Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells.

The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated. In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter. Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain. The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells. pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule. In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase. The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells. Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain. Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins. This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.

Cloning and sequencing of the Haemophilus influenzae ssb gene encoding single-strand DNA-binding protein.

The ssb gene of Haemophilus influenzae was cloned and sequenced. The deduced protein possessed 61 and 60% identity with the Serratia marcescens and Escherichia coli SSB proteins, respectively. H. influenzae ssb was also shown to complement an E. coli ssb-1 mutation.

Cloning and sequencing of the Haemophilus influenzae exbB and exbD genes.

The exbB and exbD genes of Haemophilus influenzae (Hi) were cloned and sequenced. The deduced Hi ExbB and ExbD proteins possessed 27 and 28% amino-acid identity (56 and 58% relatedness) with the Escherichia coli ExbB and ExbD proteins, respectively; two proteins which function as TonB accessory proteins during biopolymer transport. Plasmid-encoded Hi exbB and exbD partially complemented an E. coli exbB/exbD mutation.

Binding of catalase by Gardnerella vaginalis.

Previous work has demonstrated that Gardnerella vaginalis can utilize catalase as a sole source of iron. In this study, the interaction between G. vaginalis cells and catalase was investigated. G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled catalase using a solid phase dot blot assay. An increase in catalase binding was observed from cells grown under iron-restrictive conditions. Western blot analysis of G. vaginalis proteins resulted in the detection of a putative catalase-binding protein with an estimated molecular mass of 128 kDa. The 128-kDa catalase-binding protein was not detected from intact G. vaginalis cells treated with trypsin prior to Western blot analysis suggesting this protein may be surface-exposed.

The effects of cis-platinum (II)diamminodichloride, UV light and N-methyl-N'-nitro-N-nitrosoguanidine on Escherichia coli:plasmid mediated resistance to mutagens.

Protease deficient recA431 mutants of Escherichia coli are defective in their capacity for induction of SOS responses and were intermediate in their sensitivities to ultraviolet light (UV) and cis-platinum (II) diamminodichloride (cis-PDD). Survival after treatment determined as colony forming ability was greater in rec+ strains and decreased in recA13 mutants which are defective in both recA proteolytic and recombination capabilities. In contrast, recA431 mutants were as sensitive to N-methyl-N'-nitro-N-nitrosoguanidine (NTG) as the recA13 cells. When cells carried either the pKM101 or N3 plasmid, survival after treatment with the three mutagens was increased. Presence of these plasmids in cells also resulted in hypermutagenicity as indicated by reversion of the argE3 mutation using a modified Ames test. Mutagenesis by NTG and cis-PDD was increased, as was survival of cells treated with UV light, cis-PDD and NTG in both recA+ and recA431 (protease deficient) strains. No plasmid mediated enhancement of mutagenesis or cell survival was observed in recA13 mutants. Thus, the ability of the plasmids to enhance cell survival and mutagenesis was dependent on recombination proficiency of the recA gene product and not its regulatory proteolytic activity. Unlike UV or NTG, presence of one of these plasmids was needed to detect reversion of the argE3 mutation by cis-PDD.

Identification of a Gardnerella vaginalis hemoglobin-binding protein.

Previous studies have shown that Gardnerella vaginalis can utilize human hemoglobin as a sole source of iron. In this study, the interaction between human hemoglobin and G. vaginalis cells was investigated. With a solid phase dot blot assay, G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled human hemoglobin. A human hemoglobin-binding protein with an estimated molecular weight of 124 kilodaltons (kDa) was detected by Western blot analysis of G. vaginalis proteins. The hemoglobin-binding activity of this protein was found to be heat stable and was observed in G. vaginalis cells grown under iron-restrictive and iron-replete conditions. The 124-kDa hemoglobin-binding protein was not detected from intact G. vaginalis cells treated with trypsin prior to Western blot analysis, suggesting that this protein was surface exposed.

Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide.

Previous studies have shown that changes in the expression of the Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) epitope reactive with monoclonal antibody (MAb) 5G8 can be correlated with alterations in the virulence of some Hib strains. To identify the locus involved in expression of this particular LOS epitope, a genomic library was constructed in the plasmid shuttle vector pGJB103 from Hib strain DL42, which constitutively expressed LOS reactive with MAb 5G8. This library was used to transform a second Hib strain (DL180) that normally does not express this LOS epitope, and a recombinant clone was identified that bound MAb 5G8. Subcloning of different regions of the Hib DL42 DNA insert in this recombinant plasmid determined that a 1.9-kb EcoRI fragment, designated lex-2, was responsible for transforming Hib strain DL180 to reactivity with MAb 5G8. Nucleotide sequence analysis revealed the presence of two contiguous open reading frames (ORFs) in lex-2, the first of which contained 18 tandem repeats of the nucleotide tetramer GCAA near its 5' end. Sequence analysis of PCR-derived products from MAb 5G8-reactive and -nonreactive Hib DL180 colonies established that 18 GCAA repeats were associated with expression of the LOS epitope that bound MAb 5G8. Mutational analysis determined that a functional ORF 2 was essential for expression of the MAb 5G8-reactive LOS epitope. The nucleotide tetramer GCAA repeat present in ORF 1 was also detected in at least two different chromosomal regions in all Hib strains tested. The availability of the cloned lex-2 locus should facilitate future analysis of the complex regulatory mechanisms involved in expression of LOS epitopes by this pathogen.

Isolation and analysis of dominant secA mutations in Escherichia coli.

The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.

Binding of heme by Gardnerella vaginalis.

It has been previously demonstrated that Gardnerella vaginalis could acquire iron from a number of different iron-containing compounds, including heme. In this study, the direct binding of heme by G. vaginalis strains was demonstrated utilizing a liquid broth heme-binding assay. Competition studies demonstrated that pretreatment of G. vaginalis cells with other iron sources such as hemoglobin, catalase, and lactoferrin did not affect heme binding. Also, heme binding was not inhibited by preincubation of G. vaginalis cells with protoporphyrin IX. Two potential heme-binding proteins with estimated molecular weights of 30 and 70 kDa were isolated using heme-agarose batch affinity chromatography.

Identification of a human lactoferrin-binding protein in Gardnerella vaginalis.

Previous studies have shown that Gardnerella vaginalis can utilize iron-loaded human lactoferrin as a sole source of iron. In this study, G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled human lactoferrin in a dot blot assay. Using the DIG-labeled human lactoferrin, a 120-kDa human lactoferrin-binding protein was detected by Western blot analysis of G. vaginalis proteins. The lactoferrin-binding activity of this protein was found to be heat stable. Competition studies indicated that this binding activity was specific for human lactoferrin. Treatment of G. vaginalis cells with proteases suggested that this protein was surface exposed. An increase in lactoferrin binding by the 120-kDa protein was observed in G. vaginalis cells grown under iron-restrictive conditions, suggesting that this activity may be iron regulated.

SecA protein: autoregulated initiator of secretory precursor protein translocation across the E. coli plasma membrane.

Several classes of secA mutants have been isolated which reveal the essential role of this gene product for E. coli cell envelope protein secretion. SecA-dependent, in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulate secA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.

Utilization of transferrin-bound iron by Haemophilus influenzae requires an intact tonB gene.

Haemophilus influenzae can utilize iron-loaded human transferrin as an iron source for growth in vitro. H. influenzae tonB mutants, containing a chloramphenicol acetyltransferase gene within their tonB genes, could bind iron-charged human transferrin to their cell surfaces, but they were unable to utilize this serum glycoprotein as the sole source of iron for growth in vitro. In contrast, these tonB mutants were able to utilize an iron chelate (ferric ammonium citrate) for growth. Transformation of a tonB mutant with a plasmid encoding a wild-type H. influenzae tonB gene restored the ability of a tonB mutant to utilize iron-charged human transferrin. These results indicate that the uptake of iron from human transferrin by H. influenzae is a TonB-dependent process.

Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery.

Escherichia coli azi mutants, whose growth is resistant to millimolar concentrations of sodium azide, were among the earliest E. coli mutants isolated. Genetic complementation, mapping, and DNA sequence analysis now show that these mutations are alleles of the secA gene, which is essential for protein export across the E. coli plasma membrane. We have found that sodium azide is an extremely rapid and potent inhibitor of protein export in vivo and that azi mutants are more resistant to such inhibition. Furthermore, SecA-dependent in vitro protein translocation and ATPase activities are inhibited by sodium azide, and SecA protein prepared from an azi mutant strain is more resistant to such inhibition. These studies point to the utility of specific inhibitors of protein export, such as sodium azide, in facilitating the dissection of the function of individual components of the protein export machinery.

Human immune response against outer membrane proteins of Moraxella (Branhamella) catarrhalis determined by immunoblotting and enzyme immunoassay.

The role of Moraxella (Branhamella) catarrhalis as a respiratory tract pathogen is increasingly recognized. We looked at the human immune response against individual outer membrane proteins of M. catarrhalis and against the 81-kDa CopB protein, which has previously been shown to be a target for protective antibodies. Paired serum samples from six elderly patients with pneumonia were tested by Western blot (immunoblot) analysis by using outer membrane vesicles of M. catarrhalis 035E as antigen. All of the six convalescent-phase serum samples reacted with a protein which migrated at the position of the CopB protein and with a high-molecular-weight protein of M. catarrhalis; three serum samples also reacted with a 34-kDa outer membrane protein. Paired serum samples from 18 patients, 10 of which had M. catarrhalis infection on the basis of previous serology results, were tested by enzyme immunoassay (EIA) with the CopB protein and whole cells of M. catarrhalis 035E as antigens. Nine patients showed a significant rise in EIA titer between acute- and convalescent-phase sera when whole bacterial cells were used as antigens. Six (67%) patient samples that were positive by the EIA with the whole-cell antigen were also positive by the EIA with the CopB antigen, and six of nine patient samples negative by the EIA with the whole-cell antigen were also negative by the EIA with the CopB antigen. These results suggest that both the CopB and a high-molecular-weight protein are major targets of the immune response against M. catarrhalis, and further studies with greater amounts of patient materials are needed to elucidate the usefulness of CopB as an antigen in etiologic studies.

Acquisition of iron by Gardnerella vaginalis.

Six Gardnerella vaginalis strains were examined for the ability to utilize various iron-containing compounds as iron sources. In a plate bioassay, all six strains acquired iron from ferrous chloride, ferric chloride, ferrous sulfate, ferric ammonium citrate, ferrous ammonium sulfate, bovine and equine hemin, bovine catalase, and equine, bovine, rabbit, and human hemoglobin. All six strains also acquired iron from human lactoferrin, but not from human transferrin, as determined by a liquid broth growth assay. Siderophore production was detected in eight G. vaginalis strains by the chrome azurol S universal chemical assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytoplasmic membrane proteins isolated from G. vaginalis 594 grown under iron-replete and iron-restricted conditions revealed several iron-regulated proteins ranging in molecular mass from 33 to 94 kDa. These results indicate that G. vaginalis may acquire iron from iron salts and host iron compounds.

A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b.

Haemophilus influenzae is nearly unique among facultatively anaerobic bacteria in its absolute requirement for exogenously supplied heme for aerobic growth. In this study, a mutant analysis strategy was used to facilitate identification of H. influenzae cell envelope components involved in the uptake of heme. Chemical mutagenesis was employed to produce a mutant of a nontypeable H. influenzae strain unable to utilize either protein-bound forms of heme or low levels of free heme. This mutant was transformed with a plasmid shuttle vector-based genomic library constructed from the same wild-type nontypeable H. influenzae strain, and a growth selection technique was used to obtain a recombinant clone that could utilize heme. Analysis of the DNA insert in the recombinant plasmid revealed the presence of several open reading frames, one of which encoded a 28-kDa protein with significant similarity to the TonB protein of Escherichia coli. This H. influenzae gene product was able to complement a tonB mutation in E. coli, allowing the E. coli tonB mutant to form single colonies on minimal medium containing vitamin B12. When this H. influenzae gene was inactivated by insertional mutagenesis techniques and introduced into the chromosome of wild-type strains of H. influenzae type b, the resultant transformants lost their abilities to utilize heme and produce invasive disease in an animal model. Genetic restoration of the ability to express this TonB homolog resulted in the simultaneous acquisition of both heme utilization ability and virulence. These results indicate that the H. influenzae TonB protein is required not only for heme utilization by this pathogen in vitro, but also for virulence of H. influenzae type b in an animal model.

Reconstitution of a porin-deficient mutant of Haemophilus influenzae type b with a porin gene from nontypeable H. influenzae.

The major outer membrane protein (OmpP2) of nontypeable Haemophilus influenzae (NTHI) has been shown to vary markedly with respect to both size and the presence of specific surface-exposed epitopes among strains of this unencapsulated pathogen. In contrast, the OmpP2 proteins of H. influenzae type b (Hib) strains are well conserved at the level of primary protein structure and have in common several surface-exposed antigenic determinants that have not been detected in NTHI strains. The availability of an isogenic, avirulent Hib ompP2 mutant made it possible to investigate whether an NTHI OmpP2 protein could function properly in the Hib outer membrane. A plasmid shuttle vector (pGJB103) was used to clone the ompP2 gene from NTHI TN106 into a recombination-deficient H. influenzae strain in which expression of the NTHI OmpP2 protein was detected by means of an NTHI TN106 OmpP2-specific monoclonal antibody. The amino acid sequence of this NTHI OmpP2 protein, as deduced from the nucleotide sequence of the NTHI TN106 ompP2 gene, was determined to be 83% identical to that of the Hib OmpP2 protein. Transformation of this cloned NTHI ompP2 gene into the Hib ompP2 mutant yielded a Hib transformant strain that expressed the NTHI OmpP2 protein. Expression of this NTHI OmpP2 protein allowed the Hib ompP2 mutant, which normally grows poorly in vitro, to grow in a manner indistinguishable from that of the wild-type Hib strain. More importantly, the introduction of this functional NTHI ompP2 gene into the avirulent Hib ompP2 mutant restored the virulence of this strain to wild-type levels. These results indicate that an NTHI OmpP2 protein can be expressed and function properly in the Hib outer membrane.