sequences of each purified enzyme agreed with those deduced from amnC, amnF, amnE, and amnD, respectively. These genes were therefore assigned as the genes encoding these respective proteins. The tight clustering of the amn genes, which were all transcribed in the same direction, raised the possibility that these genes formed a single operon. The organization of the amn genes was entirely different from that of the atd, dmp, and xyl genes reported in the meta-cleavage pathway, although these latter genes clustered similarly.     

Evolution of enzymes involved in the photorespiratory 2-phosphoglycolate cycle from cyanobacteria via algae toward plants

The photorespiratory pathway was shown to be essential for organisms performing oxygenic photosynthesis, cyanobacteria, algae, and plants, in the present day O(2)-containing atmosphere. The identification of a plant-like 2-phosphoglycolate cycle in cyanobacteria indicated that not only genes of oxygenic photosynthesis but also genes encoding photorespiratory enzymes were endosymbiotically conveyed from ancient cyanobacteria to eukaryotic oxygenic phototrophs. Here, we investigated the origin of the photorespiratory pathway in photosynthetic eukaryotes by phylogenetic analysis. We found that a mixture of photorespiratory enzymes of either cyanobacterial or α-proteobacterial origin is present in algae and higher plants. Three enzymes in eukaryotic phototrophs clustered closely with cyanobacterial homologs: glycolate oxidase, glycerate kinase, and hydroxypyruvate reductase. On the other hand, the mitochondrial enzymes of the photorespiratory cycle in algae and plants, glycine decarboxylase subunits and serine hydroxymethyltransferase, evolved from proteobacteria. Other than most genes for proteins of the photosynthetic machinery, nearly all enzymes involved in the 2-phosphogylcolate metabolism coexist in the genomes of cyanobacteria and heterotrophic bacteria.     

Organization of Enzymes in the Polyaromatic Synthetic Pathway: Separability in Bacteria

ULTRACENTRIFUGATION IN SUCROSE DENSITY GRADIENTS WAS EMPLOYED TO ESTIMATE THE MOLECULAR WEIGHTS AND TO DETERMINE POSSIBLE PHYSICAL AGGREGATION OF THE FIVE ENZYMES CATALYZING STEPS TWO TO SIX IN THE PRECHORISMIC ACID PORTION OF THE POLYAROMATIC SYNTHETIC PATHWAY IN SIX SPECIES OF BACTERIA: Escherichia coli, Salmonella typhimurium, Aerobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa, and Streptomyces coelicolor. The five enzymes were not aggregated in extracts of any of the species examined, nor are the genes encoding these enzymes clustered in those bacterial species for which genetic evidence exists. (An initial examination of the blue-green alga Anabaena variabilis indicates nonaggregation in this species also.) This situation in bacteria is in marked contrast to that found in Neurospora crassa and other fungi in which the same five enzymes are associated as an aggregate encoded (at least in the case of N. crassa) by a cluster of five genes. In addition, also in contrast to N. crassa, no evidence was obtained for more than one kind of dehydroquinase activity in any of the bacteria examined. These comparative results are discussed in relation to the origin, evolution, and functional significance of the gene-enzyme relationships existing in the early steps of aromatic biosynthesis in bacteria and fungi.     

The dual functions of biphenyl-degrading ability of Pseudomonas sp. KKS102: energy acquisition and substrate detoxification

The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.     

Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects

In insects, chitin is a major structural component of the cuticle and the peritrophic membrane (PM). In nature, chitin is always associated with proteins among which chitin-binding proteins (CBPs) are the most important for forming, maintaining and regulating the functions of these extracellular structures. In this study, a genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin-binding domains (CBDs) was conducted. A total of 53 genes encoding 56 CBPs were identified, including 15 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 11 CPAP3s (CPAPs with 3 CBDs) and 17 PMPs (PM proteins) with a variable number of CBDs, which are structural components of cuticle or of the PM. CBDs were also identified in enzymes of chitin metabolism including 6 chitinases and 7 chitin deacetylases encoded by 6 and 5 genes, respectively. RNA-seq analysis confirmed that PMP and CPAP genes have differential spatial expression patterns. The expression of PMP genes is midgut-specific, while CPAP genes are widely expressed in different cuticle forming tissues. Phylogenetic analysis of CBDs of proteins in insects belonging to different orders revealed that CPAP1s from different species constitute a separate family with 16 different groups, including 6 new groups identified in this study. The CPAP3s are clustered into a separate family of 7 groups present in all insect orders. Altogether, they reveal that duplication events of CBDs in CPAP1s and CPAP3s occurred prior to the evolutionary radiation of insect species. In contrast to the CPAPs, all CBDs from individual PMPs are generally clustered and distinct from other PMPs in the same species in phylogenetic analyses, indicating that the duplication of CBDs in each of these PMPs occurred after divergence of insect species. Phylogenetic analysis of these three CBP families showed that the CBDs in CPAP1s form a clearly separate family, while those found in PMPs and CPAP3s were clustered together in the phylogenetic tree. For chitinases and chitin deacetylases, most of phylogenetic analysis performed with the CBD sequences resulted in similar clustering to the one obtained by using catalytic domain sequences alone, suggesting that CBDs were incorporated into these enzymes and evolved in tandem with the catalytic domains before the diversification of different insect orders. Based on these results, the evolution of CBDs in insect CBPs is discussed to provide a new insight into the CBD sequence structure and diversity, and their evolution and expression in insects.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biosynthesis and molecular genetics of cephamycins

Cephamycin C is produced in a nine steps pathway by the actinomycetes S. clavuligerus and N. lactamdurans. The genes encoding the biosynthesis enzymes are clustered in both microorganisms as well as in the cephabacin producer Lysobacter lactamgenus, a Gram negative bacterium. The clusters of genes include genes encoding enzymes common to the biosynthesis of penicillin and cephalosporin C by the eukaryotic producers Penicillium chrysogenum and Cephalosporiun acremonium and genes for steps specific for the formation of the precursor -aminoadipic acid as well as for the enzymes involved in the late modification of the cephalosporin intermediates of the pathway. Present are also genes for proteins involved in the export and/or resistance to cephamycin C. In S. clavuligerus a gene encoding a regulatory protein controlling the formation of cephamycin C and clavulanic acid is also present in the cluster.     

A cluster of three genes (dapA, orf2, and dapB) of Brevibacterium lactofermentum encodes dihydrodipicolinate synthase, dihydrodipicolinate reductase, and a third polypeptide of unknown function.

The dapA and dapB genes, encoding, respectively, dihydrodipicolinate synthase and dihydrodipicolinate reductase, the two first enzymes of the lysine branch of the aspartic amino acid family, were cloned from the DNA of the amino acid-producing bacterium Brevibacterium lactofermentum. The two genes were clustered in a 3.5-kb Sau3AI-BamHI fragment but were separated by an open reading frame of 750 nucleotides. The protein encoded by this open reading frame had little similarity to any protein in the data banks, and its function remains unknown. The three genes were translated in Escherichia coli, giving the corresponding polypeptides.