Influence of Complex Nutrient Source on Growth of and Curvacin A Production by Sausage Isolate Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of a complex nutrient source were investigated in vitro during laboratory fermentations with modified MRS medium. A modification of the nutrient depletion model (Leroy and De Vuyst, Appl. Environ, Microbiol. 67:4470-4473, 2001) was used to fit the data describing growth and bacteriocin production. Both cell growth and bacteriocin activity were influenced by changes in the complex nutrient source concentration. Standard MRS medium clearly limited the growth of L. curvatus LTH 1174. Higher nutrient concentrations, up to a certain degree, led to improved growth, a higher attainable biomass concentration, and a higher bacteriocin activity in the supernatant. A lower concentration of complex nutrient source caused severe growth inhibition, leading to a lower biomass concentration but a much higher specific bacteriocin production. When examining the separate components of the complex nutrient source, a stimulating effect of bacteriological peptone on growth was found without an adverse effect on bacteriocin production, resulting in increased curvacin A activity. Furthermore, specific depletion of the amino acids tyrosine, serine, and asparagine/aspartic acid was observed for this strain.     

Sodium Chloride Reduces Production of Curvacin A, a Bacteriocin Produced by Lactobacillus curvatus Strain LTH 1174, Originating from Fermented Sausage

Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of L. curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k_B) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a_w-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X_B) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X_B, an increase in k_B, and hence an increase in the maximum attainable bacteriocin activity.     

Effects of Different Spices Used in Production of Fermented Sausages on Growth of and Curvacin A Production by Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the listericidal bacteriocin curvacin A. The effect of different spices relevant for the production of fermented sausages was investigated in vitro through laboratory fermentations with a meat simulation medium and an imposed pH profile relevant for Belgian-type fermented sausages. The influence on the growth characteristics and especially on the kinetics of curvacin A production with L. curvatus LTH 1174 was evaluated. Pepper, nutmeg, rosemary, mace, and garlic all decreased the maximum specific growth rate, while paprika was the only spice that increased it. The effect on the lag phase was minor except for nutmeg and especially for garlic, which increased it, yet garlic was stimulatory for biomass production. The maximum attainable biomass concentration (X_max) was severely decreased by the addition of 0.40% (wt/vol) nutmeg, while 0.35% (wt/vol) garlic or 0.80% (wt/vol) white pepper increased X_max. Nutmeg decreased both growth and bacteriocin production considerably. Garlic was the only spice enhancing specific bacteriocin production, resulting in higher bacteriocin activity in the cell-free culture supernatant. Finally, lactic acid production was stimulated by the addition of pepper, and this was not due to the manganese present because an amount of manganese that was not growth limiting was added to the growth medium. Addition of spices to the sausage mixture is clearly a factor that will influence the effectiveness of bacteriocinogenic starter cultures in fermented-sausage manufacturing.     

The Curing Agent Sodium Nitrite, Used in the Production of Fermented Sausages, Is Less Inhibiting to the Bacteriocin-Producing Meat Starter Culture Lactobacillus curvatus LTH 1174 under Anaerobic Conditions

Curvacin A is a listericidal bacteriocin produced by Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.     

Sequencing and expression analysis of sakacin genes in Lactobacillus curvatus strains

In this study, we focused our investigation on two strains of Lactobacillus curvatus, L442 and LTH1174, which are able to produce bacteriocins. L. curvatus LTH1174 is widely studied for its capability to produce curvacin A, while L. curvatus L442 was isolated from traditional Greek fermented sausages and was shown to possess a strong inhibitory activity toward Listeria monocytogenes. By polymerase chain reaction, we were able to target in both strains the genes for the production of sakacin P and sakacin Q, sppA and sppQ, respectively, both encoded chromosomally. While sppA was found to be conserved when compared with other sakacin P genes, sppQ showed a deletion of about 15 nucleotides when aligned with sequences obtained from Lactobacillus sakei. This difference did not affect the activity of sakacin Q as determined by testing sensitive strains. Expression analysis highlighted that sakacin P was expressed in L. curvatus L442 but not in L. curvatus LTH1174. Curing experiments were performed on L. curvatus LTH1174 to study the effect of the megaplasmid, present in this strain. In the plasmid-cured strain, expression of the sppA gene was detected. sppQ was expressed in both plasmid-cured and wild-type L. curvatus LTH1174, although expression was higher in the plasmid-cured strain.     

Influence of complex nutrient source on growth of and curvacin a production by sausage isolate Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of a complex nutrient source were investigated in vitro during laboratory fermentations with modified MRS medium. A modification of the nutrient depletion model was used to fit the data describing growth and bacteriocin production. Both cell growth and bacteriocin activity were influenced by changes in the complex nutrient source concentration. Standard MRS medium clearly limited the growth of L. curvatus LTH 1174. Higher nutrient concentrations, up to a certain degree, led to improved growth, a higher attainable biomass concentration, and a higher bacteriocin activity in the supernatant. A lower concentration of complex nutrient source caused severe growth inhibition, leading to a lower biomass concentration but a much higher specific bacteriocin production. When examining the separate components of the complex nutrient source, a stimulating effect of bacteriological peptone on growth was found without an adverse effect on bacteriocin production, resulting in increased curvacin A activity. Furthermore, specific depletion of the amino acids tyrosine, serine, and asparagine/aspartic acid was observed for this strain.     

Effects of different spices used in production of fermented sausages on growth of and curvacin A production by Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the listericidal bacteriocin curvacin A. The effect of different spices relevant for the production of fermented sausages was investigated in vitro through laboratory fermentations with a meat simulation medium and an imposed pH profile relevant for Belgian-type fermented sausages. The influence on the growth characteristics and especially on the kinetics of curvacin A production with L. curvatus LTH 1174 was evaluated. Pepper, nutmeg, rosemary, mace, and garlic all decreased the maximum specific growth rate, while paprika was the only spice that increased it. The effect on the lag phase was minor except for nutmeg and especially for garlic, which increased it, yet garlic was stimulatory for biomass production. The maximum attainable biomass concentration (X(max)) was severely decreased by the addition of 0.40% (wt/vol) nutmeg, while 0.35% (wt/vol) garlic or 0.80% (wt/vol) white pepper increased X(max). Nutmeg decreased both growth and bacteriocin production considerably. Garlic was the only spice enhancing specific bacteriocin production, resulting in higher bacteriocin activity in the cell-free culture supernatant. Finally, lactic acid production was stimulated by the addition of pepper, and this was not due to the manganese present because an amount of manganese that was not growth limiting was added to the growth medium. Addition of spices to the sausage mixture is clearly a factor that will influence the effectiveness of bacteriocinogenic starter cultures in fermented-sausage manufacturing.     

Sodium chloride reduces production of curvacin A, a bacteriocin produced by Lactobacillus curvatus strain LTH 1174, originating from fermented sausage

Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of L. curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k(B)) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a(w)-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X(B)) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X(B), an increase in k(B), and hence an increase in the maximum attainable bacteriocin activity.     

The curing agent sodium nitrite,used in the production of fermented sausages,is less inhibiting to the bacteriocin-producing meat starter culture Lactobacillus curvatus LTH 1174 under anaerobic conditions

Curvacin A is a listericidal bacteriocin produced by Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.     

Purification and characterization of curvaticin L442, a bacteriocin produced by Lactobacillus curvatus L442

Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without the addition of starters, produces a bacteriocin, curvaticin L442, which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Partial N-terminal sequence analysis using Edman degradation revealed 30 amino acid residues, revealing high homology with the amino acid sequence of sakacin P. Curvaticin L442 is active at pH values between 4.0 and 9.0 and it retains activity even after incubation for 5 min at 121 °C with 1 atm of overpressure. Proteolytic enzymes and α-amylase inactivated this curvaticin, while the effect of lipase was not severe.     

Biochemical and Genetic Characterization of Propionicin T1, a New Bacteriocin from Propionibacterium thoenii

A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii. This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii. The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA. The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions

Summary The sequence of an mRNA encoding nitrite reductase (NiR, EC 1.7.7.1.) from the tree Betula pendula was determined. A cDNA library constructed from leaf poly(A)⁺ mRNA was screened with an oligonucleotide probe deduced from NiR sequences from spinach and maize. A 2.5 kb cDNA was isolated that hybridized to an mRNA, the steady-state level of which increased markedly upon induction with nitrate. The nucleotide sequence of the cDNA contains a reading frame encoding a protein of 583 amino acids that reveals 79% identity with NiR from spinach. The transit peptide of the NiR precursor from birch was determined to be 22 amino acids in size by sequence comparison with NiR from spinach and maize and is the shortest transit peptide reported so far. A graphical evaluation of identities found in the NiR sequence alignment revealed nine well conserved sections each exceeding ten amino acids in size. Sequence comparisons with related redox proteins identified essential residues involved in cofactor binding. A putative binding site for ferredoxin was found in the N-terminal half of the protein.These sequence data appear in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X60093     

Evidence for Production of a New Lantibiotic (Butyrivibriocin OR79A) by the Ruminal Anaerobe Butyrivibrio fibrisolvens OR79: Characterization of the Structural Gene Encoding Butyrivibriocin OR79A

The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79A was a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5′ region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.     

Biochemical and Genetic Characterization of the Two-Peptide Bacteriocin Enterocin 1071 Produced by Enterococcus faecalis FAIR-E 309

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.     

Characterization of leucocin B-Ta11a: A bacteriocin fromLeuconostoc carnosum Ta11a isolated from meat

Leuconostoc (Lc.)carnosum Ta11a, isolated from vacuum-packaged processed meats, produced a bacteriocin designated leucocin B-Ta11a. The crude bacteriocin was heat stable and sensitive to proteolytic enzymes, but not to catalase, lysozyme, or chloroform. It was active againstListeria monocytogenes and several lactic acid bacteria. Leucocin B-Ta11a was optimally produced at 25°C in MRS broth at an initial pH of 6.0 or 6.5 An 8.9-MDa plasmid inLeuconostoc carnosum Ta11a hybridized to a 36-mer oligonucleotide probe (JF-1) that was homologous to leucocin A-UAL187. A 4.9-kbSau3A fragment from a partial digest of the 8.9-MDa plasmid was cloned into pUC118. The 8.1-kb recombinant plasmid (pJF8.1) was used for sequencing and revealed the presence of two open reading frames (ORFs). ORF1 codes for a protein of 61 amino acids comprising a 37-amino-acid bacteriocin that was determined to be the leucocin B-Ta11a structural gene by virtue of its homology to leucocin A-UAL 187 (Hastings et al. 1991. J. Bacteriol 173: 7491–7500). The 24-amino-acid N-terminal extension, however, differs from that of leucocin A-UAL187 by seven residues. The predicted protein of the ORF2 has 113 amino acids and is identical with the amino acid sequence of the cognate ORF of the leucocin A-UAL 187 operon.     

Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.     

粘质沙雷氏菌几丁质酶基因ChiA克隆及生物信息学分析

通过生物信息学技术对Chi A基因序列进行分析预测,了解Chi A的基因结构及蛋白质性质。从自有菌株(粘质沙雷氏菌Serratia mareescens S68)中克隆到几丁质酶基因Chi A,利用相关软件对Chi A基因序列进行分析预测。Chi A基因全长1 714 bp,开放阅读框编码563个氨基酸,推测其编码的蛋白质分子量为60 983.8Da,等电点为6.35,是一种稳定的亲水性蛋白质。预测Chi A可能存在信号肽,切割位点在第23~24位氨基酸之间,1~23位氨基酸为其跨膜结构,其余肽链位于细胞外。Chi A主要存在3种二级结构元件,在二级、三级结构中都有体现。该Chi A是一种水溶性蛋白质,结构稳定且可以分泌到胞外。     

The First Complete cDNA Sequence of the Hemocyanin from a Bivalve,the Protobranch <Emphasis Type="Italic">Nucula nucleus</Emphasis>

By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5′utr of 75 bp, a 3′utr of 791 bp, and an open reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (M _r = 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5′utr of 47 bp, a 3′utr of 647 bp, and an open reading frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (M _r = 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million ± 50 million years ago, in conformity with fossil records from the late Cambrian. [Reviewing Editor: Dr. Rüdiger Cerff] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ786639 for NnH1 and AJ786640 for NnH2.     

Cloning and sequencing of an endo-β-1,4-glucanase genemcenA fromMicromonospora cellulolyticum 86W-16

Summary Endo--1,4-glucanase genemcenA ofMicromonospora cellulolyticum 86W-16 was cloned, and the nucleotide sequence was determined. An open reading frame (ORF) of 1374 bases, coding for a peptide (McenA) of 457 amino acids and 46742 Da, was found. It is preceded by a Gram-positive type of ribosomebinding site and followed by an imperfect inverted repeat. A putative signal peptide containing 23 amino acids is at the N-terminus and a linker region possessing 37 amino acids is in the midpart of McenA. The N-half of McenA functions as the catalytic domain and the C-half might serve as a cellulosebinding domain (CBD). Deletion of the latter did not decrease the CMCase activity of McenA. Significant similarity (70%) was found between the amino acid sequences of McenA and MbcelA, an endoglucanase fromMicrobispora bispora.