Horizontal gene transfer of glycosyl hydrolases of the rumen fungi

By combining analyses of G + C content and patterns of codon usage and constructing phylogenetic trees, we describe the gene transfer of an endoglucanase (celA) from the rumen bacteria Fibrobacter succinogenes to the rumen fungi Orpinomyces joyonii. The strong similarity between different glycosyl hydrolases of rumen fungi and bacteria suggests that most, if not all, of the glycosyl hydrolases of rumen fungi that play an important role in the degradation of cellulose and other plant polysaccharides were acquired by horizontal gene transfer events. This acquisition allows fungi to establish a habitat within a new environmental niche: the rumen of the herbivorous mammals for which cellulose and plant hemicellulose constitute the main raw nutritive substrate.     

The nature of anaerobic fungi and their polysaccharide degrading enzymes

Conclusion The discovery of anaerobic fungi has added a new member to the indigenous microorganisms that inhabit the rumen ecosystem. Anaerobic fungi do not appear essential for the survival of ruminants due to their presence in very low numbers, and sometimes absence, in ruminants fed low fiber diets, but their presence may likely be very important in the digestion of fibrous diets. The anaerobic fungi have adapted well to the rumen environment. They are able to ferment a large array of soluble carbohydrates and can synthesize cellular components in an anaerobic environment. The fungi posses hydrogenosomes for the removal of reducing equivalents in the form of molecular hydrogen and the removal of trace oxygen is a accomplished via removal by NADH oxidase. Their positive synergistic interaction with methanogenic bacteria eludes to their highly evolved role in the rumen environment. The fungi also produce resistant sporangia that allows for transfer of species to a new host in an oxygen environment. The anaerobic fungi posses a highly active array of polysaccharide degrading enzymes that may provide an advantage in the highly competitive rumen ecosystem. The production of specific enzymes that hydrolyze the lignocellulosic fraction of plant walls is unique in rumen microorganisms and allows for their attachment and growth on fibrous plant particles that are not available to the rumen bacteria.     

Evolution of beta-lactam biosynthesis genes and recruitment of trans-acting factors

Penicillins and cephalosporins belong chemically to the group of beta-lactam antibiotics. The formation of hydrophobic penicillins has been reported in fungi only, notably Penicillium chrysogenum and Emericella nidulans, whereas the hydrophilic cephalosporins are produced by both fungi, e.g., Acremonium chrysogenum (cephalosporin C), and bacteria. The producing bacteria include Gram-negatives and Gram-positives, e.g. Lysobacter lactamdurans (cephabacins) and Streptomyces clavuligerus (cephamycin C), respectively. For a long time the evolutionary origin of beta-lactam biosynthesis genes in fungi has been discussed. As often, there are arguments for both hypotheses, i.e., horizontal gene transfer from bacteria to fungi versus vertical descent. There were strong arguments in favour of horizontal gene transfer, e.g., fungal genes were clustered or some genes lack introns. The recent identification and characterisation of cis-/trans-elements involved in the regulation of the beta-lactam biosynthesis genes has provided new arguments in favour of horizontal gene transfer. In contrast to the bacterium S. clavuligerus, all regulators of fungal beta-lactam biosynthesis genes represent wide-domain regulators which were recruited to also regulate the beta-lactam biosynthesis genes. Moreover, the fungal regulatory genes are not part of the gene cluster. If bacterial regulators were co-transferred with the gene cluster from bacteria to fungi, most likely they would have been non-functional in eukaryotes and lost during evolution. Alternatively, it is conceivable that only a part of the beta-lactam biosynthesis gene cluster was transferred to some fungi, e.g., the acvA and ipnA gene without a regulatory gene.     

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria

AIMS: To investigate biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria, and to identify the fungus with the fastest biohydrogenation rate. METHODS AND RESULTS: Biohydrogenation of linoleic acid by mixed rumen fungi and mixed rumen bacteria were compared in vitro. With mixed rumen bacteria, all biohydrogenation reactions were finished within 100 min of incubation and the end product of biohydrogenation was stearic acid. With mixed rumen fungi, biohydrogenation proceeded more slowly over a 24-h period. Conjugated linoleic acid (CLA; cis-9, trans-11 C18 : 2) was an intermediate product, and vaccenic acid (VA; trans-11 C18 : 1) was the end product of biohydrogenation. Fourteen pure fungal isolates were tested for biohydrogenation rate. DNA sequencing showed that the isolate with the fastest rate belonged to the Orpinomyces genus. CONCLUSIONS: It is concluded that rumen fungi have the ability to biohydrogenate linoleic acid, but biohydrogenation is slower in rumen fungi than in rumen bacteria. The end product of fungal biohydrogenation is VA, as for group A rumen bacteria. Orpinomyces is the most active biohydrogenating fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that rumen fungi can biohydrogenate fatty acids. Fungi could influence CLA content of ruminant products.     

How modern systematics relates to the rumen fungi

The zoosporic fungi comprise a polyphyletic grouping of four classes, the Plasmodiophoromycetes, Oomycetes, Hyphochytriomycetes and Chytridiomycetes. Apart from their absorptive mode of nutrition and the presence of zoospores in some stage of their life cycle, there is little these classes have in common. The zoosporic species of rumen fungi are classified in the Class Chytridiomycetes which is a monophyletic group with extreme diversity in thallus morphology, reproduction and zoospore cytology. The rumen fungi have many characteristics in common with the Spizellomycetaceae but have been given their own family, the Neocallimasticaceae. There are arguments for reducing this family to synonymy with the Spizellomycetaceae, or elevating it to a new order, but before a rational decision can be made, more rumen fungi require detailed examination, especially their zoospore ultrastructure.     

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event

Background  

Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included.     

The xynC gene from Fibrobacter succinogenes S85 codes for a xylanase with two similar catalytic domains.

The xynC gene of Fibrobacter succinogenes S85 codes for a 66.4-kDa xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. Domains A and B of XynC code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of Ruminococcus flavefaciens, Neocallimastix patriciarum, Clostridium acetobutylicum, Bacillus pumilus, Bacillus subtilis, and Bacillus circulans. More than 88% of the xylanase activity of Escherichia coli cells carrying the original 13-kb recombinant plasmid was released from intact cells by cold water washes. The major products of hydrolysis of xylan by both domains were xylose and xylobiose, indicating that the xynC gene product exhibits catalytic properties similar to those of the XynA xylanases from R. flavefaciens and N. patriciarum. So far, these features are not shared broadly with bacteria from other environments and may indicate specific selection for this domain structure in the highly competitive environment of the rumen.     

Pathogenicity islands: the tip of the iceberg.

Pathogenicity islands represent distinct genetic elements encoding virulence factors of pathogenic bacteria. Pathogenicity islands belong to the class of genomic islands, which are common genetic elements sharing a set of unifying features. Genomic islands have been acquired by horizontal gene transfer. In recent years many different genomic islands have been discovered in a variety of pathogenic as well as non-pathogenic bacteria. Because they promote genetic variability, genomic islands play an important role in microbial evolution.     

A modular xylanase from mesophilic Cellulomonas fimi contains the same cellulose-binding and thermostabilizing domains as xylanases from thermophilic bacteria

Abstract The xynC gene from mesophilic Cellulomonas fimi encodes a large 125 kDa modular xylanase (XYLC), consisting of six distinct functional domains. In addition to a single Family 10 catalytic domain, XYLC contains a domain homologous with the nodulation protein, NodB, from nitrogen-fixing bacteria and therrnostabilizing and cellulose-binding domains found previously only in xylanases from thermophilic bacteria.     

植物病原物无毒基因及其功能

植物抗病基因与病原物无毒基因产物间直接或间接相互作用导致产生的基因对基因抗性是植物抗病性的重要形式。无毒基因已在多种植物病原物 ,包括真菌、细菌、病毒和卵菌等中得到克隆。绝大多数已克隆无毒基因之间 ,及其与已知蛋白之间 ,均无显著序列同源性。然而 ,多数已克隆植物抗病基因有较高序列一致性 ,产物往往具有相似的结构域。由序列一致性很高的抗病基因产物与没有明显序列同源性的无毒基因产物相互作用 ,介导产生的过敏性细胞坏死和抗病性 ,在产生速度、强度和组织特异性等方面均可能有显著差异。无毒基因具有双重功能 :在含互补抗病基因植物中表现无毒效应 ,而在不含互补抗病基因植物中显示小种、菌株、致病型、或种特异性毒性效应     

Intergeneric transfer of ribosomal genes between two fungi

Background  

Horizontal gene transfer, also called lateral gene transfer, frequently occurs among prokaryotic organisms, and is considered an important force in their evolution. However, there are relatively few reports of transfer to or from fungi, with some notable exceptions in the acquisition of prokaryotic genes. Some fungal species have been found to contain sequences resembling those of bacterial genes, and with such sequences absent in other fungal species, this has been interpreted as horizontal gene transfer. Similarly, a few fungi have been found to contain genes absent in close relatives but present in more distantly related taxa, and horizontal gene transfer has been invoked as a parsimonious explanation. There is a paucity of direct experimental evidence demonstrating the occurrence of horizontal gene transfer in fungi.     

Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL

Pseudomonas aeruginosa bacteriophage endolysins KZ144 (phage phiKZ) and EL188 (phage EL) are highly lytic peptidoglycan hydrolases (210 000 and 390 000 units mg(-1)), active on a broad range of outer membrane-permeabilized Gram-negative species. Site-directed mutagenesis indicates E115 (KZ144) and E155 (EL188) as their respective essential catalytic residues. Remarkably, both endolysins have a modular structure consisting of an N-terminal substrate-binding domain and a predicted C-terminal catalytic module, a property previously only demonstrated in endolysins originating from phages infecting Gram-positives and only in an inverse arrangement. Both binding domains contain conserved repeat sequences, consistent with those of some peptidoglycan hydrolases of Gram-positive bacteria. Fusions of these domains with green fluorescent protein immediately label all outer membrane-permeabilized Gram-negative bacteria tested, isolated P. aeruginosa peptidoglycan and N-acetylated Bacillus subtilis peptidoglycan, demonstrating the broad range of peptidoglycan-binding capacity by these domains. Specifically, A1 chemotype peptidoglycan and fully N-acetylated glucosamine units are essential for binding. Both KZ144 and EL188 appear to be a natural chimeric enzyme, originating from a recombination of a cell wall-binding domain encoded by a Bacillus or Clostridium species and a catalytic domain of an unknown ancestor.     

Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics

The degradation of plant cell walls by ruminants is of major economic importance in the developed as well as developing world. Rumen fermentation is unique in that efficient plant cell wall degradation relies on the cooperation between microorganisms that produce fibrolytic enzymes and the host animal that provides an anaerobic fermentation chamber. Increasing the efficiency with which the rumen microbiota degrades fiber has been the subject of extensive research for at least the last 100 years. Fiber digestion in the rumen is not optimal, as is supported by the fact that fiber recovered from feces is fermentable. This view is confirmed by the knowledge that mechanical and chemical pretreatments improve fiber degradation, as well as more recent research, which has demonstrated increased fiber digestion by rumen microorganisms when plant lignin composition is modified by genetic manipulation. Rumen microbiologists have sought to improve fiber digestion by genetic and ecological manipulation of rumen fermentation. This has been difficult and a number of constraints have limited progress, including: (a) a lack of reliable transformation systems for major fibrolytic rumen bacteria, (b) a poor understanding of ecological factors that govern persistence of fibrolytic bacteria and fungi in the rumen, (c) a poor understanding of which glycolyl hydrolases need to be manipulated, and (d) a lack of knowledge of the functional genomic framework within which fiber degradation operates. In this review the major fibrolytic organisms are briefly discussed. A more extensive discussion of the enzymes involved in fiber degradation is included. We also discuss the use of plant genetic manipulation, application of free-living lignolytic fungi and the use of exogenous enzymes. Lastly, we will discuss how newer technologies such as genomic and metagenomic approaches can be used to improve our knowledge of the functional genomic framework of plant cell wall degradation in the rumen.     

厌氧真菌生态作用及其分子生物学研究进展

厌氧真菌是瘤胃内重要的纤维降解菌,在瘤胃功能的发挥中起重要作用。目前对厌氧真菌纤维降解能力的研究较多,主要集中于对厌氧真菌纤维降解酶如纤维素酶、木聚糖酶等的研究。在瘤胃中,厌氧真菌对粗纤维的降解是其和瘤胃内其他微生物共同作用的结果,因此,瘤胃内厌氧真菌与他微生物之间相互关系的研究越来越受到重视。现代分子生物学技术的发展有利于更深入和透彻的研究厌氧真菌,利用18S rRNA、RFLPI、TS1等分子生物学方法对厌氧真菌进行系统学及进化研究成为热点。     

A comparative analysis of two cDNA clones of the cellulase gene family from anaerobic fungus Piromyces rhizinflata

Cellulase family and some other glycosyl hydrolases of anaerobic fungi inhabiting the digestive tract of ruminants are believed to form an enzyme complex called cellulosome. Study of the individual component of cellulosome may shed light on understanding the organization of this complex and its functional mechanism. We have analysed the primary sequences of two cellulase clones, cel5B and cel6A, isolated from the cDNA library of ruminal fungus, Piromyces rhizinflata strain 2301. The deduced amino acid sequences of the catalytic domain of Cel5B, encoded by cel5B, showed homology with the subfamily 4 of the family 5 (subfamily 5(4)) of glycosyl hydrolases, while cel6A encoded Cel6A belonged to family 6 of glycosyl hydrolases. Phylogenetic tree analysis suggested that the genes of subfamily 5(4) glycosyl hydrolases of P. rhizinflata might have been acquired from rumen bacteria. Cel5B and Cel6A were modular enzymes consisting of a catalytic domain and dockerin domain(s), but not a cellulose binding domain. The occurrence of dockerin domains indicated that both enzymes were cellulosome components. The catalytic domain of the Cel5B (Cel5B') and Cel6A (Cel6A') recombinant proteins were purified. The optimal activity conditions with carboxymethyl cellulose (CMC) as the substrate were pH 6.0 and 50 degrees C for Cel5B', and pH 6.0 and 37-45 degrees C for Cel6A'. Both Cel5B' and Cel6A' exhibited activity against CMC, barley beta-glucan, Lichenan, and oat spelt xylan. Cel5B' could also hydrolyse p-nitrophenyl-beta-d-cellobioside, Avicel and filter paper while Cel6A' did not show any activity on these substrates. It is apparent that Cel6A' acted as an endoglucanase and Cel5B' possessed both endoglucanase and exoglucanase activities. No synergic effect was observed for these recombinant enzymes in vitro on Avicel and CMC.     

Fungal denitrification and nitric oxide reductase cytochrome P450nor

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N(2)O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N(2)O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N(2) or N(2)O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.     

Hydrolyse enzymatique des protéines par les bactéries du rumen

The hydrolysis of proteins in the rumen is a process brought about mainly by bacteria, of which many species produce proteases. The majority of endopeptidases are cysteine proteases, whereas exopeptidases are mainly aminopeptidases. Prevotella ruminicola is distinguished from other bacterial species by its capacity to produce dipeptidases such as type I dipeptidyl aminopeptidase. The mechanisms controlling the synthesis of endo- and exopeptidases have been little studied. Enzyme production seems to depend on the concentrations of peptides, amino acids and carbohydrates. Proteolytic activity varies in relation to pH, and the concentrations of ions and phenolic compounds. Various works have shown that hydrolysis of a protein by enzymes depends on its three-dimensional structure and possible bonding to non-protein structures. These properties determine the peptide and amino acid concentrations that occur in the rumen. The molecular weight, hydrophobic property and primary structure of the peptides are the main factors that affect the hydrolysis and/or uptake of these compounds by rumen bacteria. The methodological problems inherent to assaying these compounds do however lead to current divergences of opinion concerning the physico-chemical characteristics of the peptides that escape rumen fermentation.     

Multidomain and Multifunctional Glycosyl Hydrolases from the Extreme Thermophile Caldicellulosiruptor Isolate Tok7B.1

DNA sequencing techniques have revealed widespread molecular diversity of the genomic organization of apparently closely related bacteria (as judged from SSU rDNA sequence similarity). We have previously described the extreme thermophile Caldicellulosiruptor saccharolyticus, which is unusual in possessing multi-catalytic, multidomain arrangements for the majority of its glycosyl hydrolases. We report here the sequencing of three gene clusters of glycosyl hydrolases from Caldicellulosiruptor sp. strain Tok7B.1. These clusters are not closely linked, and each is different in its organization from any described for Cs. saccharolyticus. The catalytic domains of the enzymes belong to glycosyl hydrolase families 5, 9, 10, 43, 44, and 48. The cellulose binding domains (CBDs) of these enzymes from Caldicellulosiruptor sp. Tok7B.1 are types IIIb, IIIc, or VI. A number of individual catalytic and binding domains have been expressed in Escherichia coli, and biochemical data are reported on the purified enzymes for cellulose degradation encoded by engineered derivatives of celB and celE. Received: 12 November 1999 / Accepted: 30 November 1999