Histidine starvation and adenosine 5'-triphosphate depletion in chemotaxis of Salmonella typhimurium.

Starvation for histidine prevented tumbling in Salmonella typhimurium hisF auxotrophs, including constantly tumbling strains with an additional mutation in cheB or cheZ. However, histidine-starved cheZs hisF strains were not defective in flagellar function or the tumbling mechanism since freshly starved auxotrophs tumbled in response to a variety of repellents. Tumbling in histidine-starved S. typhimurium could be restored in 13 s by addition of adenine or in 4 min by addition of histidine. Chloramphenicol did not prevent restoration of tumbling by these substances. Assays of adenosine 5'-triphosphate were performed based upon previous demonstration of adenine depletion in hisF auxotrophs starved for histidine. The adenosine 5'-triphosphate concentration dropped rapidly during the course of starvation, falling to less than 5% of the initial level as the cells ceased tumbling entirely. The change to smooth motility was prevented by 2-thiazolealanine, which inhibits phosphoribosyltransferase, thereby preventing adenine depletion during histidine starvation. These results suggest that an adenosine 5'-triphosphate deficiency was responsible for the change in tumbling frequency.     

Mutagenesis of Erwinia carotovora subsp. carotovora with bacteriophage Mu d1 (Apr lac cts62): construction of his-lac gene fusions.

The bacteriophage Mu d1(Apr lac cts62 ) obtained from an Escherichia coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid phage was utilized as a vector for phage mutagenesis in Erwinia carotovora subsp. carotovora. Among ampicillin-resistant transductants. 1.4% were auxotrophs. The synthesis of beta-galactosidase was derepressed upon starvation for histidine in two different his-lac fusion strains.     

Sequence Analysis of Mutations Arising during Prolonged Starvation of Salmonella Typhimurium

We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.     

Requirement of ATP in bacterial chemotaxis

Evidence is presented that chemotaxis requires ATP or a closely related metabolite, in addition to its known requirements of ATP for synthesis of S-adenosylmethionine (AdoMet) and maintenance of the proton motive force. Previous studies demonstrated a loss of tumbling and chemotaxis, and depletion of ATP when hisF auxotrophs of Salmonella typhimurium are starved for histidine (Galloway, R. J., and Taylor, B. L. (1980) J. Bacteriol. 144, 1068-1075). In the present study, intracellular [AdoMet], membrane potential, and [ATP] were measured in a hisF mutant of S. typhimurium. Membrane potential, determined from partitioning of [3H]tetraphenylphosphonium ion between the inside and the outside of the cell, was about -150 mV at pH 7.6, and did not decrease in histidine starvation but was slightly increased. The concentration of AdoMet decreased from 0.4 mM to 0.3 mM during starvation but when cycloleucine, an inhibitor of AdoMet synthetase, was used to decrease [AdoMet] by a similar amount in histidine-fed cells there was little change in tumbling frequency. Intracellular [ATP] was reduced from 4.5 mM to less than 0.2 mM by histidine starvation. About 0.2 mM ATP was necessary for spontaneous tumbling. A similar [ATP] was required for tumbling in arsenate-treated cells. Adenine at concentrations as low as 20 nM caused a transient increase in both tumbling frequency and [ATP] in histidine-starved cells. Thus, out of three parameters tested, only the intracellular [ATP] correlated with changes in tumbling frequency in the histidine-starved cells.     

Arginine auxotrophs of Candida albicans deficient in argininosuccinate lyase

Auxotrophic mutants of Candida albicans FC18 were induced by a combination of treatments with nitrous acid and UV irradiation. Arginine (Arg-), histidine (His-) and methionine/cysteine (MetA-) auxotrophs were recovered by this means. The Arg- auxotrophs lacked active argininosuccinate lyase (EC 4.3.2.1), the enzyme catalysing the final step in arginine biosynthesis. Thus the locus may be designated arg-4. The mutant strains bearing this mutation did not form germ tubes unless the germination medium contained arginine.     

Powerful mutator activity of the polA1 mutation within the histidine region of Escherichia coli K-12.

We examined 122 spontaneous histidine auxotrophs accumulated in overnight cultures of polA1 strains of Escherichia coli K-12 at approximate frequencies of 10(-3). One hundred and thirteen appeared to be minus frameshifts, and nine appeared to be deletions. Of the frameshift mutations, 109 affected the hisC gene, and 4 affected genes hisD, hisH, hisA, and hisI. The lack of base substitutions supported the idea that polymerase-defective polA is a minus frameshift- and deletion-type mutator. Contrary to a previous report, we did not observe superior growth of PolA auxotrophs over their prototrophic progenitors (15 auxotrophs tested). We conclude that the polA1 mutation exerts a powerful mutator activity in this specific genetic context.     

Differential Recovery of Auxotrophs After Penicillin Enrichment in Escherichia coli

Various auxotrophs are recovered from a penicillin enrichment cycle with differing efficiencies. Reconstruction experiments indicate that, under starvation conditions in the presence of penicillin, most auxotrophs undergo some death, whereas prolineless mutants are virtually immune to penicillin-induced killing.     

Partial deprivation of GTP initiates meiosis and sporulation in Saccharomyces cerevisiae

Summary We have investigated the physiological conditions under which meiosis and the ensuing sporulation of Saccharomyces cerevisiae are initiated. Initiation of sporulation occurs in response to carbon, nitrogen, phosphorus, or sulfur deprivation, and also, when met auxotrophs are partially starved for methionine, but not after starvation of other amino acid auxotrophs. It also occurs after partial starvation of pur or gua auxotrophs for guanine but not after starvation of ura auxotrophs for uracil. Under all these sporulation conditions the concentrations of both guanine nucleotides (GTP) and S-adenosylmethionine (SAM) decrease whereas those of other nucleotides show no trend. We show that the decrease of guanine nucleotides is essential for the initiation of meiosis and sporulation: when a gua auxotroph, also lacking one of the two SAM synthetases, is starved for guanine but supplemented with 0.1 mM methionine, GTP decreases while SAM slightly increases and yet the cells sporulate.     

Methionineless Death in Escherichia coli

Methionine auxotrophs of strains derived from Escherichia coli 15 lose their colony-forming ability when deprived of this amino acid. Late addition of methionine to liquid cultures did not restore plating efficiency but permitted growth of surviving cells. This phenomenon, termed methionineless death (mld), was not observed with methionine auxotrophs of E. coli strains B, W, or K(12), nor was a similar amino acidless death observed with corresponding auxotrophs of E. coli 15 for arginine, tryptophan, proline, isoleucine, and leucine. Mld was not dependent upon the genetic site determining methionine auxotrophy, nor did it affect the decarboxylation of methionine or the stability of methionyl-transfer ribonucleic acid synthetase activity of starved cells. Death was not altered by the presence of spermine or spermidine but was abolished by the methionine analogue, alpha-methylmethionine. Simultaneous starvation of another amino acid in a multiple auxotroph also significantly reduced mld, suggesting a possible role of protein synthesis. The onset of mld is correlated with a lower net increase of deoxyribonucleic acid.     

Cross-Pathway Regulation: Tryptophan-Mediated Control of Histidine and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, the starvation of tryptophan mutants for tryptophan resulted in the derepression of tryptophan, histidine, and arginine biosynthetic enzymes. This tryptophan-mediated derepression of histidine and arginine biosynthetic enzymes occurred despite the fact that the tryptophan-starved cells had a higher intracellular concentration of histidine and arginine than did nonstarved cells.     

Inducible and Constitutive β-Galactosidase Formation in Cells Recovering from Protein Synthesis Inhibition

Inducible and constitutive β-galactosidase formation and radioactive amino acid incorporation were measured in cells recovering from various treatments which inhibit protein synthesis in the cell. Undelayed β-galactosidase formation was found in stringent auxotrophs recovering from amino acid starvation, in cells recovering from glycerol or potassium starvation, and in bacteria recovering from puromycin treatment. Delayed β-galactosidase formation was found in relaxed auxotrophs recovering from amino acid starvation and in prototrophs recovering from chloramphenicol or from tetracycline treatment. The length of this delay was directly proportional to the duration of the treatment. All cells recovering from the various treatments exhibited a slightly decreased rate of β-galactosidase formation and an increase in radioactive amino acid incorporation.     

Interspecific complementation analysis by protoplast fusion of Candida tropicalis and Candida albicans adenine auxotrophs.

A protocol employing inositol starvation was used to isolate proline and adenine auxotrophs of Candida tropicalis. Interspecific hybrids between red adenine auxotrophs of C. tropicalis and Candida albicans were formed by protoplast fusion. These C. tropicalis red adenine auxotrophs were shown to fall into two complementation groups by crossing them with a known C. albicans ade1 tester strain. It is suggested that these two groups correspond to the ade1 and ade2 mutants of Saccharomyces cerevisiae and C. albicans and that these defined mutants may be useful in attempts to develop transformation systems for C. tropicalis.     

Loss of Deoxyribonucleic Acid-Thymine During Thymine Starvation of Escherichia coli

A substantial loss of deoxyribonucleic acid-thymine occurs during thymine starvation of several thymine auxotrophs derived from Escherichia coli strains B, 15, and K-12.     

Strains overproducing tRNA for histidine

Summary Hybridization analysis of total genomic DNA indicated that Escherichia coli K12 contains a single copy of the gene encoding the histidine-accepting tRNA. This gene was subcloned onto an inducible expression vector under the control of the tac promoter. Strains carrying the resulting plasmid showed five- to six-fold increased histidine-accepting activity after induction. This overproduction of tRNA^His did not effect the growth rate of the strain or lead to derepression of the histidine biosynthetic enzymes. Neither did it have an effect on mistranslation elicited by histidine starvation. However, in cells starved for histidine by the addition of -methyl histidine, the overproduction of tRNA^His interfered with the ability of the cells to recover from starvation.     

Histidine auxotrophy in commensal and disease-causing nontypeable Haemophilus influenzae

Histidine biosynthesis is one of the best studied metabolic pathways in bacteria. Although this pathway is thought to be highly conserved within and between bacterial species, a previous study identified a genetic region within the histidine operon (his) of nontypeable strains of Haemophilus influenzae (NTHI) that was more prevalent among otitis media strains than among throat commensal NTHI strains. In the present study, we further characterized this region and showed that genes in the complete his operon (hisG, -D, -C, -NB, -H, -A, -F, and -IE) are >99% conserved among four fully sequenced NTHI strains, are present in the same location in these four genomes, and are situated in the same gene order. Using PCR and dot blot hybridization, we determined that the his operon was significantly more prevalent in otitis media NTHI strains (106/121; 87.7%) than in throat strains (74/137; 54%) (prevalence ratio, 1.62; P<0.0001), suggesting a possible role in middle ear survival and/or acute otitis media. NTHI strains lacking the his operon showed attenuated growth in histidine-restricted media, confirming them as his-negative auxotrophs. Our results suggest that the ability to make histidine is an important factor in bacterial growth and survival in the middle ear, where nutrients such as histidine may be found in limited amounts. Those isolates lacking the histidine pathway were still able to survive well in the throat, which suggests that histidine is readily available in the throat environment.     

Effect of starvation on free histidine and amino acids in white muscle of milkfish Chanos chanos

Milkfish (Chanos chanos) decreased their body weight from 47 to 28 g over the 60-day period of starvation. Starvation also resulted in the reduction of muscle lipid and protein, and hepatosomatic index. The predominant free amino acid (FAA) in white muscle of milkfish was histidine, followed by taurine and glycine. In the first 25 days of starvation, no significant change in histidine was found. After 40 days of starvation, however, the histidine concentration was significantly decreased by 46%, and remained unchanged thereafter. As compared to control group fish, the 60-day-starved fish possessed only half the amount of histidine. Taurine and glycine, on the other hand, showed no significant changes throughout starvation. Taurine became the most predominant in the FAA pool after 40 days of starvation, and the concentration of 60-day-starved fish was two times higher than that of control group fish without starvation. The ratios of histidine, taurine, and glycine to total FAAs remained approximately the same although the individual contributions varied considerably to the total FAAs during starvation. The results of this study suggested that a good strategy would be to keep taurine and glycine in milkfish muscle at relatively high levels for physiological function as histidine decreased drastically for energy source under conditions of food deprivation.     

A note on auxotrophic mutants of cowpea rhizobia

We have examined a variety of common mutagens in producing auxotrophic mutants in cowpea rhizobia strains JRC23 and IRC256. While NTG (N-methyl-N-nitro-N-nitrosoguanidine), EMS (ethylmethane sulfonate), NA (nitrous acid), and UV (ultraviolet) irradiation were mutagenic with the strain JRC23, these mutagenic agents did not mutate strain IRC256. On the contrary, transposon mutagenesis with Tn5 yielded auxotrophs in strain IRC256 but not in strain JRC23, while only methionine (Met^–) auxotrophs from strain JRC23, histidine (His^–), and adenine plus thiamine (Ade^–+Thi^–) auxotrophs from strain IRC256 were isolated.     

Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), and L. Hogg. Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium. J. Bacteriol. 87:1118-1122. 1964.-When strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, is allowed to undergo thymineless death on a minimal medium, the survivors are greatly enriched in polyauxotrophic mutants. Cells were irradiated with ultraviolet light, grown in the presence of thymidine and a complete amino acid mixture, and then starved for thymidine in the absence of amino acids. Doubly auxotrophic mutants (thymine(-) amino acid(-)) may account for more than 90% of the survivors. The most reproducible results were obtained when sucrose (0.4 m) was added to both growth and starvation media. Although the percentage of mutants among the survivors increases with the time of thymine starvation, the absolute number of double auxotrophs per milliliter decreases. It is probable that the extent of cross-feeding determines both the mutant yield and the mutants types. Substrains of KM:T(-) having additional requirements for each of the following amino acids have been isolated: histidine, threonine, tyrosine, tryptophan, arginine, isoleucine, methionine, serine, and cysteine.     

Partial derepression of the isoleucine-valine enzymes during methionine starvation is Salmonella typhimurium.

Methionine starvation of methionine auxotrophs in the presence of excess branched-chain amino acids results in a partial derepression of the isoleucine and valine enzymes. Reversed-phase chromatography indicated that isoleucine, valine and leucine tRNA were altered during methionine starvation. In addition, the total tRNA isolated from cells under these conditions were undermethylated. The observed derepression may be caused by the inability of methyl-deficient tRNA's to participate adequately in normal regulatory functions.