Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol. To improve characteristics of xylose fermentation, the recombinant strain Delta xyl1 Delta xyl2-ADelta xyl2-B, with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively), was constructed and used as a recipient for co-overexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter. Xylose isomerase activities of obtained transformants amounted to approximately 80% of that of the bacterial host strain. Xylulokinase activities of the transformants increased twofold when compared with the parental strain. The recombinant strains displayed improved ethanol production during the fermentation of xylose.     

Cloning and targeted disruption of two polygalacturonase genes in Penicillium olsonii

The filamentous fungus Penicillium olsonii secretes several polygalacturonases (PGs) with molecular masses of about 47 kDa. These enzymes consist of several basic and acidic isoforms, with dominant activities at pI 4.5 and pI 7.9. Two polygalacturonase genes, pg1 and pg2, have been cloned. The corresponding enzymes, PG1 and PG2, consist of 370 and 380 amino acids, respectively, and show significant similarities to endo-polygalacturonases from other filamentous fungi. Targeted disruption of pg1 resulted in the elimination of all basic PG isoforms. In contrast, disruption of pg2 reduced, but did not eliminate the acidic PG activities. The PGs of P. olsonii must therefore be encoded by a gene family of at least three genes. Induction studies with various carbon sources revealed that the acidic and basic isoforms are differentially regulated. Pectin is the best inducer of the acidic PG isoforms. The basic isoforms, however, are best induced by monosaccharides like glucose, alpha-L-rhamnose and alpha-L-arabinose.     

Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2.

Twenty-one Xyl- mutants of Salmonella typhimurium were selected: all had lost one or more of the activities for D-xylose isomerase, C-xylulokinase, or D-xylose transport. The mutants were classified into five functional groups: xylR, pleiotropic negative (12 mutants); xylA, D-xylose isomerase defective (3 mutants); xylB, D-xylulokinase defective (2 mutants); xylT, D-xylose transport defective (1 mutant); and 3 mutants with defective D-xylose isomerase and D-xylulokinase. Some nonsense mutations were identified among the xylR mutants. Two F'xyl plasmids were isolated by selection for early transfer of xyl+ by an Hfr which transfers xyl as a terminal gene; a plasmid with a mutation in the xyl genes, F'xylR1, was also isolated. Complementation tests using F'xyl plasmids indicate that expression of the xylA, xylB, and xylT genes is under the positive control of the xylR regulatory gene. Conjugation crosses and P22-mediated transduction data indicate that all the xyl mutations tested are in a cluster of genes at 78 units on the linkage map, and that the gene order is xylT--xylR--xylB--xylA--glyS--mtlA,D.     

Polyamine analogs modulate gene expression by inhibiting lysine-specific demethylase 1 (LSD1) and altering chromatin structure in human breast cancer cells

Aberrant epigenetic repression of gene expression has been implicated in most cancers, including breast cancer. The nuclear amine oxidase, lysine-specific demethylase 1 (LSD1) has the ability to broadly repress gene expression by removing the activating mono- and di-methylation marks at the lysine 4 residue of histone 3 (H3K4me1 and me2). Additionally, LSD1 is highly expressed in estrogen receptor α negative (ER-) breast cancer cells. Since epigenetic marks are reversible, they make attractive therapeutic targets. Here we examine the effects of polyamine analog inhibitors of LSD1 on gene expression, with the goal of targeting LSD1 as a therapeutic modality in the treatment of breast cancer. Exposure of the ER-negative human breast cancer cells, MDA-MB-231 to the LSD1 inhibitors, 2d or PG11144, significantly increases global H3K4me1 and H3K4me2, and alters gene expression. Array analysis indicated that 98 (75 up and 23 down) and 477 (237 up and 240 down) genes changed expression by at least 1.5-fold or greater after treatment with 2d and PG11144, respectively. The expression of 12 up-regulated genes by 2d and 14 up-regulated genes by PG11144 was validated by quantitative RT-PCR. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that up-regulated gene expression by polyamine analogs is associated with increase of the active histone marks H3K4me1, H3K4me2 and H3K9act, and decrease of the repressive histone marks H3K9me2 and H3K27me3, in the promoter regions of the relevant target genes. These data indicate that the pharmacologic inhibition of LSD1 can effectively alter gene expression and that this therapeutic strategy has potential.     

Structure and function of the Pseudomonas putida integration host factor.

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.     

Expression of multiple forms of polygalacturonase gene during ripening in banana fruit.

The activity of polygalacturonase (PG, E.C 3.2.1.15) during ripening in climacteric fruits has been positively correlated with softening of the fruit tissue and differential expression of its gene is suspected to be regulated by the plant hormone ethylene. We have cloned four partial cDNAs, MAPG1 (acc. no. AF311881), MAPG2 (acc. no. AF311882), MAPG3 (acc. no. AF542382) and MAPG4 (acc. no. AY603341) for PG genes and studied their differential expression during ripening in banana. MAPG3 and MAPG4 are believed to be ripening related and regulated by ethylene whereas MAPG2 is associated more with senescence. MAPG1 shows constitutive expression and is not significantly expressed in fruit tissue. The genomic clone MAGPG (acc. No. AY603340) includes the complete MAPG3 gene, which consists of four exons and three introns. The structure of the gene has more similarity to tomato abscission PG rather than tomato fruit PG. It is concluded that softening during ripening in banana fruit results from the concerted action of at least four PG genes, which are differentially expressed during ripening.     

Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi

Polygalacturonases (PGs) are secreted by fungal pathogens during saprophytic and parasitic growth, and their degradation of pectin in the plant cell wall is believed to play a major role in tissue invasion and maceration. In this study, PG activity was demonstrated in culture filtrates of the oomycete plant pathogen, Phytophthora cinnamomi. A P. cinnamomi pg gene fragment amplified using degenerate primers based on conserved regions in fungal and plant PGs was used to isolate 17 complete P. cinnamomi pg genes and pseudogenes from a genomic library and partial sequence for another two genes. Gel blotting of genomic DNA indicated that there may be even more pg genes in the P. cinnamomi genome. P. cinnamomi pg gene sequences were expressed in PG-deficient yeast and found to confer PG activity, thereby confirming their functional identity. The predicted mature P. cinnamomi PGs fall into subgroups that exhibit large differences in the extent of N-glycosylation, isoelectric points, and N- and C-terminal structure. Evidence for birth-and-death and reticulate evolution in the P. cinnamomi pg gene family was obtained, and some codons for surface exposed residues in the P. cinnamomi PGs were shown to have been subject to diversifying selection. Contrary to accepted phylogenies for other proteins, phylogenetic analysis of the P. cinnamomi PGs revealed a closer relationship with PGs from true fungi than with those from plants.     

Cloning, disruption, and expression of two endo-beta 1, 4-xylanase genes, XYL2 and XYL3, from Cochliobolus carbonum.

In culture, the filamentous fungus Cochliobolus carbonum, a pathogen of maize, makes three cationic xylanases, XYL1, which encodes the major endoxylanase (Xyl1), was earlier cloned and shown by gene disruption to encode the first and second peaks of xylanase activity (P. C. Apel, D. G. Panaccione, F. R. Holden, and J. D. Walton, Mol. Plant-Microbe Interact. 6:467-473, 1993). Two additional xylanase genes, XYL2 and XYL3, have now been cloned from C. carbonum. XYL2 and XYL3 are predicted to encode 22-kDa family G xylanases similar to Xyl1. Xyl2 and Xyl3 are 60% and 42% identical, respectively, to Xyl1, and Xyl2 and Xyl3 are 39% identical. XYL1 and XYL2 but not XYL3 mRNAs are present in C. carbonum grown in culture, and XYL1 and XYL3 but not XYL2 mRNAs are present in infected plants. Transformation-mediated gene disruption was used to construct strains mutated in XYL1, XYL2, and XYL3. Xyl1 accounts for most of the total xylanase activity in culture, and disruption of XYL2 or XYL3 does not result in the further loss of any xylanase activity. In particular, the third peak of cationic xylanase activity is still present in a xyl1 xyl2 xyl3 triple mutant, and therefore this xylanase must be encoded by yet a fourth xylanase gene. A minor protein of 22 kDa that can be detected immunologically in the xyl1 mutant disappears in the xyl2 mutant and is therefore proposed to be the product of XYL2. The single xylanase mutants were crossed with each other to obtain multiple xylanase disruptions within the same strain. Strains disrupted in combinations of two and in all three xylanases were obtained. The triple mutant grows at the same rate as the wild type on xylan and on maize cell walls. The triple mutant is still fully pathogenic on maize with regard to lesion size, morphology, and rate of lesion development.     

Identification of the amidotransferase AsnB1 as being responsible for meso-diaminopimelic acid amidation in Lactobacillus plantarum peptidoglycan

The peptidoglycan (PG) of Lactobacillus plantarum contains amidated meso-diaminopimelic acid (mDAP). The functional role of this PG modification has never been characterized in any bacterial species, except for its impact on PG recognition by receptors of the innate immune system. In silico analysis of loci carrying PG biosynthesis genes in the L. plantarum genome revealed the colocalization of the murE gene, which encodes the ligase catalyzing the addition of mDAP to UDP-N-muramoyl-d-glutamate PG precursors, with asnB1, which encodes a putative asparagine synthase with an N-terminal amidotransferase domain. By gene disruption and complementation experiments, we showed that asnB1 is the amidotransferase involved in mDAP amidation. PG structural analysis revealed that mDAP amidation plays a key role in the control of the l,d-carboxypeptidase DacB activity. In addition, a mutant strain with a defect in mDAP amidation is strongly affected in growth and cell morphology, with filamentation and cell chaining, while a DacB-negative strain displays a phenotype very similar to that of a wild-type strain. These results suggest that mDAP amidation may play a critical role in the control of the septation process.     

黄瓜CsPGIP基因的克隆及表达分析

以加工型黄瓜材料NW99为对象,利用RT-PCR技术克隆黄瓜多聚半乳糖醛酸酶抑制蛋白基因(PGIP),并分析其基因编码序列、组织表达特异性和诱导表达模式。结果表明:(1)从黄瓜中克隆到一个PGIP基因,命名为CsPGIP;CsPGIP基因全长1 026bp,读码框987bp,无内含子,编码328个氨基酸残基,具有xxLxLxxNxLt/sGxIPxxLxxLxxL结构域,属于Pgip基因家族。(2)CsPGIP基因与甜瓜PGIP基因同源性最高,与十字花科Pgip基因同源性较高。(3)CsPGIP在黄瓜各个器官都表达,但表达水平具有组织特异性,在嫩叶中表达量最高,在茎中表达量最低;该基因表达明显受到水杨酸诱导,可能在抵御外界病原菌入侵过程中起重要作用。     

Functional analysis of two polygalacturonase genes in Apolygus lucorum associated with eliciting plant injury using RNA interference

Salivary enzymes of many piercing–sucking insects lead to host plant injury. The salivary enzymes, polygalacturonase (PGs), act in insect feeding. PG family genes have been cloned from the mirid bug Apolygus lucorum, a pest of cotton and other host crops in China. We investigated the function of two PG genes that are highly expressed in A. lucorum nymphs (PG3‐4) and adults (PG3‐5), using siRNA injection‐based RNA interference (RNAi). Accumulation of mRNA encoding both genes and their cognate proteins was significantly reduced (>60%) in experimental compared control green fluorescent protein (GFP) siRNA‐treated mirids at 48 h post injection. Injury levels of cotton buds were also significantly reduced after injecting saliva isolated from PG3‐4 and PG3‐5 siRNA‐treated A. lucorum. These results demonstrate that these two PG act in A. lucorum elicitation of plant injury.     

Molecular diversity of plasmids bearing genes that encode toluene and xylene metabolism in Pseudomonas strains isolated from different contaminated sites in Belarus

Twenty different Pseudomonas strains utilizing m-toluate were isolated from oil-contaminated soil samples near Minsk, Belarus. Seventeen of these isolates carried plasmids ranging in size from 78 to about 200 kb (assigned pSVS plasmids) and encoding the meta cleavage pathway for toluene metabolism. Most plasmids were conjugative but of unknown incompatibility groups, except for one, which belonged to the IncP9 group. The organization of the genes for toluene catabolism was determined by restriction analysis and hybridization with xyl gene probes of pWW0. The majority of the plasmids carried xyl-type genes highly homologous to those of pWW53 and organized in a similar manner (M. T. Gallegos, P. A. Williams, and J. L. Ramos, J. Bacteriol. 179:5024-5029, 1997), with two distinguishable meta pathway operons, one upper pathway operon, and three xylS-homologous regions. All of these plasmids also possessed large areas of homologous DNA outside the catabolic genes, suggesting a common ancestry. Two other pSVS plasmids carried only one meta pathway operon, one upper pathway operon, and one copy each of xylS and xylR. The backbones of these two plasmids differed greatly from those of the others. Whereas these parts of the plasmids, carrying the xyl genes, were mostly conserved between plasmids of each group, the noncatabolic parts had undergone intensive DNA rearrangements. DNA sequencing of specific regions near and within the xylTE and xylA genes of the pSVS plasmids confirmed the strong homologies to the xyl genes of pWW53 and pWW0. However, several recombinations were discovered within the upper pathway operons of the pSVS plasmids and pWW0. The main genetic mechanisms which are thought to have resulted in the present-day configuration of the xyl operons are discussed in light of the diversity analysis carried out on the pSVS plasmids.     

The Bacillus thuringiensis cyt genes for hemolytic endotoxins constitute a gene family

In the same way that cry genes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, the cyt genes for hemolytic toxins seem to compose another set of highly related genes in Bacillus thuringiensis. Although the occurrence of Cyt hemolytic factors in B. thuringiensis has been typically associated with mosquitocidal strains, we have recently shown that cyt genes are also present in strains with different pathotypes; this is the case for the morrisoni subspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such as B. thuringiensis subsp. israelensis and subsp. morrisoni PG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50 B. thuringiensis strains revealed that cyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of many B. thuringiensis strains.     

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyll and Pichia stipitis gene xyl2,encoding xylose reductase (XR) and xylitol dehydrogenase (XD) respectively,were amplified by PCR.The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-PI2.Subsequently the vector pYES2-P12 was transformed into S.cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12.The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%.     

Suppression of lung cancer cell invasion and metastasis by connexin43 involves the secretion of follistatin-like 1 mediated via histone acetylation

Although connexin has been recognized as a tumor suppressor in many types of cancer, the underlying mechanisms are poorly understood. We have previously shown that transfection of connexin43 (Cx43) cDNA retarded the growth of a highly metastatic human pulmonary giant cell carcinoma cell line, PG, both in vitro and in vivo. Here, we further demonstrate that the metastasis and invasion, but not the migration, of PG cells are also inhibited following Cx43 transfection. The diminishment of metastasis and invasion is associated with down-regulation of genes including MMP-2, S100A, LAMA4, and HDAC10, as well as up-regulation of genes such as MTSS1 and FSTL1 as revealed by gene chip analysis. Interestingly, the suppression effects of Cx43 are related to secreted factor(s), which are blocked by FSTL1 antibody treatment in a dose-dependent manner. Furthermore, the FSTL1 promoter was shown to be associated with acetylated histones H3 and H4 upon Cx43 transfection. These data suggest that Cx43 inhibits the invasion and metastasis of PG cells by modulating the secretion of FSTL1, which is regulated by histone acetylation. Cx43 may act as a "histone deacetylase inhibitor" to modulate gene expression and subsequent cellular functions in PG cells.     

Crystal structures of triosephosphate isomerase from methicillin resistant Staphylococcus aureus MRSA252 provide structural insights into novel modes of ligand binding and unique conformations of catalytic loop

Staphylococcus aureus is one of the most dreaded pathogens worldwide and emergence of notorious antibiotic resistant strains have further exacerbated the present scenario. The glycolytic enzyme, triosephosphate isomerase (TIM) is one of the cell envelope proteins of the coccus and is involved in biofilm formation. It also plays an instrumental role in adherence and invasion of the bacteria into the host cell. To structurally characterize this important enzyme and analyze it's interaction with different inhibitors, substrate and transition state analogues, the present article describes several crystal structures of SaTIM alone and in complex with different ligands: glycerol-3-phosphate (G3P), glycerol-2-phosphate (G2P), 3-phosphoglyceric acid (3PG) and 2-phosphoglyceric acid (2PG). Unique conformations of the catalytic loop 6 (L6) has been observed in the different complexes. It is found to be in “almost closed” conformation in both subunits of the structure complexed to G3P. However L6 adopts the open conformation in presence of G2P and 2PG. The preference of the conformation of the catalytic loop can be correlated with the position of the phosphate group in the ligand. Novel modes of binding have been observed for G2P and 3PG for the very first time. The triose moiety is oriented away from the catalytic residues and occupies an entirely different position in some subunits. A completely new binding site for phosphate has also been identified in the complex with 2PG which differs substantially from the conventional phosphate binding site of the ligand in the crystal structures of TIM determined so far.     

Substrate-Specific Gene Expression in Batrachochytrium dendrobatidis,the Chytrid Pathogen of Amphibians

Determining the mechanisms of host-pathogen interaction is critical for understanding and mitigating infectious disease. Mechanisms of fungal pathogenicity are of particular interest given the recent outbreaks of fungal diseases in wildlife populations. Our study focuses on Batrachochytrium dendrobatidis (Bd), the chytrid pathogen responsible for amphibian declines around the world. Previous studies have hypothesized a role for several specific families of secreted proteases as pathogenicity factors in Bd, but the expression of these genes has only been evaluated in laboratory growth conditions. Here we conduct a genome-wide study of Bd gene expression under two different nutrient conditions. We compare Bd gene expression profiles in standard laboratory growth media and in pulverized host tissue (i.e., frog skin). A large proportion of genes in the Bd genome show increased expression when grown in host tissue, indicating the importance of studying pathogens on host substrate. A number of gene classes show particularly high levels of expression in host tissue, including three families of secreted proteases (metallo-, serine- and aspartyl-proteases), adhesion genes, lipase-3 encoding genes, and a group of phylogenetically unusual crinkler-like effectors. We discuss the roles of these different genes as putative pathogenicity factors and discuss what they can teach us about Bd’s metabolic targets, host invasion, and pathogenesis.