Effects of temperature and incubation time on production of ochratoxin A by black aspergilli

The effects of temperature (5-45 degrees C) on the growth and production of ochratoxin A (OTA) by eighteen strains of Aspergillus section Nigri, cultured on Czapek yeast autolysate agar (CYA) and on yeast extract sucrose agar (YES), were studied for an incubation period of 30 days. Isolates were selected to include different sources and different reported abilities to produce OTA. Temperature ranges for OTA production were more restrictive than those for growth and each strain tested differed in its optimum conditions for OTA production. Aspergillus niger aggregate strains achieved maximum OTA levels in YES medium mainly at 20-25 degrees C. The A. carbonarius strains produced the highest OTA levels in CYA medium at 15 or 20 degrees C. Significant amounts of OTA were produced after only five days of incubation. Due to their ability to produce OTA at a wide range of temperatures, OTA can be continuously produced in the field. This fact has to be taken into account in commodities such as grapes, raisins and wine, where A. carbonarius and members of the A. niger aggregate are considered to be the main sources of the OTA contamination.     

Characterization of phenol and trichloroethene degradation by the rhizobium Ralstonia taiwanensis

Ralstonia taiwanensis is a root nodule bacterium originally isolated from Mimosa sp. in southern Taiwan. Some strains of R. taiwanensis demonstrated the ability to grow on medium containing phenol as the sole carbon source, especially strain TJ86, which was able to survive and grow at phenol concentrations of up to 900 mg/l. The dependence of the phenol degradation rate on the phenol concentration can be described by Haldane's model with a low KS (the apparent half-saturation constant) of 5.46 microM and an extremely high KSI (the apparent inhibition constant) 9075 microM. The optimal phenol degradation rate was 61 micromol/min/g cell, which occurred at a phenol concentration of 228 microM. The phenol-limited growth kinetics of TJ86 by Andrews's model also followed a similar trend to that of phenol degradation, indicating the close links between phenol degradation and cell growth. Strain TJ86 also achieved 100 and 40% degradation for soil samples amended with 500 and 1000 microg phenol/g soil (dry weight) within 9 days, respectively. Moreover, strain TJ86 cometabolically degraded trichloroethene (TCE) after being cultivated with media containing phenol or m-cresol as the carbon substrate. The sequence of the large-subunit phenol hydroxylase (LmPH) gene obtained from TJ86 displayed high homology to that of other phenol-utilizing bacteria. Results from kinetic and phylogenetic analyses suggest that strain TJ86 most likely belongs to group I phenol-degrading bacteria which are considered to be efficient TCE degraders. It is proposed that the symbiotic relationship between rhizobia R. taiwanensis and its host plant Mimosa sp. may have the potential for rhizoremediation of aquatic and soil environments contaminated by phenol and TCE.     

The effect of temperature on the interaction of Haemophilus ducreyi with human epithelial cells

To investigate if temperature affects the interaction of Haemophilus ducreyi with human epithelial cells, nine strains were used to evaluate the adhesion kinetics of the organism at 33 degrees C and 37 degrees C. The effect of the free toxin on the epithelial cells at those temperatures was also assessed. The cyto-adherence kinetics of H. ducreyi to the epithelial cells was significantly greater at 33 degrees C (10 times more) than at 37 degrees C in all seven clinical isolates tested. There was a significant difference in cell-associated H. ducreyi at 33 degrees C as compared with 37 degrees C. Control strains showed similar adhesion properties at both temperatures. However, the virulent strain CIP542 adhered in larger amounts than the avirulent strain A77. Electron microscopy revealed that there was more tissue necrosis at the lower than the higher temperature. The effect of the free toxin was the same at each temperature. However, strain A77 had significantly lower toxicity than strain CIP542 and the clinical isolates. These results suggest that H. ducreyi displays a temperature-dependent interaction with human epithelial cells, and this feature may play a role in the virulence of the organism in vivo. While the overall toxic effect of viable bacteria depends on the metabolic activity of the bacteria and is, therefore, higher at 33 degrees C than at 37 degrees C withthe same initial inoculum, the effect of the extracted toxin at molecular level with fixed concentrations is a temperature-independent event.     

Differential expression of actin modulated by temperature in mycelial and yeast cells of Paracoccidioides brasiliensis.

We used yeast and mycelial forms of Paracoccidioides brasiliensis to evaluate the effect of heat shock stress on actin expression. P. brasiliensis yeasts harvested during the exponential growth phase showed more expression of the actin mRNA when incubated at 40 degrees C than when incubated at 37 degrees C, the usual temperature at which these yeasts grow. In contrast, expression of actin mRNA was lower in yeasts incubated at 25 degrees C than in yeasts incubated at 37 degrees C. Mycelium harvested at 25 degrees C, an approximation of its normal growth temperature, and then exposed to 37 degrees C and 40 degrees C showed progressively higher expression of actin mRNA. Mycelial and yeast forms showed a similar pattern of response to exposure to supra-optimal temperatures: both showed the same increase in expression of actin. This suggests that actin may play a role not only in cellular differentiation but also in this species' rapid adaptive response to heat stress, a mechanism necessary to deal with a potentially hostile environment.     

Biodegradation of benzene and its derivatives by a psychrotolerant and moderately haloalkaliphilic Planococcus sp. strain ZD22

The potential for biodegradation of aromatic hydrocarbons simultaneously at low temperatures and under saline and alkaline conditions is not well understood, but such biodegradation would be useful for remediation of polluted sites. A psychrotolerant, moderately haloalkaliphilic pure culture using benzene as a sole source of carbon and energy was isolated by selective enrichment from alkaline and saline soils in the vicinity of the Daqing oil field in China. An analysis of the 16S rDNA gene sequence and morphological and physiological characteristics showed that this strain is a member of the genus Planococcus, and it was designated as strain ZD22. Strain ZD22 could grow at temperatures between 2 and 36 degrees C (pH 7.5-11) and salt concentrations from 0.5 to 25%. Its optimal conditions for biodegradation of benzene were 20 degrees C (pH 9.5) and 10% salt concentration. Strain ZD22 not only utilized benzene, toluene, ethylbenzene and o-xylene, but also degraded chlorobenzene, bromobenzene, iodobenzene and fluorobenzene. The kinetic model of strain ZD22 for benzene was solved to obtain mumax=0.34 h-1, Ks=0.041 mM, n=1.21, Sm=10.2 mM. To our knowledge, this is the first report of biodegradation of benzene and its derivatives simultaneously under multiple extreme conditions.     

Morphogenesis and pathogenicity of Histoplasma capsulatum.

The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma capsulatum, G184A and G222B, when the temperature of incubation was raised to 37 degrees C, and the block persisted even after the cultures were washed free of PCMS. Instead of transforming to yeast cells, PCMS-treated mycelia continued to grow as mycelia at the elevated temperatures. A less virulent strain (Downs) was more temperature sensitive, but it showed a similar irreversible effect at 34 degrees C. Therefore, the mycelium-to-yeast transition of H. capsulatum is not required for the adaptation of mycelia to elevated temperatures but probably results from the temperature-dependent activation of yeast-specific genes. The transition to yeast is inferred to be obligate for pathogenicity in mice because PCMS-treated mycelia failed to cause infection, and no fungi were seen in tissues after PCMS-treated mycelia were injected into mice.     

Heterogeneity of phenol oxidases in Cryptococcus neoformans.

Phenol oxidase enzymes, linked to virulence in Cryptococcus neoformans, were prepared from broken cells. More enzyme activity was found in the ultracentrifugation supernatant; less was found in the membrane fraction. Phenol oxidases were located in acrylamide gel electropherograms by activity staining with L-dihydroxyphenylalanine (DOPA). Mobility differences between soluble and solubilized membrane-bound phenol oxidases were not found. Comparison of enzymes produced at 25 and 37 degrees C revealed that the enzyme had lower activity and lower mobility at 37 degrees C. The mobility of 25 degrees C phenol oxidases from strains of C. neoformans var. gattii was lower than that of those from C. neoformans var. neoformans. Half of the phenol oxidase produced at 25 degrees C was bound by concanavalin A, while that produced at 37 degrees C was not bound. However, glucose starvation of cultures at 25 degrees C overnight resulted in increased amounts of enzyme which did not bind to concanavalin A. A given strain of C. neoformans produces different species of phenol oxidase under different culture conditions.     

Analysis of cytoplasmic antigens of the yeast and mycelial phases of Candida albicans by two-dimensional electrophoresis.

The extent of the macromolecular change accompanying yeast to mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis of the cytoplasmic proteins of the two growth forms after antibody cross-absorption experiments. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium (LSM). The two strains selected for this study were strain 4918, which produces pure mycelial (M) cultures in LSM at 37 degrees C (designated 4918-37M) and yeasts (Y) at 24 degrees C (4918-24Y), and strain 2252, which produces yeasts exclusively at both 24 and 37 degrees C in LSM (2252-24Y and 2252-37Y). The proteins of both strains were labeled at both temperatures with [35S]sulfate, and cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation. Rabbits were immunized with the 4918-24Y and 4918-37M cytoplasmic fractions to produce anti-yeast-phase and anti-mycelial-phase hyperimmune sera. Each radiolabeled cytoplasmic fraction was absorbed with anti-mycelial-phase immunoglobulin, anti-yeast immunoglobulin, and immunoglobulin from normal rabbit serum. Staphyloccal protein A was used to remove immune complexes. The labeled, nonabsorbed proteins were also analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. The specificity of the immunoglobulin hundreds of proteins in each extract. The specificity of the immunoglobulin preparations was extremely broad, and as many as 168 cytoplasmic antigens were detected. Eighty-three antigens were recognized in the mycelial-phase extract only by the anti-mycelial-phase immunoglobulin. However, comparative analysis revealed that all of these proteins were present in at least one other extract. Therefore, none of them was unique to the mycelial morphology. Eleven antigens were detected in the 2252-37Y extract that were not present in the extracts from strain 4918, which indicates that proteins obtained from different strains may express similar antigenic determinants, but differ in their physiochemical properties.     

Freeze-drying of respiratory syncytial viruses for transportation and storage.

The stabilities of eight strains of respiratory syncytial virus were compared after the strains were freeze-dried in the presence and absence of the stabilizer SPGA, which contains 218 mM sucrose, 7.1 mM dipotassium hydrogen phosphate, 3.76 mM potassium dihydrogen phosphate, 4.9 mM sodium glutamate, and 1% (wt/vol) bovine albumin. Strains freeze-dried in the presence of SPGA showed only small-to-negligible losses at 4 degrees C and losses of approximately 2.0 log10 infectious units at 25 degrees C when held for 45 weeks. Losses at 37 degrees C for one strain were approximately 10-fold greater when the strain was freeze-dried in the absence of SPGA. These results indicate that respiratory syncytial virus strains freeze-dried in the presence of a suitable stabilizer can be transported as unrefrigerated samples without undue losses in infectivity.     

Evaluation of Beauveria bassiana (hyphomycetes) strains as potential agents for control of Triatoma infestans (Hemiptera: Reduviidae)

Chagas disease constitutes a major human health problem in most Latin American countries. This endemic disease is transmitted by several species of triatomine bugs, the most important being Triatoma infestans (Klug). In this article, we report on the selection of strains of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. virulent to T. infestans for possible use as bioinsecticides. Four strains of B. hassiana isolated from Argentina (Bb 1, 10, 25, and 65) were evaluated. To calculate mortality and mean lethal time, nymphs and adults of T. infestans were treated with conidia produced on complete agar medium and wheat brain and rice husk medium (WH). The LD50 for nymphs and adults of T. infestans was calculated. The effect of different temperatures (18, 22, 26, 30, and 34 degrees C) and relative humidities (35 and 90% RH) on mortality of nymphs were studied. We evaluated the compatibility of strains with Deltamethrin and Beta-Cypermethrin. Although the strain Bb 25 failed to grow on WH, the other three strains showed similar mortality values independent of the culture medium used to grow the microorganisms. The lowest LD50 values for nymphs were obtained with the strains Bb 10 and 65 and for adults were Bb 1, 10, and 65. The highest mean mortality was obtained with strain Bb 10, and among temperatures the highest mean mortality was observed at 26 degrees C. Relative humidity did not affect the mortality of T. infestans nymphs with all strains and temperatures assayed. Deltamethrin did not affect any of the three strains for the four studied doses, and Beta-Cypermethrin could be used in combination with the fungus only at low doses. The strain Bb 10 was selected for future assays under natural climatic conditions.     

Intraspecies and temperature-dependent variations in susceptibility of Yersinia pestis to the bactericidal action of serum and to polymyxin B

Lipopolysaccharide (LPS) structure impacts the bactericidal action of cationic peptides, such as polymyxin B (PMB), and sensitivity to killing by normal human serum (NHS). Cultivation of different subspecies strains of Yersinia pestis isolated from unrelated geographic origins at various temperatures (mammals, 37 degrees C; fleas, 25 degrees C; or winter hibernation, 6 degrees C) affects LPS composition and structure. We tested the susceptibilities of various strains of Y. pestis grown at these different temperatures to PMB and serum bactericidal killing. Both properties varied significantly in response to temperature changes. In Y. pestis subsp. pestis (the main subspecies causing human plague), high levels of resistance to PMB and NHS were detected at 25 degrees C. However, at the same temperature, Y. pestis subsp. caucasica was highly sensitive to PMB. At both of the extreme temperatures, all strains were highly susceptible to PMB. At 25 degrees C and 37 degrees C, Y. pestis subsp. caucasica strain 1146 was highly susceptible to the bactericidal activity of 80% NHS. All Y. pestis strains studied were able to grow in heat-inactivated human serum or in 80% normal mouse serum. At 6 degrees C, all strains were highly sensitive to NHS. Variations in the PMB resistance of different bacterial cultures related to both the content of cationic components (4-amino-4-deoxyarabinose in lipid A and glycine in the core) and a proper combination of terminal monosaccharides in the LPS. The NHS resistance correlated with an elevated content of N-acetylglucosamine in the LPS. Structural variation in the LPS of Y. pestis correlates with the organism's ability to resist innate immunity in both fleas and mammals.     

Evaluation of a new method for identification of Cryptococcus neoformans which uses serologic tests aided by selected biological tests.

A new method for identifying Cryptococcus neoformans isolates and their serotypes by the slide agglutination test using five kinds of factor sera, with the aid of nitrate reduction, phenol oxidase, and growth at 37 degrees C tests was evaluated by using 36 reference strains and 75 clinical isolates of C. neoformans. The results showed that the reference strains were identified exactly as they were labeled, and clinical isolates were identified as C. neoformans serotypes A, D, and AD. C. neoformans could be distinguished from other Cryptococcus species that cross-reacted with factor sera by their ability to grow at 37 degrees C. These results indicate that the slide agglutination test combined the use of factor sera for isolates which grow at 37 degrees C is a useful method for identification of C. neoformans and their serotypes and that the nitrate reduction test (negative in 100% of the isolates) and the phenol oxidase test (positive in approximately 95% of the isolates) can be used to confirm that the species is C. neoformans.     

NADP+-dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium,Psychrobacter sp. strain 13A

The genes encoding dimeric and monomeric isocitrate dehydrogenase (IDH) isozymes from a psychrotrophic bacterium, strain 13A (13AIDH-D and 13AIDH-M, respectively), were cloned and sequenced. The deduced amino acid sequences of these two IDHs showed high degrees of identity with those of bacteria of genus Psychrobacter. Analysis of the 16S ribosomal RNA gene of the strain 13A revealed that this bacterium is classified to genus Psychrobacter. The optimum temperatures for activities of 13AIDH-D and 13AIDH-M were 55°C and 45°C, respectively, indicating that they are mesophilic. On the contrary, 13AIDH-D maintained 90% of its maximum activity after incubation for 10 min at 50°C, while the 13AIDH-M activity was completely lost under the same condition. In addition, 13AIDH-D showed much higher specific activity than 13AIDH-M. From northern and western blot analyses, the 13AIDH-D gene was found to be not transcribed under the growth conditions tested in this study. However, the catalytic ability of the mesophilic 13AIDH-M was concluded to be enough to sustain the growth of strain 13A at low temperatures. Therefore, a novel pattern of the contribution of IDH isozymes in cold-living bacteria to their growth at low temperatures was confirmed in strain 13A.     

Gas chromatographic assessment of alcoholyzed fatty acids from yeasts: a new chemotaxonomic method.

An alternative chemotaxonomic method to methanolysis was developed for gas chromatographic assessment of fatty acids in whole yeast cells. Clinical and reference strains of the medically important yeasts Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae were cultured for 48 h at 26 degrees C. Cellular lysis and transesterification were then performed with ethanol, propanol, butanol, or methanol. The relative recovery rates for cellular fatty acids, including the volatile acids C10:0 and C12:0, were similar after alcoholysis with ethanol, propanol, or butanol, while methanolysis gave lower recoveries of volatile fatty acids. Thus, after ethanolysis, the recovery of C10:0 acid (0.1, 1, and 10%) from a defined matrix (lyophilized Actinobacillus actinomycetemcomitans cells) varied from 97 to 102%, while the recovery of C10:0 after methanolysis varied from 49 to 75%. This indicated that with the frequently used methanolysis technique, there is a considerable loss of volatile fatty acids. These acids may be used as marker molecules for taxonomic differentiation between yeasts.     

Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells.     

Two different mechanisms of serum resistance in Escherichia coli

Fifty-three serum-resistant strains of E. coli which were all able to grow in at least 50% normal human serum (NHS) were tested in respect to binding and consumption of C3b, factor H, C5, and C6 after incubation in pooled NHS. The results of immunofluorescence tests, hemolytic assays, and binding studies using radiolabeled components were comparable. The different binding patterns allowed us to divide the strains into three different groups. The main features of group I were the attachment of C3, C5, and C6 to the bacterial cells as well as consumption of C3 and C5, whereas factor H did not bind at all or only in small amounts. In addition, released MAC was detectable in the supernatant of reaction mixtures containing bacteria of a group I strain and NHS. In group II factor H was easily bound to the bacteria, but no C3, C5, and C6 binding or C5 consumption was detectable. In addition, strains of group III bound C3 and factor H and some strains also bound and consumed C5. Because of the inhomogeneity of group III, this investigation was restricted to a comparison of groups I and II. From the results presented in this study we conclude that group I bacteria activate the whole complement cascade, whereas with bacteria of group II, complement activation is interrupted at the C3 level. These findings therefore indicate a second, alternative mechanism of serum resistance in E. coli.     

Influence of growth temperature of Escherichia coli on K1 capsular antigen production and resistance to opsonization.

When Escherichia coli strains that produce K1 capsular polysaccharide antigen at 37 degrees C were grown at 22 degrees C, K1 antigen was not detected in the supernatant or washed-cell fraction of broth cultures. Significant amounts of K1 polysaccharide were detected only when the organism was grown at temperatures of 30 degrees C or higher. Rabbits immunized with an E. coli K1 strain (serotype O18ac:K1:H7) grown at 37 degrees C produced agglutinating antibody to somatic antigen and precipitating and agglutinating antibody to capsular K1 antigen; those immunized with this strain grown at 22 degrees C produced antibody to somatic antigen, but not to K1 antigen. Antibody to somatic antigen was markedly reduced by adsorption with the organism grown at 22 degrees C, while antibody to capsular antigen was not. E. coli K1 strains grown at 37 degrees C (K1 present) resisted phagocytosis and killing if they were opsonized solely by the alternative complement pathway (ACP) using magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid-chelated serum. When these strains were grown at 22 degrees C (K1 absent), they were opsonized efficiently by the ACP (28 versus 94% killing, respectively; P less than 0.001). In addition, a non-K1 mutant of an E. coli K1 strain was opsonized efficiently by the ACP although its encapsulated K1 parent was not. Sensitivity of E. coli strains to the bactericidal activity of serum was observed in strains with and without K1 capsular antigen. These studies demonstrated that production of K1 polysaccharide antigen was regulated by environmental temperature and that K1 capsule plays an essential role in rendering the organism resistant to opsonization by the ACP.     

Differential expression of the invasion-associated locus B (ialB) gene of Bartonella bacilliformis in response to environmental cues

Bartonella bacilliformis is the causative agent of the biphasic human disease, Oroya fever. During the primary disease phase, up to 100% of the circulating erythrocytes can be parasitized and 80% lysed. During the secondary phase of this disease, bacterial invasion shifts to endothelial cells lining the vasculature. B. bacilliformis is transferred between human hosts by the sandfly, Lutzomyia verrucarum. To investigate the regulation of ialB by environmental cues signaling vector-to-host transmission; nuclease protection assays were performed to compare the amount of ialB mRNA in bacteria subjected to temperature shift, pH change, oxidative stress, or hemin limitation. The amount of ialB mRNA increased by 223-310% in acid-treated samples and decreased by 28-39% in base-treated samples as compared to bacteria kept at pH 7.2. B. bacilliformis samples showed a 56-63% and 74-80% decrease in ialB mRNA when shifted to 37 degrees C from growth temperatures of 20 and 30 degrees C, respectively. Oxidative stress (1 mM H(2)O(2)) and hemin limitation had no significant effect on mRNA levels. Determination of IalB protein amounts using SDS-PAGE and immunoblotting showed the greatest amounts of IalB under acidic conditions or at 20 degrees C. The least amount of IalB was synthesized under basic conditions or at 37 degrees C. The viability of wild-type B. bacilliformis under the various experimental culture conditions was determined and found not to affect ialB mRNA amounts in these experiments. Finally, we compared the survival of wild-type and ialB mutant B. bacilliformis and found no difference in the viability of these two strains, demonstrating that IalB does not aid bacterial survival under these conditions.     

Alkane biodegradation in Pseudomonas aeruginosa strains isolated from a polluted zone: identification of alkB and alkB-related genes

Pseudomonas aeruginosa strains that grow on crude oil as the sole source of carbon and energy were isolated from an environment in Morocco polluted by petroleum refinery effluents. The twenty isolates grew on saturated alkanes from C12 to C22. Three of the isolates were also able to grow on low molecular weight C6 to C10 n-alkanes, but the other 17 strains were not. The strains were tested for alkB and a/kB-related genes encoding alkane-1-monooxygenase (alkane hydroxylase). Oligonucleotide primers specific for the alkB gene of strain P. putida (GPo1 ) and for the alkB1 and alkB2 genes of P. aeruginosa strain PAO1 allowed amplification from the P. aeruginosa isolates of fragments similar to alkB1 and alkB2 genes of strain PAO1. Only 3 strains carried an alkB gene very similar to that of strain GPo1, and these strains were the same ones that could utilise C6 to C10 n-alkanes.     

Selection of amino-acid overproducer yeast mutants

Summary Mutants resistant to ethionine, a toxic analog of methionine, were selected by subjecting yeast cells to competition experiments under continous culture, controlled by pH, with a wide range of increasing ethionine concentrations. The mutants accumulated up to over 30 mM methionine and were able to grow in ethionine concentrations from 0.5 to 50 mM, whereas the wild-type strain stopped growing at 0.1 mM ethionine, and its free methionine pool was 0.2 mM. From ethionine-resistant mutants, strains able to overproduce threonine were isolated by selecting either in continuous or in batch cultures, the latter supplemented with 0.1–5 mM hydroxynorvaline a toxic analog of threonine. These mutants accumulated up to over 200 mM of threonine (the internal pool of threonine in the wild-type was around 5 mM), grew in minimal medium with growth rates similar to that of the wild-type, accumulated the analogs at internal concentrations proportional to the external, and accumulated other amino acids such as homoserine, aspartate, isoleucine and S-adenosyl-methionine.