Anna-Irini Koukkou

Arthrobacter phenanthrenivorans sp. nov., to accommodate the phenanthrene-degrading bacterium Arthrobacter sp. strain Sphe3.

A novel phenanthrene-degrading bacterium, designated strain Sphe3(T), was isolated from a creosote-contaminated soil in Greece. Cells were non-motile, Gram-positive, aerobic, and rod- to coccus-shaped. The strain was isolated on the basis of formation of a clear zone on agar plates sprayed with phenanthrene. Optimal growth occurred at 30 degrees C. The G+C content of the DNA was 65.7 mol%. The polar lipid pattern of strain Sphe3(T) consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant fatty acids were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(16 : 0), C(16 : 0) and anteiso-C(17 : 0), representing >86 % of the total fatty acids. The predominant isoprenoid quinone of strain Sphe3(T) was menaquinone-8 (MK-8). Based on 16S rRNA gene sequence analysis, strain Sphe3(T) showed 99 and 98.9 % similarity to the type strains of Arthrobacter oxydans and Arthrobacter polychromogenes, respectively. Strain Sphe3(T) showed 91 % similarity to homologues of A. oxydans and A. polychromogenes based on recA gene sequence analysis. Based on 16S rRNA and recA gene sequence analysis and DNA-DNA hybridization analysis, as well as physiological and chemotaxonomic characteristics, it is concluded that strain Sphe3(T) represents a novel species of the genus Arthrobacter, for which the name Arthrobacter phenanthrenivorans sp. nov. is proposed. The type strain is Sphe3(T) (=DSM 18606(T) =LMG 23796(T)).

Characterization of a Wild, Novel Nisin A-Producing Lactococcus Strain with an L. lactis subsp. cremoris Genotype and an L. lactis subsp. lactis Phenotype, Isolated from Greek Raw Milk

ABSTRACT Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis+) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijašic, and M. C. Montel, J. Food Prot. 72:783–790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897T strain, while they were distant from strains of the lactis genotype, including the LMG 6890T strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890T strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture.

Enhanced Control of Listeria monocytogenes by Enterococcus faecium KE82, a Multiple Enterocin–Producing Strain, in Different Milk Environments

Enterococcus faecium KE82, isolated from traditional Greek Graviera cheese, was identified in pure broth cultures in vitro as a multiple enterocin-producing bacterial strain possessing the structural entA, entB, and entP enterocin genes. E. faecium KE82 was further assessed for in situ antilisterial activity in raw milk (RM) and commercially thermized milk (TM; 63°C for 30 s) in the presence of the indigenous microbiota and in sterile raw milk (SRM; 121°C for 5 min) with or without the addition of two commercial starter culture (CSC) strains Streptococcus thermophilus and Lactococcus lactis . Growth of Listeria monocytogenes was completely inhibited in RM incubated at 37°C for 6 h, whereas the pathogen was significantly inactivated in RM+KE82 samples during further incubation at 18°C for 66 h. In contrast, L. monocytogenes levels increased by approximately 2 log CFU/ml in TM, but in TM+KE82 samples, pathogen growth was retarded during the first 6 h at 37°C followed by growth cessation and partial inactivation at 18°C. After 48 to 72 h, growth of L. monocytogenes in SRM+CSC samples decreased by 4 to 5 log CFU/ml compared with the SRM control, whereas additional 10-fold decreases in the pathogen were observed in SRM+CSC+KE82 samples. Reverse transcription PCR analysis of SRM+KE82 and SRM+CSC+KE82 samples confirmed that the entA and entB genes were transcribed, but entP gene transcription was not detected. All RM and SRM samples inoculated with E. faecium KE82 displayed strong in situ inhibitory activity against L. monocytogenes in well diffusion bioassays, whereas activity was weaker to undetectable in comparable or additional TM+KE82 samples; no milk sample without E. faecium KE82 had activity against L. monocytogenes . The findings of this study indicate that E. faecium KE82 is an antilisterial agent that could be used in traditional dairy foods because it concomitantly produces enterocins A and B in situ in milk.

Heterologous Expression and Characterization of Two 1-Hydroxy-2-Naphthoic Acid Dioxygenases from Arthrobacter phenanthrenivorans

ABSTRACT A protein fraction exhibiting 1-hydroxy-2-naphthoic acid (1-H2NA) dioxygenase activity was purified via ion exchange, hydrophobic interactions, and gel filtration chromatography from Arthrobacter phenanthrenivorans sp. nov. strain Sphe3 isolated from a Greek creosote-oil-polluted site. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS-MS) analysis revealed that the amino acid sequences of oligopeptides of the major 45-kDa protein species, as analyzed by SDS-PAGE and silver staining, comprising 29% of the whole sequence, exhibited strong homology with 1-H2NA dioxygenase of Nocardioides sp. strain KP7. A BLAST search of the recently sequenced Sphe3 genome revealed two putative open reading frames, named diox1 and diox2, showing 90% nucleotide identity to each other and 85% identity at the amino acid level with the Nocardia sp. homologue. diox1 was found on an indigenous Sphe3 plasmid, whereas diox2 was located on the chromosome. Both genes were induced by the presence of phenanthrene used as a sole carbon and energy source, and as expected, both were subject to carbon catabolite repression. The relative RNA transcription level of the chromosomal (diox2) gene was significantly higher than that of its plasmid (diox1) homologue. Both diox1 and diox2 putative genes were PCR amplified, cloned, and overexpressed in Escherichia coli. Recombinant E. coli cells expressed 1-H2NA dioxygenase activity. Recombinant enzymes exhibited Michaelis-Menten kinetics with an apparent K m of 35 μM for Diox1 and 29 μM for Diox2, whereas they showed similar kinetic turnover characteristics with K cat/K m values of 11 × 106 M−1 s−1 and 12 × 106 M−1 s−1, respectively. Occurrence of two diox1 and diox2 homologues in the Sphe3 genome implies that a replicative transposition event has contributed to the evolution of 1-H2NA dioxygenase in A. phenanthrenivorans.

A dialkylcyclohexadienecarbinol from the bacterium Zymomonas mobilis, a novel type of potential membrane lipid

Abstract (2-methyl-4,5-ditridecyl-1,4-cyclohexadienyl)methanol has been isolated from the lipidic fraction of the ethanologenic bacterium Zymomonas mobilis . The amphiphilic feature of this novel lipid is compatible with interactions within a phospholipid bilayers and suggests a function in membrane metabolism of the bacterium.

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G1LLKR5IKT8LL‐NH2, it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly1, Arg5, and Thr8 and lipophilic groups with different lengths in the N‐terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF5IKK8LL‐NH2 exhibited high activity against Gram‐negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N‐terminus increased the antimicrobial activity against Gram‐positive and Gram‐negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α‐helical conformation inducing better antimicrobial effects. Copyright

Major ecological shifts within the dominant nonstarter lactic acid bacteria in mature Greek Graviera cheese as affected by the starter culture type

Traditional Greek Graviera cheese is often produced from thermized milk to control undesirable bacterial contaminants. Since thermization also reduces the desirable lactic acid bacteria (LAB) microbiota of raw milk, natural undefined or commercially defined starters are used. This study evaluated effects of the type of starter added to bulk thermized milk on the microbiology of mature (day-90) Graviera cheese. Cheeses produced with a natural starter culture (NSC) in non-concentrated yogurt-like form or a commercial starter culture (CSC) containing Streptococcus thermophilus and various Lactococcus lactis strains in concentrated freeze-dried form, were analyzed microbiologically, and 200 LAB isolates (100 from each type of cheese) were identified. The LAB microbiota of the mature CSC-cheeses was dominated by nonstarter strains of Lactobacillus paracasei and Lb. plantarum whereas indigenous Enterococcus faecium and E. durans strains of high phenotypic and genotypic diversity predominated in the respective NSC-cheeses. Populations of enterococci in CSC-cheeses were subdominant by 10 to 100-fold compared with those in NSC-cheeses; E. faecium was the most frequently isolated Enterococcus species from the mature CSC-cheeses. Sporadic or no isolates of other LAB species, including the commercial S. thermophilus and Lc. lactis starter strains in the CSC-cheeses and the natural S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus starter strains plus indigenous Lactococcus, Leuconostoc and E. faecalis in the NSC-cheeses, were detected. In conclusion, the replacement of the NSC with the CSC controlled growth of dairy enterococci in favor of mesophilic nonstarter lactobacilli during ripening. While safety concerns associated with the inefficiency of NSCs to prevent outgrowth of indigenous enterococci suggest that CSCs should be preferred by traditional Greek Graviera cheese processors, panel sensory evaluations showed that the NSC-ripened cheeses were of slightly lower appearance but of occasionally higher flavor scores than the CSC-ripened cheeses.