Modulating the production of xylanase by Bacillus pumilus BS131 through optimization using waste fiber sludge / Modulando a produção de xilanase por Bacillus pumilus BS131 por meio da otimização usando lodo de fibra residual

In recent days, cheapest alternative carbon source for fermentation purpose is desirable to minimize production cost. Xylanases have become attractive enzymes as their potential in bio-bleaching of pulp and paper industry. The objective of the present study was to identify the potential ability on the xylanase production by locally isolated Bacillus pumilus BS131 by using waste fiber sludge and wheat bran media under submerged fermentation. Culture growth conditions were optimized to obtain significant amount of xylanase. Maximum xylanase production was recorded after 72 hours of incubation at 30 °C and 7 pH with 4.0% substrate concentration. In the nutshell, the production of xylanase using inexpensive waste fiber sludge and wheat-bran as an alternative in place of expensive xylan substrate was more cost effective and environment friendly.

Nos últimos dias, a fonte alternativa de carbono mais barata para fins de fermentação é desejável para minimizar o custo de produção. As xilanases têm se tornado enzimas atraentes como seu potencial no biobranqueamento da indústria de papel e celulose. O objetivo do presente estudo foi identificar a capacidade potencial na produção de xilanase por Bacillus pumilus BS131 isolado localmente usando lodo de fibra residual e farelo de trigo em meio de fermentação submersa. As condições de crescimento da cultura foram otimizadas para obter uma quantidade significativa de xilanase. A produção máxima de xilanase foi registrada após 72 horas de incubação a 30 °C e pH 7 com concentração de substrato de 4,0%. Resumindo, a produção de xilanase usando lodo de fibra residual de baixo custo e farelo de trigo como uma alternativa no lugar do substrato de xilano caro foi mais econômica e ecológica.

Deep Functional Profiling of Wild Animal Microbiomes Reveals Probiotic Bacillus pumilus Strains with a Common Biosynthetic Fingerprint.

The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.

Biosurfactant from endophytic Bacillus pumilus 2A: physicochemical characterization, production and optimization and potential for plant growth promotion.

BACKGROUND: Microbial surfactants called biosurfactants, thanks to their high biodegradability, low toxicity and stability can be used not only in bioremediation and oil processing, but also in the food and cosmetic industries, and even in medicine. However, the high production costs of microbial surfactants and low efficiency limit their large-scale production. This requires optimization of management conditions, including the possibility of using waste as a carbon source, such as food processing by-products. This papers describes the production and characterization of the biosurfactant obtained from the endophytic bacterial strain Bacillus pumilus 2A grown on various by-products of food processing and its potential applications in supporting plant growth. Four different carbon and nitrogen sources, pH, inoculum concentration and temperature were optimized within Taguchi method. RESULTS: Optimization of bioprocess within Taguchi method and experimental analysis revealed that the optimal conditions for biosurfactant production were brewer's spent grain (5% w/v), ammonium nitrate (1% w/v), pH of 6, 5% of inoculum, and temperature at 30 °C, leading to 6.8 g/L of biosurfactant. Based on gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy analysis produced biosurfactant was determined as glycolipid. Obtained biosurfactant has shown high and long term thermostability, surface tension of 47.7 mN/m, oil displacement of 8 cm and the emulsion index of 69.11%. The examined glycolipid, used in a concentration of 0.2% significantly enhanced growth of Phaseolus vulgaris L. (bean), Raphanus L. (radish), Beta vulgaris L. (beetroot). CONCLUSIONS: The endophytic Bacillus pumilus 2A produce glycolipid biosurfactant with high and long tem thermostability, what makes it useful for many purposes including food processing. The use of brewer's spent grain as the sole carbon source makes the production of biosurfactants profitable, and from an environmental point of view, it is an environmentally friendly way to remove food processing by products. Glycolipid produced by endophytic Bacillus pumilus 2A significantly improve growth of Phaseolus vulgaris L. (bean), Raphanus L. (radish), Beta vulgaris L. (beetroot). Obtained results provide new insight to the possible use of glycolipids as plant growth promoting agents.

Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria.

Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.

A novel feruloyl esterase with high rosmarinic acid hydrolysis activity from Bacillus pumilus W3.

A novel feruloyl esterase (BpFae12) with rosmarinic acid (RA) hydrolysis activity was isolated from Bacillus pumilus W3 and expressed in Escherichia coli BL21 (DE3). With RA as a substrate, the optimal pH and temperature of BpFae12 were pH 8.0 and 50 °C, respectively. The specific enzyme activity was 12.8 U·mg-1. BpFae12 showed the highest activity and substrate affinity toward RA (Vmax of 13.13 U·mg-1, Km of 0.41 mM). Moreover, it also presented strong hydrolysis performance against chlorogenic acid (190.17 U·mg-1). RA was effectively Hydrolyzed into more bioactive caffeic acid and 3,4-dihydroxyphenyllactic acid by BpFae12, which have potential applications in the food industry.

Bacillus pumilus probiotic feed supplementation mitigates Lawsonia intracellularis shedding and lesions.

The causative agent of ileitis, Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10-20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10-65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis.

Impact of a probiotic-based cleaning product on the microbiological profile of broiler litters and chicken caeca microbiota.

This study investigated for the first time the decontamination efficacy of a probiotic-based cleaning product containing Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium spores on fresh and reused broiler litters during 3 rearing cycles of 6 wk each. Moreover, the impact of reused litters treated with the cleaning product on the chicken caeca microbiota was assessed at the end of the rearing cycles in comparison to untreated litter. The Bacillus spores provided with the cleaning treatment were able to successfully colonize the reused poultry litters, decreasing the mean counts of total aerobic bacteria, Enterobacteriaceae, and coagulase positive Staphylococci. The decrease of Enterobacteriaceae, mainly represented by the genus Escherichia, was also observed in the caeca of broilers reared on reused litters treated with the cleaning product. Moreover, the treatment retained the caeca content of Ruminococcaceae and Faecalibacterium as well as the level of biodiversity among the bacteria genera colonizing the caeca of animals reared on reused litter. Overall, the results of this study highlight a positive effect of the probiotic-based cleaning strategy on the microbial decontamination of reused litters and on broiler caeca stability, thereby enhancing animal health and prevention of poultry diseases.

Bacillus pumilus SE5 originated PG and LTA tuned the intestinal TLRs/MyD88 signaling and microbiota in grouper (Epinephelus coioides).

The normal microbiota plays a key role in the health of host, but little is known of how the fish immune system recognizes and responds to indigenous bacteria/probiotics. Our previous studies have showed that heat-inactivated indigenous Bacillus pumilus SE5 activate the TLR2 signaling pathways and modulate the intestinal microbiota in grouper (Epinephelus coioides), suggesting microbial-associated molecular patterns (MAMPs) involved. In this study, whole cell wall (CW) and two possible MAMPs, peptidoglycan (PG) and lipoteichoic acid (LTA) have been extracted from B. pumilus SE5 and their effects on intestinal immune related genes expression and microbiota were evaluated in a 60 days feeding trial. Significantly elevated expression of TLR1, TLR2, TLR5 and MyD88 was observed in fish fed the CW, PG and LTA containing diets, and the highest expression was observed in groups PG and LTA. At the same time, significantly upregulated expression of antimicrobial effectors, such as antimicrobial peptides (epinecidin-1, hepcidin-1 and ß-defensin), C-type Lectin and IgM was observed in fish fed PG and LTA containing diets. This induced activation of intestinal immunity was consistent with the microbiota data showing that CW, PG and LTA originated from SE5 modulated the overall structure of intestinal microbiota, and the relative abundance of potentially pathogenic Vibrio decreased significantly while beneficial Lactobacillus increased significantly in fish fed PG and LTA. In conclusion, both the PG and LTA originated from B. pumilus SE5 could activate TLRs/MyD88 signaling and expression of wide-ranging antibacterial effectors, and therefore shape the intestinal microbiota in grouper.

Genome Sequencing and Analysis of Bacillus pumilus ICVB403 Isolated from Acartia tonsa Copepod Eggs Revealed Surfactin and Bacteriocin Production: Insights on Anti-Staphylococcus Activity.

Here we show that Bacillus pumilus ICVB403 recently isolated from copepod eggs is able to produce, after 48-72 h of growth in Landy medium, extracellular inhibitory compounds, which are active against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 43300, MRSA-S1, Staphylococcus epidermidis 11EMB, Staphylococcus warneri 27EMB, and Staphylococcus hominis 13EMB. Moreover, these extracellular inhibitory compound(s) were able to potentiate erythromycin against the aforementioned staphylococci. The minimum inhibitory concentration (MIC) of erythromycin was reduced from 32 µg/mL to 8 µg/mL for MRSA ATCC 43300 and MRSA SA-1 strains, and from 32-64 µg/mL to 4 µg/mL for S. epidermidis 11EMB and S. hominis 13EMB strains.The genome sequencing and analysis of B. pumilus ICVB403 unveiled 3.666.195 nucleotides contained in 22 contigs with a G + C ratio of 42.0%, 3.826 coding sequences, and 73 RNAs. In silico analysis guided identification of two putative genes coding for synthesis of surfactin A, a lipopeptide with 7 amino acids, and for a circular bacteriocin belonging to the circularin A/uberolysin family, respectively.

Biological control of Pseudomonas syringae pv. aptata on sugar beet with Bacillus pumilus SS-10.7 and Bacillus amyloliquefaciens (SS-12.6 and SS-38.4) strains.

AIM: Assessment of biological control of Pseudomonas syringae pv. aptata using crude lipopeptide extracts (CLEs) of two Bacillus amyloliquefaciens strains (SS-12.6 and SS-38.4) and one Bacillus pumilus strain (SS-10.7). METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of CLEs and their combinations against the pathogen and potential interaction between the extracts were determined in vitro. The most effective antibacterial activity was achieved with the CLE from B. amyloliquefaciens SS-12.6, with an MIC value of 0·63 mg ml-1 . Interactions between CLE combinations were mostly indifferent. The biocontrol potential of CLEs, mixtures of CLEs, and cell culture of B. amyloliquefaciens SS-12.6 was tested on sugar beet plants inoculated with P. syringae pv. aptata P53. The best result in inhibiting the appearance of tissue necrosis (up to 92%) was achieved with B. amyloliquefaciens SS-12.6 cell culture. CONCLUSION: This work demonstrated significant biocontrol potential of the CLE and cell culture of B. amyloliquefaciens SS-12.6 which successfully suppress leaf spot disease severity on sugar beet plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of biocontrol of sugar beet emerging pathogen will contribute to growers in terms of alternative disease control management. This study represents first assessment of biological control of P. syringae pv. aptata.

Enhancing the activity and stability of Mn-superoxide dismutase by one-by-one ligation to catalase.

Dismutation of superoxide by superoxide dismutase (SOD) generates hydrogen peroxide, which may be reduced to hydroxyl radical. The generated H2O2 during the catalysis can have an oxidative damage to SOD. Hydrogen peroxide decomposition by catalase (CAT) can help circumvent the problem. Mn-superoxide dismutase (herein referred to as SOD) and CAT are dimeric and tetrameric proteins, respectively. Herein, through intein-mediated in vivo subunit splicing, the C-terminus of the CAT subunit (CATS) has been specifically ligated to the N-terminus of the SOD subunit (SODS) with a peptide bond. Thus, the splicing product SOD&CAT combines the superoxide anion (•O2-) scavenging ability and the ability of decomposing H2O2. The in vivo subunit splicing has little effect on the secondary structures of the enzymes as confirmed by circular dichroism (CD) spectra. Fluorescence spectra showed that the splicing product SOD&CAT has a higher stability than SOD. In the splicing product SOD&CAT, the SOD subunits are in close proximity to the CAT subunits, facilitating immediate transfer of H2O2 between the enzymes and enabling efficient decomposition of H2O2. SOD&CAT exhibited a superoxide anion (•O2-) scavenging ability 244% higher than that of SOD and 46% higher than that of the mixed enzymes SOD+CAT.

Bacillamidins A⁻G from a Marine-Derived Bacillus pumilus.

Seven long-chain amides, including five previously undescribed bacillamidins A⁻E (1⁻5) and two previously reported synthetic analogs, bacillamidins F (6) and G (7), were isolated from extracts of the marine-derived Bacillus pumilus strain RJA1515. The structures of the new compounds were established by extensive analysis of 1D and 2D nuclear magnetic resonance (NMR) data as well as high resolution mass spectrometry (HRMS), and the absolute configurations of the stereogenic carbons of 1⁻4 were established by comparison of the calculated and the experimental electronic circular dichroism (ECD) spectra. The cytotoxic and antimicrobial activities of 1⁻7 were evaluated.

Global quantification of phosphoproteins combining metabolic labeling and gel-based proteomics in B. pumilus.

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post-translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two-dimensional gels after differential phosphoprotein staining or gel-free by stable isotope labeling, sequential phosphopeptide enrichment and following LC-MS analysis. In the current work, we combined in a proof-of-principle experiment these techniques using 14 N/15 N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC-MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide-based gel-free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med-fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well-known as invertebrate-active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N-terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid-cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.

Integration of Bacterial Expansin on Agarolytic Complexes to Enhance the Degrading Activity of Red Algae by Control of Gelling Properties.

Expansin act by loosening hydrogen bonds in densely packed polysaccharides. This work characterizes the biological functions of expansin in the gelling and degradation of algal polysaccharides. In this study, the bacterial expansin BpEX from Bacillus pumilus was fused with the dockerin module of a cellulosome system for assembly with agarolytic complexes. The assembly of chimeric expansin caused an indicative enhancement in agarase activity. The enzymatic activities on agar substrate and natural biomass were 3.7-fold and 3.3-fold higher respectively than that of agarase as a single enzyme. To validate the effect on the agar degradation, the regulation potential of parameters related to gel rheology by bacterial expansin was experimentally investigated to indicate that the bacterial expansin lowered the gelling temperature and viscosity of agar. Thus, these results demonstrated the possibility of advancing more efficient strategies for utilizing agar as oligo sugar source in the biorefinery field that uses marine biomass as feedstocks.

Production of spore laccase from Bacillus pumilus W3 and its application in dye decolorization after immobilization.

Given that spore laccase from the Bacillus genus is heat- and alkali-resistant, it is more suitable for industrial applications than fungal laccase. To determine the optimal culture conditions for spore laccase production, the effects of Cu2+ concentration, oxygen content, and culture time on spore laccase production from Bacillus pumilus W3 were investigated. The optimal production parameters were 0.2 mM of Cu2+, 200 rpm shaking speed, 100 mL liquid loading, and 5 days of cultivation. Spore laccase was efficiently immobilized on amino-functionalized celite. When used in dye decolorization, the immobilized spore laccase removed 84.15% of methyl green and 69.70% of acid red 1 after 48 h of treatment. Moreover, the immobilized spore laccase retained 87.04% of its initial decolorization activity after six cycles in the decolorization of acid red 1. These insights into the culture conditions and immobilization of spore laccases should be useful in the development of spore laccase as a biocatalyst in the treatment of textile wastewater.

Efficacy of viable Bacillus pumilus isolated from farmed fish on immune responses and increased disease resistance in Nile tilapia (Oreochromis niloticus): Laboratory and on-farm trials.

Applications of viable Bacillus pumilus AQAHBS01 isolated from Nile tilapia farms as probiotics were studied in both laboratory and farm conditions. In the laboratory, feeding fish (approximately 50 g) with feed containing viable B. pumilus at concentrations of 1 × 107-109 colony forming units (CFU)/kg elevated fish immune responses, as indicated by their phagocytic activity and superoxide anion levels, and led to more effective disease resistance against Streptococcus agalactiae. However, when these concentrations were applied to Nile tilapia cultures growing in cage culture systems, only B. pumilus AQAHBS01 at concentrations of 1 × 108 and 109 CFU/kg diet could effectively enhance disease resistance against S. agalactiae during the critical period of early to middle April when the temperature reached 33 °C, whereas control fish and fish that consumed B. pumilus AQAHBS01 at concentrations of 1 × 107 CFU/kg showed very rapid streptococcosis-induced mortality. However, in late April, massive levels of organic matter-containing water flowed into the culture areas, causing all fish groups to become infected with Flavobacterium columnare. Moreover, the dissolved oxygen levels in the river declined to critical levels of approximately 1.0-1.5 mg/L, causing anorectic effects in fish for long periods of time. This effect may have also gradually killed the cultured fish until the end of the experiment. This information strongly demonstrates the effective application of B. pumilus as a probiotic for streptococcosis resistance in both laboratory and field culture conditions. For on-farm cage culture practices, however, fluctuations in water quality remain a significant constraint for probiotic application, as they usually induce negative effects on fish health. This decline in health makes fish more fragile and more susceptible to problems from both infectious and non-infectious diseases, which farmers must consider carefully.

Extracellular Proteome Profiling of Bacillus pumilus SCU11 Producing Alkaline Protease for Dehairing.

Bacillus pumilus is one of the most characterized microorganisms that are used for high-level production of select industrial enzymes. A novel B. pumilus SCU11 strain possessing high alkaline protease activity was obtained in our previous work. The culture supernatant of this strain showed efficient dehairing capability with minimal collagen damage, indicating promising potential applications in the leather industry. In this study, the strain's extracellular proteome was identified by LC-MS/MS-based shotgun proteomic analysis, and their related secretory pathways were characterized by BLAST searches. A total of 513 proteins, including 100 actual secreted and 413 intracellular proteins, were detected in the extracellular proteome. The functions of these secreted proteins were elucidated and four complete secretory systems (Sec, Tat, Com, and ABC transporter) were proposed for B. pumilus. These data provide B. pumilus a comprehensive extracellular proteome profile, which is a valuable theoretical and applicative basis for future genetic modifications and development of industrial enzymes.

1-Acyl-3-0-[ß-glucopyranosyl-(1"-+ 6')-ß-glucopyranosyl]-glycerols and Cordycedipeptides B and C, New Metabolites from Bacillus pumilus.

Four 1-monoacyl-3-0-[ß-glucopyranosyl-( ->6)-ß-glucopyranosyl]-glycerols (1) and four 1,2-diacyl-3-0-[p-glucopyranosyl-(1->6)-p-glucopyranosyl]- glycerols (2a) with acyl residues consisting of 1:1 mixtures of 1-iso-pentadecanoyl- and 1 -anteiso-pentadecanoyl residues and the respective heptadecanoic acid isomers s as main components, have been characterized in the extracts of Bacillus pumilus strain DKS 1. Twenty-seven further metabolites, among them the diketopiperazines cordycedipeptide A (3), B (4), and C (5), were obtained. All compounds were elucidated by NMR and MS techniques and fully characterized and tested for antimicrobial activity against Legionella pneumophila.