Excretome of the chitinolytic bacterium <Emphasis Type="Italic">Clostridium paraputrificum</Emphasis> J4

A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-d-glucosaminide, N,N′-diacetyl-β-d-chitobiose, or N,N′,N˝-triacetyl-β-d-chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.     

Structure-function relationship of substrate length specificity of dextran glucosidase from <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Dextran glucosidase from Streptococcus mutans (SMDG), an exo-type glucosidase of glycoside hydrolase (GH) family 13, specifically hydrolyzes an α-1,6-glucosidic linkage at the non-reducing ends of isomaltooligosaccharides and dextran. SMDG shows the highest sequence similarity to oligo-1,6-glucosidases (O16Gs) among GH family 13 enzymes, but these enzymes are obviously different in terms of substrate chain length specificity. SMDG efficiently hydrolyzes both short-and long-chain substrates, while O16G acts on only short-chain substrates. We focused on this difference in substrate specificity between SMDG and O16G, and elucidated the structure-function relationship of substrate chain length specificity in SMDG. Crystal structure analysis revealed that SMDG consists of three domains, A, B, and C, which are commonly found in other GH family 13 enzymes. The structural comparison between SMDG and O16G from Bacillus cereus indicated that Trp238, spanning subsites +1 and +2, and short β → α loop 4, are characteristic of SMDG, and these structural elements are predicted to be important for high activity toward long-chain substrates. The substrate size preference of SMDG was kinetically analyzed using two mutants: (i) Trp238 was replaced by a smaller amino acid, alanine, asparagine or proline; and (ii) short β → α loop 4 was exchanged with the corresponding loop of O16G. Mutant enzymes showed lower preference for long-chain substrates than wild-type enzyme, indicating that these structural elements are essential for the high activity toward long-chain substrates, as implied by structural analysis.     

Gamma radiation induced mutagenesis in <Emphasis Type="Italic">Aspergillus niger</Emphasis> to enhance its microbial fermentation activity for industrial enzyme production

α- and β-Galactosidases find application in food processing, health and nutrition. Aspergillus niger is one of the potent producer of these enzymes and was genotypically improved using gamma-ray induced mutagenesis. The mutant-derivative produced two-fold higher α- and β-galactosidases. For testing genetic variability and its relationship with phenotypic properties of the two organisms, DNA samples of the mutant and parental strains of A. niger were amplified with 28 deca-nucleotide synthetic primers. RAPD analysis showed significantly different pattern between parental and mutant cultures. The mutant derivative yielded homogeneous while parental strain formed heterogeneous amplification patterns. Seven primers identified 42.9% polymorphism in the amplification products, indicating that these primers determined some genetic variability between the two strains. Thus RAPD was found to be an efficient technique to determine genetic variability in the mutant and wild organisms. Both wild and mutant strains were analyzed for their potential to produce galactosidases. Comparison of different carbon sources on enzyme yield revealed that wheat bran is significant (P < 0.01) effective producer and economical source followed by rice bran, rice polishing and lactose. The mutant was significantly better enzyme producer and could be considered for its prospective application in food, nutrition and health and that RAPD can be effectively used to differentiate mutant strain from the parental strain based on the RAPD patterns.     

Xylanases of anaerobic fungus <Emphasis Type="Italic">Anaeromyces mucronatus</Emphasis>

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.     

Characterization of the <Emphasis Type="Italic">Pragia fontium</Emphasis> Lipopolysaccharides

Lipopolysaccharides (LPSs) of two strains Pragia fontium 97U116 and 27480 were isolated and characterized; they were close to those of other representatives of the family Enterobacteriaceae in fatty acid composition and contained, respectively, 3-hydroxytetradecanoic acid as the predominant component (45.8 and 45.1%), tetradecanoic (23.5 and 28.9%), hexadecanoic (12.6 and 7.9%), hexadecenoic (12.6 and 7.9%), and dodecanoic (4.9 and 4.2%) fatty acids. The O-specific polysaccharides consisted of linear penta- and tetrasaccharide repeating units: →2)-α-D-Galf-(1→3)-α-L-Rhap2Ac-(1→4)-α-D-GlcpNAc-(1→2)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→ →4)-β-D-ManpNAc3NAcA-(1→2)-α-L-Rhap-(1→3)-β-L-Rhap-(1→4)-α-D-GlcpNAc-(1→ The LPSs of P. fontium 97U116 and 27480 were serologically active and belonged to different serogroups; they were less toxic than those of strain E. coli O55:B5, but more pyrogenic than the Pyrogenal preparation.     

Genotoxic activity of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> essential oil in <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> diploid cells

Eucalyptus globulus essential oil was evaluated for its genotoxic potential using a somatic segregation assay and a diploid strain of the fungus Aspergillus nidulans, heterozygous for nutritional and conidia color markers. The main compounds of the current essential oil sample were eucalyptol (49.0 %), α-pinene (8.9), β-pinene (1.5), globulol (6.9), α-eudesmol (1.12), spathulenol (1.42), γ-cadinene (1.45), trans-β-elemenone (1.23) and aromandendrene (2.3), totaling 74 % of oil. Oil at 0.12 and 0.25 μL/mL was found to increase the mitotic instability of the original diploid strain and the number of diploid mitotic recombinants of A. nidulans. The genotoxicity of the oil was associated with the induction of mitotic crossing-over or with oil-broken chromosomes.     

Variation in specificity of the PrtP extracellular proteinases in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis>

Comparison of cell-wall-bound extracellular proteinases (CEPs) from Lactobacillus paracasei (LBP) ssp. paracasei natural isolates BGHN14, BGAR75 and BGAR76 with Lactococcus lactis (LCL) ssp. cremoris Wg2, in their action on α_S1-, β- and κ-casein was done. The CEPs of LBP strains were able to degrade α_S1- and β-caseins and their caseinolytic specificity depended on the type of buffer used. These CEPs, compared with LCL Wg2, differ in four amino acid residues in small segments predicted to be involved in substrate binding. The most striking features of this comparison are the presence of Ala instead of Ser³²⁹ and the presence of Thr instead of Asn²⁵⁶ and Ala²⁹⁹, in the subtilisin-like region of the CEP in LBP natural isolates. Additional conservative amino acid substitution Leu to Ile³⁶⁴ was found.     

Antibacterial activity of cyclodextrins against <Emphasis Type="Italic">Bacillus</Emphasis> strains

Growth of alkaliphilic Bacillus halodurans C-125 both on agar plates and in liquid culture was inhibited by methyl-β-cyclodextrin (CD). Furthermore, resting cells of the strain were lysed by contact with methyl-β-CD higher than 10 mM. α-CD also showed lysis activity against Bacillus and related strains. The activity was not observed with Gram-negative and Gram-positive bacteria except for Bacillus strains. Fluorescence staining and scanning electron microscopy of cells revealed that methyl-β-CD disrupted cell membranes, and consequently, the cells were lysed. This is a novel physiological property of CDs.     

Isolation and properties of a <Emphasis Type="Italic">levo-</Emphasis>lactonase from <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> ECU2002: a robust biocatalyst for production of chiral lactones

A fungus strain ECU2002, capable of enantioselectively hydrolyzing chiral lactones to optically pure hydroxy acids, was newly isolated from soil samples through two steps of screening and identified as Fusarium proliferatum (Matsushima) Nirenberg. From the crude extract of F. proliferatum ECU2002, a novel levo-lactonase was purified to homogeneity, with a purification factor of 460-folds and an overall yield of 9.7%, by ultrafiltration, acetone precipitation, and chromatographic separation through DEAE-Toyopearl, Butyl-Toyopearl, Hydroxyapatite, Toyoscreen-Super Q, and TSK-gel columns. The purified enzyme is a monomer; with a molecular mass of ca 68 kDa and a pI of 5.7 as determined by two-dimensional electrophoresis. The catalytic performance of the partially purified levo-lactonase was investigated, giving temperature and pH optima at 50°C and 7.5, respectively, for γ-butyrolactone hydrolysis. The substrate specificity of the partially purified lactonase was also examined using several useful lactones, among which α-hydroxy-γ-butyrolactone was the best substrate, with 448-fold higher lactonase activity as compared to γ-butyrolactone. The F. proliferatum lactonase preferentially hydrolyzed the levo enantiomer of butyrolactones, including β-butyrolactone, α-hydroxy-γ-butyrolactone, α-hydroxy-β,β-dimethyl-γ-butyrolactone (pantolactone), and β-hydroxy-γ-butyrolactone, affording (+)-hydroxy acids in high (94.8∼98.2%) enantiomeric excesses (ee) and good conversions (38.2∼44.2%). A simple immobilization of the crude lactonase with glutaraldehyde cross-linking led to a stable and easy-to-handle biocatalyst for catalytic resolution of chiral lactones. The immobilized lactonase also performed quite well in repeated batch resolution of dl-pantolactone at a concentration of 35% (w/v), retaining 67% of initial activity after ten cycles of reaction (corresponding to a half life of 20 cycles) and affording the product in 94∼97% ee, which can be easily enhanced to >99% ee after the d-hydroxy acid was chemically converted into l-lactone and crystallized.     

Functional properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from dairy products

Twenty-four acid- and bile-tolerant lactobacilli isolates from dairy products were identified and further in vitro characterized for the presence of functional traits potentially useful for probiotic applications, which included desirable and undesirable traits, such as biofilm formation, ability to inhibit intestinal pathogens, antibiotic susceptibility, and enzyme activity. The majority of examined strains were susceptible to certain antimicrobial agents (streptomycin, gentamicin, clindamycin, erythromycin, tetracycline, quinupristin–dalfopristin), except for three strains of Lactobacillus rhamnosus with minimal inhibitory concentration levels for streptomycin higher than the microbiological breakpoints (≥32 μg/mL), which are considered as resistant. Undesirable traits such as α-chymotrypsin or N-acetyl-β-glucosaminidase activities were not detected, but low β-glucuronidase, and moderate and high β-glucosidase activities were recorded in nine strains, which were eliminated from further examination together with three isolates showing unsuitable antibiotic resistance. Of the remaining 12 isolates, 4 (Lactobacillus fermentum 202, Lactobacillus gallinarum 7001, L. rhamnosus 183, and Lactobacillus plantarum L2-1) manifested an outstanding potential to inhibit selected intestinal pathogens in an agar spot test, including Escherichia coli and Salmonella spp., and simultaneously demonstrated strong biofilm-forming capacity. In conclusion, the results of our in vitro experiments showed that the above four strains had a potential probiotic value and met the criteria to be identified as a possible probiotic microorganism, with the necessity of verification through well-designed in vivo experimental, clinical, and technological studies before the strains can be used as probiotics or as starter probiotic cultures.     

Synergistic effect of surfactin from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C4 and <Emphasis Type="Italic">Achyrocline satureioides</Emphasis> extracts on the viability of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>

The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE) and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 μg ml⁻¹ and the HE concentration from 32 to 4 μg ml⁻¹, values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Association Between the <Emphasis Type="Italic">cfx</Emphasis>A Gene and Transposon Tn<Emphasis Type="Italic">4555</Emphasis> in <Emphasis Type="Italic">Bacteroides distasonis</Emphasis> Strains and Other <Emphasis Type="Italic">Bacteroides</Emphasis> Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.     

Identification and Characteristics of Nisin Z-Producing <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> Isolated from Kimchi

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100°C for 1 h and at 121°C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or β-glucosidase. There were some differences in characteristics from those of nisins described previously. Received: 21 June 2002 / Accepted: 22 July 2002     

GacS-dependent regulation of enzymic and antifungal activities and synthesis of <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones in rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30–84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G⁺ bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS ⁻ mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism, polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Validation of Arsenic Resistance in <Emphasis Type="Italic">Bacillus cereus</Emphasis> Strain AG27 by Comparative Protein Modeling of <Emphasis Type="Italic">ars</Emphasis>C Gene Product

The ars gene system provides arsenic resistance to a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. Therefore, arsC gene from Bacillus cereus strain AG27 isolated from soil was amplified, cloned and sequenced. The strain exhibited a minimum inhibitory concentration of 40 and 35 mM to sodium arsenate and sodium arsenite, respectively. Homology of the sequence, when compared with available database using BLASTn search showed that 300 bp amplicons obtained possess partial arsC gene sequence which codes for arsenate reductase, an enzyme involved in the reduction of arsenate to arsenite which is then effluxed out of the cell, thereby indicating the presence of efflux mechanism of resistance in strain. The efflux mechanism was further confirmed by atomic absorption spectroscopy and scanning electron microscopy studies. Moreover, three dimensional structure of modeled arsC from Bacillus cereus strain shares significant structural similarity with arsenate reductase protein of B.subtilis, consisting of, highly similar overall fold with single α/β domain containing a central four stranded, parallel, open-twisted β-sheet flanked by α-helices on both sides. The structure harbors the arsenic binding motif AB loop or P-loop that is highly conserved in arsenate reductase family.     

Heterologous interferons synthesis in yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism.     

<Emphasis Type="Italic">QUASIMODO1</Emphasis> is expressed in vascular tissue of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> inflorescence stems,and affects homogalacturonan and xylan biosynthesis

An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan α-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4-D-xylan synthase, β-1-4-D-galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4-D-galacturonosyltransferase and β-1,4-D-xylan synthase activities.