Transcriptional and functional characterization of genetic elements involved in galacto‐oligosaccharide utilization by Bifidobacterium breve UCC2003

Several prebiotics, such as inulin, fructo‐oligosaccharides and galacto‐oligosaccharides, are widely used commercially in foods and there is convincing evidence, in particular for galacto‐oligosaccharides, that prebiotics can modulate the microbiota and promote bifidobacterial growth in the intestinal tract of infants and adults. In this study we describe the identification and functional characterization of the genetic loci responsible for the transport and metabolism of purified galacto‐oligosaccharides (PGOS) by Bifidobacterium breve UCC2003. We further demonstrate that an extracellular endogalactanase specified by several B. breve strains, including B. breve UCC2003, is essential for partial degradation of PGOS components with a high degree of polymerization. These partially hydrolysed PGOS components are presumed to be transported into the bifidobacterial cell via various ABC transport systems and sugar permeases where they are further degraded to galactose and glucose monomers that feed into the bifid shunt. This work significantly advances our molecular understanding of bifidobacterial PGOS metabolism and its associated genetic machinery to utilize this prebiotic.     

Biochemical analysis of cross-feeding behaviour between two common gut commensals when cultivated on plant-derived arabinogalactan

In this paper, we reveal and characterize cross-feeding behaviour between the common gut commensal Bacteroides cellulosilyticus (Baccell) and certain bifidobacterial strains, including Bifidobacterium breve UCC2003, when grown on a medium containing Larch Wood Arabinogalactan (LW-AG). We furthermore show that cross-feeding is dependent on the release of β-1,3-galacto-di/trisaccharides (β-1,3-GOS), and identified that the bga gene cluster of B. breve UCC2003 allows β-1,3-GOS metabolism. The product of bgaB is presumed to be responsible for the import of β-1,3-GOS, while the bgaA gene product, a glycoside hydrolase family 2 member, was shown to hydrolyse both β-1,3-galactobiose and β-1,3-galactotriose into galactose monomers. This study advances our understanding of strain-specific syntrophic interactions between two glycan degraders in the human gut in the presence of AG-type dietary polysaccharides.     

A Bifidobacterial pilus‐associated protein promotes colonic epithelial proliferation

Development of the human gut microbiota commences at birth, with certain bifidobacterial species representing dominant and early colonisers of the newborn gastrointestinal tract. The molecular basis of Bifidobacterium colonisation, persistence and presumed communication with the host has remained obscure. We previously identified tight adherence (Tad) pili from Bifidobacterium breve UCC2003 as an essential colonisation factor. Here, we demonstrate that bifidobacterial Tad pili also promote in vivo colonic epithelial proliferation. A significant increase in cell proliferation was detectable 5 days postadministration of B. breve UCC2003. Using advanced functional genomic approaches, bacterial strains either (a) producing the Tad₂₀₀₃ pili or (b) lacking the TadE or TadF pseudopilins were created. Analysis of the ability of these mutant strains to promote epithelial cell proliferation in vivo demonstrated that the pilin subunit, TadE, is the bifidobacterial molecule responsible for this proliferation response. These findings were confirmed in vitro using purified TadE protein. Our data imply that bifidobacterial Tad pili may contribute to the maturation of the naïve gut in early life through the production of a specific scaffold of extracellular protein structures, which stimulate growth of the neonatal mucosa.     

Molecular analysis of the replication functions of the bifidobacterial conjugative megaplasmid pMP7017

pMP7017 is a conjugative megaplasmid isolated from the gut commensal Bifidobacterium breve JCM7017 and was shown to encode two putative replicases, designated here as RepA and RepB. In the current work, RepB was identified as the pMP7017 replicative initiator, as the repB gene, and its surrounding region was shown to be sufficient to allow autonomous replication in two bifidobacterial species, B. breve and Bifidobacterium longum subsp. longum. RepB was shown to bind to repeat sequence downstream of its coding sequence and this region was determined to be essential for efficient replication. Based on our results, we hypothesize that pMP7017 is an iteron-regulated plasmid (IRP) under strict auto-regulatory control. Recombinantly produced and purified RepB was determined to exist as a dimer in solution, differing from replicases of other IRPs, which exist as a mix of dimers and monomers. Furthermore, a stable low-copy Bifidobacterium-E. coli shuttle vector, pRD1.3, was created which can be employed for cloning and expression of large genes, as was demonstrated by the cloning and heterologous expression of the 5.1 kb apuB gene encoding the extracellular amylopullulanase from B. breve UCC2003 into B. longum subsp. longum NCIMB8809.     

Nucleotide sequence and genetic organization of the NgoPII restriction-modification system of Neisseria gonorrhoeae

Summary The NgoPII restriction endonuclease, which recognizes the sequence 5-GGCC-3, differs from its isoschizomer HaeIII in being sensitive to methylation at the external cytosine residue. The entire nucleotide sequence of a cloned 3.3 kb segment of Neisseria gonorrhoeae strain P9 chromosomal DNA which harbours the NgoPII restriction-modification system has been determined. This data, coupled with sub-cloning experiments, indicates that the restriction endonuclease (R.NgoII) and modification (M.NgoII) genes are transcribed from separate promoters but are arranged in tandem, with the R.NgoPII gene being located on the 5 side of the M.NgoPII gene. Unlike all previously reported restriction systems the 3 end of the endonuclease open reading frame overlaps the 5 end of the methylase open reading frame by 8 codons. This overlap may have implications for the regulation of the NgoPII restriction-modification system.     

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.     

Cloning of two type II methylase genes that recognise asymmetric nucleotide sequences: FokI and HgaI

Summary The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI.The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI.Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.     

Production of SacI and SacII isoschizomers by soil streptomycetes

Five fresh soil Streptomyces spp. strains were isolated, phylogenetically characterized on the basis of 16S rDNA sequences and analyzed for the presence of restriction modification systems. Three type II site-specific endonucleases were detected and partially purified. Two isolated enzymes were isoschizomers of SacI restriction endonuclease recognizing 5′-GAGCTC-3′ sequence; the third one recognised 5′-CCGCGG-3′ sequence and it was an isoschizomer of SacII. SacII like modification was observed in other two isolates having no detectable restriction activity. The lack of correlation between restriction and modification phenotypes and phylogenetic classification of the isolates indicates efficient gene transfer mechanism in the Streptomyces genus.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

Characterization of DNA restriction and modification activities in Neisseria species

Type II restriction endonuclease activities detected in various Neisseria species were characterized for sequence specificity and precise site of cleavage. NsiCI isolated from N. sicca C351 cleaves the sequence 5′-GAT↓ATC-3′ (EcoRV isoschizomer); NmeCI from N. meningitidis C114 and NphI from N. pharyngis C245 cleave 5′-N↓GATCN-3′ (MboI isoschizomers); NgoPII and NgoPIII from N. gonorrhoeae P9-2 cleave at 5′-CC↓GCGG-3′ (SacII isoschizomer) and 5′-GG↓CC-3′ (HaeIII isoschizomer), respectively. Chromosomal DNA isolated from these strains and two other N. meningitidis strains (which lacked detectable endonuclease activities), was found to be refractive to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases.     

Isolation and characterization of the restriction endonucleasePpel fromPhormidium persicinum

PpeI is a type II restriction endonuclease isolated from cyanobacterial strainPhormidium persicinum. The endonuclease PpeI, an isoschizomer of ApaI, recognizes the hexanucleotide sequence (5’t-GGGCC/C-3’t) and cleaves, after the second C, producing four nucleotide 3’t-cohesive ends.     

Cloning the BamHI restriction modification system.

BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.     

Antirestriction Activity of ArdA Encoded by the IncI1 Transmissive Plasmid R64

The transmissive plasmid R64 (IncI1) performs an antirestriction function, reducing the efficiency of EcoKI-dependent restriction in Escherichia coli K12 cells approximately fivefold. The R64 ardA gene has been cloned and sequenced. The ArdA proteins specifically inhibit type I restriction–modification enzymes. R64 ArdA is highly homologous to ColIb-P9 ArdA: only 4 out of 166 amino acid residues differ. While ColIb-P9 inhibits both endonuclease and methylase activities of the type I restriction–modification enzyme EcoKI (R₂M₂S), R64 ArdA inhibits only its endonuclease activity. It has been assumed that R64 ArdA suppresses the binding of unmodified DNA with the R subunit, which is responsible for DNA translocation and cleavage. ColIb-P9 ArdA suppresses DNA binding not only with the R, but also with the S subunit, which contacts the sK site containing target adenines. The binding of ArdA with the specific site inhibits both endonuclease and methylase activities; the binding of ArdA with the nonspecific site of the R subunit inhibits only the endonuclease activity ofEcoKI (R₂M₂S).     

The restriction modification system of Bacillus licheniformis MS1 and generation of a readily transformable deletion mutant

Restriction modification systems (R-M systems), consisting of a restriction endonuclease and a cognate methyltransferase, constitute an effective means of a cell to protect itself from foreign DNA. Identification, characterization, and deletion of the restriction modification system BliMSI, a putative isoschizomer of ClaI from Caryophanon latum, were performed in the wild isolate Bacillus licheniformis MS1. BliMSI was produced as recombinant protein in Escherichia coli, purified, and in vitro analysis demonstrated identical restriction endonuclease activity as for ClaI. A recombinant E. coli strain, expressing the heterologous bliMSIM gene, was constructed and used as the host for in vivo methylation of plasmids prior to their introduction into B. licheniformis to improve transformation efficiencies. The establishment of suicide plasmids in the latter was rendered possible. The subsequent deletion of the restriction endonuclease encoding gene, bliMSIR, caused doubled transformation efficiencies in the respective mutant B. licheniformis MS2 (∆bliMSIR). Along with above in vivo methylation, the establishment of further gene deletions (∆upp, ∆yqfD) was performed. The constructed triple mutant (∆bliMSIR, ∆upp, ∆yqfD) enables rapid genome manipulation, a requirement for genetic engineering of industrially important strains.

     

Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extract. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5-G/GNCC-3) in this site to generate a three base 5 overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10³ viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.     

Restriction Endonuclease Sst12I from a Strain of Streptomyces Recognizing the Nucleotide Sequence 5"-CTGCAG-3"

A new restriction endonuclease Sst12I belonging to type II and recognizing the sequence 5"-CTGCAG-3" was isolated from the bacterial strain Streptomycessp. St-12. The enzyme hydrolyzes DNA between adenine and guanine residues; thus, it is a true isoschizomer of restrictase PstI. In contrast to PstI, the restriction endonuclease Sst12I hydrolyses DNA both at 37 and 55°C and remains active after long-term storage.