Trans-editing of Cys-tRNAPro by Haemophilus influenzae YbaK protein

Prolyl-tRNA synthetases (ProRSs) from all three domains of life have been shown to misactivate cysteine and to mischarge cysteine onto tRNAPro. Although most bacterial ProRSs possess an amino acid editing domain that deacylates mischarged Ala-tRNAPro, editing of Cys-tRNAPro has not been demonstrated and a double-sieve mechanism of editing does not appear to be sufficient to eliminate all misacylated tRNAPro species from the cell. It was recently shown that a ProRS paralog, the YbaK protein from Haemophilus influenzae, which is homologous to the ProRS editing domain, is capable of weakly deacylating Ala-tRNAPro. This function appears to be redundant with that of its corresponding ProRS, which contains a canonical bacterial editing domain. In the present study, we test the specificity of editing by H. influenzae YbaK and show that it efficiently edits Cys-tRNAPro and that a conserved Lys residue is essential for this activity. These findings represent the first example of an editing domain paralog possessing altered specificity and suggest that similar autonomous editing domains could act upon different mischarged tRNAs thus providing cells with enhanced proofreading potential. This work also suggests a novel mechanism of editing wherein a third sieve is used to clear Cys-tRNAPro in at least some organisms.     

Structure of the Haemophilus influenzae HMW1B translocator protein: evidence for a twin pore

Secretion of the Haemophilus influenzae HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded beta-barrel domain, the approximate size of the beta-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 A resolution. The native HMW1B structure is a dimer of beta-barrels, with each beta-barrel measuring 40 A by 50 A in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.     

Exogenous glutathione completes the defense against oxidative stress in Haemophilus influenzae

Since they are equipped with several strategies by which they evade the antimicrobial defense of host macrophages, it is surprising that members of the genus Haemophilus appear to be deficient in common antioxidant systems that are well established to protect prokaryotes against oxidative stress. Among others, no genetic evidence for glutathione (gamma-Glu-Cys-Gly) (GSH) biosynthesis or for alkyl hydroperoxide reduction (e.g., the Ahp system characteristic or enteric bacteria) is apparent from the Haemophilus influenzae Rd genome sequence, suggesting that the organism relies on alternative systems to maintain redox homeostasis or to reduce small alkyl hydroperoxides. In this report we address this apparent paradox for the nontypeable H. influenzae type strain NCTC 8143. Instead of biosynthesis, we could show that this strain acquires GSH by importing the thiol tripeptide from the growth medium. Although such GSH accumulation had no effect on growth rates, the presence of cellular GSH protected against methylglyoxal, tert-butyl hydroperoxide (t-BuOOH), and S-nitrosoglutathione toxicity and regulated the activity of certain antioxidant enzymes. H. influenzae NCTC 8143 extracts were shown to contain GSH-dependent peroxidase activity with t-BuOOH as the peroxide substrate. The GSH-mediated protection against t-BuOOH stress is most probably catalyzed by the product of open reading frame HI0572 (Prx/Grx), which we isolated from a genomic DNA fragment that confers wild-type resistance to t-BuOOH toxicity in the Ahp-negative Escherichia coli strain TA4315 and that introduces GSH-dependent alkyl hydroperoxide reductase activity into naturally GSH peroxidase-negative E. coli. Finally, we demonstrated that cysteine is an essential amino acid for growth and that cystine, GSH, glutathione amide, and cysteinylglycine can be catabolized in order to complement cysteine deficiency.     

Characterization of a ferric-binding protein mutant in Haemophilus influenzae

Ferric-binding proteins (FbpA) have been implicated in the transferrin receptor-mediated iron acquisition pathways of Haemophilus influenzae and Neisseria spp. These proteins are believed to function by shuttling iron from outer membrane transferrin receptors to a specific inner membrane permease complex. However, the role of these proteins has not been conclusively resolved, as attempts at creating isogenic mutants in the fbpA genes of both species have been unsuccessful, prompting the hypothesis that FbpA may play a critical role in H . influenzae and Neisseria spp. This study describes the construction and characterization of an H . influenzae isogenic fbpA mutant. It is demonstrated that this mutant is deficient in its ability to use human transferrin as a sole iron source, even though the strain is still competent for binding human transferrin. It is also demonstrated that this mutant is impaired in its ability to use ferric citrate as an iron source, and grows at a reduced rate relative to wild type in broth supplemented with protoporphyrin rather than haemin.     

Haemophilus influenzae biochemotyping

Biochemotyping was performed in 129 strains of Haemophilus influenzae. 95% of strains could be assigned to one of the five biochemotypes proposed by Kilian. Most of the serotype B strains isolated in meningitis belonged to biochemotype I. The biochemical differentiation of Haemophilus influenzae is regarded as a reliable technique deserving further application.     

Plasmid-to-plasmid recombination in Haemophilus influenzae.

No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec+ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec+ independent.     

Plasmid transformation in Haemophilus influenzae.

Purified 34-megadalton-plasmid deoxyribonucleic acid from antibiotic-resistant strains of Haemophilus influenzae transforms competent strains of H. influenzae more efficiently if the recipient strains contain certain other 30-megadalton plasmids.     

Plasmid transfer in Haemophilus influenzae.

Twenty-nine strains of Haemophilus influenzae highly resistant to ampicillin, chloramphenicol, or tetracycline were examined for the presence of plasmids. Agarose gel electrophoresis of ethanol-precipitated cell extracts revealed large plasmids in 11 strains, of which 7 were conjugative. Plasmid transfer by conjugation between isogenic strains was quite efficient, but transfer between different serotypes was nearly always much more inefficient. Type I or II restriction enzymes do not appear to be barriers to this transfer. Encapsulated cells can be both efficient donors and recipients. Small plasmids were seen in three strains, but only two of the three are resistance factors (RSF0885, pUB703). Thus, in 17 isolates antibiotic resistance genes are believed to be located in the bacterial chromosome. Most of these resistances could be transferred by genetic transformation into the widely used Rd strain. In some cases transfer of chromosomal resistance into conjugative plasmids was observed in both rec+ and rec host cells. Since transfer by conjugation seems to be the more efficient process, it is puzzling that in the majority of the 29 isolates studied resistance genes appeared to be in the chromosome.     

Lipoproteins of Haemophilus influenzae type b.

Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3H]palmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3H]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.     

Origin of Haemophilus influenzae R factors.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.     

A hex mutant of Haemophilus influenzae.

Summary A mutant of Haemophilus influenzae which does not discriminate between low efficiency (LE) and high efficiency (HE) markers has been isolated. The mutant does not differ wild type in its sensitivity to ultraviolet radiation, methyl methanesulfonate (MMS) mitomycin C, and nitrous acid. Spontaneous mutation frequencies for three loci studied are 10-to 30-fold higher in the mutant than in the wild type strain. Low- and high-efficiency transforming markers are equally UV-resistant when assayed on this mutant. This mutant is thus similar to the hex mutant of Streptococcus pneumoniae.     

Comparison of transformation mechanisms of Haemophilus parainfluenzae and Haemophilus influenzae.

Transformation pathways in two closely related bacterial species, Haemophilus parainfluenzae and Haemophilus influenzae, were studied. Both organisms rapidly take up transforming DNA within minutes into specialized membranous structures on the cell surface (transformasomes). DNA within transformasomes is in a protected state, inaccessible to external DNase or internal restriction and modification enzymes. However, the subsequent processing of donor DNA differs in these two organisms. In H. influenzae, linear DNA immediately undergoes degradation from one end at a constant rate, leaving a lower-molecular-weight intermediate in the transformasome. The end undergoing degradation is searching for homologous regions of the chromosome. Once pairing is initiated, the remaining lower-molecular-weight DNA exits from the transformasome, and a single strand undergoes efficient integration. In contrast, in H. parainfluenzae little degradation of donor DNA is observed, with the majority remaining intact within the transformasomes after 1 h. Thus, whereas only 10% of donor DNA molecules leave the protected state after 1 h, portions of each molecule appear to become quantitatively integrated.