Biomedical subjects

J H Crosa

Publications and source records attributed to J H Crosa.

At least 55 records · Page 3Linked to original sources

PubMed1991

Roles of DNA adenine methylation in controlling replication of the REPI replicon of plasmid pColV-K30.

DNA adenine methylation controls DNA replication of plasmids containing the prototypic REPI replicon by affecting protein recognition and by altering the helical stability of the origin. Denaturing gradient gel electrophoresis shows that adenine methylated origin DNA is more easily melted than unmethylated. However, because an added DNA adenine methylation (dam) site at the origin, whether in or out of phase with other helically aligned dam sites, actually prevents replication, we conclude that destabilization of the helix is not the exclusive function of adenine methylation in REPI replication. We find that the conformation and degree of methylation at the origin, features which are important for protein recognition, are essential for replication. In fact, RepI, a protein required for replication initiation at REPI replicons, contains a region homologous with a domain in proteins which specifically recognize and bind 5'-GATC-3'. We propose that the dam sites in the origin play a dual role: one is destabilization of the helix, and the other is protein recognition.

Adenine↗

PubMed1991

Polymorphism in the aerobactin-cloacin DF13 receptor genes from an enteroinvasive strain of Escherichia coli and pColV-K30 is associated only with a decrease in cloacin susceptibility.

We have cloned chromosomal genes mediating the aerobactin iron transport system from the enteroinvasive strain Escherichia coli 978-77. The physical map of the region spanning the siderophore biosynthesis genes and the upstream portion of the receptor gene in strain 978-77-derived clones was identical to the corresponding regions in pColV-K30, while the downstream portion was different. Recombinant plasmids derived from strain 978-77 encoded a 76-kDa outer membrane protein, in contrast to the 74-kDa polypeptide encoded by similar clones derived from pColV-K30. No differences were found in the uptake of ferric aerobactin mediated by either the 76-kDa- or the 74-kDa-encoding plasmids. In contrast, cells containing the 76-kDa-encoding plasmids showed a 16-fold decrease in susceptibility to cloacin compared with cells harboring the 74-kDa-encoding plasmids. Two classes of chimeric aerobactin receptor genes were constructed by exchanging sequences corresponding to the downstream portion from the aerobactin receptor gene of both systems. The pColV-K30-978-77 chimeric gene encoded a 76-kDa outer membrane protein which mediated a low level of cloacin susceptibility, whereas the 978-77-pColV-K30 type encoded a protein of 74 kDa determining a level of cloacin susceptibility identical to that mediated by pColV-K30.

Bacterial Outer Membrane Proteins↗

PubMed1991

Colicin V virulence plasmids.

ColV plasmids are a heterogeneous group of IncFI plasmids which encode virulence-related properties such as the aerobactin iron uptake system, increased serum survival, and resistance to phagocytosis. These plasmids have been found in invasive strains of Escherichia coli which infect vertebrate hosts including humans and livestock. Colicin V was the first colicin to be identified, in 1925, but not until the field experienced a renewed interest has the mechanism of colicin V activity been explored. As encoded by ColV plasmid pColV-K30, the aerobactin iron uptake system has been extensively investigated, but other ColV-encoded phenotypes remain largely uncharacterized. Restriction enzyme mapping of the 144-kb pColV-K30 and of the 80-kb pColV-B188 has facilitated systematic study, so that questions can be addressed by a molecular and comparative approach regarding the contributions of individual factors and plasmids to the virulence of host E. coli in model systems. The family of large ColV plasmids could be analogous to other families of large virulence plasmids, and insights gained from studying these plasmids should contribute to our understanding of cross-genetic interactions and the role of large plasmids in bacterial pathogenesis.

Bacteriocin Plasmids↗

PubMed1990

A regulatory gene, angR, of the iron uptake system of Vibrio anguillarum: similarity with phage P22 cro and regulation by iron.

The angR locus in Vibrio anguillarum encodes a trans-acting transcriptional activator which modulates several Fe2(+)-regulated loci in the anguibactin biosynthesis gene cluster. In this paper, the complete nucleotide (nt) sequence of the angR gene and deduced amino acid (aa) sequence of the AngR protein are presented. A region upstream from the angR gene is shown to have similarity with Fe2(+)-regulated operators in Escherichia coli which bind the Fur protein. The involvement of a Fur-like regulator is supported by transcription analysis which show that angR itself is Fe2(+)-regulated. The aa sequence of the AngR protein predicts a helix-turn-helix motif which shows striking homology with prokaryotic DNA-binding proteins, particularly the lambda and P22 Cro proteins. In addition, there are two 18-nt regions, upstream from the angR gene, which show similarity with the OR1 and OR2 operators of P22 cro. These regions overlap with, respectively, the -35, -10 region and the putative Fur-binding region upstream from angR. These results suggest that AngR may be a DNA-binding protein which modulates Fe2(+)-regulated transcription and is itself Fe2(+)-regulated at the transcriptional level.

Amino Acid Sequence↗

PubMed1990

Dissemination of plasmid-mediated amikacin resistance among pathogenic Klebsiella pneumoniae.

Plasmids isolated from Klebsiella pneumoniae strains that caused outbreaks in pediatric units in various geographical regions of Argentina harbored genetic determinants for resistance to amikacin. By using restriction endonuclease and Southern blot hybridization analysis it was determined that all of the strains carried plasmids with Tn1331-related elements indicating that transposition of these elements may have played an important role in the dissemination process of resistance to amikacin.

Amikacin↗

PubMed1989

CoIV plasmids pCoIV-B188 and pCoIV-K30: genetic maps according to restriction enzyme sites and landmark phenotypic characteristics.

The CoIV plasmids are large virulence plasmids of the incompatibility group IncFI. We have obtained the genetic maps of two of the most studied CoIV plasmids, pCoIV-B188 and pCoIV-K30, according to restriction enzyme sites and landmark phenotypic characteristics such as colicin V, the aerobactin iron uptake system, the transfer region, replication regions, and repeated sequences. Although the two plasmids differ in size (pCoIV-B188 is 80 kb and pCoIV-K30 is 144 kb), the maps reveal many regions which are apparently identical or very similar.

Escherichia coli↗

PubMed1989

Regulation of the iron uptake system in Vibrio anguillarum: evidence for a cooperative effect between two transcriptional activators.

We have identified a 110-kDa polypeptide that has a trans-acting regulatory activity on the expression of the pJM1 plasmid iron-uptake genes in Vibrio anguillarum. This protein is encoded by the angR gene and maps in a 3.6-kilobase-pair pJM1 DNA region located downstream of the iron transport genes. Full expression of this gene occurs under iron-limiting conditions and requires a 2.9-kilobase-pair upstream region in cis that maps within the coding region of the OM2 outer membrane protein, essential for the transport of iron into the cell cytosol. Determination of the siderophore anguibactin levels as well as analysis of specific transcripts for anguibactin biosynthetic genes demonstrated that AngR and another transcriptional activator, Taf, regulate in a synergistic fashion the level of anguibactin production by activation of transcription of the anguibactin biosynthetic genes under iron-limiting conditions.

Bacterial Proteins↗

PubMed1989

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1.

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.

Antigens, Bacterial↗

PubMed1989

Nucleotide sequence analysis and expression of the minimum REPI replication region and incompatibility determinants of pColV-K30.

We sequenced the minimum REPI replication region and the incompatibility determinants of pColV-K30. The minimum replication region contains an open reading frame which corresponds to a 35-kilodalton (kDa) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis with maxicells transformed with a number of deletion derivatives demonstrated that this replication region encodes a 39-kDa protein and also established the direction of transcription of the RepI protein gene. The 39-kDa polypeptide was identified as the trans-acting factor essential for replication of REPI-containing plasmids. A translated region of the nucleotide sequence of the RepI protein gene showed homology with the helix-turn-helix binding domains of a number of DNA-binding proteins and also with other plasmid replication proteins. Further nucleotide analysis of the REPI region revealed the presence of direct and inverted repeat sequences in the incE, incF, and ori regions. The REPI ori also contained a perfect DnaA-binding site in addition to a high frequency of occurrence of the DNA adenine methylation (dam) site 5'GATC3'.

Amino Acid Sequence↗

PubMed1989

Genetics and molecular biology of siderophore-mediated iron transport in bacteria.

The possession of specialized iron transport systems may be crucial for bacteria to override the iron limitation imposed by the host or the environment. One of the most commonly found strategies evolved by microorganisms is the production of siderophores, low-molecular-weight iron chelators that have very high constants of association for their complexes with iron. Thus, siderophores act as extracellular solubilizing agents for iron from minerals or organic compounds, such as transferrin and lactoferrin in the host vertebrate, under conditions of iron limitation. Transport of iron into the cell cytosol is mediated by specific membrane receptor and transport systems which recognize the iron-siderophore complexes. In this review I have analyzed in detail three siderophore-mediated iron uptake systems: the plasmid-encoded anguibactin system of Vibrio anguillarum, the aerobactin-mediated iron assimilation system present in the pColV-K30 plasmid and in the chromosomes of many enteric bacteria, and the chromosomally encoded enterobactin iron uptake system, found in Escherichia coli, Shigella spp., Salmonella spp., and other members of the family Enterobacteriaceae. The siderophore systems encoded by Pseudomonas aeruginosa, namely, pyochelin and pyoverdin, as well as the siderophore amonabactin, specified by Aeromonas hydrophila, are also discussed. The potential role of siderophore-mediated systems as virulence determinants in the specific host-bacteria interaction leading to disease is also analyzed with respect to the influence of these systems in the expression of other factors, such as toxins, in the bacterial virulence repertoire.

Aeromonas↗

PubMed1988

Genetic and molecular characterization of essential components of the Vibrio anguillarum plasmid-mediated iron-transport system.

The iron-transport genes from the pJM1 plasmid of Vibrio anguillarum have been cloned and sequenced. Five open-reading frames have been identified, one of which encodes the outer membrane receptor for ferric anguibactin, OM2. This coding region corresponds to a protein of 726 amino acids with a Mr of 78,777. The protein has a hydrophobic signal sequence of 35 amino acids and a potential membrane-associated hydrophobic region at the carboxyl terminus. A 2.3-kilobase iron-regulated mRNA was transcribed from this region in vivo. The four other open-reading frames were shown to be involved in the regulation of OM2 expression and in iron transport by the use of insertion mutagenesis and complementation analysis. One of these open-reading frames, ORF3, encodes a 40-kDa polypeptide which, as deduced from the amino acid sequence and the hydropathy plot, is likely to be membrane-associated and together with OM2 may play a role in the transport of iron into the cell cytosol.

Amino Acid Sequence↗

PubMed1988

Multiple intensive care unit outbreak of Acinetobacter calcoaceticus subspecies anitratus respiratory infection and colonization associated with contaminated, reusable ventilator circuits and resuscitation bags.

PURPOSE: Acinetobacter calcoaceticus subspecies anitratus (A. anitratus) can cause nosocomially and community acquired pneumonia. Source identification of the organism is often difficult. An outbreak of respiratory infection and colonization with A. anitratus affecting 93 ventilated patients in all six of a hospital's intensive care units (ICUs) over 10 months is described. PATIENTS AND METHODS: In April 1984, the infection control staff started to review positive culture results from all patients in all ICUs. At this point, information on significant isolates was recorded by patient, site, date, genus and species, and antimicrobial susceptibility. During the month of August 1984, an increased number of A. anitratus isolates from sputum began to be detected. Information was expanded to include the date of hospital admission, ICU admission, intubation, and extubation; the dates and types of all surgical procedures; the results and dates of all prior sputum cultures; and the use of nebulized bronchodilator medications. Monthly numbers of cases were compared for four months prior to the outbreak, during the outbreak, and for seven months after the outbreak. Plasmid DNA from isolates was prepared, electrophoresed, and visualized. Isolates were designated according to the molecular weights of visualized plasmids. RESULTS: Barrier precautions and improved staff handwashing did not diminish the frequency of new cases. When pasteurized, reusable ventilator circuits and resuscitation bags were cultured for the possibility of low-level contamination, 18 percent were positive for A. anitratus. Terminal ethylene oxide sterilization of these devices was associated with prompt control of the outbreak. Plasmid DNA analysis of isolates from patients involved in the outbreak, contaminated devices, and the hands of personnel responsible for device disinfection revealed two predominant plasmid profiles. After outbreak control, isolates with these profiles were found much less frequently in patient specimens. CONCLUSION: Contaminated, reusable ventilator support equipment may be a leading cause for the extent of A. anitratus in the sputum of intubated patients. This problem is potentially correctable by the use of terminal etyhlene oxide sterilization of reusable ventilator circuits and resuscitation bags.

Acinetobacter↗

PubMed1988

Plasmid DNA analysis of Staphylococcus epidermidis isolated from blood and colonization cultures in very low birth weight neonates.

We prospectively studied the course of colonization and sepsis with Staphylococcus epidermidis among 29 very low birth weight neonates undergoing prolonged umbilical catheterization. S. epidermidis bacteremia occurred in 7 patients. In 6 bacteremia was preceded by positive colonization cultures. Isolates obtained from nares, base of umbilicus, umbilical catheter entry sites, catheter tips and blood were examined for plasmid DNA profiles. In 4 patients the plasmid profiles of the catheter entry site isolates were identical with those of the blood isolates. In the other 3 bacteremic patients plasmid profiles of the catheter entry site and blood isolates were different. No correlation was observed in the plasmid DNA patterns of isolates obtained from catheter tip cultures as compared to the corresponding blood cultures. The blood isolates from bacteremic patients had different plasmid profiles.

Catheterization↗

PubMed1988

Transposon-mediated amikacin resistance in Klebsiella pneumoniae.

A multiresistant Klebsiella pneumoniae strain isolated from neonates in Mendoza, Argentina, harbored a 48-kilobase-pair (kbp) plasmid, pMET1, with genetic determinants for resistance to amikacin and also ampicillin, kanamycin, streptomycin, and tobramycin. This plasmid was compared with pJHCMW1, a previously isolated 11-kbp plasmid carrying transposon Tn1331, which encodes resistance to amikacin, as well as ampicillin, kanamycin, streptomycin, and tobramycin, and which was originally present in a K. pneumoniae strain that caused an outbreak in a hospital in Buenos Aires, Argentina. The comparison demonstrated that the replication regions of the two plasmids are unrelated. However, in pMET1 an 11-kbp transposition element, Tn1331.2, was identified; it was closely related to Tn1331, with the difference that a 3-kbp BamHI DNA fragment carrying the aminoglycoside resistance genes was duplicated in tandem.

Amikacin↗

PubMed1988

Increased production of the siderophore anguibactin mediated by pJM1-like plasmids in Vibrio anguillarum.

The virulence of the fish pathogen Vibrio anguillarum 775 is mediated by the pJM1 plasmid-specified iron uptake system which is expressed under conditions of iron limitation. Other V. anguillarum strains isolated from various geographical locations harbor plasmids that are highly related to pJM1 and that are also associated with the high-virulence phenotype of these strains. In this work, we found that a pJM1-like plasmid, pJHC1, from one of these virulent strains encoded an iron uptake system that resulted in an increased level of production of the siderophore anguibactin. The gene(s) responsible for increased anguibactin production was included within the iron uptake region of plasmid pJHC1. The cloned iron uptake regions of pJHC1 and pJM1 possessed identical restriction endonuclease maps, suggesting that the DNA region encoding those genes in pJHC1 may have diverged subtly from that in pJM1. Analysis of the iron uptake system from other V. anguillarum strains carrying pJM1-like plasmids demonstrated that strains originating from diseased fish from the Atlantic coast carry plasmids encoding an increased-siderophore-production phenotype, while strains isolated from Pacific Ocean locations behaved as the 775 strain.

Cloning, Molecular↗

PubMed1988

Divergence of the aerobactin iron uptake systems encoded by plasmids pColV-K30 in Escherichia coli K-12 and pSMN1 in Aerobacter aerogenes 62-1.

Although the aerobactin-mediated iron uptake system has been characterized genetically in Escherichia coli, the siderophore aerobactin was chemically characterized after purification from culture supernatants of Aerobacter aerogenes 62-1, a member of the Klebsielleae. We have cloned and mapped the genes encoding the aerobactin system genes of A. aerogenes 62-1 and begun characterization of the relevant proteins and enzymatic activities of this plasmid-mediated aerobactin system. Published chemical data indicate that the siderophore aerobactin of E. coli is the same molecule as the aerobactin of Aerobacter aerogenes 62-1, but we have found that both the genes and the complement of proteins making up the biosynthetic enzymes in the two systems have diverged. In contrast, the outer membrane receptors for ferric aerobactin of the two systems showed immunologic cross-reactivity, were of the same molecular size (74 kilodaltons), and were encoded by homologous DNA sequences.

Cloning, Molecular↗

PubMed1988

Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E. coli K1.

We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187. Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30. However, in the E. coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used. RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E. coli K-12. In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30. Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems. DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.

Base Sequence↗

PubMed1988

New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae.

Unlike the great majority of the aerobactin-producing enteric bacteria documented in the literature, Enterobacter cloacae EK33, isolated from a case of human neonatal meningitis, did not show any homology at the DNA level with the prototype aerobactin system encoded by the ColV-K30 plasmid. However, both the nuclear magnetic resonance spectrum and fast-atom bombardment mass spectrometry of the siderophore purified from EK33 confirmed its identity with aerobactin. Bioassay screening of a gene library of total DNA of EK33 led to the isolation of several aerobactin-positive clones. Under conditions of iron limitation, these clones expressed in Escherichia coli a protein of 72 kilodaltons that reacted with antiserum raised against the pColV-K30 74-kilodalton aerobactin receptor, while the original E. cloacae strain synthesized an 85-kilodalton protein which also cross-reacted with the antiserum. Restriction endonuclease analysis of the cloned DNA confirmed the structural differences between the two aerobactin genetic systems.

Conjugation, Genetic↗