Analysis of protein-protein interactions linked to the formation of a bacterial cytoskeleton in Mycoplasma pneumoniae

Mycoplasma pneumoniae has a cytoskeleton-like structure. Based on genetic evidence, it was proposed that the 1818 amino acids long protein HMW2 plays a central role in both formation of the cytoskeleton-like structure and adherence to its host cell (cytadherence). As gene products of the hmw2 gene (MPN310), two proteins were identified, the full-length protein HMW2 with a molar mass of 216 kDa and a smaller one (HMW2-s) with only 25 kDa. HMW2-s was considered to be the processing product of HMW2, but it could be shown by determining the N-terminus of HMW2-s and by expression studies with an artifical hmw2-s gene in M. pneumoniae that HMW2-s was synthesized by a new internal start within the hmw2 gene but in the same raster as HMW2. This internal expression unit also ensures the transcription of the two genes, MPN311 and MPN312, located immediately downstream. To characterize HMW2, it was expressed in Escherichia coli under various condition and with alternative E. coli strains, but, it was poorly expressed and degraded rapidly independent of the applied conditions. Therefore, it was impossible to isolate enough soluble full-length protein to do biochemical and structural analyses. The proposed function of HMW2 requires its interaction with other proteins of M. pneumoniae. Pilot experiments with the two-hybrid system suggested several candidates. By applying the “pairwise tests”, an internal fragment of HMW2 was found to interact with the C-terminal fragment of HMW1 (MPN447) and with the MPN297 encoded 17-kDa protein. The latter one has not yet been implicated in cytoskeleton formation. In addition, the interaction between the main adhesin P1 with HMW1 and the gene product of MPN297 was established linking indirectly HMW2 to the P1 adhesion complex consisting of at least three proteins: P1, P40 and P90. Further evidence for the interaction between HMW2 and the P1 adhesin (complex) derived from comparative protein analyses of M. pneumoniae WT and the mutant M. pneumoniae A3 (hmw2−). Western blot analyses showed that in M. pneumoniae A3 the turnover rate of the proteins of the P1 complex was significantly higher. This was interpreted as a consequence of the missing binding partner, because without HMW2, the P1 complex can not be formed and inserted properly in the membrane, making those proteins useless for the bacterium. First attempts to isolate protein complexes containing HMW2, to which a TAP tag was fused, were succesful. Twelve genes/ proteins were identified from the purified complexes: MPN015, MPN140 (ORF4 gene product), MPN141 (P1), MPN142 (P90), MPN160, MPN297, MPN392 (PdhB), MPN426 (P115), MPN430 (GAPDH), MPN447 (HMW1), MPN573 (GroEL), MPN665 (EF-Tu). These results confirm the two-hybrid analyses of proteins interacting with HMW2. Eight of them (P1, P90, PdhB, GAPDH, HMW1, GroEL, EF-tu and the gene product of MPN297) were also found in the Triton X-100 insoluble fraction, which contains almost all of the known cytoskeletal proteins including HMW2. Furthermore, the interaction of HMW2 with EF-Tu (elongation factor Tu) and PdhB (pyruvate dehydrogenase E1-beta subunit), of which a subfraction was reported to be surface exposed, provide evidence, that HMW2 might also have an important function in organizing other proteins than cytoskeletal proteins. Finally, a new antiserum against the N-terminal part of HMW2 was generated, which improved the immunocytochemistry and allowed to co-localize HMW2 with the rod structure (co-operation with Dr. Hegermann), which is one of the predominant structures seen in thin sections of M. pneumoniae

Особенности фазово-структурных превращений при отпуске низколегированных сталей для штампов горячего деформирования

Изучены особенности фазовых и структурных превращений при отпуске низколегированных сталей 20ХФ и 15ХСТ, применяемых для штампов горячего деформирования. Установлено, что при продолжительности отпуска (6500С) 60 – 240 минут сталь 15ХСТ, по сравнению со сталью 20ХФ, является более устойчивой к процессам разупрочнения и обеспечивает стабильный уровень твёрдости и ударной вязкости.Метою роботи є аналіз особливостей структурних перетворень при виробництві низьколегованих сталей 20ХВ та 15 ХСТ для штампів гарячого деформувавння. Виявлено особливості фазово-структурних перетворень під час відпускання низьколегованих сталей 20ХФ та 15ХСТ для штампів гарячого деформування. Встановлено, що сталь 15ХСТ є більш стійкою до процесів знеміцнювання, ніж сталь 20ХФ, і забезпечує стабільний рівень твердості та ударної в'язкості при тривалості відпускання (6500С) 60 – 240 хвилин.The features of the phase and structural transformations during tempering of the low-alloyed steels 20CrV and 15CrСТi for hot working dies have been studied. It has been established that the steel 15CrCTi is more resistant to the process of softening than steel 20CrV and provides a stable level of hardness and impact toughness for the duration of tempering (650 C) 60 – 240 minutes

Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.

The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases

Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.

The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases

Burkholderia pseudomallei-absent soil bacterial community results in secondary metabolites that kill this pathogen.

Burkholderia pseudomallei is a Gram-negative bacterium found in soil and the causative agent of a severe disease in humans and animals known as melioidosis. It is intrinsically resistant to many antibiotics and has been reported resistant to the drugs of choice; ceftazidime. Microbial communities in soil in the presence and absence of B. pseudomallei were investigated using metagenomics approach. The variation in bacterial species diversity was significantly higher in soil samples without B. pseudomallei. Abundances of phyla Actinobacteria and Firmicutes were found significantly higher in B. pseudomallei-negative soils. Bacillus amyloliquefaciens KKU1 in phylum Firmicutes was discovered from negative soil and its secondary metabolites could inhibit clinical, environmental and drug resistant isolates of B. pseudomallei, together with some pathogenic Gram-negative but not Gram-positive bacteria. The antimicrobial activity from KKU 1 against B. pseudomallei was abolished when treated with proteinase K, stable in a wide range of pH and remained active after heating at 100 °C for 15 min. Precipitated proteins from KKU1 were demonstrated to cause lysis and corrugated surfaces of B. pseudomallei. The minimum inhibitory concentrations and minimum bactericidal concentrations of the precipitated proteins from KKU1 against B. pseudomallei were 0.97 μg/ml and 3.9 μg/ml. Interestingly, Native SDS-PAGE showed small active compounds of less than 6 kDa, along with other information collectively suggesting the properties of antimicrobial peptides. For the first time, culture-independent information in melioidosis endemic area could lead to a suspected source of metabolites that may help defense against B. pseudomallei and other pathogenic Gram-negative bacteria

THE CONTRIBUTION OF SOIL PHYSICOCHEMICAL PROPERTIES TO THE PRESENCE AND GENETIC DIVERSITY OF BURKHOLDERIA PSEUDOMALLEI.

Burkholderia pseudomallei (Bp), the causative agent of melioidosis, is unevenly distributed in the complex soil environment. Physicochemical factors in the soil have been reported to affect microbial communities in the soil. The effect of physicochemical factors on the number and diversity of organisms in the soil has not been reported. Twenty-five each B. pseudomallei-positive and -negative soil samples were collected from a melioidosis-endemic area. The amount of Bp in each soil sample was measured by culture and quantitative PCR (qPCR). The following physicochemical properties from each soil sample were measured: pH, total organic carbon (TOC), total nitrogen (TN), carbon to nitrogen ratio (C:N ratio), exchangeable calcium (EC) and extractable iron (EI). All the physico- chemical properties measured were significantly different between the Bp-positive and -negative soil samples. The Bp-positive soil samples had lower C:N ratios and lower EC and a higher EI (p < 0.05) than the Bp-negative samples. The average pH was lower (3.7-5.0) in the Bp-negative samples. Among the Bp-positive soil samples, the EC was negatively correlated with the PCR copy number. The amount of bacteria detected with the qPCR method was higher than with the culture method, suggesting the presence of unculturable forms of bacteria that might re-grow when the environmental conditions was suitable. A total of 117 Bp isolates obtained from the soil samples were classified into 25 groups using BOX-PCR. The genetic diversity of Bp, did not correlate with the physicochemical factors investigated. A suitable pH range and C:N ratio may be important for the presence of Bp. The EI supports the needs and EC probably alters the growth of Bp. The genetic diversity of the bacteria was not influenced by the soil factors investigated in this study. This information shows the environment conducive to the growth of Bp. This gives us information about how to potentially control or decrease Bp in the soil in the future

THE CONTRIBUTION OF SOIL PHYSICOCHEMICAL PROPERTIES TO THE PRESENCE AND GENETIC DIVERSITY OF BURKHOLDERIA PSEUDOMALLEI.

Burkholderia pseudomallei (Bp), the causative agent of melioidosis, is unevenly distributed in the complex soil environment. Physicochemical factors in the soil have been reported to affect microbial communities in the soil. The effect of physicochemical factors on the number and diversity of organisms in the soil has not been reported. Twenty-five each B. pseudomallei-positive and -negative soil samples were collected from a melioidosis-endemic area. The amount of Bp in each soil sample was measured by culture and quantitative PCR (qPCR). The following physicochemical properties from each soil sample were measured: pH, total organic carbon (TOC), total nitrogen (TN), carbon to nitrogen ratio (C:N ratio), exchangeable calcium (EC) and extractable iron (EI). All the physico- chemical properties measured were significantly different between the Bp-positive and -negative soil samples. The Bp-positive soil samples had lower C:N ratios and lower EC and a higher EI (p < 0.05) than the Bp-negative samples. The average pH was lower (3.7-5.0) in the Bp-negative samples. Among the Bp-positive soil samples, the EC was negatively correlated with the PCR copy number. The amount of bacteria detected with the qPCR method was higher than with the culture method, suggesting the presence of unculturable forms of bacteria that might re-grow when the environmental conditions was suitable. A total of 117 Bp isolates obtained from the soil samples were classified into 25 groups using BOX-PCR. The genetic diversity of Bp, did not correlate with the physicochemical factors investigated. A suitable pH range and C:N ratio may be important for the presence of Bp. The EI supports the needs and EC probably alters the growth of Bp. The genetic diversity of the bacteria was not influenced by the soil factors investigated in this study. This information shows the environment conducive to the growth of Bp. This gives us information about how to potentially control or decrease Bp in the soil in the future