Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae.

The structure and function of the phoB and phoR genes of Shigella dysenteriae strains and Klebsiella pneumoniae, which are involved in regulation of the phosphate regulon, were analyzed. Complementation tests among the genes of Escherichia coli, S. dysenteriae strains, and K. pneumoniae for production of alkaline phosphatase indicate that S. dysenteriae serotype 2 and serotype 3 strains and K. pneumoniae are phoA+ phoB+ phoR+ but S. dysenteriae Sh and serotype 1 strains are phoA phoB+ phoR. Nucleotide sequences of phoB and phoR of S. dysenteriae Sh and K. pneumoniae are highly homologous to those of E. coli, except for a single base insertion found in phoR of S. dysenteriae Sh.     

Transforming activity of plasmid R6K DNA on Serratia marcescens strain 20-10. The behavior of the plasmid in the transformants]

The authors described transformation of S. marcescens, strain 20-10, of the isolated R6K plasmide DNA. As demonstrated by centrifugation in cesium chloride gradient and electrophoresis in agarose, the plasmide was present in the transformants in the form identical to R6K in E. coli K12. Analysis of the transforming activity of R6K plasmide from Serratia and E. coli K12 strains with a complete and defective restriction system showed S. marsescens, strain 20-10, to possess specific system of restriction and modification. In studying beta-lactamase activity and Serratia and E. coli strains ampicillin and streptomycin resistance revealed differences in the phenotypical expression of the plasmide signs in the heterologous and homologous host.     

Gibb's free energy as a index of the assimilation of carbohydrate sources by bacteria]

The author presents substantiation of the use of thermodynamic function--of free Gibbs' energy (--deltaG) for energy characteristics of enterobacteria periodic cultures on synthetic media. Examination of strains of E. coli K12P4X Hfr, E. coli PA-309, and Sh. newcastle 762S showed the --detlaG = f (N) curves to characterise the energy value of various carbohydrate sources more completely, and also permitted the choice of their optimal concentrations in synthetic media for each of the enterobacteria strain.     

Nucleotide sequences of the gnd genes from nine natural isolates of Escherichia coli: evidence of intragenic recombination as a contributing factor in the evolution of the polymorphic gnd locus.

Nine natural isolates of Escherichia coli were examined, and the sequence of the entire 1,404 bases of the gnd gene (6-phosphogluconate dehydrogenase, EC 1.1.1.44) was determined. These isolates, along with E. coli K-12, constitute 10 strains for analysis. (The sequence of the E. coli K-12 gnd gene is known.) A total of 184 sites were polymorphic, and up to 6% sequence divergence was observed between pairs of strains. The deduced amino acid sequences showed much more variation than had been shown by multilocus enzyme electrophoresis, and in addition the net charge calculated did not correlate strongly with electrophoretic mobility. A phylogenetic tree for the sequences that was based on maximum parsimony differed significantly from a tree for the same strains that was based on multilocus enzyme electrophoresis for 35 enzymes (R. K. Selander, D. A. Caugant, and T. S. Whittam, p. 1625-1648, in F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger, ed., Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 1987). These data, together with analysis of sequence variation between the strains, indicated that intragenic recombination and transfer of the whole of gnd have occurred in the evolution of these strains. There is evidence of one recombination event between E. coli and Salmonella typhimurium.     

Characteristics of new pKMR-plasmids detected in natural strains of Enterobacteriaceae

pKMR-plasmids controlling the antibiotic resistance and adhesive properties were isolated from clinical strains of E. coli O26 and O124, and Sh. sonnei. Two of them, i.e. pKMR 207 and pKMR 208 were conjugative. On conjugation they jointly transferred the features of the antibiotic resistance and capacity for production of the colonization antigen. The studies on transformation of E. coli K 12 802 with the plasmid DNA of E. coli O124 showed that the antibiotic resistance and colonization properties in E. coli O124 were controlled by the nonconjugative plasmid pKMR 209. It was found that plasmids pKMR 207 and pKMR 208 had the fi(-)-phenotype. None of the plasmids allotted the host cells sensitivity to the donor specific phages of the incompatibility groups F, N, P, W, and I. Probably, the plasmids did not belong to these incompatibility groups. When the cells of E. coli K 12 802 were transformed with the plasmid DNA of the wild strain to the hemolytic strain of S. typhimurium with multiple antibiotic resistance, 3 pKMR 210 plasmids with different markers of the antibiotic resistance were detected in the transformants. One of the plasmids controlled both the drug resistance and the capacity for production of hemolysin. The ability of the detected pKMR plasmids to inhibit fertility and relation to the donor specific phages was studied.     

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.     

Role of glutamate-126 and arginine-144 in the lactose permease of Escherichia coli

Several previous studies have suggested that glutamate-126 and arginine-144 in the lactose permease of Escherichia coli form an ion pair that is essential for sugar binding. To further investigate the role of these residues, E126Q, R144Q, and R144S mutants were made. The R144Q and R144S strains, which had negligible levels of transport, were used as parental strains to isolate suppressor mutations that partially restored sugar transport. The R144Q parent only yielded first-site revertants, but the R144S strain produced three types of second-site replacements: E126Q, V229A, and L330R. In downhill transport assays, the E126Q strain was able to transport lactose at low levels, with an apparent K(m) 3-fold higher than the wild-type strain but a severely depressed apparent V(max). A triple mutant, E126Q/R144S/V229A, showed a relatively robust V(max) value for downhill transport and could actively accumulate lactose against a concentration gradient. Taken together, these results indicate that Glu-126 and Arg-144 are not essential for sugar binding. An alternative explanation for their role in maintaining secondary structure is discussed.     

Antimicrobial sensitivity of Escherichia coli strains with K1 antigen isolated from pregnant women and newborns

The aim of this study was comparison of the susceptibility to antibiotics of E. coli strains with K1 antigen (E. coli K1+) and non-K1 E. coli strains (E. coli K1-). This study included 67 of E. coli K1+ and 67 of E. coli K1- strains isolated in the time period from June to September of 2008 from pregnant women and newborns hospitalized at dr. J. Biziel University Hospital number 2 L. Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń. Antimicrobial susceptibility of E. coli strains was tested by the disc-diffusion method, on the Mueller Hinton 2 Agar (Becton Dickinson). It was found that 64,2% of E. coli K1+ strains and 53,7% of E. coli K1-strains were susceptible to all tested antibiotics and chemioterapeutics. E. coli K1- strains were more often than E. coli K1+ nonsusceptible to at least one antimicrobial agent. The obtained results indicate that E. coli K1+ strains significant differed in the susceptibility to ampicillin/sulbactam (85,1% versus 95,5%) (p=0,041), cephalothin (70,1% versus 85,1%) (p=0,038) and tetracycline (91,0% versus 74,6%) (p=0,012) from E. coli K1-strains. All tested E. coli K1+ and K1-strains were sensitive to piperacillin/tazobactam, cefoperazone/sulbactam, cefotaxime, ceftazidime, cefepime, imipenem, amikacin, netilmicin and tigecycline. There weren't the ESBL-producing strains among tested E. coli K1+ and K1- rods.     

Effects of Escherichia coli ribosomal protein S12 mutations on cell-free protein synthesis.

We examined the effects of Escherichia coli ribosomal protein S12 mutations on the efficiency of cell-free protein synthesis. By screening 150 spontaneous streptomycin-resistant isolates from E. coli BL21, we successfully obtained seven mutants of the S12 protein, including two streptomycin-dependent mutants. The mutations occurred at Lys42, Lys87, Pro90 and Gly91 of the 30S ribosomal protein S12. We prepared S30 extracts from mutant cells harvested in the mid-log phase. Their protein synthesis activities were compared by measuring the yields of the active chloramphenicol acetyltransferase. Higher protein production (1.3-fold) than the wild-type was observed with the mutant that replaced Lys42 with Thr (K42T). The K42R, K42N, and K42I strains showed lower activities, while the other mutant strains with Lys87, Pro90 and Pro91 did not show any significant difference from the wild-type. We also assessed the frequency of Leu misincorporation in poly(U)-dependent poly(Phe) synthesis. In this assay system, almost all mutants showed higher accuracy and lower activity than the wild-type. However, K42T offered higher activity, in addition to high accuracy. Furthermore, when 14 mouse cDNA sequences were used as test templates, the protein yields of nine templates in the K42T system were 1.2-2 times higher than that of the wild-type.     

Effect of fermented milk intake on plasmid transfer and on the persistence of transconjugants in the digestive tract of gnotobiotic mice

Plasmid transfer occurs in the digestive tract and the transconjugants may become durably established. The aim of the present work is to investigate the effect of probiotics on plasmid transfer and on establishment of transconjugants in the gut. Plasmid transfers were carried out in the digestive tract of germ free mice associated with an E. coli K12 donor strain harboring three plasmids (R388, self-transmissible, pCE325 and pUB2380, mobilisable,) and an E. coli recipient strain, PG1, of human origin (Duval-Iflah et al., 1994). Milks fermented with either Lactobacillus bulgaricus or Streptococcus thermophilus or symbiosis, S85, of both strains were given daily as 1/3 of food diet. Fermented milks have no effect on the transfer of R388 and pUB2380 except a slight increase of TC(R388) with milk fermented with S85. Long term ingestion of milk fermented with S85 inhibited the formation and the establishment of transconjugants TC(pCE325). Milk fermented with L. bulgaricus lowered the population density of TC(pCE325) in animals where they were already established. This phenomenon was reversible, since the density of TC(pCE325) increased in the same animals after cessation of supplementation. Bacterial cultures obtained in MRS broth and given in state of drinking water were compared with fermented milks. Bacterial cultures with L. bulgaricus and with S85 favoured the establishment of TC(pCE325). These results indicate for the first time that probiotics have various effects on the formation and/or establishment of transconjugants in the gut of axenic mice. The effects depend on whether the probiotics were cultivated in milk or in MRS, indicating that bacterial metabolites and viable bacteria can be involved.     

Conjugative transfer frequency of resistance genes from ESBL-producing Enterobacteriaceae strains isolated from patients hospitalized in pediatric wards

The aim of this study was to evaluate the transfer frequency of plasmids encoding extended-spectrum beta-lactamases (ESBLs) from clinical isolates of Enterobacteriaceae to E. coli K12 C600 recipient strain. Additionally, resistance patterns to antimicrobial drugs of the isolates as well as transconjugants were analyzed. Fifty-four clinical strains belonging to the Enterobacteriaceae family were isolated from children hospitalized in Medical University Hospital in Wroc?aw. All the strains studied were identified in automatic ATB system using ID32E tests. Besides, they were ESBL-positive as was confirmed by the double-disc synergy test (DDST). The minimal inhibitory concentration (MIC) was determined for twelve selected antibiotics and chemotherapeutics. The majority of the strains (87%) were able to transfer plasmid-mediated ESBL to E. coli K12 C600 recipient strain with a frequencies ranged from 10(-5) to 10(-1) per donor cell. All the isolates studied as well as their transconjugants were susceptible to imipenem, meropenem and norfloxacin (MIC <1mg/L). On the other hand, these strains displayed high level of resistance (MIC 512 - >1024 mg/L) to cefotaxime, ceftriaxone, gentamycin, amikacin and cotrimoxazole. Genetic markers conferring resistance to aminoglycosides and cotrimoxazole were often co-transferred to recipient strain in conjugation process.     

Appearance of enterotoxigenic Escherichia coli in piglets with diarrhea in connection with feed changes

Twelve weaned piglet (3-week-old) were divided into three groups according to time of feed change and observed for diarrhea during the time they were 3 to 8 weeks of age. A total of 553 strains of Escherichia coli were isolated from rectal fecal samples and examined for heat-labile (LT) and heat-stable (ST) enterotoxins, pilus antigens (K88, K99, and 987P), hemolysin (Hly), raffinose utilization (Raf) and drug resistance. Enterotoxins and/or hemolytic E. coli strains appeared in the rectal feces of 5- to 6-week-old piglets with diarrhea in connection with feed change and changing temperatures. Most of the isolates showed multiple drug resistance to sulfonamides (Sa), streptomycin (Sp), ampicillin, and/or mercury. Enterotoxigenic E. coli isolates represented four phenotypes: K88+.LT+.Hly+.Raf+.(SaSmCpTcKmSp) (12 strains), K99+.ST+.Raf+.(TcKm) (7 strains), ST+.Raf+.(TcKm) (7 strains), and ST.+(SaSmKm) (25 strains). The drug resistance determinants were transferable concurrently and some of them mobilized the determinants for K88, LT, Hly, and Raf to an E. coli C strain.     

Dwarf colony mutants of Escherichia coli: plasmids resistant to antibiotics]

Deficient dwarf colony (DDC) mutants of E. coli K 12, harboring or no resistance plasmids, were obtained in vitro. The R plasmids of parental strains and to DDC mutants were transfered by conjugation to normal colony, and to DDC mutants of E. coli K 12; the frequencies of transfer were similar for all strains studied.     

R-plasmid transfer frequencies from environmental isolates of Escherichia coli to laboratory and fecal strains.

Multiple-drug-resistant strains of Escherichia coli were isolated from the water at an estuarine site. They represented about 8.3% of the total E. coli population. Fifty-five strains, representing each of the 32 resistance patterns identified, were mated with an E. coli K-12 F- strain. Matings were performed on membrane filters, and the cells were washed to remove any colicins produced by the donors. Thirty-one strains, about 5% of the mean E. coli density in the samples, transferred drug resistance and, hence, posessed conjugative R plasmids. Of these, 80% transferred drug resistance at a frequency of about 10(-4) or less. Nine environmental R+ strains were mated with three fecal recipients. The R-plasmid transfer frequencies to the fecal strains from the environmental donors correlated well with those from a derepressed K-12 R+ laboratory donor. The R+ X K-12 F- lac- transconjugants from 16 environmental strains were "backcrossed" to a lac+ K-12 F- strain. All transfer frequencies were higher in the backcrosses than in the original matings from the environmental donor. Furthermore, 7 of 13 different transconjugants, which accepted plasmids at repressed frequencies of less than 10(-3), donated them at frequencies greater than 10(-2). This suggests that these were derepressed plasmids in a repressed host.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

灵芝液态深层发酵产物中10种不饱和脂肪酸类化合物的鉴定

通过规模化液态深层发酵获得灵芝发酵产物,采用多种硅胶色谱柱层析及重结晶的方式,从中分离得到10个化合物。通过核磁、质谱等波谱分析,鉴定出这些化合物均属于含羟基或酮基的不饱和脂肪酸类化合物,分别为(9S,10R,11E,13R)-9,10,13-trihydroxyoctadec-11-enoic acid（1）和(9S,10R,11E,13S)-9,10,13-trihydroxyoctadec-11-enoic acid（2）的混合物、12S^*,13S^*-dihydroxy-9-oxo-10(E)- octadecenoic acid（3）、9R*,10R*-dihydroxy-13-oxo-11(E)-octadecenoic acid（4）、12S*,13R*-dihydroxy- 9-oxo-10(E)-octadecenoic acid（5）、9S*,10R*-dihydroxy-13-oxo-11(E)-octadecenoic acid（6）、10(S)-hydroxy-8(Z)-octadecenoic acid（7）、12-oxooctadeca-8,10-dienoic acid（8）、9,12-dihydroxy-10-eicosenoic acid（9）和9-oxooctadeca-10,12-dienoic acid（10）。这些化合物均为首次从灵芝发酵产物中获得,且具有不同程度的体外抗肿瘤活性。其中,化合物8和化合物10对L1210细胞增殖抑制的IC₅₀值分别为13.00μmol/L和16.88μmol/L,对K562细胞增殖亦有良好的抑制效果,是具有抗肿瘤潜力的天然产物。     

Generation of novel plasmids in Escherichia coli S17-1(pSUP106)

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn(2+) (48 m M), Cu(2+) (12 m M), Ni(2+) (12 m M), chloramphenicol (50 microg/ml), and tetracycline (25 microg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 m M Zn(2+), 12 m M Cu(2+), and 12 m M Ni(2+), but remained chloramphenicol and tetracycline resistant-the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell.     

Heterospecific expression of misrepair-enhancing activity of mucAB in Escherichia coli and Bacillus subtilis.

Enterobacterial plasmid genes mucAB, which possess error-prone repair activity, were cloned and sequenced independently of a sequence previously determined (K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, and G.C. Walker, Proc. Natl. Acad. Sci. USA 82:4331-4335, 1985). The survival- and mutation-enhancing activities of mucAB ligated to the MLSr promoter of a Bacillus subtilis plasmid in the shuttle vector pTE22R were expressed in B. subtilis as well as in Escherichia coli after mutagenic treatment. mucAB fragments with 5' deletions of various lengths up to the base sequence encoding Ala-26-Gly-27, the putative RecA-mediated cleavage site of the MucA protein, showed mutation-enhancing activity for noninducible lexA3 E. coli when ligated to the MLSr promoter in frame. This activity was lost by extending the deletion downstream. The formations of MucA and MucB proteins in B. subtilis and E. coli were demonstrated by Western blot (immunoblot) analysis. MucA cleavage in Rec+ B. subtilis was observed only after treatment with an alkylating agent and was not observed in RecA- and RecE- strains, whereas in E. coli cleavage was observed in Rec+ cells after treatment with either mitomycin C or an alkylating agent but was not detected in RecA- cells. Common activity of B. subtilis Rec and E. coli RecA in the induction of mutants is suggested.     

Biological activity of extracts isolated from a virulent strain of Sh. flexneri grown in the presence of calcium ions]

The authors carried out a comparative study of the genetically connected Sh. flexneri cultures (3 virulent strains, 3 clones of an avirulent mutant selected in the flux of an oblique light from the virulent strain, and lac+ Kcp A-hybrids obtained by crossing the initial virulent cultures with the E. coli K12 Hfr strains). The absence of any correlation between the virulence of the strains under study and the lipopolysaccharide (by rhamnose) content in the extracts from them in growing the cultures in the presence of calcium ions was noted. Toxicity of the extracts from the virulent cultures was demonstrated on a model of developing chick embryos. No such property was possessed by the extracts from avirulent strains. The extracts from the virulent cultures in nontoxic doses possessed the capacity to decrease LD50 of shigella strains used for the infection. The biologically active factor determined in the extracts from the virulent cultures apparently was not lipopolysaccharide.