The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.     

An ampD gene in Pseudomonas aeruginosa encodes a negative regulator of AmpC beta-lactamase expression

The ampD and ampE genes of Pseudomonas aeruginosa PAO1 were cloned and characterized. These genes are transcribed in the same orientation and form an operon. The deduced polypeptide of P. aeruginosa ampD exhibited more than 60% similarity to the AmpD proteins of enterobacteria and Haemophilus influenzae. The ampD product transcomplemented Escherichia coli ampD mutants to wild-type beta-lactamase expression.     

Virulence properties of Pseudomonas aeruginosa lacking the extreme-stress sigma factor AlgU (sigmaE)

A discerning feature of Pseudomonas aeruginosa strains causing chronic endobronchial infections in cystic fibrosis is their conversion into the mucoid, exopolysaccharide alginate-overproducing phenotype. This morphologically prominent change is caused by mutations which upregulate AlgU (sigma(E)), a novel extreme-stress sigma factor with functional equivalents in gram-negative organisms. In this work, we investigated the role of algU in P. aeruginosa sensitivity to reactive oxygen intermediates, killing by phagocytic cells, and systemic virulence of this bacterium. Inactivation of algU in P. aeruginosa PA01 increased its susceptibility to killing by chemically or enzymatically generated halogenated reactive oxygen intermediates and reduced its survival in bactericidal assays with J774 murine macrophages and human neutrophils. Surprisingly, inactivation of algU caused increased systemic virulence of P. aeruginosa in mouse models of acute infection. The increased lethality of the algU-deficient strain was also observed in the endotoxin-resistant C3H/HeJ mice. Only minor differences between algU+ and algU mutant cells in their sensitivity to human serum were observed, and no differences in their lipopolysaccharide profiles were detected. Intriguingly, while inactivation of algU downregulated five polypeptides it also upregulated the expression of seven polypeptides as determined by two-dimensional gel analyses, suggesting that algU plays both a positive and a negative role in gene expression in P. aeruginosa. While the observation that algU inactivation increases systemic virulence in P. aeruginosa requires further explanation, this phenomenon contrasts with the apparent selection for strains with upregulated AlgU during colonization of the cystic fibrosis lung and suggests opposing roles for this system in chronic and acute infections.     

Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.     

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant DeltaF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.     

The RFC2 gene, encoding the third-largest subunit of the replication factor C complex, is required for an S-phase checkpoint in Saccharomyces cerevisiae

Replication factor C (RF-C), an auxiliary factor for DNA polymerases delta and epsilon, is a multiprotein complex consisting of five different polypeptides. It recognizes a primer on a template DNA, binds to a primer terminus, and helps load proliferating cell nuclear antigen onto the DNA template. The RFC2 gene encodes the third-largest subunit of the RF-C complex. To elucidate the role of this subunit in DNA metabolism, we isolated a thermosensitive mutation (rfc2-1) in the RFC2 gene. It was shown that mutant cells having the rfc2-1 mutation exhibit (i) temperature-sensitive cell growth; (ii) defects in the integrity of chromosomal DNA at restrictive temperatures; (iii) progression through cell cycle without definitive terminal morphology and rapid loss of cell viability at restrictive temperatures; (iv) sensitivity to hydroxyurea, methyl methanesulfonate, and UV light; and (v) increased rate of spontaneous mitotic recombination and chromosome loss. These phenotypes of the mutant suggest that the RFC2 gene product is required not only for chromosomal DNA replication but also for a cell cycle checkpoint. It was also shown that the rfc2-1 mutation is synthetically lethal with either the cdc44-1 or rfc5-1 mutation and that the restrictive temperature of rfc2-1 mutant cells can be lowered by combining either with the cdc2-2 or pol2-11 mutation. Finally, it was shown that the temperature-sensitive cell growth phenotype and checkpoint defect of the rfc2-1 mutation can be suppressed by a multicopy plasmid containing the RFC5 gene. These results suggest that the RFC2 gene product interacts with the CDC44/RFC1 and RFC5 gene products in the RF-C complex and with both DNA polymerases delta and epsilon during chromosomal DNA replication.     

Deletion of the alternative sigma factor sigmaB in Staphylococcus aureus reveals its function as a global regulator of virulence genes

A deletion of the sigB operon was constructed in three genetically distinct Staphylococcus aureus strains, and the phenotypes of the resulting mutants were analyzed. Compared to the corresponding wild-type strains, the DeltasigB mutants showed reduced pigmentation, accelerated sedimentation, and increased sensitivity to hydrogen peroxide during the stationary growth phase. A cytoplasmic protein missing in the DeltasigB mutants was identified as alkaline shock protein 23, and an extracellular protein excreted at higher levels in one of the DeltasigB mutants was identified as staphylococcal thermonuclease. Interestingly, most sigB deletion phenotypes were only seen in S. aureus COL and Newman and not in 8325, which was found to contain an 11-bp deletion in the regulator gene rsbU. Taken together, our results show that sigmaB is a global regulator which modulates the expression of several virulence factors in S. aureus and that laboratory strain 8325 is a sigmaB-defective mutant.     

The Aspergillus nidulans gltA gene encoding glutamate synthase is required for ammonium assimilation in the absence of NADP-glutamate dehydrogenase

Mutants of Aspergillus nidulans lacking NADP-glutamate dehydrogenase activity grow more poorly than wild-type strains on ammonium as a sole nitrogen source. The leaky growth of these mutants is indicative of an alternative pathway of ammonium assimilation and glutamate biosynthesis. We have PCR-amplified a portion of the A. nidulans gene encoding glutamate synthase and used this sequence to inactivate the genomic copy. This gene, designated gltA, was found to be dispensable for growth on ammonium in the presence of NADP-glutamate dehydrogenase activity. However, a strain carrying the gltA inactivation together with an NADP-glutamate dehydrogenase structural gene mutation (gdhA) was unable to grow on ammonium or on nitrogen sources metabolized via ammonium. The gltA gene was located to linkage group V of the A. nidulans genetic map.     

Pyoverdin is essential for virulence of Pseudomonas aeruginosa

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.     

The Kluyveromyces lactis equivalent of casein kinase I is required for the transcription of the gene encoding the low-affinity glucose permease

BACKGROUND: Sonic muscle fibers intrinsic to the swim bladder of the oyster toadfish Opsanus tau proliferate throughout adult life and have an unusual radial morphology: alternating ribbons of sarcoplasmic reticulum (SR) and myofibrils surround a central core of sarcoplasm. Large fibers in adults form multiple cores, fragment, and appear to divide into smaller, more energy efficient units. METHODS: We examined embryonic to adult development of sonic muscle using electron and light microscopy and focused on the incidence of satellite cells (SC). RESULTS: Muscle fibers form late in the larval period from myoblasts, which do not appear to fuse into myotubes, but enlarge and differentiate myofibrils in a single patch. The SR differentiates from the outside inward, separating the myofibrils into bundles of varying thickness, which often exceed the thickness seen in adults. SCs in juveniles and adults have a sparse cytoplasm and a heterochromatic nucleus. The % SC nuclei (SC nuclei/total nuclei) decreases from a high of 88% in larvae to a low of 1% in adults although the adult average is 10%. No embryonic type fibers in the process of differentiating myofibrils were seen in adults. Small immature fibers, which had not yet formed the central core, have a complete radially organized contractile cylinder. CONCLUSIONS: Immature muscle fibers formed embryonically in the larval period have a different morphology from immature fibers in adults, suggesting that splitting rather than SCs is a major source of new fibers in adults.     

A gene encoding the plant-like alternative oxidase is present in Phytomonas but absent in Leishmania spp

OBJECTIVE: To develop a unique strain of Pasteurella haemolytica, selectable from nasopharyngeal respiratory tract secretions, that retains the ability to efficiently colonize the respiratory tract of calves. ANIMALS: 26 calves that each weighed approximately 200 kg. PROCEDURE: Rifampicin-resistant mutants of P haemolytica were developed and tested for in vitro growth rate and leukotoxin production. After instillation into the tonsils of calves, an isolate that was efficient at colonizing was selected and transformed, using electroporation, with a 4.2-kilobase (kb) plasmid encoding for streptomycin resistance. This isolate was instilled into the tonsils of 4 of 14 commingled calves to examine transmission of organisms. Nasal secretion and tonsil wash specimens were collected, cultured, and examined for P haemolytica. Serum antibody concentration was measured by means of indirect hemagglutination. RESULTS: Selected P haemolytica organisms colonized the tonsils and nasal passages for more than 2 weeks. Exposed calves and contact calves shed the organism, which was recovered from specimens of nasal secretions and tonsil washes. The 4.2-kb plasmid was lost during in vivo colonization. CONCLUSIONS AND CLINICAL RELEVANCE: The selected rifampicin-resistant P haemolytica organism colonized tonsils and nasal passages in a manner similar to the wild-type organisms. Selective media suppressed other bacterial flora to the extent that a single colony-forming unit was detectable from 200 microl of specimen, a 100-fold improvement in detection sensitivity. The selectable strain spread rapidly among commingled calves. A 4.2-kb plasmid marker was unstable when P haemolytica replicated in vivo.     

The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase which is specifically involved in rhamnolipid synthesis

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent beta-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-beta-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase absolutely required for the synthesis of the beta-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.