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July 7, 2019

Genome sequencing and analysis of the first complete genome of Lactobacillus kunkeei strain MP2, an Apis mellifera gut isolate

Background. The honey bee (Apis mellifera) is the most important pollinator in agriculture worldwide. However, the number of honey bees has fallen significantly since 2006, becoming a huge ecological problem nowadays. The principal cause is CCD, or Colony Collapse Disorder, characterized by the seemingly spontaneous abandonment of hives by their workers. One of the characteristics of CCD in honey bees is the alteration of the bacterial communities in their gastrointestinal tract, mainly due to the decrease of Firmicutes populations, such as the Lactobacilli. At this time, the causes of these alterations remain unknown. We recently isolated a strain of Lactobacillus kunkeei (L. kunkeei strain MP2) from the gut of Chilean honey bees. L. kunkeei, is one of the most commonly isolated bacterium from the honey bee gut and is highly versatile in different ecological niches. In this study, we aimed to elucidate in detail, the L. kunkeei genetic background and perform a comparative genome analysis with other Lactobacillus species. Methods. L. kunkeei MP2 was originally isolated from the guts of Chilean A. mellifera individuals. Genome sequencing was done using Pacific Biosciences single-molecule real-time sequencing technology. De novo assembly was performed using Celera assembler. The genome was annotated using Prokka, and functional information was added using the EggNOG 3.1 database. In addition, genomic islands were predicted using IslandViewer, and pro-phage sequences using PHAST. Comparisons between L. kunkeei MP2 with other L. kunkeei, and Lactobacillus strains were done using Roary. Results. The complete genome of L. kunkeei MP2 comprises one circular chromosome of 1,614,522 nt. with a GC content of 36,9%. Pangenome analysis with 16 L. kunkeei strains, identified 113 unique genes, most of them related to phage insertions. A large and unique region of L. kunkeei MP2 genome contains several genes that encode for phage structural protein and replication components. Comparative analysis of MP2 with other Lactobacillus species, identified several unique genes of L. kunkeei MP2 related with metabolism, biofilm generation, survival under stress conditions, and mobile genetic elements (MGEs). Discussion. The presence of multiple mobile genetic elements, including phage sequences, suggest a high degree of genetic variability in L. kunkeei. Its versatility and ability to survive in different ecological niches (bee guts, flowers, fruits among others) could be given by its genetic capacity to change and adapt to different environments. L. kunkeei could be a new source of Lactobacillus with beneficial properties. Indeed, L. kunkeei MP2 could play an important role in honey bee nutrition through the synthesis of components as isoprenoids.

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July 7, 2019

Isolation and complete genome sequence of the thermophilic Geobacillus sp. 12AMOR1 from an Arctic deep-sea hydrothermal vent site.

Members of the genus Geobacillus have been isolated from a wide variety of habitats worldwide and are the subject for targeted enzyme utilization in various industrial applications. Here we report the isolation and complete genome sequence of the thermophilic starch-degrading Geobacillus sp. 12AMOR1. The strain 12AMOR1 was isolated from deep-sea hot sediment at the Jan Mayen hydrothermal Vent Site. Geobacillus sp. 12AMOR1 consists of a 3,410,035 bp circular chromosome and a 32,689 bp plasmid with a G?+?C content of 52 % and 47 %, respectively. The genome comprises 3323 protein-coding genes, 88 tRNA species and 10 rRNA operons. The isolate grows on a suite of sugars, complex polysaccharides and proteinous carbon sources. Accordingly, a versatility of genes encoding carbohydrate-active enzymes (CAZy) and peptidases were identified in the genome. Expression, purification and characterization of an enzyme of the glycoside hydrolase family 13 revealed a starch-degrading capacity and high thermal stability with a melting temperature of 76.4 °C. Altogether, the data obtained point to a new isolate from a marine hydrothermal vent with a large bioprospecting potential.

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July 7, 2019

The absence of a mature cell wall sacculus in stable Listeria monocytogenes L-form cells is independent of peptidoglycan synthesis.

L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan.

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July 7, 2019

Complete genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, two phylogenetically distinct probiotics.

Several spore-forming strains of Bacillus are marketed as probiotics due to their ability to survive harsh gastrointestinal conditions and confer health benefits to the host. We report the complete genomes of two commercially available probiotics, Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, and compare them with the genomes of other Bacillus and Lactobacillus. The taxonomic position of both organisms was established with a maximum-likelihood tree based on twenty six housekeeping proteins. Analysis of all probiotic strains of Bacillus and Lactobacillus reveal that the essential sporulation proteins are conserved in all Bacillus probiotic strains while they are absent in Lactobacillus spp. We identified various antibiotic resistance, stress-related, and adhesion-related domains in these organisms, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival during antibiotic treatment and harsh conditions.

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July 7, 2019

Evaluation of an optimal epidemiologic typing scheme for Legionella pneumophila with whole genome sequence data using validation guidelines.

Sequence-based typing (SBT), analogous to multi-locus sequence typing (MLST), is the current gold-standard typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila However, as common sequence types (STs) cause many infections, some investigations remain unresolved. Here, various whole genome sequencing (WGS)-based methods were evaluated according to published guidelines, including: i) single nucleotide polymorphism (SNP)-based; ii) extended multi-locus sequence typing (MLST) using different numbers of genes; iii) gene presence/absence, and iv) kmer-based. L. pneumophila serogroup 1 isolates (n=106) from the standard “typing panel”, previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI) were tested together with another 229 isolates.Over 98% isolates were considered typable using the mapping- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50-gene) to 86.8% (1455-gene) whilst only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP-based), and all values are higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ~50 genes provides optimal epidemiological concordance whilst substantially improving the discrimination offered by SBT, and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila. Copyright © 2016 David et al.

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July 7, 2019

Bacillus pumilus SAFR-032 genome revisited: sequence update and re-annotation.

Bacillus pumilus strain SAFR-032 is a non-pathogenic spore-forming bacterium exhibiting an anomalously high persistence in bactericidal environments. In its dormant state, it is capable of withstanding doses of ultraviolet (UV) radiation or hydrogen peroxide, which are lethal for the vast majority of microorganisms. This unusual resistance profile has made SAFR-032 a reference strain for studies of bacterial spore resistance. The complete genome sequence of B. pumilus SAFR-032 was published in 2007 early in the genomics era. Since then, the SAFR-032 strain has frequently been used as a source of genetic/genomic information that was regarded as representative of the entire B. pumilus species group. Recently, our ongoing studies of conservation of gene distribution patterns in the complete genomes of various B. pumilus strains revealed indications of misassembly in the B. pumilus SAFR-032 genome. Synteny-driven local genome resequencing confirmed that the original SAFR-032 sequence contained assembly errors associated with long sequence repeats. The genome sequence was corrected according to the new findings. In addition, a significantly improved annotation is now available. Gene orders were compared and portions of the genome arrangement were found to be similar in a wide spectrum of Bacillus strains.

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July 7, 2019

Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment.

Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism’s physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota (‘Nanopusillus acidilobi’) and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of ‘Nanopusillus’ are among the smallest known cellular organisms (100-300?nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea.

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July 7, 2019

Lactobacillus rhamnosus GG outcompetes Enterococcus faecium by mucus-binding pili – Evidence for a novel probiotic mechanism on a distance.

Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections. IMPORTANCE Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of Lactobacillus rhamnosus GG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intact L. rhamnosus GG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state.

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July 7, 2019

Complete genome sequence of Cyanobium sp. NIES-981, a marine strain potentially useful for ecotoxicological bioassays.

Cyanobium sp. NIES-981 is a marine cyanobacterium isolated from tidal flat sands in Okinawa, Japan. Here, we report the complete 3.0-Mbp genome sequence of NIES-981, which is composed of a single chromosome, and its annotation. This sequence information may provide a basis for developing an ecotoxicological bioassay using this strain. Copyright © 2016 Yamaguchi et al.

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July 7, 2019

Vibrio anguillarum is genetically and phenotypically unaffected by long-term continuous exposure to the antibacterial compound tropodithietic acid.

Minimizing the use of antibiotics in the food production chain is essential for limiting the development and spread of antibiotic-resistant bacteria. One alternative intervention strategy is the use of probiotic bacteria, and bacteria of the marine Roseobacter clade are capable of antagonizing fish-pathogenic vibrios in fish larvae and live feed cultures for fish larvae. The antibacterial compound tropodithietic acid (TDA), an antiporter that disrupts the proton motive force, is key in the antibacterial activity of several roseobacters. Introducing probiotics on a larger scale requires understanding of any potential side effects of long-term exposure of the pathogen to the probionts or any compounds they produce. Here we exposed the fish pathogen Vibrio anguillarum to TDA for several hundred generations in an adaptive evolution experiment. No tolerance or resistance arose during the 90 days of exposure, and whole-genome sequencing of TDA-exposed lineages and clones revealed few mutational changes, compared to lineages grown without TDA. Amino acid-changing mutations were found in two to six different genes per clone; however, no mutations appeared unique to the TDA-exposed lineages or clones. None of the virulence genes of V. anguillarum was affected, and infectivity assays using fish cell lines indicated that the TDA-exposed lineages and clones were less invasive than the wild-type strain. Thus, long-term TDA exposure does not appear to result in TDA resistance and the physiology of V. anguillarum appears unaffected, supporting the application of TDA-producing roseobacters as probiotics in aquaculture.It is important to limit the use of antibiotics in our food production, to reduce the risk of bacteria developing antibiotic resistance. We showed previously that marine bacteria of the Roseobacter clade can prevent or reduce bacterial diseases in fish larvae, acting as probiotics. Roseobacters produce the antimicrobial compound tropodithietic acid (TDA), and we were concerned regarding whether long-term exposure to this compound could induce resistance or affect the disease-causing ability of the fish pathogen. Therefore, we exposed the fish pathogen Vibrio anguillarum to increasing TDA concentrations over 3 months. We did not see the development of any resistance to TDA, and subsequent infection assays revealed that none of the TDA-exposed clones had increased virulence toward fish cells. Hence, this study supports the use of roseobacters as a non-risk-based disease control measure in aquaculture. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

E. coli O157:H7 strain EDL933 harbors multiple functional prophage-associated genes necessary for the utilization of 5-N-acetyl-9-O-acetyl neuraminic acid as a growth substrate.

Enterohemorrhagic E. coli (EHEC) O157:H7 strain EDL933 harbors multiple prophage-associated open reading frames (orfs) in its genome, which are highly homologous to the chromosomal nanS gene. The latter is part of the nanCMS-operon, which is present in most E. coli strains and encodes an esterase, which is responsible for the mono-deacetylation of 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). Whereas one prophage-borne orf (z1466) has been characterized in previous studies, the functions of the other nanS-homologous orfs are unknown.In the current study, the nanS-homologous orfs of EDL933 were initially studied in silico Due to their homology to the chromosomal nanS gene and their location in prophage genomes, we designated them nanS-p, and numbered the different nanS-p alleles consecutively from 1-10. The two alleles nanS-p2 and nanS-p4 were selected for production of recombinant proteins, their enzymatic activities were investigated and differences in their temperature optima were found. Furthermore, a function of these enzymes in substrate utilization could be demonstrated using an E. coli C600?nanS mutant in a growth medium with Neu5,9Ac2 as carbon source and supplementation with the different recombinant NanS-p proteins. Moreover, generation of sequential deletion of all nanS-p alleles in strain EDL933, and subsequent growth experiments demonstrated a gene-dose-effect on the utilization of Neu5,9Ac2Since Neu5,9Ac2 is an important component of human and animal gut mucus, and the nutrient availability in the large intestine is limited, we hypothesize that the presence of multiple Neu5,9Ac2-esterases provides them a nutrient supply under certain conditions in the large intestine, even if particular prophages get lost.In this study, a group of homologous prophage-borne nanS-p alleles and two of the corresponding enzymes of enterohemorrhagic E. coli (EHEC) O157:H7 strain EDL933 are characterized that may be important to provide alternative genes for substrate utilization. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

SAR11 bacteria linked to ocean anoxia and nitrogen loss.

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world’s largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth’s most abundant organismal group.

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July 7, 2019

Comparative genomic analysis of isoproturon-mineralizing sphingomonads reveals the isoproturon catabolic mechanism.

The worldwide use of the phenylurea herbicide, isoproturon (IPU), has resulted in considerable concern about its environmental fate. Although many microbial metabolites of IPU are known and IPU-mineralizing bacteria have been isolated, the molecular mechanism of IPU catabolism has not been elucidated yet. In this study, complete genes that encode the conserved IPU catabolic pathway were revealed, based on comparative analysis of the genomes of three IPU-mineralizing sphingomonads and subsequent experimental validation. The complete genes included a novel hydrolase gene ddhA, which is responsible for the cleavage of the urea side chain of the IPU demethylated products; a distinct aniline dioxygenase gene cluster adoQTA1A2BR, which has a broad substrate range; and an inducible catechol meta-cleavage pathway gene cluster adoXEGKLIJC. Furthermore, the initial mono-N-demethylation genes pdmAB were further confirmed to be involved in the successive N-demethylation of the IPU mono-N-demethylated product. These IPU-catabolic genes were organized into four transcription units and distributed on three plasmids. They were flanked by multiple mobile genetic elements and highly conserved among IPU-mineralizing sphingomonads. The elucidation of the molecular mechanism of IPU catabolism will enhance our understanding of the microbial mineralization of IPU and provide insights into the evolutionary scenario of the conserved IPU-catabolic pathway. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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