Structural and Functional Differences in Two Cyclic Bacteriocins with the Same Sequences Produced by Lactobacilli

Kawai, Yasushi; Ishii, Yasuyuki; Arakawa, Kiyoshi; Uemura, Koichiro; Saitoh, Boku; Nishimura, Junko; Kitazawa, Haruki; Yamazaki, Yukiko; Tateno, Yoshio; Itoh, Takatoshi; Saito, Tadao

doi:10.1128/aem.70.5.2906-2911.2004

Cited by 84 publications

(68 citation statements)

References 38 publications

(38 reference statements)

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“…1A). A previous study established the insolubility of gassericin A in aqueous solution (44), similar to what was encountered with acidocin B. In the same study, CD data showed that gassericin A is ␣-helical in 60% isopropanol.…”

Section: Discussionsupporting

confidence: 70%

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Solution Structure of Acidocin B, a Circular Bacteriocin Produced by Lactobacillus acidophilus M46

Acedo

Belkum

Lohans

et al. 2015

Appl Environ Microbiol

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Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four ␣-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ϳ7 or lower.Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four ␣-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation. C ircular bacteriocins are antimicrobial peptides that are ribosomally synthesized by bacteria and are posttranslationally modified to release a leader peptide and form a peptide bond between the N and C termini. These peptides exhibit antimicrobial activity against a broad range of Gram-positive bacteria, including Listeria spp. and Clostridium spp., which are common pathogens causing food-borne diseases (1). In addition, the circular nature of these bacteriocins imparts enhanced stability against proteolytic degradation and denaturation due to extreme temperature and pH conditions relative to linear forms (2). They thus serve as promising alternatives to traditional antimicrobial agents for food, medical, and industrial applications (3).A number of circular bacteriocins that are composed of 58 to 70 amino acid residues have been identified to date, including enterocin AS-48 (4), gassericin A (5), circularin A (6), butyrivibriocin AR10 (7), uberolysin (8), carnocyclin A (9), lactocyclicin Q (10), garvicin ML (11), leucocyclicin Q (12), amylocyclicin (13), and aureocyclicin 4185 (14). Another peptide exhibiting Nto C-terminal cyclization is subtilosin A (15). It is, however, considered a member of the sactipeptides, which represent a class of peptides containing cross-links between cysteine sulfurs and ␣-carbons (16). The structure and genetics of circular bacteriocins were reviewed previously (2). More recently, a re...

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Section: Discussionsupporting

confidence: 70%

“…CD spectroscopy was used to screen solvent conditions suitable for further characterization of acidocin B via NMR spectroscopy. A previous report revealed that the high degree of hydrophobicity of gassericin A rendered it insoluble in aqueous solutions (44). Similarly, acidocin B could not be dissolved at a concentration required for NMR …”

Section: Resultsmentioning

confidence: 99%

Solution Structure of Acidocin B, a Circular Bacteriocin Produced by Lactobacillus acidophilus M46

Acedo

Belkum

Lohans

et al. 2015

Appl Environ Microbiol

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“…It has recently been established that the gassericin A and reutericin 6 class II bacteriocins possess D-alanines, apparently arising from L-alanine to D-alanine conversion (12). For quite some time, however, the presence of D-alanines in lacticin 3147 and lactocin S (13)(14)(15), members of a group of highly posttranslationally modified antimicrobial peptides called lantibiotics (class I bacteriocins), has been the subject of much interest because the chirality and the identity of the relevant amino acids are altered.…”

mentioning

confidence: 99%

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

Cotter

O’Connor²,

Draper³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

117

140

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As a general rule, ribosomally synthesized polypeptides contain amino acids only in the L-isoform in an order dictated by the coding DNA͞RNA. Two of a total of only four examples of L to D conversions in prokaryotic systems occur in posttranslationally modified antimicrobial peptides called lantibiotics. In both examples (lactocin S and lacticin 3147), ribosomally encoded L-serines are enzymatically converted to D-alanines, giving rise to an apparent mistranslation of serine codons to alanine residues. It has been suggested that this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the gene-encoded L-serine to dehydroalanine (dha). By using lacticin 3147 as a model system, we report the identification of an enzyme, LtnJ, that is responsible for the conversion of dha to D-alanine. Deletion of this enzyme results in the residues remaining as dha intermediates, leading to a dramatic reduction in the antimicrobial activity of the producing strain. The importance of the chirality of the three D-alanines present in lacticin 3147 was confirmed when these residues were systematically substituted by L-alanines. In addition, substitution with L-threonine (ultimately modified to dehydrobutyrine), glycine, or L-valine also resulted in diminished peptide production and͞or relative activity, the extent of which depended on the chirality of the newly incorporated amino acid(s).antimicrobial ͉ bacteriocin ͉ chirality T he presence of D-amino acids in ribosomally synthesized peptides was first observed in dermorphins, peptides found in the skin secretions of species of a subfamily of South American tree frogs, Phyllomedusinae. Subsequently, D-amino acids have been identified in a number of other ribosomally synthesized peptides of eukaryotic origin (1, 2). -Agatoxin IVb and bombinin H, produced by the funnel-web spider and the skin secretions of the frog Bombina variegata, respectively, represent the only instances in which the responsible enzymes, both peptide epimerases, have been identified (1, 2). For -agatoxin IVb the relevant L-serine to D-serine conversion is thought to involve a deprotonation͞reprotonation reaction (3). The presence of D-amino acids in many of these peptides is crucial, as demonstrated by the creation of synthetic analogues with Lrather than D-amino acids at the relevant locations, which are devoid of activity (4). The artificial incorporation of D-amino acids can also be beneficial. For example, a growing number of synthetic peptides͞peptide libraries containing D-amino acids have been assembled to capitalize on the ability of such residues to provide improved protease stability [enhanced pharmokinetic profile (5-7)], alter tertiary structure (new structural elements can be assembled that cannot be built solely from L-amino acids), and affect the activity of bioactive peptides [enhanced antibacterial activity with concomitant reduction in cell cytotoxicity (5, 6, 8-11)].In prokaryotic systems, there are only a few examples of L-to D-amino acid conversions. It...

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“…Although we (Y. K. and T. S.) cannot at this stage exclude the presence of other anti-microbial components in the bacteriocin preparations, we are also examining the possibility that the difference is caused by distinguished post-translational modification mechanisms in the two producers (eg. D-/Lamino acid conversions) [48].…”

Section: Discussionmentioning

confidence: 99%

The Circular Bacteriocins Gassericin A and Circularin A

Kawai

Kemperman

Kok

et al. 2004

CPPS

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Gassericin A, a bacteriocin produced by Lactobacillus gasseri LA39, shows antibacterial activity against a number of Gram-positive food-borne pathogenic bacteria. Circularin A produced by Clostridium beijerinckii ATCC25752 is active against C. tyrobutyricum, a known cheese-spoilage bacterium. Both bacteriocins were purified to homogeneity from culture supernatants by reverse-phase chromatography and the subsequently determined amino acid sequences were used to clone the bacteriocin structural genes. Mature gassericin A and circularin A are class V circular bacteriocins comprised of 58 and 69 amino acid residues, respectively. Both bacteriocins are resistant to several peptidases and proteases, as are other cyclic bacteriocins. Heterologous expression of gassericin A in Escherichia coli was used to produce a non-cyclic mature peptide, which was shown to have a specific activity 173-fold lower than the circular molecule. The minimal region for production and secretion of active circularin A is comprised of five genes, as was deduced by heterologous gene expression in Enterococcus faecalis . Gassericin A and circularin A have limited mutual similarity in their primary sequences. Unlike most bacteriocins, including gassericin A, circularin A has a three-aminoacid-leader sequence.

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Structural and Functional Differences in Two Cyclic Bacteriocins with the Same Sequences Produced by Lactobacilli

Cited by 84 publications

References 38 publications

Solution Structure of Acidocin B, a Circular Bacteriocin Produced by Lactobacillus acidophilus M46

Solution Structure of Acidocin B, a Circular Bacteriocin Produced by Lactobacillus acidophilus M46

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

The Circular Bacteriocins Gassericin A and Circularin A

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