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J H Crosa

Publications and source records attributed to J H Crosa.

At least 73 records · Page 4Linked to original sources
PubMed1988

Genetic analysis of the iron uptake region of the Vibrio anguillarum plasmid pJM1: molecular cloning of genetic determinants encoding a novel trans activator of siderophore biosynthesis.

Clones carrying the iron uptake region of the Vibrio anguillarum plasmid pJM1 were subjected to insertion mutagenesis, using transposon Tn3::HoHo1 which carries a promoterless lacZ gene and can thus generate lacZ transcriptional fusions if inserted downstream from an indigenous promoter. Four classes of insertion mutants were obtained based on the level of expression of components of the iron uptake system, and six genetic units were defined according to the phenotype of the mutants. Five of the six genetic units were crucial for biosynthesis of the siderophore anguibactin. Insertions in the remaining genetic unit led to an iron uptake-deficient phenotype and showed either reduced levels of the outer membrane protein OM2 as well as anguibactin activity or a complet shutoff of both OM2 and anguibactin biosyntheses. Analysis of beta-galactosidase production by cells carrying the lacZ fusion derivatives identified iron-regulated and constitutive transcriptional units as well as their orientation in the genetic units. Molecular cloning of pJM1 plasmid DNA noncontiguous to the iron uptake region also identified genetic determinants for a trans-acting factor required for full expression of anguibactin activity. Evidence obtained from bioassays, spectrophotometric measurements, and the lacZ fusion mutants suggested that the trans-acting factor is a novel activator of siderophore biosynthesis at the transcriptional level.

Cloning, Molecular↗
PubMed1988

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.

Animals↗
PubMed1988

Sequencing and expression of the 6'-N-acetyltransferase gene of transposon Tn1331 from Klebsiella pneumoniae.

Plasmid-mediated amikacin resistance in Klebsiella pneumoniae resides on a 1.5-kilobase BamHI fragment which is part of the Tn3-related multiresistance transposon Tn1331. In this work, we present the complete nucleotide sequence of the amikacin resistance gene and the neighboring sequences. Maxicell experiments detected only one polypeptide of 23 kilodaltons, the product of one of the open reading frames identified as ORF I. Comparison of the complete sequence with that of Tn3 indicated that 396 base pairs located just upstream from ORF I are identical to a region between the end of the tnpR gene and the first six amino acids of the beta-lactamase transcript. Sequences which may act as hot spots for recombination were identified. One was located just after amino acid 6 of beta-lactamase, and the other was located at the end of the amikacin resistance gene.

Acetyltransferases↗
PubMed1987

Flanking and internal regions of chromosomal genes mediating aerobactin iron uptake systems in enteroinvasive Escherichia coli and Shigella flexneri.

We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.

Bacterial Outer Membrane Proteins↗
PubMed1987

Tn1331, a novel multiresistance transposon encoding resistance to amikacin and ampicillin in Klebsiella pneumoniae.

A 7.5-kilobase-pair multiresistance transposon, Tn1331, harboring amikacin resistance was identified as part of Klebsiella pneumoniae plasmid pJHCMW1. Restriction mapping, hybridization, and transposition complementation experiments demonstrated that Tn1331 belongs to the Tn3 family. Its structure is similar to that of Tn3 with the insertion of a DNA fragment encoding resistance to amikacin, kanamycin, and tobramycin.

Amikacin↗
PubMed1987

Novel incompatibility and partition loci for the REPI replication region of plasmid ColV-K30.

The minimum pColV-K30 REPI region necessary for replication was located within a ca. 1.3-kilobase DNA segment. Adjacent to the essential replication sequences, there are two DNA regions that express incompatibility with plasmids containing the F secondary replicon of the F EcoRI fragment f7. One of these regions corresponds to incE, already described in that F plasmid fragment which expresses incompatibility with f7-containing plasmids. The other is a novel sequence that we designated incF, which confers incompatibility with REPI, P307, and f7 derivatives, cis-acting pColV-K30 sequences conferring stability to REPI-containing plasmids were also identified and localized noncontiguous to REPI, ca. 20 kilobases downstream from the aerobactin iron transport genes, which were thus flanked by REPI and its partition (par) sequences.

Cloning, Molecular↗
PubMed1987

Antimicrobic susceptibility and plasmid profile analysis as identity tests for multiple blood isolates of coagulase-negative staphylococci.

We compared a disk diffusion antimicrobic susceptibility panel with plasmid DNA profiles as tests for identity of 106 isolates of coagulase-negative staphylococci cultured from the blood of 45 patients on multiple occasions. The antimicrobic panel included penicillin, oxacillin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, tobramycin, kanamycin, and gentamicin. Nineteen patterns of antimicrobic susceptibility were found. The most common pattern was present in 25% of the isolates, and at least one isolate from 31% of the patients had this pattern. Forty-seven distinct plasmid DNA profiles were found. The most common plasmid profile was present in 8.5% of the isolates, and at least one isolate from 15% of the patients had this profile. Twenty-eight patients had multiple isolates that were identical by plasmid profile analysis. Twenty-seven (96%) of these patients had isolates that were also identical by antimicrobic susceptibility. Nineteen patients had multiple isolates that were different by plasmid profile analysis. In 18 (95%) of these patients, the isolates were also different by antimicrobic susceptibility. Although plasmid DNA profile analysis is a more discriminating tool, these data confirm that a selected disk diffusion antimicrobic susceptibility panel may be used to screen multiple blood isolates of coagulase-negative staphylococci for identity or differences.

Anti-Bacterial Agents↗
PubMed1986

The characterization of a conjugative R-plasmid isolated from Aeromonas salmonicida.

We have analyzed two related R-plasmids isolated from Aeromonas salmonicida: pAr32 and pJA8102-1, which encode resistance to chloramphenicol, streptomycin, and sulfonamides (Su). The genetic map of pJA8102-1 indicated that this plasmid consists of at least three functionally different regions. One is the region conferring ability to transfer (tra), one is the region coding for drug resistance genes (r-det), and one is the region encoding the replication functions (rep). Both plasmids contain repeated sequences (RS); pJA8102-1 has four copies of this RS, while pAr-32 has three copies. The RS sequence of pJA8102-1 are located adjacent to each of the drug-resistance genes. These sequences might have been responsible for the duplication of the Su-resistance gene on this plasmid.

Aeromonas↗
PubMed1986

Evidence for the role of a siderophore in promoting Vibrio anguillarum infections.

Vibrio anguillarum strain 775 harbouring the virulence plasmid pJM1 produces a plasmid-mediated siderophore that can crossfeed siderophore-deficient, receptor-proficient mutants of V. anguillarum in vitro. Experimental infections of salmonid fishes with mixtures consisting of the wild-type strain and a siderophore-deficient, receptor-proficient mutant resulted in recovery of both the wild-type and the mutant strain, while in infections with mixtures consisting of the wild-type strain and a siderophore-deficient, receptor-deficient mutant only the wild-type strain could be recovered. These results suggest that the V. anguillarum plasmid-mediated siderophore is produced in vivo in a diffusible form and that it is an important factor of virulence.

Animals↗
PubMed1986

Plasmid-encoded amikacin resistance in multiresistant strains of Klebsiella pneumoniae isolated from neonates with meningitis.

Two multiresistant Klebsiella pneumoniae strains isolated from cerebrospinal fluid of human neonates were analyzed for their plasmid content. Two of the plasmids harbored by these strains, pJHCMW1 (11 kilobase pairs) and pJHCMW4 (75 kilobase pairs), carried genetic determinants for amikacin resistance. These plasmids also encoded resistance to kanamycin, tobramycin, and ampicillin which could be transferred to Escherichia coli by conjugation. Extracts from transconjugant derivatives carrying pJHCMW4 produced an acetyltransferase activity that acetylated all three aminoglycosides. Transconjugant derivatives carrying pJHCMW1 encoded both acetylating and phosphorylating activities. Southern blot hybridization analysis indicated considerable DNA homology between these two plasmids.

Acetyltransferases↗
PubMed1986

Molecular cloning of amikacin resistance determinants from a Klebsiella pneumoniae plasmid.

A multiresistant Klebsiella pneumoniae strain, JHCK1, harbored several plasmids. One of these, plasmid pJHCMW1, carried determinants for resistance to amikacin in addition to kanamycin, tobramycin, and ampicillin. The amikacin resistance determinant(s) was cloned and studied by restriction mapping, insertion, and deletion analysis. The amikacin resistance gene(s) was localized in a 1.5-kilobase DNA fragment. This pJHCMW1 DNA region was responsible for not only the resistance to amikacin but also the resistance to kanamycin and tobramycin. The cloned DNA fragment specified both an acetyltransferase activity and a low level of phosphotransferase activity. The two activities were absent from mutants that did not confer resistance to amikacin, kanamycin, and tobramycin.

Acetyltransferases↗
PubMed1986

Occurrence of chromosome- or plasmid-mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichia coli K1.

The incidence of the aerobactin system and the genetic location of aerobactin genes were investigated in Escherichia coli K1 neonatal isolates belonging to different clonal groups. A functional aerobactin system was found in all members of the O7 MP3, O1 MP5, O1 MP9, and O18 MP9 clonal groups examined and also in K1 strains having O6, O16, and O75 lipopolysaccharide types, which are less frequently associated with neonatal infections. In contrast, the aerobactin system was not detected in strains from the O18 MP6 clone. The combined results of plasmid and colony hybridization experiments showed that the aerobactin genes were located on the chromosome in the majority (75%) of the aerobactin-producing K1 isolates, the genetic location of the aerobactin genes was closely correlated with the outer membrane protein profile rather than the O lipopolysaccharide type, the K1 strains harboring a chromosome-mediated aerobactin system did not possess colicin V genes, and five of six K1 isolates possessing a plasmid-borne aerobactin system contained colicin V genes which were located on the same plasmids carrying the aerobactin genes. The comparison of hemolysin production with possession of the aerobactin system in virulent clones of E. coli K1 strains showed that all of the aerobactin-producing strains from the O18 MP9 and O7 MP3 clonal groups did not synthesize hemolysin, whereas 11 of 12 aerobactin-nonproducing O18 MP6 isolates were hemolytic. Of the K1 strains examined, 92.5% possessed either the aerobactin system or the ability to produce hemolysin or both.

Bacterial Outer Membrane Proteins↗
PubMed1986

Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1).

Anguibactin, a siderophore produced by cells of Vibrio anguillarum 775 harboring the pJM1 plasmid, has now been isolated from the supernatants of iron-deficient cultures. This iron-reactive material was purified by adsorption onto an XAD-7 resin and subsequent gel filtration on a Sephadex LH-20 column. The resulting neutral compound produced an ion at m/z 348 in mass spectrometry and contained one sulfur, four oxygen, and four nitrogen atoms as determined by elemental analysis. Its strong UV absorbance and blue fluorescence were suggestive of a phenolic moiety. In colorimetric reactions anguibactin behaved like a catechol. The catechol assignment was supported by the appearance of a new absorption band at 510 nm in the ferric complex and by the appearance of peaks at 1,367, 1,447, 1,469, and 1,538 cm-1 in the resonance Raman spectrum. In addition, the infrared spectrum gave evidence of a secondary amide function, but no free carboxylic acid or hydroxamic acid groups were observed. A third iron-ligating group was suggested by the liberation of three protons during iron binding; mass spectrometry of the resulting material yielded a molecular ion characteristic of a 1:1 complex of ferric anguibactin. The purified anguibactin exhibited specific growth-promoting activity under iron-limiting conditions for a siderophore-deficient mutant of V. anguillarum 775(pJM1). A novel structure for anguibactin was indicated by the failure of a large number of known siderophores and synthetic chelators to yield a similar type of specific cross-feeding in the V. anguillarum bioassay.

Bacterial Outer Membrane Proteins↗
PubMed1986

DNA environment of the aerobactin iron uptake system genes in prototypic ColV plasmids.

The aerobactin iron uptake system genes in the prototypic plasmid pColV-K30 are flanked by inverted copies of insertion sequence IS1 and by two distinct replication regions. To address the question of how these flanking regions may facilitate the maintenance and spread of the aerobactin system among the plasmids and chromosomes of enteric species, we investigated the DNA environment of 12 ColV plasmids. We found that the aerobactin system-specific genes are conserved in every plasmid phenotypically positive for the aerobactin system. The upstream IS1 and its overlapping replication region (REPI) are also conserved. This replication region was cloned from several ColV plasmids and found to be functional by transforming these cloned derivatives into a polA bacterial host. In contrast, the downstream flanking region is variable. This includes the downstream copy of IS1 and the downstream replication region (REPII). We infer from these results that sequences in addition to the two flanking copies of IS1, in particular the upstream region including REPI, have been instrumental in the preservation and possible spread of aerobactin genes among ColV plasmids and other members of the FI incompatibility group.

Bacteriocin Plasmids↗
PubMed1985

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain.

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.

Animals↗
PubMed1985

Molecular characterization of chloramphenicol-resistant Haemophilus parainfluenzae and Haemophilus ducreyi.

We examined chloramphenicol-resistant Haemophilus parainfluenzae and Haemophilus ducreyi strains isolated in various parts of the world. The antibiotic resistance determinants were located on conjugative plasmids in H. ducreyi, but were chromosomally located in H. parainfluenzae. Both species produced chloramphenicol acetyltransferases (CATs) that were sensitive to 5,5'-dithiobis(2-nitrobenzoic acid) like the enteric type II and Haemophilus influenzae CAT enzymes, but differed from these enzymes in elution patterns and subunit molecular weight. Southern blot analysis showed the H. parainfluenzae and H. ducreyi CAT genes were molecularly related to the enteric type II class as well as the H. influenzae CAT. Heterogeneity of the physiochemical properties of the CATs was observed; however, the data suggested that all three Haemophilus spp. have a common ancestral source for the CATs.

Acetyltransferases↗
PubMed1985

Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid pJM1.

Vibrio anguillarum 775 harboring the virulence plasmid pJM1 synthesized an outer membrane protein of 86 kilodaltons, OM2, that was inducible under conditions of iron limitation. pJM1 DNA fragments obtained by digestion with restriction endonucleases were cloned into cosmid vectors and transferred into Escherichia coli. The OM2 protein was synthesized in E. coli, demonstrating that it is actually encoded by the pJM1 plasmid. Mobilization of the recombinant plasmids to V. anguillarum was accomplished by using the transfer factor pRK2013. A V. anguillarum exconjugant harboring the recombinant derivative pJHC-T7 and synthesizing the OM2 protein took up 55Fe3+ and grew under iron-limiting conditions, only in presence of the pJM1-mediated siderophore. Exconjugants harboring recombinant plasmids, such as pJHC-T2 which did not encode the OM2 protein, were transport negative. Membrane protein iodination experiments, together with protease treatment of whole cells, indicated that the OM2 protein is exposed to the outside environment of the V. anguillarum cells.

Bacterial Outer Membrane Proteins↗