Development of a real-time PCR assay for the specific detection and identification of Streptococcus pseudopneumoniae using the recA gene.

We sequenced the evolutionarily conserved genes 16S rRNA, atpD, tuf, and recA from Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis. Phylogenetic analysis revealed that recA provided good resolution between these species, including discrimination of the novel species S. pseudopneumoniae. By contrast, the more conserved 16S rRNA, tuf and atpD are not sufficiently discriminatory. Therefore, recA sequences were used to develop a real-time PCR assay with a locked nucleic acid-mediated TaqMan probe for the specific detection and identification of S. pseudopneumoniae. The PCR assay showed excellent specificity and a detection limit of <10 genome copies for the detection and identification of S. pseudopneumoniae strains, which makes it a promising tool for molecular identification and epidemiological studies. In conclusion, this article describes for the first time a PCR assay for the specific identification of S. pseudopneumoniae.

Clostridium lavalense sp. nov., a glycopeptide-resistant species isolated from human faeces.

Two vancomycin-resistant, strictly anaerobic, Gram-positive, rod-shaped, spore-forming organisms (strains CCRI-9842(T) and CCRI-9929) isolated from human faecal specimens in Québec, Canada, and Australia were characterized using phenotypic, biochemical and molecular taxonomic methods. Pairwise analysis of the 16S rRNA gene sequences showed that both strains were closely related to each other genetically (displaying 99.2 % sequence similarity) and represented a previously unknown subline within the Clostridium coccoides rRNA group of organisms (rRNA cluster XIVa of the genus Clostridium). Strains CCRI-9842(T) and CCRI-9929 used carbohydrates as fermentable substrates, producing acetic acid as the major product of glucose metabolism. The novel strains were most closely related to Clostridium asparagiforme, Clostridium bolteae and Clostridium clostridioforme, but morphological, biochemical and phylogenetic studies demonstrated that they represent a previously unidentified species of the genus Clostridium. This was confirmed by the unique cellular fatty acid composition of strains CCRI-9842(T) and CCRI-9929. Therefore, on the basis of data from the polyphasic taxonomic analysis, it is proposed that strains CCRI-9842(T) and CCRI-9929 represent a novel species of the genus Clostridium, for which the name Clostridium lavalense sp. nov. is proposed. The type strain is CCRI-9842(T) (=CCUG 54291(T)=JCM 14986(T)=NML 03-A-015(T)).

Ruminococcus gauvreauii sp. nov., a glycopeptide-resistant species isolated from a human faecal specimen.

A novel strictly anaerobic, vancomycin-resistant, Gram-positive coccus (strain CCRI-16,110(T)) was isolated from a human faecal specimen. This strain was characterized using morphological, biochemical and molecular taxonomic methods. The organism was unable to hydrolyse aesculin and failed to produce acid from cellobiose, d-lactose and alpha-raffinose. Acetic acid was the sole product of glucose fermentation by the organism. On the basis of 16S rRNA and tuf gene sequence comparison, strain CCRI-16,110(T) was most closely related to species of the genus Ruminococcus and formed a hitherto unknown sublineage within the Clostridium coccoides rRNA cluster of organisms (cluster XIVa). Based on phenotypic and phylogenetic evidence, a novel species, Ruminococcus gauvreauii sp. nov., is proposed. The type strain is CCRI-16,110(T) (=NML 060141(T) =CCUG 54,292(T) =JCM 14987(T)).

vanD and vanG-like gene clusters in a Ruminococcus species isolated from human bowel flora.

A vancomycin-resistant, anaerobic, gram-positive coccus containing the vanD and vanG-like genes (strain CCRI-16110) was isolated from a human fecal specimen during a hospital surveillance program to detect carriers of vancomycin-resistant enterococci. Comparison of the 16S rRNA gene sequence of strain CCRI-16110 with databases revealed a potentially novel Ruminococcus species that was most similar (<94% identity) to Clostridium and Ruminococcus species. Strain CCRI-16110 was highly resistant to vancomycin and teicoplanin (MICs of >256 microg/ml). The complete DNA sequence of the vanD cluster was most similar (98.2% identity) to that of Enterococcus faecium BM4339, containing the vanD1 allele. An intD gene with 99% identity with that of this E. faecium strain was found to be associated with the vanD gene cluster of this novel anaerobic bacterium. Strain CCRI-16110 also harbors genes encoding putative VanS(G), VanG, and VanT(G) proteins displaying 56, 73.6, and 55% amino acid sequence identity, respectively, compared to the corresponding proteins encoded by the vanG1 and vanG2 operons of Enterococcus faecalis BM4518 and N03-0233. This study reports for the first time an anaerobic bacterium containing the vanD gene cluster. This strain also harbors a partial vanG-like gene cluster. The presence of vanD- and vanG-containing anaerobic bacteria in the human bowel flora suggests that these bacteria may serve as a reservoir for the vanD and vanG vancomycin resistance genes.

High prevalence of glycopeptide resistance genes vanB, vanD, and vanG not associated with enterococci in human fecal flora.

The presence of Enterococcus-associated vancomycin resistance genes vanA, vanB, vanD, vanE, and vanG in rectal swabs was investigated in two hospitals using PCR. All vanA genes detected were associated with vancomycin-resistant enterococci (VRE), whereas VRE-associated vanB genes were detected in only one hospital (4.7%). However, in both hospitals, high prevalences of vanB (6.2 and 2.3%), vanD (43.8 and 26.7%), and vanG (10.5 and 6.9%) genes not associated with enterococci were found.

Characterization of a Tn5382-like transposon containing the vanB2 gene cluster in a Clostridium strain isolated from human faeces.

OBJECTIVES: During a hospital surveillance programme to detect VRE carriers, an anaerobic vancomycin-resistant bacterial strain CCRI-9842 containing a vanB gene was isolated from a human faecal specimen. In this study, we have characterized this strain and its vanB-containing element. METHODS: Strain CCRI-9842 was characterized by 16S rDNA sequencing and susceptibility testing. PCR mapping and sequencing of the vanB-containing element, as well as plasmid extraction and mating experiments, were carried out to investigate the genetic basis of vancomycin resistance in this strain. RESULTS: Strain CCRI-9842 was identified as a Clostridium species closely related to Clostridium bolteae (96.8% 16S rDNA identity). This strain was resistant to a high level of vancomycin (MIC of 256 mg/L), but was susceptible to teicoplanin and ampicillin. The complete sequence of the CCRI-9842 vanB gene exhibited 99.1% identity with that of vanB2. PCR mapping and sequencing showed that the genetic element carrying vanB2 was similar to transposon Tn5382/Tn1549. This Tn5382-like transposon forms circular intermediates and is flanked on the left and right ends by repeat sequences of at least 700 bp in the opposite direction. No plasmid was detected in this strain, suggesting that the Tn5382-like transposon was integrated into the chromosome. The vancomycin resistance was not transferable to enterococci. CONCLUSIONS: Our report shows for the first time the presence of a Tn5382-like transposon carrying vanB2 in a Clostridium species of the human intestinal flora. This suggests that the vanB2 Tn5382-like transposon is an important vector for the spread of vancomycin resistance in several bacterial species.

New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci.

Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was approximately 25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.

Evidence for horizontal gene transfer in evolution of elongation factor Tu in enterococci.

The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.

Inactivation of the ampD gene in Pseudomonas aeruginosa leads to moderate-basal-level and hyperinducible AmpC beta-lactamase expression.

It has been shown in enterobacteria that mutations in ampD provoke hyperproduction of chromosomal beta-lactamase, which confers to these organisms high levels of resistance to beta-lactam antibiotics. In this study, we investigated whether this genetic locus was implicated in the altered AmpC beta-lactamase expression of selected clinical isolates and laboratory mutants of Pseudomonas aeruginosa. The sequences of the ampD genes and promoter regions from these strains were determined and compared to that of wild-type ampD from P. aeruginosa PAO1. Although we identified numerous nucleotide substitutions, they resulted in few amino acid changes. The phenotypes produced by these mutations were ascertained by complementation analysis. The data revealed that the ampD genes of the P. aeruginosa mutants transcomplemented Escherichia coli ampD mutants to the same levels of beta-lactam resistance and beta-lactamase expression as wild-type ampD. Furthermore, complementation of the P. aeruginosa mutants with wild-type ampD did not restore the inducibility of beta-lactamase to wild-type levels. This shows that the amino acid substitutions identified in AmpD do not cause the altered phenotype of AmpC beta-lactamase expression in the P. aeruginosa mutants. The effects of AmpD inactivation in P. aeruginosa PAO1 were further investigated by gene replacement. This resulted in moderate-basal-level and hyperinducible expression of beta-lactamase accompanied by high levels of beta-lactam resistance. This differs from the stably derepressed phenotype reported in AmpD-defective enterobacteria and suggests that further change at another unknown genetic locus may be causing total derepressed AmpC production. This genetic locus could also be altered in the P. aeruginosa mutants studied in this work.

Structure-function studies of Ser-289 in the class C beta-lactamase from Enterobacter cloacae P99.

Site-directed mutagenesis of Ser-289 of the class C beta-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in beta-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant beta-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC beta-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the kcat of four of the five beta-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant beta-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

An ampD gene in Pseudomonas aeruginosa encodes a negative regulator of AmpC beta-lactamase expression.

The ampD and ampE genes of Pseudomonas aeruginosa PAO1 were cloned and characterized. These genes are transcribed in the same orientation and form an operon. The deduced polypeptide of P. aeruginosa ampD exhibited more than 60% similarity to the AmpD proteins of enterobacteria and Haemophilus influenzae. The ampD product transcomplemented Escherichia coli ampD mutants to wild-type beta-lactamase expression.

Characterization of the penA and penR genes of Burkholderia cepacia 249 which encode the chromosomal class A penicillinase and its LysR-type transcriptional regulator.

Burkholderia cepacia is recognized as an important pathogen in the lung infections of patients with cystic fibrosis. An inducible beta-lactamase activity has been associated with increased resistance to beta-lactam antibiotics in clinical isolates of B. cepacia. In this study, we report the revised sequence of the penA gene, which encodes the inducible penicillinase of B. cepacia, and show that it belongs to the molecular class A beta-lactamases and exhibits a high degree of similarity to the chromosomal beta-lactamase of Klebsiella oxytoca. Analysis of the nucleotide sequence of the DNA region directly upstream of the penA coding sequence revealed an open reading frame (penR), the transcription of which was oriented opposite to that of penA and whose initiation was 130 bp away from that of penA. Two potential ribosome-binding sites and two overlapping -10 and -35 promoter sequences were identified in the intercistronic region. The predicted translation product of penR was a polypeptide of 301 amino acids with an estimated molecular size of 33.2 kDa. The deduced polypeptide of penR showed a high degree of similarity with AmpR-like transcriptional activators of class A and C beta-lactamases, with identities of 59 and 58.7% with Pseudomonas aeruginosa PAO1 AmpR and Proteus vulgaris B317 CumR, respectively. The N-terminal portion of B. cepacia PenR was predicted to include a helix-turn-helix motif, which may bind the LysR motif identified in the intercistronic region. Induction of PenA by imipenem was shown to be dependent upon the presence of PenR. Expression of the cloned B. cepacia penA and penR genes in Escherichia coli SNO302 (ampD) resulted in a high basal and hyperinducible PenA activity. These results suggest that the regulation of the PenA penicillinase of B. cepacia 249 is similar to that observed in other class A and class C beta-lactamases that are under the control of a divergently transcribed AmpR-like regulator.

Sequence of the ponA gene and characterization of the penicillin-binding protein 1A of Pseudomonas aeruginosa PAO1.

The nucleotide sequence of the ponA gene encoding the high molecular-mass penicillin-binding protein 1A (PBP1A) of Pseudomonas aeruginosa (Pa) PAO1 was determined and characterized. The predicted PBP1A protein of 822 amino acids (aa) has a calculated molecular mass of 91.2 kDa corresponding to the size of the protein expressed in vitro and in vivo. A penicillin-binding (PB) assay showed that the Pa ponA gene product covalently binds penicillin. The deduced PBP1A aa sequence has features typical of class-A high-molecular-mass PBPs: a highly hydrophobic N-terminus portion containing a potential transmembrane segment which might anchor the protein to the cytoplasmic membrane; an N-terminal module with the conserved boxes 1 (E86D(DN)F(AN)H(Y)G), 2 (G117GS(T)I(TM)Q), 3 (R139K(IN)E(ILL)AL) and 4 (R221R(NW)IL); a PB module with the conserved boxes 5 (S461SFK), (S520RN) and (K695TG); an internal extension at aa 297-407 between the N-terminal and PB modules; and a C-extension at the end of the PB module at aa 742 to 822. The highest percentage of similarity (62.8%) was found with the class A high-molecular-mass PBP1A of Escherichia coli (Ec) and Haemophilus influenzae. The observed extensive homology in the modular design of the Pa PBP1A with the bifunctional Ec PBP1A suggests structural and functional relationships between these proteins and refutes the proposed correspondence between Pa PBP1A and Ec PBP1B.

Role of Ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling.

A growing number of extended spectrum SHV-type beta-lactamases capable of hydrolyzing third-generation cephalosporins such as cefotaxime and ceftazidime have been reported. These new enzymes differ by a few amino acids from SHV-1, an enzyme incapable of hydrolyzing these drugs. Two of these substitutions, Gly-238-->Ser and Glu-240-->Lys, are in a key beta-strand of the catalytic site of class A beta-lactamases. To understand the structural basis of these new activities, we first subcloned the DNA region coding for SHV-1 and SHV-2 and did site-directed mutagenesis to create two mutant SHV-1 proteins containing Ser and Glu or Gly and Lys and two mutant SHV-2 proteins containing Gly and Glu or Ser and Lys in positions 238 and 240, respectively. Phenotypic analysis (antibiograms and minimum inhibitory concentrations) and activity spectra of mutant enzymes showed that Ser-238 is critical for cefotaxime hydrolysis whereas both Ser-238 and Lys-240 are needed for strong ceftazidime hydrolysis. A three-dimensional model for SHV beta-lactamase complexes was constructed using the crystallographic structure of the homologous Bacillus licheniformis beta-lactamase, the complex of cefotaxime with the Streptomyces sp. R61 D-alanyl-D-alanine peptidase, and the complex of aztreonam with the Citrobacter freundii beta-lactamase. The modeling of SHV beta-lactamase complexes showed that factors which are most likely to correlate with binding and kinetic data are the size of the relatively buried amino acid at position 238 and the electrostatic charge of the exposed group at position 240.

Nucleotide sequence and phylogeny of SHV-2 beta-lactamase.

We determined the nucleotide sequence of the blaSHV-2(pBP60-1) gene from Klebsiella ozaenae which confers resistance to broad-spectrum cephalosporins. The structural gene encodes a polypeptide product of 286 amino acids, and the estimated molecular weight of the mature protein is 28,900. Amino acid sequence comparison of the SHV-2pBP60-1 enzyme with all known class A beta-lactamases and homology studies showed that the residues were highly conserved. Furthermore, SHV-2pBP60-1 was clearly related to SHV-1, LEN-1, and OHIO-1. The SHV-2pBP60-1 enzyme differed from SHV-1 isolated from Klebsiella pneumoniae by seven amino acid substitutions. One of these substitutions, the Gly----Ser substitution at position 234, is probably a key region for the novel activity of cefotaxime hydrolysis. A phylogenetic tree was constructed by using all class A beta-lactamases of known sequences by a progressive alignment method. The data suggested that the beta-lactamases of gram-positive Streptomyces, Staphylococcus, and Bacillus species appeared early in evolution, followed by the PSE and CARB enzymes of Pseudomonas species and, more recently, by the SHV-type and TEM-type enzymes found in enteric bacteria. Larger evolutionary distances separated clusters of the gram-positive beta-lactamases than separated clusters of the gram-negative enzymes. Results of this phylogenetic study suggested that extended-spectrum enzymes are recent derivatives that are selected by the use of new cephalosporins.

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly(ADP-ribosyl)ation on core nucleosome structure.

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin was analyzed by gel electrophoresis, electron microscopy, and velocity sedimentation. In parallel, the interaction of automodified poly(ADP-ribose) polymerase with native and H1-depleted chromatin was analyzed. In H1-depleted chromatin histone H2B becomes the major poly(ADP-ribose) histone acceptor protein, whereas in native chromatin histone H1 was the major histone acceptor. Poly(ADP-ribosyl)ation of H1-depleted chromatin prevented the recondensation of polynucleosomes reconstituted with exogenous histone H1. This is probably due to the presence of modified poly(ADP-ribose) polymerase and hyper(ADP-ribosyl)ated histone H2B. Indeed, about 40% of the modified enzyme remained associated with H1-depleted chromatin, while less than 1% of the modified enzyme was bound to native chromatin. The influence of poly(ADP-ribosyl)ation on the chromatin conformation was also studied at the level of nucleosome in using monoclonal and polyclonal antibodies specific for individual histones and synthetic peptides of histones. In native chromatin incubated in the presence of Mg2+ there was a drop in the accessibility of histone epitopes to monoclonal and polyclonal antibodies whereas upon poly(ADP-ribosyl)ation their accessibility was found to remain even in the presence of Mg2+. In poly(ADP-ribosyl)ated H1-depleted chromatin an increased accessibility of some histone tails to antibodies was observed.

Modulation of chromatin structure by poly(ADP-ribosyl)ation.

Poly(ADP-ribose) polymerase is a nuclear enzyme that is highly conserved in eucaryotes. Its activity is totally dependent on the presence of DNA containing single or double stranded breaks. We have shown that this activation results in a decondensation of chromatin superstructure in vitro, which is caused mainly by hyper(ADP-ribosy)ation of histone H1. In core particles, the modification of histone H2B leads to a partial dissociation of DNA from core histones. The conformational change of native chromatin by poly(ADP-ribosyl)ation is reversible upon degradation of the histone H1-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. We propose that cuts produced in vivo on DNA during DNA repair activate poly(ADP-ribose) polymerase, which then synthesizes poly(ADP-ribose) on histone H1, in particular, and contributes to the opening of the 25-nm chromatin fiber, resulting in the increased accessibility of DNA to excision repair enzymes. This mechanism is fast and reversible.

Modulation of chromatin superstructure induced by poly(ADP-ribose) synthesis and degradation.

It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.

Poly(ADP-ribosyl)ation of chromatin: kinetics of relaxation and its effect on chromatin solubility.

We have studied the kinetics of relaxation of poly(ADP-ribosyl)ated polynucleosomes produced by endogenous enzyme activity by comparing the generation of hyper(ADP-ribosyl)ated histone H1 and its effect on the chromatin structure as revealed by electron microscopy. A correlation can be established between the appearance of histone H1 modified forms and the localized relaxation of the chromatin. We have also noticed, in parallel, that poly(ADP-ribosyl)ated chromatin showed increased solubility in the presence of Mg2+ and 0.2 M NaCl. Electron microscopic examination of the solubilized chromatin produced by poly(ADP-ribosyl)ation shows polynucleosomes exhibiting more relaxed conformation, whereas an increasing amount of hyper(ADP-ribosyl)ated histone H1 is found in the pellet, as shown by acid-urea-polyacrylamide electrophoretic separation of histone extracts.

Sequential ADP-ribosylation pattern of nucleosomal histones. ADP-ribosylation of nucleosomal histones.

The pattern of nucleosomal histones poly(ADP-ribosyl)ation is changed under conditions which affect the poly(ADP-ribosyl)ation state of the enzyme. At low NAD concentrations the enzyme can poly(ADP-ribosyl)ate histones H1 and H1, H2A, A2A, and H2B. However at NAD concentrations above 10 microM the enzyme preferentially poly(ADP-ribosyl)ates histone H1 to a hyper ADP-ribosylated form. Furthermore we have observed hyper ADP-ribosylation of histone H2B at NAD concentrations of 10 microM suggesting that histone H2B can undergo the same type of ADP-ribosylation pattern as histone H1. Also at higher NAD concentrations an elongation of the polymer attached to the enzyme and other nuclear proteins takes place.