Fate, Inducibility, and Behavior of Latilactobacillus curvatus Temperate Phage TMW 1.591 P1 during Sausage Fermentation.

AIMS: Temperate phages insert their genome into the host's chromosome. As prophages, they remain latent in the genome until an induction event leads to lytic phage production. When this occurs in a starter culture that has been added to food fermentation, this can impair the fermentation success. This study aimed to analyze prophage inducibility in the Latilactobacillus curvatus TMW 1.591 strain during meat fermentation and investigate whether an induction signal before cryopreservation is maintained during storage and can lead to phage-induced lysis after culture activation. METHODS AND RESULTS: A prophage-free isogenic derivative of the model starter organism, L. curvatus TMW 1.591, was developed as a negative control (L. curvatus TMW 1.2406). Raw meat fermentation was performed with the wild-type (WT) and phage-cured strains. The WT strain produced high numbers of phages (5.2 ± 1.8 × 107 plaque-forming units g-1) in the meat batter. However, the prophage did not significantly affect the meat fermentation process. Induction experiments suggested an acidic environment as a potential trigger for prophage induction. Phage induction by ultraviolet light before strain cryopreservation remains functional for at least 10 weeks of storage. CONCLUSIONS: Intact prophages are active during meat fermentation. However, in this study, this has no measurable consequences for fermentation, suggesting a high resiliency of meat fermentation against phages. Inadequate handling of lysogenic starter strains, even before preservation, can lead to phage introduction into food fermentation and unintended host lysis.

Characterization of the major autolysin (AtlC) of Staphylococcus carnosus.

BACKGROUND: Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated. RESULTS: AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC. CONCLUSIONS: In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.

Distribution, inducibility, and characteristics of Latilactobacillus curvatus temperate phages.

Aim: Temperate phages are known to heavily impact the growth of their host, be it in a positive way, e.g., when beneficial genes are provided by the phage, or negatively when lysis occurs after prophage induction. This study provides an in-depth look into the distribution and variety of prophages in Latilactobacillus curvatus (L. curvatus). This species is found in a wide variety of ecological niches and is routinely used as a meat starter culture. Methods: Fourty five L. curvatus genomes were screened for prophages. The intact predicted prophages and their chromosomal integration loci were described. Six L. curvatus lysogens were analysed for phage-mediated lysis post induction via UV light and/or mitomycin C. Their lysates were analysed for phage particles via viral DNA sequencing and transmission electron microscopy. Results: Two hundred and six prophage sequences of any completeness were detected within L. curvatus genomes. The 50 as intact predicted prophages show high levels of genetic diversity on an intraspecies level with conserved regions mostly in the replication and head/tail gene clusters. Twelve chromosomal loci, mostly tRNA genes, were identified, where intact L. curvatus phages were integrated. The six analysed L. curvatus lysogens showed strain-dependent lysis in various degrees after induction, yet only four of their lysates appeared to contain fully assembled virions with the siphovirus morphotype. Conclusion: Our data demonstrate that L. curvatus is a (pro)phage-susceptible species, harbouring multiple intact prophages and remnant sequences thereof. This knowledge provides a basis to study phage-host interaction influencing microbial communities in food fermentations.

Glycoside hydrolase family 32 enzymes from Bombella spp. catalyze the formation of high-molecular weight fructans from sucrose.

AIMS: Acetic acid bacteria of the genus Bombella have not been reported to produce exopolysaccharides (EPS). In this study, the formation of fructans by B. apis TMW 2.1884 and B. mellum TMW 2.1889 was investigated. METHODS AND RESULTS: Out of eight strains from four different Bombella species, only B. apis TMW 2.1884 and B. mellum TMW 2.1889 showed EPS formation with 50 g l-1 sucrose as substrate. Both EPS were identified as high-molecular weight (HMW) polymers (106-107 Da) by asymmetric flow field-flow fractionation coupled to multi angle laser light scattering and UV detecors (AF4-MALLS/UV) and high performance size exclusion chromatography coupled to MALLS and refractive index detectors (HPSEC-MALLS/RI) analyses. Monosaccharide analysis via trifluoroacetic acid hydrolysis showed that both EPS are fructans. Determination of glycosidic linkages by methylation analysis revealed mainly 2,6-linked fructofuranose (Fruf) units with additional 2,1-linked Fruf units (10%) and 2,1,6-Fruf branched units (7%). No glycoside hydrolase (GH) 68 family genes that are typically associated with the formation of HMW fructans in bacteria could be identified in the genomes. Through heterologous expression in Escherichia coli Top10, an enzyme of the GH32 family could be assigned to the catalysis of fructan formation. The identified fructosyltransferases could be clearly differentiated phylogenetically and structurally from other previously described bacterial fructosyltransferases. CONCLUSIONS: The formation of HMW fructans by individual strains of the genus Bombella is catalyzed by enzymes of the GH32 family. Analysis of the fructans revealed an atypical structure consisting of 2,6-linked Fruf units as well as 2,1-linked Fruf units and 2,1,6-Fruf units.

Bombella pluederhausensis sp. nov., Bombella pollinis sp. nov., Bombella saccharophila sp. nov. and Bombella dulcis sp. nov., four Bombella species isolated from the environment of the western honey bee Apis mellifera.

Four strains of members of the genus Bombella were isolated from samples associated with the western honey bee Apis mellifera, which could not be assigned to a species with a validly published name. Strains TMW 2.2543T, TMW 2.2556T, TMW 2.2558T and TMW 2.2559T exhibit in silico DNA-DNA hybridisation (isDDH) and orthologous average nucleotide identity (orthoANI) values below species delineation thresholds compared with all described species of the genus Bombella and with each other. TMW 2.2556T and TMW 2.2558T form their own clade within the genus. The major respiratory quinone of all strains was Q-10. The composition of cellular fatty acids was diverse between strains. All strains stained Gram-negative, were rod-shaped, strictly aerobic, pellicle-forming, catalase-positive, oxidase-negative, mesophilic and grew over a wide pH range; they were halosensitive but glucose-tolerant. Unlike the other studied strains, TMW 2.2558T was non-motile. Phylogenetic, chemotaxonomic and physiological analyses revealed a clear distinction between all the strains and species with validly published names. All the data support the proposition of four novel species within the genus Bombella, namely Bombella pluederhausensis sp. nov., Bombella pollinis sp. nov., Bombella saccharophila sp. nov. and Bombella dulcis sp. nov., with the respective type strains Bombella pluederhausensis sp. nov. TMW 2.2543T (= DSM 114872T, = LMG 32791T), Bombella pollinis sp. nov. TMW 2.2556T (= DSM 114874T, = LMG 32792T), Bombella saccharophila sp. nov. TMW 2.2558T (= DSM 114875T, = LMG 32793T) and Bombella dulcis sp. nov. TMW 2.2559T (= DSM 114877T, = LMG 32794T). Moreover, three genomes available in the NCBI database that have not yet been described as species with validly published names could be assigned to the proposed species. Bombella sp. ESL0378 and Bombella sp. ESL0385 to Bombella pollinis sp. nov. and Bombella sp. AS1 to Bombella saccharophila sp. nov.

Monitoring the growth dynamics of Tetragenococcus halophilus strains in lupine moromi fermentation using a multiplex-PCR system.

OBJECTIVE: The microbiota of a seasoning sauce fermentation process is usually complex and includes multiple species and even various strains of one species. Moreover, composition and cell numbers of individual strains vary over the course of the entire fermentation. This study demonstrates the applicability of a multiplex PCR system to monitor growth dynamics of Tetragenococcus (T.) halophilus strains in order to evaluate their performance and help to select the most competitive starter strain. RESULTS: In a previous study we isolated T. halophilus strains from multiple lupine moromi fermentation processes and characterized them. In this study we wanted to monitor the growth dynamics of these strains in a competitive lupine moromi model fermentation process using a multiplex PCR system. Therefore, pasteurized lupine koji was inoculated with eight different T. halophilus strains, six from lupine moromi, one from an experimental buckwheat moromi fermentation process and the type strain DSM 20,339T, to create the inoculated lupine moromi pilot scale fermentation process. With the multiplex PCR system, we could detect that all strains could grow in lupine moromi but, that TMW 2.2254 and TMW 2.2264 outperformed all other strains. Both strains dominated the fermentation after three weeks with cell counts between 4 × 106 to 4 × 107 CFU/mL for TMW 2.2254 and 1 × 107 to 5 × 107 CFU/mL for TMW 2.2264. The pH dropped to value below 5 within the first 7 days, the selection of these strains might be related to their acid tolerance.

Characterization of the Staphylococcus xylosus methylome reveals a new variant of type I restriction modification system in staphylococci.

Restriction modification (RM) systems are known to provide a strong barrier to the exchange of DNA between and within bacterial species. Likewise, DNA methylation is known to have an important function in bacterial epigenetics regulating essential pathways such as DNA replication and the phase variable expression of prokaryotic phenotypes. To date, research on staphylococcal DNA methylation focused mainly on the two species Staphylococcus aureus and S. epidermidis. Less is known about other members of the genus such as S. xylosus, a coagulase-negative commensal of mammalian skin. The species is commonly used as starter organism in food fermentations but is also increasingly considered to have an as yet elusive function in bovine mastitis infections. We analyzed the methylomes of 14 S. xylosus strains using single-molecular, real-time (SMRT) sequencing. Subsequent in silico sequence analysis allowed identification of the RM systems and assignment of the respective enzymes to the discovered modification patterns. Hereby the presence of type I, II, III and IV RM systems in varying numbers and combinations among the different strains was revealed, clearly distinguishing the species from what is known for other members of the genus so far. In addition, the study characterizes a newly discovered type I RM system, encoded by S. xylosus but also by a variety of other staphylococcal species, with a hitherto unknown gene arrangement that involves two specificity units instead of one (hsdRSMS). Expression of different versions of the operon in E. coli showed proper base modification only when genes encoding both hsdS subunits were present. This study provides new insights into the general understanding of the versatility and function of RM systems as well as the distribution and variations in the genus Staphylococcus.

Carbohydrate Sources Influence the Microbiota and Flavour Profile of a Lupine-Based Moromi Fermentation.

Lupine-based seasoning sauce is produced similarly to soy sauces and therefore generates a comparable microbiota and aroma profile. While the koji state is dominated by Aspergillus oryzae, the microbiome of the moromi differs to soy moromi, especially in yeast composition due to the absence of Zygosaccharomyces rouxii and Debaryomyces hansenii as the dominant yeast. In this study, we monitored the addition of a carbohydrate source on the microbiome and aroma profile of the resulting sauce. Compared to previous studies, the usage of a yeast starter culture resulted in a sparsely diverse microbiota that was dominated by D. hansenii and T. halophilus. This led to a pH below 5 even after four months of incubation and most of the measured aroma compounds were pyrazines and acids. The addition of wheat and buckwheat resulted in a temporary change in the yeast consortium with the appearance of Z. rouxii and additional bacterial genera. The aroma profile differs in the presence of pyrazines and esters. Since no significant differences in the taste and odour of wheat-added and buckwheat-added sauce was sensed, both substrates influence the lupine sauce in a similar way.

Transcriptomic profiling reveals differences in the adaptation of two Tetragenococcus halophilus strains to a lupine moromi model medium.

BACKGROUND: Tetragenococcus (T.) halophilus is a common member of the microbial consortia of food fermented under high salt conditions. These comprises salty condiments based on soy or lupine beans, fish sauce, shrimp paste and brined anchovies. Within these fermentations this lactic acid bacterium (LAB) is responsible for the formation of lactic and other short chain acids that contribute to the flavor and lower the pH of the product. In this study, we investigated the transcriptomic profile of the two T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine moromi model medium supplied with galactose. To get further insights into which genomic trait is important, we used a setup with two strains. That way we can determine if strain dependent pathways contribute to the overall fitness. These strains differ in the ability to utilize L-arginine, L-aspartate, L-arabinose, D-sorbitol, glycerol, D-lactose or D-melibiose. The lupine moromi model medium is an adapted version of the regular MRS medium supplied with lupine peptone instead of casein peptone and meat extract, to simulate the amino acid availabilities in lupine moromi. RESULTS: The transcriptomic profiles of the T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine peptone-based model media supplied with galactose, used as simulation media for a lupine seasoning sauce fermentation, were compared to the determine potentially important traits. Both strains, have a great overlap in their response to the culture conditions but some strain specific features such as the utilization of glycerol, sorbitol and arginine contribute to the overall fitness of the strain TMW 2.2256. Interestingly, although both strains have two non-identical copies of the tagatose-6P pathway and the Leloir pathway increased under the same conditions, TMW 2.2256 prefers the degradation via the tagatose-6P pathway while TMW 2.2254 does not. Furthermore, TMW 2.2256 shows an increase in pathways required for balancing out the intracellular NADH/NADH+ ratios. CONCLUSIONS: Our study reveals for the first time, that both versions of tagatose-6P pathways encoded in both strains are simultaneously active together with the Leloir pathway and contribute to the degradation of galactose. These findings will help to understand the strain dependent features that might be required for a starter strain in lupine moromi.

Distribution, inducibility, and characterisation of prophages in Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria (LAB) are used as starters in a wide variety of food fermentations. While the number of reports of phages infecting other LAB steadily increased over the years, information about phage associated with Latilactobacillus sakei, a frequently used meat starter, remains scarce. RESULTS: In this study, a predictive genomic analysis of 43 Latilactobacillus sakei genomes revealed the presence of 26 intact, eleven questionable and 52 incomplete prophage sequences across all analysed genomes with a range of one to five predicted prophage sequences per strain. Screening 24 sakei strains for inducible prophages by utilising UV light or mitomycin C, we identified seven lysogenic strains showing lysis after induction during subsequent growth monitoring. Electron microscopic analysis revealed fully assembled virions in the purified lysates of four samples, thus confirming successful prophage induction. All virions featured icosahedral, isomeric heads and long, most likely non-contractile tails indicating siphoviruses. By performing phylogenetic analyses with various marker genes as well as full prophage sequences, we displayed a remarkably high diversity of prophages, that share a similar gene module organisation and six different chromosomal integration sites were identified. By sequencing viral DNA purified from lysates of Latilactobacillus sakei TMW 1.46, we demonstrate that simultaneous induction of multiple prophages is possible. CONCLUSIONS: With this work, we not only provide data about the incidence of prophages harboured by the meat starter Latilactobacillus sakei, we also demonstrated their potential to impact growth of their host after induction, as well as forming seemingly fully assembled virions.

Fructobacillus cardui sp. nov., isolated from a thistle (Carduus nutans) flower.

A Fructobacillus strain was isolated from the flower of a nodding thistle (Carduus nutans) collected in Bavaria, Germany. The strain is Gram-positive, rod-shaped, non-motile, non-sporulating, catalase- and oxidase-negative, and facultatively anaerobic. Growth can be detected at 10-37 °C and pH 4 to 9. The genome size is about 1.56 Mbp and the G+C content is 43.76 mol%. Assignment to the genus Fructobacillus was done by average nucleotide identity (ANI), 16S rRNA gene sequence and multilocus sequence analyses. Calculations of ANI and digital DNA-DNA hybridization values indicate a novel species with Fructobacillus tropaeoli DSM 23246T (93.58% ANI and 57.9 % dDDH) being its closest relative. Therefore, a new species named Fructobacillus cardui sp. nov. with TMW 2.2452T (=DSM 113480T=CECT 30515T) as type strain is proposed.

Genome Sequence of the Diploid Yeast Debaryomyces hansenii TMW 3.1188.

Debaryomyces hansenii TMW 3.1188 is a halotolerant diploid yeast that was isolated from lupine moromi fermentation. Here, we report on the 24.77-Mbp genome of a diploid strain of the species D. hansenii.

Characterisation of recombinant GH 3 ß-glucosidase from ß-glucan producing Levilactobacillus brevis TMW 1.2112.

Levilactobacillus (L.) brevis TMW 1.2112 is an isolate from wheat beer that produces O2-substituted (1,3)-ß-D-glucan, a capsular exopolysaccharide (EPS) from activated sugar nucleotide precursors by use of a glycosyltransferase. Within the genome sequence of L. brevis TMW 1.2112 enzymes of the glycoside hydrolases families were identified. Glycoside hydrolases (GH) are carbohydrate-active enzymes, able to hydrolyse glycosidic bonds. The enzyme ß-glucosidase BglB (AZI09_02170) was heterologous expressed in Escherichia coli BL21. BglB has a monomeric structure of 83.5 kDa and is a member of the glycoside hydrolase family 3 (GH 3) which strongly favoured substrates with ß-glycosidic bonds. Km was 0.22 mM for pNP ß-D-glucopyranoside demonstrating a high affinity of the recombinant enzyme for the substrate. Enzymes able to degrade the (1,3)-ß-D-glucan of L. brevis TMW 1.2112 have not yet been described. However, BglB showed only a low hydrolytic activity towards the EPS, which was measured by means of the D-glucose releases. Besides, characterised GH 3 ß-glucosidases from various lactic acid bacteria (LAB) were phylogenetically analysed to identify connections in terms of enzymatic activity and ß-glucan formation. This revealed that the family of GH 3 ß-glucosidases of LABs comprises most likely exo-active enzymes which are not directly associated with the ability of these LAB to produce EPS.

Comparative Genomics of Acetic Acid Bacteria within the Genus Bombella in Light of Beehive Habitat Adaptation.

It is known that the bacterial microbiota in beehives is essential for keeping bees healthy. Acetic acid bacteria of the genus Bombella colonize several niches in beehives and are associated with larvae protection against microbial pathogens. We have analyzed the genomes of 22 Bombella strains of different species isolated in eight different countries for taxonomic affiliation, central metabolism, prophages, bacteriocins and tetracycline resistance to further elucidate the symbiotic lifestyle and to identify typical traits of acetic acid bacteria. The genomes can be assigned to four different species. Three genomes show ANIb values and DDH values below species demarcation values to any validly described species, which identifies them as two potentially new species. All Bombella spp. lack genes in the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle, indicating a focus of intracellular carbohydrate metabolism on the pentose phosphate pathway or the Entner-Doudoroff pathway for which all genes were identified within the genomes. Five membrane-bound dehydrogenases were identified that catalyze oxidative fermentation reactions in the periplasm, yielding oxidative energy. Several complete prophages, but no bacteriocins, were identified. Resistance to tetracycline, used to prevent bacterial infections in beehives, was only found in Bombella apis MRM1T. Bombella strains exhibit increased osmotolerance in high glucose concentrations compared to Gluconobacter oxydans, indicating adaption to high sugar environments such as beehives.

Chromohalobacter moromii sp. nov., a moderately halophilic bacterium isolated from lupine-based moromi fermentation.

Three moderately halophilic strains, TMW 2.2308T, TMW 2.2299 and TMW 2.2304, were isolated from a lupine-based moromi fermentation. Initial identification based on their low molecular sub-proteome using mass spectrometry showed relation to the genus Halomonas, however, low score values indicated novelty. The comparison of 16S rRNA gene sequences placed these strains within the genus Chromohalobacter with C. japonicus CECT 7219T (99.67% 16S rRNA sequence similarity to strain TMW2.2308T), C. canadensis DSM 6769T (99.54%) and C. beijerinckii LMG 2148T (99.32%) being their closest relatives. However, average nucleotide highest identity values of TMW 2.2308T to C. beijerinckii LMG 2148T of 93.12% and 92.88% to C. japonicus CECT 7219T demonstrate that it represents a novel species within the genus Chromohalobacter with additional strains TMW 2.2299 (96.91%) and TMW 2.2304 (96.98%). The isolated strains were non-spore-forming, motile and able to grow at temperatures from 5 to 45 °C with an optimum at 37 °C. Growth of TMW 2.2308T occurs at 5 to 25% (w/v) NaCl with optimum growth between 10and 12.5%. The genome of TMW 2.2308T has a size of 3.47 Mb and a G + C content of 61.0 mol%. The polyphasic evidence lead to the classification of TMW 2.2308T, TMW 2.2299 and TMW 2.2304 as members of a novel species of the genus Chromohalobacter. We propose a novel species as Chromohalobacter moromii sp. nov., with TMW 2.2308T (=DSM113153T =CECT30422T) as the type strain.

Use of the ß-Glucan-Producing Lactic Acid Bacteria Strains Levilactobacillus brevis and Pediococcus claussenii for Sourdough Fermentation-Chemical Characterization and Chemopreventive Potential of In Situ-Enriched Wheat and Rye Sourdoughs and Breads.

The aim of the present study was to examine ß-glucan production and the potential prebiotic and chemopreventive effects of wheat and rye sourdoughs and breads generated with wild-type and non-ß-glucan-forming isogenic mutant strains of Levilactobacillus brevis and Pediococcus claussenii. Sourdough and bread samples were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) and pellets (FP) were analyzed (pH values, short-chain fatty acids (SCFA), ammonia, bacterial taxa) and the effects of FS on LT97 colon adenoma cell growth, viability, caspase-2 and -3 activity, genotoxic and antigenotoxic effects and on gene and protein expression of p21, cyclin D2, catalase and superoxide dismutase 2 (SOD2) were examined. Concentrations of SCFA were increased and concentrations of ammonia were partly reduced in the FS. The relative abundance of Bifidobacteriaceae was increased in all FPs. Treatment with FS reduced the growth and viability of LT97 cells and significantly increased caspase-2 and -3 activities without exhibiting genotoxic or antigenotoxic effects. The p21 mRNA and protein levels were increased while that of cyclin D2 was reduced. Catalase and SOD2 mRNA and protein expression were marginally induced. The presented results indicate a comparable chemopreventive potential of wheat and rye sourdoughs and breads without an additional effect of the formed ß-glucan.

Proteomic Analysis Reveals Enzymes for ß-D-Glucan Formation and Degradation in Levilactobacillus brevis TMW 1.2112.

Bacterial exopolysaccharide (EPS) formation is crucial for biofilm formation, for protection against environmental factors, or as storage compounds. EPSs produced by lactic acid bacteria (LAB) are appropriate for applications in food fermentation or the pharmaceutical industry, yet the dynamics of formation and degradation thereof are poorly described. This study focuses on carbohydrate active enzymes, including glycosyl transferases (GT) and glycoside hydrolases (GH), and their roles in the formation and potential degradation of O2-substituted (1,3)-ß-D-glucan of Levilactobacillus (L.) brevis TMW 1.2112. The fermentation broth of L. brevis TMW 1.2112 was analyzed for changes in viscosity, ß-glucan, and D-glucose concentrations during the exponential, stationary, and early death phases. While the viscosity reached its maximum during the stationary phase and subsequently decreased, the ß-glucan concentration only increased to a plateau. Results were correlated with secretome and proteome data to identify involved enzymes and pathways. The suggested pathway for ß-glucan biosynthesis involved a ß-1,3 glucan synthase (GT2) and enzymes from maltose phosphorylase (MP) operons. The decreased viscosity appeared to be associated with cell lysis as the ß-glucan concentration did not decrease, most likely due to missing extracellular carbohydrate active enzymes. In addition, an operon was discovered containing known moonlighting genes, all of which were detected in both proteome and secretome samples.

SxsA, a novel surface protein mediating cell aggregation and adhesive biofilm formation of Staphylococcus xylosus.

Biofilm formation of staphylococci has been an emerging field of research for many years. However, the underlying molecular mechanisms are still not fully understood and vary widely between species and strains. The aim of this study was to identify new effectors impacting biofilm formation of two Staphylococcus xylosus strains. We identified a novel surface protein conferring cell aggregation, adherence to abiotic surfaces, and biofilm formation. The S. xylosus surface protein A (SxsA) is a large protein occurring in variable sizes. It lacks sequence similarity to other staphylococcal surface proteins but shows similar structural domain organization and functional features. Upon deletion of sxsA, adherence of S. xylosus strain TMW 2.1523 to abiotic surfaces was completely abolished and significantly reduced in TMW 2.1023. Macro- and microscopic aggregation assays further showed that TMW 2.1523 sxsA mutants exhibit reduced cell aggregation compared with the wildtype. Comparative genomic analysis revealed that sxsA is part of the core genome of S. xylosus, Staphylococcus paraxylosus, and Staphylococcus nepalensis and additionally encoded in a small group of Staphylococcus cohnii and Staphylococcus saprophyticus strains. This study provides insights into protein-mediated biofilm formation of S. xylosus and identifies a new cell wall-associated protein influencing cell aggregation and biofilm formation.

Optimization of a cultivation procedure to selectively isolate lactic acid bacteria from insects.

AIMS: Natural niches and transmission routes of lactic acid bacteria (LAB) are highly versatile. Proposed routes of transmission to food fermentations are from plant material via insects or vice versa. This study aimed to establish a method for the selective isolation of LAB from insects. METHODS AND RESULTS: Varied parameters that influence growth and selectivity are temperature, type of carbohydrate and atmosphere. Additionally, the effects of antibiotics to suppress non-LAB species were evaluated. A model consortium consisting of 12 species representing different lifestyles was inoculated in a growth medium to identify conditions for the highest diversity and recovery rate. The method was applied to isolate LAB from Drosophila melanogaster, Sitotroga cerealella, Tribolium castaneum and Tenebrio molitor. Isolated species were Leuconostoc mesenteroides, Paucilactobacillus vaccinostercus and Lactiplantibacillus plantarum from D. melanogaster and L. mesenteroides, Pediococcus pentosaceus and Latilactobacillus curvatus from T. molitor. No LAB could be isolated from T. castaneum and S. cerealella. 16S rDNA amplicon sequencing of DNA obtained from insects corroborated part of our results. CONCLUSION: A combination of different enrichment conditions ensures a high probability to isolate LAB species from insects and can be helpful above already known non-cultivation methods. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel method allows to selectively isolate LAB from insects and the strategy of the method is of interest to study other niches.

Bap-Independent Biofilm Formation in Staphylococcus xylosus.

The biofilm associated protein (Bap) is recognised as the essential component for biofilm formation in Staphylococcus aureus V329 and has been predicted as important for other species as well. Although Bap orthologs are also present in most S. xylosus strains, their contribution to biofilm formation has not yet been demonstrated. In this study, different experimental approaches were used to elucidate the effect of Bap on biofilm formation in S. xylosus and the motif structure of two biofilm-forming S. xylosus strains TMW 2.1023 and TMW 2.1523 was compared to Bap of S. aureus V329. We found that despite an identical structural arrangement into four regions, Bap from S. xylosus differs in key factors to Bap of S. aureus, i.e., isoelectric point of aggregation prone Region B, protein homology and type of repeats. Disruption of bap had no effect on aggregation behavior of selected S. xylosus strains and biofilm formation was unaffected (TMW 2.1023) or at best slightly reduced under neutral conditions (TMW 2.1523). Further, we could not observe any typical characteristics of a S. aureus Bap-positive phenotype such as functional impairment by calcium addition and rough colony morphology on congo red agar (CRA). A dominating role of Bap in cell aggregation and biofilm formation as reported mainly for S. aureus V329 was not observed. In contrast, this work demonstrates that functions of S. aureus Bap cannot easily be extrapolated to S. xylosus Bap, which appears as non-essential for biofilm formation in this species. We therefore suggest that biofilm formation in S. xylosus follows different and multifactorial mechanisms.