Interplay of OpdP Porin and Chromosomal Carbapenemases in the Determination of Carbapenem Resistance/Susceptibility in Pseudomonas aeruginosa.

Carbapenem resistance in Pseudomonas aeruginosa strains responsible for chronic lung infections in cystic fibrosis (CF) patients is mainly due to loss of the OprD protein and, limited to meropenem and doripenem, to overexpression of efflux pumps. However, recent reports of isolates showing inconsistent genotype-phenotype combinations (e.g., susceptibility in the presence of resistance determinants and vice versa) suggest the involvement of additional factors whose role is not yet fully elucidated. Among them, the OpdP porin as an alternative route of entry for carbapenems other than OprD and the overexpression of two chromosomal carbapenemases, the Pseudomonas-derived cephalosporinase (PDC) and the PoxB oxacillinase, have recently been reconsidered and studied in specific model strains. Here, the contribution of these factors was investigated by comparing different phenotypic variants of three strains collected from the sputum of colonized CF patients. Carbapenem uptake through OpdP was investigated both at the functional level, by assessing the competition exerted by glycine-glutamate, the OpdP's natural substrate, against imipenem uptake, and at the molecular level, by comparing the expression levels of opdP genes by quantitative real-time PCR (qRT-PCR). Moreover, overexpression of the chromosomal carbapenemases in some of the isolates was also investigated by qRT-PCR. The results showed that, even if OprD inactivation remains the most important determinant of carbapenem resistance in strains infecting the CF lung, the interplay of other determinants might have a nonnegligible impact on bacterial susceptibility, being able to modify the phenotype of part of the population and consequently complicating the choice of an appropriate therapy. IMPORTANCE This study examines the interplay of multiple factors in determining a pattern of resistance or susceptibility to carbapenems in clinical isolates of Pseudomonas aeruginosa, focusing on the role of previously poorly understood determinants. In particular, the impact of carbapenem permeability through OprD and OpdP porins was analyzed, as well as the activity of the chromosomal carbapenemases AmpC and PoxB, going beyond the simple identification of resistance determinants encoded by each isolate. Indeed, analysis of the expression levels of these determinants provides a new approach to determine the contribution of each factor, both individually and in coexistence with the other factors. The study contributes to understanding some phenotype-genotype discordances closely related to the heteroresistance frequently detected in P. aeruginosa isolates responsible for pulmonary infections in cystic fibrosis patients, which complicates the choice of an appropriate patient-specific therapy.

blaOXA -731 , a new chromosome-encoded blaOXA -48 -like variant in Shewanella sp. from the aquatic environment in Myanmar.

Shewanella sp., the progenitors of blaOXA-48 -like genes are increasingly reported with the possession of different blaOXA-48 -like variants. This study aims to characterize blaOXA-731 , a new variant of a blaOXA-48 -like gene identified in Shewanella sp. isolated from the aquatic environment in Myanmar. Phylogenetic analysis of the blaOXA-731 sequence with other blaOXA-48 -like variants showed that it has the highest nucleotide identity of 86.09% with blaOXA-48 . However, the active site motifs in OXA-731 were 100% identical to that in OXA-48. Whole-genome sequencing analysis showed that blaOXA-731 is not surrounded by any mobile genetic elements. The genetic context of blaOXA-731 was found as similar to other blaOXA-48 -like genes previously identified in Shewanella sp. S1 nuclease pulsed-field gel electrophoresis followed by Southern blotting confirmed the location of blaOXA-731 in the chromosome of the Shewanella genome. Cloning and expression studies showed that OXA-731 has ß-lactamase activity similar to OXA-48 and OXA-181, but it has no significant carbapenemase activity. Our results showed the significance of blaOXA-48 -like-carrying Shewanella sp. in the spreading of blaOXA-48 -like genes in the community.

Molecular Basis of Class A ß-Lactamase Inhibition by Relebactam.

ß-Lactamase production is the major ß-lactam resistance mechanism in Gram-negative bacteria. ß-Lactamase inhibitors (BLIs) efficacious against serine ß-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum ß-lactamases L2 (inhibition constant 3 µM) and CTX-M-15 (21 µM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 µM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates ß-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-ß-lactam combinations.

Tuned SMC Arms Drive Chromosomal Loading of Prokaryotic Condensin.

SMC proteins support vital cellular processes in all domains of life by organizing chromosomal DNA. They are composed of ATPase "head" and "hinge" dimerization domains and a connecting coiled-coil "arm." Binding to a kleisin subunit creates a closed tripartite ring, whose ∼47-nm-long SMC arms act as barrier for DNA entrapment. Here, we uncover another, more active function of the bacterial Smc arm. Using high-throughput genetic engineering, we resized the arm in the range of 6-60 nm and found that it was functional only in specific length regimes following a periodic pattern. Natural SMC sequences reflect these length constraints. Mutants with improper arm length or peptide insertions in the arm efficiently target chromosomal loading sites and hydrolyze ATP but fail to use ATP hydrolysis for relocation onto flanking DNA. We propose that SMC arms implement force transmission upon nucleotide hydrolysis to mediate DNA capture or loop extrusion.

Genetic diversity of chromosomal metallo-beta-lactamase genes in clinical isolates of Elizabethkingia meningoseptica from Korea.

This study was performed to characterize the chromosomal metallo-beta-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5 alpha. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the bla (GOB) genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of bla (GOB) gene, including 10 novel variants (bla (GOB-8) to bla (GOB-17)). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient E. coli DH5 alpha. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino acid similarities.

Analysis by mutagenesis of a chromosomal integron integrase from Shewanella amazonensis SB2BT.

Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the beta-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the beta-4 strand is less important for the excision activity.

How to identify Raoultella spp. including R. ornithinolytica isolates negative for ornithine decarboxylase? The reliability of the chromosomal bla gene.

Although Raoultella planticola and Raoultella ornithinolytica were described more than 20 years ago, identifying them remains difficult. The reliability of the chromosomal bla gene for this identification was evaluated in comparison with that of the 16S rDNA and rpoB genes in 35 Raoultella strains from different origins. Of the 26 strains previously identified as R. planticola by biochemical tests alone or in association with molecular methods, 21 harboured a bla gene with 99.8% identity with the bla gene of two reference R. ornithinolytica strains (bla(ORN) gene) and 5 harboured a bla gene with 99.2% identity with the bla gene of two reference R. planticola strains (bla(PLA) gene). The 9 isolates previously identified as R. ornithinolytica harboured a bla(ORN) gene. The bla gene-based identification was confirmed by 16S rDNA and rpoB sequencing. The 21 isolates newly identified as R. ornithinolytica had a test negative for ornithine decarboxylase (ODC). Molecular experiments suggested one copy of ODC-encoding gene in both ODC-negative R. ornithinolytica and R. planticola strains and two copies in ODC-positive R. orninthinolytica strains. Analysis of the 35 bla genes allowed us (i) to confirm an identity of only 94% between the bla genes of the two Raoultella species while this identity was > 98% for rpoB and > 99% for 16S rDNA genes and (ii) to develop and successfully apply a bla PCR RFLP assay for Raoultella spp. identification. Overall, this study allowed us to discover ODC-negative R. ornithinolytica and to provide a reliable Raoultella identification method widely available as not requiring sequencing equipment.

Interaction between the co-inherited TraG coupling protein and the TraJ membrane-associated protein of the H-plasmid conjugative DNA transfer system resembles chromosomal DNA translocases.

Bacterial conjugation is a DNA transfer event that requires three plasmid-encoded multi-protein complexes: the membrane-spanning mating pair formation (Mpf) complex, the cytoplasmic nucleoprotein relaxosome complex, and a homo-multimeric coupling protein that links the Mpf and relaxosome at the cytoplasmic membrane. Bacterial two-hybrid (BTH) technology and immunoprecipitation were used to demonstrate an interaction between the IncH plasmid-encoded transfer protein TraJ and the coupling protein TraG. TraJ is essential for conjugative transfer but is not required for the formation of the conjugative pilus, and is therefore not regarded as an Mpf component. Fractionation studies indicated that TraJ shared a similar cellular domain to that of TraG at the cellular membrane. Protein blast analyses have previously identified TraJ homologues encoded in a multitude of plasmid and chromosomal genomes that were also found to encode an adjacent TraG homologue, thus indicating co-inheritance. BTH analysis of these TraJ and cognate TraG homologues demonstrated conservation of the TraJ-TraG interaction. Additional occurrences of the traJ-traG module were also detected in genomic sequence data throughout the Proteobacteria, and phylogenetic comparison of these IncH-like TraG proteins with the coupling proteins encoded by other conjugative transfer systems (including IncP, IncW and IncF) that lack TraJ homologues indicated that the H-like coupling proteins were distinct. Accordingly, the IncP, IncW and IncF coupling proteins were unable to interact with TraJ, but were able to interact with IncH plasmid-encoded TrhB, an Mpf component known to complex with its cognate coupling protein TraG. The divergence of the IncH-type coupling proteins may partly be due to the requirement of TraJ interaction, and notably, TraG and TraJ cumulatively represent the domain architecture of the known translocase family FtsK/SpoIIIE. It is proposed that TraJ is a functional part of the IncH-type coupling protein complex required for translocation of DNA through the cytoplasmic membrane.

Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant beta-lactamase-producing Klebsiella pneumoniae strain.

Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of beta-lactamase-producing K. pneumoniae strains highlight the need to develop new therapeutic strategies. In this study, we developed antisense peptide nucleic acids (PNAs) conjugated to the (KFF)(3)K peptide and investigated whether they could mediate gene-specific antisense effects in K. pneumoniae. No outer membrane permeabilization was observed with antisense PNAs when used alone. Antisense peptide-PNAs targeted at two essential genes, gyrA and ompA, were found to be growth inhibitory at concentrations of 20 muM and 40 muM, respectively. Mismatched antisense peptide-PNAs with sequence variations of the gyrA and ompA genes when used as controls were not growth inhibitory. Bactericidal effects exerted by peptide-anti-gyrA PNA and peptide-anti-ompA PNA on cells were observed within 6 h of treatment. The antisense peptide-PNAs specifically inhibited expression of DNA gyrase subunit A and OmpA from the respective targeted genes in a dose-dependent manner. Both antisense peptide-PNAs cured IMR90 cell cultures that were infected with K. pneumoniae (10(4) CFU) in a dose-dependent manner without any noticeable toxicity to the human cells.

Differential and dynamic localization of topoisomerases in Bacillus subtilis.

Visualization of topoisomerases in live Bacillus subtilis cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcellular topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the B. subtilis nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.

Stepwise upregulation of the Pseudomonas aeruginosa chromosomal cephalosporinase conferring high-level beta-lactam resistance involves three AmpD homologues.

Development of resistance to the antipseudomonal penicillins and cephalosporins mediated by hyperproduction of the chromosomal cephalosporinase AmpC is a major threat to the successful treatment of Pseudomonas aeruginosa infections. Although ampD inactivation has been previously found to lead to a partially derepressed phenotype characterized by increased AmpC production but retaining further inducibility, the regulation of ampC in P. aeruginosa is far from well understood. We demonstrate that ampC expression is coordinately repressed by three AmpD homologues, including the previously described protein AmpD plus two additional proteins, designated AmpDh2 and AmpDh3. The three AmpD homologues are responsible for a stepwise ampC upregulation mechanism ultimately leading to constitutive hyperexpression of the chromosomal cephalosporinase and high-level antipseudomonal beta-lactam resistance, as shown by analysis of the three single ampD mutants, the three double ampD mutants, and the triple ampD mutant. This is achieved by a three-step escalating mechanism rendering four relevant expression states: basal-level inducible expression (wild type), moderate-level hyperinducible expression with increased antipseudomonal beta-lactam resistance (ampD mutant), high-level hyperinducible expression with high-level beta-lactam resistance (ampD ampDh3 double mutant), and very high-level (more than 1,000-fold compared to the wild type) derepressed expression (triple mutant). Although one-step inducible-derepressed expression models are frequent in natural resistance mechanisms, this is the first characterized example in which expression of a resistance gene can be sequentially amplified through multiple steps of derepression.

Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome.

Chromatin immunoprecipitation and high-density microarrays have been used to monitor the distribution of the global transcription regulator Escherichia coli cAMP-receptor protein (CRP) and RNA polymerase along the E. coli chromosome. Our results identify targets occupied by CRP and genes transcribed by RNA polymerase in vivo. Many of the loci of CRP binding are at known CRP regulated promoters. However, our results show that CRP also interacts with thousands of weaker sites across the whole chromosome and that this "background" binding can be used as a probe for organization within the E. coli folded chromosome. In rapidly growing cells, we show that the major sites of RNA polymerase binding are approximately 90 transcription units that include genes needed for protein synthesis. Upon the addition of rifampicin, RNA polymerase is distributed among >500 functional promoters. We show that the chromatin immunoprecipitation and high-density-microarrays methodology can be used to study the redistribution of RNA polymerase induced by environmental stress, revealing previously uncharacterized aspects of RNA polymerase behavior and providing an alternative to the "transcriptomics" approach for studying global transcription patterns.

Cloning and characterization of a chromosomal class C beta-lactamase and its regulatory gene in Laribacter hongkongensis.

Laribacter hongkongensis, a newly discovered bacterium recently shown to be associated with community-acquired gastroenteritis, is generally resistant to most beta-lactams except the carbapenems. We describe the cloning and characterization of a novel chromosomal class C beta-lactamase and its regulatory gene in L. hongkongensis. Two genes, ampC and ampR, were cloned by inserting restriction fragments of genomic DNA from L. hongkongensis strain HLHK5 into pBK-CMV to give the recombinant plasmid pBK-LHK-5. The ampR and ampC genes and their promoters were divergently oriented, with the ampR gene immediately upstream of the ampC gene and an intercistronic Lys-R motif, typical of inducible ampC-ampR regulatory systems. The deduced amino acid sequence of the cloned AmpC beta-lactamase (pI 8.1) contained consensus motifs characteristic of class C beta-lactamases but had identities no greater than 46% to known class C beta-lactamases. The kinetic properties of this AmpC were also compatible with those of a class C beta-lactamase. PCR of 20 clinical isolates of L. hongkongensis, including HLHK5, showed the presence of both ampC and ampR genes in all isolates. Southern hybridization suggested that the ampC gene of HLHK5 was chromosomally encoded. Subcloning experiments showed that the expression of the ampC gene of HLHK5 was regulated by its ampR gene, which acts as a repressor. The beta-lactamase characterized from strain HLHK5 was named LHK-5 (gene, bla(LHK-5)) and represents the first example of AmpC beta-lactamase in the beta subdivision of proteobacteria.

OXA-60, a chromosomal, inducible, and imipenem-hydrolyzing class D beta-lactamase from Ralstonia pickettii.

A chromosomally encoded oxacillinase, OXA-22, had been characterized from Ralstonia pickettii PIC-1 that did not explain by itself the resistance profile of this strain to beta-lactams. Thus, further analysis of the genetic background of this species led to the identification of another oxacillinase, OXA-60, that was expressed only after beta-lactam induction. This chromosomally encoded oxacillinase shared 19% amino acid identity with OXA-22. It has a narrow-spectrum hydrolysis profile that includes imipenem. OXA-60-like enzymes were identified in several R. pickettii strains. Gene inactivation and induction studies of the bla(OXA-60) and bla(OXA-22) genes in R. pickettii identified the relative contribution of each oxacillinase to the resistance profile of R. pickettii to beta-lactams.

Evolutionary mapping of the SHV beta-lactamase and evidence for two separate IS26-dependent blaSHV mobilization events from the Klebsiella pneumoniae chromosome.

OBJECTIVES: To determine the most likely evolutionary pathway that has led to the development of extended-spectrum SHV derivatives, and to the mobilization of blaSHV. MATERIALS AND METHODS: Evolutionary mapping used a shortest-path analysis of aligned blaSHV variants, and other basic bioinformatic approaches, such as CLUSTAL W and Blast were employed. RESULTS: Two main branches of the blaSHV evolutionary tree were located; both are derived from variant blaSHV-1 alleles. Identical mutations, responsible for extended-spectrum SHV substrate profiles, have been selected independently in each branch. There is evidence for a pool of non-mobile blaSHV framework sequences. Analysis of the genome sequence of Klebsiella pneumoniae confirms the chromosomal origin of blaSHV, whose mobilization has occurred at least twice, once for each of the main evolutionary branches. Both these mobilization events have been catalysed by IS26. Evolution of blaSHV to give common extended-spectrum variants is most likely to have occurred following mobilization. CONCLUSIONS: These data shed new light on the evolution and mobilization of blaSHV, and these observations may be useful in predicting what might happen in future, both for blaSHV, and for other beta-lactamase genes.

Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis and chronic lung infection. Mechanism of antibiotic resistance and target of the humoral immune response.

The intensive antibiotic treatment of cystic fibrosis (CF) patients with chronic lung infection with Pseudomonas aeruginosa has improved the survival rate and the clinical condition of Danish patients. Acquirement of resistance to anti-pseudomonal antibiotics is one of the main drawbacks of this therapeutic strategy and our results showed the development of resistance of P. aeruginosa to several antibiotics during 25 years of intensive antibiotic treatment. Our studies have been concentrating on the development of resistance to beta-lactam antibiotics. We have shown an association between the development of resistance to beta-lactam antibiotics and the occurrence of high beta-lactamase producing strains and between the MIC of the beta-lactams and the levels of beta-lactamase expression. Partially derepressed mutants, characterized by high basal levels of beta-lactamase with the possibility of induction to even higher levels during treatment with beta-lactam antibiotics, were the most frequent phenotype found among resistant Danish P. aeruginosa CF isolates. We have also shown that the high alginate producing P. aeruginosa isolates, that characterize the chronic lung infection in CF patients, are more susceptible to antibiotics and produce less beta-lactamase than the non-mucoid paired isolates. We propose that the non-mucoid isolates are exposed to a relatively higher antibiotic pressure than the mucoid isolates and therefore, they become easily antibiotic resistant and in consequence produce high levels of beta-lactamase. The beta-lactamase produced by the non-mucoid isolates might play a protective role in the biofilm, defending the mucoid isolates from the action of beta-lactam antibiotics and helping them to maintain their antibiotic susceptibility. We have also shown that beta-lactamase, which is a periplasmic enzyme, can be secreted extracellulary packed in membrane vesicles liberated by high beta-lactamase-producing P. aeruginosa. The continuos presence in the CF lungs of bacteria producing high basal levels of beta-lactamase (partial derepressed) induces a humoral immune response to beta-lactamase. We have shown that antibodies against the chromosomally encoded beta-lactamase (a beta ab) might be considered a marker of the development of resistance to beta-lactam antibiotics. We investigated the humoral immune response to beta-lactamase by quantifying a beta ab specific IgG and IgG subclass antibodies, by investigating the influence of the allotypes on the IgG subclass response and by measuring the avidity of the IgG a beta ab. We found that CF patients with good lung function had in the early stages of the chronic lung infection higher titers of a beta ab of good avidity than patients with poor lung function. Therefore, we raised the hypothesis that some of the a beta ab might have beta-lactamase neutralizing effect, playing a beta-lactamase inhibitor role and improving the effect of the treatment with beta-lactam antibiotics. Finally, we tested our hypothesis in the rat model of chronic lung infection by assessing the effect of a beta ab raised by vaccination with purified chromosomal beta-lactamase on the outcome of the treatment with ceftazidime of bacteria resistant to beta-lactam antibiotics. Our results showed that significantly lower bacterial load and better lung pathology were found in rats with neutralizing antibodies compared to non-immunized rats or rats without neutralizing antibodies. Our findings might be of potential importance for the improvement of the treatment with beta-lactam antibiotics of resistant P. aeruginosa hyperproducing chromosomal beta-lactamase that represent a threat especially for patients with CF and chronic lung infection.

Identification of a chromosome-borne expanded-spectrum class a beta-lactamase from Erwinia persicina.

From whole-cell DNA of an enterobacterial Erwinia persicina reference strain that displayed a penicillinase-related antibiotic-resistant phenotype, a beta-lactamase gene was cloned and expressed in Escherichia coli. It encoded a clavulanic-acid-inhibited Ambler class A beta-lactamase, ERP-1, with a pI value of 8.1 and a relative molecular mass of ca. 28 kDa. ERP-1 shared 45 to 50% amino acid identity with the most closely related enzymes, the chromosomally encoded enzymes from Citrobacter koseri, Kluyvera ascorbata, Kluyvera cryocrescens, Klebsiella oxytoca, Proteus vulgaris, Proteus penneri, Rahnella aquatilis, Serratia fonticola, Yersinia enterocolitica, and the plasmid-mediated enzymes CTX-M-8 and CTX-M-9. The substrate profile of the noninducible ERP-1 was similar to that of these beta-lactamases. ERP-1 is the first extended-spectrum beta-lactamase from an enterobacterial species that is plant associated and plant pathogenic.

Chromosome-encoded beta-lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (formerly Flavobacterium odoratum), new members of the lineage of molecular subclass B1 metalloenzymes.

Myroides odoratus and Myroides odoratimimus (formerly designated in a single species as Flavobacterium odoratum) are gram-negative aerobes and sources of nosocomial infections in humans. They have variable susceptibility to beta-lactams and a decreased susceptibility to carbapenems. Using genomic DNAs of M. odoratus CIP 103105 and M. odoratimimus CIP 103073 reference strains, shotgun cloning of beta-lactamase genes was performed, followed by protein expression in Escherichia coli. The deduced amino acid sequences of these beta-lactamase genes revealed that TUS-1 and MUS-1 from M. odoratus CIP 103105 and M. odoratimimus CIP 103073, respectively, shared 73% amino acid identity. Mature proteins TUS-1 and MUS-1, with pI values of 7.8 and 5.2, respectively, had relative molecular masses of ca. 26 kDa. These beta-lactamases are members of the subclass B1 of metallo-beta-lactamases and are distantly related to other metalloenzymes, being most closely related to IND-1 from Chryseobacterium indologenes (42% amino acid identity). However, phylogenic analysis showed that TUS-1 and MUS-1 belong to the same phylogenic lineage of subclass B1 enzymes that groups the subclass B1 beta-lactamases of Flavobacterium species. Kinetic parameters of purified beta-lactamases TUS-1 and MUS-1 detailed their hydrolysis spectra, which encompass most beta-lactams except aztreonam. beta-Lactamases TUS-1 and MUS-1 were classified in functional subgroup 3a of metalloenzymes. This work further characterizes chromosome-encoded metalloenzymes from Flavobacteriaceae species that explain at least part of their intrinsic resistance to beta-lactams.

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.

Overexpression and characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.

The gene coding for aminoglycoside 2'-N-acetyltransferase Ia [AAC(2')-Ia] from Providencia stuartii was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2'-N-acetyltransferase were 20 degrees C and pH 6.0, respectively, while the enzymes were most stable at 15 degrees C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20 degrees C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K(m) value of 8.75 microM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both K(m) and k(cat)/K(m) values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for P. stuartii PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.