Construction of a urocanic acid-producing strain of Serratia marcescens by transduction.

In Serratia marcescens, the mutation responsible for triazolealanine (TRA) resistance was transferred from a TRA-resistant mutant to a urocanase-less mutant by PS20-mediated transduction. The two crosses were performed using as donors two TRA-resistant mutants, whose phenotypes included increased levels of histidine-biosynthetic enzymes and feedback-insensitive phosphoribosyltransferase. In one cross, TRA-resistant transductants were urocanase-less mutants having only increased levels of the enzymes and barely detectable levels of urocanic acid. In the other cross, the transductants were urocanase-less mutants having both phenotypes of the donor, and most produced high concentrations (10.5 mg/ml) of urocanic acid.     

Regulation of hut enzymes and intracellular protease activities in Vibrio alginolyticus hut mutants

The production of alkaline protease, collagenase and histidine utilization (Hut) enzymes by Vibrio alginolyticus wild-type, hutH1 and hutU1 strains was investigated. Alkaline protease synthesis was stimulated by histidine and urocanic acid in the wild-type and hutU1 strains. The hutH1 mutant alkaline protease production was stimulated by urocanic acid and not by histidine. The Hut enzymes in the wild-type strain were coordinately induced by histidine. Urocanase and formimino-hydrolase were induced by histidine in the hutH1 mutant which lacked histidase and was not able to convert histidine to urocanic acid. Collagenase production in peptone medium was inhibited in the hut mutants. It is concluded that in V. alginolyticus urocanic acid regulates alkaline protease synthesis but that the Hut enzymes are induced by histidine. The involvement of the Hut genetic system in the regulation of alkaline protease and collagenase synthesis is discussed.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

Ribitol catabolic pathway in Klebsiella aerogenes

In Klebsiella aerogenes W70, there is an inducible pathway for the catabolism of ribitol consisting of at least two enzymes, ribitol dehydrogenase (RDH) and d-ribulokinase (DRK). These two enzymes are coordinately controlled and induced in response to d-ribulose, an intermediate of the pathway. Whereas wild-type K. aerogenes W70 are unable to utilize xylitol as a carbon and energy source, mutants constitutive for the ribitol pathway are able to utilize RDH to oxidize the unusual pentitol, xylitol, to d-xylulose. These mutants are able to grow on xylitol, presumably by utilization of the d-xylulose produced. Mutants constitutive for l-fucose isomerase can utilize the isomerase to convert d-arabinose to d-ribulose. In the presence of d-ribulose, RDH and DRK are induced, and such mutants are thus able to phosphorylate the d-ribulose by using the DRK of the ribitol pathway. Derivatives of an l-fucose isomerase-constitutive mutant were plated on d-arabinose, ribitol, and xylitol to select and identify mutations in the ribitol pathway. Using the transducing phage PW52, we were able to demonstrate genetic linkage of the loci involved. Three-point crosses, using constitutive mutants as donors and RDH(-), DRK(-) double mutants as recipients and selecting for DRK(+) transductants on d-arabinose, resulted in DRK(+)RDH(+)-constitutive, DRK(+)RDH(+)-inducible, and DRK(+)RDH(-)-inducible transductants but no detectable DRK(+)RDH(-) constitutive transductants, data consistent with the order rbtC-rbtD-rbtK, where rbtC is a control site and rbtD and rbtK correspond to the sites for the sites for the enzymes RDH and DRK, respectively.     

Genetic Analysis of an Escherichia coli Syndrome

A mutant strain of Escherichia coli that fails to recover from prolonged (72 hr) starvation also fails to grow at 43 C. Extracts of this mutant strain show an increased ribonuclease II activity as compared to extracts of the parental strain, and stable ribonucleic acid is degraded to a larger extent in this strain during starvation. Ts(+) transductants and revertants were tested for all the above-mentioned phenotypes. All the Ts(+) transductants and revertants tested behaved like the Ts(+) parental strain, which suggests that all the observed phenotypes are caused by a single sts (starvation-temperature sensitivity) mutation. The reversion rate from sts(-) to sts(+) is rather low but is within the range of reversion rates for other single-site mutations. Three-point transduction crosses located this sts mutation between the ilv and rbs genes. The properties of sts(+)/sts(-) merozygotes suggested that the Ts(-) phenotype of this mutation is recessive.     

Auxotrophic mutations related to symbiotic properties of Rhizobium meliloti strain L5-30.

Mutants isolated from effective R. meliloti strain L5-30 which required histidine (his-240), arginine+uracil (arg-55) and cysteine (cys-243, cys-244 and cys-246) showed also loss of effectiveness. Mutant requiring isoleucine+valine (ilv-74) was non-infective. Relation of the metabolic deficiency to the symbiotic properties of these mutants was tested comparing symbiotic response of their prototrophic revertants and transductants. It was found that all revertants and transductants of the strain his-240 were effective which suggests that histidine deficiency was the cause of their ineffectiveness. All revertants and transductants of the cysteine mutants were still ineffective. This result indicates two independent mutations which were not cotransductible. Prototrophic revertants of the mutant arg-55 were ineffective whereas 56.9 percent of transductants appeared effective suggesting close linkage of two mutations. i.e. auxotrophic and the other concerned with symbiotic effectiveness. Though one of 69 prototrophic transductants obtained from the non-nodulating mutant ilv-74 remained non-nodulating, it seems that changes in nodulating ability of the mutant are related to the auxotrophic requirements.     

Characterization of Lac+ Transductants of Streptococcus lactis

A phage-mediated transducing system was used in studying certain physiological characteristics of S. lactis C2 wild type, lactose-negative mutants, and lactose-positive transductants. Lac(-) mutants, obtained by acriflavine treatment of the wild type, were similar to the wild type in all characteristics tested except they lacked beta-D-phosphogalactoside galactohydrolase (beta-Pgal) and could not transport [(14)C]lactose; they also had approximately 10% of the proteolytic ability than wild-type cells. The lactose-fermenting characteristic was transduced from the wild type to Lac(-) mutants. The Lac(+) transductants obtained were similar to the wild-type parent with respect to lactose fermentation and level of beta-Pgal activity (0.186 U of protein per mg). These transductants, however, had not regained full proteolytic ability and were similar to the Lac(-) mutant in this respect. Lactic acid production of the transductants in milk was approximately two-thirds that of the wild type. Data suggest that both the lactose-fermenting and proteolytic characters are carried on extrachromasomal particles (plasmids).     

Segregation for endosperm lysine in F2, F3 and F4 progeny from a cross of in vitro-selected and unselected cultivar of rice

Summary Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F₂ populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R² 0.52 and 0.56). The F₂ segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F₃ and F₄ data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility.Research done under the auspices of the USDA, ARS, Plant Sciences Institute, Plant Molecular Biology Laboratory, Beltsville, MD 20705, USA     

Mutant identifying a third recombination group in a bunyavirus.

Only two recombination groups have been reported in genetic analyses of ts mutants of 10 different bunyaviruses from the Bunyamwera and California encephalitis serogroups, although three groups are expected from the tripartite structure of the genome of all members of the family Bunyaviridae. We describe now a ts mutant of Maguari virus, MAGts23(III), which recombined in both vertebrate (BHK-21) and invertebrate (Aedes albopictus) cells with mutants representing recombination groups I and II of this Bunyamwera serogroup virus. In addition, MAGts23(III) recombined with two mutants MAGts20 and MAGts21, provisionally identified as double mutants by their failure to recombine with group I or group II mutants, Mutant MAGts23(III) therefore represents a third bunyavirus recombination group. Mutant MAGts23(III) differed phenotypically from other bunyavirus mutants by growth restriction in BS-C-1 cells. Wild-type recombinants were obtained in the heterologous cross of MAGts23(III) and a group II mutant of Bunyamwera virus, but not in a cross with a group I mutant. The recombinants had the G protein of the Maguari virus parent and the N protein of the Bunyamwera virus parent. Analysis of the phenotypes of clones isolated at permissive temperature from the progeny of the other cross [MAGts23(III) and a group I mutant of Bunyamwera virus] indicated that recombination occurred in this cross, but that the possible recombinant phenotypes were not recovered with equal frequency. As a consequence, it has not been possible to obtain a gene assignment for group III from genetic data alone.     

Efficiency of Nitrous Acid as An Inactivating and Mutagenic Agent of Intact Tobacco Mosaic Virus and Its Isolated Nucleic Acid

When tobacco mosaic virus (TMV) and its isolated nucleic acid (TMV-RNA) were treated with nitrous acid, the nucleic acid was inactivated six times faster than the intact virus. Inactivation of both the infectious entities was exponential with treatment time to 0.1% level of survival. Eight different mutant phenotypes were scored after inactivation of TMV and TMV-RNA to 50, 10, 1.0, and 0.1% survival levels. Significantly more mutants in relation to unaltered isolates were induced at all levels of survival upon nitrous acid treatment of TMV than of TMV-RNA. Furthermore, the proportion of two specific mutant phenotypes was significantly greater in treated TMV than in treated TMV-RNA. No qualitative differences, however, were observed between the mutational spectra of nitrous acid-treated TMV and TMV-RNA. These results indicate that, in the intact virus, the viral capsid protects some of the sites involved in lethality; thus, proportionately more mutants are induced on nitrous acid treatment of TMV versus TMV-RNA.     

Disrupting the Acyl Carrier Protein/SpoT interaction in vivo: identification of ACP residues involved in the interaction and consequence on growth

In bacteria, Acyl Carrier Protein (ACP) is the central cofactor for fatty acid biosynthesis. It carries the acyl chain in elongation and must therefore interact successively with all the enzymes of this pathway. Yet, ACP also interacts with proteins of diverse unrelated function. Among them, the interaction with SpoT has been proposed to be involved in regulating ppGpp levels in the cell in response to fatty acid synthesis inhibition. In order to better understand this mechanism, we screened for ACP mutants unable to interact with SpoT in vivo by bacterial two-hybrid, but still functional for fatty acid synthesis. The position of the selected mutations indicated that the helix II of ACP is responsible for the interaction with SpoT. This suggested a mechanism of recognition similar to one used for the enzymes of fatty acid synthesis. Consistently, the interactions tested by bacterial two-hybrid of ACP with fatty acid synthesis enzymes were also affected by the mutations that prevented the interaction with SpoT. Yet, interestingly, the corresponding mutant strains were viable, and the phenotypes of one mutant suggested a defect in growth regulation.     

Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants

Degradation of active C(19)-gibberellins (GAs) by dioxygenases through 2beta-hydroxylation yields inactive GA products. We identified two genes in Arabidopsis (AtGA2ox7 and AtGA2ox8), using an activation-tagging mutant screen, that encode 2beta-hydroxylases. GA levels in both activation-tagged lines were reduced significantly, and the lines displayed dwarf phenotypes typical of mutants with a GA deficiency. Increased expression of either AtGA2ox7 or AtGA2ox8 also caused a dwarf phenotype in tobacco, indicating that the substrates for these enzymes are conserved. AtGA2ox7 and AtGA2ox8 are more similar to each other than to other proteins encoded in the Arabidopsis genome, indicating that they may constitute a separate class of GA-modifying enzymes. Indeed, enzymatic assays demonstrated that AtGA2ox7 and AtGA2ox8 both perform the same GA modification: 2beta-hydroxylation of C(20)-GAs but not of C(19)-GAs. Lines containing increased expression of AtGA2ox8 exhibited a GA dose-response curve for stem elongation similar to that of the biosynthetic mutant ga1-11. Double loss-of-function Atga2ox7 Atga2ox8 mutants had twofold to fourfold higher levels of active GAs and displayed phenotypes associated with excess GAs, such as early bolting in short days, resistance to the GA biosynthesis inhibitor ancymidol, and decreased mRNA levels of AtGA20ox1, a gene in the GA biosynthetic pathway.     

Mutational analysis of the Helicobacter pylori carbonic anhydrases

In the gastric microenvironment, Helicobacter pylori is exposed to bicarbonate, urea and acid. Here it is demonstrated that both H. pylori carbonic anhydrases (CAs) are required for maintaining urease activity and therefore influence H. pylori urea resistance at neutral pH. Furthermore, the beta-CA is required for acid resistance as indicated by a growth defect of the corresponding mutant at low pH. The alpha- and beta-CA mutants as well as the double mutant were more resistant to bicarbonate, indicating that both enzymes are involved in bicarbonate metabolism. These phenotypes support important CA-functions in H. pylori urea and bicarbonate metabolism and acid resistance. Thus, both CA enzymes might be required for survival in the gastric niche.     

The Saccharomyces cerevisiae YBR159w gene encodes the 3-ketoreductase of the microsomal fatty acid elongase

The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes required for very long-chain fatty acid (VLCFA) synthesis. Mutants lacking the YBR159w gene display many of the phenotypes that have previously been described for mutants with defects in fatty acid elongation. These phenotypes include reduced VLCFA synthesis, accumulation of high levels of dihydrosphingosine and phytosphingosine, and accumulation of medium-chain ceramides. In vitro elongation assays confirm that the ybr159Delta mutant is deficient in the reduction of the 3-ketoacyl intermediates of fatty acid elongation. The ybr159Delta mutant also displays reduced dehydration of the 3-OH acyl intermediates of fatty acid elongation, suggesting that Ybr159p is required for the stability or function of the dehydratase activity of the elongase system. Green fluorescent protein-tagged Ybr159p co-localizes and co-immunoprecipitates with other elongating enzymes, Elo3p and Tsc13p. Whereas VLCFA synthesis is essential for viability, the ybr159Delta mutant cells are viable (albeit very slowly growing) and do synthesize some VLCFA. This suggested that a functional ortholog of Ybr159p exists that is responsible for the residual 3-ketoreductase activity. By disrupting the orthologs of Ybr159w in the ybr159Delta mutant we found that the ybr159Deltaayr1Delta double mutant was inviable, suggesting that Ayr1p is responsible for the residual 3-ketoreductase activity.     

Simultaneous enhancement of thermostability and catalytic activity of phospholipase A(1) by evolutionary molecular engineering

The thermal stability and catalytic activity of phospholipase A(1) from Serratia sp. strain MK1 were improved by evolutionary molecular engineering. Two thermostable mutants were isolated after sequential rounds of error-prone PCR performed to introduce random mutations and filter-based screening of the resultant mutant library; we determined that these mutants had six (mutant TA3) and seven (mutant TA13) amino acid substitutions. Different types of substitutions were found in the two mutants, and these substitutions resulted in an increase in nonpolar residues (mutant TA3) or in differences between side chains for polar or charged residues (mutant TA13). The wild-type and mutant enzymes were purified, and the effect of temperature on the stability and catalytic activity of the enzymes was investigated. The melting temperatures of the TA3 and TA13 enzymes were increased by 7 and 11 degrees C, respectively, compared with the melting temperature of the wild-type enzyme. Thus, we found that evolutionary molecular engineering was an effective and efficient approach for increasing thermostability without compromising enzyme activity.     

串珠镰孢霉硝酸盐还原途径酶的遗传研究*

通过对菌株突变体有性杂交后代的检测方法,对串珠镰孢霉Fusariummoniliforme氮代谢过程中硝酸盐还原途径相关酶基因间关系进行研究。串珠镰孢霉含钼协同因子突变体缺陷型(nitB)与亚硝酸盐还原酶缺陷型突变体(nitC)间的杂交结果显示,在不同的交配群以及在相同交配群不同寄主上的分离菌株中,控制这两种酶的基因有两种类型,并据此提出细胞核基因和细胞质基因共同调控硝酸盐还原途径酶的假说。当杂交后代出现四种表型(nitD:nitB:nitC:wt=1:1:1:1)时,表明这两种酶的遗传受核基因调控,分离时两种基因自由组合;当杂交后代仅有两种表型时,表现为父本表型隐藏,双基因缺陷型(表型同主氮调节基因缺陷型nitD)表型不出现,即母本表现型:野生型为1:1,表明这种遗传类型除受核基因的控制外,还存在细胞质基因的影响。     

Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions

In plants, peroxisomes are the primary site of fatty acid β-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4(Col) and acx1acx3acx4(Col) mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial β-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.