Multiple nutritional requirements of lactobacilli: genetic lesions affecting amino acid biosynthetic pathways.

Genetic lesions responsible for amino acid requirements in several species of multiple auxotrophic lactobacilli were investigated. Systematic attempts were made to isolate mutants that could grow in the absence of each of the amino acids required by the parental strains of Lactobacillus plantarum, L. casei, L. helveticus, and L. acidophilus. After treatment with appropriate mutagens, such mutants could be obtained with respect to many but not all required amino acids. Successful isolation of mutants for a given amino acid means that a minor genetic lesion reparable by single-step mutations affects its biosynthesis; a failure to isolate mutants suggests the involvement of more extensive lesions. Analysis of these results as well as the specific requirements exhibited by the parental strains revealed certain regularities; some of the biosynthetic pathways for individual amino acids were virtually unaffected by more extensive lesions in at least species tested, whereas others were affected by more extensive lesions in at least some species. Both the number and the kind of pathways affected by extensive lesions differed appreciably among different species. Furthermore, the growth response of the parental strains to some putative amino acid precursors revealed a clear correlation between the extent of genetic lesions and the occurrence and location of a genetic block(s) for a given pathway. These findings are discussed in relation to the phylogeny, ecology, and evolution of lactic acid bacteria.     

Nucleotide sequences and genomic constitution of five tryptophan genes of Lactobacillus casei

Five trp genes, trpD, trpC, trpF, trpB, and trpA, of Lactobacillus casei were cloned by transformation of tryptophan auxotrophic mutants of the respective trp genes in Escherichia coli. These trp genes appear to constitute an operon and are located in the above order in a segment of DNA of 6,468 base pairs. The entire nucleotide sequence of this DNA segment was determined. Five contiguous open reading frames in this segment can encode proteins consisting of 341, 260, 199, 406, and 266 amino acids, respectively, in the same direction. The amino acid sequences of these proteins exhibit 25.5-50.2% homology with the amino acid sequences of the corresponding trp enzymes of E. coli. Two trp genes, trpC and trpF, from L. casei can complement mutant alleles of the corresponding genes of E. coli. However, neither the trpA gene nor the trpB gene of L. casei can complement mutations in the E. coli trpA gene and the trpB gene, respectively, suggesting that the protein products of the L. casei and E. coli trpA and trpB genes, respectively, cannot form heterodimers of tryptophan synthetase with activity. Other features of the coding and flanking regions of the trp genes are also described.     

Mutants of Escherichia coli with an Altered Tryptophanyl-Transfer Ribonucleic Acid Synthetase

Fourteen mutant strains of Escherichia coli were examined, each of which requires tryptophan for growth but is unaltered in any of the genes of the tryptophan biosynthetic operon. The genetic lesions responsible for tryptophan auxotrophy in these strains map between str and malA. Extracts of these strains have little or no ability to charge transfer ribonucleic acid (tRNA) with tryptophan. We found that several of the mutants produce tryptophanyl-tRNA synthetases which are more heat-labile than the enzyme of the parental wild-type strain. Of these heat-labile synthetases, at least one is protected against thermal inactivation by tryptophan, magnesium, and adenosine triphosphate. Two other labile synthetases which are not noticeably protected against heat inactivation by substrate have decreased affinity for tryptophan. On low levels of supplied tryptophan, these mutants exhibit markedly decreased growth rates but do not contain derepressed levels of the tryptophan biosynthetic enzymes. This suggests that the charging of tryptophan-specific tRNA is not involved in repression, a conclusion which is further substantiated by our finding that 5-methyltryptophan, a compound which represses the tryptophan operon, is not attached to tRNA by the tryptophanyl-tRNA synthetase of E. coli.     

Isolation and symbiotic characterization of aromatic amino acid auxotrophs of Sinorhizobium meliloti

Ten aromatic amino acid auxotrophs of Sinorhizobium meliloti (previously called Rhizobium meliloti) Rmd201 were generated by random mutagenesis with transposon Tn5 and their symbiotic properties were studied. Normal symbiotic activity, as indicated by morphological features, was observed in the tryptophan synthase mutants and the lone tyrosine mutant. The trpE and aro mutants fixed trace amounts of nitrogen whereas the phe mutant was completely ineffective in nitrogen fixation. Histology of the nodules induced by trpE and aro mutants exhibited striking similarities. Each of these nodules contained an extended infection zone and a poorly developed nitrogen fixation zone. Transmission electron microscopic studies revealed that the bacteroids in the extended infection zone of these nodules did not show maturation tendency. A leaky mutant, which has a mutation in trpC, trpD, or trpF gene, was partially effective in nitrogen fixation. The histology of the nodules induced by this strain was like that of the nodules induced by the parental strain but the inoculated plants were stunted. These studies demonstrated the involvement of anthranilic acid and at least one more intermediate of tryptophan biosynthetic pathway in bacteroidal maturation and nitrogen fixation in S. meliloti. The alfalfa plant host seems to provide tryptophan and tyrosine but not phenylalanine to bacteroids in nodules.     

Isoleucine Auxotrophy Due to Feedback Hypersensitivity of Biosynthetic Threonine Deaminase

Isoleucine-deficient mutants of Salmonella typhimurium were isolated. Three groups of mutants can be discerned by their nutritional requirements and enzyme patterns. (i) Mutants which grow with isoleucine alone are devoid of biosynthetic threonine deaminase (TD). (ii) Mutants growing with isoleucine and valine are devoid of transaminase B. (iii) Mutants growing with either isoleucine or threonine have normal levels of TD. However, the sensitivity of this enzyme to feedback inhibition by isoleucine is greatly enhanced. The inhibitory effect of isoleucine can be counterbalanced by high concentrations of threonine. These results indicate that the production of isoleucine in the mutants is restricted to a low level not sufficient to support the growth of the cells. This hypothesis is confirmed by studies with revertants of an isoleucine-threonine mutant. In nine revertants, wild-type properties of TD have been restored. In four revertants, the hypersensitivity of TD is unchanged, but the strains produce a greatly enhanced quantity of threonine, which is excreted into the culture medium. It follows, that hypersensitivity of TD to inhibition by isoleucine is the cause of the nutritional requirement of isoleucine-threonine mutants.     

Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants.

The ability of yeasts to grow in the presence of weak organic acid preservatives is an important cause of food spoilage. Many of the determinants of acetate resistance in Saccharomyces cerevisiae differ from the determinants of resistance to the more lipophilic sorbate and benzoate. Interestingly, we show in this study that hypersensitivity to both acetate and sorbate results when the cells have auxotrophic requirements for aromatic amino acids. In tryptophan biosynthetic pathway mutants, this weak acid hypersensitivity is suppressed by supplementing the medium with high levels of tryptophan or, in the case of sorbate sensitivity, by overexpressing the Tat2p high affinity tryptophan permease. Weak acid stress therefore inhibits uptake of aromatic amino acids from the medium. This allows auxotrophic requirements for these amino acids to strongly influence the resistance phenotypes of mutant strains. This property must be taken into consideration when using these phenotypes to attribute functional assignments to genes. We show that the acetate sensitivity phenotype previously ascribed to yeast mutants lacking the Pdr12p and Azr1p plasma membrane transporters is an artefact arising from the use of trp1 mutant strains. These transporters do not confer resistance to high acetate levels and, in prototrophs, their presence is actually detrimental for this resistance.     

Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum,L. paraplantarum,L. pentosus,and L. casei: prevalence of CO(2)-dependent auxotrophs and characterization of deficient arg genes in L. plantarum

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO(2). We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO(2) requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.     

Dominant expression of an amino-acid uptake deficiency in carrot somatic fusion hybrids

Somatic hybrids were selected previously by their ability to grow in medium containing normally inhibitory levels of the two amino acid analogs aminoethylcysteine (AEC) and 5-methyltryptophan (5MT) following fusion of protoplasts from a cell strain resistant to AEC with protoplasts resistant to 5MT. The hybrid nature of the selected clones was shown by several criteria including the presence of another resistance, azetidine-2-carboxylate (A2C), carried by one of the parental strains which was not selected for in the initial hybrid selection scheme. The characterization presented here shows that the AEC resistance in the parental strain, as well as the two somatic hybrids, was due to decreased AEC uptake. Also the 5MT resistance in the hybrids, as in the parent was caused by a feedback altered form of the tryptophan biosynthetic control enzyme, anthranilate synthase which leads to increases in free tryptophan. The A2C resistance was caused by the accumulation of free proline by a mechanism which has not been studied. These studies confirm that AEC resistance caused by decreased uptake can be expressed dominantly in protoplast fusion hybrids.Abbreviations A2C Azetidine-2-carboxylate - AEC Aminoethylcysteine - 5MT 5-methyltryptophan     

A relationship between L-serine degradation and methionine biosynthesis in Escherichia coli K12

While wild-type Escherichia coli K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can. The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source. Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine. These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine beta-lyase. The second type is exemplified by two newly isolated strains carrying mutations mapping between 89.6 and 90 min. These mutants use L-serine as carbon source, and also require methionine for growth with glucose at 37 degrees C and above. The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them.     

Regulation of tryptophan metabolism in Coprinus cinereus: Isolation and characterisation of mutants resistant to 5-fluoroindole

171 mutations conferring resistance to the indole analogue 5-fluoroindole (5 FI) were isolated in the filamentous basidiomycete fungus Coprinus cinereus. 5 FI is thought to be toxic because it is converted intracellularly to 5-fluorotryptophan (5 FT) which feedback inhibits the first enzyme of the tryptophan biosynthetic pathway, anthranilate synthase. Mutations were assigned to five loci, iar-1-iar-5 on the basis of functional analyses and mapping experiments. iar-5 mutations mapped in the anthranilate synthase structural gene and gave rise to an enzyme feedback resistant to tryptophan and its analogue. Mutants at other loci had regulatory changes. iar-1 and iar-3 mutants had elevated levels of two pathway enzymes measured (anthranilate synthase and tryptophan synthase) and were cross resistant to analogues of other aromatic amino acids suggesting that the entire aromatic pathway was derepressed. iar-3 mutants were unable to degrade metabolically derived typtophan to anthranilic acid unlike iar-1 mutants which excreted high levels of anthranilic acid. iar-2 mutants appeared to have a constitutive degradative pathway. iar-4 mutants had a blocked degradative pathway and unusual levels of tryptophan pathway enzymes.Abbreviations 5 FI 5-fluoroindole - 5 FT 5-fluorotryptophan - pFP para-fluorophenylalanine - mFT meta-fluoro-tyrosine     

An Allelic Series of Blue Fluorescent Trp1 Mutants of Arabidopsis Thaliana

Nine blue fluorescent mutants of the flowering plant Arabidopsis thaliana were isolated by genetic selections and fluorescence screens. Each was shown to contain a recessive allele of trp1, a previously described locus that encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase (PAT, called trpD in bacteria). The trp1 mutants consist of two groups, tryptophan auxotrophs and prototrophs, that differ significantly in growth rate, morphology, and fertility. The trp1 alleles cause plants to accumulate varying amounts of blue fluorescent anthranilate compounds, and only the two least severely affected of the prototrophs have any detectable PAT enzyme activity. All four of the trp1 mutations that were sequenced are G to A or C to T transitions that cause an amino acid change, but in only three of these is the affected residue phylogenetically conserved. There is an unusually high degree of sequence divergence in the single-copy gene encoding PAT from the wild-type Columbia and Landsberg erecta ecotypes of Arabidopsis.     

Analysis of the temperature-sensitive mutation of Escherichia coli pantothenate kinase reveals YbjN as a possible protein stabilizer

Pantothenate kinase (PanK), a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway, catalyzes the rate-limiting phosphorylation of pantothenic acid to form phosphopantothenate during CoA biosynthesis. Escherichia coli ts9 strain manifests temperature-sensitive phenotype on LB media due to its mutation in the coaA gene (coaA1). Sequencing analysis revealed that coaA1 arises from a single base pair mutation that results in an amino acid change, L236F. This change, located proximate to the ATP binding site of CoaA, destabilizes both enzymatic activity and structural integrity or stability of the mutant protein in vitro. Spontaneously, revertants of ts9 were occasionally found on LB medium plates. Two groups of revertants were isolated: for those that can grow at 40 degrees C, a reversion of the original amino acid mutation L236F to L236L or other amino acid (such as L236C) occurs; for those that can grow at 37 degrees C but not 40 degrees C, a mutation at another gene or intergenic suppression is strongly indicated. Towards genetic identification of genes that might interact with coaA1, ybjN, which encodes a putative sensory transduction regulator protein, and whose over-expression is capable of ameliorating the temperature-sensitive phenotype of the structurally unstable CoaA1 or CoaA[L236F], was isolated. Over-expression of ybjN appears to suppress the temperature-sensitive phenotype of several other temperature-sensitive mutations, including coaA14 (carried by DV51 strain), coaA15 (carried by DV70 strain), and ilu-1, suggesting it not only helps CoaA1, but possibly works as a general stabilizer for some other unstable proteins.     

Polypeptide Composition of Urea- and Heat-Resistant Mutants of Poliovirus Types 1 and 2

Five urea-resistant and two heat-resistant mutants of poliovirus types 1 and 2 were isolated and their structural and nonstructural polypeptides compared to those of their wild-type, parental strains in an attempt to correlate mutant phenotypes with alterations in specific capsid polypeptides. Four of the seven mutants were found to contain polypeptides which differed in molecular weight from their respective parental viruses. However, resistance of virions to heat- or urea-inactivation could not be attributed to changes in particular capsid polypeptides because alterations were detected in all but one of the capsid components. For two of the urea-resistant mutants and one heat-resistant mutant, no differences were found in the molecular weights of the capsid and noncapsid polypeptides. These results, and the fact that at least 12 selective treatments were required to obtain stable mutants, indicate that: (i) such phenotypes probably can be expressed by mutations affecting one or more of the larger capsid polypeptides, and (ii) such phenotypes reflect multiple mutational steps.     

Polar and Antipolar Mutants in the Tryptophan Operon of Salmonella typhimurium

Polar mutations in trpA, the first structural gene of the tryptophan operon of Salmonella typhimurium, have an uncoordinate effect on the expression of the distal genes, with trpB, the second gene, being more drastically affected than the last three. A number of these polar mutant strains grow very poorly on anthranilic acid-supplemented minimal medium. By selecting for more rapid growth in the presence of anthranilic acid, secondary mutant clones showing a correction of the polar effect were isolated. A few of these were analyzed and shown to contain deletions of various segments of the trpA gene. Ten randomly isolated deletion mutants missing various segments of the trp operon were analyzed for possible pleiotropic effects. Five of them showed a pleiotropic effect of some sort and five did not. Of those showing pleiotropic effects, one had lost the promotor-like elements necessary to initiate expression of the operon, three showed possible antipolar effects, and one showed both polar and antipolar effects simultaneously.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in amino acid transport System L

The Chinese hamster ovary cell line CHO-tsH1 is a temperature-sensitive leucyl-tRNA synthetase mutant that shows temperature-dependent regulation of the amino acid transport responsible for accumulating leucine, System L. At nonpermissive temperatures, CHO-tsH1 cells are unable to grow because they are unable to incorporate leucine into protein. As a result, System L activity is increased. We have isolated mutants from CHO-tsH1 that have constitutively de-repressed System L activity. These mutants are temperature-resistant as a result of increased intracellular steady-state accumulations of System L-related amino acids, which compensates for the defective synthetase activity. In this study, we have subjected one of these regulatory mutant cell lines (C11B6) to a tritium-suicide selection, in which L-[3H]leucine was used as a toxic substrate. Three mutant cell lines, C4B4, C5D9, and C9D9 that showed reduced System L transport activity were isolated. The decreases in the initial rates of System L transport activity lead to reduced steady-state accumulations of System L-related amino acids. In contrast to the parental cell line, C11B6, the transport-defective mutants are temperature-sensitive because the reduced intracellular pool of leucine can no longer compensate for the defective synthetase activity.     

Structure of the Escherichia coli 0104 polysaccharide and its identity with the capsular K9 polysaccharide

Spontaneous mutants of Rhizobium leguminosarum biovar viciae strain C1204b were selected for their ability to tolerate 0.2 M NaCl, a growth-inhibiting level of salt for the parental strain. Transposon-mediated salt-sensitive mutants of strain C1204b were screened for their inability to grow in 0.08 M NaCl. Quantitation of the free-amino acid pools in the mutants grown in NaCl revealed a dramatic increase in glutamine, serine, glutamate and proline, and to a lesser extent alanine and glycine in the salt-tolerant mutants in comparison with the parental strain exposed to NaCl; but only glutamate and proline increased in the salt-sensitive mutants under NaCl stress. Extracellular polysaccharide levels were quantitated for the salt-tolerant mutants and determined to be approximately two-fold higher than for the parental strain. Although the mutations that occurred in the NaCl-tolerant and NaCl-sensitive strains did not interfere with nodule formation, no nitrogenase activity could be observed in the NaCl tolerant mutants as evaluated by acetylene reduction.     

Genetic Transformation of Biosynthetically Defective Neisseria gonorrhoeae Clinical Isolates

Deoxyribonucleic acid (DNA) and chemically defined media were used in transformation tests of 51 strains of Neisseria gonorrhoeae which exhibited various biosynthetic defects when isolated from patients. These auxotrophic gonococci had one or more nutritional requirements involving proline, methionine, arginine, hypoxanthine, uracil, and thiamine pyrophosphate (THPP). DNA from a clinical isolate which did not require these compounds for growth on defined medium transformed each of the auxotrophic markers of all 51 recipient populations. Ten isolates had defects involving the synthesis of THPP; four strains (designated Thp(-)) had a growth requirement that was satisfied only by THPP, whereas the requirement of six strains (designated Thi(-)) was satisfied by either thiamine or THPP. DNA from Thp(-) donors elicited transformation of Thp(-) as well as Thi(-) recipients. Reciprocally, DNA from a Thi(-) donor transformed both Thi(-) and Thp(-) recipients. Furthermore, DNA from other auxotrophic gonococci had transforming activity for some phenotypically similar auxotrophic recipients. The findings indicate the existence of various nonidentical genetic defects which block reactions in the biosynthesis of proline, methionine, arginine, hypoxanthine, and THPP. Routine cultures from patients with gonorrhea were the source of these auxotrophic strains of N. gonorrhoeae; the various nutritional requirements were identified by a recently described system of gonococcal auxotyping. The transformation test results verify the hereditary basis of the auxotypes, establish that many different mutations exist in potentially virulent gonococci, and illustrate the value of these auxotrophic mutants for studies of the genetic structure and evolution of natural populations of gonococci.     

Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit.

Suppressors of the blue fluorescence phenotype of the Arabidopsis trp1-100 mutant can be used to identify mutations in genes involved in plant tryptophan biosynthesis. Two recessive suppressor mutations define a new gene, TRP4. The trp4 mutant and the trp1-100 mutant are morphologically normal and grow without tryptophan, whereas the trp4; trp1-100 double mutant requires tryptophan for growth. The trp4; trp1-100 double mutant does not segregate at expected frequencies in genetic crosses because of a female-specific defect in transmission of the double mutant genotype, suggesting a role for the tryptophan pathway in female gametophyte development. Genetic and biochemical evidence shows that trp4 mutants are defective in a gene encoding the beta subunit of anthranilate synthase (AS). Arabidopsis AS beta subunit genes were isolated by complementation of an Escherichia coli anthranilate synthase mutation. The trp4 mutation cosegregates with one of the genes, ASB1, located on chromosome 1. Sequence analysis of the ASB1 gene from trp4-1 and trp4-2 plants revealed different single base pair substitutions relative to the wild type. Anthranilate synthase alpha and beta subunit genes are regulated coordinately in response to bacterial pathogen infiltration.