Chemical changes in cell envelope and poly-β-hydroxybutyrate during short term starvation of a marine bacterial isolate

Qualitative and quantitative changes were observed in lipids, poly--hydroxybutyrate (PHB), and a cell wall peptidoglycan consitutent in a marine bacterial isolate during starvation for 24 h in an energy and nutrient-free medium. While the amount and composition of the membrane fatty acids fluctuated within the first hours of starvation, the total amount of fatty acids decreased during the starvation period. Furthermore, the ratio of monounsaturated to saturated fatty acids decreased and the proportion of short chain fatty acids increased. In the very early phase of starvation the bacteria contained PHB, which had been accumulated during the growth phase, but after 3 h no PHB was detected. Cells starved for phosphorus showed a different pattern as PHB was initially accumulated and did not decrease until 5 h of starvation. Synthesis of the cell wall amino acid d-alanine was initiated during the first phase of starvation. The effects of these changes on membrane fluidity and uptake of substrates as well as the use of fatty acids and PHB as energy resources during starvation are discussed.Non-common abbreviations FID flame ionization detector - GC gas chromatography - HFBA heptafluorobutyric anhydride - MS mass spectrometry - NSS nine salt solution - PHB poly--hydroxybutyrate - PFB pentafluorobenzylbromide     

Ilyobacter delafieldii sp. nov., a metabolically restricted anaerobic bacterium fermenting PHB

Enrichments from an estuarine sediment with crotonate as substrate resulted in the isolation of a motile, gram-negative, obligately anaerobic rod with pointed ends, designated strain 10cr1. The organism was asporogenous, did not reduce sulfur, sulfate, thiosulfate, nitrate, oxygen or fumarate, and had a mol %G+C ratio of 29. Strain 10cr1 was able to ferment crotonate, 3-hydroxybutyrate, lactate, pyruvate, and poly--hydroxybutyric acid (PHB). Acetate, propionate, butyrate, CO₂ and H₂ were the fermentation products. When grown on PHB there was accumulation of 3-hydroxybutyrate once growth had ceased, indicating degradation of PHB to the monomer. The 3-hydroxybutyrate formed during growth of the culture was fermented to acetate, butyrate and H₂. Experimental evidence suggested the production of an extracellular PHB depolymerase. The cells were not attached to the PHB granules. This is the first isolation of an anaerobic bacterium capable of degrading exogenous PHB. This strain is described as a new species, Ilyobacter delafieldii sp. nov., and strain 10cr1 (=DSM 5704) is designated as the type (and at present, only) strain.Abbreviations G+C guanine plus cytosine - OD optical density - PHB poly--hydroxybutyric acid - specific growth rate - HPLC high-performance liquid chromatography - YE yeast extract     

Rapid and simple analysis of poly-β-hydroxybutyrate content by capillary isotachophoresis

Summary We present a rapid method for the direct analysis of poly--hydroxybutyrate (PHB) content in the soil bacteria Alcaligenes eutrophus. PHB from the fresh cells was converted by sulfuric acid to the crotonic acid and measured by capillary isotachophoresis after the neutralization by CaCO₃. The method can be used for rapid and routine monitoring of the fermentation processes in samples containing 0.001 to 20 mg of PHB.     

Pathway of anaerobic poly-β-hydroxybutyrate degradation byIlyobacter delafieldii

Ilyobacter delafieldii produced an extracellular poly--hydroxybutyrate (PHB) depolymerase when grown on PHB; activity was not detected in cultures grown on 3-hydroxybutyrate, crotonate, pyruvate or lactate. PHB depolymerase activity was largely associated with the PHB granules (supplied as growth substrate), and only 16% was detected free in the culture supernatant. Monomeric 3-hydroxybutyrate was detectable as a product of depolymerase activity. The monomer was fermented to acetate, butyrate and H₂. After activation by coenzyme A transfer from acetyl-CoA or butyryl-CoA, the resultant 3-hydroxybutyryl-CoA was oxidized to acetoacetyl-CoA (producing NADH), followed by thiolytic cleavage to yield acetyl-CoA which was further metabolized to acetyl-phosphate, then to acetate with concomitant ATP production. The reducing equivalents (NADH) could be disposed of by the evolution of H₂, or by a reductive pathway in which 3-hydroxybutyryl-CoA was dehydrated to crotonyl-CoA and reduced to butyryl-CoA. In cocultures ofI. delafieldii withDesulfovibrio vulgaris on PHB, the H₂ partial pressure was much lower than in the pure cultures, and sulfide was produced. Thus interspecies hydrogen transfer caused a shift to increased acetate and H₂ production at the expense of butyrate.     

Production of poly-β-hydroxybutyrate by thermophilic cyanobacterium, Synechococcus sp. MA19, under phosphate-limited conditions

Synechococcus sp. MA19, grown autotrophically under phosphate-limited conditions at 50 °C, produced poly--hydroxybutyrate (PHB) when intracellular phosphate content was 0.043–0.076mmol per g of cellular components. In the culture for 260h using Ca₃(PO₄)₂ as a phosphate source, strain MA19 accumulated PHB at 55% (w/w) of the dry cells and the amount of PHB produced was 2.4gl^–1 which was almost twice that without Ca₃(PO₄)₂ addition.     

Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa

The mycolic acid compositions of Nocardia rubra and related bacteria grown in media containing different concentrations of antituberculous isonicotinic acid hydrazide (INH) were determined in detail by gas chromatography-mass spectrometry. On the basis of molecular species composition, average carbon numbers of mycolic acids were calculated. In Nocardia rubra, N. lutea and Rhodococcus rhodochrous IFO-13161, the ratio of mycolic to non-mycolic fatty acids and the average carbon numbers of mycolic acids were decreased at the INH concentrations of higher than 1 g/ml, paralleling with the significant inhibition of growth. In above three species the synthesis of longer chain mycolic acids (longer than C₄₄ or C₄₆) was inhibited more significantly than shorter homologues such as C₃₈ or C₄₀. In contrast, neither growth inhibition nor change in corynomycolic acid composition was observed in Corynebacteria xerosis and Rhodococcus rhodochrous IFO-13165 at the concentration region of INH up to 100 g/ml. The direct mass fragmentographic analysis of the trimethylsilylated (TMS) derivatives of mycolic acid methyl esters, monitoring [M-15] ions of individual molecular species, revealed that the chain shortening of total mycolic acid molecule by INH occurred more greatly in more highly unsaturated subclasses than in less unsaturated subclasses. Furthermore, mass fragmentographic analysis, monitoring fragment ions (A) and (B), due to straight chain and branched chain alkyl units, respectively, demonstrated the inhibition of mycolic acids was not attributed to the shortening of -alkyl chain, but to the inhibition of chain elongation of C₂₈ to C₃₂ straight chain meromycolic acids. It was also indicated the amounts of trehalose mono- and di-mycolate (cord factor) decreased significantly with the addition of INH (1 to 20 g/ml) in the above strains. From the results obtained above, INH appeared to inhibit the synthesis of mycolic acids longer than C₄₄ or C₄₆ specifically by inhibiting chain elongation or desaturation of precursor long chain fatty acids longer than C₂₈ or C₃₀.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

Lipids of the Green Alga BotryococcusCultured in a Batch Mode

The lipid fraction of the green alga Botryococcuscultured in a batch mode was found to contain polar lipids (more than 50% of the total lipids), di- and triacylglycerols, sterols and their esters, free fatty acids, and hydrocarbons. In aging culture, the content of polar lipids somewhat decreased and that of triacylglycerols increased by more than four times. The content of hydrocarbons in the algal biomass did not exceed 0.9% and depended little on the culture age. Intracellular lipids contained saturated and unsaturated (mono-, di-, and trienoic) fatty acids. The maximum content of C_{16 : 3}and -C_{18 : 3}fatty acids (up to 35% of the total fatty acids) was detected in the phase of active growth. The extracellular and intracellular lipids of the alga differed in the proportion of particular lipids and in the fatty acid pattern.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus transformants harbouring cloned phbCAB genes

Summary Three transformants of Alcaligenes eutrophus harbouring the recombinant plasmids containing phbCAB, phbAB, and phbC genes, were cultivated to investigate the effect of cloned genes on cell growth and poly--hydroxybutyrate accumulation. Both in the nutrient-rich and minimal media, the increased PHB accumulation in the transformants was observed compared to the parent strain, and this was the result of the increased enzyme activities in the transformants. Low carbon concentration and high C/N molar ratio favored higher PHB accumulations in the transformants. The transformant harbouring the phbC gene showed the highest PHB accumulation, which indicated that PHB synthase was the most critical enzyme for PHB biosynthesis in the transformant.     

Adenine nucleotide contents and energy charge of Azotobacter vinelandii grown at low phosphate concentration

The effect of a low phosphate concentration on intracellular adenine nucleotide content, oxygen consumption and poly--hydroxybutyrate deposition was investigated with N-free and NH ₄ ⁺ batch cultures of Azotobacter vinelandii. When the microorganisms were cultured under low-phosphate concentrations the cells contained much larger amounts of poly--hydroxybutyrate, but displayed lower oxygen consumption activities and energy charge values than did control cells. Also, the ratio ATP to ADP was much higher in control cells and the intracellular levels of ATP were lower in low-phosphate cells.     

Determination of C20-C30 fatty acids by reversed-phase chromatographic techniques: An efficient method to quantitate minor fatty acids in serum of patients with adrenoleukodystrophy

An analytical method for the determination of saturated very long chain (VLC) fatty acids in the serum has been devised. Free fatty acids obtained after hydrolysis of total lipid extracts were converted intop-bromophenacyl esters. The derivatives were purified in two sequential steps by clean-up on C₁₈ reversed-phase cartridge and fractionation by reversed-phase thin-layer chromatography (TLC), and then quantitated by high performance liquid chromatography (HPLC) analysis. This technique provides a reliable and alternative method for the biochemical identification of patients and carriers of an inherited metabolic disease characterized by the accumulation of saturated VLC fatty acids (C₂₄–C₂₆) such as Adrenoleukodystrophy (ALD). In four cases of diagnosed ALD the fatty acid composition of serum total lipids was dramatically enriched in saturated VLC fatty acids compared to controls. The ratio of hexacosanoic acid (C₂₆₀) to docosanoic acid (C₂₂₀) in ALD patients was approximately six-fold higher than that of healthy controls or patients affected by metabolic or neurological disorders other than ALD.     

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

Summary The use of Cytophaga lysing enzymes was investigated for the liberation of poly--hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.     

The influence of poly-β-hydroxybutyrate accumulation on cell volume and buoyant density in alcaligenes eutrophus

Accumulation of poly--hydroxybutyrate (PHB) was studied in Alcaligenes eutrophus strain N9A. Under nitrogen limitation and heterotrophic conditions, the cells accumulated PHB at a rate of 50 fg cell^-1 h^-1. Volume increased from 1.208 to 3.808 m³ and buoyant density from 1.110 to 1.145 pg m^-3 with an increase in PHB from 0 up to 1.699 pg cell^-1. Volume was found to change linearly with PHB content. The changes were due to increases in cell width and not in cell length. PHB explained 93% of the changes in cellular volume. The relationship between density and PHB was hyperbolic. PHB explained 96% of the changes in density. When a mutant strain unable to accumulate PHB was analyzed together with the wild type, the PHB-less mutant and the wild type showed densities of 1.100 pg m^-3 and 1.120 pg m^-3, respectively, in gradients of 65% Percoll. In sucrose gradients, nevertheless, the results were reversed. This discrepancy was explained by the high osmolarity of sucrose which gives artificial results. Thus, we conclude that Percoll is a more suitable medium than sucrose to measure the density of live bacterial cells.Abbreviation PHB poly--hydroxybutyrate     

Metabolite concentrations in Alcaligenes eutrophus H 16 and a mutant defective in poly-β-hydroxybutyrate synthesis

Intracellular concentrations of hexose phosphates, phosphoenolpyruvate, pyruvate, NAD(H) and NADP(H) as well as the protein and poly--hydroxybutyrate (PHB) content were measured in suspensions of autotrophically grown cells of Alcaligenes eutrophus H 16 and compared with those in a mutant unable to synthesize poly--hydroxybutyrate. The parent strain was subjected to successive changes in conditions, and new steady states were rapidly (20 min) attained. When the parent strain was provided with carbon and energy but no nitrogen source, it fixed CO₂ and accumulated large amounts of PHB. When the mutant PHB^-4 was exposed to identical conditions, no accumulation of PHB occurred, but pyruvate, malate and citrate were excreted, and a 6-fold accumulation of hexose monophosphate (over the levels in the parent) was observed: in contrast, cofactors in intermediates between fructose-1,6-phosphate and phosphoenolpyruvate reached steady state as in the parent strain. When ammonium ion was then supplied, growth started and the metabolite concentrations in the mutant returned to the levels observed in the parent strain.     

Effects of environmental conditions on cell growth and poly-β-hydroxybutyrate accumulation in Alcaligenes eutrophus

Influences of the control of glucose and oxygen concentrations on cell growth and poly--hydroxybutyrate (PHB) accumulation in Alcaligenes eutrophus were studied. Glucose affects both biosynthesis and glycolysis directly and the other pathways indirectly. PHB accumulation could also be stimulated under oxygen limitation conditions, but the final PHB content within the cells was less than in the case of nitrogen limitation. When the culture was shifted from the PHB accumulation state to balanced growth conditions, PHB degradation occurred in the cells. The cell growth was inhibited by high PHB content within the cells.     

Nitrogen-limited continuous culture ofRhodopseudomonas capsulata growing photosynthetically or heterotrophically under low oxygen tensions

A continuous culture apparatus is described, which allows cultivation of photosynthetic bacteria anaerobically in the light and semiaerobically in the dark at constant oxygen tensions. The growth-parameters 1. substrate concentration at half maximum growth rate (K _s) and 2. yield (Y) forRhodopseudomonas capsulata are calculated.An automatic control system of the oxygen partial pressure in the culture medium is elaborated. It is shown, that the discontinuous regulation with a control unit in connection with two magnetic valves, which give short pulses of either oxygen-free gas or gas mixed with oxygen, is an economic, practicable and reliable method.The yield coefficientY during growth limited by ammonium sulfate is variable due to the synthesis of nitrogen independent metabolites, such as poly--hydroxybutyrate. The storage of poly--hydroxybutyrate in continuous culture is a function of both the actual concentration of ammonia and of theC/N ratio. At very low growth rates (1/6 µ _m ) the poly--hydroxybutyrate content increases amounting to 33% of the dry weight.In semiaerobically dark grown cells (pO₂: 5 mm Hg) the protein and bacteriochlorophyll content increased definitely on dry weight basis with increasing growth rates. In contrast, in anaerobic light cultures only a small increase of the bacteriochlorophyll level but no change of the protein content per dry weight of cells was observed at increasing growth rates.     

Development of nodules of Glycine max infected with an ineffective strain of Rhizobium japonicum

Bacteroids in ineffective (nitrogenase negative) nodules of Glycine max, infected with Rhizobium japonicum 61-A-24, as compared to those in effective nodules are characterized by reduced specific activities of alanine dehydrogenase to 15%, of 3-hydroxybutyrate dehydrogenase to 50%, and an increase of glutamine synthetase to 400%. In the plant cytoplasm of ineffective nodules, glutamine synthetase activity is reduced to 10–30%, glutamate dehydrogenase to 50–70%, and the aspartate aminotransferase and alanine aminotransferase are enhanced to 120–200%, depending on the age of the nodules. The total pool of soluble amino acids is reduced to 52 mol per g nodule fresh weight, as compared to 186 mol in effective nodules, with a replacement of asparagine (42 mol% of the amino acids) by an unknown amino compound. This compound is absent in nitrogenase, repressed and derepressed, free-living Rhizobium japonicum cells and in the uninfected root tissue. In nitrogenase derepressed, as compared to the repressed free-living cells of Rhizobium japonicum 61-A-101, arginine shows the most obvious change with a reduction to less than one tenth. The ultrastructure of the ineffective nodule is different from the effective organ even in the early stages. The membrane envelopes of the infection vacuoles are decomposing in heavily infected cells within 18 to 20 d after infection. In lightly infected cells very large vacuoles develop with only a few bacteroids inside. No close associations of cristae-rich mitochondria with amyloplasts are observed as in effective nodules. The uninfected cells keep their large starch granules even 40 d after infection. Some poly--hydroxybutyrate accumulation in the bacteroids is observed but only in the early stages, and it is almost absent in old nodules (40 d). At this age the infected cells are obviously compressed by uninfected cells, whereas in effective nodules with nitrogenase activity and leghaemoglobin formation, the infected cells have a much higher osmotic pressure than the neighbouring uninfected cells.Abbreviations PHBA poly--hydroxybutyric acid Prof. Dr. A. Pirson on the occasion of his 70th birthday     

Microscopic examination and fatty acid characterization of filamentous bacteria colonizing substrata around subtidal hydrothermal vents

Microscopic examination of the whitish mat that covered the substrata around subtidal hydrothermal vents at White Point in southern California revealed a Thiothrix-like bacterium containing sulfur inclusions as the dominant filamentous form in this microbial community. The matlike appearance developed as a result of the closely-packed manner inwhich the basal ends of the filaments were anchored to the substrate. The dominant phospholipid fatty acids of these filaments (16:0, 16:1w7c, 18:0, 18:1w7c) were similar to those recovered from a sample of Beggiatoa isolated from a spring in Florida. Filaments from both sources contained small quantities of C₁₈ and C₂₀ polyunsaturated fatty acids, as well. A larger but less abundant sheathless, filamentous form, which also contained sulfur inclusions and displayed a cell wall structure similar to a previously described Thioploca strain, also colonized the substrata around the subtidal mat. The preservation methods used in the preparation of thin-sections of the subtidal mat material were found to be inadequate for defining some key cellular structures of the large filaments. Nevertheless, the results demonstrate that the filamentous bacteria comprising the microbial mat in the vicinity of the subtidal vents exhibit some of the features of the free-living filamentous microorganisms found in deep-water hydrothermal areas.Published as Technical Report of the Southern California Ocean Studies Consortium, Long Beach, CA, USA     

Content of fatty acids ofChlorella kessleri in a deep tank fermentation

Chlorella kessleri cultivated in a deep tank contained 4.8% of non-polar lipid; 51% of this fraction represents saturated fatty acids, 7% unsaturated fatty acids. Our investigation of the fatty acids profile demonstrated even- and odd-numbered saturated and unsaturated fatty acids ranging from C₁₂ to C₂₀. Unlike in otherChlorella species, stearic acid was the dominant fatty acid found. Also shown was an elevated C_16:0 fatty acid content and a reduced level of unsaturated fatty acids.     

Increased poly-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli

A second promoter (P1) was inserted to the PHA (poly--hydroxyalkanoate) operon (pSP2) of Esherichia coli DH5 with an optimal E. coli ribosome binding site and a trc strong promoter (pSJS1) to obtain poly--hydroxybutyrate (PHB) with long chain length. When the inducer, IPTG was added to the culture at 0.4 mM, the average molecular weight was 1.1 × 10⁶ Da. However, an even greater increase of the PHB average molecular weight to 2.5 × 10⁷ Da was observed without IPTG being added.