Phylogenetic assessment of heterotrophic bacteria from a water distribution system using 16S rDNA sequencing.

Determination of a heterotrophic plate count (HPC) for drinking-water samples alone is not enough to assess possible health hazards associated with sudden changes in the bacterial count. Speciation is very crucial to determine whether the population includes pathogens and (or) opportunistic pathogens. Most of the isolates recovered from drinking water samples could not be allocated to a specific phylogenetic branch based on the use of conventional diagnostic methods.

Ligation of arabinogalactan to peptidoglycan in the cell wall of Mycobacterium smegmatis requires concomitant synthesis of the two wall polymers.

To study the late events of cell wall assembly in Mycobacterium smegmatis, specific in vivo radiolabelling of exponentially growing liquid cultures over periods of less than one cell generation were carried out. N-Acetyl-[(14)C]glucosamine was used to label peptidoglycan and [(14)C]glucose to label arabinogalactan and arabinomannan. Over periods of several generations, radioactive cell wall material was turned over as soluble autolysis products into the culture fluid.

'The day of the soft towel?': comparison of the current bed-bathing method with the soft towel bed-bathing method.

The impressions of 200 patients (both medical and surgical) and 200 nursing staff (registered, enrolled and trainee enrolled nurses) in relation to two bed-bathing methods were compared by means of questionnaires and semi-structured interviews. Data regarding costs were obtained from appropriate cost centre managers. The results of the study found the soft towel bed-bathing method to be more cost effective and provide more patient and nurse satisfaction than the current bed-bathing method.

A lead-absorbing protein with superoxide dismutase activity from Streptomyces subrutilus.

A lead binding protein was purified from the culture filtrate of Streptomyces subrutilus P5. The subunit and native molecular weights were estimated to be 28 and 55 kDa, respectively, indicating that the protein was composed of two identical subunits. The inhibition pattern, the metal content analysis and the EPR spectrum confirmed that the protein was a superoxide dismutase containing Fe and Zn (FeZnSOD).

Influence of Bacillus subtilis phoR on cell wall anionic polymers.

In Bacillus subtilis the Pho regulon is controlled by a sensor and regulator protein pair, PhoR and PhoP, that respond to phosphate concentrations. To facilitate studies of the Pho regulon, a strain with an altered PhoR protein was isolated by in vitro mutagenesis. The mutation in this strain (phoR12) leads to the production of a PhoR sensor kinase that, unlike the wild-type, is functionally active in phosphate-replete conditions.

Biosynthesis of the linkage region of the mycobacterial cell wall.

The "core" structure of the cell wall of Mycobacterium and related genera is unique among prokaryotes, consisting of a covalently linked complex of mycolic acids, D-arabinan and D-galactan (mycolylarabinogalactan, mAG), which, in turn, is linked to peptidoglycan via a special linkage unit, -alpha-L-Rhap(1-->3)-D-GlcNAc-P-. Little is known of the biosynthesis of this complex, although it is the site of action of several common anti-tuberculosis drugs. Isolated cell membranes of Mycobacterium smegmatis catalyzed the incorporation of [14C]GlcNAc from UDP-[14C]GlcNAc into two glycolipids (1 and 2) and of [14C]Rha from TDP-[14C]Rha into glycolipid 2.

Region II of the Haemophilus influenzae type be capsulation locus is involved in serotype-specific polysaccharide synthesis.

The central (serotype-specific) Region II of the Haemophilus influenzae Type b capsulation locus cap is 8.3 kb long and contains a cluster of four genes. We show that these genes, designated orf1 to orf4, are involved in the biosynthetic steps required for the formation of the Type b capsular polysaccharide and that orf1 probably encodes a CDP-ribitolpyrophosphorylase.

The capsular turnover product of Staphylococcus aureus strain Smith.

The capsular polysaccharide released from the bacterial surface by cell wall turnover during growth exhibited less size heterogeneity and a higher average molecular mass than the polysaccharide extracted from the cell by treatment with lysostaphin or low pH. Treatment of turnover polysaccharide, radiolabelled by growth of the bacteria in the presence of N-acetyl-[3H]-glucosamine, with muramidase B from Chalaropsis released a low molecular weight product chromatographically identical to the peptidoglycan degradation products released from the peptidoglycan-teichoic acid complex by the same treatment. It is concluded that some or all of the capsular polysaccharide released into the culture fluid during growth is derived from peptidoglycan-linked capsular material, solubilised by cell wall turnover.

Cell wall assembly in Bacillus megaterium: incorporation of new peptidoglycan by a monomer addition process.

The pattern of cross-linking in the peptidoglycan of Bacillus megaterium has been studied by the pulsed addition of radiolabeled diaminopimelic acid. The distribution of label in muropeptides, generated by digestion with Chalaropsis muramidase and separated by high-performance liquid chromatography, stabilized after 0.15 of a generation time.

Turnover of cell surface-bound capsular polysaccharide in Staphylococcus aureus.

The capsular polysaccharide (CPS) of Staphylococcus aureus strain Smith was labelled by growth of bacteria in the presence of radioactive N-acetylglucosamine and was separated from labelled cell wall components by affinity chromatography on wheat germ agglutinin following dissolution of the cells by lysostaphin. The products were partially characterised chemically and immunochemically. Similar labelled components were found in the culture fluid during growth.

Encapsulation of coagulase-negative staphylococci.

It is becoming clear that encapsulation is frequent among coagulase negative staphylococci and is unrelated to the formation of extracellular polysaccharide slime by many strains. Crude slime may contain capsular polysaccharides or proteins, as well as cell wall components, but this is probably the result of cell wall turnover in growing bacteria. As in coagulase-positive staphylococci the capsules confer resistance to phagocytosis and can be regarded as important virulence factors.

Cell wall assembly in Bacillus subtilis: partial conservation of polar wall material and the effect of growth conditions on the pattern of incorporation of new material at the polar caps.

The use of phage SP50 as marker for cell wall containing teichoic acid in Bacillus subtilis showed clear differences in the rates at which new wall material becomes exposed at polar and cylindrical regions of the wall, though the poles were not completely conserved. Following transition from phosphate limitation to conditions that permitted synthesis of teichoic acid, old polar caps fairly rapidly incorporated enough teichoic acid to permit phage binding. Electron microscopy suggested that the new receptor material spread towards the tip of the pole from cylindrical wall so that phages bound to an increasing proportion of the pole area until only the tip lacked receptor.

Cell wall assembly in Bacillus subtilis: visualization of old and new wall material by electron microscopic examination of samples stained selectively for teichoic acid and teichuronic acid.

Uranyl acetate staining of thin sections allowed a distinction to be made between cell wall material that contains teichoic acid and that which contains teichuronic acid. The stain was used to study the pattern of wall assembly in Bacillus subtilis undergoing transitions between growth conditions leading to incorporation of the different anionic polymers. The results showed that new material is incorporated along the inner surface of the cylindrical region of the wall confirming, by a more direct method, results obtained earlier with teichoic acid specific phages.

Activation and inactivation of synthesis of secondary wall polymers in Bacillus subtilis W23.

The progress of activation and inactivation of synthesis of the wall polymers, teichoic acid and teichuronic acid, in response to changes in the phosphate content of the growth medium has been examined using toluenised cells of B. subtilis W23. Activation of teichoic acid synthesis from nucleotide precursors was independent of protein synthesis, but chloramphenicol prevented activation when DL-glycerol 3-phosphate and CTP replaced CDP-glycerol as one of the substrates of the reaction.

Control of synthesis of wall teichoic acid during balanced growth of Bacillus subtilis W23.

Enzymes involved in the synthesis of teichoic acid and its linkage to the wall in Bacillus subtilis W23 were measured in chemostat cultures growing at equilibrium at a dilution rate of 0.2 h-1 in different concentrations of inorganic phosphate. All the enzymes, except teichoic acid glucosyl transferase, which was insensitive to changes in phosphate concentration, were almost undetectable at 0.

Synthesis of teichoic acid by Bacillus subtilis protoplasts.

Protoplasts of Bacillus subtilis W23 readily synthesized ribitol teichoic acid from nucleotide precursors in the surrounding medium. With cytidine diphosphate-ribitol they made poly(ribitol phosphate), presumably attached to lipoteichoic acid carrier; when cytidine diphosphate-glycerol and uridine diphosphate-N-acetylglucosamine were also present a 10-fold increase in the rate of polymer synthesis occurred, and the product contained both the main chain and the linkage unit. Synthesis was inhibited by trypsin or p-chloromercuribenzenesulfonate in the medium, and we concluded that it occurred at the outer surface of the membrane.

The biosynthesis of wall teichoic acid by toluenised cells of Bacillus subtilis W23.

Toluenised cells of Bacillus subtilis W23 synthesized the teichoic acid, poly(ribitol phosphate), from exogenous precursors. The synthesis was dependent on concomitant synthesis of the linkage unit that joins teichoic acid to peptidoglycan. Under conditions that reduced cell autolytic activity, a large proportion of the teichoic acid became linked to the cell wall, independently of peptidoglycan synthesis.

Lipid intermediate in the synthesis of the linkage unit that joins teichoic acid to peptidoglycan in Bacillus subtilis.

Membranes from Bacillus subtilis W23 synthesized a lipid precursor of the linkage unit that attaches teichoic acid to the cell wall. It contained glycerophosphoryl-N-acetylglucosamine, linked through an acid-labile bond to a lipid.

Fractionation studies of the enzyme complex involved in teichoic acid synthesis.

The membrane-bound enzymes participating in the syntheses of the teichoic acid main chain and linkage unit have been solubilized with Triton X-100 and fractionated by sucrose density gradient centrifugation. Two main fractions were obtained: a heavy fraction, containing enzymes effecting synthesis of the main chain attached to the linkage unit, which was associated with only a small amount of lipid, and a light fraction which was rich in prenyl phosphate and catalyzed only linkage-unit synthesis. The separation by density was not based entirely on polypeptide chain length, as some of the shortest chains appeared in the denser fractions and some relatively high-molecular-weight peptides occurred in the lightest fraction.

Attachment of the main chain to the linkage unit in biosynthesis of teichoic acids.

The main chain of teichoic acids can be assembled in cell-free membrane preparations by the transfer of residues from the appropriate nucleotide precursors to an incompletely characterized amphiphilic molecule, lipoteichoic acid carrier (LTC). However, in the cell wall, the main chain is attached to peptidoglycan through a linkage unit which is synthesized independently. It is believed that, in these cell-free systems, lipid intermediates carrying linkage units are also able to accept residues directly from nucleotide precursors to build up the main chain.

Identification of surface proteins of a bacterial membrane using thiolactone-activated polyacrylamide beads.

A new method for the determination of protein accessibility in membranes and membrane fractions using a resin, 'Enzacryl' polythiolactone, is described. Enzacryl polythiolactone is a hydrophilic polymer of acrylamide and acrylamide derivatives with thiolactone ring substituents. The binding of enzymes and proteins to this resin is accomplished very simply by mixing them together in a simple aqueous buffer.