Vaborbactam (formerly RPX7009) is a new beta-lactamase inhibitor based on a cyclic boronic acid pharmacophore. The spectrum of beta-lactamase inhibition by vaborbactam and the impact of bacterial efflux and permeability on its activity were determined using a panel of strains with beta-lactamases cloned from various classes and a panel of Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing isogenic strains with various combinations of efflux and porin mutations. Vaborbactam is a potent inhibitor of class A carbapenemases, such as KPC, as well as an inhibitor of other class A (CTX-M, SHV, TEM) and class C (P99, MIR, FOX) beta-lactamases. Vaborbactam does not inhibit class D or class B carbapenemases. When combined with meropenem, vaborbactam had the highest potency compared to the potencies of vaborbactam in combination with other antibiotics against strains producing the KPC beta-lactamase. Consistent with broad-spectrum beta-lactamase inhibition, vaborbactam reduced the meropenem MICs for engineered isogenic strains of K. pneumoniae with increased meropenem MICs due to a combination of extended-spectrum beta-lactamase production, class C beta-lactamase production, and reduced permeability due to porin mutations. Vaborbactam crosses the outer membrane of K. pneumoniae using both OmpK35 and OmpK36, but OmpK36 is the preferred porin. Efflux by the multidrug resistance efflux pump AcrAB-TolC had a minimal impact on vaborbactam activity. Investigation of the vaborbactam concentration necessary for restoration of meropenem potency showed that vaborbactam at 8 μg/ml results in meropenem MICs of ≤2 μg/ml in the most resistant engineered strains containing multiple mutations. Vaborbactam is a highly active beta-lactamase inhibitor that restores the activity of meropenem and other beta-lactam antibiotics in beta-lactamase-producing bacteria, particularly KPC-producing carbapenem-resistant Enterobacteriaceae.
Despite major advances in the β-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzymes of Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts were directed toward expanding the spectrum to allow treatment of a wider range of organisms. Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably broad spectrum of inhibition, including class B and class D enzymes, and is little affected by porin modifications and efflux. Compound 35 is a promising agent for use in combination with a β-lactam antibiotic for the treatment of a wide range of multidrug resistant Gram-negative bacterial infections, by both intravenous and oral administration.
During sporulation of Bacillus subtilis, two identical genomes segregate in two compartments, the forespore and mother cell. These genomes are expressed differentially, with some genes such as sspE turned on only in the forespore. In vitro transcription of sspE was obtained only with RNA polymerase extracted from sporulating cells. Fractionation of factors associated with this enzyme and reconstitution with core RNA polymerase from vegetative cells generated an enzyme accurately transcribing sspE in vitro and led to purification of a polypeptide with the amino-terminal sequence of the spolllG product. Inactivation of spolIIG abolished expression of sspE and five other forespore-specific genes, whereas synthesis of the spolIIG product in vegetative cells rapidly turned these genes on. Therefore, spollIG encodes a o-factor, o G, which controls the expression of multiple genes in the forespore compartment.
The Bacillus subtilis spoIIIG gene codes for a sigma factor termed sigma G which directs transcription of genes expressed only in the forespore compartment of the sporulating cell. Use of spoIIIG-lacZ transcriptional fusions showed that spoIIIG is cotranscribed with the spoIIG operon beginning at t0.5-1 of sporulation. However, this large mRNA produced little if any sigma G, and transferring the spoIIIG gene without the spoIIG promoter into the amyE locus resulted in a Spo+ phenotype. Significant translation of spoIIIG began at t2.5-3 with use of an mRNA whose 5' end is just upstream of the spoIIIG coding sequence. Synthesis of this spoIIIG-specific mRNA was not abolished by a deletion in spoIIIG itself. Similar results were obtained when a spoIIIG-lacZ translational fusion lacking the spoIIG promoter was integrated at the amyE locus. These data suggest that synthesis of sigma G is dependent neither on transcription from the spoIIG promoter nor on sigma G itself but can be due to another transcription factor. This transcription factor may be sigma F, the product of the spoIIAC locus, since a spoIIAC mutation blocked spoIIIG expression, and sequences upstream of the 5' end of the spoIIIG-specific mRNA agree well with the recognition sequence for sigma F. RNA polymerase containing sigma F (E sigma F) initiated transcription in vitro on a spoIIIG template at the 5' end found in vivo, as did E sigma G. However, E sigma F showed a greater than 20-fold preference for spoIIIG over a known sigma G-dependent gene compared with the activity of E sigma G.
The Bacilus subtilis divIVBI mutation causes aberrant positioning of the septum during cell division, resulting in the formation of small, anucleate cells known as minicells. We report the cloning of the wild-type allele of divIVBI and show that the mutation lies within a stretch of DNA containing two open reading frames whose predicted products are in part homologous to the products of the Escherichia coli minicell genes minC and minD. Just upstream of minC and minD, and in the same orientation, are three genes whose products are homologous to the products of the E. coli shape-determining genes mreB, mreC, and mreD. The B. subtilis mreB, mreC, and mreD genes are the site of a conditional mutation (rodBI) that causes the production of aberrantly shaped cells under restrictive conditions. Northern (RNA) hybridization experiments and disruption experiments based on the use of integrational plasmids indicate that the mre and min genes constitute a five-cistron operon. The possible involvement of min gene products in the switch from medial to polar placement of the septum during sporulation is discussed.Cells of the gram-positive soil bacterium Bacillus subtilis are capable of entering an alternative developmental pathway that is characterized by the formation of a transverse septum. During vegetative growth, the formation of a septum at the center of the cell partitions the bacterium into identical daughter cells which separate and undergo further cycles of binary fission. The hallmark of the process of sporulation, in contrast, is the formation of a septum that is sited near one pole of the cell. This asymmetrically positioned septum partitions the bacterium into unequal-sized cellular compartments, of which one, the forespore, undergoes metamorphosis into a spore and the other, the mother cell, participates in the formation of the spore but is eventually discarded by lysis. The binary fission septum and the sporulation septum are produced by similar processes (17), involving in both cases the action of B. subtilis homologs to the Escherichia coli septation genes ftsA and ftsZ (2, 3). However, little is known about the mechanisms that govern the alternative placement of the septa at medial or polar positions within the cell.In the non-spore-forming bacterium E. coli, placement of the septum is governed by genes at the minB locus. Cells of E. coli grow by binary fission and are normally capable of producing only medially sited septa. However, certain mutations at the minB locus allow septa to form at a polar position, thereby generating small, anucleate cells with intact cell walls and membranes which except for their lack of DNA appear to be metabolically normal (1). Minicell production occurs as an alternative to normal division, and consequently, the sister cell is filamentous and carries two or more copies of the chromosome. Because the minicell division process appears to be identical to that of the wild type, the defect is apparently with site selection and not with septum formation. As with sporulation in B....
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