Valerie R. Abratt

Sucrose utilisation in bacteria: genetic organisation and regulation

Sucrose is the most abundant disaccharide in the environment because of its origin in higher plant tissues, and many Eubacteria possess catalytic enzymes, such as the sucrose-6-phosphate hydrolases and sucrose phosphorylases, that enable them to metabolise this carbohydrate in a regulated manner. This review describes the range of gene architecture, uptake systems, catabolic activity and regulation of the sucrose-utilisation regulons that have been reported in the Eubacteria to date. Evidence is presented that, although there are many common features to these gene clusters and high conservation of the proteins involved, there has been a certain degree of gene shuffling. Phylogenetic analyses of these proteins supports the hypothesis that these clusters have been acquired through horizontal gene transfer via mobile elements and transposons, and this may have enabled the recipient bacteria to colonise sucrose-rich environmental niches.

Induction of Sucrose Utilization Genes from Bifidobacterium lactis by Sucrose and Raffinose

ABSTRACT The probiotic organism Bifidobacterium lactis was isolated from a yoghurt starter culture with the aim of analyzing its use of carbohydrates for the development of prebiotics. A sucrose utilization gene cluster of B. lactis was identified by complementation of a gene library in Escherichia coli. Three genes, encoding a sucrose phosphorylase (ScrP), a GalR-LacI-type transcriptional regulator (ScrR), and a sucrose transporter (ScrT), were identified by sequence analysis. The scrP gene was expressed constitutively from its own promoter in E. coli grown in complete medium, and the strain hydrolyzed sucrose in a reaction that was dependent on the presence of phosphates. Primer extension experiments with scrP performed by using RNA isolated from B. lactis identified the transcriptional start site 102 bp upstream of the ATG start codon, immediately adjacent to a palindromic sequence resembling a regulator binding site. In B. lactis, total sucrase activity was induced by the presence of sucrose, raffinose, or oligofructose in the culture medium and was repressed by glucose. RNA analysis of the scrP, scrR, and scrT genes in B. lactis indicated that expression of these genes was influenced by transcriptional regulation and that all three genes were similarly induced by sucrose and raffinose and repressed by glucose. Analysis of the sucrase activities of deletion constructs in heterologous E. coli indicated that ScrR functions as a positive regulator.

Twenty-eight divergent polysaccharide loci specifying within- and amongst-strain capsule diversity in three strains of Bacteroides fragilis

Comparison of the complete genome sequence of Bacteroides fragilis 638R, originally isolated in the USA, was made with two previously sequenced strains isolated in the UK (NCTC 9343) and Japan (YCH46). The presence of 10 loci containing genes associated with polysaccharide (PS) biosynthesis, each including a putative Wzx flippase and Wzy polymerase, was confirmed in all three strains, despite a lack of cross-reactivity between NCTC 9343 and 638R surface PS-specific antibodies by immunolabelling and microscopy. Genomic comparisons revealed an exceptional level of PS biosynthesis locus diversity. Of the 10 divergent PS-associated loci apparent in each strain, none is similar between NCTC 9343 and 638R. YCH46 shares one locus with NCTC 9343, confirmed by mAb labelling, and a second different locus with 638R, making a total of 28 divergent PS biosynthesis loci amongst the three strains. The lack of expression of the phase-variable large capsule (LC) in strain 638R, observed in NCTC 9343, is likely to be due to a point mutation that generates a stop codon within a putative initiating glycosyltransferase, necessary for the expression of the LC in NCTC 9343. Other major sequence differences were observed to arise from different numbers and variety of inserted extra-chromosomal elements, in particular prophages. Extensive horizontal gene transfer has occurred within these strains, despite the presence of a significant number of divergent DNA restriction and modification systems that act to prevent acquisition of foreign DNA. The level of amongst-strain diversity in PS biosynthesis loci is unprecedented.

Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.

Bacteroides fragilis RecA protein overexpression causes resistance to metronidazole

Bacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with metronidazole (Mtz), a DNA damaging agent. This study examined the role of the DNA repair protein, RecA, in maintaining endogenous DNA stability and its contribution to resistance to Mtz and other DNA damaging agents. RT-PCR of B. fragilis genomic DNA showed that the recA gene was co-transcribed as an operon together with two upstream genes, putatively involved in repairing oxygen damage. A B. fragilis recA mutant was generated using targeted gene inactivation. Fluorescence microscopy using DAPI staining revealed increased numbers of mutant cells with reduced intact double-stranded DNA. Alkaline gel electrophoresis of the recA mutant DNA showed increased amounts of strand breaks under normal growth conditions, and the recA mutant also showed less spontaneous mutagenesis relative to the wild type strain. The recA mutant was sensitive to Mtz, ultraviolet light and hydrogen peroxide. A B. fragilis strain overexpressing the RecA protein exhibited increased resistance to Mtz compared to the wild type. This is the first study to show that overexpression of a DNA repair protein in B. fragilis increases Mtz resistance. This represents a novel drug resistance mechanism in this bacterium.

The Bifidobacterium longum NCIMB 702259 T ctr Gene Codes for a Novel Cholate Transporter

ABSTRACT Preexposure of Bifidobacterium longum NCIMB 702259T to cholate caused increased resistance to cholate, chloramphenicol, and erythromycin. The B. longum ctr gene, encoding a cholate efflux transporter, was transformed into the efflux-negative mutant Escherichia coli KAM3, conferring resistance to bile salts and other antimicrobial compounds and causing the efflux of [14C]cholate.

A functional analysis of the Bifidobacterium longum cscA and scrP genes in sucrose utilization

The role of genes involved in sucrose catabolism was investigated with a view to designing effective prebiotic substrates to encourage the growth of Bifidobacterium in the gut. Two gene clusters coding for sucrose utilisation in Bifidobacterium longum NCC2705 were identified in the published genome. The genes encoding putative sucrose degrading enzymes, namely, the scrP (sucrose phosphorylase) and the cscA (β-fructofuranosidase), were cloned from B. longum NCIMB 702259T and expressed in Escherichia coli DH5α. Both complemented the sucrase negative phenotype of untransformed cells and showed specific sucrase activity. Transcriptional analysis of the expression of the genes in B. longum grown in the presence of various carbohydrate substrates showed induction of scrP gene expression in the presence of sucrose and raffinose, but not in the presence of glucose. The cscA gene showed no increased transcription in B. longum grown in the presence of any of the carbohydrates tested. Phylogenetic analysis indicates that the B. longum CscA protein belongs to a distinct phylogenetic cluster of intracellular fructosidases, which specifically cleave the shorter fructose oligosaccharides.

Two Multidrug-Resistant Clinical Isolates of Bacteroides fragilis Carry a Novel Metronidazole Resistance nim Gene (nimJ)

ABSTRACT Two multidrug-resistant Bacteroides fragilis clinical isolates contain and express a novel nim gene, nimJ, that is not recognized by the “universal” nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. HMW615, an appendiceal isolate, contains at least two copies of nimJ on its genome, while HMW616, an isolate from a patient with sepsis, contains one genomic copy of nimJ. B. fragilis NimJ is phylogenetically closer to Prevotella baroniae NimI and Clostridium botulinum NimA than to the other known Bacteroides Nim proteins. The predicted protein structure of NimJ, based on fold recognition analysis, is consistent with the crystal structures derived for known Nim proteins, and specific amino acid residues important for substrate binding in the active site are conserved. This study demonstrates that the “universal” nim primers will not detect all nim genes with the ability to confer metronidazole resistance, but nimJ alone cannot account for the very high metronidazole MICs of these resistant clinical isolates.

Crystal Structure of Type III Glutamine Synthetase: Surprising Reversal of the Inter-Ring Interface

Glutamine synthetases are ubiquitous, homo-oligomeric enzymes essential for nitrogen metabolism. Unlike types I and II, which are well described both structurally and functionally, the larger, type IIIs are poorly characterized despite their widespread occurrence. An understanding of the structural basis for this divergence and the implications for design of type-specific inhibitors has, therefore, been impossible. The first crystal structure of a GSIII enzyme, presented here, reveals a conservation of the GS catalytic fold but subtle differences in protein-ligand interactions suggest possible avenues for the design GSIII inhibitors. Despite these similarities, the divergence of the GSIII enzymes can be explained by differences in quaternary structure. Unexpectedly, the two hexameric rings of the GSIII dodecamer associate on the opposite surface relative to types I and II. The diversity of GS quaternary structures revealed here suggests a nonallosteric role for the evolution of the double-ringed architecture seen in all GS enzymes.

Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron

Objectives The aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance. Methods The genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays. Results A metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose. Conclusions These data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron