- Volume 147, Issue 10, 2001

Streptococcus mutans produces a number of extracellular sucrose-metabolizing enzymes that contribute to the ability of the organism to cause dental caries, including three glucosyltransferases, the products of the gtfB, gtfC and gtfD genes, and a fructosyltransferase, encoded by the ftf gene. To better understand the regulation of the expression of these genes under environmental conditions that more closely mimic those in dental plaque, two strains of S. mutans harbouring fusions of the gtfBC (SMS102) and ftf (SMS101) promoters to a chloramphenicol acetyltransferase (CAT) gene were examined in biofilms formed in vitro. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium. CAT specific activity in biofilm cells was measured at quasi-steady state or following additions of 25 mM sucrose or glucose, with or without pH control. After approximately 10 generations of biofilm growth, the ftf and gtfBC genes of S. mutans were found to be expressed at levels different from those reported for planktonic cells growing under otherwise similar conditions. The expression of these genes was induced by the addition of sucrose to the quasi-steady-state cultures. Expression of the gtfBC genes was influenced by environmental pH, since CAT specific activities in quasi-steady-state biofilms of strain SMS102 grown without pH control were twice those produced by cells grown with pH control. Moreover, addition of glucose to quasi-steady-state biofilms resulted in increased expression of the gtfBC–cat fusion, although the magnitude of the induction was less than that seen with sucrose. The effect of pH on ftf expression was negligible. A modest and transient induction of ftf was observed in biofilms pulsed with excess glucose and the kinetics and level of induction of ftf by excess carbohydrate were dependent on the pH of the biofilms. This study demonstrates that the type and amount of carbohydrate and the environmental pH have a major influence on transcription of the gtfBC and ftf genes when the organisms are growing in biofilms, and provides evidence for previously undisclosed regulatory circuits for exopolysaccharide gene expression in S. mutans.

In silico subtractive/differential genome analysis is a powerful approach for identifying genus- or species-specific genes, or groups of genes that are responsible for a unique phenotype. By this method, one searches for genes present in one group of bacteria and absent in another group. A software package has been developed, named FindTarget, that has a user-friendly web interface to facilitate differential genome analysis. The user chooses the genomes to compare, the similarity criteria and the thresholds to decide if a gene has a counterpart in another genome. The searches are based on BLASTP comparisons of proteomes. FindTarget also includes access to sequences, coloured multiple alignments, phylogenetic trees of conserved proteins and links to public annotated databases which provide a means for validation of the results. To illustrate this approach, a FindTarget search for genes putatively involved in the specificity of cell envelope synthesis of Gram-negative bacteria is presented. The results show that most of the identified genes are clearly involved in cell wall processes, underlining the power of such an approach in general and that of FindTarget in particular.

Adherence of Burkholderia cepacia to cells of the respiratory tract of patients with cystic fibrosis (CF) appears to be a necessary precondition for colonization and infection. To date, no effective anti-adhesive strategy has been devised for preventing B. cepacia infection in CF patients. It was found in this study that B. cepacia adhered to respiratory epithelial cells both in vitro and in vivo. However, strains with cable-like pili (Cbl) exhibited the typical clump formation on pneumocytes, whereas non-cable piliated strains predominantly showed single-cell adherence. Dextrans (nominally 4000–10000 Da) significantly inhibited adhesion of B. cepacia to A549 pneumocytes. When compared on an equal weight basis, the nominally 10000 Da dextran was most inhibitory. A dose-dependent inhibitory effect (up to 80 mg ml−1) was observed for most strains. Dextran exerted less of an anti-adhesive effect on the two Cbl+ strains than on the others which were Cbl−. Dextrans appeared to block the adherence in a non-specific fashion, as shown by the observations that the inhibitory effect was readily reversible and oligosaccharides composed of 2–4 glucose units with the same α-1,6 linkage were not inhibitory. The mean molecular masses of dextrans used in this study, as determined by gel filtration and MS, were approximately 10-fold lower than those indicated by the manufacturers. Our data suggest that dextran of nominal molecular mass 4000 Da at a concentration of 40 mg ml−1 (10 mM according to manufacturer’s quoted molecular mass) or more may be useful in patients with CF to prevent colonization and infection with B. cepacia.

Listeria monocytogenes is a pathogen whose distribution in a range of foodstuffs requires the development of methods for sensitive and rapid detection. Molecular biological methods usually rely on specific detection of L. monocytogenes rDNA directly amplified by the application of PCR to DNA extracts. Information on the metabolic status of L. monocytogenes populations would be valuable and can, in theory, be provided by quantitative detection of rRNA itself. Both fluorometry and oligonucleotide probe assays were applied to L. monocytogenes cultures to quantify RNA and DNA and produced more meaningful data than previous estimates for bacteria based on eukaryotic nucleic acid standards. In batch culture, the RNA–DNA ratio was found to be greatest at the end of exponential growth, after which RNA became degraded in accordance with the rapid decrease in viability. When the pH of the medium was controlled at neutrality, culture viability was dramatically extended and although RNA was degraded, intact DNA was maintained for the duration of the experiment. Ribosome numbers per cell were estimated to decrease from about 25000 observed during mid-exponential growth to about 600 during stationary phase, under pH-controlled conditions. Like Escherichia coli, therefore, L. monocytogenes loses viability and rRNA rapidly once exponential growth has ceased in batch culture. However, much improved survival of a culturable L. monocytogenes population when pH is controlled has clear implications for the persistence of this species in buffered environments such as dairy products.

The genetic diversity of Pasteurella multocida, the aetiological agent of fowl cholera, was investigated. The strain collection comprised 69 clinical isolates representing a wide spectrum of hosts and geographic origin. The three type strains for the subspecies of P. multocida were also included. Avian isolates of P. multocida subsp. multocida and P. multocida subsp. septica did not represent separate lines by HpaII ribotyping and the two type strains of mammalian origin (porcine and cat bite) seemed to be representative of avian strains of P. multocida subspp. multocida and septica. By ribotyping, all P. multocida subsp. gallicida strains, except one chicken isolate and the type strain, clustered together. This indicated that the bovine type strain was not representative of this subspecies and that most strains of P. multocida subsp. gallicida are genetically related and may be distantly related to other P. multocida isolates, including those of avian origin. By 16S rRNA and atpD sequence comparisons of selected strains, including both P. multocida isolated from birds and mammals and selected distantly related Pasteurella species associated with birds and mammals, it was found that P. multocida is monophyletic. Extended DNA–DNA hybridizations are highly indicated since strains may exist which would connect the existing subspecies at species level. The considerable genetic diversity of P. multocida fowl cholera isolates is probably related to the clonal nature of this organism, resulting in many divergent lines.