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At least 1,315 records · Page 73Linked to original sources
PubMed2003

A glutamate mutase is involved in the biosynthesis of the lipopeptide antibiotic friulimicin in Actinoplanes friuliensis.

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin. This antibiotic is active against gram-positive bacteria such as multiresistant Enterococcus and Staphylococcus strains. It consists of 10 amino acids that form a ring structure and 1 exocyclic amino acid to which an acyl residue is attached. By a reverse genetic approach, biosynthetic genes were identified that are required for the nonribosomal synthesis of the antibiotic. In close proximity two genes (glmA and glmB) were found which are involved in the production of methylaspartate, one of the amino acids of the peptide core. Methylaspartate is synthesized by a glutamate mutase mechanism, which was up to now only described for glutamate fermentation in Clostridium sp. or members of the family ENTEROBACTERIACEAE: The active enzyme consists of two subunits, and the corresponding genes overlap each other. To demonstrate enzyme activity in a heterologous host, it was necessary to genetically fuse glmA and glmB. The resulting gene was overexpressed in Streptomyces lividans, and the fusion protein was purified in an active form. For gene disruption mutagenesis, a host-vector system was established which enables genetic manipulation of Actinoplanes spp. for the first time. Thus, targeted inactivation of biosynthetic genes was possible, and their involvement in friulimicin biosynthesis was demonstrated.

Amino Acid Sequence↗
PubMed2005

Generation and characterization of recombinant influenza A (H1N1) viruses harboring amantadine resistance mutations.

The emergence of resistance to amantadine in influenza A viruses has been shown to occur rapidly during treatment as a result of single-amino-acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the matrix-(M)-2 protein. In this study, reverse genetics was used to generate and characterize recombinant influenza A (H1N1) viruses harboring L26F, V27A, A30T, S31N, G34E, and V27A/S31N mutations in the M2 gene. In plaque reduction assays, all mutations conferred amantadine resistance, with drug concentrations resulting in reduction of plaque number by 50% (IC(50)s) 154- to 3,300-fold higher than those seen for the wild type (WT). M2 mutants had no impairment in their replicative capacities in vitro on the basis of plaque size and replication kinetics experiments. In addition, all mutants were at least as virulent as the WT in experimentally infected mice, with the highest mortality rate being obtained with the recombinant harboring a double V27A/S31N mutation. These findings could help explain the frequent emergence and transmission of amantadine-resistant influenza viruses during antiviral pressure in the clinical setting.

Amantadine↗
PubMed1999

Monounsaturated but not polyunsaturated fatty acids are required for growth of the deep-sea bacterium Photobacterium profundum SS9 at high pressure and low temperature.

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.

Cell Membrane↗
PubMed1999

Characterization of the binding protein-dependent cellobiose and cellotriose transport system of the cellulose degrader Streptomyces reticuli.

Streptomyces reticuli has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH2-terminal part of an intracellular beta-glucosidase (BglC). The gene for the ATP binding protein MsiK is not linked to the ceb operon. We have shown earlier that MsiK is part of two different ABC transport systems, one for maltose and one for cellobiose and cellotriose, in S. reticuli and Streptomyces lividans. Transcription of polycistronic cebEFG and bglC mRNAs is induced by cellobiose, whereas the cebR gene is transcribed independently. Immunological experiments showed that CebE is synthesized during growth with cellobiose and that MsiK is produced in the presence of several sugars at high or moderate levels. The described ABC transporter is the first one of its kind and is the only specific cellobiose/cellotriose uptake system of S. reticuli, since insertional inactivation of the cebE gene prevents high-affinity uptake of cellobiose.

ATP-Binding Cassette Transporters↗
PubMed2001

The glycogen-bound polyphosphate kinase from Sulfolobus acidocaldarius is actually a glycogen synthase.

Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya, Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162-5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.

Amino Acid Sequence↗
PubMed2004

Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp. strain VS and its environmental distribution.

Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.

Amino Acid Sequence↗
PubMed2005

L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321.

An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.

Amidohydrolases↗
PubMed2004

Potential serological use of a recombinant protein that is a replica of a Mycobacterium tuberculosis protein found in the urine of infected mice.

The recent availability of numerous well-characterized Mycobacterium tuberculosis recombinant proteins has revived interest in the serological diagnosis of tuberculosis. Several promising results have been reported, particularly when more than one antigen is used in the test. However, thus far these antigens have not been used in routine diagnostic tests because they lack sufficient sensitivity. In addition, with the exception of one antigen, most recombinant M. tuberculosis proteins do not identify the majority of tuberculosis patients coinfected with human immunodeficiency virus (HIV). Here, we report a newer M. tuberculosis protein that is a promising candidate for increasing the sensitivity of the serological tests, in particular for patients coinfected with HIV. The protein was found in the urine of mice during the early stages of infection with M. tuberculosis (10 to 14 days), thus suggesting that the antigen is abundantly released during the in vivo growth of the mycobacterium. Reverse genetics was used to produce the recombinant protein, which we named U1 (for urine protein 1). Using a conventional enzyme-linked immunosorbent assay (ELISA), antibody to U1 could be detected in 60% of patients with pulmonary tuberculosis with no signs of coinfection with HIV (n = 83). Conversely, anti-U1 antibody was detected in 87% of the sera from tuberculosis patients coinfected with HIV (n = 47). Out of 12 HIV-infected nontuberculosis patients' sera, 9 did not react with U1 and three sera gave borderline ELISA signals (signal/cutoff of < or =1.75). These results suggest that the high efficiency of U1 in identifying tuberculosis patients coinfected with HIV may be related to abundant release of this protein during the initial phase of the HIV coinfection. The immediate availability of the antigen at a time point in which the patient's immune system is still competent would lead to a secondary immune response to U1 that persists for months in the patient's serum.

Animals↗
PubMed2002

Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a new group of proteins important for chromosome segregation in Saccharomyces cerevisiae.

Previously, antibodies were raised against a nuclear envelope-enriched fraction of yeast, and the essential gene NNF1 was cloned by reverse genetics. Here it is shown that the conditional nnf1-17 mutant has decreased stability of a minichromosome in addition to mitotic spindle defects. I have identified the novel essential genes DSN1, DSN3, and NSL1 through genetic interactions with nnf1-17. Dsn3p was found to be equivalent to the kinetochore protein Mtw1p. By indirect immunofluorescence, all four proteins, Nnf1p, Mtw1p, Dsn1p, and Nsl1p, colocalize and are found in the region of the spindle poles. Based on the colocalization of these four proteins, the minichromosome instability and the spindle defects seen in nnf1 mutants, I propose that Nnf1p is part of a new group of proteins necessary for chromosome segregation.

Alleles↗
PubMed2002

Isocitrate lyase is essential for pathogenicity of the fungus Leptosphaeria maculans to canola (Brassica napus).

A pathogenicity gene has been identified in Leptosphaeria maculans, the ascomycetous fungus that causes blackleg disease of canola (Brassica napus). This gene encodes isocitrate lyase, a component of the glyoxylate cycle, and is essential for the successful colonization of B. napus. It was identified by a reverse genetics approach whereby a plasmid conferring hygromycin resistance was inserted randomly into the L. maculans genome. Twelve of 516 transformants tested had reduced pathogenicity on cotyledons of B. juncea and B. napus, and 1 of these 12 had a deletion of the isocitrate lyase gene, as well as an insertion of the hygromycin resistance gene. This mutant was unable to grow on fatty acids, including monolaurate, and the isocitrate lyase transcript was not detected. When the wild-type gene was reintroduced into the mutant, growth on monolaurate was restored and pathogenicity was partially restored. L. maculans isocitrate lyase is produced during infection of B. napus cotyledons, while the plant homologue is not. When 2.5% glucose was added to the inoculum of the isocitrate lyase mutant, lesions of sizes similar to those caused by wild-type isolate M1 developed on B. napus cotyledons. These findings suggest that the glyoxylate pathway is essential for disease development by this plant-pathogenic fungus, as has been shown recently for a fungal and bacterial pathogen of animals and a bacterial pathogen of plants. Involvement of the glyoxylate pathway in pathogenesis in animals and plants presents potential drug targets for control of diseases.

Amino Acid Sequence↗
PubMed2004

Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretion of amino acids.

Excretion of amino acids by yeast cells was reported long ago but has not been characterized in molecular terms. It is typically favored by overproduction of the amino acid and/or impairment of its uptake. Here we describe the construction of a yeast strain excreting threonine and homoserine. Using this excretor strain, we then applied a reverse-genetics approach and found that the transporter encoded by the YNL065w/AQR1 gene, a protein thought to mediate H(+) antiport, is involved in homoserine and threonine excretion. Furthermore, overexpression of AQR1 led to increased excretion of several amino acids (alanine, aspartate, and glutamate) known to be relatively abundant in the cytosol. Transcription of the AQR1 gene is induced severalfold by a number of amino acids and appears to be under the negative control of Gcn4. An Aqr1-green fluorescent protein fusion protein is located in multiple internal membrane structures and appears to cycle continuously between these compartments and the plasma membrane. The Aqr1 sequence is significantly similar to the vesicular amine transporters of secretory vesicles of neuronal cells. We propose that Aqr1 catalyzes transport of excess amino acids into vesicles, which then release them in the extracellular space by exocytosis.

Alanine↗
PubMed2005

Cryptococcus neoformans mitochondrial superoxide dismutase: an essential link between antioxidant function and high-temperature growth.

Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2Delta mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2Delta mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 muM) at 30 degrees C, but not at 37 degrees C. Furthermore, sod2Delta mutant cells that were incubated for 24 h at 37 degrees C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25 degrees C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.

Animals↗
PubMed1992

Role of Yersinia enterocolitica Yst toxin in experimental infection of young rabbits.

We constructed a Yst-negative mutant of Yersinia enterocolitica W1024 by reverse genetics, and we compared the virulence of the yst+ and yst isogenic strains in an experimental oral infection of the young rabbit. The rabbits infected with the yst+ strain suffered from the diarrhea and lost weight, and most of them died. By contrast, the occurrence of diarrhea, weight loss, and death in the group of rabbits infected with the yst mutant was as low as that in the group of uninfected rabbits. Bacteria from both strains were excreted in the feces and induced a serum antibody response against Yop proteins. The yst mutant disappeared more rapidly from the feces. We conclude that the enterotoxin Yst is a major factor involved in the Y. enterocolitica-associated diarrhea in the young rabbit. Given the similarity with the symptoms observed for children, this result suggests that Yst could also be an important factor in diarrhea in young children infected with Y. enterocolitica.

Animals↗
PubMed1996

Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant.

The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.

Amino Acid Sequence↗
PubMed1998

Induced expression of the Legionella pneumophila gene encoding a 20-kilodalton protein during intracellular infection.

The eukaryotic protein synthesis inhibitor cycloheximid has been used by many investigators to selectively radiolabel intracellular bacteria. Although cycloheximide has no direct effect on bacterial gene expression, there are concerns that long-term inhibition of the host cell protein synthesis may have secondary effects on bacterial gene expression. Therefore, prior to further identification and cloning of the macrophage-induced (MI) genes of Legionella pneumophila, the effects of cycloheximide on L. pneumophila-infected U937 cells were evaluated by transmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the host cell, on the formation of rough endoplasmic reticulum-surrounded replicative phagosome, or on initiation of intracellular bacterial replication. In contrast, by 15 h of cycloheximide treatment, there was profound deterioration in the host cell as well as in the phagosome. To examine protein synthesis by L. pneumophila during the intracellular infection, U937 macrophage-like cells were infected with L. pneumophila, and intracellular bacteria were radiolabeled during a 2-h cycloheximide treatment or following 12 h of cycloheximide treatment. Comparison by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein profile of radiolabeled in vitro-grown L. pneumophila to that of intracellularly radiolabeled bacteria showed that 23 proteins were induced in response to the intracellular environment during 2 h of inhibition of host cell protein biosynthesis. Twelve MI proteins of L. pneumophila were artifactually induced due to prolonged inhibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analysis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion to a promoterless lacZ gene showed that compared to in vitro-grown bacteria, expression of the pyrophosphatase gene (ppa) was induced fourfold throughout the intracellular infection. There was no detectable induction in transcription of the ppa promoter during exposure to stress stimuli in vitro. The ppa gene of L. pneumophila is the first example of a regulated ppa gene which is selectively induced during intracellular infection and which may reflect enhanced capabilities of macromolecular biosynthesis by intracellular L. pneumophila. The data indicate caution in the long-term use of inhibition of host cell protein synthesis to selectively examine gene expression by intracellular bacteria.

Amino Acid Sequence↗
PubMed1999

Effects of interleukin-1 receptor antagonist overexpression on infection by Listeria monocytogenes.

Interleukin-1 receptor antagonist (IL-1ra) is a naturally occurring cytokine whose only known function is the inhibition of interleukin-1 (IL-1). Using a reverse genetic approach in mice, we previously showed that increasing IL-1ra gene dosage leads to reduced survival of a primary listerial infection. In this study, we characterize further the role of endogenously produced IL-1ra and, by inference, IL-1 in murine listeriosis. IL-1ra overexpression inhibits, but does not eliminate, primary immune responses, reducing survival and increasing bacterial loads in the target organs. We demonstrate that IL-1ra functions in the innate immune response to regulate the peak leukocyte levels in the blood, the accumulation of leukocytes at sites of infection, and the activation of macrophages during a primary infection. Reduced macrophage class II major histocompatibility complex expression was observed despite increased gamma interferon (IFN-gamma) levels, suggesting that IL-1 activity is essential along with IFN-gamma for macrophage activation in vivo. We also show that IL-1ra plays a more limited role during secondary listeriosis, blunting the strength of the delayed-type hypersensitivity response to listerial antigen while not significantly altering cellular immunity to a second infectious challenge. When these results are compared to those for other mutant mice, IL-1ra appears to be unique among the cytokines studied to date in its regulation of leukocyte migration during primary listeriosis.

Animals↗
PubMed2000

The response regulator PhoP is important for survival under conditions of macrophage-induced stress and virulence in Yersinia pestis.

The two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in Salmonella species. We have identified, cloned, and sequenced PhoP orthologues from Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. To investigate the role of PhoP in the pathogenicity of Y. pestis, an isogenic phoP mutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and phoP mutant strains, grown at either 28 or 37 degrees C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in Y. pestis. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the phoP mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of Y. pestis.

Animals↗
PubMed2002

Identification of major outer surface proteins of Streptococcus agalactiae.

To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.

Amino Acid Sequence↗