Lichenicidin rational site-directed mutagenesis library: A tool to generate bioengineered lantibiotics

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that arise as an alternative to the traditional antibiotics. Lichenicidin is active against clinically relevant bacteria and it was the first lantibiotic to be fully produced in vivo in the Gram-negative host Escherichia coli. Here, we present the results of a library of lichenicidin mutants, in which the mutations were generated based on the extensive bibliographical search available for other lantibiotics. The antibacterial activity of two-peptide lantibiotics, as is lichenicidin, requires the synergistic activity of two peptides. We established a method that allows screening for bioactivity which does not require the purification of the complementary peptide. It is an inexpensive, fast and user-friendly method that can be scaled up to screen large libraries of bioengineered two-peptide lantibiotics. The applied system is reliable and robust because, in general, the results obtained corroborate structure–activity relationship studies carried out for other lantibiotics.     

Reparation of the damages induced by UV light in salmonellae]

The survival of Salmonella typhimurium wild type strains after UV-irradiation is studied. It is demonstrated that many of these are more sensitive to UV-irradiation than Escherichia coli of the wild type. Alkaline sucrose density gradient centrifugation has demonstrated a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a feature of Salmonella genus, because a strain is found of the same resistance and reparation ability as E. coli wild type strain.     

Cloning and expression of the thermostable pullulanase gene from Thermus sp. strain AMD-33 in Escherichia coli

Abstract The gene coding for a thermostable pullulanase from a thermophile, Thermus sp. strain AMD-33, was cloned in Escherichia coli using pDR540 as a vector. A restriction map was determined for the plasmid pTPS131 which contained the fragment carrying the pullulanase gene. DNA-DNA hybridisation analysis showed that the DNA fragment contained the gene from Thermus sp. strain AMD-33. The strain of E. coli harbouring the plasmid pTPS131 produced most of the pullulanase protein cellularly, whereas Thermus sp. strain AMD-33 produced pullulanase extracellularly. Comparative studies of the enzyme from the thermophile and the plasmid-encoded enzyme in E. coli demonstrated that the optimum temperature and pH of the enzymes were closely similar.     

A new cytolethal distending toxin (CDT) from Escherichia coli producing CNF2 blocks HeLa cell division in G2/M phase

Escherichia coli strain 1404, isolated from a septicaemic calf, carries a transferable plasmid called pVir which codes for the cytotoxic necrotizing factor type 2 (CNF2). A 4 h interaction between strain 1404 and HeLa cells induced the formation of giant mononucleated cells blocked in G2/M phase. Mating experiments between strain 1404 and a non-pathogenic recipient strain demonstrated that the factor(s) encoded by pVir mediated the cell-cycle arrest. A 3.3 kb DNA fragment isolated from a DNA bank of pVir was shown to code for the factor(s) causing the cell-cycle arrest. Nucleotide sequence analysis revealed the presence of three genes encoding proteins sharing significant amino acid homology with the cytolethal distending toxins (CDTs) previously isolated from E. coli , Campylobacter jejuni and Shigella dysenteriae . Southern hybridization experiments demonstrated that the pVir of other CNF2-producing E. coli strains contained sequences related to cdt . Although the amino acid sequences amongst CDT diverged significantly, the two other CDTs previously isolated from E. coli were also able to block the HeLa cell cycle. In conclusion, this study demonstrates the mode of action of CDT and will help us to elucidate the role of this emerging toxin family in microbial pathogenesis.     

Use of virulence factor-specific egg yolk-derived immunoglobulins as a promising alternative to antibiotics for prevention of attaching and effacing Escherichia coli infections

Using a porcine ileal in vitro organ culture model, we have demonstrated that egg yolk-derived antibodies specific for the attaching and effacing Escherichia coli (AEEC) virulence factors intimin and translocated intimin receptor (Tir), but not those specific for the AEEC-secreted proteins EspA, EspB and EspD, significantly reduced the bacterial adherence of the porcine enteropathogenic E. coli strain ECL1001, formerly 86-1390. Moreover, antibodies specific for intimin and Tir also significantly reduced bacterial adherence of heterologous AEEC strains, including human, bovine and canine enteropathogenic E. coli strains, as well as of O157:H7 Shiga toxin-producing E. coli strains in this model. In addition, we demonstrated that the oral administration of these anti-intimin antibodies significantly reduced the extent of attaching and effacing lesions found in the small intestine of weaned pigs challenged with the porcine enteropathogenic E. coli strain ECL1001. Overall, our results underline the potential use of specific egg yolk-derived antibodies as a novel approach for the prevention of AEEC infections.     

Characterization of the integration site of Yersinia high-pathogenicity island in Escherichia coli

The high-pathogenicity island (HPI) of virulent Yersiniae consists of (i) a functional core encoding for biosynthesis and uptake of the siderophore yersiniabactin and (ii) a 5- to 13-kb AT-rich region of unknown function. This Yersinia HPI has been shown to be widely distributed among different pathotypes of Escherichia coli. In this study, the insertion site of the HPI was defined in three different E. coli strains: The enteroaggregative E. coli (EAggEC) strain 17-2, the uropathogenic (UPEC) E. coli strain 536, and the probiotic E. coli DSM6601. We demonstrated that in all three E. coli isolates the HPI is associated with the asnT tRNA (5'-extremity) and truncated in the AT-rich region (3'-extremity) since the 17-bp direct repeat (DR) of the asn tRNA that flanks the HPI in Yersinia is missing in E. coli. Moreover, in comparison to the HPI-negative E. coli K-12 strain, a uniform deletion must have taken place in the E. coli chromosome adjacent to the 3'-border of the HPI.     

肠毒素源性定居因子CS6在减素伤寒沙门氏菌中表达及免疫原性的研究

将编码肠毒素源性大肠杆菌定居因子抗原ＣＳ６基因克隆到ｐＸＬ６７０,转化ａｓｄ基因突变的Ｅ．ｃｏｌｉ Ｘ６０９７,获得重组质粒ｐＳＳ６４,再将后者转化至减毒的△ａｒｏＡ、△ａｒｏＣ、△ａｓｄ伤寒沙门氏菌,构建了无药物抗性且稳定的大肠杆菌和伤寒双价菌苗候选株。小鼠腹腔免疫和攻击实验表明,该菌株对伤寒沙门氏菌毒株的攻击具有良好的保护作用。家兔免疫实验证明,该菌株能产生抗ＣＳ６和伤寒菌Ｖｉ抗原的血清抗体。     

Holo-(acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli

Holo-(acyl carrier protein) synthase (AcpS) post-translationally modifies apoacyl carrier protein (apoACP) via transfer of 4'-phosphopantetheine from coenzyme A (CoA) to the conserved serine 36 gamma-OH of apoACP. The resulting holo-acyl carrier protein (holo-ACP) is then active as the central coenzyme of fatty acid biosynthesis. The acpS gene has previously been identified and shown to be essential for Escherichia coli growth. Earlier mutagenic studies isolated the E. coli MP4 strain, whose elevated growth requirement for CoA was ascribed to a deficiency in holoACP synthesis. Sequencing of the acpS gene from the E. coli MP4 strain (denoted acpS1) showed that the AcpS1 protein contains a G4D mutation. AcpS1 exhibited a approximately 5-fold reduction in its catalytic efficiency when compared with wild type AcpS, accounting for the E. coli MP4 strain phenotype. It is shown that a conditional acpS mutant accumulates apoACP in vivo under nonpermissive conditions in a manner similar to the E. coli MP4 strain. In addition, it is demonstrated that the gene product, YhhU, of a previously identified E. coli open reading frame can completely suppress the acpS conditional, lethal phenotype upon overexpression of the protein, suggesting that YhhU may be involved in an alternative pathway for phosphopantetheinyl transfer and holoACP synthesis in E. coli.     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23.

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.     

Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria

We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1. In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E. coli and other indole-producing bacteria. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium. Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected. At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E. coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains. In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species. Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors.     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.     

A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.     

霍乱cT-B和LPs-o双价抗原工程菌苗株的构建

作者将霍乱弧菌O抗原及毒素B亚单位基因片段,经DNA体外重组技术,得到了能表达双价抗原的工程菌株1046(pMG305)。经GM1-ELISA分析表明该菌株能够表达特异的霍乱CT-B抗原,且能分泌到胞外,通过菌体凝集,全细胞O抗原酶联分析和血凝抑制试验表明在1046(pMG305)菌体表面表达了霍乱的O抗原,它的脂多糖O抗原通过SDS-PAGE电泳分析,显示它表达了霍乱LPS的特征区带。小鼠腹腔免疫后用霍乱弧菌毒株攻击表明,有良好的保护作用,因此1046(pMG305)可望成为霍乱活疫苗的候选株。     

BipA is required for growth of Escherichia coli K12 at low temperature

The bipA gene encodes a ribosome-associated GTPase postulated to be involved in regulatory functions in enteropathogenic Escherichia coli. Previous studies demonstrated that BipA is tyrosine phosphorylated in EPEC strains, but not in E. coli strain K12. Results presented here indicate that BipA function is required at low temperatures in E. coli K12, suggesting a regulatory role independent of phosphorylation and of pathogenicity.     

Cloning the DdeI restriction-modification system using a two-step method.

DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.     

Isolation of mutants with impaired retroinhibition of histidine biosynthesis

The Escherichia coli, strain possessing purF, deoD and add mutations converts exogenous adenine into guanine nucleotides exclusively by the pathway coupled with histidine biosynthesis. When grown on adenine, this strain demonstrated sensitivity to histidine, thus making it possible to select histidine-resistant hisGR mutants with ATP-phosphoribosyltransferase desensibilized for histidine. The hisGR mutations were obtained in two his operons introduced into the his operon-sensitive E. coli strain: his operon of Salmonella typhimurium incorporated in DNA and his operon of E. coli on the F'episome. In both cases, the hisGR mutants obtained were shown to excrete histidine.     

Investigation of the Antimicrobial Activity of Bacillus licheniformis Strains Isolated from Retail Powdered Infant Milk Formulae

This study investigated the potential antimicrobial activity of ten Bacillus licheniformis strains isolated from retail infant milk formulae against a range of indicator (Lactococcus lactis, Lactobacillus bulgaricus and Listeria innocua) and clinically relevant (Listeria monocytogenes, Staphylococcus aureus, Streptococcus agalactiae, Salmonella Typhimurium and Escherichia coli) microorganisms. Deferred antagonism assays confirmed that all B. licheniformis isolates show antimicrobial activity against the Gram-positive target organisms. PCR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses indicated that four of the B. licheniformis isolates produce the bacteriocin lichenicidin. The remaining six isolates demonstrated a higher antimicrobial potency than lichenicidin-producing strains. Further analyses identified a peptide of ~1,422 Da as the most likely bioactive responsible for the antibacterial activity of these six isolates. N-terminal sequencing of the ~1,422 Da peptide from one strain identified it as ILPEITXIFHD. This peptide shows a high homology to the non-ribosomal peptides bacitracin and subpeptin, known to be produced by Bacillus spp. Subsequent PCR analyses demonstrated that the six B. licheniformis isolates may harbor the genetic machinery needed for the synthesis of a non-ribosomal peptide synthetase similar to those involved in production of subpeptin and bacitracin, which suggests that the ~1,422 Da peptide might be a variant of subpeptin and bacitracin.     

Expression of the gene for Bacillus subtilis aspartokinase II in Escherichia coli

The gene coding for the subunits of aspartokinase II from Bacillus subtilis has been identified in a B. subtilis DNA library and cloned in a bacterial plasmid (Bondaryk, R. P., and Paulus, H. (1984) J. Biol. Chem. 259, 585-591). The introduction of a plasmid carrying the aspartokinase II gene into an auxotrophic Escherichia coli strain lacking all three aspartokinases restored its ability to grow in the absence of L-lysine, L-threonine, and L-methionine. The B. subtilis aspartokinase gene could thus be functionally expressed in E. coli and substitute for the E. coli aspartokinases. Measurement of aspartokinase levels in extracts of aspartokinaseless E. coli transformed with the B. subtilis aspartokinase II gene revealed an enzyme level comparable to that in a genetically derepressed B. subtilis strain. In spite of the high level of aspartokinase, the growth of the transformed E. coli strain was severely inhibited by the addition of L-lysine but could be restored by also adding L-homoserine. This apparently paradoxical sensitivity to lysine was due to the allosteric inhibition of B. subtilis aspartokinase II by that amino acid, a property which was also observed in extracts of the transformed E. coli strain. The synthesis and degradation of the aspartokinase II subunits were measured by labeling experiments in E. coli transformed with the B. subtilis aspartokinase II gene. In contrast to exponentially growing cells of B. subtilis which contained equimolar amounts of the aspartokinase alpha and beta subunits, the transformed E. coli strain contained a 3-fold molar excess of beta subunit. Pulse-chase experiments showed that the disproportionate level of beta subunit was not due to more rapid turnover of alpha subunit, both subunits being quite stable, but presumably to a more rapid rate of synthesis. After the addition of rifampicin, the synthesis of alpha subunit declined much more rapidly than that of beta subunit, indicating that the two subunits were translated independently from mRNA species that differ in functional stability. In conjunction with the results described in the preceding paper which demonstrated that the aspartokinase subunits are encoded by a single DNA sequence, these observations imply that the alpha and beta subunits of B. subtilis aspartokinase II are the products of in-phase overlapping genes.     

Mechanism of assembly of the Bis(Molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase

A fully defined in vitro system has been developed for studying the mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase (DMSOR). R. sphaeroides DMSOR expressed in a mobA(-) Escherichia coli strain lacks molybdopterin and molybdenum but contains a full complement of guanine in the form of GMP and GDP. Escherichia coli MobA, molybdopterin-Mo, GTP, and MgCl(2) are required and sufficient for the in vitro activation of purified DMSOR expressed in the absence of MobA. High levels of MobA inhibit the in vitro activation. A chaperone is not required for the in vitro activation process. The reconstituted DMSOR can exhibit up to 73% of the activity observed in recombinant DMSOR purified from a wild-type strain. The use of radiolabeled GTP has demonstrated incorporation of the guanine moiety from the GTP into the activated DMSOR. No role was observed for E. coli MobB in the in vitro activation of apo-DMSOR. This work also represents the first time that the MobA-mediated conversion of molybdopterin to molybdopterin guanine dinucleotide has been demonstrated directly without using the activation of a molybdoenzyme as an indicator for cofactor formation.