Intracellular Storage Compounds Research Articles

Acyltransferases in Bacteria

Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue.

Polyhydroxyalkanoates (PHAs) Production Through Conversion of Glycerol by Selected Strains of Pseudomonas Mediterranea and Pseudomonas Corrugata

Polyhydroxyalkanoates (PHAs) are polyesters synthesized by numerous bacteria as an intracellular carbon and energy storage compounds in the cytoplasm of cells. To cut down the cost of production and optimize the PHA/dried cell weight different concentrations (1, 2 and 5 %) of refined glycerol and a crude glycerol from biodiesel process, were used as carbon source in a batch fermentation process. Pseudomonas mediterranea 9.1 as well as Pseudomonas corrugata 388 and A1 reference strains, grew and synthesized a medium-chain-length poly(3-hy-droxyalkanoates) elastomer both on crude and refined glycerol carbon sources. The mcl-PHA produced by P. mediterranea 9.1 grown on refined glycerol was apparently different as a result of a different metabolic pathway. Gas chromatography PHA’s Different monomeric units of side chains, which ranged from C12 to C19 in length in mcl-PHA(CG) and from C5 to C16 in mcl-PHA(RG) were obtained.

Heterogeneity of Intracellular Polymer Storage States in Enhanced Biological Phosphorus Removal (EBPR) – Observation and Modeling

A number of agent-based models (ABMs) for biological wastewater treatment processes have been developed, but their skill in predicting heterogeneity of intracellular storage states has not been tested against observations due to the lack of analytical methods for measuring single-cell intracellular properties. Further, several mechanisms can produce and maintain heterogeneity (e.g., different histories, uneven division) and their relative importance has not been explored. This article presents an ABM for the enhanced biological phosphorus removal (EBPR) treatment process that resolves heterogeneity in three intracellular polymer storage compounds (i.e., polyphosphate, polyhydroxybutyrate, and glycogen) in three functional microbial populations (i.e., polyphosphate-accumulating, glycogen-accumulating, and ordinary heterotrophic organisms). Model predicted distributions were compared to those based on single-cell estimates obtained using a Raman microscopy method for a laboratory-scale sequencing batch reactor (SBR) system. The model can reproduce many features of the observed heterogeneity. Two methods for introducing heterogeneity were evaluated. First, biological variability in individual cell behavior was simulated by randomizing model parameters (e.g., maximum acetate uptake rate) at division. This method produced the best fit to the data. An optimization algorithm was used to determine the best variability (i.e., coefficient of variance) for each parameter, which suggests large variability in acetate uptake. Second, biological variability in individual cell states was simulated by randomizing state variables (e.g., internal nutrient) at division, which was not able to maintain heterogeneity because the memory in the internal states is too short. These results demonstrate the ability of ABM to predict heterogeneity and provide insights into the factors that contribute to it. Comparison of the ABM with an equivalent population-level model illustrates the effect of accounting for the heterogeneity in models.

Study on Production, Extraction and Analysis of Polyhydroxyalkanoate (PHA) from Bacterial Isolates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by various bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we have screened the best micro organism among Bacillus subtilis and Pseudomonas aeruginosa and also have screened the best cheap carbon sources among Rice bran, Paddy husk, pigeon pea waste, Sugarcane baggasse, and waste frying oil. The study of production of PHA is done in combination of the best screened substrate as well as the micro organism.

Waste rapeseed oil as a substrate for medium‐chain‐length polyhydroxyalkanoates production

AbstractMedium‐chain‐length polyhydroxyalkanoates (mcl‐PHAs) are biodegradable, biocompatible polyesters produced by many bacteria as intracellular storage compounds of energy and carbon. They have gained great attention as a new green alternative to petrochemical plastic such as polypropylene. The present study was focused on mcl‐PHA production by two Pseudomonas strains (Gl01 and Gl06). To make this process more economical, waste rapeseed oil was used as a carbon source. The levels of PHAs synthesized by Pseudomonas sp. Gl01 strain and Gl06 strain were 21.0%, and 19.3% of CDW, respectively. The polyester accumulation increased until nitrogen and oxygen were depleted from the medium. Reverse transcription real‐time PCR approach was applied to quantitatively evaluate phaC1, phaC2, and phaZ gene expression. A positive correlation was found between the cellular PHA content and the gene expression of the PHA synthases. The transcriptional analysis revealed that the phaC1 and phaZ genes could be co‐transcribed. The overexpressed phaC2 gene was only observed in the Gl06 strain. The monomeric composition of the obtained mcl‐PHA was dependent on the strain: 3‐hydroxyoctanoic acid (3HO) and 3‐hydroxydecanoic acid (3HD) were dominant in strain Gl01, whereas 3‐hydroxyhexanoic (3HHx) was present in significant amounts in strain Gl06.Practical applications: This article presents the procedure of medium‐chain‐length polyhydroxyalkanoates (mcl‐PHAs) synthesis by the Pseudomonas Gl01 and Gl06 strains from waste rapeseed oil as a feedstock. Beside the microbial production of these environmentally friendly plastics, this approach also represents a new way of utilizing waste oils. The knowledge of the key enzymes involved in mcl‐PHAs accumulation is necessary in order to understand the mechanisms of their synthesis, and to use them effectively in biotechnological applications.

Is the whole the sumof its parts? Agent-basedmodelling of wastewater treatment systems

Agent-based models (ABMS) simulate individual units within a system, such as the bacteria in a biological wastewater treatment system. This paper outlines past, current and potential future applications of ABMs to wastewater treatment. ABMs track heterogeneities within microbial populations, and this has been demonstrated to yield different predictions of bulk behaviors than the conventional, "lumped" approaches for enhanced biological phosphorus removal (EBPR) completely mixed reactors systems. Current work included the application of the ABM approach to bacterial adaptation/evolution, using the model system of individual EBPR bacteria that are allowed to evolve a kinetic parameter (maximum glycogen storage) in a competitive environment. The ABM approach was successfully implemented to a simple anaerobic-aerobic system and it was found the differing initial states converged to the same optimal solution under uncertain hydraulic residence times associated with completely mixed hydraulics. In another study, an ABM was developed and applied to simulate the heterogeneity in intracellular polymer storage compounds, including polyphosphate (PP), in functional microbial populations in enhanced biological phosphorus removal (EBPR) process. The simulation results were compared to the experimental measurements of single-cell abundance of PP in polyphosphate accumulating organisms (PAOs), performed using Raman microscopy. The model-predicted heterogeneity was generally consistent with observations, and it was used to investigate the relative contribution of external (different life histories) and internal (biological) mechanisms leading to heterogeneity. In the future, ABMs could be combined with computational fluid dynamics (CFD) models to understand incomplete mixing, more intracellular states and mechanisms can be incorporated, and additional experimental verification is needed.

Effect of copper ion on the anaerobic and aerobic metabolism of phosphorus-accumulating organisms linked to intracellular storage compounds

The shock load effect of heavy metals (Cu (II)) on the behavior of poly-phosphate-accumulating organisms (PAOs) was investigated with respect to the transformations of poly-P, intracellular polyhydroxyalkanoates (PHAs) and glycogen. The PAOs biomass was exposed to different concentrations of Cu (II) at various pH and biomass levels. The results showed that when the mixed liquor suspended solid (MLSS) concentration was 2500–4000mg/L, the P removal was not adversely affected by spiking with 2mgCu2+/L; however, it deteriorated completely after a Cu (II) shock concentration of 4mg/L. Nevertheless, the tolerance of PAOs biomass to Cu (II) shock could be enhanced by increasing the MLSS. Moreover, in the presence of 2mgCu2+/L, the P removal efficiency was highest at an initial pH of 6.2 and lowest at an initial pH of 6.9, indicating that the Cu inhibitory effect was reduced by increasing the pH to 7.6. The inhibition by Cu (II) was related to the transformation of intracellular storage compounds of PAOs. Specifically, poly-P degradation might be inhibited, which reduced the energy available for PHA production and eventually led to poor P removal.

Factors controlling bacterial attachment and biofilm formation on medium-chain-length polyhydroxyalkanoates (mcl-PHAs)

Polyhydroxyalkanoate (PHA) is a versatile class of biodegradable and biocompatible biopolyesters accumulated by many bacteria as intracellular carbon storage compound. The largest subclass consists of medium-chain-length (mcl) PHA which has a large potential in medical applications where flexible or elastomeric materials are required. Different extraction and purification methods for mcl-PHA are known to result in variations of polymer purities. In this study it was assessed whether this difference in quality may increase risk of failure of the implant material by enhanced colonization by Gram-negative and Gram-positive model pathogens ( Staphylococcus aureus and Escherichia coli curli). Two types of mcl-PHA copolymers, poly(3-hydroxyoctanoate) (PHO) and the less known poly(3-hydroxyundecanoate) (PHUA), were evaluated for an impact of monomeric unit composition and degree of polymer purity on colonization by the model pathogens. It was found that film formation by bacteria on purified PHO and PHUA coatings was lower or similar to the one observed for polystyrene controls. The presence of proteins and lipopolysaccharide impurities originating from biotechnological production contributed to an increase in biofilm development probably by triggering the attachment of bacterial cells. In addition it was found that the model strains used differed significantly in colonizing the surfaces. For both types of mcl-PHAs E. coli curli was always a better biofilm former than S. aureus, which could be explained by the presence of curli (protein) fibers and a less hydrophilic cell wall.

Biocompatibility of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) modified by silk fibroin

The materials used as tissue scaffolds play an important role for guiding the tissue to create a new functional structure when the damaged tissue is no longer regenerated naturally [1]. Whether or not the material is suitable for tissue scaffold depends on its physical and chemical properties, such as mechanical properties, degradability and, importantly, biocompatibility. Most of biomaterials used in tissue engineering are poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) [2–4]. But their poor hydrophilicity [5, 6] and relatively high crystallinity [7, 8] restrict their application in some degree. For example, previous experiments of tissue engineered heart valves fabricated by PGA only successfully acted at the low-pressure pulmonary circulation [9]. Recently, polyhydroxyalkanoate (PHA), a thermoplastic elastomor class of biopolyesters synthesized by the bacteria as an intracellular carbon and energy storage compound [10–12], is widely applied as biodegradable plastics. Moreover, it is recognized as a candidate for tissue engineered heart valve and cartilage [13] because of its elastic and easily moldable properties [14]. Of PHAs family, the most common member as biomaterial

Optimization of nutrient feed concentration and addition time for production of poly(β-hydroxybutyrate)

PHB is an intracellular storage compound produced by many organisms under conditions of nutrient limitation (N, P, etc.) when carbon source is present in excess. Model-based fed-batch cultivation is one of the techniques to induce desired nutrient limitations intelligently for over production of such partially growth-associated polymeric products. In the present study, batch kinetics of PHB synthesis by Wautersia eutropha was established in a bioreactor. Batch kinetics featured a biomass concentration of 19.68 g/l with a PHB content of 10.89 g/l in 60 h. A mathematical model was then developed, which adequately described the batch kinetics. The model equations were extrapolated to fed-batch cultivation by incorporating the dilution terms due to fresh nutrient (carbon and nitrogen) feeding. Model-based fed-batch fermentation was conducted by feeding nitrogen (7 g/l) from 20–30 h at the rate of 70 ml/h and fructose (300 g/l) from 24–34 h at the rate of 80 ml/h constantly which yielded 32 g/l biomass containing 14 g/l PHB in 50 h. The productivity was 1.5-times greater than that obtained in batch increasing from 0.18 to 0.28 g/l-h. The model proved to be highly instrumental in the design of suitable fed-batch cultivation.

Cloning and expression of the PHA synthase genes phaC1 and phaC1AB into Bacillus subtilis

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB. The two plasmids were inserted into Bacillus subtilis DB104 and generated Bacillus subtilis/pBE2C1 and Bacillus subtilis/pBE2C1AB. The two recombinant strains were subjected to fermentation and showed PHA accumulation, the first reported example of medium-chain-length-PHA production in Bacillus subtilis. GC analysis identified the compound produced by Bacillus subtilis/pBE2C1 was a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer while that produced by Bacillus subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. The results also showed that the recombinant B. subtilis could utilize the malt waste in the medium as a carbon source better than that of glucose and thus could substantially lower the cost of production of PHA.

Aerobic phosphorus release linked to acetate uptake: Influence of PAO intracellular storage compounds

The mechanisms of polyphosphate accumulating organisms (PAO) are not fully established yet under conditions that differ from the classical anaerobic/aerobic conditions. Recently, some research in the field of biological phosphorus removal has been focused on studying systems where the electron donor (substrate) and the electron acceptor (nitrate or oxygen) are present simultaneously. The aim of this study was to assess the influence of the levels of intracellular storage compounds in the net phosphorus removal process occurring under continuous aerobic conditions. An enhanced biological phosphorus removal (EBPR) system was enriched in a sequencing batch reactor working under alternating anaerobic/aerobic conditions and the percentage of “ Candidatus Accumulibacter phosphatis” (the PAO in this study) was 45%. Three different batch experiments were performed to obtain a better insight of this process: one under standard anaerobic–aerobic conditions and two experiments under strictly aerobic conditions. One of the aerobic experiments was developed with the EBPR biomass withdrawn at the end of the anaerobic period and the other one with biomass withdrawn at the end of the aerobic period of the standard process. Although net phosphorus removal was achieved in both aerobic experiments, the ratios of phosphorus released and glycogen degraded versus acetate taken up were higher when the sludge was withdrawn at the end of the aerobic period, where higher levels of polyphosphate and glycogen were present in the biomass.

A thermostable #x003B2;-ketothiolase of polyhydroxyalkanoates (PHAs) in Thermus thermophilus: Purification and biochemical properties

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a beta-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 degrees C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. beta-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 microM and 25 microM for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of beta-ketothiolase.

Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants.

The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization. The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass. The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures. Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate. They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously. Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources. Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed. Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation. The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor. Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor. This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants.

Recent advances in microbial polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as an intracellular carbon and energy storage compound. These are accumulated in the cytoplasm of cells. A number of bacteria including Alcaligenes, Pseudomonas, recombinant Escherichia coli and methylotrophs have been used for the production of PHAs and high productivities have been achieved. Production of PHA by transgenic plants has been demonstrated on a laboratory-scale and large-scale production will be feasible in the near future. By controlling the monomer composition of PHA, it has been indicated that the physical properties of polymers can be regulated to a great extent. Even though PHAs have been recognised as a good candidate for biodegradable polymers, their high production cost limits their industrial application. It is hoped that with improvement in fermentation and downstream processing techniques, development of new recombinant strains and large-scale production by transgenic plants will reduce the cost of production of PHAs thereby making them competitive with conventional plastics.

Bacterial polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. More than 80 HAs have been detected as constituents of PHAs, which allows these thermoplastic materials to have various mechanical properties resembling hard crystalline polymer or elastic rubber depending on the incorporated monomer units. Even though PHAs have been recognized as good candidates for biodegradable plastics, their high price compared with conventional plastics has limited their use in a wide range of applications. A number of bacteria including Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinelandii, methylotrophs, pseudomonads, and recombinant Escherichia coli have been employed for the production of PHAs, and the productivity of greater than 2 g PHA/L/h has been achieved. Recent advances in understanding metabolism, molecular biology, and genetics of the PHA-synthesizing bacteria and cloning of more than 20 different PHA biosynthesis genes allowed construction of various recombinant strains that were able to synthesize polyesters having different monomer units and/or to accumulate much more polymers. Also, genetically engineered plants harboring the bacterial PHA biosynthesis genes are being developed for the economical production of PHAs. Improvements in fermentation/separation technology and the development of bacterial strains or plants that more efficiently synthesize PHAs will bring the costs down to make PHAs competitive with the conventional plastics.

Intracellular storage compounds, oxygen uptake rates and biomass yield with readily and slowly degradable substrates

An experimental study was undertaken to quantify the effects of intracellular storage polymers on the Oxygen Uptake Rate (OUR) profile and biomass yield coefficients for readily and slowly degradable substrates using a sludge from anaerobic-aerobic Sequencing Batch Reactor (SBR). Glucose and acetate were chosen as readily biodegradable substrates and soluble starch served as a model polymeric slowly degradable substrate. In case of acetate as substrate, PHA was the major storage polymer while polysaccharide acted as the main storage compound in case of glucose and starch. The accumulation of storage compounds even in the case of starch suggests that storage may be an integral part of the metabolic process for the sludge having storage capabilities. The maximum storage amounted to about 45%, 68% and 36% of total acetate, glucose and starch COD removed from bulk respectively. A new approach to calculate the true biomass yield coefficients based on exogenous OUR and considering intracellular storage is proposed. The relative independence of the calculated yield values to the amount of storage makes them suitable for application in growth kinetics. The calculated yield coefficients based on the proposed approach worked out to be 0.24–0.28, 0.58–0.50 and 0.64 for acetate, glucose and starch respectively and were significantly lower than the yield coefficients calculated based on conventional approach of not accounting storage. Further, the average degradation rate of intracellular glycogen were estimated to be 15 mg carbohydrate COD/g. MLSS. hr. with an average biomass yield of 0.45 mg biomass COD/mg carbohydrate COD.

Intracellular storage compounds, oxygen uptake rates and biomass yield with readily and slowly degradable substrates

An experimental study was undertaken to quantify the effects of intracellular storage polymers on the Oxygen Uptake Rate (OUR) profile and biomass yield coefficients for readily and slowly degradable substrates using a sludge from anaerobic-aerobic Sequencing Batch Reactor (SBR). Glucose and acetate were chosen as readily biodegradable substrates and soluble starch served as a model polymeric slowly degradable substrate. In case of acetate as substrate, PHA was the major storage polymer while polysaccharide acted as the main storage compound in case of glucose and starch. The accumulation of storage compounds even in the case of starch suggests that storage may be an integral part of the metabolic process for the sludge having storage capabilities. The maximum storage amounted to about 45%, 68% and 36% of total acetate, glucose and starch COD removed from bulk respectively. A new approach to calculate the true biomass yield coefficients based on exogenous OUR and considering intracellular storage is proposed. The relative independence of the calculated yield values to the amount of storage makes them suitable for application in growth kinetics. The calculated yield coefficients based on the proposed approach worked out to be 0.240.28, 0.58-0.50 and 0.64 for acetate, glucose and starch respectively and were significantly lower than the yield coefficients calculated based on conventional approach of not accounting storage. Further, the average degradation rate of intracellular glycogen were estimated to be 15 mg carbohydrate COD/g. MLSS. hr. with an average biomass yield of 0.45 mg biomass COD/mg carbohydrate COD.

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum.

Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 micrograms/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 micrograms/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 micrograms/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) inMycobacterium neoaurum

Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 micrograms/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 micrograms/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 micrograms/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.