Effects of streptomycin and novobiocin on Staphylococcus aureus gene expression.

Streptomycin and novobiocin induced production of protein A and inhibited production of alpha- and beta-hemolysins in mutants of Staphylococcus aureus strains RN450 and RN1 resistant to these antibiotics. Streptomycin, but not novobiocin, also inhibited propagation of bacteriophages of serological group B, whereas phages of group A were unaffected. Streptomycin had to be present at adsorption of the phage, and 10 mM CACL2 reversed the inhibitory effect. Lysogenization and competence induction occurred in the presence of streptomycin, suggesting that some early phage genes were expressed.     

Effect of the Prophage and Penicillinase Plasmid of the Recipient Strain Upon the Transduction and the Stability of Methicillin Resistance in Staphylococcus aureus

Transduction of a methicillin-resistance determinant (mec) in Staphylococcus aureus RN450 was dependent on its prior lysogenization with an appropriate temperate phage. In addition, an appropriate transduced penicillinase plasmid was usually required. Some phage 80-resistant variants of RN450 or of its parental lysogenic strain, NCTC 8325, were also effective recipients for transduction of mec. Elimination of prophage from RN450 abrogated its effectiveness as a transductional recipient of mec. Elimination of prophage from a methicillin-resistant transductant of RN450 reduced resistance to undetectable levels in six of seven phage-eliminated strains. In four of these a variable number of clones again became phenotypically resistant after lysogenization alone or lysogenization combined with reintroduction of a penicillinase plasmid. In two prophage-eliminated strains, no evidence of residual mec could be adduced. The establishment, expression, or stability of the transduced mec in strain RN450 appeared to depend on some function determined by a prophage or a prophage and a penicillinase plasmid.     

Transformation in Staphylococcus aureus: role of bacteriophage and incidence of competence among strains.

When used in a helper phage capacity, phages 29, 52, 52A, 79, 80, 55, 71, 53, 83A, 85, 95, 96, phi11, and 80 alpha, all serological group B Staphylococcus phages, conferred competence for transformation to S. aureus 8325-4, a strain that does not normally become competent. Of the serological group A phages tested, only phage 3A showed significant competence-conferring activity. Phages 29, 55, 53, 83A, .85, 95, phi11, and 80 alpha showed an enhancement of competence-conferring activity if exposure to the cells occurred in the presence of nromal rabbit serum. All of the propagating strains for the Staphylococcus reference typing phages were rendered competent for transformation by exposure to at least one of these helper phages. The use of a helper phage to confer competence to S. aureus did not result in distortion of the genetic linkages observed in an inherently competent strain. Lysogenization by phages phi11 or 83A is shown not to be required for the expression of competence, and evidence is presented which indicates that competence in the inherently competent 8325 strain is due to a helper phage effect initiated by the adsorption to cells of phi11 virion parts [or phi11 particles in the case of the single lysogen 8325-4(phi11)] that have been liberated by prophage induction.     

Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages

The Sau1 type I restriction-modification system is found on the chromosome of all nine sequenced strains of Staphylococcus aureus and includes a single hsdR (restriction) gene and two copies of hsdM (modification) and hsdS (sequence specificity) genes. The strain S. aureus RN4220 is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from Escherichia coli. We show that it carries a mutation in the sau1hsdR gene and that complementation restored a nontransformable phenotype. Sau1 was also responsible for reduced conjugative transfer from enterococci, a model of vancomycin resistance transfer. This may explain why only four vancomycin-resistant S. aureus strains have been identified despite substantial selective pressure in the clinical setting. Using a multistrain S. aureus microarray, we show that the two copies of sequence specificity genes (sau1hsdS1 and sau1hsdS2) vary substantially between isolates and that the variation corresponds to the 10 dominant S. aureus lineages. Thus, RN4220 complemented with sau1hsdR was resistant to bacteriophage lysis but only if the phage was grown on S. aureus of a different lineage. Similarly, it could be transduced with DNA from its own lineage but not with the phage grown on different S. aureus lineages. Therefore, we propose that Sau1 is the major mechanism for blocking transfer of resistance genes and other mobile genetic elements into S. aureus isolates from other species, as well as for controlling the spread of resistance genes between isolates of different S. aureus lineages. Blocking Sau1 should also allow genetic manipulation of clinical strains of S. aureus.     

Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in Staphylococcus aureus

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two Staphylococcus aureus strains, RN4220 (methicillin-sensitive S. aureus) and RN450M (methicillin-resistant S. aureus). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in S. aureus.     

A slipped-mispairing mutation in AgrA of laboratory strains and clinical isolates results in delayed activation of agr and failure to translate delta- and alpha-haemolysins

agr is a global regulator of staphylococcal virulence and other accessory gene functions, especially including the haemolysins. Lack of haemolysin production therefore generally represents a defect in agr function. An example of this is Staphylococcus aureus strain RN4220, a widely used laboratory strain that carries a nitrosoguanidine (MNNG)-induced mutation enabling it to accept DNA from Escherichia coli and other bacteria. We show here that the non-haemolytic phenotype of RN4220 is caused by an extra A residue in a run of seven As at the 3' end of agrA (agrA-8A). This causes a frameshift that results in the addition of three amino acyl residues to the C-terminal end of the protein. The 8A mutation does not inactivate the agr locus, but rather delays agr activation by 2-3 h, which results in failure to translate alpha- and delta-haemolysins, and hence, in a non-haemolytic phenotype. This mutation turned out not to be an adventitious consequence of MNNG mutagenesis, but rather had arisen in RN450, the immediate parent of RN4220. RN450 had become haemolytically heterogeneous in storage, and its non-haemolytic variants had the 8A mutation. The same mutation was also identified in a clinical isolate in which a non-haemolytic variant had arisen during the course of infection. Haemolytic activity in the mutant laboratory strains could be restored by the addition of auto-inducing peptide (AIP) early in growth, indicating that delayed production of RNAIII is responsible for the failure to translate alpha- and delta-haemolysins. Discovery of the 8A mutation has revealed the basis of the dissociation between agr activity and the non-haemolytic phenotype of RN4220, and has solved the long-standing mystery of the variable non-haemolytic phenotype of its immediate parent, RN450. The occurrence of this mutation in a clinical isolate indicates that it is not simply a laboratory phenomenon, and may represent a naturally occurring mechanism for the modulation of agr activity.     

Factors Affecting Transfection in Bacillus stearothermophilus

The conditions for the infection of Bacillus stearothermophilus 4S with TP-1C phage deoxyribonucleic acid (DNA) are described. Cells from log-phase cultures are the most susceptible to phage DNA infection (transfection). A cellular component (competence factor) which enhances transfection is released into the culture medium during the transition period between the log and stationary phase of growth. Transfection is stimulated in the order of decreasing effectiveness, by Fe(3+), Mn(2+), and Mg(2+). The efficiency of transfection is the highest in cells growing at 60.5 C and does not occur in cells growing at 67 C although the cells are growing normally. A cellular component (competence factor) of this organism, which is released into the culture medium, advances by 40 min some step in the uptake of phage DNA.     

Deoxyribonucleic acid homologies among staphylococci: Coagulase-positive reference strains

DNA hybridization results confirm the proposed separation of coagulase-positive staphylococci into two distinct species. Strains ofStaphylococcus aureus representing the various biotypes and different phage typing groups of the human biotype gave high values of reassociation with DNA fromS. aureus reference strain RN 450, at both optimal and restrictive reassociation temperatures. Similar results were obtained between strains ofS. intermedius and its reference strain K 3. Interspecific reassociation between the two coagulase-positive species was low, and each reference strain showed low DNA sequence homology with 10 coagulase-negative species.S. staphylolyticus, strain PS 73, and putative pleiotropic mutants ofS. aureus were shown to be unrelated toS. aureus.     

The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus

Tst , the gene for toxic shock syndrome toxin-1 (TSST-1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST-1-negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA ⁻ recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages φ13 and 80α and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage-like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB , is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80α. It is designated SaPI2. These PIs are the first in any Gram-positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST-1 production among S. aureus strains.     

Restriction and Modification of Bacteriophage S2 in Haemophilus influenzae

The major conclusion from these studies is that variants of Haemophilus influenzae Rd which restrict and modify phage S2 are metastable and capable of giving rise to one another with high frequency. Nonrestrictive RdS cells segregate spontaneously to the restricting, modifying phenotype in about 5% of the progeny of a single clone. The restrictive cells derived from RdS revert to the nonrestrictive phenotype in 15 to 25% of the progeny of a single clone. These frequencies are not appreciably affected by treatment with acriflavine or ethidium bromide, compounds which affect plasmid stability, or by nitrosoguanidine, a powerful mutagen. The genetic locus for restriction and modification of bacteriophage S2 is found to have a chromosomal position between the biotin and proline loci. Restriction-modification of phage S2 has been shown to be a function of its deoxyribonucleic acid (DNA) in that transfection with S2 phage DNA or prophage DNA is subject to host restriction and modification. An enzyme preparation, which contains endodeoxyribonuclease but no appreciable exonuclease activity, from mutant H. influenzae com(-10) did not restrict phage S2.RdS DNA or prophage DNA transfecting activity, indicating that this endodeoxyribonuclease is not responsible for phage restriction. A new restriction enzyme isolated from H. influenzae Rd was found to be the major enzyme involved in the restriction of bacteriophage S2. The enzyme inactivated the transfecting activity of unmodified phage DNA but did not attack modified phage DNA. Unlike endodeoxyribonuclease R, this enzyme requires adenosine triphosphate and S-adenosylmethionine.     

Genetic transformation in Staphylococcus aureus: demonstration of a competence-conferring factor of bacteriophage origin in bacteriophage 80 alpha lysates.

A virion component that is responsible for conferring competence to Staphylococcus aureus was demonstrated in lysates of bacteriophage 80 alpha, a serological group B phage. Isolated particles of 80 alpha could not be shown to confer significant levels of competence. The phage component had a density of about 1.3 g/cm3, was inactivated by pronase, and was inhibited by antiserum prepared against isolated infectious particles of a serological group B phage. Centrifugation through a Ficoll gradient resulted in separation of competence-conferring activity and plaque-forming units. It is concluded that this proteinaceous subvirion component constitutes a bona fide competence factor of bacteriophage origin.     

Properties of natural 19S antibodies in normal pig serum against the ΦX 174 and T2 phages

The physicochemical properties of natural phage-neutralizing antibodies were studied. Natural neutralizing antibodies against phages ΦX 174 and T 2 were found in the 19 S fraction isolated from normal pig serum by gel filtration on a Bio-Gel P-300 column. The 7 S fraction of normal pig serum possessed no neutralizing activity. The neutralizing activity of the 19 S fraction against phage ΦX 174 was not modified either by inactivation or by the addition of neutralizing cofactor; its activity against phage T 2 was lost by inactivation, but was restored by the addition of cofactor. The neutralizing activity of the 19 S fraction of normal pig serum was completely destroyed by 2-mercaptoethanol and was not restored by the subsequent addition of antibody cofactor. The results of attempts to release phage ΦX 174 from the neutralization complex with normal porcine serum 19 S macroglobulin antibody by the dilution method were the same as those of attempts to dissociate phage from the complex with 7 S type hyperimmune antibody. The virus particle was firmly and irreversibly adsorbed to both types of antibody and was not released by dilution. It is concluded from the results that neutralization of phage ΦX 174 by 19 S macroglobulin molecule of antibody is a simple, irreversible process, for which the thermolabile nonspecific serum components are not required.     

Minicell production and bacteriophage superinducibility of thymidine-requiring strains of Haemophilus influenzae.

Aminopterin- or trimethoprin-resistant thymidine-requiring strains of Haemophilus influenzae produce minicells, and the ratio of minicells to cells increases during the stationary phase of growth. Strain LB11, isolated after mutagenesis of a thymidine-requiring strain (Rd thd), produces more minicells than the parent strain. The mutations involved in high frequency minicell production have been transferred into the wild type (strain Rd) by transformation. The thymidine requirement in the resulting strain, MCl, is essential for minicell production, since spontaneous revertants of MCl to prototrophy do not produce minicells. The ratio of minicells to cells was increased more than 10(3)-fold by differential centrifugation. The minicells contain little or no deoxyribonucleic acid (DNA). Phage HPlcl apparently cannot attach to minicells. Competent cells of LB11 and its thymidine-requiring parent strain produce defective phage as a result of exposure to transforming DNA, whereas only LB11 produces many defective phage in response to the competence regime alone. Competent HP1c1 and S2 lysogens of MC1 and Rd thd are also superinducible by transforming DNA, but competent LB11 lysogens produced about the same amount of HP1c1 or S2 phage with or without exposure to transforming DNA possibly because of competition between the induced defective phage and Hp1c1 or S2 phage.     

设施菜田不同碳氮管理对反硝化菌结构和功能的影响

【目的】通过6年长期定位试验,比较设施菜田不同碳氮管理下反硝化菌结构和功能的差异。【方法】采用末端限制性片段多态性(T-RFLP)和变性梯度凝胶电泳(DGGE)方法分别分析nir K/nir S和nos Z型反硝化菌群结构特征,利用自动连续在线培养监测体系(Robot系统)测定分析NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比,并通过乙炔抑制法测定反硝化酶活性。【结果】传统施肥处理(CN)显著改变了nir K和nos Z型反硝化菌的结构,增加了NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比。nir S型菌受碳氮管理影响较小。减氮(RN)和添加秸秆处理(RN+S)的nir K和nos Z型反硝化菌结构与CN处理的差异性显著,且会显著降低NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比;与CN和RN相比,RN+S显著增加反硝化酶活性。【结论】设施菜田长期传统施肥措施改变了反硝化菌的结构和功能,增加土壤自身的NO产生能力并减弱了N2O还原N2的能力。减氮和添加秸秆管理能形成自身的反硝化菌群结构,并降低NO和N2O排放风险;秸秆的添加会促进反硝化潜在速率,降低菜田NO3-淋洗风险。     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Reaction of monosubstituted hydrazines and diazenes with rat-liver cytochrome P450. Formation of ferrous-diazene and ferric sigma-alkyl complexes

The alkyldiazenes RN = NH (R = CH3 or C2H5) react with reduced microsomal cytochrome P450 leading to complexes exhibiting a Soret peak at 446 nm. Upon oxidation of the [cytochrome P450-Fe(II)(CH3N = NH)] complex with limited amounts of dioxygen, a new complex characterized by a Soret peak at 486 nm is formed. The latter complex was also formed upon slow reaction of methyldiazene with microsomal cytochrome P450-Fe(III) or in situ oxidation of methylhydrazine by limited amounts of O2 or ferricyanide. This complex is rapidly destroyed by O2 or ferricyanide in excess and more slowly by excess dithionite in the presence of CO. Reactions of ethyldiazene or benzyldiazene with cytochrome P450-Fe(III) afforded similar complexes characterized by Soret peaks around 480 nm. These results, when compared to those recently described on reactions of monosubstituted hydrazines RNHNH2 and diazenes RN = NH with hemoglobin and iron-porphyrins, are consistent with a [cytochrome P450-Fe(II)(RN = NH)] structure for the 446-nm-absorbing complexes and a sigma-alkyl cytochrome P450-Fe(III)-R structure for the complexes characterized by a Soret peak around 480 nm. They also suggest a sigma-cytochrome P450-Fe(III)-Ph structure for the complex derived from phenylhydrazine oxidation, recently described in the literature. Finally, they provide the first evidence that cytochrome P450-Fe(III)-R complexes are formed upon microsomal oxidation of alkyl or phenylhydrazines.     

A second function of the S gene of bacteriophage lambda

Infection of Escherichia coli by bacteriophage lambda caused an immediate inhibition of uptake by members of all three classes of E. coli active transport systems and made the inner membrane permeable to sucrose and glycine; however, infection stimulated alpha-methyl glucoside uptake. Phage infection caused a dramatic drop in the ATP pool of the cell, but the membrane did not become permeable to nucleotides. Infection by only one phage per cell was sufficient to cause transport inhibition. However, adsorption of phage to the lambda receptor did not cause transport inhibition; DNA injection was required. The inhibition of transport caused by lambda phage infection was transient, and by 20 min after infection, transport had returned to its initial level. The recovery of transport activity appeared to require a lambda structural protein with a molecular weight of 5,500. This protein was present in wild-type phage and at a reduced level in S7 mutant phage but was missing in S2 and S4 mutant phage. Cells infected with S7 phage had a partial recovery of active transport, whereas cells infected with S2 or S4 phage did not recover active transport. Neither the inhibition of transport caused by phage infection nor its recovery were affected by the protein synthesis inhibitors chloramphenicol and rifampin.     

Isolation and characterization of a novel cytochrome P-450-like pseudogene

A rabbit liver P-450-like pseudogene has been isolated from a lambda phage genomic library. Sequence analysis revealed structural homology with respect to the rat P-450b and P-450e genes as well as a similar intron-exon organization. A 5'-proximal TATA box-like sequence and two 3'-distal putative polyadenylation signals were identified, and all putative intron-exon boundaries except at the 3'-splice site of intron 2 were found to follow the GT/AG rule. With allowance for apparent deletions and insertions, the structural homology of the amino acid sequence deduced from the pseudogene with respect to rabbit P-450 isozyme 2 is lower for exons 1 through 4 (18-28%) than for exons 5 through 9 (42-65%). S1 nuclease mapping showed that mRNAs complementary to the DNA sequence of exon 9 are expressed. However, due to the alterations in the pseudogene, it appears that functional P-450 would not be produced from such mRNAs.     

Cloning of genes affecting capsule expression in Staphylococcus aureus strain M

Staphylococcus aureus strain M produces large amounts of capsular polysaccharide. It produces a non-encapsulated variant at a frequency of 0.01% at 37 degrees C. At high temperature (43 degrees C), the frequency of capsule loss was shown to be 1-38%. A 19 kb plasmid and a prophage were found to be carried by the M strain, but curing of these elements did not affect capsular production. To clone the capsular (cap) genes, a plasmid library of S. aureus M was constructed directly in S. aureus RN4200. The library was then infected with phage 80 alpha. After transduction of the phage lysates to a Cap- mutant derived from M strain, a recombinant plasmid was obtained which complemented the mutant to a Cap+ phenotype. Chromosomal walking experiments were used to clone additional nearby cap genes. Complementation tests using a collection of Cap- mutants showed that most of the mutants were complemented by a 19.4 kb DNA fragment, suggesting that the majority of the cap genes affecting capsule production are clustered together.