Sharon J. Reid

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.

A Clostridium acetobutylicum regulator gene (regA) affecting amylase production in Bacillus subtilis

Plasmid pMET7C containing a 6.05 kb DNA insert from Clostridium acetobutylicum P262 made Escherichia coli F19 cells sensitive to metronidazole. The nucleotide sequence of the C. acetobutylicum DNA controlling metronidazole sensitivity in E. coli F19 revealed an ORF of 972 bp which encoded a protein of 324 amino acids with a calculated Mr of 35,000. The amino acid sequence encoded by the ORF contained a helix-turn-helix DNA-binding domain and was homologous to the catabolite control protein, CcpA, from Bacillus subtilis and Bacillus megaterium, a tRNA repressor of E. coli encoded by the shl gene, and the GalR, Lacl and PurR repressors of E. coli. The C. acetobutylicum ORF, which was termed regA, complemented a B. subtilis ccpA mutant and an E. coli shl mutant, but was unable to complement E. coli galR, lacl or purR mutants. To determine whether the regA gene product was involved in the regulation of amylase gene expression in C. acetobutylicum, a starch-degrading enzyme gene (staA) from C. acetobutylicum NCIMB 8052 was cloned. The RegA protein inhibited the degradation of starch by the C. acetobutylicum staA gene product in E. coli.

The genes controlling sucrose utilization in Clostridium beijerinckii NCIMB 8052 constitute an operon

The sucrose operon of Clostridium beijerinckii NCIMB 8052 comprises four genes, which encode a sucrose-specific enzyme IIBC(Scr) protein of the phosphotransferase system (ScrA), a transcriptional repressor (ScrR), a sucrose hydrolase (ScrB) and an ATP-dependent fructokinase (ScrK). The scrARBK operon was cloned in Escherichia coli in three stages. Initial isolation was achieved by screening a C. beijerinckii genomic library in E. coli for clones able to utilize sucrose, while the remainder of the operon was isolated by inverse PCR and by plasmid rescue of flanking regions from a scrB mutant constructed by targeted gene disruption. Substrate specificity assays confirmed that the sucrose hydrolase was a beta-fructofuranosidase, able to hydrolyse sucrose and raffinose but not inulin or levans, and that the scrK gene encoded an ATP/Mg2+-dependent fructokinase. Both enzyme activities were induced by sucrose in C. beijerinckii. Disruption of the scr operon of C. beijerinckii by targeted plasmid integration into either the scrR or the scrB gene resulted in strains unable to utilize sucrose, indicating that this was the only inducible sucrose catabolic pathway in this organism. RNA analysis confirmed that the genes of the scr operon were co-transcribed on a 5 kb mRNA transcript and that transcription was induced by sucrose, but not by glucose, fructose, maltose or xylose. Primer extension experiments identified the transcriptional start site as lying 44 bp upstream of the scrA ATG start codon, immediately adjacent to the imperfect pelindrome sequence proposed to be a repressor binding site. Disruption of the scrR gene resulted in constitutive transcription of the upstream scrA gene, suggesting that ScrR encodes a transcriptional repressor which acts at the scrA operator sequence. The scrR gene is therefore itself negatively autoregulated as part of the polycistronic scrARBK mRNA

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.

Transcriptional regulation of an endoglucanase and a cellodextrinase gene in Ruminococcus flavefaciens FD-1

A gene which encodes a 35 kDa protein with both carboxymethylcellulase and xylanase activity was cloned from Ruminococcus flavefaciens FD-1 and the nucleotide sequence determined. The FD-1 gene, celE, and the celA gene, which encodes a cellodextrinase, were used as probes to analyse transcription in R. flavefaciens grown under different conditions. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose, although the effect was not as pronounced as induction by cellulose and was greater for the celA gene than for the celE gene.

The development of a flagellin surface display expression system in a moderate thermophile, Bacillus halodurans Alk36

This study relates to the development of an alkaliphilic, thermotolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium continuously over-produces flagellin. To harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene, which codes for flagellin, was inactivated on the chromosome giving rise to the B. halodurans BhFC01 mutant. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-1 subtype C gp120 epitope were, respectively, incorporated into this site as in-frame fusions. The peptides were found to be successfully displayed on the cell surface and functional through metal binding and immunological studies, respectively.

A comparison of Clostridium difficile diagnostic methods for identification of local strains in a South African centre.

Accurate diagnosis of Clostridium difficile infection is essential for disease management. A clinical and molecular analysis of C. difficile isolated from symptomatic patients at Groote Schuur Hospital, South Africa, was conducted to establish the most suitable clinical test for the diagnosis and characterization of locally prevalent strains. C. difficile was detected in stool samples using enzyme-based immunoassays (EIA) and nucleic acid amplification methods, and their performance was compared with that of C. difficile isolation using direct selective culture combined with specific PCR to detect the C. difficile tpi gene, toxin A and B genes and binary toxin genes. Toxigenic isolates were characterized further by ribotyping. Selective culture isolated 32 C. difficile strains from 145 patients (22 %). Of these, the most prevalent (50 %) were of ribotype 017 (toxin A- B+) while 15.6 % were ribotype 001 (toxin A+B+). No ribotype 027 strains or binary toxin genes (cdtA and cdtB) were detected. The test sensitivities and specificities, respectively, of four commercial clinical diagnostic methods were as follows: ImmunoCard Toxins A & B (40 % and 99.1 %), VIDAS C. difficile Toxin A & B (50 % and 99.1 %), GenoType CDiff (86.7 % and 88.3 %) and Xpert C. difficile (90 % and 97.3 %). Ribotype 001 and 017 strains had a 100 % detection rate by Xpert C. difficile, 100 % and 93.3 % by GenoType CDiff, 75 % and 53.3 % by ImmunoCard and 75 % and 60 % by VIDAS, respectively. The overall poor performance of EIA suggests that a change to PCR-based testing would assist diagnosis and ensure reliable detection of locally prevalent C. difficile 017 strains.

Prevalence of gastrointestinal pathogenic bacteria in patients with diarrhoea attending Groote Schuur Hospital, Cape Town, South Africa

BACKGROUND Diarrhoea due to gastrointestinal infections is a significant problem facing the South African (SA) healthcare system. Infections can be acquired both from the community and from the hospital environment itself, the latter acting as a reservoir for potential pathogenic bacteria. OBJECTIVES To examine the prevalence of a panel of potential diarrhoea-causing bacteria in patients attending a tertiary healthcare facility in Cape Town, SA. METHODS Polymerase chain reaction (PCR) primers specific for Clostridium difficile, Shigella spp., Salmonella spp., Klebsiella oxytoca, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC), Staphylococcus aureus, enterotoxigenic Bacteroides fragilis and Campylobacter spp. were used to screen total bacterial genomic DNA extracted from stool samples provided by 156 patients with diarrhoea attending Groote Schuur Hospital, Cape Town, SA. RESULTS C. difficile was the most frequently detected pathogen (16% of cases) in the 21-87-year-old patient range, but was not present in samples from the 16-20-year-old range. K. oxytoca (6%), EPEC/EHEC strains (9%) and S. aureus (6%) were also detected. The remaining pathogens were present at low frequencies (0-2.9%), and the occurrence of mixed infections was 5%. The majority of non-C. difficile-related diarrhoeas were community acquired. CONCLUSION C. difficile was the main cause of infectious diarrhoea in the sampled patients, while K. oxytoca and EPEC/EHEC strains were present as relatively minor but potentially significant pathogens.