Interaction of gene <Emphasis Type="Italic">HSM3</Emphasis> with genes of the epistatic <Emphasis Type="Italic">RAD6</Emphasis> group in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In eukaryotes, damage tolerance of matrix DNA is mainly determined by the repair pathway under the control of the RAD6 epistatic group of genes. This pathway is also a main source of mutations generated by mutagenic factors. The results of our recent studies show that gene HSM3 participating in the control of adaptive mutagenesis increases the frequency of mutations induced by different mutagens. Mutations rad18, rev3, and mms2 controlling various stages of the RAD6 pathway are epistatic with mutation hsm3 that decreases UV-induced mutagenesis to the level typical for single radiation-sensitive mutants. The level of mutagenesis in the double mutant srs2 hsm3 was lower than in both single mutants. Note that a decrease in the level of mutagenesis relative to the single mutant srs2 depends on the mismatch repair, since this level in the triple mutant srs2 hsm3 pms1 corresponds to that in the single mutant srs2. These data show that the mutator phenotype hsm3 is probably determined by processes occurring in a D loop. In a number of current works, the protein Hsm3 was shown to participate in the assembly of the proteasome complex S26. The assembly of proteasomes is governed by the N-terminal domain. Our results demonstrated that the Hsm3 protein contains at least two domains; the N-terminal part of the domain is responsible for the proteasome assembly, whereas the C-terminal portion of the protein is responsible for mutagenesis.     

Synthesis of <Emphasis Type="SmallCaps">dl</Emphasis>-tryptophan by modified broad specificity amino acid racemase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.     

The mouse mutants <Emphasis Type="Italic">recoil wobbler</Emphasis> and <Emphasis Type="Italic">nmf373</Emphasis> represent a series of <Emphasis Type="Italic">Grm1</Emphasis> mutations

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP₃). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.     

Chloroplast genome aberration in micropropagation-derived albino <Emphasis Type="Italic">Bambusa edulis</Emphasis> mutants, <Emphasis Type="Italic">ab1</Emphasis> and <Emphasis Type="Italic">ab2</Emphasis>

Two albino mutants (ab1 and ab2) have been derived from long-term shoot proliferation of Bambusa edulis. Based on transmission electronic microscopy data, the chloroplasts of these mutants were abnormal. To study the mutation of gene regulation in the aberrant chloroplasts, we designed 19 pairs of chloroplast-encoded gene primers for genomic and RT-PCR. Only putative NAD(P)H-quinone oxidoreductase chain 4L (ndhE; DQ908943) and ribosomal protein S7 (rps7; DQ908931) were conserved in both the mutant and wild-type plants. The deletions in the chloroplast genome of these two mutants were different: nine genes were deleted in the chloroplast genomic aberration in ab1 and 11 genes in ab2. The chloroplast genes, NAD(P)H-quinone oxidoreductase chain 4 (ndhD; DQ908944), chloroplast 50S ribosomal protein L14 (rpl14; DQ908934), and ATP synthase beta chain (atpB; DQ908948) were abnormal in both mutants. The gene expressions of 18 of these 20 genes were correlated with their DNA copy number. The two exceptions were: ATP synthase CF0 A chain (atpI; DQ908946), whose expression in both mutants was not reduced even though the copy number was reduced; ribosomal protein S19 (rps19; DQ908949), whose expression was reduced or it was not expressed at all even though there was no difference in genomic copy number between the wild-type and mutant plants. The genomic PCR results showed that chloroplast genome aberrations do occur in multiple shoot proliferation, and this phenomenon may be involved in the generation of albino mutants.     

Toluene biodegradation by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1: targeting culture stability in long-term operation

The stability of Pseudomonas putida F1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h⁻¹). Each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the TOL plasmid. At all tested conditions, P. putida F1 exhibited stable performance as shown by the constant values of the specific toluene degradation yield, CO₂ produced versus toluene degraded yield, and biomass concentration within each steady state. Benzyl alcohol, a curing agent causing TOL plasmid deletion in Pseudomonas strains, was present in the cultivation medium as a result of the monooxygenation of toluene by the diooxygenase system of P. putida F1. However, no mutant population growing at the expense of the extracellular excreted carbon or lysis products was established in the chemostat as confirmed by the constant dissolved total organic carbon (TOC) concentration and fraction of toluene degrading cells (approx. 100%). In addition, batch experiments conducted with both lysis substrate and toluene simultaneously confirmed that P. putida F1 preferentially consumed toluene rather than extracellular excreted carbon.     

Luciferase and fluorescent protein as dual reporters analyzing the effect of <Emphasis Type="Italic">n</Emphasis>-dodecyltrimethylammonium bromide on the physiology of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

With the growing interest in using surfactants to improve microbial cell performance for whole-cell biocatalysis and bioremediation, understanding the interactions between surfactants and bacteria is of great importance. By using cyanine fluorescent protein (CFP) and bacterial luciferase (LUX) as dual bioreporters, the effects of n-dodecyltrimethylammonium bromide (DTAB) on the whole cells and intracellular proteins in Pseudomonas putida cultures were quantitatively and systematically studied. The dual reporter system was shown to be a useful indicator to assess the effect of DTAB treatment on whole-cell metabolic activity, membrane permeability, and cellular enzyme activity. CFP was useful to assess the leakage of intracellular enzymes and the lysis of cells and was able to reflect the activities of most cellular enzymes, while LUX reflected the permeability of cell membranes and cellular metabolic activity. The validity of CFP–LUX dual bioreporters was further confirmed by detecting changes in extracellular proteins, membrane potential, oxygen consumption rate (OUR), and intracellular catechol 2,3-dioxygenase (C23O) activity with the addition of DTAB. The dual LUX–CFP bioreporter is a useful tool for analyzing the surfactant–bacterium interactions for biotechnological applications.     

Utilization of <Emphasis Type="Italic">fadA</Emphasis> knockout mutant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> for overproduction of medium chain-length-polyhydroxyalkanoate

The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l⁻¹ stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l⁻¹ with a cell dry weight of 10.3 g l⁻¹.     

Biocontrol of <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> by <Emphasis Type="Italic">Rhizobium</Emphasis> and plant growth-promoting rhizobacteria on lentil

Biocontrol of the root-knot nematode Meloidogyne javanica was studied on lentil using plant growth-promoting rhizobacteria (PGPR) namely Pseudomonas putida, P. alcaligenes, Paenibacillus polymyxa and Bacillus pumilus and root nodule bacterium Rhizobium sp. Pseudomonas putida caused greater inhibitory effect on the hatching and penetration of M. javanica followed by P. alcaligenes, P. polymyxa and B. pumilus. Inoculation of any PGPR species alone or together with Rhizobium increased plant growth both in M. javanica-inoculated and -uninoculated plants. Inoculation of Rhizobum caused greater increase in plant growth than caused by any species of plant growth-promoting rhizobacteria in nematode-inoculated plants. Among PGPR, P. putida caused greater increase in plant growth and higher reduction in galling and nematode multiplication followed by P. alcaligenes, P. polymyxa and B. pumilus. Combined use of Rhizobium with any species of PGPR caused higher reduction in galling and nematode multiplication than their individual inoculation. Use of Rhizobium plus P. putida caused maximum reduction in galling and nematode multiplication followed by Rhizobium plus P. alcaligens. Pseudomonas putida caused greater root colonization and siderophore production followed by P. alcaligenes, P. polymyxa and B. pumilus. Analysis of the protein bands of these four species by SDS-PAGE revealed that P. putida had a different protein band profile compared to the protein profiles of P. alcaligenes, P. polymyxa and B. pumilus. However, the protein profiles of P. acaligenes, P. polymyxa and B. pumilus were similar.     

Detection and characterization of an ABC transporter in <Emphasis Type="Italic">Clostridium hathewayi</Emphasis>

An ABC transporter gene from Clostridium hathewayi is characterized. It has duplicated ATPase domains in addition to a transmembrane protein. Its deduced amino acid sequence has conserved functional domains with ATPase components of the multidrug efflux pump genes of several bacteria. Cloning this transporter gene into C. perfringens and E. coli resulted in decreased sensitivities of these bacteria to fluoroquinolones. It also decreased the accumulation and increased the efflux of ethidium bromide from cells containing the cloned gene. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) inhibited both accumulation and efflux of ethidium bromide from these cells. The ATPase mRNA was overexpressed in the fluoroquinolone-resistant strain when exposed to ciprofloxacin. This is the first report of an ABC transporter in C. hathewayi. An erratum to this article can be found at     

Overproduction of Delta-Endotoxins by Sporeless <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">thuringiensis</Emphasis> Mutants Obtained by Nitrous Acid Mutagenesis

Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

Ethylene biosynthesis in a chilling-sensitive<Emphasis Type="Italic">Arabidopsis</Emphasis> mutant,<Emphasis Type="Italic">chs4-2</Emphasis>

We investigated chilling-induced changes in ethylene levels in Arabidopsis to find plants with distinct patterns of ethylene production in the cold-related biosynthetic pathway. The sensitive mutants identified here includedchs1-2,chs4-2, andchs6-2. Among these, plants of thechs4-2 mutant produced more ethylene than did the wild type after both were transferred from 4°C or 10°C to 22°C. This mutant also showed less freezing tolerance and more electrolyte leakage than the wild-type plants. Our results suggest a relationship between ethylene biosynthesis and chilling sensitivity in the mutant To determine which of the enzymes involved in ethylene biosynthesis were induced by chilling, we tested the activities of ACC synthase and ACC oxidase in both mutant and wild-type plants, and found greater activity by ACC synthase as well as a higher ACC content in the mutants after all the plants were transferred from 10°C to 22°C. However, ACC oxidase activity did not differ between mutant and wild-type plants in response to chilling treatment Therefore, we conclude thatchs4-2 mutants produce more ethylene than do other mutants or the wild type during their recovery from chilling conditions. Furthermore, we believe that ACC synthase is the key enzyme involved in this response.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

Microbial production of <Emphasis Type="Italic">cis</Emphasis>-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L⁻¹ h⁻¹. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.     

ANK repeat-domain of SHN-1 Is indispensable for <Emphasis Type="Italic">in vivo</Emphasis> SHN-1 function in <Emphasis Type="Italic">C. elegans</Emphasis>

Shank protein is one of the postsynaptic density (PSD) proteins which play a major role in proper localization of proteins at membranes. The shn-1, a homolog of Shank in Caenorhabditis elegans, is expressed in neurons, pharynx, intestine, vulva and sperm. We have previously reported a possible genetic interaction between Shank and IP₃ receptor by examining shn-1 RNAi in IP₃ receptor (itr-1) mutant background. In order to show the direct interaction of Shank and IP₃ receptor as well as to show the direct in vivo function of Shank, we have characterized two different mutant alleles of shn-1, which have different deletions in the different domains. shn-1 mutants were observed for Ca²⁺-related behavioral defects with itr-1 mutants. We found that only shn-1 mutant defective in ANK repeat-domain showed significant defects in defecation, pharyngeal pumping and fertility. In addition, we found that shn-1 regulates defecation, pharyngeal pumping and probably male fertility with itr-1. Thus, we suggest that Shank ANK repeat-domain along with PDZ may play a crucial role in regulating Ca²⁺-signaling with IP₃ receptor.     

Isolation and characterization of ethylbenzene degrading <Emphasis Type="Italic">Pseudomonas putida</Emphasis> E41

Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethyl-benzene as the sole carbon and energy source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (μ_max) under the optimum conditions were 0.19+0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87+0.13 h⁻¹, respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium (each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70–80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene.