Why are the inner and outer sides of many flower petals differently coloured?

The flower perianth has various, non-mutually exclusive functions, such as visual signalling to pollinators and protecting the reproductive organs from the elements and from florivores, but how different perianth structures and their different sides play a role in these functions is unclear. Intriguingly, in many species there is a clear colour difference between the different sides of the perianth, with colour patterns or pigmentation present on only one side. Any adaptive benefit from such colour asymmetry is unclear, as is how the asymmetry evolved. In this viewpoint paper, we address the phenomenon of flowers with differently coloured inner and outer perianth sides, focusing on petals of erect flowers. Guided by existing literature and our own observations, we delineate three non-mutually exclusive evolutionary hypotheses that may explain the factors underlying differently coloured perianth sides. The pollen-protection hypothesis predicts that the outer side of petals contributes to protect pollen against UV radiation, especially during the bud stage. The herbivore-avoidance hypothesis predicts that the outer side of petals reduces the flower's visibility to herbivores. The signalling-to-pollinators hypothesis predicts that flower colours evolve to increase conspicuousness to pollinators. The pollen-protection hypothesis, the herbivore-avoidance hypothesis, and the signalling-to-pollinators hypothesis generate largely but not entirely overlapping predictions about the colour of the inner and outer side of the petals. Field and laboratory research is necessary to disentangle the main drivers and adaptive significance of inner-outer petal side colour asymmetry.

Surprising absence of association between flower surface microstructure and pollination system.

The epidermal cells of flowers come in different shapes and have different functions, but how they evolved remains largely unknown. Floral micro-texture can provide tactile cues to insects, and increases in surface roughness by means of conical (papillose) epidermal cells may facilitate flower handling by landing insect pollinators. Whether flower microstructure correlates with pollination system remains unknown. Here, we investigate the floral epidermal microstructure in 29 (congeneric) species pairs with contrasting pollination system. We test whether flowers pollinated by bees and/or flies feature more structured, rougher surfaces than flowers pollinated by non-landing moths or birds and flowers that self-pollinate. In contrast with earlier studies, we find no correlation between epidermal microstructure and pollination system. The shape, cell height and roughness of floral epidermal cells varies among species, but is not correlated with pollinators at large. Intriguingly, however, we find that the upper (adaxial) flower surface that surrounds the reproductive organs and often constitutes the floral display is markedly more structured than the lower (abaxial) surface. We thus conclude that conical epidermal cells probably play a role in plant reproduction other than providing grip or tactile cues, such as increasing hydrophobicity or enhancing the visual signal.

Competition for pollinators and intra-communal spectral dissimilarity of flowers.

Competition for pollinators occurs when, in a community of flowering plants, several simultaneously flowering plant species depend on the same pollinator. Competition for pollinators increases interspecific pollen transfer rates, thereby reducing the number of viable offspring. In order to decrease interspecific pollen transfer, plant species can distinguish themselves from competitors by having a divergent phenotype. Floral colour is an important signalling cue to attract potential pollinators and thus a major aspect of the flower phenotype. In this study, we analysed the amount of spectral dissimilarity of flowers among pollinator-competing plants in a Dutch nature reserve. We expected pollinator-competing plants to exhibit more spectral dissimilarity than non-competing plants. Using flower visitation data of 2 years, we determined the amount of competition for pollinators by different plant species. Plant species that were visited by the same pollinator were considered specialist and competing for that pollinator, whereas plant species visited by a broad array of pollinators were considered non-competing generalists. We used principal components analysis to quantify floral reflectance, and found evidence for enhanced spectral dissimilarity among plant species within specialist pollinator guilds (i.e. groups of plant species competing for the same pollinator). This is the first study that examined intra-communal dissimilarity in floral reflectance with a focus on the pollination system.

NOTCH1 and/or FBXW7 mutations predict for initial good prednisone response but not for improved outcome in pediatric T-cell acute lymphoblastic leukemia patients treated on DCOG or COALL protocols.

Aberrant activation of the NOTCH1 pathway by inactivating and activating mutations in NOTCH1 or FBXW7 is a frequent phenomenon in T-cell acute lymphoblastic leukemia (T-ALL). We retrospectively investigated the relevance of NOTCH1/FBXW7 mutations for pediatric T-ALL patients enrolled on Dutch Childhood Oncology Group (DCOG) ALL7/8 or ALL9 or the German Co-Operative Study Group for Childhood Acute Lymphoblastic Leukemia study (COALL-97) protocols. NOTCH1-activating mutations were identified in 63% of patients. NOTCH1 mutations affected the heterodimerization, the juxtamembrane and/or the PEST domains, but not the RBP-J-κ-associated module, the ankyrin repeats or the transactivation domain. Reverse-phase protein microarray data confirmed that NOTCH1 and FBXW7 mutations resulted in increased intracellular NOTCH1 levels in primary T-ALL biopsies. Based on microarray expression analysis, NOTCH1/FBXW7 mutations were associated with activation of NOTCH1 direct target genes including HES1, DTX1, NOTCH3, PTCRA but not cMYC. NOTCH1/FBXW7 mutations were associated with TLX3 rearrangements, but were less frequently identified in TAL1- or LMO2-rearranged cases. NOTCH1-activating mutations were less frequently associated with mature T-cell developmental stage. Mutations were associated with a good initial in vivo prednisone response, but were not associated with a superior outcome in the DCOG and COALL cohorts. Comparing our data with other studies, we conclude that the prognostic significance for NOTCH1/FBXW7 mutations is not consistent and may depend on the treatment protocol given.

Burkholderia cenocepacia ZmpB is a broad-specificity zinc metalloprotease involved in virulence.

In previous studies we characterized the Burkholderia cenocepacia ZmpA zinc metalloprotease. In this study, we determined that B. cenocepacia has an additional metalloprotease, which we designated ZmpB. The zmpB gene is present in the same species as zmpA and was detected in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria, and B. pyrrocinia but was absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The zmpB gene was expressed, and ZmpB was purified from Escherichia coli by using the pPROEXHTa His(6) Tag expression system. ZmpB has a predicted preproenzyme structure typical of thermolysin-like proteases and is distantly related to Bacillus cereus bacillolysin. ZmpB was expressed as a 63-kDa preproenzyme precursor that was autocatalytically cleaved into mature ZmpB (35 kDa) and a 27-kDa prepropeptide. EDTA, 1,10-phenanthroline, and Zn(2+) cations inhibited ZmpB enzyme activity, indicating that it is a metalloprotease. ZmpB had proteolytic activity against alpha-1 proteinase inhibitor, alpha(2)-macrogobulin, type IV collagen, fibronectin, lactoferrin, transferrin, and immunoglobulins. B. cenocepacia zmpB and zmpA zmpB mutants had no proteolytic activity against casein and were less virulent in a rat agar bead chronic infection model, indicating that zmpB is involved in B. cenocepacia virulence. Expression of zmpB was regulated by both the CepIR and CciIR quorum-sensing systems.

Distribution and expression of the ZmpA metalloprotease in the Burkholderia cepacia complex.

The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA protease-deficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extracellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression.

Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease.

Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His(6) tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His(6)-pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H(465) with G(465) or A(465), E(377) with A(377) or D(377), or H(380) with P(380) or A(380). Mutagenesis of H(465), E(377), or H(380) resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H(380) was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil alpha-1 proteinase inhibitor, alpha(2)-macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.

The CepIR quorum-sensing system contributes to the virulence of Burkholderia cenocepacia respiratory infections.

The cepIR genes encode an N-acyl homoserine lactone (AHL)-dependent quorum-sensing system consisting of an AHL synthase that directs the synthesis of N-octanoyl-L-homoserine lactone (ohl) and n-hexanoyl-L-homoserine lactone and a transcriptional regulator. The virulence of cepIR mutants was examined in two animal models. Rats were infected with agar beads containing Burkholderia cenocepacia K56-2, K56-I2 (cepI : : Tp(r)) or K56-R2 (cepR : : Tn5-OT182). At 10 days post-infection, the extent of lung histopathological changes was significantly lower in lungs infected with K56-I2 or K56-R2 compared to the parent strain. Intranasal infections were performed in Cftr((-/-)) mice and their wild-type siblings. K56-2 was more virulent in both groups of mice. K56-I2 was the least virulent strain and was not invasive in the Cftr((-/-)) mice. OHL was readily detected in lung homogenates from Cftr((-/-)) mice infected with K56-2 but was only detected at levels slightly above background in a few mice infected with K56-I2. Lung homogenates from mice infected with K56-2 had significantly higher levels of the inflammatory mediators murine macrophage inflammatory protein-2, KC/N51, interleukin-1beta and interleukin-6 than those from K56-I2-infected animals. These studies indicate that a functional CepIR quorum-sensing system contributes to the severity of B. cenocepacia infections. A zinc metalloprotease gene (zmpA) was shown to be regulated by CepR and may be one of the factors that accounts for the difference in virulence between the cepI mutant and the parent strain.

An extracellular zinc metalloprotease gene of Burkholderia cepacia.

Burkholderia cepacia produces at least one extracellular zinc metalloprotease that may be involved in virulence. A B. cepacia zinc metalloprotease gene was cloned using a Burkholderia pseudomallei zinc metalloprotease gene as a probe. The predicted amino acid sequences of these B. cepacia and a B. pseudomallei extracellular zinc metalloproteases indicate that they are similar to the thermolysin-like family of metalloproteases (M4 family of metalloendopeptidases) and they are likely to be secreted via the general secretory pathway. zmpA isogenic mutants were constructed in B. cepacia genomovar III strains Pc715j and K56-2 by insertional inactivation of the zmpA genes. The zmpA mutants produced less protease than the parent strains. The B. cepacia strain K56-2 zmpA mutant was significantly less virulent than its parent strain in a chronic respiratory infection model; however, there was no difference between the virulence of B. cepacia strain Pc715j and a Pc715j zmpA mutant. The results indicate that this extracellular zinc metalloprotease may play a greater role in virulence in some strains of B. cepacia.

Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase.

Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin mononucleotide. In Escherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase. In addition, domains homologous to flavodoxin are components of the multidomain flavoproteins cytochrome P450 reductase, nitric oxide synthase, and methionine synthase reductase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions. We address this issue by using NMR chemical shift mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase and methionine synthase. We find that these physiological partners bind to unique overlapping sites on flavodoxin, precluding the formation of ternary complexes. We infer that the flavodoxin-like domains of the cytochrome P450 reductase family form mutually exclusive complexes with their electron-donating and -accepting partners, complexes that require conformational changes for interconversion.

In vitro activity of trovafloxacin against Bacteroides fragilis in mixed culture with either Escherichia coli or a vancomycin- resistant strain of Enterococcus faecium determined by an anaerobic time-kill technique.

To determine the efficacy of trovafloxacin as a possible treatment for intra-abdominal abscesses, we have developed an anaerobic time-kill technique using different inocula to study the in vitro killing of Bacteroides fragilis in pure culture or in mixed culture with either Escherichia coli or a vancomycin-resistant strain of Enterococcus faecium (VREF). With inocula of 5 x 10(5) CFU/ml and trovafloxacin concentrations of /=6.1 (log(10) CFU/ml) was attained with all pure and mixed cultures within 24 h. With inocula of 10(8) CFU/ml, a similar E(max) and a similar concentration to produce 50% of E(max) (EC(50)) for B. fragilis were found in both pure cultures and mixed cultures with E. coli. However, to produce a similar killing of B. fragilis in the mixed cultures with VREF, a 14-fold increase in the concentration of trovafloxacin was required. A vancomycin-susceptible strain of E. faecium and a trovafloxacin-resistant strain of E. coli were also found to confer a similar "protective" effect on B. fragilis against the activity of trovafloxacin. Using inocula of 10(9) CFU/ml, the activity of trovafloxacin was retained for E. coli and B. fragilis and was negligible against VREF. We conclude that this is a useful technique to study the anaerobic killing of mixed cultures in vitro and may be of value in predicting the killing of mixed infections in vivo. The importance of using mixed cultures and not pure cultures is clearly shown by the difference in the killing of B. fragilis in the mixed cultures tested. Trovafloxacin will probably be ineffective in the treatment of infections involving large numbers of enterococci. However, due to its ability to retain activity against large cultures of B. fragilis and E. coli, trovafloxacin could be beneficial in the treatment of intra-abdominal abscesses.

Identification of a siderophore receptor required for ferric ornibactin uptake in Burkholderia cepacia.

Ornibactins are linear hydroxamate siderophores produced by Burkholderia cepacia with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads. The gene encoding the outer membrane receptor (orbA) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms. The orbA precursor was predicted to be a protein with a molecular mass of 81 kDa. An orbA mutant was constructed and demonstrated to be unable to take up (59)Fe-ornibactins or to grow in medium supplemented with ornibactins. Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56orbA::tp mutant. When this mutant harbored a plasmid containing the orbA gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron. The orbA mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of B. cepacia respiratory infections.

Immunization with a Pseudomonas aeruginosa elastase peptide reduces severity of experimental lung infections due to P. aeruginosa Or Burkholderia cepacia.

Pseudomonas aeruginosa and Burkholderia cepacia produce metalloproteases that effect lung injury. Two epitopes (peptides 15 and 42) previously identified on P. aeruginosa elastase induce the production of antibodies that neutralize protease activity. The effects of immunization with synthetic peptides based on these epitopes on experimental lung infections due to P. aeruginosa or B. cepacia were examined. Rats were immunized with peptides conjugated to keyhole limpet hemocyanin or tetanus toxoid before infection. Immunization with peptide 15 (pep15) resulted in a decrease in total cells and polymorphonuclear leukocytes in bronchoalveolar lavage (BAL) fluid and a 50%-70% decrease in lung histopathologic changes, compared with findings in controls. Immunization with peptide 42 decreased cells in BAL fluid but did not decrease lung pathologic changes. Immunization with pep15 alone was just as effective in protecting against lung injury as immunization with a combination of both peptides. These studies suggest that immunization with pep15 can reduce the severity of lung infections due to P. aeruginosa or B. cepacia.

Magnetization transfer via residual dipolar couplings: application to proton-proton correlations in partially aligned proteins.

A novel three-dimensional NMR experiment is reported that allows the observation of correlations between amide and other protons via residual dipolar couplings in partially oriented proteins. The experiment is designed to permit quantitative measurement of the magnitude of proton-proton residual dipolar couplings in larger molecules and at higher degree of alignments. The observed couplings contain data valuable for protein resonance assignment, local protein structure refinement, and determination of low-resolution protein folds.

Structural insights into substrate binding by the molecular chaperone DnaK.

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.

Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase.

Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa. I117 contained an insertion in a pvdA homolog, the gene for the enzyme L-ornithine N(5)-oxygenase, which catalyzes the hydroxylation of L-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursor L-N(5)-OH-Orn was added to the culture medium. T10 contained an insertion in a pvdD homolog, which is a peptide synthetase involved in pyoverdine synthesis. beta-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn5-OT182 contains a lacZ gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The B. cepacia pvdA homolog was approximately 47% identical and 59% similar to L-ornithine N(5)-oxygenase from P. aeruginosa. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of (59)Fe-ornibactin uptake in I117. A chromosomal pvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. The pvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functional pvdA gene appears to be required for effective colonization and persistence in B. cepacia lung infections.

Line narrowing in spectra of proteins dissolved in a dilute liquid crystalline phase by band-selective adiabatic decoupling: application to 1HN-15N residual dipolar coupling measurements.

Residual heteronuclear dipolar couplings obtained from partially oriented protein samples can provide unique NMR constraints for protein structure determination. However, partial orientation of protein samples also causes severe 1H line broadening resulting from residual 1H-1H dipolar couplings. In this communication we show that band-selective 1H homonuclear decoupling during data acquisition is an efficient way to suppress residual 1H-1H dipolar couplings, resulting in spectra that are still amenable to solution NMR analysis, even with high degrees of alignment. As an example, we present a novel experiment with improved sensitivity for the measurement of one-bond 1HN-15N residual dipolar couplings in a protein sample dissolved in magnetically aligned liquid crystalline bicelles.

Identification of neutralizing epitopes on Pseudomonas aeruginosa elastase and effects of cross-reactions on other thermolysin-like proteases.

Monoclonal antibodies (MAbs) to a Burkholderia (Pseudomonas) cepacia 36-kDa protease (PSCP) which neutralize PSCP and Pseudomonas aeruginosa elastase but not P. aeruginosa alkaline protease have been isolated (C. Kooi et al., Infect. Immun. 62:2811-2817, 1994). These MAbs, designated 36-6-6 and 36-6-8, react with N-chlorosuccinimide cleavage products of P. aeruginosa elastase, consistent with the recognition of a 13.9-kDa fragment which contains the active site. Overlapping 9-mer peptides that span this region were synthesized. Neutralizing MAbs to PSCP reacted strongly with two peptides (341HGFTEQNSG349 and 395RYM DQPSRD403). Peptide 341HGFTEQNSG349 overlaps the motif 337HEXXH341, which has been found in many zinc-dependent endopeptidases. Peptide 395RYMDQPSRD403 lies between E361, which binds a zinc atom, and H420, which acts as a proton donor at the active site. Polyclonal rabbit sera raised against these peptides reacted with elastase on Western immunoblots and by enzyme-linked immunosorbent assay. With hide powder azure as the substrate, antisera to either HGFTEQNG and RYMDQPSRD completely neutralized the activities of elastase, thermolysin, Vibrio cholerae hemagglutinin/protease, and PSCP but had no effect on P. aeruginosa alkaline protease or the Serratia marcescens major protease. These results suggest that the MAbs recognize two different epitopes on P. aeruginosa elastase and that antibodies raised against synthetic peptides corresponding to either of these epitopes neutralize proteolytic activity.

Differentiation of thermolysins and serralysins by monoclonal antibodies.

Two monoclonal antibodies (MAbs) to a 36-kDa extracellular metalloprotease (PSCP) from Burkholderia (Pseudomonas) cepacia were found to react with thermolysin, Pseudomonas aeruginosa elastase, alkaline protease (Apr) and LasA, Serratia marcescens protease (SMP), Aeromonas hydrophila protease (AhP), and both the lethal factor (LF) and protective antigen (PA) of Bacillus anthracis on immunoblots. The MAbs were capable of neutralising the proteolytic activity of thermolysin, P. aeruginosa elastase and PSCP but not that of Apr, SMP, and AhP. These results suggest that these MAbs may be able to differentiate between the thermolysin and serralysin family of metalloproteases on the basis of their neutralisation capability and could, therefore, be useful tools in the characterisation of new bacterial proteases.

Characterization of monoclonal antibodies to Yersinia enterocolitica iron-regulated proteins.

Yersinia enterocolitica 1165 (0:8) expressed several iron-regulated proteins with molecular masses of 240, 194, 80, 79, 70, and 67 kDa. These proteins were not detected in cells grown in iron-rich conditions. Cell surface iodination indicated that the 240- and 190-kDa proteins (HMWPs) were not surface exposed, whereas the 67- and 70-kDa proteins appeared to be exposed to the cell surface. Incubation with iron protected the 67- and 70-kDa proteins from proteinase K treatment, suggesting that they may be involved in iron acquisition. Monoclonal antibodies (MAbs) were produced against the HMWPs and the 67-kDa iron-regulated protein. MAbs to the HMWPs not only recognized the 240- and 194-kDa proteins but also reacted with the 67- and 70-kDa iron-regulated proteins. Similarly, MAbs to the 67-kDa protein reacted with the 67- and 70-kDa proteins and the HMWPs, suggesting that these iron-regulated proteins are related immunologically. In addition, the MAbs recognized the 67- and 70-kDa proteins and HMWPs from other Y. enterocolitica serotypes, suggesting that the antigenic sites recognized on these iron-regulated proteins are conserved. The MAbs examined did not inhibit iron binding or iron uptake and did not provide protection against a Y. enterocolitica 1165 (0:8) infection in a systemic mouse infection model. Although these MAbs were not protective in this model, these iron-regulated proteins may play a role in iron acquisition and virulence, but the MAbs examined are probably not directed against epitopes involved in iron acquisition or virulence.