Cell envelope growth of Gram-negative bacteria proceeds independently of cell wall synthesis.

All bacterial cells must expand their envelopes during growth. The main load-bearing and shape-determining component of the bacterial envelope is the peptidoglycan cell wall. Bacterial envelope growth and shape changes are often thought to be controlled through enzymatic cell wall insertion. We investigated the role of cell wall insertion for cell shape changes during cell elongation in Gram-negative bacteria. We found that both global and local rates of envelope growth of Escherichia coli remain nearly unperturbed upon arrest of cell wall insertion-up to the point of sudden cell lysis. Specifically, cells continue to expand their surface areas in proportion to biomass growth rate, even if the rate of mass growth changes. Other Gram-negative bacteria behave similarly. Furthermore, cells plastically change cell shape in response to differential mechanical forces. Overall, we conclude that cell wall-cleaving enzymes can control envelope growth independently of synthesis. Accordingly, the strong overexpression of an endopeptidase leads to transiently accelerated bacterial cell elongation. Our study demonstrates that biomass growth and envelope forces can guide cell envelope expansion through mechanisms that are independent of cell wall insertion.

Bacterial growth - from physical principles to autolysins.

For bacteria to increase in size, they need to enzymatically expand their cell envelopes, and more concretely their peptidoglycan cell wall. A major task of growth is to increase intracellular space for the accumulation of macromolecules, notably proteins, RNA, and DNA. Here, we review recent progress in our understanding of how cells coordinate envelope growth with biomass growth, focusing on elongation of rod-like bacteria. We first describe the recent discovery that surface area, but not cell volume, increases in proportion to mass growth. We then discuss how this relation could possibly be implemented mechanistically, reviewing the role of envelope insertion for envelope growth. Since cell-wall expansion requires the well-controlled activity of autolysins, we finally review recent progress in our understanding of autolysin regulation.

The role of cell-envelope synthesis for envelope growth and cytoplasmic density in Bacillus subtilis.

All cells must increase their volumes in response to biomass growth to maintain intracellular mass density within physiologically permissive bounds. Here, we investigate the regulation of volume growth in the Gram-positive bacterium Bacillus subtilis. To increase volume, bacteria enzymatically expand their cell envelopes and insert new envelope material. First, we demonstrate that cell-volume growth is determined indirectly, by expanding their envelopes in proportion to mass growth, similarly to the Gram-negative Escherichia coli, despite their fundamentally different envelope structures. Next, we studied, which pathways might be responsible for robust surface-to-mass coupling: We found that both peptidoglycan synthesis and membrane synthesis are required for proper surface-to-mass coupling. However, surprisingly, neither pathway is solely rate-limiting, contrary to wide-spread belief, since envelope growth continues at a reduced rate upon complete inhibition of either process. To arrest cell-envelope growth completely, the simultaneous inhibition of both envelope-synthesis processes is required. Thus, we suggest that multiple envelope-synthesis pathways collectively confer an important aspect of volume regulation, the coordination between surface growth, and biomass growth.

Robust surface-to-mass coupling and turgor-dependent cell width determine bacterial dry-mass density.

During growth, cells must expand their cell volumes in coordination with biomass to control the level of cytoplasmic macromolecular crowding. Dry-mass density, the average ratio of dry mass to volume, is roughly constant between different nutrient conditions in bacteria, but it remains unknown whether cells maintain dry-mass density constant at the single-cell level and during nonsteady conditions. Furthermore, the regulation of dry-mass density is fundamentally not understood in any organism. Using quantitative phase microscopy and an advanced image-analysis pipeline, we measured absolute single-cell mass and shape of the model organisms Escherichia coli and Caulobacter crescentus with improved precision and accuracy. We found that cells control dry-mass density indirectly by expanding their surface, rather than volume, in direct proportion to biomass growth-according to an empirical surface growth law. At the same time, cell width is controlled independently. Therefore, cellular dry-mass density varies systematically with cell shape, both during the cell cycle or after nutrient shifts, while the surface-to-mass ratio remains nearly constant on the generation time scale. Transient deviations from constancy during nutrient shifts can be reconciled with turgor-pressure variations and the resulting elastic changes in surface area. Finally, we find that plastic changes of cell width after nutrient shifts are likely driven by turgor variations, demonstrating an important regulatory role of mechanical forces for width regulation. In conclusion, turgor-dependent cell width and a slowly varying surface-to-mass coupling constant are the independent variables that determine dry-mass density.

Integrative transcriptomic and proteomic analysis of the mutant lignocellulosic hydrolyzate-tolerant Rhodosporidium toruloides.

The oleaginous yeast Rhodosporidium toruloides has been considered as an economical lipid producer because it transforms carbohydrates from lignocellulosic hydrolyzate into triglycerides; however, R. toruloides cannot survive in hydrolyzate due to the inhibitors co-produced by hydrolysis. We have previously reported a plasma mutagenesis-generated mutant strain M18 that had strong tolerance for the stress environments of hydrolyzate. Here, we applied transcriptomic and proteomic approaches to analyze the global metabolic responses to the stress in hydrolyzate of R. toruloides and elucidate the tolerant mechanism of the mutant strain. The results showed that 57% genes matched and correlated well with their corresponding proteins. Five hundred and seven genes and 366 proteins had their transcription and expression levels changed, respectively, and 39 key genes with significantly changed transcription and expression levels (≥5-fold changes) were identified. The results demonstrated that four cellular processes and their key genes are likely related to the mechanism of tolerance of M18 strain. Enhanced expression of the key genes in R. toruloides could improve the cellular stress tolerance to lignocellulosic hydrolyzate, while the altered expression of most key genes is probably not caused by mutagenesis, but induced by stressful environments of the hydrolyzate.

Isolation of oleaginous yeast (Rhodosporidium toruloides) mutants tolerant of sugarcane bagasse hydrolysate.

Rhodosporidium toruloides is a lipid-producing yeast, the growth of which is severely suppressed when hydrolysates of lignocellulosic biomass are used as carbon source. This is probably due to the toxic substances, such as organic acids, furans, and phenolic compounds produced during the preparation of the hydrolysates. In order to solve this problem, R. toruloides cultures were subjected to atmospheric room-temperature plasma mutagenesis, resulting in the isolation of mutants showing tolerance to sugarcane bagasse hydrolysate (SBH). Three mutant strains, M11, M13, and M18, were found to grow with producing lipids with SBH as carbon source. M11 in particular appeared to accumulate higher levels (up to 60% of dry cell weight) of intracellular lipids. Further, all three mutant strains showed tolerance of vanillin, furfural, and acetic acid, with different spectra, suggesting that different genetic determinants are involved in SBH tolerance.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum.

BACKGROUND: Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. RESULTS: The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. CONCLUSION: There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system.

Thermal analysis of polyethylene glycol: evolved gas analysis with ion attachment mass spectrometry.

The thermal decomposition of polyethylene glycol was investigated by using a technique combining evolved gas analysis (time-resolved pyrolysis) with ion-attachment mass spectrometry. This technique allows the detection of intact pyrolysis products and, therefore, offers the opportunity for direct real-time monitoring of thermal by-products. Unstable products can thus be detected; for instance, many highly reactive organic peroxides, such as CH(3)OOH and HOCH(2)OOH, were found in this study. Classification analysis revealed 10 major compositional formulas among the product species: C(n)H(2)(n)(+2)O, C(n)H(2)(n)(+2)O(2), C(n)H(2)(n)(+2)O(3), C(n)H(2)(n)(+2)O(4), C(n)H(2)(n)(+2)O(5), C(n)H(2)(n)(+2)O(6), C(n)H(2)(n)(+2)O(7), C(n)H(2)(n)O, C(n)H(2)(n)O(2), and HO(CH(2)CH(2)O)(n)H ethylene glycol oligomers. The Li(+) ion adduct mass spectra showed a characteristic profile in terms of both the appearance of unique components and the distribution of pyrolysis products. Among the products of the thermal decomposition of PEG, formaldehyde (HCHO) and organic peroxides were particularly interesting. Formaldehyde, one of the 10 most abundant products, is a known human carcinogen. The detection of peroxides suggests that they may form during the incineration of PEG, which may have important environmental implications. The existence of peroxide products may have implications for chemical evolution in incinerator systems.

Thermal decomposition of pyridoxine: an evolved gas analysis-ion attachment mass spectrometry study.

RATIONALE: Pyridoxine is an important vitamer in food and pharmaceutical products. Heat treatments applied during preparation or storage of the products cause the decomposition of pyridoxine. Identification and understanding of the degradation products of pyridoxine and studying its decomposition kinetics are essential in the preparation and preservation of pyridoxine-containing foods and pharmaceuticals. METHODS: Real-time, non-isothermal decomposition of pyridoxine was studied using evolved gas analysis-Li(+) ion attachment mass spectrometry (EGA-Li(+) IAMS). Arrhenius parameters for the thermal decomposition of pyridoxine were obtained via the total ion monitoring (TIM) curve. RESULTS: Most of the pyridoxine evaporated in molecular form, but the formation of pyridoxal and o-quinone methide, both biologically important species, was also observed from the solid-phase degradation of pyridoxine. The observation of o-quinone methide, a species possessing anticancer activity, was particularly noteworthy due to its chemical instability. The activation energy (E(a) ) for pyridoxine decomposition determined by EGA-IAMS was found to be 20.0 kcal mol(-1) , and the pre-exponential factor (A) was 5.7 × 10(9) min(-1) . CONCLUSIONS: The calculated kinetic parameters are important for predicting the thermal stability of pyridoxine vitamer. The estimated lifetime (t(90%,25°C) ) of 1.7 × 10(-2) years in nitrogen was also obtained from the EGA-IAMS experiment.

Thermal stability of vitamin C: thermogravimetric analysis and use of total ion monitoring chromatograms.

The thermal decomposition kinetics and shelf life of vitamin C in nitrogen or air were studied by using thermogravimetric analysis (TGA) and evolved-gas analysis-lithium-ion attachment mass spectrometry (EGA-Li⁺IAMS). Arrhenius parameters obtained via TGA were reported for thermal decomposition. For vitamin C in a nitrogen atmosphere, the activation energy (E(a)) was 25.1 kcal/mol and the pre-exponential factor (A) was 2.5 × 10¹¹ min⁻¹. The kinetic parameters estimated via TGA agreed with values estimated from a pyrogram when the weight loss observed by TGA was shown to be due to gas evolution as a result of decomposition of the compound. Thermal stability was expressed by calculating the time for 10% of the vitamin C to decompose at 25 °C (t(90%,25 °C)). The t(90%,25 °C) for vitamin C obtained via TGA or EGA-Li⁺IAMS was higher in nitrogen (2.0 and 2.0 years, respectively) than in air (1.3 and 1.6 years, respectively). This indicates that the type of atmosphere influences vitamin C stability.

Evolved gas analysis of Ti(C5H5)2Cl2 by means of Li+ ion attachment mass spectrometry.

Characterization of the compound Ti(C(5)H(5))(2)Cl(2) was studied using Li(+) ion attachment mass spectrometry (IAMS) as an analytical methodology. Since this target compound is used as an anticancer drug in the treatment of leukemia, accurate and rapid monitoring methods for the determination of titanium drugs in a hospital environment are desirable. A quadrupole mass spectrometry system along with a Li(+) ion attachment technique and a direct inlet probe (DIP) produced the Li(+) adduct of Ti(C(5)H(5))(2)Cl(2), Ti(C(5)H(5))(2)Cl(2)Li(+). The DIP also was used to study the temperature-resolved behavior of this compound. The slope of the plot of signal intensity of Ti(C(5)H(5))(2)Cl(2)Li(+) versus temperature for Ti(C(5)H(5))(2)Cl(2) sublimation from 60 to 100 °C was used to determine an apparent activation energy (E(a)) of 124.43 kJ/mol for the sublimation of Ti(C(5)H(5))(2)Cl(2). This value is comparable to the reported value of 118.8 kJ/mol for molar enthalpy of sublimation of Ti(C(5)H(5))(2)Cl(2). These results demonstrate that the IAMS methodology can be used to study the enthalpy of sublimation for d-metal complex materials.

Is the bisphenol A biradical formed in the pyrolysis of polycarbonate?

An unknown species has been detected in the analysis of the products in a pyrolysis of polycarbonate using Li(+) ion-attachment mass spectrometry (IAMS). The mass spectra exhibited a Li(+) adduct peak at m/z 233 that was tentatively assigned to bisphenol A (BPA) biradical. Experimentally, this assignment was supported by the observation that the production rate increased under an inert nitrogen atmosphere. To further confirm the assignment, the stability of the BPA biradical to intramolecular rearrangement reactions as well as unimolecular decomposition has been analyzed via density functional theory calculations [B3LYP/6-311+G(3df,2p)]. The results show that the bisphenol A biradical is an open-shell biradical singlet that is stable to unimolecular decomposition. Although some of the proposed intramolecular rearrangement products have lower energies than those of the BPA diradical, these pathways have large reaction barriers and the kinetic lifetime of the radical is expected to be of the order of hours under the conditions of the experiment. The calculations also reveal that the bisphenol A diradical has large Li(+) affinities supporting the fact that these Li(+) complexes could be detected in the Li(+) ion attachment mass spectrometry. On the basis of these results the Li(+) adduct peak at m/z 233 detected in the pyrolysis of polycarbonate is assigned to the bisphenol A biradical.

Thermal decomposition of vitamin C: An evolved gas analysis-ion attachment mass spectrometry study.

Evolved gas analysis-ion attachment mass spectrometry (EGA-IAMS) was utilised to study the real-time non-isothermal decomposition of vitamin C. Dehydro-l-ascorbic acid, which has until this study been undetectable from the solid phase degradation of vitamin C, was observed as a decomposition product. While it is an important compound because it possesses some biological activity, dehydro-l-ascorbic acid is difficult to measure due to its chemical instability. In the present study using EGA-IAMS, we were able to detect dehydro-l-ascorbic acid from the thermal degradation of vitamin C. Our EGA-IAMS results obtained from the thermal decomposition of vitamin C were compared with a previous study employing pyrolysis-gas chromatography-mass spectrometry (Pyr-GC-MS). The observed quantitative and qualitative differences of the pyrolysis products obtained by the two techniques (EGA-IAMS vs. Pyr-GC-MS) are in part due to the difference in transportation time of the products out of the pyrolysis chamber.

Design and performance of an evolved gas analysis ion attachment mass spectrometer.

We designed a simple evolved gas analysis (EGA) system to act as a sampler between solid samples at atmospheric pressure and the high vacuum inside a mass spectrometer. The newly designed stainless steel system is simple, small and rugged and fulfills all the basic requirements for EGA. The temperature is programmable with 60 degrees C/min as the maximum heating rate and the temperature range is up to 600 degrees C. With this system coupled with lithium ion attachment mass spectrometry (IAMS), it is possible to study the temperature-programmed decomposition of a number of solid materials by detecting any chemical species on a real-time basis. For illustrative purposes, EGA-IAMS experiments of polyethylene polymers have been conducted.

Formation of bisphenol A by thermal degradation of poly(bisphenol A carbonate).

The thermal decomposition of poly(bisphenol A carbonate) (PoC) results in the formation of the endocrine disruptor bisphenol A (BPA). In the present work, we investigated the kinetics of the thermal decomposition of PoC, and the subsequent decomposition of BPA, under pyrolysis conditions and in the presence of oxygen by using infrared image furnace-ion attachment mass spectrometry. The decomposition of PoC obeyed Arrhenius kinetics, which allowed us to determine the activation energy (E(a)) for thermal decomposition to BPA from Arrhenius plots. From the selected ion monitoring curves for BPA, E(a) for thermal decomposition in a nitrogen atmosphere was calculated to be 133.2 kcal mol(-1), whereas E(a) for oxidative thermal decomposition was calculated to be approximately 35% lower (86.5 kcal mol(-1)).

Temperature-resolved thermal analysis of cisplatin by means of Li+ ion attachment mass spectrometry.

Li(+) ion attachment mass spectrometry (IAMS) was evaluated as an analytical methodology for measurement of the thermally labile, nonvolatile, and insoluble compound cisplatin, which is used as an anticancer agent in the treatment of testicular and ovarian cancers. We aimed to develop an improved method for the mass spectrometric determination of cisplatin, particularly in its molecular ion form. A uniquely designed quadrupole mass spectrometry system along with a Li(+) ion attachment technique and a direct inlet probe provided cisplatin molecular ions as Li(+) ion adducts; to our knowledge this is the first reported instance of cisplatin Li(+) ion adducts. Full-scan spectra were obtained with approximately 10 microg samples. Infrared image furnace-ion attachment mass spectrometry (IIF-IAMS) also was used to study the temperature-programmed decomposition of this drug. The slope of the plot of signal intensity versus temperature for cisplatin decomposition from 225 to 249 degrees C was used to determine an apparent activation energy (E(a)) of 38.0 kcal mol(-1) for the decomposition of cisplatin. This decomposition parameter is useful for predicting drug stability (shelf life). In this study, we have demonstrated that IAMS can be a valuable technique for the direct mass spectral analysis and kinetic study of d-metal complex platinum anticancer agents.

Effect of atmosphere and catalyst on reducing bisphenol A (BPA) emission during thermal degradation of polycarbonate.

Bisphenol A (BPA), a well-known endocrine disruptor, is one of the major products in the thermal degradation of polycarbonate (PC) and is also leached out from various PC products. Because of the high acute toxicity of BPA, reducing BPA production during degradation of PC is an important topic. A combined Infrared Image Furnace with Ion attachment mass spectrometry technique was used to investigate the evolution of BPA from a PC sample during heating in either nitrogen or air atmosphere and with or without a CuCl(2) catalyst. Thermal treatment in the presence of CuCl(2), in nitrogen atmospheres and at lower degradation temperatures, substantially reduced the BPA emission.

Ion attachment mass spectrometry combined with infrared image furnace for thermal analysis: evolved gas analysis studies.

A well-established ion attachment mass spectrometer (IAMS) is combined with an in-house single-atom infrared image furnace (IIF) specifically for thermal analysis studies. Besides the detection of many chemical species at atmospheric pressure, including free radical intermediates, the ion attachment mass spectrometer can also be used for the analysis of products emanating from temperature-programmed pyrolysis. The performance and applicability of the IIF-IAMS is illustrated with poly(tetrafluoroethylene) (PTFE) samples. The potential of the system for the analysis of oxidative pyrolysis is also considered. Temperature-programmed decomposition of PTFE gave constant slopes of the plots of temperature versus signal intensity in a defined region and provided an apparent activation energy of 28.8 kcal/mol for the PTFE decomposition product (CF(2))(3). A brief comparison with a conventional pyrolysis gas chromatography/mass spectrometry system is also given.

Latanoprost does not affect immune privilege of corneal allografts.

The present study determined whether topical latanoprost, a prostaglandin (PG) F(2alpha) analog, influences the induction of anterior chamber-associated immune deviation (ACAID), corneal neovascularization (NV) or survival of corneal allografts in mice. BALB/c mice received topical latanoprost or PGE(2) once or multiple times daily starting 4 weeks prior to or the day of anterior chamber injection of C57BL/6 splenocytes. Induction of allo-specific ACAID was assessed by ear challenge with C57BL/6 splenocytes 1 week after subcutaneous immunization. In a separate experiment, orthotopic corneal transplantation was performed using C57BL/6 mice as donors and BALB/c mice as recipients. Recipients were randomized in a masked fashion to receive topical latanoprost or PGE(2). Graft fate was assessed clinically under surgical microscopy. Presence of MHC class II(+) CD11c(+) or CD11b(+) cells in normal BALB/c mouse eyes following latanoprost or PGE(2) administration was assessed immunohistochemically. Control mice received topical 20% dimethyl sulfoxide or no treatment. Allo-specific ACAID was induced after 2 or 6 weeks of once daily treatment with latanoprost, and was induced even after 6 weeks of multiple treatments with latanoprost. Conversely, mice receiving PGE(2) failed to develop ACAID. Opacity and corneal NV scores for allografts treated with latanoprost were statistically indistinguishable from those for control allografts (p>0.05), whereas all allografts treated with PGE(2) were rejected. Opacity and NV scores were significantly higher in these allografts than in controls (p<0.05). A number of MHC class II(+) CD11c(+) cells were present in the central cornea after PGE(2) treatment. Topical application of latanoprost does not influence induction of ACAID or graft outcomes including opacity and NV, whereas PGE(2) does. Immune privilege of corneal allograft is maintained after topical latanoprost application in mice.