Targeted gene engineering in Clostridium cellulolyticum H10 without methylation

Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative MspI endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C. cellulolyticum mutant H10ΔmspI lost the MspI endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10ΔmspI without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10ΔmspI, double mutants H10ΔmspIΔldh and H10ΔmspIΔack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10ΔmspI, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10ΔmspI constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level.     

Esp1396I restriction-modification system: structural organization and mode of regulation

Esp1396I restriction–modification (RM) system recognizes an interrupted palindromic DNA sequ ence 5′-CCA(N)₅TGG-3′. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp. strain RFL1396. The nucleotide sequence of the entire 5622 bp pEsp1396 plasmid was determined on both strands. Identified genes for DNA methyltransferase (esp1396IM) and restriction endonuclease (esp1396IR) are transcribed convergently. The restriction endonuclease gene is preceded by the small ORF (esp1396IC) that possesses a strong helix-turn-helix motif and resembles regulatory proteins found in PvuII, BamHI and few other RM systems. Gene regulation studies revealed that C.Esp1396I acts as both a repressor of methylase expression and an activator of regulatory protein and restriction endonuclease expression. Our data indicate that C protein from Esp1396I RM system activates the expression of the Enase gene, which is co-transcribed from the promoter of regulatory gene, by the mechanism of coupled translation.     

Hjc resolvase is a distantly related member of the type II restriction endonuclease family

Hjc resolvase is an archaeal enzyme involved in homologous DNA recombination at the Holliday junction intermediate. However, the structure and the catalytic mechanism of the enzyme have not yet been identified. We performed database searching using the amino acid sequence of the enzyme from Pyrococcus furiosus as a query. We detected 59 amino acid sequences showing weak but significant sequence similarity to the Hjc resolvase. The detected sequences included DpnII, HaeII and Vsr endonuclease, which belong to the type II restriction endonuclease family. In addition, a highly conserved region was identified from a multiple alignment of the detected sequences, which was similar to an active site of the type II restriction endonucleases. We substituted three conserved amino acid residues in the highly conserved region of the Hjc resolvase with Ala residues. The amino acid replacements inactivated the enzyme. The experimental study, together with the results of the database searching, suggests that the Hjc resolvase is a distantly related member of the type II restriction endonuclease family. In addition, the results of our database searches suggested that the members of the RecB domain superfamily are evolutionarily related to the type II restriction endonuclease family.     

A mobile restriction–modification system provides phage defence and resolves an epigenetic conflict with an antagonistic endonuclease

Epigenetic DNA methylation plays an important role in bacteria by influencing gene expression and allowing discrimination between self-DNA and intruders such as phages and plasmids. Restriction–modification (RM) systems use a methyltransferase (MTase) to modify a specific sequence motif, thus protecting host DNA from cleavage by a cognate restriction endonuclease (REase) while leaving invading DNA vulnerable. Other REases occur solitarily and cleave methylated DNA. REases and RM systems are frequently mobile, influencing horizontal gene transfer by altering the compatibility of the host for foreign DNA uptake. However, whether mobile defence systems affect pre-existing host defences remains obscure. Here, we reveal an epigenetic conflict between an RM system (PcaRCI) and a methylation-dependent REase (PcaRCII) in the plant pathogen Pectobacterium carotovorum RC5297. The PcaRCI RM system provides potent protection against unmethylated plasmids and phages, but its methylation motif is targeted by the methylation-dependent PcaRCII. This potentially lethal co-existence is enabled through epigenetic silencing of the PcaRCII-encoding gene via promoter methylation by the PcaRCI MTase. Comparative genome analyses suggest that the PcaRCII-encoding gene was already present and was silenced upon establishment of the PcaRCI system. These findings provide a striking example for selfishness of RM systems and intracellular competition between different defences.     

DNA methylases of Hemophilus influenzae Rd. I. Purification and properties

Hemophilus influenzae strain Rd DNA contains small amounts of 5-methylcytosine (0.012%) and significantly greater amounts of N-6-methyladenine (0.34%). Four DNA adenine methylases have been identified and purified from crude extracts of H. influenzae Rd by means of phosphocellulose chromatography. Each of the four enzymes requires (S-adenosyl-l-methionine as a methyl group donor and each differs in its ability to methylate various DNAs in vitro. DNA methylase I is related to the genetically described modification-restriction system in H. influenzae Rd, and is presumably the modification enzyme for that system. DNA methylase II introduces approximately 130 methyl groups into a phage T7 DNA molecule and protects T7 DNA from the H. influenzae Rd restriction enzyme, endonuclease R, described by Smith and Wilcox (1970). These findings indicate that DNA methylase II is the modification enzyme corresponding to endonuclease R. A third modification-restriction system, which does not affect T7 DNA, has been detected in H. influenzae Rd. DNA methylase III is apparently the modification enzyme for this system. The biological function of DNA methylase IV remains unknown.     

The Fsp4HI restriction-modification system: Gene cloning, comparison of protein structures, and biochemical properties of recombinant DNA methyltransferase M.Fsp4HI

Genes coding for the Flavobacterium sp. 4H restriction-modification (RM) system, which recognizes the sequence 5′-GCNGC-3′, were cloned in Escherichia coli ER2267 and sequenced. The Fsp4HI RM system includes two genes: one for DNA methyltransferase (M.) and the other for restriction endonuclease (R.), immediately following the former in the same direction. The genes partly overlap. According to the deduced amino acid sequences, M.Fsp4HI belongs to C5 DNA methyltransferases, whereas R.Fsp4HI is only slightly similar to some restriction enzymes recognizing similar sequences. M.Fsp4HI was purified by column chromatography. The optimal conditions for the enzyme are 30°C and pH 7.5. M.Fsp4HI modifies the first cytosine in 5′-GCNGC-3′.     

PsasM2I,a Type II Restriction–Modification System in Pseudomonas savastanoi pv. savastanoi: Differential Distribution of Carrier Strains in the Environment and the Evolutionary History of Homologous RM Systems in the Pseudomonas syringae Complex

A type II restriction–modification system was found in a native plasmid of Pseudomonas savastanoi pv. savastanoi MLLI2. Functional analysis of the methyltransferase showed that the enzyme acts by protecting the DNA sequence CTGCAG from cleavage. Restriction endonuclease expression in recombinant Escherichia coli cells resulted in mutations in the REase sequence or transposition of insertion sequence 1A in the coding sequence, preventing lethal gene expression. Population screening detected homologous RM systems in other P. savastanoi strains and in the Pseudomonas syringae complex. An epidemiological survey carried out by sampling olive and oleander knots in two Italian regions showed an uneven diffusion of carrier strains, whose presence could be related to a selective advantage in maintaining the RM system in particular environments or subpopulations. Moreover, carrier strains can coexist in the same orchards, plants, and knot tissues with non-carriers, revealing unexpected genetic variability on a very small spatial scale. Phylogenetic analysis of the RM system and housekeeping gene sequences in the P. syringae complex demonstrated the ancient acquisition of the RM systems. However, the evolutionary history of the gene complex also showed the involvement of horizontal gene transfer between related strains and recombination events.     

The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model.     

Restriction and modification systems of ruminal bacteria

A high frequency of type II restriction endonuclease activities was detected inSelenomonas ruminantium but not in other rumen bacteria tested. Eight different restriction endonucleases were characterized in 17 strains coming from genetically homogeneous local population. Chromosomal DNA isolated fromS. ruminantium strains was found to be refractory to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases. The presence of Dam methylation was detected inS. ruminantium strains as well as in several other species belonging to theSporomusa subbranch of low G+C Gram-positive bacteria (Megasphaera elsdenii, Mitsuokella multiacidus).     

Resonance assignments of cohesin and dockerin domains from Clostridium acetobutylicum ATCC824

Cohesin and dockerin domains are critical assembling components of cellulosome, a large extracellular multienzyme complex which is used by anaerobic cellulolytic bacteria to efficiently degrade lignocellulose. According to sequence homology, cohesins can be divided into three major groups, whereas cohesins from Clostridium acetobutylicum are beyond these groups and emanate from a branching point between the type I and type III cohesins. Cohesins and dockerins from C. acetobutylicum show low sequence homology to those from other cellulolytic bacteria, and their interactions are specific in corresponding species. Therefore the interactions between cohesins and dockerins from C. acetobutylicum are meaningful to the studies of both cellulosome assembling mechanism and the construction of designer cellulosome. Here we report the NMR resonance assignments of one cohesin from cellulosome scaffoldin cipA and one dockerin from a cellulosomal glycoside hydrolase (family 9) of C. acetobutylicum for further structural determination and functional studies.     

FspAI, a unique type II restriction endonuclease that recognizes the octanucleotide sequence 5′-RTGC↓GCAY-3′

A new type II restriction endonuclease designated FspAI has been partially purified from a Flexibacter species Tv-m21K. FspAI recognizes the octanucleotide sequence 5′-RTGC↓GCAY-3′ and cleaves it in the center generating blunt-ended DNA fragments.     

Complexes formed between the restriction endonuclease EcoK and heteroduplex DNA

The complexes between the Escherichia coli K restriction endonuclease and heteroduplex DNA (one strand methylated and one unmethylated) have been characterized and shown to have different properties from those formed with unmodified DNA. The nature of the heteroduplex complex appears to commit the enzyme to its methylase mode.     

Horizontal gene transfer contributes to the wide distribution and evolution of type II restriction-modification systems

Restriction modification (RM) systems serve to protect bacteria against bacteriophages. They comprise a restriction endonuclease activity that specifically cleaves DNA and a corresponding methyltransferase activity that specifically methylates the DNA, thereby protecting it from cleavage. Such systems are very common in bacteria. To find out whether the widespread distribution of RM systems is due to horizontal gene transfer, we have compared the codon usages of 29 type II RM systems with the average codon usage of their respective bacterial hosts. Pronounced deviations in codon usage were found in six cases:EcoRI,EcoRV,KpnI,SinI,SmaI, andTthHB81. They are interpreted as evidence for horizontal gene transfer in these cases. As the methodology is expected to detect only one-fourth to one-third of all horizontal gene transfer events, this result implies that horizontal gene transfer had a considerable influence on the distribution and evolution of RM systems. In all of these six cases the codon usage deviations of the restriction enzyme genes are much more pronounced than those of the methyltransferase genes. This result suggests that in these cases horizontal gene transfer had occurred sequentially with the gene for the methyltransferase being first acquired by the cell. This can be explained by the fact that an active restriction endonuclease is highly toxic in cells whose DNA is not protected from cleavage by a corresponding methyltransferase.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

Solvent Production and Morphological Changes in Clostridium acetobutylicum

The morphological and cytological changes which occurred in Clostridium acetobutylicum P262 during the production of acetone, butanol, and ethanol in an industrial fermentation medium were identified and correlated with the growth and physiological changes. The swollen, cigar-shaped clostridial forms were involved in the conversion of acids to neutral solvents, and there was a correlation between the number of clostridial forms and the production of solvents. Sporulation mutants which were unable to form clostridial stages (cls mutants) did not produce solvents. Oligosporogenous mutants which showed reduced clostridial stage formation produced intermediate levels of solvents. Sporulation mutants blocked after the clostridial stage, which were unable to form mature spores (spo mutants), produced normal levels of solvents.     

A functional bacteria-derived restriction modification system in the mitochondrion of a heterotrophic protist

The overarching trend in mitochondrial genome evolution is functional streamlining coupled with gene loss. Therefore, gene acquisition by mitochondria is considered to be exceedingly rare. Selfish elements in the form of self-splicing introns occur in many organellar genomes, but the wider diversity of selfish elements, and how they persist in the DNA of organelles, has not been explored. In the mitochondrial genome of a marine heterotrophic katablepharid protist, we identify a functional type II restriction modification (RM) system originating from a horizontal gene transfer (HGT) event involving bacteria related to flavobacteria. This RM system consists of an HpaII-like endonuclease and a cognate cytosine methyltransferase (CM). We demonstrate that these proteins are functional by heterologous expression in both bacterial and eukaryotic cells. These results suggest that a mitochondrion-encoded RM system can function as a toxin–antitoxin selfish element, and that such elements could be co-opted by eukaryotic genomes to drive biased organellar inheritance.

This study reveals that a functional type II restriction modification system of flavobacterial ancestry has been horizontally transferred into the mitochondrion of a marine protist and is capable of encoding potent function, perhaps allowing it to play a role in inter-organellar warfare or protection against further integration of foreign DNA.     

Isolation of a Degeneration-Resistant Mutant of Clostridium acetobutylicum NCIMB 8052

Unless periodically grown from germinated spores, Clostridium acetobutylicum tends to degenerate (that is, to spontaneously lose the capacity both to produce solvents and to develop into spores). To obtain mutants that are deficient in degeneration, C. acetobutylicum NCIMB 8052 was mated with Enterococcus faecalis BM4110 harboring transposon Tn1545. We developed a degeneration resistance assay based on a secondary effect of degeneration, the production of toxic levels of acetic and butyric acids. Erythromycin-resistant transconjugant clones were tested individually for longevity by repeated and timely subculturing. One long-lived mutant, A10, survived 18 ± 3 transfers (mean ± standard deviation; n = 20) before extinction, while the wild type (parental cells) survived 6.6 ± 1.5 transfers (n = 11). The three-fold difference in longevity is statistically significant. In a batch culture in a rich medium, the wild-type cells degenerated within 24 h after inoculation with 1% of an overnight culture derived from germinated spores. In contrast, A10 cells were able to switch to solventogenesis and to sporulate. In a minimal medium with greater buffering capacity, both cell types produced solvents and spores. Southern blots of EcoRI and HindIII restriction digests of A10 chromosomal DNA (but not parental DNA) showed that only one copy of Tn1545 was inserted into the clostridial chromosome. Our findings are consistent with the hypothesis that there was an alteration at a regulatory locus that was effected by the insertion of the transposon.