Enhanced chitinase production by Chitinolyticbacter meiyuanensis SYBC-H1 using staged pH control.

The pH of a microbiological culture is important for both cell growth and chitinase accumulation, but the optimal pH is not normally the same for both. The objective of this study was to investigate the effect of pH on chitinase production by Chitinolyticbacter meiyuanensis strain SYBC-H1 (ATCC BAA-2140) in a mineral medium. The results of batch culture at different pH values showed that the optimum pH for cell growth and chitinase production varied with time, although KOH produced the best results for cell growth and chitinase production, NaOH was chosen because of cost considerations. We designed a three-stage pH control strategy using NaOH as the neutralizing agent. Maximum cell growth (1.07 g dry cell weight/l) and maximum chitinase activity (13.6 U/ml) were observed after culture at 26°C for 72 h in a mineral medium. These values were greater by 129% and 162%, respectively, and the length of time to attain maximum chitinase activity was decreased by 12 h, compared with results from an earlier study (Hao et al., 2011b).

Cloning, Expression and 3D Structure Prediction of Chitinase from Chitinolyticbacter meiyuanensis SYBC-H1.

Two CHI genes from Chitinolyticbacter meiyuanensis SYBC-H1 encoding chitinases were identified and their protein 3D structures were predicted. According to the amino acid sequence alignment, CHI1 gene encoding 166 aa had a structural domain similar to the GH18 type II chitinase, and CHI2 gene encoding 383 aa had the same catalytic domain as the glycoside hydrolase family 19 chitinase. In this study, CHI2 chitinase were expressed in Escherichia coli BL21 cells, and this protein was purified by ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G-100 chromatography. Optimal activity of CHI2 chitinase occurred at a temperature of 40 °C and a pH of 6.5. The presence of metal ions Fe(3+), Fe(2+), and Zn(2+) inhibited CHI2 chitinase activity, while Na⁺ and K⁺ promoted its activity. Furthermore, the presence of EGTA, EDTA, and ß-mercaptoethanol significantly increased the stability of CHI2 chitinase. The CHI2 chitinase was active with p-NP-GlcNAc, with the Km and Vm values of 23.0 µmol/L and 9.1 mM/min at a temperature of 37 °C, respectively. Additionally, the CHI2 chitinase was characterized as an N-acetyl glucosaminidase based on the hydrolysate from chitin. Overall, our results demonstrated CHI2 chitinase with remarkable biochemical properties is suitable for bioconversion of chitin waste.

Chiral amine synthesis using ω-transaminases: an amine donor that displaces equilibria and enables high-throughput screening.

The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by-product removal using only one equivalent of a diamine donor (ortho-xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone). Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity.

Heme utilization by pathogenic bacteria: not all pathways lead to biliverdin.

The eukaryotic heme oxygenases (HOs) (E.C. 1.14.99.3) convert heme to biliverdin, iron, and carbon monoxide (CO) in three successive oxygenation steps. Pathogenic bacteria require iron for survival and infection. Extracellular heme uptake from the host plays a critical role in iron acquisition and virulence. In the past decade, several HOs required for the release of iron from extracellular heme have been identified in pathogenic bacteria, including Corynebacterium diphtheriae, Neisseriae meningitides, and Pseudomonas aeruginosa. The bacterial enzymes were shown to be structurally and mechanistically similar to those of the canonical eukaryotic HO enzymes. However, the recent discovery of the structurally and mechanistically distinct noncanonical heme oxygenases of Staphylococcus aureus and Mycobacterium tuberculosis has expanded the reaction manifold of heme degradation. The distinct ferredoxin-like structural fold and extreme heme ruffling are proposed to give rise to the alternate heme degradation products in the S. aureus and M. tuberculosis enzymes. In addition, several "heme-degrading factors" with no structural homology to either class of HOs have recently been reported. The identification of these "heme-degrading proteins" has largely been determined on the basis of in vitro heme degradation assays. Many of these proteins were reported to produce biliverdin, although no extensive characterization of the products was performed. Prior to the characterization of the canonical HO enzymes, the nonenzymatic degradation of heme and heme proteins in the presence of a reductant such as ascorbate or hydrazine, a reaction termed "coupled oxidation", served as a model for biological heme degradation. However, it was recognized that there were important mechanistic differences between the so-called coupled oxidation of heme proteins and enzymatic heme oxygenation. In the coupled oxidation reaction, the final product, verdoheme, can readily be converted to biliverdin under hydrolytic conditions. The differences between heme oxygenation by the canonical and noncanonical HOs and coupled oxidation will be discussed in the context of the stabilization of the reactive Fe(III)-OOH intermediate and regioselective heme hydroxylation. Thus, in the determination of heme oxygenase activity in vitro, it is important to ensure that the reaction proceeds through successive oxygenation steps. We further suggest that when bacterial heme degradation is being characterized, a systems biology approach combining genetics, mechanistic enzymology, and metabolite profiling should be undertaken.

A regio- and stereoselective ω-transaminase/monoamine oxidase cascade for the synthesis of chiral 2,5-disubstituted pyrrolidines.

Biocatalytic approaches to the synthesis of optically pure chiral amines, starting from simple achiral building blocks, are highly desirable because such motifs are present in a wide variety of important natural products and pharmaceutical compounds. Herein, a novel one-pot ω-transaminase (TA)/monoamine oxidase (MAO-N) cascade process for the synthesis of chiral 2,5-disubstituted pyrrolidines is reported. The reactions proceeded with excellent enantio- and diastereoselectivity (>94 % ee; >98 % de) and can be performed on a preparative scale. This methodology exploits the complementary regio- and stereoselectivity displayed by both enzymes, which ensures that the stereogenic center established by the transaminase is not affected by the monoamine oxidase, and highlights the potential of this multienzyme cascade for the efficient synthesis of chiral building blocks.

Heterologous expression and functional characterization of a novel chitinase from the chitinolytic bacterium Chitiniphilus shinanonensis.

Chitiniphilus shinanonensis strain SAY3(T) is a chitinolytic bacterium isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. Fifteen genes encoding putative chitinolytic enzymes (chiA-chiO) have been isolated from this bacterium. Five of these constitute a single operon (chiCDEFG). The open reading frames of chiC, chiD, chiE, and chiG show sequence similarity to family 18 chitinases, while chiF encodes a polypeptide with two chitin-binding domains but no catalytic domain. Each of the five genes was successfully expressed in Escherichia coli, and the resulting recombinant proteins were characterized. Four of the recombinant proteins (ChiC, ChiD, ChiE, and ChiG) exhibited endo-type chitinase activity toward chitinous substrates, while ChiF showed no chitinolytic activity. In contrast to most endo-type chitinases, which mainly produce a dimer of N-acetyl-D-glucosamine (GlcNAc) as final product, ChiG completely split the GlcNAc dimer into GlcNAc monomers, indicating that it is a novel chitinase.

Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase.

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 ß-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.

Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis.

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced ß-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.

Sequence-based predictions of lipooligosaccharide diversity in the Neisseriaceae and their implication in pathogenicity.

Endotoxin [Lipopolysaccharide (LPS)/Lipooligosaccharide (LOS)] is an important virulence determinant in gram negative bacteria. While the genetic basis of endotoxin production and its role in disease in the pathogenic Neisseria has been extensively studied, little research has focused on the genetic basis of LOS biosynthesis in commensal Neisseria. We determined the genomic sequences of a variety of commensal Neisseria strains, and compared these sequences, along with other genomic sequences available from various sequencing centers from commensal and pathogenic strains, to identify genes involved in LOS biosynthesis. This allowed us to make structural predictions as to differences in LOS seen between commensal and pathogenic strains. We determined that all neisserial strains possess a conserved set of genes needed to make a common 3-Deoxy-D-manno-octulosonic acid -heptose core structure. However, significant genomic differences in glycosyl transferase genes support the published literature indicating compositional differences in the terminal oligosaccharides. This was most pronounced in commensal strains that were distally related to the gonococcus and meningococcus. These strains possessed a homolog of heptosyltransferase III, suggesting that they differ from the pathogenic strains by the presence a third heptose. Furthermore, most commensal strains possess homologs of genes needed to synthesize lipopolysaccharide (LPS). N. cinerea, a commensal species that is highly related to the gonococcus has lost the ability to make sialyltransferase. Overall genomic comparisons of various neisserial strains indicate that significant recombination/genetic acquisition/loss has occurred within the genus, and this muddles proper speciation.

Detection of Deefgea chitinilytica in freshwater ornamental fish.

AIM: To identify and characterize six chitinolytic bacterial strains isolated from ornamental fish. METHODS AND RESULTS: Six different isolates of Deefgea chitinilytica were detected in healthy as well as diseased ornamental fish in Germany over a period of 2 years. Bacterial strains were identified using 16S rRNA partial gene sequencing and further characterized using different biochemical microtest systems and additional standard biochemical tests. CONCLUSION: We show that commercially available biochemical microtest systems are useful for identification of D. chitinilytica, supplemented by 16S rRNA partial gene sequencing. Furthermore, this study provides new information about the occurrence of D. chitinilytica, as this is the first isolation of D. chitinilytica from animals and first described isolation in Europe. SIGNIFICANCE AND IMPACT OF THE STUDY: Deefgea chitinilytica may be isolated regularly in fish diagnostic laboratories. Therefore, accurate identification of this bacterial species is important. Involvement of D. chitinilytica in opportunistic infections of aquatic organisms cannot be excluded and has to be further investigated.

Chitiniphilus shinanonensis gen. nov., sp. nov., a novel chitin-degrading bacterium belonging to Betaproteobacteria.

A bacterial strain capable of degrading chitin, strain SAY3T, was isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. The strain was gram-negative, curved rod-shaped, facultatively anaerobic, and motile with a single polar flagellum. It grew well with chitin as a sole carbon source. The cellular fatty acids profiles showed the presence of C16:1 omega7c and C16:0 as the major components. The G+C content of DNA was 67.6 mol% and Q-8 was the major respiratory quinone. A 16S rRNA gene sequence-based phylogenetic analysis showed the strain belonged to the family Neisseriaceae but was distantly related (94% identity) to any previously known species. Since the strain was clearly distinct from closely related genera in phenotypic and chemotaxonomic characteristics, it should be classified under a new genus and a new species. We propose the name Chitiniphilus shinanonensis gen. nov., sp. nov. The type strain is SAY3T (=NBRC 104970T=NICMB 14509T).

Clinical interpretation of beta-lactamase-producing strains of Branhamella catarrhalis in sputum Gram's stain and culture.

Branhamella catarrhalis has been implicated previously as a cause of bronchopulmonary infections. Sputum Gram's stain and culture results suggesting significant infection with beta-lactamase-producing strains of B. catarrhalis were correlated with a retrospective chart review of eight pediatric and ten adult patients. Preexisting pulmonary disease was observed in 12 patients; 5 had a history of aspiration; and 13 were intubated. Clinically, ten patients had pneumonia, five had bronchitis, and three manifested no disease. Only three sputum specimens grew a pure culture of B. catarrhalis, and six specimens yielded B. catarrhalis in the presence of normal upper respiratory flora. Analysis of broth microdilution susceptibility test results showed that 90% of the strains were inhibited at the following minimum inhibitory concentrations (MICs90): ampicillin, 8 micrograms/mL; cefotaxime, 0.5 microgram/mL; cefoxitin, 0.5 microgram/mL; cephalexin, 4 micrograms/mL; cephalothin, 8 micrograms/mL; chloramphenicol, 1 microgram/mL; clindamycin, 4 micrograms/mL; erythromycin, 0.25 microgram/mL; methicillin, 16 micrograms/mL; mezlocillin, 16 micrograms/mL; moxalactam less than or equal to 0.6 microgram/mL; penicillin, 16 micrograms/mL; piperacillin, 8 micrograms/mL; tetracycline, less than or equal to 0.3 microgram/mL; and trimethoprim/sulfamethoxazole, 1.6/30 micrograms/mL. Therapy may have been adequate in only eight (44%) of the cases. However, all but four of the patients, who died of unrelated causes, exhibited resolution of disease. The data indicate that Gram's stain and culture results of sputum specimens suggesting B. catarrhalis bronchopulmonary infection should be interpreted with caution by clinicians.

Acute otitis media caused by Branhamella catarrhalis: biology and therapy.

Since 1980, we have observed an epidemic of otitis media caused by Branhamella catarrhalis. This event was characterized by studying the nasopharyngeal colonization of infants and children with B. catarrhalis and the clinical presentation and therapeutic outcome of acute otitis media caused by this organism. Pharyngeal colonization with B. catarrhalis was commoner in winter than summer. B. catarrhalis was present in middle-ear fluid (MEF) of 17% of children with otitis media, and was commoner in fall and winter (20%) than in spring and summer (11%, P less than .05). Seventy-five percent of isolates produced beta-lactamase (Ravasio type). In five of 20 patients, treatment with beta-lactamase-susceptible agents failed to sterilize B. catarrhalis-infected MEF. All of these five patients were infected with beta-lactamase-producing strains. The increasing prominence of antibiotic-resistant B. catarrhalis in acute otitis media may lead to a reevaluation of initial antibiotic therapy for acute otitis media, particularly in winter or in areas where colonization with such strains is prevalent.

Enzymatic modification of aminoglycoside antibiotics by Branhamella catarrhalis carrying an R factor.

Fifteen out of 89 clinical strains of Branhamella catarrhalis isolated from patients at the University Hospital of Zaragoza were resistant to aminoglycosides and other antimicrobials. In two strains, B. catarrhalis 220 and B. catarrhalis 115, the resistance to aminoglycosides was associated with synthesis of aminoglycoside-modifying enzymes, namely 3"-O-phosphotransferase [APH(3")] and 3'-O-phosphotransferase [APH(3')]. B. catarrhalis 115 was resistant to ampicillin, streptomycin, kanamycin, neomycin, butirosin, lividomycin, ribostamycin, paromomycin and trimethoprim-sulfamethoxazole and harboured a 32 megadalton (Md) plasmid. The resistance determinants of the latter were transferred to Neisseria subflava by conjugation and to Escherichia coli by transformation. The transconjugant strain presented an antibiotic resistance pattern similar to the donor strain and carried the same plasmid. The transformant strain acquired the 32 Md plasmid but presented, besides the resistance pattern already mentioned, resistance to tetracycline, gentamicin and tobramycin. Resistance to gentamicin and tobramycin was mediated by the synthesis of a 3-N-acetyltransferase. This resistance and the related enzyme were expressed neither in the donor B. catarrhalis strain nor in the transconjugant N. subflava strain.

In vitro activity of amoxicillin plus clavulanic acid against Haemophilus influenzae and Branhamella catarrhalis.

The in vitro activity of amoxicillin in the presence of clavulanic acid against clinical isolates of Haemophilus influenzae and Branhamella catarrhalis was assessed in comparison with ampicillin, amoxicillin, cefaclor and erythromycin. The isolates were selected so as to yield equal numbers of beta-lactamase producing and non-beta-lactamase producing strains of the two species. MICs obtained by agar dilution indicated that amoxicillin in the presence of clavulanic acid was the most active of the drugs tested. Clavulanic acid potentiated the activity of amoxicillin against beta-lactamase-producing strains of both Haemophilus influenzae and Branhamella catarrhalis. Further studies on a few strains of each species revealed that the beta-lactamase of Haemophilus influenzae (TEM-1) rapidly inactivated ampicillin and slowly inactivated cefaclor but not cefuroxime. The Branhamella catarrhalis enzyme rapidly inactivated cefaclor, ampicillin and to some extent cefuroxime. Clavulanic acid afforded protection against the beta-lactamase action of both species when beta-lactam antibiotics were added to bacterial cultures.

Preliminary evaluation of a rapid colorimetric method for identification of pathogenic Neisseria.

A rapid colorimetric method for the identification of pathogenic Neisseria (Identicult-Neisseria; Scott Laboratories, Inc.) based on beta-galactosidase, gamma-glutamylaminopeptidase, and gamma-prolylaminopeptidase is described. All 82 clinical isolates of Neisseria gonorrhoeae, 9 clinical isolates of N. meningitidis, and 5 clinical isolates of N. lactamica were correctly determined to the species level, as were 4 isolates of Branhamella catarrhalis. Reactions were prompt and easily interpreted. The system should be extremely useful in clinical laboratories.

Lack of immunoglobulin A1 protease production by Branhamella catarrhalis.

Clinical isolates of Branhamella catarrhalis from the sputum of 20 patients with acute bronchopulmonary infection were examined for synthesis of immunoglobulin A1 protease by immunoelectrophoresis. Ten strains produced beta-lactamase, and 10 were beta-lactamase negative. None of the strains demonstrated immunoglobulin A1 protease activity despite the fact that three different culture media were used.

The incidence and antibiotic susceptibility of Branhamella catarrhalis in respiratory infections.

The incidence of Branhamella catarrhalis in respiratory infections at City Hospital, Edinburgh from January 1981 to April 1984 is described. Beginning in January 1982 there was an increased incidence associated with a high proportion of beta-lactamase-producing strains. The number of these strains increased: from January 1981 to April 1983, 61% of strains produced beta-lactamase, and 83% produced beta-lactamase from January to April 1984. 53% of patients were infected in hospital. Environmental studies showed that 7% of staff and 8% of patients were carriers; there was also circumstantial evidence of ward and patient-to-patient infection. The antimicrobial susceptibility of 54 clinical strains was tested: all strains were resistant to trimethoprim but were susceptible to clavulanic acid plus amoxycillin, chloramphenicol, erythromycin, co-trimoxazole, cefotaxime and cefuroxime. beta-Lactamase-negative strains were uniformly susceptible to penicillin and ampicillin.

Incidence of Branhamella catarrhalis in the sputa of patients with chronic lung disease.

The incidence of Branhamella catarrhalis in the respiratory tract of adults, especially in the United States, is not known. During the 30-month period from January 1983 to June 1985, 4180 sputum and endotracheal samples from patients in a hospital for chest diseases were evaluated. All samples were acceptable for Gram-stain analysis and/or culture based on published cellular criteria. Using primarily Gram-stain directed cultures, 220 isolates of B. catarrhalis were identified in 180 patients, being present in 5.3% of all sputum cultures and 11.5% of those positive for a pathogen. B. catarrhalis was the fourth most common pathogen identified. It was found in pure culture (124) and mixed culture (96), the latter usually in association with Haemophilus influenzae or Streptococcus pneumoniae. Of the 220 B. catarrhalis isolates, 158 (71.8%) were positive for beta-lactamase. The number and incidence of B. catarrhalis varied, with the organism being most prevalent during the winter months. Despite its frequent presence in sputum, B. catarrhalis was not recovered from pleural fluid or blood during the same period. This study demonstrates the frequent presence of B. catarrhalis in the sputum of adults with chronic lung disease, although the role of this organism as a pathogen was not determined.

Upper respiratory tract infections. Ecological and therapeutic aspects of beta-lactamase production with special reference to Branhamella catarrhalis.

Available data indicate that the most common beta-lactamase produced by Branhamella catarrhalis is plasmid mediated. The same enzyme occurs in Moraxella nonliquefaciens, a commensal in the upper respiratory tract. The ability to produce the enzyme, which is known as BRO-1, can be transferred by conjugation from M. nonliquefaciens to B. catarrhalis. Since the first beta-lactamase-producing strains of B. catarrhalis appeared in 1977, the frequency of beta-lactamase production has increased rapidly; figures as high as 76% have been reported. The plasmid-mediated beta-lactamase TEM-1 occurs in several species of the genus Haemophilus. While the frequency of beta-lactamase production in H. influenzae is reported to be 10-15%, the incidence is significantly higher in non-pathogenic Haemophilus species. Both phenoxymethyl-penicillin and ampicillin promote the occurrence of beta-lactamase-producing strains, but the selective pressure exerted by ampicillin seems to be more pronounced. It may be possible to reduce the ecological effects of the penicillins by avoiding overdiagnosis of the most common bacterial infections of the respiratory tract, and by shortening the courses of antibiotic treatment.