GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter

The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens.     

Validation of MALDI-TOF MS for rapid classification and identification of lactic acid bacteria, with a focus on isolates from traditional fermented foods in Northern Vietnam

Aims: To evaluate the potential use of MALDI-TOF MS for fast and reliable classification and identification of lactic acid bacteria (LAB) from traditional fermented foods. Methods and Results: A total of 119 strains of LAB from fermented meat (nem chua) were analysed with both (GTG)(5) -PCR fingerprinting and MALDI-TOF MS. Cluster analysis of the profiles revealed five species represented by a single isolate both in (GTG)(5) -PCR and in MALDI-TOF MS; five species grouped alike for (GTG)(5) -PCR and for MALDI-TOF MS; however, differences in minimal similarity between the delineated (GTG)(5) -PCR and MALDI-TOF MS clusters could be observed; three species showed more heterogeneity in their MALDI-TOF MS profiles compared to their (GTG)(5) -PCR profiles; two species, each represented by a single MALDI-TOF cluster, were subdivided in the corresponding (GTG)(5) -PCR dendrogram. As proof of the identification potential of MALDI-TOF MS, LAB diversity from one fermented mustard sample was analysed using MALDI- TOF MS. PheS gene sequencing was used for validation. Conclusions: MALDI-TOF MS is a powerful, fast, reliable and cost-effective technique for the identification of LAB associated with the production of fermented foods. Significance and Impact of the Study: Food LAB can be identified using MALDI-TOF MS, and its application could possibly be extended to other food matrices and/or other food-derived micro-organisms.     

Dimerization of SHBG by gelatin and dithiothreitol. Implications for the measurement of SHBG binding capacity in human serum

In individual serum samples, the sex hormone-binding globulin (SHBG) binding capacity for dihydrotestosterone (DHT) was systematically found to be decreased by 30-60% when either gelatin or dithiothreitol (DTT) was present in the assay buffer. The presence of gelatin in the buffer prevented DTT from further decreasing the SHBG binding capacity of serum samples, suggesting a similar mechanism of action on SHBG for both of these substances. This observation led us to compare the molecular forms of SHBG by high performance liquid chromatography on a TSK G 3000 SW column, in the presence or absence of DTT. When undiluted serum previously incubated with [3H]DHT was chromatographed, only monomeric SHBG could be detected, independently of the presence or absence of DTT in the elution buffer. When the serum was diluted, incubated and chromatographed with buffer devoid of DTT, a dimeric SHBG peak was progressively observed, as a function of the sample dilution. Furthermore, for a given serum dilution, the relative size of the dimeric SHBG peak was also dependent on the steroid concentration present in the sample. By contrast, when serum was diluted, incubated and chromatographed with DTT-supplemented buffer, only the SHBG dimer peak could be detected. These results suggest that in serum, in vitro at least, SHBG is present in its monomeric form. Serum dilution with buffer devoid of DTT or gelatin induces the progressive dimerization of the protein, resulting in a progressive decrease of its apparent binding capacity. This could explain the great discrepancies of SHBG levels as reported in the literature. Because serum dilution with buffer supplemented with DTT or gelatin induces the complete dimerization of SHBG, independently of the sample dilution, we suggest that these substances be routinely used for the measurement of SHBG binding capacity. The SHBG binding capacity obtained in these latter conditions reflects however half the binding capacity of undiluted serum.     

Evaluation of MALDI-TOF MS as a tool for high-throughput dereplication

The present study examined the suitability of matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid grouping of bacterial isolates, i.e. dereplication. Dereplication is important in large-scale isolation campaigns and screening programs since it can significantly reduce labor intensity, time and costs in further downstream analyses. Still, current dereplication techniques are time consuming and costly. MALDI-TOF MS is an attractive tool since it performs fast and cheap analyses with the potential of automation. However, its taxonomic resolution for a broad diversity of bacteria remains largely unknown. To verify the suitability of MALDI-TOF MS for dereplication, a total of 249 unidentified bacterial isolates retrieved from the rhizosphere of potato plants, were analyzed with both MALDI-TOF MS and repetitive element sequence based polymerase chain reaction (rep-PCR). The latter technique was used as a benchmark. Cluster analysis and inspection of the profiles showed that for 204 isolates (82%) the taxonomic resolution of both techniques was comparable, while for 45 isolates (18%) one of both techniques had a higher taxonomic resolution. Additionally, 16S rRNA gene sequence analysis was performed on all members of each delineated cluster to gain insight in the identity and sequence similarity between members in each cluster. MALDI-TOF MS proved to have higher reproducibility than rep-PCR and seemed to be more promising with respect to high-throughput analyses, automation, and time and cost efficiency. Its taxonomic resolution was situated at the species to strain level. The present study demonstrated that MALDI-TOF MS is a powerful tool for dereplication.     

Influence of storage conditions on the growth of Pseudomonas species in refrigerated raw milk

The refrigerated storage of raw milk throughout the dairy chain prior to heat treatment creates selective conditions for growth of psychrotolerant bacteria. These bacteria, mainly belonging to the genus Pseudomonas, are capable of producing thermoresistant extracellular proteases and lipases, which can cause spoilage and structural defects in pasteurized and ultra-high-temperature-treated milk (products). To map the influence of refrigerated storage on the growth of these pseudomonads, milk samples were taken after the first milking turn and incubated laboratory scale at temperatures simulating optimal and suboptimal preprocessing storage conditions. The outgrowth of Pseudomonas members was monitored over time by means of cultivation-independent denaturing gradient gel electrophoresis (DGGE). Isolates were identified by a polyphasic approach. These incubations revealed that outgrowth of Pseudomonas members occurred from the beginning of the dairy chain (farm tank) under both optimal and suboptimal storage conditions. An even greater risk for outgrowth, as indicated by a vast increase of about 2 log CFU per ml raw milk, existed downstream in the chain, especially when raw milk was stored under suboptimal conditions. This difference in Pseudomonas outgrowth between optimal and suboptimal storage was already statistically significant within the farm tank. The predominant taxa were identified as Pseudomonas gessardii, Pseudomonas gessardii-like, Pseudomonas fluorescens-like, Pseudomonas lundensis, Pseudomonas fragi, and Pseudomonas fragi-like. Those taxa show an important spoilage potential as determined on elective media for proteolysis and lipolysis.     

Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria

Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.     

Significance of bile salt hydrolytic activities of lactobacilli

Bile salt hydrolase (BSH) activity was shown to be constitutive and substrate-specific: the BSH isogenic Lactobacillus plantarum wild type (LP80 WT) and BSH overproducing LP80 (pCBH1) strains preferentially hydrolysed glycodeoxycholic acid (GDCA), whereas the hamster Lact. animalis isolates H362 and H364 showed a higher affinity for taurodeoxycholic acid (TDCA). In viability studies in the presence of nutrients, it was demonstrated that GDCA exerted a higher toxicity than TDCA in a pH-dependent manner. This toxicity was inversely proportionate to the BSH activity level of the strains tested, indicating that BSH activity contributed towards bile salt resistance when appropriate nutrients were available. The high toxicity of GDCA relative to TDCA was suggested to be caused by their weak and strong acid properties respectively. It was therefore hypothesized that the protonated form of bile salts exhibited toxicity as it imported protons in the cell. This puts an energy-burden on BSH⁻ lactobacilli which undergo intracellular acidification. BSH⁺ cells primarily protect themselves through the formation of the weaker DCA compound, which can help negate the pH-drop by recapturing and exporting the co-transported proton. However, since DCA is more toxic than its conjugated counterparts, an additional energy-dependent detoxification of DCA is suggested.     

The N-cadherin/catenin complex in colon fibroblasts and myofibroblasts.

Fibroblasts and myofibroblasts were isolated respectively from normal colon mucosa and from colon cancers. Immunostaining with an antibody against alpha-smooth muscle actin (alpha-SMA) of the tissues of origin and of early passage cultures showed equal proportions of alpha-SMA positive myofibroblasts in vivo as in vitro. Immunocytochemistry, immunoprecipitation of metabolically labelled cells followed by Western blotting and RT-PCR of RNA isolates demonstrated the presence of a N-cadherin/catenin complex in both fibroblasts and myofibroblasts. This complex was found preferentially at the cell cell boundaries. Immunocytochemistry and, to a lesser extent, co-immunoprecipitation indicated partial colocalisation of catenins and alpha-SMA. Transforming growth factor beta1 (TGF-beta1) greatly enhanced the expression of alpha-SMA, but left the N-cadherin/catenin complex unaltered. We speculate that the N-cadherin/catenin complex may have different functions in myofibroblasts than in fibroblasts because of its interaction with alpha-SMA.