Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanXB dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.     

Induction of surfactin production in Bacillus subtilis by gsp, a gene located upstream of the gramicidin S operon in Bacillus brevis

The deduced amino acid sequence of the gsp gene, located upstream of the 5' end of the gramicidin S operon (grs operon) in Bacillus brevis, showed a high degree of similarity to the sfp gene product, which is located downstream of the srfA operon in B. subtilis. The gsp gene complemented in trans a defect in the sfp gene (sfpO) and promoted production of the lipopeptide antibiotic surfactin. The functional homology of Gsp and Sfp and the sequence similarity of these two proteins to EntD suggest that the three proteins represent a new class of proteins involved in peptide secretion, in support of a hypothesis published previously (T. H. Grossman, M. Tuckman, S. Ellestad, and M. S. Osburne, J. Bacteriol. 175:6203-6211, 1993).     

Morbidity and mortality following preoperative radiation therapy and total pelvic exenteration for primary rectal adenocarcinoma

Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 23-kb chromosomal segment, which covers the region between the iol and hut operons, has been cloned and sequenced, creating a 99-kb contig from the gnt operon to the wapA locus. This region (23351 bp) contains 25 complete open reading frames (ORFs; genes) including deoR, dra, nupC and pdp and two partial ones. The region (5140 bp) containing these four genes, being also sequenced by H. H. Saxild et al., was sequenced by subjecting a long polymerase chain reaction product to random sequencing using phage M13mp19. However, we could detect no conflict, between two independently determined sequences, which could be attributed to our sequencing method. A homology search for the 24 newly identified gene products revealed significant homology to known proteins in 14 of them. It was notable that three proteins, encoded by the successive genes (yxeMNO), exhibited meaningful homology to the E. coli GlnHPQ products constituting a periplasmic ATP-dependent transport system for glutamine.     

Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600

A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotide sequence contained three major open reading frames (ORFs). Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components. We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B). The ntaA and ntaB genes could be expressed in Escherichia coli DH5alpha, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible. The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases. These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression.     

Randomized, placebo-controlled, multicenter trial of granulocyte-macrophage colony-stimulating factor as infection prophylaxis in oncologic surgery. Leukine Surgical Prophylaxis Research Group

The EUO gene (for early upstream open reading frame) of Chlamydia psittaci was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with C. psittaci 6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp -200 to +67 promoter region of the cysteine-rich envelope protein (crp) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects crp DNA from DNase I from approximately bp -60 to -9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the crp operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the crp operon with high affinity in vitro (Kd of about 15 nM), it is not known whether the protein binds the crp DNA in vivo.     

Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate

An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.     

Identification of novel intergenic repetitive units in a mycobacterial two-component system operon

Mycobacterial interspersed repetitive units (MIRUs), a novel class of repeated sequences, were identified within the intercistronic region of an operon coding for a mycobacterial two-component system, named senX3-regX3. Southern blot analysis and homology searches revealed the presence of several homologous sequences in intergenic regions dispersed throughout the genomes of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae. These could be grouped into three major families, containing elements of 77-101 bp, 46-53 bp and 58-101 bp. Based on the available mycobacterial sequences, the total number of MIRUs is estimated to be about 40-50 per genome. Similar to previously identified small repetitive sequences, the MIRUs of the two-component operon are transcribed on a polycistronic mRNA. Unlike previously identified small repetitive sequences, however, MIRUs do not contain dyad symmetries, comprise small open reading frames (ORFs) whose extremities overlap those of the contiguous ORFs and are oriented in the same translational direction as those of the adjacent genes. Analyses of the sequences at the insertion sites suggest that MIRUs disseminate by transposition into DTGA sites involved in translational coupling in polycistronic operons.     

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(+) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.     

Cloning and characterization of a human DEAH-box RNA helicase, a functional homolog of fission yeast Cdc28/Prp8

During the splicing process, spliceosomal snRNAs undergo numerous conformational rearrangements that appear to be catalyzed by proteins belonging to the DEAD/H-box superfamily of RNA helicases. We have cloned a new RNA helicase gene, designated DBP2 (DEAH-boxprotein), homologous to the Schizosaccaromyces pombe cdc28(+)/prp8(+) gene involved in pre-mRNA splicing and cell cycle progression. The full-length DBP2 contains 3400 nucleotides and codes for a protein of 1041 amino acids with a calculated mol. wt of 119 037 Da. Transfection experiments demonstrated that the GFP-DBP2 gene product, transiently expressed in HeLa cells, was localized in the nucleus. The DBP2 gene was mapped by FISH to the MHC region on human chromosome 6p21.3, a region where many malignant, genetic and autoimmune disease genes are linked. Because the expression of DBP2 gene in S.pombe prp8 mutant cells partially rescued the temperature-sensitive phenotype, we conclude that DBP2 is a functional human homolog of the fission yeast Cdc28/Prp8 protein.