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. The present study had to use phylogenetic analysis, which was simplified by determining and using the first 500-bp sequence of the 16S rDNA, to successfully identify the type and species of bacteria found in the samples. Gram-positive bacteria alpha-, beta-, and gamma-Proteobacteria were found to be the major groups representing the heterotrophic bacteria in drinking water. The study also revealed that the presence of sphingomonads in drinking water supplies may be much more common than has been reported so far and thus further studies are merited. The intermittent mode of supply, mainly characterized by water stagnation and flow interruption associated possibly with biofilm detachment, raised the possibility that the studied bacterial populations in such systems represented organisms coming from 2 different niches, the biofilm and the water column.

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. However, turnover of newly synthesized and labelled cell wall was delayed for about one cell generation, implying inside-to-outside growth of the wall as observed in Bacillus. Little radioactive wall material was released into the culture fluid during the first generation of labelling in growing cultures, but the addition of amoxicillin plus the beta-lactamase inhibitor clavulanic acid, at the minimum inhibitory concentration of amoxicillin, led to the release of radioactive peptidoglycan that could be isolated by gel filtration chromatography and contained nearly 3 mol alanine per glutamic acid residue, indicating that it was linear, un-crosslinked peptidoglycan that had never been substantially cross-linked to the cell wall due to inhibition of transpeptidation by amoxicillin. This peptidoglycan had no covalently attached arabinogalactan. Radioactive arabinogalactan was synthesized and released from the amoxicillin-treated bacteria without attachment to peptidoglycan. The results indicate that during growth, incorporation of arabinogalactan into the cell wall requires its ligation to newly synthesized peptidoglycan and that the peptidoglycan must be undergoing concomitant cross-linking to the inner surface of the cell wall. Inhibition of peptidoglycan transpeptidation prevents ligation of arabinogalactan to peptidoglycan and its consequent incorporation into the wall.

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. The lesion in PhoR12 was shown to be a single base change that results in an Arg to Ser substitution in a region of PhoR that is highly conserved in histidine sensor kinases. While a phoR-negative mutant was unable to induce the synthesis of cell wall teichuronic acid under phosphate-limited conditions, the phoR12 mutant showed a relative increase in teichuronic acid and a decrease in teichoic acid, even under phosphate-replete conditions. The latter suggests that some or all of the genes required for teichuronic acid synthesis are members of the Pho regulon.