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What disease does Saccharopolyspora erythraea cause?

What disease does Saccharopolyspora erythraea cause?


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For an examination assignment I have to find a disease caused by the bacterial species Saccharopolyspora erythraea, but I have searched the internet and have found no report of patients being infected with Saccharopolyspora erythraea. The only information I found on this bacteria is about its production of erythromycin.


Saccharopolyspora rectivirgula is a Gram-positive thermophilic sporulating actinomycete (1) that causes farmer’s lung disease (FLD), a type of hypersensitivity pneumonitis (2). FLD can develop into a chronic disease and lead to irreversible lung damage and even death (3). S. rectivirgula is often found in high concentrations in the air in barns where wet hay is stored (4). Wet hay may reach temperatures of 55 to 60ଌ, which promotes growth of S. rectivirgula (5). Hay can generate dust containing S. rectivirgula, which can lead to FLD after inhalation.

Only 1 to 15% of farmers exposed to S. rectivirgula develop FLD (6𠄸). There seems to be a genetic component protecting against the hyperreactive allergic response that is associated with FLD (6, 9, 10). Interestingly, smokers seem to have a lower incidence of the disease (2, 11). By use of a mouse model, it was shown that nicotine reduced the allergic response and lung damage (11). In contrast, other studies have shown that the allergic response gets stronger after a recent viral infection (12, 13).

The only efficient long-term treatment of FLD is removal of the antigen (6, 9). For the individual, this may have large economic and social consequences. Knowing the genome sequence of S. rectivirgula may provide a better understanding of the cause of the hyperreactive allergic reaction, which can be used to develop better tools to monitor and detect this working environmental hazard and lead to new methods of treatment.

The S. rectivirgula type strain DSM 43747 was obtained from and grown under conditions recommended by the Deutsche Sammlung von Mikroorganismen und Zellkulturen in Germany. Genomic DNA was isolated by lysis using bead beating in equal volumes DNAzol (Invitrogen) and Tris-EDTA (TE) buffer (10 mM Tris [pHਇ.9] and 1 mM EDTA) using conditions previously described (14), and the pellet was resuspended in TE buffer. The DNA was treated with RNase A and proteinase K according to standard protocols. Whole-genome sequencing of S. rectivirgula was performed at the SNP&SEQ Technology Platform of Uppsala University on a HiSeq2000 (Illumina) platform. A total of 9.3 million paired-end reads were generated, with an average read length of 100 nucleotides. Assembly of the reads was done using the A5 assembly pipeline (15). The reads were assembled into 182 contigs making up a total genome size of 3,977,051਋p with a GC content of 68.9% and an N50 value of 52,597. The average coverage was 469×. The contigs were annotated using the RAST server (16). This analysis resulted in 3,840 predicted protein-coding genes, 50 tRNA genes, and 2 ribosomal RNA operons. Interestingly, the S. rectivirgula genome is considerably smaller than those of the previously sequenced Saccharopolyspora erythraea (8.21 Mb) (17) and Saccharopolyspora spinosa (8.58 Mb) (18). By use of PanOCT v 1.9 (19), it was predicted that only 1,951 genes are shared among all three genomes and 1,467 genes are present only in S. rectivirgula.

Nucleotide sequence accession numbers.


Results and Discussion

Protein Binding to the Promoters of the Erythromycin Biosynthetic Gene Cluster.

Sustained, coordinate expression of the biosynthetic gene cluster of erythromycin by the OVP strain indicated the existence of a global regulator of these genes (8). To begin to identify the protein, we prepared fluorescent DNA probes of the five regions containing ery promoters (Fig. 1A) (10, 11). When incubated with crude lysates of both WT and OVP strains after 22 and 65 h of growth in liquid medium, all five promoter probes showed shifts in electrophoretic mobility shift assays (EMSAs), with eryAI-BIV and eryBVI probes producing the strongest shifts with the 22 h lysates (Fig. 1B, lanes c and e). For both WT and OVP strains, there was no shift observed after 65 h of growth (Fig. 1B, lanes d and f). The eryK and eryCI-ermE probes exhibited only modest shifts, whereas a smeared shift was observed for the eryBI-BIII probe with all lysates tested [supporting information (SI) Fig. S1]. Moreover, we obtained similar EMSA results by using DNA probes prepared from genomic DNA of both WT and OVP strains (data not shown).

EMSAs with promoters of the ery cluster and lysates of S. erythraea. (A) The biosynthetic gene cluster of erythromycin (ery). Gray lines show regions that contain promoters and were used as probes for EMSAs. For eryBVI and eryK, probes included the start site of transcription and the start codon. For divergent promoters (eryCI-ermE, eryBI-BIII, and eryAI-BIV), probes included start sites of transcription and start codons of the divergent genes. (B) EMSAs for promoters eryAI-BIV and eryBVI. Each probe has lanes b–f. Lanes show DNA ladder (a), probe only (b), probe + WT 22-hr lysate (c), probe + WT 65-hr lysate (d), probe + overproducer 22-hr lysate (e), and probe + overproducer 65-hr lysate (f). (C) EMSAs with the eryBVI probe and lysates of the WT and OVP strains at 12, 24, 30, and 48 h. Results are a representative example of three independent experiments.

Several tests indicated that the protein(s) causing the shifts bound to ery promoters specifically. In competition with the eryBVI probe, excess unrelated DNA (a fragment of a plasmid that contains an oriT site) did not affect the shift of the probe, but excess unlabeled eryBVI probe effectively competed with the labeled probe for binding to BldD (data not shown). In addition, incubation of a crude lysate of S. erythraea with a promoter from S. coelicolor (actVI-Ap) produced no shift (data not shown). Together, these initial results suggested that the protein(s) causing the EMSA shifts bind specifically to promoters of the ery cluster.

Presence of BldD Correlates with Expression of the ery Cluster.

A time course revealed more precisely when the WT and OVP strains produce the protein(s) causing the EMSA shifts in vegetative cultures. The two strains had similar growth curves in a liquid, rich medium (Fig. S2). In EMSAs with the eryBVI probe, lysates from both strains produced a shift at 12 h, the earliest time point (Fig. 1C). For the WT strain, the shift was observed until 24 h. In contrast, for the OVP strain, the shift remained detectable at 30 h (Fig. 1C), roughly correlating with the lengthened expression of the ery cluster (8).

Regulator Is a BldD Ortholog.

A protein designated BldD (see above) was purified based on its ability to cause an EMSA shift by using the eryBVI probe, and its structural gene was cloned as described in Materials and Methods. The bldD gene encodes a protein of 162 aa and molecular mass of 17.7 kDa (Fig. S3). The predicted BldD sequence yielded strong matches to the BldD homologs of several actinomycetes. For instance, BldD is 77% identical to the BldD of Streptomyces coelicolor. This finding was unexpected because, although mutations in S. coelicolor bldD influence the synthesis of secondary metabolites (12), we found no published evidence indicating that a member of the BldD family directly regulates antibiotic biosynthetic genes. In S. coelicolor, BldD negatively regulates expression of the developmental σ factor genes bldN and whiG during vegetative growth (13), in addition to repressing vegetative expression of the stress response σ factor gene sigH (14) and an as-yet uncharacterized regulator termed bdtA (13). It also negatively regulates its own synthesis (15). Thus, all of the BldD targets so far identified in S. coelicolor are repressed by BldD, whereas the ery genes are positively regulated by BldD in S. erythraea.

BldD Resides Apart from the ery Cluster in the Circular Chromosome of S. erythraea.

In the 8.2-Mb S. erythraea chromosome, the ery cluster is centrally located within the core region, which encodes most of the essential genes (9), whereas bldD lies near the edge of the core region, ≈1.5 Mb from the ery cluster (Fig. 2A). The separate positions of bldD and the ery cluster in the chromosome of S. erythraea contrast with many biosynthetic clusters of antibiotics such as actinorhodin (4) and undecylprodigiosin (5) that contain regulatory genes. Analysis of sequences for Aeromicrobium erythreum, which also synthesizes erythromycin, and Micromonospora megalomicea, which synthesizes the related molecule megalomicin, suggests that the ery cluster once might have contained a regulatory gene. Both species have biosynthetic gene clusters extremely similar to the ery cluster in S. erythraea but contain putative regulatory genes. In A. erythreum, a transcriptional regulator of the MarR family (ery-ORF25) is encoded at one end of the ery cluster adjacent to eryCI (16), and in M. megalomicea, a putative regulator is encoded at the end adjacent to megDVI (17).

Chromosomal locus of bldD. (A) Location of bldD in the chromosome of S. erythraea. Gray bar indicates the core region. (B) Alignment of the locus of bldD (S. ery) to the locus of bldD in S. coelicolor (S. coe). Black arrows show bldD (S. ery) and bldD (S. coe). Alignments represent genes homologous between the two bacteria. The dotted line represents a discontinuity of one gene in S. coelicolor in the alignment. The numbers above the genes are gene numbers in the genome sequence for both species.

The gene organization around the bldD gene is very similar to that of bldD in S. coelicolor (Fig. 2B). In particular, the three genes downstream of bldD (SACE_2074–2076) resemble three genes downstream of S. coelicolor bldD (SCO1492–1490), and the six genes upstream of bldD (SACE_2078–2084) resemble six genes upstream of S. coelicolor bldD (SCO1488–1483 and SCO1481). Combined with the bald phenotype of the bldD mutant (see above), this synteny reinforces the idea that S. erythraea BldD and S. coelicolor BldD are orthologous.

Recombinant BldD Binds to ery Promoters and to Its Own Promoter.

To confirm that bldD encodes a DNA binding protein, we expressed recombinant BldD in Escherichia coli BL21(DE3). In EMSAs, a lysate of E. coli induced to express BldD shifted the eryBVI probe completely, whereas a lysate of an uninduced strain caused no shift (data not shown). Further, a 56-bp footprint of BldD binding at the eryBVIp region was obtained (Fig. 3 and Fig. S4). The footprint includes the transcriptional start site of eryBVIp (11), and although it is an unusual binding location for an activator, it has been observed before (18). By visual inspection of the sequence within this protected region, we identified a possible binding sequence of AGTGC(n)9TCGAC for BldD, based on the S. coelicolor BldD consensus binding sequence of AGTgA(n)mTCACc (13). Consistent with our data, S. coelicolor BldD binds upstream or across the transcriptional start sites of its targets (13 ⇓ –15). However, S. coelicolor BldD acts as a transcriptional repressor of each of these target genes (12 ⇓ –14), whereas S. erythraea BldD appears to be acting as a transcriptional activator of the ery gene cluster. The mechanism by which BldD can act as a transcriptional activator in some cases and a transcriptional repressor in others is unclear and warrants further investigation.

Identification of the sequence of the BldD-protected regions of the eryBVI promoter by DNase I protection footprinting. (A) Electrophoregram for DNase I (0.1U) digest of the eryBVI probe without BldD. (B) Electrophoregram for DNase I (0.1U) digest of the eryBVI probe after incubation with 54 μM recombinant BldD. The boxed region indicates a region protected by BldD. (C) Underlined sequence indicates footprint. Gray nucleotides show putative BldD binding sites. Asterisk denotes start sites of transcription for eryBVI, and bracketed sequences indicate putative −10 and −35 promoter sequences (11).

BldD shifted all five probes of the ery promoters (Fig. 4), indicating that BldD regulates the entire biosynthetic cluster of erythromycin. To assess the affinity of BldD for each probe, we purified recombinant BldD by using a Ni-NTA resin (data not shown). Titrations of BldD with a fixed concentration of each probe, followed by measurements of the fraction of probe bound in EMSAs, yielded equilibrium dissociation constants (Kd) (Table 1 and Fig. S5). BldD binds with similar affinities to the probes of eryAI-BIV, eryBVI, and eryBI-BIII, and ≈3- to 5-fold less strongly to the probes of eryK and eryCI-ermE.

EMSAs with recombinant BldD and the five regions of ery promoters, designated eryCI-ermE, eryBI-BIII, eryAI-BIV, eryBVI, and eryK. − , probe only +, probe and BldD.

Dissociation constants for BldD binding its own promoter and the five regions with promoters of the ery cluster

Because S. coelicolor BldD binds to its own promoter (15), we asked whether S. erythraea BldD bound to a sequence upstream of bldD. EMSAs with the S. erythraea bldD promoter and purified recombinant BldD resulted in two shifted fragments (Fig. 5A). Both shifts required the presence of BldD, but we were unable to determine whether they arose from different conformations or multimers of BldD. Note that S. coelicolor BldD binds to its own promoter as a dimer, also resulting in two distinct bands in EMSA experiments (7). In addition, Kd of 0.32 μM for BldD binding to its own promoter was determined, which approximates the Kd of BldD for its own promoter (19). Therefore, BldD binds to the bldD promoter an order of magnitude more strongly than it does to promoters of the ery cluster.

EMSAs with the bldD promoter and purified, recombinant BldD (A) and lysates of the WT and OVP strains of S. erythraea (B). See text for details. (C) Western blot of lysates (4 μg of total protein) of the WT and OVP strains with polyclonal antibody for BldD. As a positive control, the far right lane shows a Western blot of purified, recombinant BldD. Results are a representative example of three independent experiments. (D) Titers of erythromycin of the WT (black diamonds) and OVP (open squares) strains grown in a liquid, rich medium.

S. erythraea Overproducer Strain Has More BldD.

Because BldD binds to its own promoter with submicromolar Kd, we used the bldD promoter to examine how BldD binding varied with a time course of lysates from S. erythraea. Cultures of the WT and OVP strains in a liquid, rich medium were sampled at 27, 40, 62, 87, and 111 h. EMSAs using lysates from both strains revealed two shifted probe fragments at 27 h, the initial time point (Fig. 5B). By 40 h, the WT strain lysate failed to produce most of the upper band, whereas the OVP strain lysate still revealed that band at the last time point tested (111 h). Also, the reactions with WT-strain lysates resulted in more unbound probe than with the OVP strain lysates at all times (Fig. 5B). To determine whether the greater shifts observed with OVP strain lysates were because of more abundant BldD, we assessed the expression of BldD in the WT and OVP strains by using Western blots. Polyclonal anti-BldD antibody detected BldD in the first four lysates of each previous time course. BldD was observed in both strains after 27 h of growth, when they were producing erythromycin (Fig. 5 C and D). However, BldD abundance in the WT strain decreased after 40 h, coinciding with a decrease in erythromycin production. In contrast, the OVP strain maintained relatively constant BldD levels up to 87 h, as production of erythromycin continued. The amounts of BldD detected by Western blot analysis matched well with the intensities of shifts in EMSAs (Fig. 5B), indicating that a higher abundance of BldD, rather than a more active form, caused stronger shifts from the OVP strain. How the OVP strain acquires the phenotype of extended BldD expression is not known, but we postulate that during classical strain improvement, mutations were introduced in genes that regulate BldD expression, as the sequences for the promoter and coding region of bldD are identical for the WT and OVP strains.

Deletion of bldD Generates a “Bald” Phenotype.

We deleted bldD in S. erythraea strain AML315–638 (which derives from the WT strain) (20) as described in Materials and Methods. The deletion strain (ΔbldD) failed to form aerial mycelium and to sporulate on three different media, M1 (21), SFM, and R5 agar (Fig. 6A and Fig. S6). This bald phenotype, which is also characteristic of S. coelicolor ΔbldD strains (7, 22), suggests that S. erythraea BldD and S. coelicolor BldD have similar functions. Complementation of the bldD deletion with a single copy of bldD restored the WT phenotype, whereas complementation with a plasmid lacking bldD maintained the bald phenotype (Fig. 6A). When grown in a liquid, rich medium for 5 days, the ΔbldD strain produced 7-fold less erythromycin than the WT strain (Fig. 6B). Complementation of the bldD deletion with a single copy of bldD restored normal titers, whereas complementation with a plasmid lacking bldD left titers low (data not shown). Together, these data suggest that BldD positively regulates the ery genes.

Phenotypes and titers of different S. erythrea strains. (A) Phenotypes of S. erythraea lacking bldD. Strains were grown on M1 agar. WT, wild-type S. erythraea ΔbldD, WT strain with bldD deleted ΔbldD::bldD, ΔbldD strain with bldD integrated on a vector next to the ery cluster (see text for details) and ΔbldD::empty vector, ΔbldD strain with an empty vector integrated next to the ery cluster, which served as a negative control. (B) Titers of the WT (black diamonds) and ΔbldD (open squares) strains.

However, because the mutation left some synthesis of erythromycin intact, it differs from deletions of activators in many streptomycetes that abolish the production of an antibiotic completely (3, 23). Our observations more closely resemble the case of S. noursei, in which a strain with a deletion of a nystatin synthesis regulator still produced some antibiotic (24).

Our data show that not only is BldD important for erythromycin biosynthesis, it is also necessary for morphological differentiation. We report the discovery of a developmental transcription factor that directly activates expression of the enzymes of an antibiotic biosynthetic pathway. The findings here provide a starting point for understanding the regulation of erythromycin biosynthesis. The complex mechanisms that generate antibiotics in actinomycetes involve factors such as small signaling molecules (25) and hierarchies of transcriptional proteins (26). Attempts to understand how proteins such as BldD work with these factors should promote progress toward new strategies for strain improvement. The same efforts should reveal further how bacteria connect the synthesis of small molecules to their morphogenesis.


Introduction

The megalomicins are 6-O-glycosides of erythromycin C with acetyl or propionyl groups esterified to the 3′′′ or 4′′′ hydroxyls of the mycarose sugar (Fig. 1). They were discovered in 1969 as antibacterial agents produced by Micromonospora megalomicea sp. n. ( Weinstein et al., 1969 ). The deoxyamino sugar at C-6 was named ‘megosamine’ ( Nakagawa and Omura, 1984 ) and is identical in structure to l -rhodosamine (or N-dimethyldaunosamine). Although the initial structural assignment of megalomicin was in error, a thorough reassessment of NMR data coupled with an X-ray crystal structure of a megalomicin A derivative ( Nakagawa and Omura, 1984 ) established the current structures.

Structures of megalomicins and erythromycin A.

Therapeutic interest in megalomicin stems from its several different observed biological activities. As antibacterials, they act like erythromycin, which inhibits protein synthesis through selective binding to the bacterial 50S ribosomal RNA. The potency, spectrum of activity and toxicity of the megalomicins is very similar to that of erythromycin A ( Weinstein et al., 1969 ). Megalomicin also affects protein trafficking in eukaryotic cells ( Bonay et al., 1996 , 1997 ). Although the mechanism of this action is not entirely clear, it appears to involve inhibition of vesicular transport between the medial and trans Golgi, resulting in undersialylation of proteins. They also strongly inhibit the ATP-dependent acidification of lysosomes in vivo ( Bonay et al., 1997 ) and cause an anomalous glycosylation of viral proteins, which may be responsible for their antiviral activity against herpes ( Alarcon et al., 1984 , 1988 ). Strikingly, the megalomicins are also potent antiparasitic agents, effective against Plasmodium falciparum, Trypanosoma sp. and Leishmania donovani ( Bonay et al., 1998 ). As erythromycin does not have antiparasitic activity, the antiparasitic action of megalomicin is most probably related to the presence of the deoxyamino sugar at C-6. In animal studies, megalomicin was shown to have exceedingly low toxicity, raising the prospects that derivatives could be developed into potent drugs for treatment of malarial disease.

The aglycone backbone of both megalomicin and erythromycin is the complex polyketide 6-deoxyerythronolide B (6-dEB), produced from the successive condensations of a propionyl-CoA starter unit and 6 methylmalonyl-CoA extender units (Fig. 2). Complex polyketides are assembled by modular polyketide synthases (PKSs), which are composed of multifunctional polypeptides that contain the activities (as enzymatic domains) for the condensation and subsequent reductions required to produce the polyketide chain ( Katz, 1997 Cane et al., 1998 ). In modular PKSs, each module contains the required catalytic domains for a single round of condensation and reduction steps 6-dEB synthesis requires six modules ( Donadio et al., 1991 ). During assembly, the nascent polyketide is tethered to the PKS through thioester linkages. The prototypical modular PKS is 6-deoxyerythronolide B synthase (DEBS) ( Cortés et al., 1990 Donadio et al., 1991 ), encoded by the eryA genes from Saccharopolyspora erythraea.

Erythromycin and proposed megalomicin biosynthetic pathways.

Making derivatives of megalomicin through genetic engineering would require either the ability to manipulate genetically the producing host M. megalomicea or the outcloning of the megalomicin biosynthetic genes in a more genetically tractable host. The proposed biosynthetic pathway of megalomicin and its relationship to that of erythromycin is shown in Fig. 2. Both pathways are identical through erythromycin C, the penultimate intermediate of erythromycin A and megalomicin A. We believed therefore that the genes for synthesis of the polyketide, l -mycarose and d -desosamine ( Gaisser et al., 1997 Summers et al., 1997 ) would be similar in the corresponding hosts and that we could use the erythromycin (ery) genes as probes to locate the megalomicin (meg) biosynthesis cluster. Furthermore, we believed that the meg cluster would contain an additional set of genes for synthesis of the unique deoxysugar l -megosamine and that expression of these genes in S. erythraea would result in the production of megalomicin.

We describe the characterization of a major portion of the megalomicin biosynthesis gene cluster containing the megalomicin PKS, genes for the complete megosamine pathway and portions of the mycarose and desosamine pathways. Transfer of the megosamine genes to S. erythraea resulted in production of megalomicins. In contrast to M. megalomicea, S. erythraea is a well-characterized host with established genetic engineering tools and extensive fermentation development. Thus, the conversion of S. erythraea into a megalomicin producer enables the prospect of generating new antiparasitic compounds by genetic engineering.


RESULTS

A collection of mutants from a single cycle of strain improvement

Thirty-five mutants influencing erythromycin production were obtained from the screening of 1048 transposon-generated mutants of S. erythraea representing ∼7% of the genes in the genome. DNA sequence analysis of the transposon insertion sites revealed 15 unique genotypes siblings and multiple mutations in the same gene accounted for the duplicate genotypes. Of the 15 knockout mutant strains found, 13 showed a >25% improved yield and 1 genotype had reduced yield and 1 genotype was neutral but showed reduced yield upon later scale-up analysis (Fig. 2). The mutants from the first screen showed mean increases in erythromycin yield of 34%–109%.

DNA sequence analysis revealed 15 gene targets affecting erythromycin production with transposons falling into coding regions in 13 cases, and promoter regions in 2 cases nucleotide numbers of the transposon insertion sites are given (Table 1). Nine mutants showed sporulation or pigmentation defects in addition to influencing erythromycin production. The 15 targeted genes fell into six general functional categories determined by BLASTP analysis: transcriptional regulators (acrR and rho1), cell wall biogenesis (cps2I, cwh1 and possibly gtf1), hydrolases (tsp1, dppII and cwh1), metabolism, (citA4, hpcH, fabG, cysH, arsC), antibiotic biosynthesis (eryAII and possibly gtf1) and unknown (unk1597) (Table 1). Tn5 insertion into the eryAII gene produced the expected complete blockage in erythromycin production. A neutral phenotype was produced by insertion into the cccA gene coding for a cytochrome-c related protein involved in energy production however, in shake flasks this mutant showed reduced erythromycin yield (data not shown).

Transposon mutations from this study influencing erythromycin biosynthesis in S. erythraea.

Gene . Mutant # . Transposon nucleotide (nt) insertion site . Ery 1 . Spo 2 . Pig 3 . Predicted Function (reference where known) .
FL2302 Control + + Parental strain control
acrR S6.18–36 In SACE_0303 339,794 + + + acrR, regulator of multidrug efflux pump (Wu et al. 2014)
citA4S6.18–32 In SACE_0632 696,384 + + + citA4 citrate synthase (Viollier et al. 2001)
hpcHS7.11–58 In SACE_0699 769,528 + hpcH 2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase
fabGS6.07–125 In SACE_0700 770,333 + fabG, short-chain dehydrogenase/reductase family
eryAIIS5.17–06 In SACE_0723 802,912 eryAII erythromycin polyketide synthase
cysHS6.18–04 In SACE_1474 1,626,645 + + + cysH, sulfate adenylyltransferase subunit 2
unk1597S6.07–72 In SACE_1597 1,752,052 + +/– unk1597, hypotheical protein, polar effects expected on SACE_1598
cwh1S6.07–03 In SACE_1598 1,752,509 + +/– cwh1, cell-wall-associated hydrolase, NlpC/P60 superfamily (Anantharaman and Aravind 2003)
cccAS5.17–12 In SACE_1685 1,852,856 cccA - cytochrome c mono-and diheme variants
gtf1S7.05–53 Upstream of SACE_2010 2,197,132 + gtf1, glycosyltransferase- GT1_Gtf_like family (Liang and Qiao 2007)
cps2IS6.15–149 In SACE_3177 3,507,747 + bld + cps2I, nucleotide-sugar-dependent glycosyltransferase, group 1 (Lakkitjaroen et al. 2014)
arsCS6.18–12 In SACE_5143 5,750,836 + arsC, arsenical resistance protein, arsenate reductase, arsenic transporter (EC 1.20.4.1)
tsp1S6.18–17 In SACE_5967 6,700,519 + + + tsp1, secreted trypsin-like serine protease
rho1S7.12–136 In SACE_6295 7,045,657 + + + rho1, rho1-like transcription terminator (Cardinale et al. 2008)
dppIIS6.15–143 Upstream of SACE_6505 7,295,927 + + + dppII, X-Pro dipeptidyl-peptidase (Maes, Scharpé and De Meester 2007)
Gene . Mutant # . Transposon nucleotide (nt) insertion site . Ery 1 . Spo 2 . Pig 3 . Predicted Function (reference where known) .
FL2302 Control + + Parental strain control
acrR S6.18–36 In SACE_0303 339,794 + + + acrR, regulator of multidrug efflux pump (Wu et al. 2014)
citA4S6.18–32 In SACE_0632 696,384 + + + citA4 citrate synthase (Viollier et al. 2001)
hpcHS7.11–58 In SACE_0699 769,528 + hpcH 2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase
fabGS6.07–125 In SACE_0700 770,333 + fabG, short-chain dehydrogenase/reductase family
eryAIIS5.17–06 In SACE_0723 802,912 eryAII erythromycin polyketide synthase
cysHS6.18–04 In SACE_1474 1,626,645 + + + cysH, sulfate adenylyltransferase subunit 2
unk1597S6.07–72 In SACE_1597 1,752,052 + +/– unk1597, hypotheical protein, polar effects expected on SACE_1598
cwh1S6.07–03 In SACE_1598 1,752,509 + +/– cwh1, cell-wall-associated hydrolase, NlpC/P60 superfamily (Anantharaman and Aravind 2003)
cccAS5.17–12 In SACE_1685 1,852,856 cccA - cytochrome c mono-and diheme variants
gtf1S7.05–53 Upstream of SACE_2010 2,197,132 + gtf1, glycosyltransferase- GT1_Gtf_like family (Liang and Qiao 2007)
cps2IS6.15–149 In SACE_3177 3,507,747 + bld + cps2I, nucleotide-sugar-dependent glycosyltransferase, group 1 (Lakkitjaroen et al. 2014)
arsCS6.18–12 In SACE_5143 5,750,836 + arsC, arsenical resistance protein, arsenate reductase, arsenic transporter (EC 1.20.4.1)
tsp1S6.18–17 In SACE_5967 6,700,519 + + + tsp1, secreted trypsin-like serine protease
rho1S7.12–136 In SACE_6295 7,045,657 + + + rho1, rho1-like transcription terminator (Cardinale et al. 2008)
dppIIS6.15–143 Upstream of SACE_6505 7,295,927 + + + dppII, X-Pro dipeptidyl-peptidase (Maes, Scharpé and De Meester 2007)

Erythromycin production phenotype increased production +, reduced production –, compared to parent strain FL2302.

Sporulation phenotype. +, wild type sporulation –, makes aerial mycelium but no spores bld, makes no aerial mycelium or spores.

Pigmentation. +, normal red pigmentation –, no red pigmentation +/–, reduced red pigmentation.

Transposon mutations from this study influencing erythromycin biosynthesis in S. erythraea.

Gene . Mutant # . Transposon nucleotide (nt) insertion site . Ery 1 . Spo 2 . Pig 3 . Predicted Function (reference where known) .
FL2302 Control + + Parental strain control
acrR S6.18–36 In SACE_0303 339,794 + + + acrR, regulator of multidrug efflux pump (Wu et al. 2014)
citA4S6.18–32 In SACE_0632 696,384 + + + citA4 citrate synthase (Viollier et al. 2001)
hpcHS7.11–58 In SACE_0699 769,528 + hpcH 2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase
fabGS6.07–125 In SACE_0700 770,333 + fabG, short-chain dehydrogenase/reductase family
eryAIIS5.17–06 In SACE_0723 802,912 eryAII erythromycin polyketide synthase
cysHS6.18–04 In SACE_1474 1,626,645 + + + cysH, sulfate adenylyltransferase subunit 2
unk1597S6.07–72 In SACE_1597 1,752,052 + +/– unk1597, hypotheical protein, polar effects expected on SACE_1598
cwh1S6.07–03 In SACE_1598 1,752,509 + +/– cwh1, cell-wall-associated hydrolase, NlpC/P60 superfamily (Anantharaman and Aravind 2003)
cccAS5.17–12 In SACE_1685 1,852,856 cccA - cytochrome c mono-and diheme variants
gtf1S7.05–53 Upstream of SACE_2010 2,197,132 + gtf1, glycosyltransferase- GT1_Gtf_like family (Liang and Qiao 2007)
cps2IS6.15–149 In SACE_3177 3,507,747 + bld + cps2I, nucleotide-sugar-dependent glycosyltransferase, group 1 (Lakkitjaroen et al. 2014)
arsCS6.18–12 In SACE_5143 5,750,836 + arsC, arsenical resistance protein, arsenate reductase, arsenic transporter (EC 1.20.4.1)
tsp1S6.18–17 In SACE_5967 6,700,519 + + + tsp1, secreted trypsin-like serine protease
rho1S7.12–136 In SACE_6295 7,045,657 + + + rho1, rho1-like transcription terminator (Cardinale et al. 2008)
dppIIS6.15–143 Upstream of SACE_6505 7,295,927 + + + dppII, X-Pro dipeptidyl-peptidase (Maes, Scharpé and De Meester 2007)
Gene . Mutant # . Transposon nucleotide (nt) insertion site . Ery 1 . Spo 2 . Pig 3 . Predicted Function (reference where known) .
FL2302 Control + + Parental strain control
acrR S6.18–36 In SACE_0303 339,794 + + + acrR, regulator of multidrug efflux pump (Wu et al. 2014)
citA4S6.18–32 In SACE_0632 696,384 + + + citA4 citrate synthase (Viollier et al. 2001)
hpcHS7.11–58 In SACE_0699 769,528 + hpcH 2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase
fabGS6.07–125 In SACE_0700 770,333 + fabG, short-chain dehydrogenase/reductase family
eryAIIS5.17–06 In SACE_0723 802,912 eryAII erythromycin polyketide synthase
cysHS6.18–04 In SACE_1474 1,626,645 + + + cysH, sulfate adenylyltransferase subunit 2
unk1597S6.07–72 In SACE_1597 1,752,052 + +/– unk1597, hypotheical protein, polar effects expected on SACE_1598
cwh1S6.07–03 In SACE_1598 1,752,509 + +/– cwh1, cell-wall-associated hydrolase, NlpC/P60 superfamily (Anantharaman and Aravind 2003)
cccAS5.17–12 In SACE_1685 1,852,856 cccA - cytochrome c mono-and diheme variants
gtf1S7.05–53 Upstream of SACE_2010 2,197,132 + gtf1, glycosyltransferase- GT1_Gtf_like family (Liang and Qiao 2007)
cps2IS6.15–149 In SACE_3177 3,507,747 + bld + cps2I, nucleotide-sugar-dependent glycosyltransferase, group 1 (Lakkitjaroen et al. 2014)
arsCS6.18–12 In SACE_5143 5,750,836 + arsC, arsenical resistance protein, arsenate reductase, arsenic transporter (EC 1.20.4.1)
tsp1S6.18–17 In SACE_5967 6,700,519 + + + tsp1, secreted trypsin-like serine protease
rho1S7.12–136 In SACE_6295 7,045,657 + + + rho1, rho1-like transcription terminator (Cardinale et al. 2008)
dppIIS6.15–143 Upstream of SACE_6505 7,295,927 + + + dppII, X-Pro dipeptidyl-peptidase (Maes, Scharpé and De Meester 2007)

Erythromycin production phenotype increased production +, reduced production –, compared to parent strain FL2302.

Sporulation phenotype. +, wild type sporulation –, makes aerial mycelium but no spores bld, makes no aerial mycelium or spores.

Pigmentation. +, normal red pigmentation –, no red pigmentation +/–, reduced red pigmentation.

The transposon insertions were mapped broadly around the S. erythraea chromosome (Fig. 3A). Insertions influencing erythromycin production were most frequently found in the upper-half ‘core-metabolism’ region of the genome (0–2.5 and 5.5–8.0 Mb) and less frequently in the lower half ‘non-core’ region (2.5–5.5 Mb) (Oliynyk et al. 2007). Sibling mutants were found for six of the genes (acrR, fabG, eryAII, cwh1, gtf1 and rho1) and three insertion events occurred into the cell wall hydolase gene cwh1 (Fig. 3B). For 11 of the 15 genes in the collection, the closest NCBI GenBank homologs were found in S. spinosa.

(A) Genome map of S. erythraea showing transposon insertion sites of Library 3 mutants. Map is based on data generated by Oliynyk et al. ( 2007). Knockouts of genes highlighted in green gave increases in yield, and red shading is for decreases in yield. Map positions are shown in megabase pairs. (B) Map of the cwh1 (SACE_1598) region of the S. erythraea genome (Oliynyk et al. 2007). Transposon insertion and orientation are indicated by directional flags. Numbers above the flags refer to mutant numbers all mutant numbers shown are from the S6.07 pool, for example, ‘03’ indicates mutant number S6.07–03. Map positions are shown in megabase pairs.

(A) Genome map of S. erythraea showing transposon insertion sites of Library 3 mutants. Map is based on data generated by Oliynyk et al. ( 2007). Knockouts of genes highlighted in green gave increases in yield, and red shading is for decreases in yield. Map positions are shown in megabase pairs. (B) Map of the cwh1 (SACE_1598) region of the S. erythraea genome (Oliynyk et al. 2007). Transposon insertion and orientation are indicated by directional flags. Numbers above the flags refer to mutant numbers all mutant numbers shown are from the S6.07 pool, for example, ‘03’ indicates mutant number S6.07–03. Map positions are shown in megabase pairs.

Interaction between genotype and environment

The fermentation growth environment determines which phenotypes can be scaled-up and which cannot. Genotypes influencing increased erythromycin production are potentially commercially viable. In this study, only one of the genotypes, mutant cwh1, showed increased erythromycin production after scale-up in shake flasks (Fig. 2C). Two independent cwh1 insertion mutants were tested: mutant S6.07–03 whose insertion was near the middle of the gene and mutant S6.07–06 whose insertion was near the 5 ′ end of the gene (Fig. 3B). Both mutant strains showed statistically significant increases in erythromycin production (Fig. 2C). Upstream of the cwh1 gene one insertion (‘72’, Fig. 3B) showed increased erythromycin production similar to that with insertions in cwh1 downstream of cwh1 one insertion (‘14’) was found that had a neutral phenotype—evidence that the Cwh1 phenotype is not due to an effect on the downstream gene. Based on DNA sequence data, Cwh1 is predicted to be cell-wall associated and to hydrolyze cell walls (see Supplementary Results and Fig. S1, Supporting Information). Therefore, it was not surprising that the Cwh1 phenotype included a growth defect (Fig. 4A) and non-sporulation (Fig. 4B) on solid medium.

The parental and Cwh1 phenotypes are shown at 48 and 144 h (32°C, E20A agar). The parental phenotype of normal growth and sporulation was displayed by FL2302 as well as by mutants with insertions downstream of cwh1 (mutant S6.07–14 is shown). The Cwh1 phenotype of slower growth and no sporulation was displayed by mutants with insertions in cwh1 (S6.07–03 and −06), as well as by mutants with insertions upstream of cwh1 (S6.07–72 shown).

The parental and Cwh1 phenotypes are shown at 48 and 144 h (32°C, E20A agar). The parental phenotype of normal growth and sporulation was displayed by FL2302 as well as by mutants with insertions downstream of cwh1 (mutant S6.07–14 is shown). The Cwh1 phenotype of slower growth and no sporulation was displayed by mutants with insertions in cwh1 (S6.07–03 and −06), as well as by mutants with insertions upstream of cwh1 (S6.07–72 shown).


Immunology of Infection

Diane J. Ordway , lan M. Orme , in Methods in Microbiology , 2010

3 Harvesting of CFPs

The bacilli in 500 or 1000 ml cultures are allowed to settle and the supernatant is filtered through a 0.2 mm ZapCap filter into 4-1 bottles. To minimize plugging of the filter it is important to use ZapCap with a prefilter and decant a minimal amount of cells along with the supernatant. Sodium azide is added to the filtrate to a final concentration of 0.04% w/v and this material is stored at 4° C. After filtration, the culture supernatant is considered sterile. Nevertheless, before further use, a 1 ml aliquot of this filtrate is plated on Middlebrook 7Hll agar and incubated for 3 weeks to ensure the absence of viable bacilli. The filtrate is concentrated to approximately 2% of its original volume using an Amicon apparatus with a low protein binding, 10 kDa molecular weight cut off membrane. This concentrate is dialysed extensively against 10 mM ammonium bicarbonate and the protein concentration estimated by the BCA protein assay (Pierce, Rockford, IL, USA). The final culture filtrate protein (CFP) preparation is aliquoted and stored at –70° C. Typically, 4–5 mg of CFPs is obtained from 1 l of a 2 week culture of M. tuberculosis.


Therapeutic Areas II: Cancer, Infectious Diseases, Inflammation & Immunology and Dermatology

7.18.5.7.1 C11-Tethered ketolides: telithromycin

Researchers at Abbott Laboratories were the first to develop the chemistry 238 and notice the advantages of C11/C12 cyclic carbamates in overcoming Erm-resistant streptococci. Starting with clarithromycin (6 ), the desosamine and cladinose sugar hydroxy groups were protected with acetyl and benzyloxycarbonyl groups respectively ( Scheme 5 ). Treatment with excess carbonyldiimidazole and sodium hexamethyldisilazide in one step resulted in elimination of the C11-hydroxy group and the formation of the O12 acyl imidazole 33, a key electrophilic intermediate used for the genesis of C11/C12 carbamates. Upon reacting with a series of amines in aqueous acetonitrile the imidazole of 33 was displaced and the resulting carbamate cyclized in Michael fashion stereoselectively, forming the fused C11/C12 ring and equilibration of the methyl group at C10 to the β-epimer (R) predominantly. Under conditions of insufficient equilibration the α-epimer (S) was isolated and shown to be less active. Thus, the overall sequence enabled not only formation of the C11/C12 carbamate, but also introduction of a variety of tethered aromatic rings attached to the C11 position through the nitrogen (N11) of the carbamate without disturbing the stereochemistry of the natural system by reintroduction of the C10/C11 stereogenic centers. After removal of the protecting groups on the sugars, analogs with a four-atom tether terminated by a phenyl ring, exemplified by 34, were found to have in vitro potencies 8–16-fold higher than erythromycin against both the inducible and constitutive MLS S. pyogenes strains. Significantly, analogs without the aryl ring at the end of the tether did not show this advantage, indicating the importance of the tethered aryl group in achieving unique interactions with the ribosome.

Although it was later than the Roussel–Uclaf disclosure of what came to be known as the ketolides, Taisho disclosed C11/C12 carbamates on the clarithromycin template with a ketone at C3, in which the cladinose ring was cleaved by acidic hydrolysis and the resulting C3 hydroxy oxidized to a ketone. 239,240 Similar to the Abbott approach described above, the synthetic route involved formation of the acyl imidazole intermediate 35 with excess carbonyldiimidazole and base. When reacted with ethylenediamine, the cyclic carbamate 36 was formed and the primary amine was then condensed in the presence of acetic acid with the ketone at C9 to give the tricyclic intermediate 37 ( Scheme 6 ). Significantly, the cladinose sugar was then cleaved leaving the naked hydroxy group at C3 38, which was subsequently oxidized under Pfitzner–Moffatt conditions giving, after 2'-acetyl removal, the C3 keto analog TE-802 (39). The discovery of 39 was important in that it demonstrated very good activity against the S. pneumoniae Mef phenotype, and suggested that the cladinose may be a recognition element for macrolide efflux. However, because it lacked a tethered aryl group, 39 did not exhibit activity against the Erm phenotype and furthermore had relatively poor H. influenzae potency. 241

Researchers at Roussel-Uclaf (subsequently Aventis and currently Sanofi-Aventis) were the first to disclose the hybrid of a N-substituted C11/C12 carbamate with a ketone at C3 to yield compounds that were active against both Mef and Erm S. pneumoniae, providing a major breakthrough in the fight to overcome resistance in the macrolide class ( Scheme 7 ). 12,242–244 The sequence begins with the removal of the cladinose sugar from clarithromycin (6) followed by acetylation of the desosamine 2'-hydroxy group to give 40. Chemoselective Pfitzner–Moffatt oxidation of the C3 hydroxy then yielded 41, which was further dehydrated to the enone 42 by selective mesylation of O11 and elimination with DBU (1,8-diazabicyclo[2,2,2]undec-7-ene). After conversion of 42 to the central acyl imidazole intermediate (43), different amine-containing fragments were used to displace the imidazole and subsequently form the C11/C12 ring fusion. One variation on the tether came with the C11/C12 carbazates, in which hydrazine hydrate was employed to give intermediate 44. It should be noted that a 10-fold excess of hydrazine and heating for several hours are both important for the equilibration of the C10 methyl group to the more active R configuration. 245 Reductive amination of aldehydes with 44 subsequently yielded a number of analogs, the most promising of which was the quinolin-4-yl ketolide, HMR-3004 (45). The in vitro activity of HMR-3004 against macrolide-sensitive and -resistant S. pneumoniae and against H. influenzae were well within the range of potential therapeutic usefulness. Oral in vivo efficacy was demonstrated in several intraperitoneal murine models of infection against macrolide resistant pneumococci (50% protective dose (PD50) values ranging from 15 to 42 mg kg −1 ) where clarithromycin and azithromycin failed (PD50>100 mg kg −1 ), and HMR-3004 was subsequently chosen as the first ketolide clinical candidate. The second clinical candidate to emerge and eventually surpass HMR-3004 came out of the carbamate series, in which the n-butyl tether is substituted with the 4-(3-pyridinyl)-imidazol-1-yl heteroaryl (telithromycin, HMR-3647, (8)). 11,246–248 Telithromycin is the first ketolide approved for clinical use. It is important to note the Aventis chemists also demonstrated that previous reports of C3 keto macrolide derivatives of erythromycin (1) (rather than clarithromycin, 6) were in fact equilibrated to the O6/C3 hemiacetals 49 ( Scheme 8 ) and that these compounds were devoid of antibacterial activity. The hemiacetal formation can only be avoided by employing a template with groups other than hydrogen at O6, making this a necessary feature of any ketolide.


Erythromycin

Adults: 250 mg P.O. q 6 hours, or 333 mg P.O. q 8 hours, or 500 mg P.O. q 12 hours (base, estolate, or stearate) or 400 mg P.O. q 6 hours or 800 mg P.O. q 12 hours (ethylsuccinate) or 250 to 500 mg I.V. (up to 1 g) q 6 hours (gluceptate or lactobionate)

Children: 30 to 50 mg/kg/day (base, estolate, ethylsuccinate, or lactobionate) I.V. or P.O., in divided doses q 6 hours when giving I.V. and q 6 to 8 hours when giving P.O. Maximum dosage is 2 g/day for base or estolate, 3.2 g/day for ethylsuccinate, and 4 g/day for lactobionate.

Adults: 250 mg (base, estolate, or stearate) or 400 mg (ethylsuccinate) P.O. q 6 hours for 10 to 14 days

Children: 30 to 50 mg/kg/day (base, estolate, ethylsuccinate, or stearate) P.O. in divided doses over 10 to 14 days

➣ Prophylaxis of ophthalmia neonatorum caused by Neisseria gonorrhoeae or Chlamydia trachomatis

Neonates: 0.5- to 1-cm ribbon of ointment into each lower conjunctival sac once

➣ Treatment of conjunctivitis of the newborn caused by susceptible organisms


<p>This section provides any useful information about the protein, mostly biological knowledge.<p><a href='/help/function_section' target='_top'>More. </a></p> Function i

Catalyzes the reversible conversion of 2-phosphoglycerate into phosphoenolpyruvate. It is essential for the degradation of carbohydrates via glycolysis.

<p>Manual validated information which has been generated by the UniProtKB automatic annotation system.</p> <p><a href="/manual/evidences#ECO:0000255">More. </a></p> Manual assertion according to rules i


The impact of early-in-life macrolide treatment on gut microbial communities and host health.

A story about perturbations, maturity, community, and resilience.

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Find the full story in Nature Communications: http://go.nature.com/2h0Lo27

The first macrolide, erythromycin was isolated in the 1950’s from the soil bacterium Streptomyces erythraeus now known as Saccharopolyspora erythraea. Decades later, the combination of naturally derived and synthetic antibiotics have dramatically altered the course of human health. The macrolide azithromycin is commonly prescribed to both children and adults for ear, respiratory, and skin infections and is the common alternative to β-lactam antibiotics such as penicillin azithromycin alone now accounts for more than 50 million antibiotic courses in the United States each year (1). Although these drugs have had a transformative impact on society as a whole, their unintended consequences have been highlighted in many publications over the years, specifically their role in altering our intestinal microbiota, the aggregate of the microorganisms that live in the gut (2-4).

This current body of work builds on the work of others and demonstrates that the timing of the antibiotic course can have a lasting impact on the host. Using a mouse model to mimic therapeutic doses given to children, we show that one macrolide antibiotic course given early-in-life is sufficient to alter the healthy microbial community that lives in our gut and leads to sustained effects on the host immune system. Although this work was performed in mice with the macrolide used widely in animals, tylosin the proxy of the human erythromycin, clarithromycin, and azithromycin, it may have implications to host health. Previous studies have demonstrated the lasting effects of macrolides on the developing microbiota in human children for the first 24 months of life (5). Based on our studies this disruption of the microbiota may also have an impact long after the antibiotic has been administered, especially if the perturbed microbiota was initially immature. Many epidemiological studies have demonstrated a strong correlation between early-life antibiotic use and obesity, asthma, and atopic diseases and the importance of the acquisition of a healthy microbiota at birth (6-8). Inappropriate microbial cues may alter immune tolerance mechanisms or affect energy homeostasis and general host physiology. We hope to continue to define and characterize key bacterial assemblages necessary to increase the capability of the microbiota to recover quickly after an antibiotic insult. The ultimate goal of our work is to develop new therapeutic strategies to increase microbial community resilience or to restore a “healthy” microbiota to improve overall host health.

The body of work in this paper was a culmination of hours of research, sleepless nights, doggedness, and numerous conversations with lab members and visiting scientists. However, for this part of “Behind the paper”, I wanted to discuss the beginnings of this project. The body of work in this article stemmed after the disaster of Hurricane Sandy. I was just months into my postdoctoral work and in less than 24 hours, 30 years of work was at risk of being lost. The storm brought an unprecedented surge directly to the hospital and we lost all electrical power, the contents of our twelve -80C freezers were melting along with our current samples and reagents. The day after Sandy, our lab ran up and down six flights of stairs, sometimes with up to 25 pounds of dry ice to save a massive quantity of samples. We worked tirelessly to not only save our own work but the work of others. We were displaced from our lab for almost a year, but again as a team, we continued to pursue the various experiments we planned before Sandy hit. Almost a year after superstorm Sandy, we moved back to our lab where the rest of this work was eventually completed and made into the article mentioned above.

Rescuing experiments after Hurricane Sandy. Superstorm sandy caused massive power outages throughout Lower Manhattan. With all 12 -80°C freezers steadily increasing in temperature lab members walked, biked, and carpooled to the lab to help savage valuable samples.

Performing experiments post Hurricane Sandy. Preparation for 16s rRNA library prep (left) and intestinal lamina propria lymphocytes isolation (right) in a new laboratory setting.

I have learned that perturbations are inevitable, whether it is in the micro or macro sense. The ability to properly react and recover marks the difference between success and failure. Unlike an antibiotic-perturbed immature microbiota, our lab was able to recover from a massive perturbation, even after being displaced for a year. Many factors promoted resiliency including leadership, institutional and government support, and most importantly the cooperation, collaboration, and sheer will of a determined group of scientists, physicians, and students.

Blaser lab members back at work (or fun) after perturbation.

Hicks, L. A., Taylor, T. H., Jr. & Hunkler, R. J. U.S. outpatient antibiotic prescribing, 2010. N Engl J Med 368, 1461-1462, doi:10.1056/NEJMc1212055651 (2013).

Nobel, Y. R. et al. Metabolic and metagenomic outcomes from early-life pulsed antibiotic treatment. Nat Commun 6, 7486, doi:10.1038/ncomms8486 (2015).

Brandl, K. et al. Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits. Nature 455, 804-807, doi:10.1038/nature07250 (2008).

Morgun, A. et al. Uncovering effects of antibiotics on the host and microbiota using transkingdom gene networks. 1732-1743, doi:10.1136/gutjnl-2014-308820

Korpela, K. et al. Intestinal microbiome is related to lifetime antibiotic use in Finnish pre-school children. Nat Commun 7, 10410, doi:10.1038/ncomms10410 (2016).