[PCR--a new era in clinical microbiologic diagnosis and epidemiologic studies? (Part II)].
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Bacterial Infections↗
Biomedical subjects
J Kur
Publications and source records attributed to J Kur.
Genomic species typing of acinetobacters by polymerase chain reaction amplification of the recA gene.
The recA gene has been used as a target in screening for the presence of acinetobacters on the genospecies level and differentiation of relevant acinetobacter species from one another by PCR. Primers deduced from known recA gene sequences of Acinetobacter calcoaceticus and Neisseria gonorrhoeae allowed the amplification of DNAs from all Acinetobacter genospecies. The size of the amplified DNA fragment from all genospecies tested was approximately 435-500 bp relative to DNA size markers. The amplified products were examined further by restriction fragment length polymorphism (RFLP) analysis. Restriction analysis with only two enzymes, MboI and HinfI, enabled us to identify all known genospecies. Since this method uses conserved recA gene sequences for primers, it is expected to be applicable for the identification of most bacterial species.
Acinetobacter↗
Mutations in HU and IHF affect bacteriophage T4 growth: HimD subunits of IHF appear to function as homodimers.
The Escherichia coli nucleoid-associated DNA-binding proteins HU and IHF are required for numerous biological processes, including phage growth (e.g., lambda, phi 80, Mu and f1) and DNA replication. Here, we show that growth of T4 phage is inhibited both in hupA hupB and himA himD double mutants. The growth profile of triple mutants (hupA hupB himA and hupA hupB himD) suggests that HimD subunits can form homodimers, which are functionally competent for supporting in vivo growth of phage T4.
Bacterial Proteins↗
Purification and characterization of two restriction endonucleases isolated from Phormidium inundatum.
We have isolated two restriction endonucleases, PinBI and PinBII, from the cyanobacterial strain Phormidium inundatum, and identified them as isoschizomers of AvaIII and BspMII, respectively.
Bacteriophage lambda↗
PCR differentiation of seventeen genospecies of Acinetobacter.
In the present study, strains of 17 reference Acinetobacter genospecies were investigated by using the polymerase chain reaction (PCR). We used primers to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the tested acinetobacters into 15 groups. The genospecies 5 (Acinetobacter junii), 7 (Acinetobacter johnsonii) and 10 produced the same characteristic PCR patterns, suggesting the identity of these three genospecies. A preliminary evaluation of the proposed scheme for PCR diagnostics was carried out. Using the proposed scheme, tested clinical strains were identified correctly to the genospecies level, and the identifications confirmed by conventional biochemical tests. On the basis of our results, PCR amplification of the 16S-23S spacer region shows significant promise as a tool for the simple identification of genospecies belonging to Acinetobacter sp. The nucleotide sequences of our primers are sufficiently highly conserved among these organisms as to permit PCR reactions to be carried out with a single set of reaction conditions and amplification parameters irrespective of species or genus.
Acinetobacter↗
Binding of Escherichia coli integration host factor (IHF) to the origin segment of p15A plasmid.
The integration host factor (IHF) is a sequence-specific, histone-like, multi-functional DNA-binding and -bending protein of Escherichia coli. Characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. In this paper we report data concerning the binding of IHF protein to the plasmid orip15A region. IHF binds to the single site of the DNA fragment containing the orip15A, as shown by the gel mobility shift assays and footprinting experiment. On the basis of the ihf consensus sequences published, we have been able to identify one sequence of putative ihf site into the orip15A sequence with two mismatches in relation to the consensus sequence of Kur et al., 1989, Gene 81, 1-15. One ihf binding site was also found in the oriColE1 region sequence with three mismatches in relation to this consensus sequence.
Bacterial Proteins↗
Identification of Pseudomonas aeruginosa on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.
In the present study, 40 clinical strains of Pseudomonas aeruginosa were investigated by using the polymerase chain reaction (PCR). We used primers to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. When the spacer amplification products were resolved by electrophoresis, the resulting patterns were characteristic for all tested strains. Only one specific PCR fragment of 580 bp was formed. This product was digested with HaeIII, HinfI and AluI restriction endonucleases. Restriction fragments produced by all three restriction endonucleases were characteristic and have the same sizes for all strains tested. The amplification product contains a conserved, internal single HaeIII restriction site. On the basis of our results, PCR amplifications of the 16S-23S spacer region for Pseudomonas aeruginosa and subsequent RFPL analysis show significant promise as a tool for the simple identification of this bacteria.
Base Sequence↗
Identification of Serratia marcescens on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.
The aim of the present study was to develop a method for identification of strains of Serratia marcescens that is also suitable for use in the epidemiologic studies. 40 clinical strains of Serratia marcescens were investigated by using the polymerase chain reaction (PCR). We used primers to amplify spacer regions between the 16S and 23S genes in the prokaryotic rDNA genetic loci. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the strains tested into 3 groups (group A-35, group B-3 and group C-2 isolates). Digestion of PCR fragments of the group A strains with HinfI enzyme enabled separation of organisms into 3 distinct groups (A1-A3).
Base Sequence↗
Characterization of restriction endonuclease AjoI from Acinetobacter johnsonii.
A new type II restriction endonuclease, named AjoI, was detected in Acinetobacter johnsonii. The enzyme AjoI, an isoschizomer of PstI, recognized the hexanucleotide sequence [5'-CTGCA/G-3'], with a cleavage site generating fragments of DNA with protruding cohesive 3' termini.
Acinetobacter↗
Integration host factor, a histone-like Escherichia coli protein, binds to at least four sites of the DNA fragment containing the recA gene.
The integration host factor (IHF) is a sequence-specific, histone-like, multifunctional DNA-binding and -bending protein of Escherichia coli. The characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. Less is known concerning the role of IHF in E. coli, although it has been implicated in a number of processes in this organism including DNA replication, site-specific recombination, and gene expression. In this paper we report data concerning the binding of IHF protein to the recA gene region. IHF binds to at least four sites of the DNA fragment containing the recA gene, as shown by gel mobility shift assays. On the basis of the ihf consensus sequences published, we have been able to identify two sequences of putative ihf sites (ihf 1 and ihf2) into the 1390 bp long sequence containing the recA gene, but only the ihf2 site was able to bind IHF, as measured by gel mobility shift experiments. The nonfunctional ihf1 sequence was found to overlap the -35 region of the recA promoter and the functional ihf2 sequence was found within the recA gene structure at nt +780 to +807 (both with three mismatches according to the consensus sequence of Kur et al., 1989). This confirms our earlier results that the IHF-DNA interaction does not depend on any very rigid sequence, but also on the suitable sequences of the neighbouring regions, together with the proper DNA conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial Proteins↗
Use of IHF--mediated Achilles' heel cleavage (IHF-AC) method for mapping ihf sites.
We have shown that Integration Host Factor of E. coli can successfully be used in the IHF-mediated Achilles' Heel Cleavage (IHF-AC) technique (Kur et al., 1992b), for generating rare natural cleavage sites. The first step of this procedure is methylation of DNA in the presence of IHF, when the overlapping ihf/restriction sites are protected from methylation, and in the second step the DNA is cut by the cognate restriction enzyme. The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature. The aim of the present study is to demonstrate that IHF-AC procedure might serve as a useful tool for finding new protein-binding sites which overlap known restriction sites. I have used this approach in conjunction with several MTases to find several other unknown IHF-binding sites.
Bacterial Proteins↗
Integration host factor (IHF) applied for partial digestion by restriction endonucleases in large DNA molecules (IARC procedure).
The IHF protein of Escherichia coli was successfully used in IHF-mediated Achilles' Heel Cleavage (IHF-AC) technique (Koob et al., 1988; Kur et al., 1992), and leads to the generation of very rare restriction sites in large DNA molecules. The first step of this procedure is methylation of DNA in the presence of IHF, when the overlapping ihf/restriction sites are protected from methylation, and in the second step the DNA is cut by the cognate restriction enzyme. The aim of the present study is to develop a very exact and reproducible procedure to obtain only a few well-defined cuts with the IHF-pre-treated DNA, depending on the variety of all parameters. This technique (IARC, i.e., IHF-assisted rare cutters) employs the restriction enzyme and only one auxiliary protein (IHF). The advantage of the IARC procedure is that no methylation is required (as opposed to the IHF-AC method). Using the IARC approach, the effects of various IHF concentrations were evaluated on cleaving the activity of the DraI, PacI, PmeI, and SwaI enzymes using DNA of phage lambda or the entire genomic 4.7-Mb DNA of E. coli. At low IHF concentrations only a few cut sites were eliminated by IHF binding, but at high IHF concentration, enzymes were able to cut in only one or several specific sites.
Amino Acid Sequence↗
[Test of using plasmid DNA profile analysis for identification of Acinetobacter species].
The analysis regarded 32 strains of Acinetobacter genus isolated from a variety of samples from human and animal sources (hospital environment, nonhospital source, water, burns). The genus Acinetobacter is heterogeneous and has a complex taxonomy. For this reason, plasmid profile analysis has been used as a method of identification to study the genetic diversity of natural populations. Strains having an identical plasmid profile were pooled in the same plasmid group. According to these criteria, 32 isolates were grouped in 11 classes. Within the 11 plasmid groups, 2 were found in A. baumannii, 3 in A. lwoffii, 2 in A. johnsonii, 3 in A. haemolyticus and 1 in A. junii. Most frequently isolated species of Acinetobacter from burns was A. baumannii (11 out of the 13 isolates). Plasmid profile analysis of those strains revealed a presence of only one plasmid group. Plasmid profile analysis of Acinetobacter strains can be an useful technique for characterizing isolates in epidemiologic studies as a complementary method. It can be used directly as a very rapid and convenient technique to type Acinetobacter strains.
Acinetobacter↗
Isolation and identification of the restriction endonuclease PtaI from Phormidium tadzschicicum, an isoschizomer of BspMII.
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Base Sequence↗
RecA-AC: single-site cleavage of plasmids and chromosomes at any predetermined restriction site.
We have developed a novel version of the Achilles' Cleavage (AC) reaction in which virtually any restriction site on DNA of any size can be converted to a unique cleavage site. We first polymerized RecA protein on a synthetic oligodeoxyribonucleotide (oligo) in the presence of a nonhydrolyzable ATP analogue to generate oligo:RecA nucleoprotein filaments. These filament were then incubated with plasmid or intact chromosomal DNA from Saccharomyces cerevisiae to form stable complexes in the yeast LEU2 gene at the target sequence identical (or complementary) to that of the oligo. When HhaII (HinfI) methyltransferase (M.HhaII) was added, all of the recognition sites for HhaII with the exception of the one protected by the RecA filament were methylated and thus no longer cleaved by the cognate restriction endonuclease (HinfI). After inactivation of the RecA and the M.HhaII, HinfI was used to efficiently cleave the plasmid or chromosome specifically at the targeted restriction site. Since oligos specific for any sequence can be easily synthesized and the other reagents necessary to perform RecA-mediated AC (RecA-AC) reactions on both plasmids and intact chromosomes are readily available, this procedure can be applied immediately to the precise dissection and analysis of genomic DNA from any source and to any other research problem requiring efficient, highly specific cleavage of DNA at predetermined sites.
Base Sequence↗
Integration host factor (IHF) binds to many sites in the A + T-rich b2 region of phage lambda DNA.
Computer analysis of almost the entire b2 region of lambda phage (nt 22346-27475) revealed 23 consensus-like ihf sites, with eleven pointing in one direction and twelve in the opposite direction [27 bp; Kur et al., Gene 81 (1989) 1-15]. To confirm the significance of this finding experimentally, the region was subdivided into 21 fragments and examined for integration host factor (IHF) binding by gel retardation and a variety of footprinting methods. Out of 21 fragments examined 13 were found to be retarded on gels in the presence of IHF and to contain one to three ihf sites each. All sites which differ by up to 2 bp from our 27-bp consensus ihf sequence can bind IHF in vitro. However, three of the computer-predicted sites overlap with sites of opposite orientation; therefore we could not determine at the present time which of the two antiparallel sequences binds IHF. We have compared the predictive values of various kinds of consensus sequences and show that our 27-bp consensus ihf sequence agrees best with the experimental data. It was demonstrated by Kur et al. [Virology 168 (1989) 236-244] that the IHF protein represses transcription from promoters located close to the right terminus of the b2 region, within the phage lambda attachment site. We discuss the possibility that some of the IHF-binding sites could be instrumental in repressing in vivo transcription from the A + T-rich b2 region during the lambda prophage state [Rosenvold et al., Virology 107 (1980) 476-487].
Amino Acid Sequence↗
A novel method for converting common restriction enzymes into rare cutters: integration host factor-mediated Achilles' cleavage (IHF-AC).
Integration host factor (IHF)-mediated protection against enzymatic methylation at ihf-overlapping sites provides the basis for this novel application of the Achilles' cleavage (AC) technique [Koob et al., Science 241 (1988) 1084-1086] for generating rare natural cleavage sites. When applying IHF-AC to plasmid, phage lambda, Escherichia coli and yeast genomes, only a few of the EcoRI, HinfI, and MboI sites (which overlapped the ihf sites) remained cleavable after prior methylation with the cognate M.EcoRI, M.HinfI, or Dam methyltransferases in the presence of IHF. Thus, IHF-AC essentially converted these enzymes into very rare cutters. The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature. Moreover, the method permits the genomic location and strength of the ihf sites to be determined.
Bacterial Proteins↗
Alterations in the p'R promoter of coliphage lambda modify both its activity and interaction with the integration host factor (IHF).
A limited number of deletion/insertions and a point mutation in the -35 region of the p'R promoter of phage lambda were examined and found to influence both transcription and its repression by the integration host factor (IHF). Positive effects on transcription (in the absence of IHF) are small (up to 1.4-fold) and are caused by a deletion-substitution upstream of the -35/ihf site. Up to three base changes in the -35 promoter element seem to be tolerated, with only a small negative effect on transcription. In some cases, effective transcription requires supercoiling of such mutant template. Since an ihf sequence overlaps the -35 region of p'R, IHF represses transcription. Repression is correlated with IHF binding and consequent DNA bending, as assessed by gel retardation experiments. Nine p'R mutants were tested for their IHF binding and repression; the results confirm the consensus sequence, 5'-W2WWWWN7WWWWCARNWN2TTR derived from the hydroxyl radical footprinting, where the bold letters indicate the IHF-protected bases and W is A or T, R is A or G and N represents A, T, G or C. Perhaps surprisingly, some mutations just upstream or downstream of this ihf sequence also affect IHF binding, as observed not only for the pR'/ihf but also for the att H' site of lambda. Supercoiling in some cases permits the IHF-mediated repression to be overcome, probably by increasing the RNA polymerase binding and/or decreasing the interaction with IHF. All our data are consistent with a model which assumes that IHF initially binds to one or two ihf contact points depending on preexisting DNA topology, bends DNA, and completes the remaining contacts while finally adjusting the DNA conformation to establish the best fit within the minor groove of the double helix. Effective IHF repression of transcription would thus depend on several factors, including: (1) the sequence, and (2) the initial conformation of the ihf site, together with (3) the capacity of IHF to compete with RNA polymerase for the overlapping binding sites.
Bacterial Proteins↗