The Biosynthesis of UDP-d-FucNAc-4N-(2)-oxoglutarate (UDP-Yelosamine) in Bacillus cereus ATCC 14579: Pat AND Pyl,AN AMINOTRANSFERASE AND AN ATP-DEPENDENT Grasp PROTEIN THAT LIGATES 2-OXOGLUTARATE TO UDP-4-AMINO-SUGARS*

Surface glycan switching is often observed when micro-organisms transition between different biotic and abiotic niches, including biofilms, although the advantages of this switching to the organism are not well understood. Bacillus cereus grown in a biofilm-inducing medium has been shown to synthesize an unusual cell wall polysaccharide composed of the repeating subunit →6)Gal(α1–2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1–6)GlcNAc(β1→, where galactose is linked to the hydroxyglutarate moiety of FucNAc-4-amido-(2)-hydroxyglutarate. The molecular mechanism involved in attaching 2-hydroxyglutarate to 4-amino-FucNAc has not been determined. Here, we show two genes in B. cereus ATCC 14579 encoding enzymes involved in the synthesis of UDP-FucNAc-4-amido-(2)-oxoglutarate (UDP-Yelosamine), a modified UDP-sugar not previously reported to exist. Using mass spectrometry and real time NMR spectroscopy, we show that Bc5273 encodes a C4″-aminotransferase (herein referred to as Pat) that, in the presence of pyridoxal phosphate, transfers the primary amino group of l-Glu to C-4″ of UDP-4-keto-6-deoxy-d-GlcNAc to form UDP-4-amino-FucNAc and 2-oxoglutarate. Pat also converts 4-keto-xylose, 4-keto-glucose, and 4-keto-2-acetamido-altrose to their corresponding UDP-4-amino-sugars. Bc5272 encodes a carboxylate-amine ligase (herein referred as Pyl) that, in the presence of ATP and Mg(II), adds 2-oxoglutarate to the 4-amino moiety of UDP-4-amino-FucNAc to form UDP-Yelosamine and ADP. Pyl is also able to ligate 2-oxoglutarate to other 4-amino-sugar derivatives to form UDP-Yelose, UDP-Solosamine, and UDP-Aravonose. Characterizing the metabolic pathways involved in the formation of modified nucleotide sugars provides a basis for understanding some of the mechanisms used by bacteria to modify or alter their cell surface polysaccharides in response to changing growth and environmental challenges.     

Induction of cadmium tolerance in Escherichia coli K-12

Abstract Cadmium ions are bacteriocidal, resulting in exponential killing that starts immediately after exposure. We have shown that pretreatment with sublethal concentrations of cadmium induces tolerance. Protection against cadmium killing can also be obtained by preincubation at elevated temperatures, known to induce the heat-shock response. However, in contrast to pretreatment at elevated temperatures, exposure to sublethal cadmium concentrations does not induce thermotolerance.     

Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase⁺ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca²⁺ and Mg²⁺) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.     

A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12

Abstract Five different c -type cytochromes have been detected during anaerobic growth of various Escherichia coli strains in different media. None of these cytochromes was detectable in aerobically-grown cultures. Only a single, 43 kDa cytochrome was synthesized in response to the presence of trimethylamine-N-oxide: synthesis of this cytochrome was unaffected by the presence of nitrate or nitrite, was repressed by oxygen, but was dependent upon a funtional tor operon located at minute 22 (coordinate 1070 kb) on the E. coli chromosome. The other four cytochromes, masses 16, 18, 24 and 50 kDa, were induced by nitrite coordinately with formate-dependent nitrite reductase activity, but repressed by oxygen and nitrate. As only the 18 kDa and 50 kDa cytochromes are encoded by the nrf operon located at minute 92 (coordinate 4366 kb), there must be other loci, possibly essential for formate-dependent nitrite reduction, encoding the 16 kDa and 24 kDa cytochromes. No other c -type cytochrome was detected under any growth condition tested.     

Purification and Characterization of Aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinyl-glycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni²⁺, Mn²⁺, Co²⁺, and Cd²⁺, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidase B is a metallopeptidase. It was stabilized against heat in the presence of Mn²⁺ or Co²⁺. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The k_cat/K_m for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

The LIV-I/LS system as a determinant of azaserine sensitivity of Escherichia coli K-12

The growth of Escherichia coli is inhibited by an antibiotic compound, azaserine (O-diazoacetyl-L-serine). Previous studies revealed the biochemical properties of azaserine, which involves inhibition of various enzymatic reactions as well as introduction of DNA breakage. However, genetically, nothing has been elucidated except that all the azaserine-resistant strains isolated so far carry lesions in the aroP gene as a primary determinant. Here, we demonstrate that, in addition to AroP, the LIV-I/LS system, an ATP-binding cassette type transporter, is involved in azaserine sensitivity of E. coli, by genetic analysis and transport studies, in which Ki value for azaserine was determined to be approximately 10(-3) M.     

Inhibition of the cytochrome bd-terminated NADH oxidase system in Escherichia coli K-12 by divalent metal cations

Abstract Co(II), Zn(II) and Cd(II) ions inhibited NADH oxidase activity in membranes prepared from two cytochrome bo' -deficient mutants of Escherichia coli K-12 with the following order of potency: Zn ( II ) > Cd ( II ) > > Co ( II ). The degree of inhibition exhibited by these metal ions was not diminished in membranes which contained elevated levels of the cytochrome bd complex, suggesting that the most sensitive site precedes this complex in the aerobic respiratory chain. For each of the metal ions studied, inhibition was determined to be of the non-competitive type. Based upon the efficacy with which EDTA alleviated inhibition, Co(II), Zn(II) and Cd(II) ions are proposed to inhibit NADH oxidase activity by binding to at least two sites in the respiratory chain with significantly different affinities.     

'Receptor-independent' infection of Mu G(−) phage in Escherichia coli K-12

Abstract Bacteriophage Mu with its invertible G segment in G(−) orientation does not make plaques on Escherichia coli K-12, due to the absence of a suitable lipopolysaccharide receptor. Plaques formed by Mu G(−) were found, however, when the infected E. coli K-12 strain harbours a plasmid with the cloned DNA inversion function Gin which converts the infecting G(−) phage to G(+). Under overproducing conditions, where Gin expression is placed under the control of the tac promoter, the infectivity of Mu G(−) can be estimated as approximately 1% of that in the presence of the receptor. Furthermore, interaction of Mu G(−) with the E. coli K-12 cell wall leads to interference with the plating of a Mu G(+) variant which has the new phenotype Pen⁻ (penetration-negative).     

Inactivation of the Escherichia coli K-12 twin-arginine translocation system promotes increased hydrogen production

The effect of deleting the genes encoding the twin-arginine translocation (Tat) system on H2 production by Escherichia coli strain MC4100 and its formate hydrogenlyase upregulated mutant (DeltahycA) was investigated. H2 evolution tests using two mutant strains defective in Tat transport (DeltatatC and DeltatatA-E) showed that the rate doubled from 0.88+/-0.28 mL H2 mg dry weight-1 L culture-1 in the parental strain, to 1.70+/-0.15 and 1.75+/-0.18 mL H2 mg dry weight-1 L culture-1, respectively, in the DeltatatC and DeltatatA-E strains. This increase was comparable to that of a previously characterized hydrogen over-producing E. coli strain carrying a DeltahycA allele. Construction of a tatC, DeltahycA double deletion strain did not increase hydrogen production further. Inactivation of the Tat system prevents correct assembly of the uptake hydrogenases and formate dehydrogenases in the cytoplasmic membrane and it is postulated that the subsequent loss of basal levels of respiratory-linked hydrogen and formate oxidation accounts for the observed increases in formate-dependent hydrogen evolution.     

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus ) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.     

大肠杆菌K-12转醛醇酶基因talB的克隆及在运动发酵单胞菌CP4中的表达

研究了E.coliK-12转醛醇酶基因(talB)在自身启动子和在Z.mobilisCP4eno基因启动子的启动下在E.coliDH5α和Z.mobilisCP4中的表达情况。首先克隆了E.coliK-12talB基因,并连接到穿梭载体pZB1上构建成pZB1-talB;然后利用PCR重叠延伸技术将E.coliK-12talB自身的启动子换成Z.mobilisCP4eno的启动子,构建得到pZB1-Peno-talB。将这两个质粒分别转化E.coliDH5α和Z.mobilisCP4。对转化子粗酶液进行的转醛醇酶酶活力测定结果表明,E.coli talB自身启动子和Z.mobilis eno启动子能以基本相同的效率启动talB基因在E.coli和Z.mobilis中的表达。     

A Simple Method for Multiple Modification of the Escherichia coli K-12 Chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by λ Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.     

A rapid screening procedure to identify mini-Tn10 insertion mutants of Escherichia coli K-12 with altered adhesion properties

Abstract A rapid screening procedure was developed for detection of Escherichia coli mutants with altered adhesion abilities using polystyrene 96-well microtiter plates as attachment surfaces. During this assay, bacterial strains grew and adhered simultaneously, and attached cells were measured after crystal violet staining. Starting with a total of 7000 W3110::Tn10 insertion mutants of E. coli K-12 W3110, 50 adhesion-deficient mutants were isolated which showed less than 40% attachment, and 22 mutants were found with an attachment of 40–75%. Motility assays were performed on these 72 mutants, and 34 displayed altered motility.     

Identification of the rhaA, rhaB and rhaD gene products from Escherichia coli K-12

Washed cells of Peptostreptococcus products (strain Marburg), which were incubated in the presence of CO/CO2/N2 (50%/17%/33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis-Menten kinetics. The apparent Km for sodium was determined to be about 2 mmol/l. Sodium also stimulated acetate synthesis from H2 plus CO2. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO2.     

On the enthalpy of formation of Escherichia coli K-12 cells

An examination is made of five methods for obtaining values of the enthalpy of formation of a unit mass of living Escherichia coli K-12 cells. The values obtained by these methods ranged from -88.95 kJ to -99.55 kJ, the gross average being 96.01 kJ, per unit carbon formula weight equivalent of living, hydrated cells. Although theoretically the growth of this organism in a microcalorimeter should provide the best value, the value obtained by this method (-88.95 kJ per UCFW equivalent) is not in close agreement with those of the other four methods, the values from which form a cluster averaging -97.8 +/- 1.0 kJ (-23.4 +/- 0.2 kcal)/UCFW equivalent. Calculations using this value indicate that the enthalpy change accompanying anabolism (as this is represented) is zero, or very nearly so, and that the heat of growth is that from catabolism alone.     

大肠杆菌σ70启动子的识别

应用多样性增量结合二次判别分析（Increment of Diversity with Quadratic Discriminant analysis,IDQD）方法,对大肠杆菌σ^70启动子进行识别。使用受试者操作特性（receiver operating characteristic,ROC）曲线和精度召回率曲线（Precision Recall Curves,PRC）进行性能评估。10-fold交叉检验给出,在正负集之比为1：1时,ROC曲线下面积和PRC曲线下面积均为95％。结果表明,IDQD算法有能力应用于原核启动子的识别。识别精度高于现有算法。     

Protein localization in Escherichia coli K-12: an analysis of ompC-lacZ gene fusions

Abstract Two conditionally expressed lacZU131 gene fusions were constructed in vivo to the ompC gene which encodes a major outer membrane protein in Escherichia coli . The resulting hybrid molecules contained approximately 25% and 50% of the mature OmpC protein fused to the LacZ. Export analysis showed that under nonoverproducing conditions essentially all synthesized OmpC-LacZ hybrid protein was effectively processed in vivo unless the signal peptide cleavage was inhibited by ethanol addition. Also, the hybrid proteins were highly accessible to solid phase iodination of whole cells under conditions where cytoplasmic proteins remained unlabelled. Thus, hybrids containing large portions of the OmpC protein were clearly recognized by the cellular export machinery, and probably all synthesized hybrid protein was partially translocated through the cytoplasmic membrane.     

Peptidase D of Escherichia coli K-12, a metallopeptidase of low substrate specificity

Abstract Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity. The pure enzyme was stable at 4°C or −20°C and had a pH optimum at pH 9, and a p I of 4.7; the temperature optimum was at 37°C. As the enzyme was activated by Co²⁺ and Zn²⁺, and deactivated by metal chelators, it appears to be a metallopeptidase. By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified. Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the α or β position. Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed. K _m values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.     

紫锥菊多糖抑制致病性大肠埃希菌细胞黏附的试验研究

目的研究紫锥菊多糖能否影响致病性大肠埃希菌对细胞的黏附。方法使用PK-15细胞进行黏附试验及黏附抑制试验。结果发现紫锥菊多糖浓度为1.6 mg/ml时,对细菌黏附细胞的抑制作用最好,黏附率由50个细菌/细胞降低到6.8个细菌/细胞。结论紫锥菊多糖对致病性大肠埃希菌的细胞黏附具有抑制作用,提示该多糖具有调节肠道微生态的潜在应用价值。