The fundamental question in this study is concerned with whether the increase of unsaturated fatty acids in the cell membrane is a general response of certain thermotolerant strains or species when exposed to superoptimal temperatures, and in combination with other stresses, especially oxidative stress. A strain of Lactobacillus helveticus, a species widely used as a starter in the dairy industry and able to tolerate high temperature and NaCl concentrations as well as acidic conditions, was chosen for this study. Cells of strain CNBL 1156, grown in its natural medium (i.e. milk whey), were exposed for 100 min to sublethal combinations of temperature, NaCl, H2O2 and pH, modulated according to a Central Composite Design. The fatty acid composition of cell lipid extract was identified by GC/MS. Polynomial equations, able to describe the individual interactive and quadratic effects of the independent variables on cell fatty acid composition, were obtained. The results and the mathematical models relative to the individual fatty acids indirectly suggest that desaturase activation or hyperinduction play an important role in the response to heat stress. In fact, the relative proportions of oleic, linoleic and palmitic acids increased with temperature in a range between 38 and 54 °C. The fatty acid profiles included vernolic acid (up to 37% of total fatty acids), an epoxide of linoleic acid not previously reported in microbial cells. In particular, this epoxide was present in cells exposed to low pH in combination with high temperatures and oxidative stress. In conclusion, these results provide experimental support to the hypothesis that the increase of an oxygen-consuming desaturase system, with a consequent increase in fatty acid desaturation, is a cellular response to environmental stresses able to protect the cells of this anaerobic micro-organism from toxic oxygen species and high temperatures.

P.S. COCCONCELLI, D. PORRO, S. GALANDINI AND L. SENINI. 1995. A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.

The novel multi-million read generating sequencing technologies are very promising for resolving the immense soil 16S rRNA gene bacterial diversity. Yet they have a limited maximum sequence length screening ability, restricting studies in screening DNA stretches of single 16S rRNA gene hypervariable (V) regions. The aim of the present study was to assess the effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. Using an in silico approach, the performance of each V region was compared with the complete 16S rRNA gene stretch. We assessed related properties of the soil derived bacterial sequence collection of the Ribosomal Database Project (RDP) database and concomitantly performed simulations based on published datasets. Results indicate that overall the most prominent V region for soil bacterial diversity studies was V3, even though it was outperformed in some of the tests. Despite its high performance during most tests, V4 was less conserved along flanking sites, thus reducing its ability for bacterial diversity coverage. V5 performed well in the non-redundant RDP database based analysis. However V5 did not resemble the full-length 16S rRNA gene sequence results as well as V3 and V4 did when the natural sequence frequency and occurrence approximation was considered in the virtual experiment. Although, the highly conserved flanking sequence regions of V6 provide the ability to amplify partial 16S rRNA gene sequences from very diverse owners, it was demonstrated that V6 was the least informative compared to the rest examined V regions. Our results indicate that environment specific database exploration and theoretical assessment of the experimental approach are strongly suggested in 16S rRNA gene based bacterial diversity studies.

Mutations in the genes encoding enzymes responsible for the incorporation of d -Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase ( alr ) mutant is strictly dependent on an external supply of d -Ala to be able to synthesize peptidoglycan and to incorporate d -Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when d -Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of d -Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the d -alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that d -alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis , LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking d -Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis , however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, d -Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis . Second, reduced amounts of d -Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.