Constitutive mutants for dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Constitutive mutants for dextransucrase were isolated from cells of Leuconostoc mesenteroides NRRL B-512F by treatment with N-methyl-N′-nitro-N-nitrosoguanidine, growing on an agar plate containing glucose as a carbon source and overlaying a soft agar with sucrose and tetracycline. These mutants were able to produce the enzyme in a liquid media containing sugars other than sucrose, such as glucose, fructose and maltose, without simultaneous synthesis of dextran. The enzyme activity of one mutant strain, SH 3002, was 2- to 3-fold higher than that of the wild strain grown on sucrose. When the concentration of glucose in the medium was increased from 2 to 4%, a 1.7-fold increase of enzyme activity was obtained for the mutant, whereas only a slight increase of the activity was observed on sucrose for both the wild strain and the mutant.     

Improvement of ethanol production of themophilic Clostridium sp. by Mutation

Three thermophilic Clostridium strains were isolated from soil as cellulose-fermenting bacteria wich produced ethanol, lactic acid, and acetic acid from cellulose at 60°C. To increase ethanol productivity, strains no. 187 was mutated with N-methyl-N′-nitro-N-nitrosguanidine. The resultant mutant, no 187-102-27, was superior to the original strain in ethanol production, and produced less lactic and acetic acid. The activities of some enzymes involved in the biosynthesis of the lactic and acetic acid of mutant no. 187-102-27 were lower than those of the original strain. These results are highly consistent with the acid production of the mutant strain being low.     

Ribonucleic acid-permeable mutant of Escherichia coli

An RNA-permeable mutant was isolated from a tryptophan amber auxotrophic strain of Escherichia coli after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rationale of the isolation was based on the suppression of an amber mutation. A strain was selected, which could grow in minimal medium supplemented with transfer RNA prepared from an suI-carrying strain but not from an su⁻ strain. This mutant incorporated³H-labeled bulk RNA into the cells at a rate 40 times higher than did the parent strain. The level of tryptophan requirement, susceptibility to the lytic action of lysozyme and RNase activity in the culture medium of the mutant strain did not differ from those of the parent strain. The mutant strain incorporated ³H-labeled ribosomal RNA equally as well as it incorporated ³H-labeled transfer RNA and the incorporation was competitively inhibited by any species of cold RNA. However, the fate of ³H-labeled rRNA after incorporation resulted in degradation to yield acid-soluble fragments whereas tRNA after incorporation remained intact in the cell.     

Cell wall mutants of Saccharomyces cerevisiae with increased digestibility by cell wall lytic enzymes and protein extractability

Yeast cell wall mutants were obtained by mutagenesis of Saccharomyces cerevisiae X2180-1A, a haplid strain, with N-methyl-N′-nitro-N-nitrosoguanidine. The two S. cerevisiae mutants showed considerable morphological changes and digestibilities by lytic enzymes. Sequential extractions of proteins and polysaccharides from the mutant and wild type cells indicate that the mutants have high protein extractability and lack some wall proteins as well as some polysaccharide fractions.     

Isolation and Characterization of an Oxygen Sensitive Mutant of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 was isolated by NTG mutagenesis. It was defective in N₂ fixation under 3% O₂ level, while under 1% O₂ it was almost as active as the parent strain IRBG 46. The mutant was also found to be a slow grower with reduced respiratory activity, low azide tolerance and no catalase activity. However, it did not differ from its parent strain with respect to nitrate respiration. Under symbiotic condition the mutant formed smaller, light green nodules as compared to bigger, dark green nodules formed by the wild type strain. The mutant was also defective in N₂ fixation under symbiotic condition. Complementation analysis showed that the mutation might be in either fixL or fixJ gene which are involved in O₂ regulation of nif/fix gene expression. A possible role of all these factors in conferring a highly O₂ tolerant nitrogen fixing system in the organism, has been discussed.     

Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA.     

Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition

Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition.     

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.     

Isolation and partial characterization of a mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase.

A mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase (EC 1.6.1.1) was isolated by assaying activity in clones of cells mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine. The mutant is missing both energy-independent and energy-dependent transhydrogenase, but has normal NADH dehydrogenase and ATPase activities. Compared to the parental strain, the mutant has normal growth rates with glucose, glycerol, or succinate aerobically and with glucose or glycerol plus fumarate anaerobically. The aerobic growth yield with limiting glucose concentrations is also normal. These growth properties indicate that the enzyme is not an essential source of NADPH or ATP in vivo.     

Comparisons of Characteristics of Mercury-Tolerant Bacterium Pseudomonas oleovorans G-1 and Its Mercury-Sensitive Mutant Strain

An Hg²⁺-sensitive mutant strain was isolated from an Hg²⁺-tolerant bacterium Pseudomonas oleovorans G-1 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. The Hg²⁺-sensitive mutant strain was about 10-times as sensitive to Hg²⁺ as the parent strain. Moreover, the mutant strain was considerably more sensitive to Cr⁶⁺ than the parent strain, but it did not show an appreciable change in sensitivity to Cd²⁺ and Cu²⁺. The mutant strain was considerably more sensitive to antibiotics achromycin, chloramphenicol and streptomycin than the parent strain. A more rigid structure was observed in the cell envelope of the mutant strain than the parent strain under transmission electron microscope. Higher amounts of DNA but less protein and RNA were found in the mutant strain compared to the parent strain. Disc electrophoretic patterns showed some differences in protein bands between the parent and mutant strain.     

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.     

Differential Expression of Uptake Hydrogenase Activity in Free Living Cells and Nodule Bacteroids of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 in which N₂ fixation ability was affected, was previously isolated by NTG mutagenesis. Now, the mutation has been shown to affect H₂- uptake hydrogenase (Hup) activity under symbiotic conditions. However, free living Hup activity remained unaffected. Thus the mutant is Hup^- under symbiotic conditions and Hup⁺ under free living condtions. A possible regulatory link between N₂ fixation and H₂ uptake system has been discussed.     

Characterization of heterotrophic nitrifying bacteria with respiratory ammonification and denitrification activity – Description of Paenibacillus uliginis sp. nov., an inhabitant of fen peat soil and Paenibacillus purispatii sp. nov., isolated from a spacecraft assembly clean room

In the course of studying the influence of N-fertilization on N₂ and N₂O flux rates in relation to soil bacterial community composition of a long-term fertilization experiment in fen peat grassland, a strain group was isolated that was related to a strain isolated from a spacecraft assembly clean room during diversity studies of microorganisms, which withstood cleaning and bioburden reduction strategies. Both the fen soil isolates and the clean room strain revealed versatile physiological capacities in N-transformation processes by performing heterotrophic nitrification, respiratory ammonification and denitrification activity.     

Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus

The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestris pv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from the xpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into the xpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsN gene or by introducing extra copies of wild-type xpsL or xpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL or xpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with the xpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of the xpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

pbpB, a gene coding for a putative penicillin-binding protein, is required for aerobic nitrogen fixation in the cyanobacterium Anabaena sp. strain PCC7120

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N₂, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.     

Molecular cloning and characterization of the gene encoding N′-[(5′-phosphoribosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (BBM II) isomerase from Arabidopsis thaliana

We have isolated an Arabidopsis BBM II isomerase cDNA from an Arabidopsis cDNA library, by means of functional complementation of the E. coli hisA mutant strain HfrG6. The isolated cDNA encodes a polypeptide of 304 amino acids with a calculated molecular weight of 33?363. Sequence comparison with the HIS6 proteins of yeasts revealed that Arabidopsis BBM II isomerase contains an N-terminal extension of approximately 40 amino acids that shows the general properties of chloroplast transit peptides. This finding is consistent with the localization of other histidine biosynthetic enzymes, such as imidazoleglycerolphosphate dehydratase and histidinol dehydrogenase, in the chloroplasts in higher plants. The primary structure of the mature protein was 50% and 42% identical, respectively, to the HIS6 proteins of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, while no prominent sequence similarity to the bacterial BBM II isomerase was found. That the isolated Arabidopsis cDNA actually encodes a functionally active BBM II isomerase activity was confirmed in an in vitro enzyme assay using a crude extract prepared from strain HfrG6 transformed with the Arabidopsis BBM II isomerase cDNA.     

Maize Root Lectins Mediate the Interaction with Herbaspirillum seropedicae via N-Acetyl Glucosamine Residues of Lipopolysaccharides

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.     

Extracellular production of palmitoleic triglycerides by a yeast,Trichosporon

A yeast belonging to Trichosporon which produces triglycerides extracellularly was isolated. This strain accumulated palmitoleic triglycerides from ethyl palmitate used as a sole carbon source. To increase the level of extracellular palmitoleic triglycerides, mutant strains which supported growth of unsaturated-fatty-acid-auxotrophic cells (Saccharomyces cerevisiae KD115) layered on the mutant colonies were screened. The mutant strain excreted palmitoleic acid as triglyceride form at a significantly high level, corresponding to about double level of the parental strain.