An H(+)-coupled multidrug efflux pump, PmpM, a member of the MATE family of transporters, from Pseudomonas aeruginosa

We cloned the gene PA1361 (we designated the gene pmpM), which seemed to encode a multidrug efflux pump belonging to the MATE family, of Pseudomonas aeruginosa by the PCR method using the drug-hypersensitive Escherichia coli KAM32 strain as a host. Cells of E. coli possessing the pmpM gene showed elevated resistance to several antimicrobial agents. We observed energy-dependent efflux of ethidium from cells possessing the pmpM gene. We found that PmpM is an H(+)-drug antiporter, and this finding is the first reported case of an H(+)-coupled efflux pump in the MATE family. Disruption and reintroduction of the pmpM gene in P. aeruginosa revealed that PmpM is functional and that benzalkonium chloride, fluoroquinolones, ethidium bromide, acriflavine, and tetraphenylphosphonium chloride are substrates for PmpM in this microorganism.     

Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae

The name Enterobacter kobei sp. nov. is proposed for a group of organisms referred to as NIH Group 21 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, motile rods conforming to the definition of the family Enterobacteriaceae. The DNA relatedness of 23 strains of NIH Group 21 to the representative proposed as the type strain of this species averaged 82% at 70°C, whereas the relatedness to other species within the family Enterobacteriaceae was less than 42%. Because the phenotypic resemblance to Enterobacter cloacae is very close and the DNA relatedness (12–42%) is closer to species of the genus Enterobacter than to other species of the family, the members of NIH Group 21 were placed in the genus Enterobacter. Close phenotypic and genetic relationships were also found between NIH Group 21 and a member of a group of organisms referred to as Enteric Group 69 at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. It is suggested that the latter could be regarded as a subspecific rank of E. kobei, though this is subject to study of further strains. The majority of strains of E. kobei were isolated from clinical specimens. A culture of the type strain (NIH 1485-79) has been deposited in the Japan Collection of Microorganisms as JCM 8580. Received: 22 March 1996 / Accepted: 19 April 1996     

A staphylococcal multidrug resistance gene product is a member of a new protein family.

The complete nucleotide sequence (321 bp) of smr (staphylococcal multidrug resistance), a gene coding for efflux-mediated multidrug resistance of Staphylococcus aureus, was determined by using two different plasmids as DNA templates. The smr gene product (identical to products of ebr and qacC/D genes) was shown to be homologous to a new family of small membrane proteins found in Escherichia coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris. The smr gene was subcloned and expressed in S. aureus and E. coli and its ability to confer the multidrug resistant phenotype was demonstrated for two different lipophilic cation classes: phosphonium derivatives and quarternary amines. Expression of smr gene leads to the efflux of tetraphenylphosphonium and to a net decrease in the uptake of lipophilic cations. The deduced polypeptide sequence (107 amino acid residues, 11,665 kDa) has 46% hydrophobic residues (Phe, Ile, Leu, and Val) and 20% hydroxylic residues (Ser and Thr). Four transmembrane segments are predicted for smr gene product. Of the charged amino acid residues, only Glu 13 is located in a transmembrane segment. This Glu 13 is conserved in all members of the family of small membrane proteins. We propose a mechanism whereby exchange of protons at the Glu 13 is a key in the efflux of the lipophilic cation. This mechanism includes the idea that protons are transported to the Glu 13 via an appropriate chain of hydroxylic residues in the transmembrane segments of Smr.     

多重耐药阴沟肠杆菌质粒型AmpC酶DHA-1基因的检出

目的测定56株同时耐头孢噻肟、庆大霉素和环丙沙星的阴沟肠杆菌质粒型AmpC酶基因型。方法先后用头孢西丁纸片法、三维试验、等电聚焦及酶抑制试验和微量稀释法进行表型检测。接合试验证实酶基因的转移性。多重聚合酶链反应以及基因测序等方法鉴定质粒型AmpC酶基因型。结果受试的56株细菌中有5株三维试验阳性,其中1株能转移接合,接合子多重聚合酶链反应扩增结果呈阳性,等电点为7．8,基因测序表明和DHA-1型AmpC酶一致。结论我国的多重耐药阴沟肠杆菌已获得质粒型DHA基因,DHA基因可通过转化、接合等方式转移给其他细菌且易于传播,因此应加强监控以防DHA基因在革兰阴性菌中流行。     

Composition of O-antigenic lipopolysaccharides from Enterobacter cloacae

Analyses have been carried out on lipopolysaccharides (LPS) from 14 strains of Enterobacter cloacae representing different O serotypes. All of the products appeared to have a composition and architecture typical of enterobacterial LPS, but points of interest include the absence of phosphate residues from the core oligosaccharide, the presence of both L-glycero-D-mannoheptose and D-glycero-D-mannoheptose (ratio usually about 4:1), and the presence in lipid A of small amounts of fatty acids with odd numbers of carbon atoms (mainly C13) in addition to tetradecanoic acid and 3-hydroxytetradecanoic acid. Monosaccharides identified as components of polymeric fractions from the LPS were glucose, galactose, mannose, rhamnose, glucosamine, galactosamine, fucosamine, and galacturonic acid. Most polymeric fractions also probably contained an O-acetyl substituent. Closely similar chemotypes found for the polymeric fractions from the LPS of cross-reacting serotypes support the view that these fractions contain the O-antigenic determinants and represent the side chains of the LPS.     

Zebrafish tenascin-W,a new member of the tenascin family

A cDNA clone encoding tenascin-W, a novel member of the tenascin family, was isolated from a 20- to 28-h postfertilization (hpf) zebrafish cDNA library on the basis of the conserved epidermal growth factor-like domains represented in all tenascin molecules. An open reading frame of 2796 base pairs encodes a mature protein consisting of heptad repeats, a cysteine-rich amino terminal region, 3.5 epidermal growth factor-like repeats, five fibronectin type III homologous repeats, and a domain homologous to fibrinogen. These domains are the typical modular elements of molecules of the tenascin family. Sequence comparison demonstrated that TN-W shares homologies with the members of the tenascin family but is not a species homolog of any identified tenascin. The expression pattern of tn-w was analyzed by in situ hybridization in 1-day-old embryos, in 3-day-old larvae, and in juvenile zebrafish. At 24–25 hpf, tn-w mRNA was expressed in the lateral plate mesoderm, most conspicuously in the presumptive sclerotome. Migrating cells of sclerotomal and neural crest origins also showed high levels of expression. At 3 days, expression by sclerotomal and neural crest cells continued to be observed while expression in the somitic mesoderm was decreased. In juvenile fish, tn-w was expressed weakly by cells in the myosepta and, more strongly, by presumably nonneuronal cells in the dorsal root ganglia. In these tissues and at the same developmental stages, the expression of tn-w partially overlapped with the distribution of tn-c mRNA. In addition, tn-c was expressed in the central nervous system (CNS) and in the axial mesoderm, neither of which expressed tn-w at any of the age stages examined. The expression pattern of tn-w suggests an involvement in neural crest and sclerotome cell migration and in the formation of the skeleton. Similar and possibly overlapping functions could also be performed by tn-c, which appears to have additional functions during the development of the CNS. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 1–16, 1998     

Raver2, a new member of the hnRNP family

Raver2 was identified as a novel member of the hnRNP family based on sequence homology within three RNA recognition motifs and its general domain organization reminiscent of the previously described raver1 protein. Like raver1, raver2 contains two putative nuclear localization signals and a potential nuclear export sequence, and also displays nucleo-cytoplasmic shuttling in a heterokaryon assay. In glia cells and neurons, raver2 localizes to the nucleus. Moreover, the protein interacts with polypyrimidine tract binding protein (PTB) suggesting that it may participate in PTB-mediated nuclear functions. In contrast to ubiquitously expressed raver1, raver2 exerts a distinct spatio-temporal expression pattern during embryogenesis and is essentially restricted to brain, lung, and kidney in the adult mouse.     

Properties of Purine Nucleoside Phosphorylase from Enterobacter cloacae

Purine nucleoside phosphorylase from Enterobacter cloacae KY3074 was partially purified by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and DEAE-Sephadex A-50, and gel filtration on Sephadex G-100 and Sepharose 4B. The molecular weight of the enzyme was calculated to be about 87,000 by a gel filtration method on Sephadex G-200. The enzyme was found to be most active at pH 7.5 to 8.5 and 50°C, stable between pH 7.0 and 7.3, and the activity was nearly lost above 70°C. The enzyme split 2´-deoxyinosine and ribonucleosides. Lineweaver-Burk plots for phosphate were non-linear, showing substrate activation. The break-down of inosine approached an equilibrium when approximately 14% of inosine was phosphorylated.     

Attachment as a factor in the protection of Enterobacter cloacae from chlorination

Enterobacter cloacae attached to drinking water distribution particles was subjected to chlorination. Attachment resulted in the protection of these organisms from disinfection. This effect was found to be dependent upon both the level of chlorine in the system and attachment time. The results obtained in this study indicate that attached organisms may play an important role in coliform outbreaks.     

Purification and Properties of Uricase from Enterobacter cloacae

Uricase activity was found in Enterobacter cloacae KY3074 grown on guanine, hypoxanthine, uric acid, and xanthine media. The enzyme was purified from cells grown on uric acid as a source of nitrogen. The purification procedure included ammonium sulfate fractionation, gel filtration on Sephadex G-150, and column chromatography on DEAE-cellulose and DEAE-Sephadex. The enzyme had a molecular weight of about 105,000 and was specific for uric acid. The optimum pH was around 9.5, and the activity was inhibited by the presence of potassium cyanide, Ag⁺ or Cu²⁺. This uricase can be used for estimation of uric acid.     

Characterization of IS26‐composite transposons and multidrug resistance in conjugative plasmids from Enterobacter cloacae

SHV‐12 is the most widespread resistance determinant of Enterobacter cloacae in Taiwan; however, bla_SHV‐12 has rarely been mobilized. Six multidrug‐resistant E. cloacae isolates were collected. After conjugal transfer, plasmid profiling and analysis of incompatibility groups was performed to characterize the genetic context of bla_SHV‐12‐containing fragments. The presence of mobile genetic elements was demonstrated by PCR, cloning, sequencing and bioinformatics analyses. Four different β‐lactamase genes (bla_TEM‐1, bla_SHV‐12, bla_CTX‐M‐3 and/or bla_CTX‐M‐14) were observed in the conjugative plasmids belonging to the IncHI2 (n = 4), IncI1 or IncP incompatibility groups. The IS26‐bla_SHV‐12‐IS26 locus was located in five different genetic environments. A novel structural organization of a class 1 integron with the aac(6')‐IIc cassette truncated by IS26 was identified in one isolate. Thus, bla_SHV‐12 was obtained from different plasmids through IS26‐mediated homologous recombination. IS26 plays a vital role in the distribution of mobile resistance elements between different plasmids found in multidrug‐resistant E. cloacae isolates.     

Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae

The "classical" nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously described. Analysis of the 5'-untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.     

Gene cloning and characterization of four MATE family multidrug efflux pumps from Vibrio cholerae non-O1

There are six putative genes for multidrug and toxic compound extrusion (MATE) family multidrug efflux pumps in the chromosome of Vibrio cholerae. We have so far analyzed two MATE family pumps in V. cholerae non-O1 NCTC4716. Here we cloned four remaining genes for putative MATE family efflux pumps by the PCR method from this microorganism and designated them as vcmB, vcmD, vcmH and vcmN. Each one of the four genes was introduced and expressed in the drug hypersusceptible host Escherichia coli KAM32 cells. We observed elevated MICs of multiple antimicrobial agents, such as fluoroquinolones, aminoglycosides, ethidium bromide and Hoechst 33342 in the transformants. Energydependent efflux of substrate was observed with the transformed cells. We found that efflux activities of VcmB, VcmD and VcmH were Na+-dependent, but that of VcmN was Na+-independent. Thus, all six of the MATE family multidrug efflux pumps of V. cholerae non-O1 have been characterized. We also found that all six genes were expressed in cells of V. cholerae non-O1.     

Archaeal shikimate kinase, a new member of the GHMP-kinase family

Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: K(m,shikimate) = 414 +/- 33 microM, K(m,ATP) = 48 +/- 4 microM, and k(cat) = 57 +/- 2 s(-1) for the predicted shikimate kinase and K(m,homoserine) = 188 +/- 37 microM, K(m,ATP) = 101 +/- 7 microM, and k(cat) = 28 +/- 1 s(-1) for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.     

VapI, a new member of the Rhodococcus equi Vap family

Rhodococcus equi is a facultative intracellular bacterium which can cause bronchopneumonia in foals and AIDS patients. In this report we show that the ORF13-protein coded by the virulence associated plasmid of R. equi is clearly homologous to VapE. Nucleotide sequence analysis revealed frame shift mutations that shorten the sequence of the ORF13-protein. A theoretical extension of the sequence of ORF13 by the introduction of a single nucleotide yields a translated amino acid sequence that is highly homologous to VapE and other members of the␣Vap family. The data provided in this study indicate that the ORF13-protein is a novel member of the Vap family and is therefore designated VapI.     

Cerato-platanin, the first member of a new fungal protein family

The ascomcete Ceratocystis fimbriata, the causal agent of “canker stain disease,” secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP simlar to proteins of the hydrophobin family.     

Isolation and characterisation of a new mosquitocidal bacterium strain of Enterobacter cloacae VCRC-B519 from marine soil

A novel mosquitocidal bacterium was isolated from marine soil. 16S rRNA gene sequence alignment depicted that this isolate belonged to the strain, Enterobacter cloacae VCRC-B519 (NCBI: KC119193). Biochemical studies such as bacterial growth, biomass production and protein (toxin) synthesis showed that the strain is plausibly useful for mosquito control. It showed an increasing pattern of toxicity for Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti, without negative effects for non-targeted organisms Chironomus riparius, Daphnia cephalata and Notonecta glauca. The qualitative analysis of the E. cloacae showed that three polypeptides (M.wt: 25, 30 and 50 kDa) were associated to the toxicity observed. The characterisation of these polypeptides (M/S MALDI-TOF)showed that they are enzymatic in nature. Consequently, the peptide sequences are identified to be polysugar degrading enzymes (25 kDa), cell wall associated hydrolases (30 kDa) and amino peptidase (50 kDa). Phylogenetic analyses of 16S rDNA gene sequence of E. cloacae revealed the occurrence of homology with closely related Enterobacter species. Therefore, it is concluded that the marine bacterium (Enterobacter cloacae)is possibly of use for the biological control of mosquito immatures.