Search

Alanine-scanning mutagenesis was applied to the aminoglycoside 6'-N-acetyltransferase type Ib conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42 degrees C (D. Panaite and M. Tolmasky, Plasmid 39:123-133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.

Aminoglycosides (Ags) are a group of antibiotics that exert their bactericidal activity primarily by inhibition of protein synthesis. Aminoglycoside (Ag) molecules bind to the bacterial 30S ribosomal subunit rendering the ribosomes unavailable for translation, which results in cell death. Although these antibiotics are and have been very useful to treat a variety of bacterial infections, in recent years the number of Ag resistant and multiresistant isolates has seriously increased. Mechanisms of resistance to Ag include enzymatic inactivation by acetyltransferases, nucleotidyltransferases (adenylyltransferases), and phosphotransferases, ribosomal alterations, and reduced permeability. Of all Ags, amikacin (Ak) is the most resistant to the action of Ag-modifying enzymes. However, AAC(6')-I type enzymes (a group of 6'-N-acetyltransferases) can utilize Ak as substrate and confer resistance to this antibiotic in addition to other Ags. The gene aac(6')-Ib was found in various bacterial species and various research groups performed mutagenesis studies on this or related enzymes. In one case, aac(6')-Ib was identified in a transposable element, Tn1331, included in pJHCMW1, a plasmid isolated from a clinical K. pneumoniae strain. Tn1331 includes genes encoding two Ag-modifying enzymes (aac(6')-Ib and ant(3")-Ia) and two beta-lactamases (blaTEM and blaOXA-9). Characterization of other functions of the pJHCMW1 plasmid showed the presence of an RNA-regulated replication origin and a functional oriT. Stability by multimer resolution is achieved by the Tn1331 resolvase.

Substitutions at position F171 of 6'-N-acetyltransferase type Ib cause variable loss of aminoglycoside resistance, indicating that this residue plays an important role in the structure and/or function of the enzyme.

Plasmids are self-replicating extrachromosomal DNA molecules found in Gram-negative and Gram-positive bacteria as well as in some yeast and other fungi. Although most of them are covalently closed circular double-stranded DNA molecules, recently linear plasmids have been isolated from different bacteria. In general, plasmids are not essential for the survival of bacteria, but they may nevertheless encode a wide variety of genetic determinants, which permit their bacterial hosts to survive better in an adverse environment or to compete better with other microorganisms occupying the same ecological niche. The medical importance of plasmids that encode for antibiotic resistance, as well as specific virulence traits has been well documented and demonstrated the important role these bacterial genetic elements play in nature. Although they encode specific molecules required for initiation of their replication, plasmids rely on host-encoded factors for their replication. Plasmid replication initiates in a predetermined cis-site called ori and can proceed either by a rolling circle or a theta replication mechanism. Some of the plasmid-encoded elements required for their replication, such antisense RNA molecules and DNA repeated sequences located close to ori, determine plasmid attributes like copy number and incompatibility.

The enzyme OXA-9, an oxacillinase-carbenicillinase, is encoded by the blaOXA-9 gene which was originally found within the structure of Tn1331, a multiresistance transposon first isolated from a clinical Klebsiella pneumoniae strain. Studies to characterize OXA-9 demonstrated that it has a pI of 6.9 and the optimal pH for enzyme activity was between 7.7 and 8.2. When total soluble extracts were preincubated at 37 degrees C and at 42 degrees C, enzyme activity decayed to approximately 56% of the original value after 6 hrs. at 37 degrees C and to 50% after 30 min. at 42 degrees C. Enzymatic activity of OXA-9 was inhibited in the presence of p-chloromercuribenzoate, cloxacillin and clavulanic acid, but not by 200 mM sodium chloride. The inhibition by p-chloromercuribenzoate may indicate the presence of a cysteine residue playing a role in the catalytic activity of the enzyme. The OXA-9 enzyme was released by osmotic shock of E. coli cells harboring a recombinant clone including the blaOXA-9 gene indicating that it is located in the periplasmic space of the cells. The OXA-9 enzyme was purified from soluble protein extracts of E. coli cells carrying a recombinant clone including the blaOXA-9 by ion exchange chromatography.

The Klebsiella pneumoniae plasmid pJHCMW1 harbors a copy of Tn1331, a multiresistance transposon that includes the aac(6')-Ib gene which encodes a 6'-N-aminoglycoside acetyltransferase. This gene was mutagenized using the mutator Escherichia coli XL1-Red. Two plasmids with a single nucleotide mutation in aac(6')-Ib were selected for further analysis: pDP1 and pDP6. Plasmid pDP1 codes for a mutant enzyme, AAC(6')-IbDP1, that has the Phe171 replaced by a Leu residue. This mutant derivative showed a lower specific activity than the wild-type enzyme when either kanamycin (Km) or its semisynthetic derivative amikacin (Ak) were used as substrates in enzymatic assays performed at 30 degrees C. Furthermore, AAC(6')-IbDP1 showed a change of specificity of substrate when incubated at 42 degrees C. While its acetylating activity for Km was higher at this temperature than at 30 degrees C, it had its ability to utilize Ak as substrate for acetylation considerably reduced. Accordingly, minimal inhibitory concentration assays showed that E. coli(pDP1) was resistant to Ak at 37 degrees C but susceptible at 42 degrees C. The same assays showed that E. coli(pDP1) was highly resistant to Km at either 37 degrees C or 42 degrees C. A high level of resistance to Ak was observed for E. coli(pJHCMW1) which harbors the wild-type AAC(6')-Ib at either 37 or 42 degrees C. Extension of the analyses to other aminoglycosides showed that the enzymatic activity of AAC(6')-IbDP1 as well as the E. coli(pDP1) MICs for netilmicin dropped at 42 degrees C as was the case for Ak. These results could indicate that at 42 degrees C the mutant adopts a conformation that makes it unable to efficiently acetylate aminoglycoside molecules substituted in the C-1amino group of the deoxystreptamine moiety. Plasmid pDP6 encodes the mutant AAC(6')-IbDP6 which has the Tyr80 substituted by a Cys residue. E. coli(pDP6) exhibited reduced MICs for Ak, Km, tobramycin, and netilmicin. Analysis of the acetylating activity of AAC(6')-IbDP6 showed only marginal levels of activity when either Ak, Km, tobramycin, or netilmicin were used as substrates.

A 2.4-kb EcoRI fragment including the replication and origin of transfer regions of the Klebsiella pneumoniae multiresistance plasmid pJHCMW1 has been cloned and sequenced. The isolated replication region was sufficient for stable maintenance of the plasmid and shares homology with RNA-regulated replicons. Homology was highest with the replication region of the plasmid p15A. Incompatibility experiments, however, determined that pJHCMW1 is compatible with pACYC177, a plasmid harboring the p15A replicon. Differences in their RNA I nucleotide sequences may account for their compatibility. A mobilization origin was also found in the 2.4-kb EcoRI pJHCMW1 DNA fragment analyzed. Conjugation experiments showed that although non-self-transmissible, the recombinant clone including the 2.4-kb EcoRI pJHCMW1 fragment could be mobilized in the presence of the helper plasmid pRK2073.

Vibrio anguillarum virulence is associated with the presence of a plasmid-mediated iron-uptake system expressed under iron-limiting conditions, which consists of the siderophore anguibactin and specific iron-transport proteins. This system is maximally expressed under iron-limiting conditions and requires the AngR protein that acts as a positive regulator of anguibactin biosynthesis and also possess an EntE-like enzymatic function that may play a role in anguibactin biosynthesis. In this work, we demonstrate that in addition to possessing these functions related to anguibactin production, AngR also positively regulated transcription of the iron-transport genes fatA and fatB. We also show that transcription of angR is repressed by Fur under iron-rich conditions. In addition, we present evidence that anguibactin itself enhanced transcription of the iron-transport genes fatA and fatB, independently of AngR and the trans-acting factor (TAF) product(s). The presence of either AngR (together with the TAF product(s)) or anguibactin alone led to a partial level of expression of the iron-transport genes fatA and fatB, while full expression is achieved when AngR, the TAF products and anguibactin are all present.

The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa proFatB precursor protein.

The pJM1 genes encoding the proteins involved in iron transport in the anguibactin iron uptake system were found to be flanked by insertion sequences in a composite transposonlike structure. These Vibrio anguillarum insertion sequences, ISV-A1 and ISV-A2, are related to IS903, IS102, and the ISVs found in Vibrio parahaemoliticus, Vibrio mimicus, and non-O1 Vibrio cholerae flanking various tdh (thermostable direct hemolysin) genes. The inverted repeats at the ends of ISV-A1 and ISV-A2 have no more than three mismatches when compared to the inverted repeats of the other ISVs or IS903 and IS102. ISV-A1 and ISV-A2 are flanked by 9-bp direct repeats, which is the number of bases that are duplicated upon IS903 or IS102 transposition. The similarities found between the V. anguillarum ISVs and the other ISVs as well as IS903 and IS102 suggest that they derive from a common ancestral insertion sequence. At the end of ISV-A1 there is a -35 sequence region followed by a -10 sequence found in the pJM1 sequence immediately outside the ISV. This promoter region is followed by an open reading frame with the potential to encode a polypeptide of 26,985 Da whose function is still unknown. The functionality of this promoter has been demonstrated and expression analysis showed that the promoter is regulated by the iron concentration of the media.

We have identified and sequenced an hdc gene in the Vibrio anguillarum plasmid pJM1 which encodes a histidine decarboxylase enzyme and is an essential component for the biosynthesis of anguibactin. The open reading frame corresponds to a protein of 386 amino acids with a calculated molecular mass of 44,259.69 Da. The amino acid sequence has extensive homology with the pyridoxal-P-dependent histidine decarboxylases of Morganella morganii, Klebsiella planticola, and Enterobacter aerogenes. Tn3-HoHo1 transposition mutagenesis of the hdc gene present in a recombinant clone carrying the entire pJM1 iron uptake region produced two derivatives, one with the lacZ gene in the same orientation as the direction of hdc transcription and the other with the lacZ gene in the opposite orientation. A. V. anguillarum strain harbouring one of the mutated derivatives was unable to grow under iron-limiting conditions and did not produce anguibactin. Therefore, the hdc gene must play a role in the biosynthetic pathway of this siderophore and consequently in conferring the high virulence phenotype to this bacterium. The role of histidine decarboxylase in biosynthesis of anguibactin was confirmed by the fact that growth under iron starvation was restored by addition of histamine to the medium. The presence of anguibactin was also demonstrated in supernatants from cultures of the hdc mutant strains grown under iron starvation with the addition of histamine, further confirming that histamine is a precursor in the biosynthesis of the siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of the 86-kDa FatA outer membrane protein is repressed under iron-rich conditions. Complementation of transposition mutants derived from clones containing the pJM1 iron uptake region revealed the existence of an antisense RNA, RNA alpha. This RNA is only expressed under iron-rich conditions and acts as a negative regulator of FatA synthesis, with slight but discernible decrease in the steady-state level of fatA mRNA determined by RNase protection and by Northern blot analysis. Primer extension experiments revealed that the level of several possible fatA transcripts was reduced in the presence of RNA alpha. In addition, we found that fatA mRNA expression is slightly reduced in the presence of Escherichia coli Fur. We have identified and cloned a chromosomally encoded fur-like gene in Vibrio anguillarum.

The multiresistance transposon Tn1331 has two 520 bp direct repeats flanking a DNA region similar to the variable portion of the integrons. This region harbors three antibiotic resistance genes, aac(6')-Ib, aadA, and a beta-lactamase gene which encodes an enzyme that was characterized as an oxacillinase-carbenicillinase and was named OXA-9. Within the direct repeat located upstream of these genes there is a copy of the TEM beta-lactamase promoter. Determination of ampicillin resistance levels of Escherichia coli harboring various recombinant clones and an insertion mutant showed that all three genes are transcribed from this promoter in a polycistronic mRNA. These results also indicated that the oxa9 gene possesses another functional promoter, located immediately upstream of this gene. The transcription start site of the mRNA driven by this promoter was identified by primer extension. Within the DNA sequence harboring all three antibiotic resistance genes it is possible to distinguish insert units as defined before in the variable region of the integrons (R. Hall, D. Brookes, and H. W. Stokes (1991) Mol. Microbiol. 5, 1941-1959).

We have cloned and sequenced the recA gene from two strains, 775 and 531A, of the fish pathogen, Vibrio anguillarum. Although both strains showed different sensitivities to methyl methanesulfonate (MMS), the recA genes were identical. In vitro expression of the V. anguillarum recA gene produced a polypeptide of about 40 kDa, in agreement with the value obtained from the nucleotide sequence. We identified the transcription start point by primer extension. The promoter for the recA gene mapped to an SOS regulatory element. The presence of an SOS box suggests that a LexA-like mediated response system may exist in V. anguillarum. The deduced RecA amino acid sequence is highly homologous with Escherichia coli RecA and other RecA proteins. Domains important in RecA function are conserved. We provide a comparative analysis of the activities and features of RecA analogs from a variety of species. We observed that certain residues that could be important in protein conformation are conserved in RecA proteins across a diverse range of bacterial species.

Complementation of insertion mutants showed that the polypeptides FatD, FatC, FatB, and FatA are essential for the iron-transport process encoded by pJM1. Sequence analysis followed by homology studies indicated that transport of ferric anguibactin into Vibrio anguillarum 775 follows the same mechanism as reported for transport of Fe(3+)-hydroxamates, Fe(3+)-catecholates, ferric dicitrate, and vitamin B12 into Escherichia coli. Homology of FatA, part of the receptor complex, to seven E. coli receptor proteins involved in uptake of siderophores and vitamin B12 supports the idea of a common ancestral gene. A "TonB-Box" was found in FatA suggesting the existence of a TonB-like protein function in V. anguillarum. A high homology in the primary structure of FatB to FhuD, FecB, FepB, and BtuE suggests that FatB is the anguibactin-binding protein located in the periplasmic space. FatD and FatC are polytopic integral membrane proteins. According to their homologies to other proteins from other transport systems, they may be involved in the translocation of ferric anguibactin across the cytoplasmic membrane.

The structural gene (hsd) of the Pseudomonas testosteroni encoding the 17 beta-hydroxysteroid dehydrogenase has been cloned using the cosmid vector pVK102. Escherichia coli carrying recombinant clones of hsd, isolated by immunological screening, were able to express the biologically active enzyme, as measured by the conversion of testosterone into androstenedione. Subcloning experiments, restriction and deletion analysis, and site-directed insertion mutagenesis showed that the hsd gene is located within a 1.3-kb HindIII-PstI restriction fragment. A 26.5-kDa protein encoded by a recombinant plasmid containing this Ps. testosteroni DNA restriction fragment was detected by SDS-PAGE analysis of in vitro [35S]methionine-labeled polypeptides.

Iron is essential for bacterial growth and metabolism. In vertebrates this metal is complexed by high-affinity iron-binding proteins, such as transferrin in serum. The fish pathogen Vibrio anguillarum possesses a very efficient iron-uptake system which is encoded in the virulence plasmid pJM1. This allows the bacterium to utilize the otherwise unavailable iron in the fish host, resulting in the septicemic disease vibriosis. This system includes the siderophore anguibactin and transport components. We have cloned this iron-uptake system and have defined several genetic units by transposition mutagenesis. Nucleotide sequence analysis identified four open reading frames in the transport region, one of these corresponding to the gene for the outer membrane protein OM2 and another to a 40-kDa polypeptide. Complementation analysis indicated that products from all four reading frames are required for the transport of iron-anguibactin complexes. We have also identified positive and negative-acting regulatory elements that modulate in concert the expression of anguibactin biosynthetic genes and iron transport. The deletion or mutation of the positive-acting regulatory genes results in an iron-uptake-deficient phenotype and leads to an attenuation of virulence, underscoring the importance of this iron-uptake system as a virulence attribute of V. anguillarum.

A fragment of Tn1331 including tnpR, aac, aadA, and a bla gene which encodes lower levels of resistance to ampicillin and carbenicillin as compared to those mediated by the TEM beta-lactamase was sequenced. The polypeptide encoded by the bla gene has homology with the OXA-1, PSE-2, and OXA-2 proteins. Genes aac and bla are upstream and downstream respectively of aadA, and are both flanked by recombinational hot spots. Tn1331 has 520-bp direct repeats which include parts of the tnpR and TEM bla genes. Evolutionary models for the genesis of Tn1331 are proposed.

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.