Summary Microbial fructosyltransferases are polymerases that are involved in microbial fructan (levan, inulin and fructo‐oligosaccharide) biosynthesis. Structurally, microbial fructosyltransferase proteins share the catalytic domain of glycoside hydrolases 68 family and are grouped in seven phylogenetically related clusters. Fructosyltransferase‐encoding genes are organized in operons or in clusters associated with other genes related to carbohydrate metabolism or fructosyltransferase secretion. Fructosyltransferase gene expression is mainly regulated by two‐component systems or phosphorelay mechanisms that respond to sucrose availability or other environmental signals. Microbial fructans are involved in conferring resistance to environmental stress such as water deprivation, nutrient assimilation, biofilm formation, and as virulence factors in colonization. As a result of the biological and industrial importance of fructans, fructosyltransferases have been the subject of extensive research, conducted to improve their enzymatic activity or to elucidate their biological role in nature.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane, which expresses levansucrase, a fructosyltransferase exoenzyme with sucrose hydrolytic and levan biosynthetic activities. As a result of their physical properties, the levan can provide protection against stress caused by abiotic or biotic factors and participate in the formation of biofilms. In this study, we investigated the construction and function of a levansucrase-defective mutant of G. diazotrophicus. The lsdA mutant showed a decreased tolerance (65.5%) to 50-150 mM NaCl and a decrease of 89% in 876 mM (30%) sucrose, a reduction (99%) in tolerance to desiccation after 18 h, and a decrease (36.9-58.5%) in the ability to form cell aggregates on abiotic surfaces. Complementation of the mutant with the complete lsdA gene leads to a recovery of the ability to grow on sucrose-containing medium and to form slimy colonies, the ability to form the cell aggregates on abiotic surfaces and the tolerance to NaCl. This report demonstrates the importance of levansucrase in environmental adaptation of G. diazotrophicus under high osmotic stress and in biofilm formation.

tobacco and Hyoscyamus muticus inducible sesquiterpene cy-Phytoalexins play an important role in the inducible defense clase genes were used with the system for rapid amplification responses of plants against diseases caused by fungi. Some of cDNA ends (3%-RACE) technique to prepare pepper EAS-enzymes, involved in the respective biosynthetic pathways, catalyze key steps. Farnesyl pyrophosphate (FPP) is a key cDNA fragments (PEAS-cDNA). Three specific PEASintermediate in the biosynthesis of terpenes. It may be con-cDNAs were isolated (PEAS1, PEAS18, and PEAS55). verted into several cyclic and some acyclic sesquiterpenic Northern blots of total RNA samples from pepper plant derivatives, sterol precursors, or to geranylgeranyl pyrophos-tissues challenged with cellulase or Phytophthora capsici phate according to the requirements of the plant cells. Specific showed different expression levels of the respective transcripts. cyclization reactions of FPP are catalyzed by particular PEAS1 was used to identify the corresponding elicitor-stimulated gene (gPEAS1). The nucleotide sequence of the pro-sesquiterpene cyclases. The 5-epi-aristolochene synthase posed gPEAS1 promoter showed putative stimuli-and (EAS) enzyme of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) produce the 5-epi-aristolochene, which is tissue-specific responsive elements. A pepper 5-epi-aristolochene synthase gene family of 5 -8 members was demonstrated the immediate precursor of the bicyclic phytoalexin capsidiol. Oligonucleotides homologous to 3%-end specific regions of by Southern blot.

The pUM505 plasmid was isolated from a clinical strain of Pseudomonas aeruginosa. This plasmid contains a genomic island with sequence similar to islands found in chromosomes of virulent P. aeruginosa clinical isolates. The objective of this work was to determine whether pUM505 increases the virulence of P. aeruginosa and to identify the genes responsible for this property. First, using the lettuce-leaf model, we found that pUM505 significantly increases the virulence of P. aeruginosa reference strain PAO1. pUM505 also increased the PAO1 virulence in a murine model and increased cytotoxicity of this strain toward HeLa cells. Thus, we generated a pUM505 gene library of 103 clones in the pUCP20 binary vector. The library was transferred to Escherichia coli TOP10 and P. aeruginosa PAO1 to identify genes. The lettuce-leaf model allowed us to identify three recombinant plasmids that increased the virulence of both E. coli and P. aeruginosa strains. These recombinant plasmids also increased the virulence of the PAO1 strain in mice and induced a cytotoxic effect in HeLa cells. Eleven genes were identified in the virulent transformants. Of these genes, only the pUM505 ORF 2 has homology with a gene previously implicated in virulence. These results indicate that pUM505 contains several genes that encode virulence factors, suggesting that the plasmid may contribute directly to bacterial virulence.

The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA 2 mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA 2 mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA 2 mutant, relative to transcription of the corresponding genes in the lexA + strain, but decreased significantly in the lexA 2 /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 59 regions of SOS genes, suggesting an indirect mechanism of regulation.