Ultrastructure of the capsule of Klebsiella pneumoniae and slime of Enterobacter aerogenes revealed by freeze etching

Summary The capsule of Klebsiella pneumoniae type I and slime of Enterobacter aerogenes strain A3 (SL) was examined by electron microscopy using the freeze etch technique. The capsules of K. pneumoniae were found to be composed of several layers of polysaccharide 10 nm thick; while the polysaccharide slime of E. aerogenes strain A3 (SL) was found to be composed of a diffuse network of fibrils. This work represents the first effort to visualize the replica of the unfixed, partially hydrated bacterial capsule or slime in the electron microscope. The slime of E. aerogenes strain A3 (SL) which was purified, and then freeze etched, resembled the layered structure of the capsule of K. pneumoniae. It is suggested that the charge or dielectric constant of the slime polysaccharide polymers was altered during purification, thereby permitting the layering to occur.Paper presented at the Annual Meeting of the American Society for Microbiology, Philadelphia, Pa. (U.S.A.), 1972.     

Primary structure of the chromosomal origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae: comparisons and evolutionary relationships

The nucleotide sequences of the Enterobacter aerogenes and Klebsiella pneumoniae DNA replication origins (oriC) were determined and compared with those of Escherichia coli and Salmonella typhimurium. Four interrelated, 9-base-pair repeats were identified from the conserved regions within the minimal origin. Evolutionary rates calculated from the minimal origin sequences yielded a quantitative phylogenic tree which agreed with the taxonomic classification of these genera.     

Chromosomal replication origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae are functional in Escherichia coli.

The chromosomal DNA replication origins (oriC) from two members of the family Enterobacteriaceae, Enterobacter aerogenes and Klebsiella pneumoniae, have been isolated as functional replication origins in Escherichia coli. The origins in the SalI restriction fragments of 17.5 and 10.2 kilobase pairs, cloned from E. aerogenes and K. pneumoniae, respectively, were found to be between the asnA and uncB genes, as are the origins of the E. coli and Salmonella typhimurium chromosomes. Plasmids containing oriC from E aerogenes, K. pneumoniae, and S. typhimurium replicate in the E. coli cell-free enzyme system (Fuller, et al., Proc. Natl. Acad. Sci. U.S.A. 78:7370--7374, 1981), and this replication is dependent on dnaA protein activity. These SalI fragments from E. aerogenes and K. pneumoniae carry a region which is lethal to E. coli when many copies are present. We show that this region is also carried on the E. coli 9.0-kilobase-pair EcoRI restriction fragment containing oriC. The F0 genes of the atp or unc operon, when linked to the unc operon promoter, are apparently responsible for the lethality.     

Ethyl Violet. A Selective Dye for the Isolation of Gram-Negative Bacteria

Minimal inocula of Gram-negative and positive bacteria were seeded into tryptose broth containing varying concentrations of dyes. Three dyes were used, namely crystal violet, brilliant green and ethyl violet. Growth rates were determined for 2, 4 and 6 hours incubation. All three dyes were equally effective in inhibiting Gram positive bacteria. Ethyl violet showed markedly less toxicity toward Gram negative bacteria than did either crystal violet or brilliant green.     

Ethyl Violet. A Selective Dye for the Isolation of Gram-Negative Bacteria

Minimal inocula of Gram-negative and positive bacteria were seeded into tryptose broth containing varying concentrations of dyes. Three dyes were used, namely crystal violet, brilliant green and ethyl violet. Growth rates were determined for 2, 4 and 6 hours incubation. All three dyes were equally effective in inhibiting Gram positive bacteria. Ethyl violet showed markedly less toxicity toward Gram negative bacteria than did either crystal violet or brilliant green.     

Purification and properties of pyridoxal-5''-phosphate-dependent histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes.

Histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes were purified to homogeneity and compared with the histidine decarboxylase from Morganella morganii. All three enzymes required pyridoxal 5'-phosphate as a coenzyme, showed optimal activity at pH 6.5, decarboxylated only histidine among the amino acids derived from protein, and were tetramers or dimers of identical subunits. Amino-terminal sequences of the three enzymes showed up to 81% homology through residue 33, but the enzymes differed sufficiently in amino acid composition and sequence so that no cross-reaction occurred between the K. planticola or E. aerogenes enzymes and antibodies to the decarboxylase from M. morganii. All three enzymes were inhibited by carbonyl reagents; by amino-, carboxyl-, and some methyl-substituted histidines; and by alpha-fluoromethylhistidine. These decarboxylases, all from gram-negative organisms, differed greatly in subunit structure, biogenesis, and other properties from the pyruvoyl-dependent histidine decarboxylases from gram-positive organisms described previously.     

Isolation of a Polyvalent Bacteriophage for Escherichia coli, Klebsiella pneumoniae, and Aerobacter aerogenes

A lytic bacteriophage isolated from sewage was found to attack strains of Aerobacter aerogenes, Escherichia coli, and Klebsiella pneumoniae, but not members of the genera Salmonella, Proteus, and Serratia. The phage, designated phimp, contained deoxyribonucleic acid with a 50% guanine plus cytosine ratio and a molecular weight of 23.1 x 10(6) daltons. Single-step growth experiments of phimp plated at 37 C on A. aerogenes A2 gave a mean latent period of 20 min, an average burst size of 103 plaque-forming units/infected cell, and an average adsorption rate constant of 3 x 10(-10) ml/min. Electron microscopy of phimp revealed a phage with a flexible tail (165 nm long and 6 nm wide). The phage head had a hexagonal outline (62 nm in diameter).     

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.     

Purification and properties of a phosphohydrolase from Enterobacter aerogenes.

A phosphohydrolase from Enterobacter aerogenes which hydrolyzes phosphate mono- and diesters has been purified approximately 50-fold to apparent homoeneity and crystallized. The enzyme is produced when the bacteria utilize phosphate diesters as sole phosphorus source. From sedimentation equilibrium experiments the molecular weight of the native enzyme is 173,000; from sodium dodecyl sulfate polyacrylamide gel electrophoresis the subunit molecular weight is 29,000, indicating that the enzyme is hexameric. The hydrolytic activity of the enzyme using both mono- and diesters is maximal at pH 5; THE Km of the enzyme for bis-p-nitrophenyl phosphate is constant from pH 5 to 8.5 whereas that for p-nitrophenyl phosphate increases about 40-fold as the pH increases over the same range. The phosphodiesterase activity is not inhibited by chelating agents but is inhibited by several divalent metal ions. 31-P NMR spectroscopy was used to identify the hydrolysis products of glycoside cyclic phosphates. The enzyme-catalyzed hydrolysis of methyl beta-D-ribofuranoside cyclic 3:5-phosphate yields exclusively the 5-phosphate whereas that of adenosine 3:5-monophosphate yields a 4:1 mixture of 3- and 5- AMP.     

Action of iron and iron-complexes onKlebsiella pneumoniae (Klebsiella aerogenes)

Iron in the Fe(III) oxidation state had a negligible effect on the growth ofKlebsiella pneumoniae even at the highest concentration (0.45mm) obtainable without precipitation in a minimal medium containing glucose and inorganic salts together with Tris as the buffer and glycerol 2-phosphate as the phosphorus source. Nevertheless in its presence the toxic action of Cd²⁺, Zn²⁺ and Cu²⁺ was antagonized while that of Co²⁺ and Ni²⁺ was potentiated. Higher iron levels were obtained by supplementing the minimal medium with fructose, glycine, gluconate, tartrate and citrate at a range of concentrations. With fructose and glycine all of the resulting solutions were red-brown and non-toxic. This was also found with the other complexing agents when the ligand:iron ratios were low, but at higher ligand:iron ratios the solutions were green and toxic. Iron-citrate systems were especially toxic but resistance developed and was of the graded type. The results are discussed with particular reference to earlier physico-chemical studies by other workers and it is concluded that the red-brown colour is characteristic of the presence of polymers of high molar mass and that the green colour signifies the formation of low molar mass species.     

Incidence of antibiotic-resistant Klebsiella pneumoniae and Enterobacter species in freshwater wetlands

AIMS: The aim of this study was to assess the incidence of Enterobacteriaceae (potential human and animal pathogens) in wetlands. METHODS: Enterobacteriaceae, selected from the sediments and rhizosphere of wetland plant Juncus effusus L., were analysed using classical microbiological methods, API20E, API20NE, fatty acid analyses, and 16S rRNA sequencing. Assessed virulence factors include antibiotic resistance, presence of plasmids and capsules. RESULTS: Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter asburiae, known human pathogens, were identified. K. pneumoniae 16S rRNA gene sequence showed the significant hit (E < 0.001) with the unculturable bacteria obtained from faeces of elderly individuals (accession number AB099804) when Genbank database was used. Ent. asburiae 16S rRNA gene sequence showed the significant hit with (E < 0.001) with the unculturable bacteria obtained from the pig gastrointestinal tract (accession number AF371852). The rate of antibiotic resistance (<50 microg ml(-1)) was high for ampicillin and cephalosporins for the most strains (75.7%) yet low (>10 to 20 microg ml(-1)) for kanamycin, tetracycline and chloramphenicol for all strains tested. Capsules were detected in all investigated strains. PCR detected membrane protein but not chromosomally encoded beta-lactamase. Significance and Impact of the Study: The antibiotic resistance of tested strains and presence of capsules (protect micro-organisms from phagocytosis) suggest that wetland sediments and rhizosphere present a potential reservoirs for enteric human and animal pathogens.     

Conservation and DNA sequence arrangement of the DNA polymerase I gene region from Klebsiella aerogenes, Klebsiella pneumoniae and Escherichia coli

Linked multiple mutation is observed after treatment of Escherichia coli with methyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and N-ethyl-N-nitro-N-nitrosoguanidine but not ultraviolet light. Induction of linked multiple mutations requires the uvrE⁺ gene product indicating the involvement of the mismatch repair system. The observation of linked multiple mutations is not due to mutagenesis occurring in a subpopulation of cells. Growing point mutagenesis also occurs after treatment with these mutagens but not with ultraviolet light. It is likely that the excess of mutations observed with these mutagens at growing points is at least partly a relative effect, rather than one due to an absolute increase of reactivity at the DNA growing point region. This relative effect may result from the operation of an inducible repair mechanism which removes O⁶-alkylguanine residues from the DNA distal to the bacterial growing point. The adaptive response, first described by Robins &; Cairns (1979) prefers O⁶-methylguanine over O⁶-ethylguanine.     

产气肠杆菌EAM-Z1尿苷磷酸化酶的分离纯化及性质研究

从产气肠杆菌 (Enterobacteraerogenes)突变株EAM Z1中分离出一种具有较高转移酶活性的尿苷磷酸化酶 (UPase)。经测定这种Upase的分子量为 1 2 .8× 1 0 4,亚基分子量为 4 .3×1 0 4,由 3个同型亚基组成。N端氨基酸序列为 :MRMVDLIATKRDGGE。等电点为 4 .46。对尿苷的Km为 0 .2 9mmol L。酶反应的最适pH为 7.8,最适温度为 50℃。该酶能磷酸化尿苷、胸苷、5 氟尿苷、2′ 脱氧 5 氟尿苷及尿嘧啶 β D 阿拉伯呋喃糖 ,且具有较高的转移酶活性 ,能将尿苷和 5 氟尿嘧啶转化成 5 氟尿苷 (一种抗癌药物的中间体 ) ,其转化率为 47%。该酶的这些特性对于酶法合成核苷类抗肿瘤药物和抗病毒药物是十分有用的。     

产气肠杆菌EAM-Z1尿苷磷酸化酶的分离纯化及性质研究

从产气肠杆菌（Enterobacter aerogenes）突变株EAMZ1中分离出一种具有较高转移酶活性的尿苷磷酸化酶(Upase)。经测定这种Upase的分子量为12.8×104,亚基分子量为4.3×10.4,由３个同型亚基组成。N端氨基酸序列为：MRMVDLIATKRDGGE。等电点为4.46。对尿苷的Km为0.29mmol/L。酶反应的最适pH为7.8,最适温度为50℃。该酶能磷酸化尿苷、胸苷、5氟尿苷、2′脱氧5氟尿苷及尿嘧啶βD阿拉伯呋喃糖,且具有较高的转移酶活性,能将尿苷和5氟尿嘧啶转化成5氟尿苷(一种抗癌药物的中间体),其转化率为47％。该酶的这些特性对于酶法合成核苷类抗肿瘤药物和抗病毒药物是十分有用的。     

Isolation of a poly-α-l-guluronate lyase from Klebsiella aerogenes

The bacterium Klebsiella aerogenes type 25 produces an extracellular alginolyase which has been partly purified. The enzyme is specific for the α-l-guluronosyl linkages in whole alginate and fractions therefrom. The end products of its action on polyguluronic acid blocks are mainly the unsaturated di- and tri-saccharides, with a smaller proportion of the homologous tetrasaccharide. Some general properties of the enzyme are reported.     

Biosynthesis and secretion of pullulanase, a lipoprotein from Klebsiella aerogenes

We constructed, by site-directed mutagenesis, a mutant pullulanase gene in which the cysteine residue in a pentapeptide sequence, Leu16-Leu-Ser-Gly-Cys20 within the NH2-terminal region of pullulanase from Klebsiella aerogenes, is replaced by serine (Ser20). The modification, processing, and subcellular localization of the mutant pullulanase were studied. Labeling studies with [3H]palmitate and immunoprecipitation with mouse antiserum raised against pullulanase showed that the wild form of both the extracellular and intracellular pullulanases contained lipids, whereas the mutant enzyme was not modified with lipids. Only the Cys20 was modified with glyceryl lipids. The bulk of the mutant pullulanase was located in the periplasm, but a portion of the unmodified, mutant pullulanase was secreted into the medium. Mutant pullulanases from the extracellular and the periplasm were purified and their NH2-terminal sequences were determined. Both the mutant pullulanases were cleaved between residues of Ser13 and Leu14 which is 6-amino acid residues upstream of the lipid modified pullulanase cleavage site. This new cleavage was resistant to globomycin, an inhibitor of the prolipoprotein signal peptidase of Escherichia coli. These results indicate that the pentapeptide sequence plays an important role in maturation and translocation of pullulanase in K. aerogenes. However, the modification of pullulanase with lipids seems to be not essential for export of the enzyme across the outer membrane.     

Isolation and characterization of an alginate lyase from Klebsiella aerogenes

The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37°C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acidrich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.Abbreviations poly(ManA) (1–4)--D-mannuronan - poly(GulA) (1–4)--L-guluronan - TBA 2-thiobarbituric acid     

Purification and characterization of a bacteriocin from Klebsiella pneumoniae 158

Klebocin, a bacteriocin produced by Klebsiella pneumoniae 158, was purified to homogeneity by ammonium sulphate fractionation and sequential DEAE-Sephacel and Sephadex G-150 column chromatography. The purified preparation had an Mr of approximately 40 000 on SDS-PAGE. Chemical analysis of the purified preparation showed it to be a protein, and it was sensitive to digestion by various proteolytic enzymes.