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HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5'-GCG↓C-3' in duplex DNA and cleaves ('↓') to produce fragments with 2-base, 3'-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and cleave the two strands of duplex DNA simultaneously, in a single binding event. HhaI, in contrast, acts as a monomer with only one catalytic site, and cleaves the DNA strands sequentially, one after the other. HhaI comprises three domains, each consisting of a mixed five-stranded β sheet with a defined function. The first domain contains the catalytic-site; the second contains residues for sequence recognition; and the third contributes to non-specific DNA binding. The active-site belongs to the 'PD-D/EXK' superfamily of nucleases and contains the motif SD-X11-EAK. The first two domains are similar in structure to two other monomeric restriction enzymes, HinP1I (G↓CGC) and MspI (C↓CGG), which produce fragments with 5'-overhangs. The third domain, present only in HhaI, shifts the positions of the recognition residues relative to the catalytic site enabling this enzyme to cleave the recognition sequence at a different position. The structure of M.HhaI, the biological methyltransferase partner of HhaI, was determined earlier. Together, these two structures represent the first natural pair of restriction-modification enzymes to be characterized in atomic detail., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: The objectives of this study were to establish the frequency of Haemophilus haemolyticus in clinical samples, to determine the antimicrobial resistance rate and to identify the mechanisms of resistance to β-lactams and quinolones., Methods: An updated database was used to differentiate between MALDI-TOF MS results for Haemophilus influenzae and H. haemolyticus. Antimicrobial susceptibility was studied by microdilution, following EUCAST criteria. The β-lactamase types were identified by PCR analysis of isolates that tested positive for nitrocefin hydrolysis. Mutations in the ftsI gene were identified in isolates with ampicillin MICs ≥0.25 mg/L. Mutations in the quinolone resistance-determining region (QRDR) were identified in isolates with ciprofloxacin MICs ≥0.5 mg/L., Results: Overall, we identified 69 H. haemolyticus isolates from 1706 clinical isolates of Haemophilus spp. from respiratory, genital, invasive, and other infection sources. The frequency of H. haemolyticus was low in respiratory samples compared with that of H. influenzae, but in genital-related samples, the frequency was similar to that of H. influenzae. We found low antimicrobial resistance rates among H. haemolyticus isolates, with 8.7% for ampicillin, 8.7% for co-trimoxazole, 7.2% for tetracycline and 4.3% for ciprofloxacin. Mutations in the ftsI gene classified the isolates into four groups, including the newly described Group Hhae IV, which presents mutations in the ftsI gene not identified in H. influenzae and H. haemolyticus type strains. Three ciprofloxacin-resistant H. haemolyticus isolates with mutations affecting GyrA and ParC were identified., Conclusions: The frequency of H. haemolyticus was low, especially in respiratory samples, where H. influenzae is the main pathogen of this genus. Although antimicrobial resistance rates were low, three ciprofloxacin-resistant H. haemolyticus clinical isolates have been identified for the first time., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The Haemophilus cryptic genospecies (HCG) causes genital tract infections in pregnant and postpartum women and respiratory infections in neonates. The major surface adhesin in HCG is called Cha, which mediates bacterial adherence to cultured human epithelial cells. In this study, we report that there are two antigenically distinct variants of Cha, dubbed Cha1 and Cha2. These variants are encoded by the same genetic locus in diverse strains and have nearly identical N-terminal export and C-terminal surface anchoring domains but significantly different internal adhesive domains. Based on the comparison of derivatives of a laboratory strain of Haemophilus influenzae expressing either surface-associated Cha1 or surface-associated Cha2, Cha1 mediates a higher level of adherence to cultured human epithelial cells and Cha2 mediates a higher level of adherence to abiotic surfaces. We hypothesize that variation in the Cha1 and Cha2 internal region results in changes in binding specificity or binding affinity and may be associated with adaptation to different host environments during colonization and disease.

Generally accepted laboratory methods that have been used for decades do not reliably distinguish between H. influenzae and H. haemolyticus isolates. H. haemolyticus strains are often incorrectly identified as nontypeable Haemophilus influenzae (NTHi). To distinguish H. influenzae from H. haemolyticus we have created a new database on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) bio-typer 2 and compared the results with routine determination of Haemophilus (growth requirement for X and V factor), and multilocus sequence typing (MLST). In total we have tested 277 isolates, 244 H. influenzae and 33 H. haemolyticus. Using MLST as the gold standard, the agreement of MALDI-TOF MS was 99.6 %. MALDI-TOF MS allows reliable and rapid discrimination between H. influenzae and H. haemolyticus.

β-barrel membrane proteins are essential for nutrient import, signalling, motility and survival. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β-barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Here we describe the structure of BamA, the central and essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features shed light on the mechanism by which BamA catalyses β-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the β-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane.

Background: Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level., Methodology: A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction., Principal Findings: Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system's database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients.

Nontypable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus exhibit different pathogenicities, but to date, there remains no definitive and reliable strategy for differentiating these strains. In this study, we evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a potential method for differentiating NTHi and H. haemolyticus. The phylogenetic analysis of concatenated 16S rRNA and recombinase A (recA) gene sequences, outer membrane protein P6 gene sequencing and single-gene PCR were used as reference methods. The original reference database (ORD, provided with the Biotyper software) and new reference database (NRD, extended with Chinese strains) were compared for the evaluation of MALDI-TOF MS. Through a search of the ORD, 76.9% of the NTHi (40/52) and none of the H. haemolyticus (0/20) strains were identified at the species level. However, all NTHi and H. haemolyticus strains used for identification were accurately recognized at the species level when searching the NRD. From the dendrogram clustering of the main spectra projections, the Chinese and foreign H. influenzae reference strains were categorized into two distinct groups, and H. influenzae and H. haemolyticus were also separated into two categories. Compared to the existing methods, MALDI-TOF MS has the advantage of integrating high throughput, accuracy and speed. In conclusion, MALDI-TOF MS is an excellent method for differentiating NTHi and H. haemolyticus. This method can be recommended for use in appropriately equipped laboratories.

Background: How DNA 5-cytosine methyltransferases (DCMTases) select their substrate nucleobase for extrusion from DNA duplex is poorly understood., Results: The crystal structure of a pre-extrusion M.HaeIII DCMTase-substrate DNA complex is reported here., Conclusion: M.HaeIII selects its substrate cytosine for extrusion by selectively interfering with its stacking and hydrogen bonding interactions within the DNA duplex., Significance: This is the first structural elucidation of the target cytosine selection by a DCMTase. Epigenetic methylation of cytosine residues in DNA is an essential element of genome maintenance and function in organisms ranging from bacteria to humans. DNA 5-cytosine methyltransferase enzymes (DCMTases) catalyze cytosine methylation via reaction intermediates in which the DNA is drastically remodeled, with the target cytosine residue extruded from the DNA helix and plunged into the active site pocket of the enzyme. We have determined a crystal structure of M.HaeIII DCMTase in complex with its DNA substrate at a previously unobserved state, prior to extrusion of the target cytosine and frameshifting of the DNA recognition sequence. The structure reveals that M.HaeIII selects the target cytosine and destabilizes its base-pairing through a precise, focused, and coordinated assault on the duplex DNA, which isolates the target cytosine from its nearest neighbors and thereby facilitates its extrusion from DNA.

Haemophilus parainfluenzae is a Gram-negative bacterium that colonizes the upper respiratory tract of humans and is a part of normal flora. In this study, we investigated the lipopolysaccharide (LPS) expressed by H. parainfluenzae strain 20. Using NMR and MS techniques on LPS, oligosaccharide samples and lipid A, the structures for O-antigen, core oligosaccharide and lipid A could be established. It was found that the biological repeating unit of the O-antigen is →4)-α-D-GalpNAc-(1→P→6)-β-D-Glcp-(1→3)-α-D-FucpNAc4N-(1→, in which D-FucpNAc4N is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. This sugar is in β-configuration when linked to O-4 of the glucose residue of β-D-Galp-(1→2)-L-α-D-Hepp-(1→2)-[PEtn→6]-L-α-D-Hepp-(1→3)-[β-D-Glcp-(1→4)]-L-α-D-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. LPS from a wbaP mutant of H. parainfluenzae strain 20 did not contain an O-antigen, consistent with the wbaP gene product being required for expression of O-antigen in fully extended LPS., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid and accurate tool for the identification of many microorganisms. We assessed this technology for the identification of 103 Haemophilus parainfluenzae, Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae (HACEK) clinical isolates and 20 Haemophilus influenzae clinical isolates. Ninety-three percent of HACEK organisms were identified correctly to the genus level using the Bruker database, and 100% were identified to the genus level using a custom database that included clinical isolates.

Background: Although non-typeable (NT) Haemophilus influenzae and Haemophilus haemolyticus are closely related human commensals, H. haemolyticus is non-pathogenic while NT H. influenzae is an important cause of respiratory tract infections. Phase-variable phosphorylcholine (ChoP) modification of lipooligosaccharide (LOS) is a NT H. influenzae virulence factor that, paradoxically, may also promote complement activation by binding C-reactive protein (CRP). CRP is known to bind more to ChoP positioned distally than proximally in LOS, and the position of ChoP within LOS is dictated by specific licD alleles (designated here as licDI, licDIII, and licDIV) that are present in a lic1 locus. The lic1 locus contains the licA-licD genes, and ChoP-host interactions may also be influenced by a second lic1 locus that allows for dual ChoP substitutions in the same strain, or by the number of licA gene tetranucleotide repeats (5'-CAAT-3') that reflect phase-variation mutation rates., Results: Using dot-blot hybridization, 92% of 88 NT H. influenzae and 42.6% of 109 H. haemolyticus strains possessed a lic1 locus. Eight percent of NT H. influenzae and none of the H. haemolyticus strains possessed dual copies of lic1. The licDIII and licDIV gene alleles were distributed similarly (18-22%) among the NT H. influenzae and H. haemolyticus strains while licDI alleles were present in 45.5% of NT H. influenzae but in less than 1% of H. haemolyticus strains (P < .0001). NT H. influenzae had an average of 26.8 tetranucleotide repeats in licA compared to 14.8 repeats in H. haemolyticus (P < .05). In addition, NT H. influenzae strains that possessed a licDIII allele had increased numbers of repeats compared to NT H. influenzae with other licD alleles (P < .05)., Conclusions: These data demonstrate that genetic similarities and differences of ChoP expression exist between NT H. influenzae and H. haemolyticus and strengthen the hypothesis that, at the population level, these differences may, in part, provide an advantage in the virulence of NT H. influenzae.

Structural patterns of bacterial capsular antigens including capsular polysaccharides and exoglycans are given in this review. In addition, the immunological activity of capsular antigens and their role in type specificity of bacteria are discussed.

The structure for the carbohydrate moiety of the lipooligosaccharide (LOS) from the commensal Haemophilus somnus strain 129Pt was elucidated. The structure of the core oligosaccharide and O-deacylated LOS was established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the major fully extended carbohydrate glycoform of the LOS was determined on the basis of the combined data from these experiments. [Carbohydrate structure: see text]. In the structure Kdo is 3-deoxy-D-manno-octulosonic acid, Hep is L-glycero-D-manno-heptose and PEtn is phosphoethanolamine. Minor amounts of glycoforms containing nonstoichiometric substituents glycine and phosphate at the distal heptose residue were also identified.

The structure of the lipooligosaccharide (LOS) from the commensal Haemophilus somnus strain 1P was elucidated. The structure of the O-deacylated LOS was established by monosaccharide analysis, NMR spectroscopy and mass spectrometry. The following structure for the O-deacylated LOS was determined on the basis of the combined data from these experiments. [chemical structure: see text] In the structure Kdo is 3-deoxy-D-manno-octulosonic acid, Hep is L-glycero-D-manno-heptose and lipid A-OH refers to O-deacylated Lipid A. The elucidation of this structure has increased our understanding of the relationship between the variability in LOS structure and the pathogenic potential of this organism. Specifically, the inability of this commensal strain to sialylate its LOS suggests that LOS sialylation could be a crucial virulence factor for H. somnus.

On the basis of a species-specific PCR assay, a RFLP analysis for typing of Haemophilus parasuis strains was developed and evaluated. Amplification was based on the gene tbpA, encoding a transferrin-binding protein. RFLP analysis of the 1.9-kb tbpA-amplicon using TaqI, AvaI and RsaI endonucleases produced 12 different patterns for the reference strains of the 15 known H. parasuis serovars, and showed a high heterogeneity (33 RFLP groups) for 101 H. parasuis clinical isolates tested. The sensitivity, typeability (100% versus 65% for immunodiffusion), high degree of discrimination (0.93 versus 0.84 for immunodiffusion), simplicity and low cost per test make this PCR-RFLP assay a useful method for typing H. parasuis and, therefore, for studying the epidemiology of outbreaks of Glässer's disease.

Mutations in the gene encoding for a de novo methyltransferase, DNMT3B, lead to an autosomal recessive Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome. To analyse the protein structure and consequences of ICF-causing mutations, we modelled the structure of the DNMT3B methyltransferase domain based on Haemophilus haemolyticus protein in complex with the cofactor AdoMet and the target DNA sequence. The structural model has a two-subdomain fold where the DNA-binding region is situated between the subdomains on a surface cleft having positive electrostatic potential. The smaller subdomains of the methyltransferases differ in length and sequences and therefore only the target recognition domain loop was modelled to show the location of an ICF-causing mutation. Based on the model, the DNMT3B recognizes the GC sequence and flips the cytosine from the double-stranded DNA to the catalytic pocket. The amino acids in the cofactor and target cytosine binding sites and also the electrostatic properties of the binding pockets are conserved. In addition, a registry of all known ICF-causing mutations, DNMT3Bbase, was constructed. The structural principles of the pathogenic mutations based on the modelled structure and the analysis of chi angle rotation changes of mutated side chains are discussed.

Polyserositis caused by Haemophilus parasuis is an important disease that affects mostly weaned pigs. Recent studies have shown that virulence can differ among strains recovered from distinct body sites and also that it may be related to the presence of certain outer membrane proteins (OMPs). The objective of this study was to compare the OMP and DNA profiles of H. parasuis strains isolated from systemic and respiratory sites from diseased and healthy pigs. Strains evaluated in this study were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and repetitive-PCR techniques. Two experiments were conducted in order to better define the relationship among genotype, phenotype, and site of isolation. Experiment 1 included 53 H. parasuis isolates recovered from healthy and diseased pigs from unrelated herds. Experiment 2 included 31 isolates of H. parasuis obtained from diseased pigs involved in an outbreak in a large, multifarm system. Results showed that strains recovered from systemic sites had more homogeneous OMP and DNA profiles than those isolated from respiratory sites. Evaluation of isolates involved in the multifarm outbreak showed that only two H. parasuis strains were causing disease. These strains had homogeneous OMP and DNA profiles. However, it was noted that these two parameters were unrelated, since strains classified in the same genotype group expressed different OMP profiles. The homogeneity of OMP and DNA profiles of strains isolated from systemic sites strongly suggests the existence of clonal relationships between virulent strains and also suggests that expression of certain OMP profiles may be related to virulence.

One of the plasmids present in a Haemophilus somnus strain isolated from nasal discharge of a cattle with respiratory disease was purified and cloned for DNA sequencing. The plasmid was found to be 1065 base pairs long with 39.2% G + C content, and showed no homology to any DNA sequenced so far. It has no capacity to code any protein longer than 43 residues. It is not clear yet if this plasmid codes Haemophilus somnus specific factors.

Cellular levels of diaminopropane, putrescine and cadaverine, and decarboxylase activities to produce these diamines in six species (16 strains) of Haemophilus and four species (5 strains) of Actinobacillus belonging to the family Pasteurellaceae of the gamma subclass of the class Proteobacteria, were determined by high performance liquid chromatography (HPLC). Diaminopropane was ubiquitously distributed within all Haemophilus and Actinobacillus species, and L-2,4-diaminobutyric acid decarboxylase activity was detected in them. Putrescine and ornithine decarboxylase activity were found in H. aphrophilus, H. parainfluenzae and H. influenzae (type a, b, d, e and f except for type c) but not detected in H. aegyptius, H. parahaemolyticus, H. ducreyi and Actinobacillus species. Cadaverine occurred in H. aphrophilus, H. aegyptius, H. influenzae, H. parainfluenzae, A. actinomycetemcomitans, A. equuli and A. lignieresii, whereas their lysine decarboxylase activity was scarcely detected. Cadaverine was not found in H. parahaemolyticus, H. ducreyi and A. suis. The diamine profile serves as a phenotypic marker for the chemotaxonomic classification of the family Pasteurellaceae.

Many pathogens of the Pasteurellaceae and Neisseriaceae possess a surface receptor that binds transferrin (Tf) as an initial step in an iron acquisition process. This receptor is comprised of two proteins, transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB). Since the ability to recognize the iron-loaded form of Tf preferentially would be a useful attribute of these receptors, we examined this property in a number of bacterial species. In solid-phase binding assays with isolated membranes, only the receptor from Moraxella catarrhalis was capable of preferentially binding iron-loaded Tf. In a competitive affinity isolation assay which enabled us to resolve TbpA and TbpB, TbpA from all tested species was shown to bind both apo and iron-loaded Tf. Under these assay conditions TbpB from M. catarrhalis, Haemophilus somnus and Pasteurella haemolytica discriminated between apo and holo Tf, whereas TbpB from Neisseria meningitidis showed no discrimination. The ability of TbpB from N. meningitidis to bind iron-saturated hTf preferentially became evident in a TbpA- background or by using recombinant TbpB. In binding assays with recombinant fusion proteins, both intact TbpB and the N-terminal half of TbpB from all the tested species preferentially bound Fe-loaded Tf, indicating that this may be a conserved mechanism by which these organisms optimize their ability to acquire iron., (Copyright 1998 Academic Press.)

Several species of the genus Haemophilus are well known etiological agents of pneumonia, meningitis, conjunctivitis, epiglottitis and chancroid. However, identification and speciation of Haemophilus is both time consuming and labor intensive. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) has been used by several investigators to profile proteins from intact and disrupted bacteria; consequently, MALDI/TOF-MS has emerged as a powerful tool in diagnostic bacteriology. This paper reports the use of MALDI/TOF-MS as a technique for the rapid identification and speciation of Haemophilus. This technique was used to not only identify the pathogen, H. ducreyi, but also to determine strain differences from different isolates. Mass spectral 'fingerprints' were obtained which permitted the rapid speciation of not only pathogenic forms of Haemophilus, but also those bacteria which are normally regarded as non-pathogenic and members of the normal flora. MALDI/TOF mass spectra can be acquired in 10 min, allowing the identification of Haemophilus spp. within 24 h rather than the 48 h or more needed for traditional bacteriological methods. In addition, these are the first mass spectral fingerprints available in the literature for many of these organisms.

A fourth lipoprotein gene from Pasteurella haemolytica A1 was cloned and characterized. The plpD gene encodes a 31-kDa lipoprotein (Plp4) which could be recognized in Western immunoblot by sera from calves immunized with the culture supernatant vaccine Presponse. This suggests that Plp4 is one of the immunogenic molecules in the P. haemolytica A1 culture supernatant. The lipoprotein nature of Plp4 was confirmed by labelling with [3H]palmitate and inhibition of leader peptide cleavage with globomycin. A homology search with databanks showed extensive homology between Plp4 and a 31-kDa antigen from Haemophilus somnus and a 19.2-kDa antigen from Neisseria meningitidis. Additional homology of the distal half of Plp4 was identified with a number of bacterial outer membrane proteins belonging to the OmpA family. Plp4 appears to be a novel type of outer membrane protein that contains motifs typical of OmpA but which is also lipid modified.

The structure of the phase variable lipooligosaccharide (LOS) from Haemophilus somnus strain 738 was elucidated. The LOS was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the two major components were determined on the basis of the combined data from these experiments. [structure in text]. In the structures Kdo is 3-deoxy-D-manno-octulosonic acid, PEtn is phosphoethanolamine, PCho is phosphocholine, Hep is L-glycero-D-manno-heptose, and the remaining glucose units have the D configuration. The elucidation of these structures has increased our understanding of the relationship between the phase-variable LOS and the pathogenic potential of this organism.

Members of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135-148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2-D protein profile of the reference nontypable strain of H. influenzae (HI-64443) used as the basis for the 2-D protein database. A composite reference 2-D protein profile of HI-64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI-64443 proteins from the reference 2-D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI-64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2-D protein profile of the Rd strain of H. influenzae was compared to that of HI-64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI-64443 when analysed in parallel. The capacity of 2-D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A "knock-out" mutant in the sodA gene which encodes [Mn]-SOD was characterised with respect to protein synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2-D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.

Oral flora was classificated by SDS-PAGE combined with Comassie blue staining and silver staining. The results showed that each species of the test oral bacteria expressed its specific electrophorectic protein pattern, the way of sample preparation, staining did not change the protein pattern. This indicated that SDS-PAGE could be used to classificate oral flora. The protein pattern was more obvious among the genus than that of within the genus. This showed that different relationship was among the bacteria.

Bacterial pathogens have evolved various strategies to acquire iron from the iron-restricted environment found in mammalian hosts. Borrelia burgdorferi should be no different with regard to its requirement for ferric iron, and previous studies have suggested that transferrin (Tf) may be a source of iron in vivo. By probing blots with Tf conjugated to horseradish peroxidase, we have identified an outer membrane protein (28 kDa) from B. burgdorferi B31 that bound holo-Tf but not apo-Tf. The 28-kDa protein bound human, rat, or mouse Tf and was produced only by low-passage (less than passage 5), virulent isolates of strain B31. In addition, the Tf-binding protein (Tbp) from strain B31 retained the ability to bind Tf after treatment with 2% sodium dodecyl sulfate-1% beta-mercaptoethanol and heating to 100 degrees C for 5 min. These properties are remarkably similar to those of the Tbp of Staphylococcus aureus and Tbp2 from Neisseria meningitidis. B. burgdorferi Sh-2-82 produced an outer membrane protein different in size, i.e., 26 kDa, but with properties similar to those of to the protein from strain B31, suggesting variation in B. burgdorferi Tbps. The exact role of the 28-kDa protein in iron acquisition by B. burgdorferi remains to be determined.

Horse serum-supplemented Todd-Hewitt broth (STH) in use at Hôpital Ste-Justine for the last 12 years was compared to the recently proposed Haemophilus test medium (HTM), for broth microdilution susceptibility testing of Streptococcus pneumoniae. One hundred and twenty S. pneumoniae isolates from pediatric clinical specimens were used in this study. In general, the minimum inhibitory concentrations (MICs) in STH for 15 antimicrobial agents were quite comparable to those determined in HTM but tended to be higher. Drugs which generated MICs within +/- 1 log2 concentration differences in both media included penicillin, ampicillin, oxacillin, cefuroxime, cefotaxime, cefixime, clindamycin, chloramphenicol, trimethoprim-sulfamethoxazole, rifampin, ciprofloxacin and vancomycin. Cefaclor and tetracycline MICs tended to be > or = 2 log2 dilutions higher with STH for most of the isolates tested, while erythromycin MICs were often 2 log2 dilutions lower with STH than with HTM. Despite some differences in MICs noted above, few very major (0.4%), major (0.2%) and minor interpretive category errors (4.4%) were observed. The visual reading of the MICs for most of the 120 clinical isolates tested was generally easier in STH which was superior in supporting best the bacterial growth as detected by spectrophotometry. The risk of false susceptibility is thus decreased by using STH rather than HTM; furthermore, STH is free of the technical problems of the lysed horse blood Mueller-Hinton (LHB-MH) recommended by the NCCLS.

The present study was initiated to identify the region(s) of ovotransferrin involved in binding to the bacterial transferrin receptors from Haemophilus paragallinarum and Haemophilus avium. Ovotransferrin was digested with either trypsin or thermolysin to obtain its N-lobe and C-lobe fragments. The individual fragments were then purified by a combination of gel exclusion and ion-exchange chromatography. Solid phase binding experiments with the individual fragments demonstrated that the C-lobe fragments blocked the binding of horse radish peroxidase-conjugated ovotransferrin to the transferrin receptors and that much higher concentrations of the N-lobe fragment were required for any detectable blocking. Affinity isolation of the bacterial transferrin receptor from the two Haemophilus species revealed that both native ovotransferrin and its C-lobe fragment were capable of isolating two iron repressible outer membrane proteins. These 95 and 60 kDa outer membrane proteins correspond to Tbp1 and Tpb2, respectively. In contrast, the N-lobe fragment was capable of isolating Tbp2 of H. paragallinarum but not that of H. avium. The inability of the N-lobe and C-lobe fragments from ovotransferrin and human transferrin to support the growth of iron-limited cultures of H. paragallinarum and Neisseria meningitidis, respectively, suggested that interaction with both lobes is necessary for efficient iron acquisition.

A polyclonal antibody against the 35 kDa major outer-membrane protein of Pasteurella multocida cross-reacted with the 40 kDa major outer-membrane protein of Actinobacillus pleuropneumoniae and the 42 kDa major outer-membrane protein of Haemophilus parasuis. The N-terminal amino-acid sequences of these proteins revealed a strong homology with the putative 35 kDa porin protein of Pasteurella multocida (66.7 and 76.2%, respectively). Significant homologies were also evident between the 40 kDa and the 42 kDa protein (76.2%), and with non-specific porins of gram-negative bacteria.

The whole-cell carbohydrate patterns of 14 Haemophilus-like strains isolated from diseased birds were examined by capillary gas chromatography/mass spectrometry. The analysis of the peracetylated aldononitrile and O-methyloxime derivatives allowed the differentiation between the phenotypically and genetically different isolates. Starting from a pure culture the procedure needs only 5 hours for the preparation of the samples and 30 minutes for subsequent analysis and is of special value for rapid diagnosis.

The LPS patterns of 231 H.p. strains were studied by using SDS-PAGE. The strains were isolated from the nasal mucous membrane of clinical healthy animals, from animals with GK and from animals with pneumonia without any symptoms of GK. The LPS patterns of H.p. strains consists of 2 to 4 bands of high electrophoretical mobility. In all it was possible to distinguish seven different LPS electrophoretic profiles. The distribution of the H.p. isolates from clinically healthy animals and animals with GK or pneumonia to the 7 LPS electrophoretic profiles shows a similar picture. Variation in the growth conditions showed a process of a standardization of the LPS structure as a result of an increased CO2 atmosphere or lack of O2 respectively.

We report a 1.432-kb DNA sequence at 59 min on the Escherichia coli chromosome that connects the published sequences of the pcm gene for the isoaspartyl protein methyltransferase and that of the katF or rpoS (katF/rpoS) gene for a sigma factor involved in stationary-phase gene expression. Analysis of the DNA sequence reveals an open reading frame potentially encoding a polypeptide of 379 amino acids. The polypeptide sequence includes a consensus bacterial lipidation sequence present at residues 23 to 26 (Leu-Ala-Gly-Cys), four octapeptide proline- and glutamine-rich repeats of consensus sequence QQPQIQPV, and four heptapeptide threonine- and serine-rich repeats of consensus sequence PTA(S,T)TTE. The deduced amino acid sequence, especially in the C-terminal region, is similar to that of the Haemophilus somnus LppB lipoprotein outer membrane antigen (40% overall sequence identity; 77% identity in last 95 residues). The LppB lipoprotein binds Congo red dye and has been proposed to be a virulence determinant in H. somnus. Utilizing a plasmid construct with the E. coli gene under the control of a phage T7 promoter, we demonstrate the lipidation of this gene product by the incorporation of [3H]palmitic acid into a 42-kDa polypeptide. We also show that treatment of E. coli cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 46-kDa precursor. We thus designate the protein NlpD (new lipoprotein D). E. coli cells overexpressing NlpD bind Congo red dye, suggesting a common function with the H. somnus LppB protein. Disruption of the chromosomal E. coli nlpD gene by insertional mutagenesis results in decreased stationary-phase survival after 7 days.

Many members of the Actinobacillus-Haemophilus-Pasteurella group (family Pasteurellaceae) have been misclassified. This article reviews the chemotaxonomic characters that recently have been provided to improve the taxonomy of Pasteurellaceae. These include fatty acids of whole cells, of lipopolysaccharides and of single colonies, together with sugar contents of whole cells, of whole defatted cells, of lipopolysaccharides and of single colonies. This article also reviews taxonomy aided by distribution of proteins in whole cells and outer membranes, distribution of enzymes in outer membrane vesicles and in whole cells, bacteriolysis induced by ethylenediaminetetraacetic acid and hen eggwhite lysozyme and the distribution of respiratory quinones. Furthermore, an overview of characters obtained through studies on genetic transformation, restriction enzyme analysis, restriction fragment length polymorphism, DNA-DNA hybridization, DNA-rRNA hybridization, and 16S rRNA sequencing is given.

Chemical analysis by us has indicated that Actinobacillus actinomycetemcomitans is not a homogeneous species. The present study used chemometric methods and a multitude of chemical characters to examine this further. Strains were characterized by cell sugar and fatty acid contents, lysis kinetics during EDTA and EDTA plus lysozyme exposure, methylene blue reduction, and API ZYM enzymatic assessment of whole cells and outer membrane vesicles/fragments. In total, 41 quantitative variables were analyzed from each of 9 strains and treated with principal component analysis and soft independent modeling of class analogy. These methods divided A. actinomycetemcomitans into 2 strain groups. One group contained ATCC 33384, ATCC 29522, FDC 2112 and FDC 2043; the other comprised ATCC 29524, ATCC 29523, FDC 2097, FDC 511 and FDC Y4. With an F-test, the groups (classes) of A. actinomycetemcomitans strains could be distinguished at 95% confidence limits. Both groups were distinct from members of the genera Haemophilus and Pasteurella (Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Pasteurella multocida and Pasteurella haemolytica).

Selected strains representing established and newly described taxa in the family Pasteurellaceae were investigated for their cellular lipid and carbohydrate composition to clarify the taxonomic significance of such features. Methylated cellular fatty acids and acetylated derivatives of the cellular carbohydrates were determined by capillary gas chromatography using a flame ionization detector. In part the carbohydrates were identified by mass spectrometry. Phospholipids were determined by thin layer chromatography, the lipoquinones by high pressure liquid chromatography. The cellular fatty acid patterns proved to be uniform with minor variations, but the separation from the Neisseriaceae and from Moraxella was possible. Also the distribution of the phospholipids was uniform within the family. The lipoquinone contents were useful for the discrimination of groups within the family not necessarily reflecting the degree of genomic relatedness. The analysis of the cellular carbohydrates resulted in a common sugar pattern with all members of the family and characteristic carbohydrate profiles discriminating groups, often to the species level. All of the cytochemical features considered were useful for the characterization of the family Pasteurellaceae.

In immunoblot analysis, a murine monoclonal antibody (MAb), 27-1, which was produced to an outer membrane protein (OMP) of Haemophilus somnus, showed that a major OMP is heat modifiable, having a molecular mass of 28 kDa when the N-lauroylsarcosine-insoluble OMP preparation was solubilized at 60 degrees C and a mass of 37 kDa when the OMP preparation was solubilized at 100 degrees C. The heat-modifiable OMP reacted intensely with convalescent sera obtained from calves with experimental H. somnus pneumonia in immunoblot analysis. Immunoelectron microscopic and antibody absorption studies revealed that the MAb 27-1 epitope was not surface exposed on the intact bacterium. However, a decrease in antibody reactivity to the heat-modifiable OMP in immunoblot analysis after absorption of convalescent serum with intact bacterial cells of H. somnus suggests that a surface-exposed portion of the heat-modifiable OMP is expressed on the intact bacterium. MAb 27-1 reacted with 45 of 45 strains of H. somnus tested in immunoblot analysis. The apparent molecular mass of the antigen varied among strains, and five reactivity patterns demonstrated by MAb 27-1 were observed. MAb 27-1 also reacted with six species in the family Pasteurellaceae, Escherichia coli, and Salmonella dublin, but not with the other eight species of gram-negative bacteria. The heat-modifiable OMP of H. somnus showed immunological cross-reactivity with the OmpA protein of E. coli K-12 and significant N-terminal amino acid sequence homology with the OmpA proteins of gram-negative bacteria. We conclude that a major, 37-kDa heat-modifiable OMP of H. somnus, which elicits an antibody response in H. somnus-infected animals, is a common antigen among H. somnus strains tested and is structurally related to the OmpA protein of E. coli.

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophilus and Neisseria species is evident from the multiplicity of components observed with electrophoretic analyses. Knowledge of the precise structures that make up these diverse LOS molecules is clearly the key to reaching an understanding of pathogenic processes such as phase variation and molecular mimicry. Except for a few cases, little is known about the specific structural features of LOS that underlie phase variation and molecular mimicry, partly because of the inherent difficulties in the structural elucidation of these complex glycolipids. In the lipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia coli, rough, or R-type, mutants have been isolated that have provided insight into the biosynthetic pathways and associated genetics that control LPS expression. Nonetheless, recent work has shown that these R-type LPS are more complex than originally thought, and significant heterogeneity is still observed, primarily in their phosphorylation states. In order to investigate the structures of LPS and LOS in a more rapid fashion, we have determined the precise molecular weights of LOS (and LPS) preparations from various Haemophilus, Neisseria, and Salmonella species by electrospray ionization-mass spectrometry. The LOS (or LPS) were first O-deacylated under mild hydrazine conditions to remove O-linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M-nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Mass spectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heterogeneity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the variable phosphorylation of the core of S. typhimurium LPS have afforded insights into the biosynthetic pathways used by these organisms. Information of this type is important for understanding the underlying genetic and environmental factors controlling LOS and LPS expression.

A gene of Haemophilus somnus encoding the major 40,000-molecular-weight antigen (LppA) was cloned on a 2-kb Sau3AI fragment. The nucleotide sequence of the entire DNA insert was determined. One open reading frame, encoding a 247-residue polypeptide with a calculated molecular weight of 27,072, was identified. This reading frame was confirmed by sequencing the fusion joint of two independent IppA::TnphoA gene fusions. The 21 amino-terminal amino acids of the deduced polypeptide showed strong sequence homology to the signal peptide of secreted proteins, and the sequence Leu-Leu-Ala-Ala-Cys at the putative cleavage site is identical to the consensus cleavage sequence of lipoproteins from gram-negative bacteria. The presence of the lipid moiety on the protein was shown by incorporation of radioactive palmitic acid into the natural H. somnus protein. Palmitic acid could also be incorporated into the recombinant protein in Escherichia coli. Synthesis of the mature LppA lipoprotein was inhibited by globomycin, showing that cleavage of the signal peptide is mediated by signal peptidase II in both organisms. By using site-directed mutagenesis, the cysteine residue at the cleavage site was changed to glycine. Radiolabelled palmitate was not incorporated into the mutated protein, showing that lipid modification occurs at the Cys-22 residue.

Haemophilus parainfluenzae synthesizes an outer membrane protein adhesin which mediates binding to oral streptococci, salivary pellicle, and neuraminidase-treated erythrocytes. An indirect gold labeling technique and immunoelectron microscopy verified the location of this outer membrane protein. Further, a clustering of gold particles was observed in irregular patches at the cell surface.

Laboratory strains and fresh isolates of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus were examined for cell surface hydrophobicity and for electrokinetic properties under different experimental conditions. Fresh isolates of A. actinomycetemcomitans and H. aphrophilus were hydrophobic. Laboratory strains of A. actinomycetemcomitans were 20-30% less hydrophobic than fresh isolates. No difference was observed between laboratory and fresh isolates of H. aphrophilus. The pH of the suspending buffer, growth phase or incubation atmosphere did not significantly affect the hydrophobicity of the 2 species, whereas agar cultures of H. aphrophilus were less hydrophobic than broth cultures. All A. actinomycetemcomitans strains treated with sterile filtered saliva showed a concentration-dependent decrease in hydrophobicity of at most 30%. H. aphrophilus strains were not affected by the same treatment. Laboratory strains of H. aphrophilus were more negatively charged than A. actinomycetemcomitans, whereas fresh isolates of the 2 species exhibited similar surface charge. In the presence of saliva the mean cell surface charge of laboratory strains decreased by 56% for A. actinomycetemcomitans and by 73% for H. aphrophilus. The results indicate that the 2 species differ in expression of cell structures accounting for hydrophobicity and surface charge and that environmental factors might differently influence the physical properties of the two species analyzed.