A new type of Alcaligenes eutrophus CH34 zinc resistance generated by mutations affecting regulation of the cnr cobalt-nickel resistance system.

Spontaneous mutants that were resistant to zinc were isolated from Alcaligenes eutrophus CH34 containing either the native plasmid pMOL28 or a derivative derepressed for its self-transfer, pMOL50. With the cured plasmid-free derivative of CH34, strain AE104, such mutants were not detected. The mutations, which were shown to be located in the plasmid, increased the level of the nickel and cobalt resistance determined by the cnr locus. The chromate resistance closely linked to the cnr locus was not affected by these mutations. In the Znr mutants, the resistance to zinc and nickel was constitutively expressed. Uptake studies showed that the zinc resistance in a Znr mutant resulted from reduced accumulation of zinc ions in comparison with that in the plasmid-free strain. Reduced accumulation of zinc was also observed to a lesser degree in the parental strain induced with nickel, suggesting that zinc interferes with the Ni2+ and Co2+ efflux system. A 12.2-kb EcoRI-XbaI restriction endonuclease fragment containing the cnr locus was cloned from plasmid pMOL28 harboring the mutation and shortened to an 8.5-kb EcoRI-PstI-PstI fragment conferring resistance to zinc, nickel, and cobalt. The 12.2-kb EcoRI-XbaI fragment was also reduced to a 9.7-kb BamHI fragment still encoding weak resistance to nickel and cobalt but not to zinc. Complementation studies demonstrated the recessivity of the cnr mutations with a Znr phenotype. Such mutations thus allow positive selection of mutants affected in the expression of the cnr operon.     

Epidermolytic toxin serotype B of Staphylococcus aureus is plasmid-encoded

Abstract The gene coding for epidermolytic toxin serotype B ( etb ) was cloned in a plasmid expression vector in Escherichia coli . Its expression was dependent on the vector plasmid's trc promoter. A polypeptide immunochemically indistinguishable from the purified staphylococcal toxin and with the same molecular weight was predominantly localized in the periplasm of E. coli . The etb gene resides on a 1.7 kb Hin dIII fragment of the 42 kb plasmid pTC142 present in the parental Staphylococcus aureus strain.     

Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli.

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.     

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8kb DNA fragment in a plasmid which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1. None of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in a strain containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3' terminus of the mutR coding sequence.     

黑曲霉pepB基因缺失菌株的构建及其功能分析

以黑曲霉(Aspergillus niger)GICC2773基因组DNA为模板,用PCR方法分别扩增pepB基因中的上游约1．4kb和下游约1．3kb两段DNA序列,将此两段序列按同一方向分别插入质粒pMW1中潮霉素抗性基因(hph)表达单元的5′和3′端,构建成重组质粒pMW1-pepB,用于通过同源重组靶向破坏基因组中的pepB基因。同源重组则采用原生质体-PEG方法,将酶切pMW1-pepB得到的线性片段转化A．niger GICC2773菌株,通过潮霉素选择平板得到62个Hgy抗性转化子,然后采用PCR方法从这些抗性转化子中筛选到1个由于同源重组产生的pepB基因缺失突变菌株pepB29。功能分析显示该突变株的酸性蛋白酶活性有明显下降,外源蛋白漆酶的分泌表达有所提高。     

Changes in Staphylococcus aureus susceptibility to bactericidal action of teicoplanin in the presence of different prophages in the bacterial genome

The influence exerting by lysogeny state on the susceptibility of Staphylococcus aureus to bactericidal action of teicoplanin was studied. In this aim the standard, non-lysogenic, bacteriophage-free S. aureus NCTC 8325-4 strain was lysogenized with 10 different, bacteriophages obtained in our laboratory. All bacteriophages were derived from multiresistant S. aureus strains and all were able to convert staphylokinase. For all derivatives MBCs and MICs of teicoplanin were determined. In the case of four strains the ratio MBC/MIC showed the presence of tolerance to teicoplanin (MBC/MIC > or = 32) and was significantly higher than in the case of the parent strain NCTC8325-4. In the case of two strains this ratio was smaller than for parent strain. Only small correlation with our previous results obtained for vancomycin was observed.     

耐甲氧西林金黄色葡萄球菌MRSA252辅酶A二硫化物还原酶缺失延缓苯唑西林的次级损伤效应

【目的】耐甲氧西林金黄色葡萄球菌在苯唑西林作用下,其辅酶A二硫化物还原酶表达上调2.3倍。本文研究苯唑西林对该酶缺失的金黄色葡萄球菌的杀菌效应。【方法】利用同源重组双交换技术对金黄色葡萄球菌进行基因敲除,并用质粒p OS1构建回补株;采用分光光度法检测菌株体外增殖能力;以时间-杀菌法考察苯唑西林对菌株杀菌效应;以2’,7’-二氯荧光黄双乙酸盐为探针检测胞内活性氧水平。【结果】辅酶A二硫化物还原酶基因敲除株较亲株生长缓慢(P0.05);20倍MIC浓度苯唑西林下敲除株的时间-杀菌曲线及胞内活性氧水平与亲株无显著性差异,5倍MIC浓度下敲除株的致死速率及胞内活性氧水平均较亲株下降。【结论】在较低浓度苯唑西林作用下,辅酶A二硫化物还原酶基因缺失降低胞内活性氧水平,减小杀菌速率,延缓次级损伤效应。     

Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant

Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid.     

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones.

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.     

The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli

In Escherichia coli , lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn²⁺. In the presence of 0.5 mM EGTA, only the parental strain was able to take up ⁶⁵Zn²⁺. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI ) and localized at 42 min on the genetic map of E. coli . At high Zn²⁺ concentrations, the znu mutants took up more ⁶⁵Zn²⁺ than the parental strain. The high-affinity ⁶⁵Zn²⁺ uptake was repressed by growth in the presence of 10 μM Zn²⁺. A znuA–lacZ operon fusion was repressed by 5 μM Zn²⁺ and showed a more than 20-fold increase in β-galactosidase activity when Zn²⁺ was bound to 1.5 μM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn²⁺-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli . A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity ⁶⁵Zn²⁺ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB .     

Essential role of the czc determinant for cadmium, cobalt and zinc resistance in Gluconacetobacter diazotrophicus PAl 5

The mechanisms of cadmium, cobalt and zinc resistance were characterized in the plant-growth-promoting bacterium Gluconacetobacter diazotrophicus PAl 5. The resistance level of the wild-type strain was evaluated through the establishment of minimum inhibitory concentrations (MIC) of the soluble compounds CdCl2·H2O, CoCl2·6H2O and ZnCl2. Gluconacetobacter diazotrophicus PAl 5 was resistant to high concentrations of Cd, Co and Zn, with MICs of 1.2, 20 and 20 mM, respectively. Screening of an insertion library from transposon EZ-Tn5 in the presence of ZnO revealed that the mutant GDP30H3 was unable to grow in the presence of the compound. This mutant was also highly sensitive to CdCl2·H2O, CoCl2·6H2O and ZnCl2. Molecular characterization established that the mutation affected the czcA gene, which encodes a protein involved in metal efflux. In silico analysis showed that czcA is a component of the czcCBARS operon together with four other genes. This work provides evidence of the high tolerance of G. diazotrophicus PAl 5 to heavy metals and that czc is a determinant for metal resistance in this bacterium.     

Nickel and cobalt resistance of various bacteria isolated from soil and highly polluted domestic and industrial wastes

Abstract From enrichment cultures in the presence of 1 mM NiCl₂ 200 strains of aerobic bacteria were isolated from 50 samples collected in the metal-processing industry, waste water treatment plants and from solid waste, highly polluted by heavy metals. The strains isolated were characterized with respect to their substrate spectrum and resistance to nickel, cobalt, zinc and cadmium salts and assigned to 21 groups. One representative of each group was described with respect to cell morphology. All strains were Gram-negative, non-sporing rods or cocci. The highest concentrations of nickel, cobalt, zinc, cadmium, copper, mercury, and silver allowing growth on solid media were estimated. Two strains were able to grow at 20 mM NiCl₂ and CoCl₂, one strain tolerated 12 mM and one 7.5 mM concentrations of these salts.
Fifteen out of 21 strains contained at least one plasmid two contained two plasmids. The plasmid sizes varied between 50 and 340 kbp, except strain 10A, which contained a miniplasmid (2.6 kbp). Attempts to cure four selected strains by exposure to mitomycin C or growth at elevated temperature failed.
By helper-assisted and unassisted conjugation the plasmids of strain 31A were shown to carry nickel and cobalt resistance determinants. Alcaligenes eutrophus strains H16 and N9A and denative of strain CH34 lacking one or both of its native metal resistance plasmids were used as recipients. Both plasmids, p TOM8 and pTOM9, of strain 31A carried resistance properties which were expressed in all recipients except. A. eutrophus H16, in which only nickel resistance was expressed.
Plasmid pTOM3 residing in strain 10A could not be transferred as such. However, transconjugants derived from helper (pULB113)-assisted matings carried co-integrates of various sizes and were resistant to nickel and cobalt.     

Cloning of the Bacillus subtilis DNA fragment containing the genes for lysine and riboflavin biosynthesis

The SalI fragment of chromosomal DNA of Bacillus subtilis carrying the gene for lysine biosynthesis and the regulatory operator region (ribO) from the riboflavin gene was cloned into Escherichia coli cells. This fragment was shown to contain the gene coding for lysine synthesizing enzyme. Localization of this gene in Bac. subtili was determined. New plasmids pLRS33 and pLRB4 were constructed using pBR322; they carry a fragment homologous to pLP102 plasmid containing the operon for riboflavin biosynthesis.     

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.