Preparation and antimicrobial assessment of 2-thioether-linked quinolonyl-carbapenems.

This reports the synthesis and in vitro antimicrobial properties of a series of 2-thioether-linked quinolonyl-carbapenems. Although the title compounds exhibited broad spectrum activity, the MICs were generally higher than those observed for selected benchmark carbapenems, quinolonyl-penems, and quinolones. Enzyme assays suggested that the title compounds are potent inhibitors of penicillin binding proteins and inefficient inhibitors of bacterial DNA-gyrase. Uptake studies indicated that the new compounds are not substrates for the norA encoded quinolone efflux pump.

Genetic linkage in Pseudomonas aeruginosa of algT and nadB: mutation in nadB does not affect NAD biosynthesis or alginate production.

The 68-min region of the chromosome of Pseudomonas aeruginosa (Pa) contains the gene algT, encoding a putative alternate sigma factor similar to sigma E in Escherichia coli, that is required for the expression of several genes in the alginate biosynthetic regulon. Sequences immediately upstream from algT were found to contain a divergently expressed open reading frame encoding a 60-kDa protein with 64 and 36% identity to the nadB gene products of E. coli and Bacillus subtilis, respectively. The nadB gene encodes L-aspartate oxidase and has been shown in several bacteria to be essential for de novo nicotinamide-adenine dinucleotide (NAD) biosynthesis. Pa nadB complemented the growth requirement for nicotinic acid in a nadB mutant strain of E. coli, suggesting that this gene encodes a functional homologue of L-aspartate oxidase. A nadB::Tn501 mutant was constructed by gene replacement in the alginate-producing strain, Pa FRD. This NadB- mutant still produced alginate and appeared normal with respect to the regulation of alginate synthesis. Interestingly, the NadB- mutant did not have an auxotrophic phenotype for nicotinic acid, indicating that this nadB was not essential for NAD biosynthesis in Pa. These results suggest the possibility that Pa has an alternate mechanism for de novo NAD biosynthesis.

Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation.

The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis and is due to overproduction of an exopolysaccharide called alginate. However, the mucoid phenotype is unstable in vitro, especially when the cells are incubated under low oxygen tension. Spontaneous conversion to the nonmucoid form is typically due to mutations (previously called algS) that are closely linked to the alginate regulatory gene algT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alternate sigma factors in bacteria, suggesting that AlgT (also known as AlgU) interacts directly with RNA polymerase core to activate the promoters of alginate genes. AlgT showed striking sequence similarity (79%) to sigma E of Escherichia coli, an alternate sigma factor involved in high-temperature gene expression. Our analysis of the molecular basis for spontaneous conversion from mucoid to nonmucoid, in the cystic fibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at codons 18 and 29. RNase protection assays showed that spontaneous nonmucoid strains with the algT18 and algT29 alleles have a four- to fivefold reduction in the accumulation of algT transcripts compared with the wild-type mucoid strain. Likewise, a plasmid-borne algT-cat transcriptional fusion was about 3-fold less active in the algT18 and algT29 backgrounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::Tn501 background. These data indicate that algT is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, and the two genes may be cotranscribed. A primer extension analysis showed that algT transcription starts 54 bp upstream of the start of translation. Although the algT promoter showed little similarity to promoters recognized by the vegetative sigma factor, it was similar to the algR promoter. This finding suggests that AlgT may function as a sigma factor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were readily detectable in the typical nonmucoid strain PAO1, which was in contrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-borne algT gene in PAO1 resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that AlgT controls its own gene expression and expression of genes of the alginate regulon.