The OmpR Regulator of Burkholderia multivorans ControlsMucoid-to-Nonmucoid Transition and Other Cell EnvelopeProperties Associated with Persistence in the Cystic FibrosisLung

Inês N. Silva,a Filipa D. Pessoa,a Marcelo J. Ramires,a Mário R. Santos,a Jörg D. Becker,b Vaughn S. Cooper,c

Leonilde M. Moreiraa,d

aIBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon,Portugal

bInstituto Gulbenkian de Ciência, Oeiras, PortugalcDepartment of Microbiology and Molecular Genetics, Center for Evolutionary Biology and Medicine,University of Pittsburgh, Pittsburgh, Pennsylvania, USA

dDepartment of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

ABSTRACT Bacteria from the Burkholderia cepacia complex grow in different natu-ral and man-made environments and are feared opportunistic pathogens that causechronic respiratory infections in cystic fibrosis patients. Previous studies showed thatBurkholderia mucoid clinical isolates grown under stress conditions give rise to non-mucoid variants devoid of the exopolysaccharide cepacian. Here, we determinedthat a major cause of the nonmucoid morphotype involves nonsynonymous muta-tions and small indels in the ompR gene encoding a response regulator of a two-component regulatory system. In trans complementation of nonmucoid variants(NMVs) with the native gene restored exopolysaccharide production. The loss offunctional Burkholderia multivorans OmpR had positive effects on growth, adhesionto lung epithelial cells, and biofilm formation in high-osmolarity medium, as well asan increase in swimming and swarming motilities. In contrast, phenotypes such asantibiotic resistance, biofilm formation at low osmolarity, and virulence in Galleriamellonella were compromised by the absence of functional OmpR. Transcriptomicstudies indicated that loss of the ompR gene affects the expression of 701 genes,many associated with outer membrane composition, motility, stress response, ironacquisition, and the uptake of nutrients, consistent with starvation tolerance. Sincethe stresses here imposed on B. multivorans may strongly resemble the ones foundin the cystic fibrosis (CF) airways and mutations in the ompR gene from longitudi-nally collected CF isolates have been found, this regulator might be important forthe production of NMVs in the CF environment.

IMPORTANCE Within the cystic fibrosis (CF) lung, bacteria experience high-osmolarityconditions due to an ion unbalance resulting from defects in CF transmembrane con-ductance regulator (CFTR) protein activity in epithelial cells. Understanding how bacterialCF pathogens thrive in this environment might help the development of new therapeu-tic interventions to prevent chronic respiratory infections. Here, we show that the OmpRresponse regulator of one of the species found in CF respiratory infections, Burkholderiamultivorans, is involved in the emergence of nonmucoid colony variants and is impor-tant for osmoadaptation by regulating several cell envelope components. Specifically,genetic, phenotypic, genomic, and transcriptomic approaches uncover OmpR as a regu-lator of cell wall remodeling under stress conditions, with implications in several pheno-types such as exopolysaccharide production, motility, antibiotic resistance, adhesion, andvirulence.

Received 18 April 2018 Accepted 14 June2018

Accepted manuscript posted online 18June 2018

Citation Silva IN, Pessoa FD, Ramires MJ,Santos MR, Becker JD, Cooper VS, Moreira LM.2018. The OmpR regulator of Burkholderiamultivorans controls mucoid-to-nonmucoidtransition and other cell envelope propertiesassociated with persistence in the cysticfibrosis lung. J Bacteriol 200:e00216-18. https://doi.org/10.1128/JB.00216-18.

Editor Yves V. Brun, Indiana UniversityBloomington

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Leonilde M.Moreira, lmoreira@tecnico.ulisboa.pt.

RESEARCH ARTICLE

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KEYWORDS osmotic stress, OmpR protein, biofilm, Burkholderia multivorans,envelope remodeling, exopolysaccharide cepacian, cystic fibrosis, chronic respiratoryinfections

Acommon property of many clinical and environmental isolates within the Burk-holderia cepacia complex (Bcc), a group of opportunistic pathogens causing

chronic lung infections in cystic fibrosis (CF) patients, is the production of the exopo-lysaccharide (EPS) cepacian (1). This carbohydrate polymer consists of acetylatedheptasaccharide repeat units (2) that give rise to high-molecular-weight polysaccharidechains through polymerization. The proteins required for the synthesis of activatedsugar-nucleotide precursors, repeat unit assembly, polymerization, and export to theextracellular milieu are encoded by the bce genes (bce-I and bce-II regions) (3, 4). Exceptfor the regulator �54 and NtrC response regulator, which have been shown to positivelyregulate EPS production in Burkholderia cenocepacia grown under nitrogen limitation(5, 6), the regulation of transcription of those bce-I and bce-II gene clusters remainsmostly unknown. Many studies have linked EPS biosynthesis to virulence. For example,the EPS produced by Bcc bacteria has been shown to inhibit neutrophil chemotaxis, toneutralize reactive oxygen species in vitro (7), to affect the phagocytosis of bacteria byhuman neutrophils, and to facilitate persistent bacterial infections in mice (8, 9).Furthermore, cepacian has been shown to contribute to biofilm formation (10, 11) andto protect bacterial cells exposed to metal ion stress and desiccation (3).

Among several studies assessing the capacity for EPS production by Bcc clinicalisolates (10, 12, 13), the most comprehensive involved 560 isolates sampled from 100chronically infected CF patients and showed that strains from all species of the Bcc canexpress the mucoid phenotype via EPS production (14). These authors also showed thatsequential clonal isolates from CF patients frequently differed in this phenotype, oftenswitching from mucoid to nonmucoid. Moreover, in another retrospective clinical studyof CF patients, it was reported that patients infected with nonmucoid Bcc isolatesshowed a more dramatic decline in lung function than those infected with mucoidbacteria, suggesting that the nonmucoid morphotype is associated with increaseddisease severity and the mucoid morphotype with persistence in the lungs (15). Westudied this mucoid-to-nonmucoid morphotype transition in multiple Bcc speciesunder laboratory conditions and found it was triggered by several stresses, such asprolonged incubation, subinhibitory concentration of antibiotics, extreme tempera-tures, and osmotic, nitrosative and oxidative stresses (16). Under these stress condi-tions, mucoid strains gave rise to smaller nonmucoid colonies. These nonmucoidvariants were shown to be more susceptible to antibiotics and less virulent in Galleriamellonella (16). Colony morphology variation was also characterized in another CFpathogen, B. cenocepacia K56-2, where bacteria spontaneously experienced the con-version from a rough to a shiny colony morphotype on agar medium after a shaken orstatic incubation in liquid culture for 24 h (17). These shiny colony variants produce lessbiofilm, have less extracellular matrix surrounding the cells, and display reducedvirulence but have increased production of N-acyl-homoserine lactones (17). TheLysR-type transcriptional regulator ShvR is involved in this morphotype variation, sinceseveral point mutations were found in the shvR gene isolated from the shiny colonymorphotype.

Since the mucoid-to-nonmucoid morphotype transition occurs during chronic lunginfections of CF patients, we hypothesize that Bcc bacteria undergo a process ofadaptation involving altered expression of metabolic and surface determinants thatmight facilitate bacterial survival in vivo. To gain insights into the possible molecularmechanisms leading to this phenotypic transition, Burkholderia multivorans clinicalisolates producing mucoid colonies were exposed to stress conditions to obtainnonmucoid ones. The genome sequences of these nonmucoid colonies revealedmultiple mutations in the ompR gene encoding a response regulator as the cause of thenonmucoid colonies. Since mutations in this gene also evolved in several B. multivorans

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and B. cenocepacia isolates recovered from CF patients (18, 19), it is a likely target ofselection in vivo deserving further characterization. Here, we provide evidence of theOmpR protein being essential for cepacian biosynthesis, as well as for several otherphenotypes, such as antibiotic resistance, motility, osmotic adaptation, biofilm forma-tion, and virulence.

RESULTSMutations triggering B. multivorans mucoid-to-nonmucoid morphotype varia-

tion map within the ompR gene locus. It was previously known that stresses likeprolonged incubation, subinhibitory concentration of antimicrobials, temperature, andoxidative stress, among others, induce the mucoid-to-nonmucoid morphotype transi-tion in different Bcc species (16). Therefore, to study the appearance of colony mor-phology variants in Burkholderia, we analyzed 20 mucoid isolates of B. multivoranssequentially sampled from a CF patient over 20 years (Fig. 1A) (18). These isolatesdisplaying the mucoid morphotype due to the biosynthesis of cepacian were exposedto prolonged stationary phase in salts medium with 2% D-mannitol (SM medium)statically for 21 days at 42°C. After plating the cultures in yeast extract-mannitol (YEM)agar medium, which enhances EPS production, many colonies remained large andmucoid, but we observed, for all isolates, that 10% to 60% were small coloniesdepending on the isolate (see examples in Fig. 1B). Among these smaller colonies, mostdisplayed reduced mucoidy in YEM agar but were still shiny and, when grown in YEM

FIG 1 Selection of B. multivorans NMVs by exposure to stress conditions. (A) Temporal distribution of the B.multivorans mucoid clinical isolates recovered from a single CF patient with indication of the clade inferred byphylogenetic analysis (18). The four clades are C1 (red), C2 (yellow), C3 (blue), and C4 (green). Clinical isolate BM11was a mixture of two mucoid colony sizes, with the larger ones displaying a substitution of aspartate at position204 of BMD20_RS04295 by alanine. BMD20_RS04295 protein is the putative subunit beta of acetyl-CoA carboxy-lase. This isolate with larger colonies was named BM11L. Isolates with bold labels were the ones further studied inthis work. (B) When the 20 mucoid clinical isolates were incubated at 42°C statically for 21 days and plated on YEMagar, the majority of the colonies of each isolate were large and mucoid, whereas a smaller number displayed areduced size and mucoidy. Only one representative isolate from each clade is shown. (C) YEM agar colonymorphology of the NMV BM11L-nmv1 complemented with the empty pBBR1MCS vector or with the vectorexpressing the ompR gene. (D) Stability of the nonmucoid morphology in YEM agar after three passages of theNMVs incubated in liquid SM medium at 37°C for 2 days. Only results from two of the NMVs are shown. (E) B.multivorans genomic region where ompR is located. Gene coordinates correspond to chromosome 1 of B.multivorans D2095 (BM11) (20). MECDP, 2-C-methyl-D-erythritol 2,4-cyclo diphosphate synthase; nt, nucleotides.

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broth, were able to produce EPS. Between 1% and 5% of the small colonies wereopaque, unable to produce EPS in YEM broth, and therefore were considered nonmu-coid (named nonmucoid variants [NMVs]).

Fourteen randomly chosen NMVs derived from B. multivorans isolates BM6, BM7,BM9, BM11, and BM11L from this and a previous study (16) were isolated to study themolecular mechanisms involved in this mucoid-to-nonmucoid morphotype transition(see Table S1 in the supplemental material). To identify the genes involved in thismorphotype switch, the whole-genome sequences of the NMVs were determined andcompared with the previously sequenced genome of the respective B. multivoransancestor (18, 20). BM11L-nmv2 accumulated a frameshift mutation in the bceF gene(BMD20_RS03565) encoding a tyrosine kinase involved in the biosynthesis of cepacian(Table 1) (11). The remaining 13 NMVs (BM6-nmv1 and -nmv2, BM7-nmv1, BM9-nmv1and -nmv2, BM11-nmv6 and -nmv9, and BM11L-nmv1, -nmv3, -nmv4, -nmv5, -nmv7,and -nmv8) acquired single mutations (single nucleotide substitutions and small inser-tions and deletions) in a gene with the locus tag BMD20-RS11675 (Fig. 1E and Table 1).This gene encodes a putative response regulator of a two-component signal transduc-tion system from the OmpR family. As the homologue Bmul_1333 from B. multivoransATCC 17616 is annotated as ompR, we kept this designation. Except for NMVs BM7-nmv1 and BM11L-nmv3, which had additional mutations in other genomic regions,ompR was the only gene with mutations in the remaining 11 NMVs.

Mutations in the highly conserved ompR locus map in both the receiver andthe DNA-binding domains. The gene BMD20-RS11675 is located in chromosome 1 ofB. multivorans BM11, and downstream of its transcription is BMD20-RS11670, encodinga sensor histidine kinase tentatively named envZ (Fig. 1E). Not only is this geneticorganization highly conserved among more than 800 Burkholderia strains with availablegenome sequences (www.burkholderia.com), but the amino acid identities of all OmpRproteins also range from 95% to 100%. The closest homologs of B. multivorans OmpR(OmpR_BURMU) in well-studied bacteria include the OmpR proteins from Escherichiacoli, Salmonella enterica, and Shigella flexneri, with 68% identity (80% similarity) (Fig. 2).Proteins such as AruR, ArcA, WalR, VirG, and PetR were in the range of 38% to 45%identity (57% to 64% similarity). These proteins share conserved domains such as theN-terminal receiver domain housing the site of phosphorylation (D57), the site of Mg2�

coordination (13DD14), T85 and K107 that interact with the phosphoryl group, andY104, which functions as a rotamer in E. coli OmpR (21) (Fig. 2). The C-terminal effector

TABLE 1 Mutations found in the NMVs derived from the indicated B. multivorans clinical isolates exposed to stress conditions

Isolate Mutated gene

Type ofmutation/nucleotidepositiona Protein effectb Additional mutationsc

BM6-nmv1 BMD20_RS11675 (ompR) Point mutation/697 Nonsynonymous Y233N NoBM6-nmv2 BMD20_RS11675 (ompR) Point mutation/697 Nonsynonymous Y233N NoBM7-nmv1 BMD20_RS11675 (ompR) 3-bp deletion/29–31 11V, deletion Δ12 bp BMD20_RS07800; point mutation

in BMD20_RS28910BM9-nmv1 BMD20_RS11675 (ompR) 2-bp insertion/518 172L, frameshift NoBM9-nmv2 BMD20_RS11675 (ompR) 2-bp insertion/518 172L, frameshift NoBM11L-nmv1 BMD20_RS11675 (ompR) 23-bp insertion/64 21L, frameshift NoBM11L-nmv2 BMD20_RS03565 (bceF) 10-bp deletion/1938 646A, frameshift NoBM11L-nmv3 BMD20_RS11675 (ompR) 15-bp deletion/160–174 54LVLDL58, deletion 81-bp deletion in BMD20_RS14730BM11L-nmv4 BMD20_RS11675 (ompR) 28-bp insertion/67 22L, frameshift NoBM11L-nmv5 BMD20_RS11675 (ompR) 41-bp insertion/49 16P, frameshift NoBM11-nmv6 BMD20_RS11675 (ompR) 8-bp insertion/131 43N, frameshift NoBM11L-nmv7 BMD20_RS11675 (ompR) 1-bp deletion/300 99 M, frameshift NoBM11L-nmv8 BMD20_RS11675 (ompR) 1-bp deletion/167 55V, frameshift NoBM11-nmv9 BMD20_RS11675 (ompR) Point mutation/158 Nonsynonymous L53P NoBM11-nmv9r BMD20_RS11675 (ompR) Point mutation/158 Nonsynonymous L53P No

Point mutation/38 Nonsynonymous D13VaNumbers indicate positions in the wild-type gene sequence.bNumbers indicate positions in the wild-type protein sequence.cBMD20_RS07800 encodes a multidrug transporter; BMD20_RS28910 encodes histidinol dehydrogenase; BMD20_RS14730 encodes translation initiation factor IF-2.

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DNA-binding domain is also well conserved among homologs and contains the hydro-phobic core of OmpR from E. coli (Fig. 2, blue characters). In addition, amino acidsknown to be important for DNA binding in E. coli are mostly conserved in the otherhomologs, including OmpR_BURMU.

The evolved mutations in ompR producing NMVs form three logical groups. In thefirst group (G-I), the DNA-binding domain was disrupted in mutants BM6-nmv1/-nmv2(Y233N) and BM9-nmv1/-nmv2 (frameshift mutation) (Fig. 3). The second group (G-II)acquired mutations in the receiver domain. BM7-nmv1 has a 3-bp deletion, removingvaline at position 11, which is located in the vicinity of the aspartate residues involvedMg2� ion coordination during the phosphorylation reaction. BM11L-nmv3 acquired a15-bp deletion, removing amino acids 52DLLVL56, which form a beta sheet togetherwith the aspartate at position 57, where phosphorylation takes place. BM11-nmv9(L53P) might affect the phosphorylation of the aspartate residue at position 57. Thethird group (G-III, including BM11-nmv6 and BM11L-nmv1, -nmv4, -nmv5, -nmv7, and-nmv8) acquired indels of different sizes that cause frameshift mutations and truncatedor aberrant proteins (Fig. 3; Table 1). To summarize, the mucoid-to-nonmucoid transi-

FIG 2 Sequence alignment of OmpR from B. multivorans BM11 and several family members showing the conserved features required for phosphorylation andDNA binding. The sequences are for protein OmpR from B. multivorans D2095 (BM11) (KHS13833), AruR from Pseudomonas aeruginosa PAO1 (Q9HUI2.1), ArcAfrom Haemophilus influenzae Rd KW20 (P44918.1), WalR from Staphylococcus epidermidis RP62A (Q5HK18), ResD from Bacillus subtilis subsp. subtilis strain 168(P35163.2), SrrA Staphylococcus aureus subsp. aureus COL (Q5HFT0.1), VirG from Agrobacterium rhizogenes (P13359.2), MprA from Mycobacterium sp. strain MCS(Q1B3X8.1), PetR from Rhodobacter capsulatus SB 1003 (P31079.3), and OmpR from Escherichia coli K-12 (P0AA16.1). The amino acid positions are indicated bythe numbers in parentheses. A schematic diagram of the secondary structure of OmpR from B. multivorans is indicated above with arrows indicating the betasheets and cylinders indicating the alpha helices. Residues highlighted in red correspond to the ones important for phosphorylation of OmpR_ECOLI (63).Residues corresponding to the hydrophobic core of OmpR_ECOLI are in blue, and the vertical arrows above the OmpR_BURMU sequence correspond tomutations of E. coli OmpR that affected DNA binding (21). Asterisks indicate the amino acid residues that are identical in all the proteins; one or two dots indicatesemiconserved or conserved substitutions, respectively. Identity/similarity at the amino acid level between OmpR_BURMU and its best homologues is alsoshown.

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tion was caused by different loss-of-function mutations in ompR, affecting either thereceiver domain or the DNA-binding domain of the encoded protein.

In trans complementation of the NMVs restored the mucoid morphotype. Toassess if the nonmucoid morphotype of the NMVs could be reverted to the mucoid oneby expressing the native form of the mutated gene, genetic constructs were prepared(Table S1). The expression of bceF from the pBBR1MCS vector restored the mucoidmorphotype of the BM11L-nmv2 nonmucoid variant, while the expression of the vectoralone did not (not shown). Similarly, the 13 NMVs with ompR mutations recovered themucoid morphotype following complementation with plasmid pLM014-5 expressingthe wild-type ompR gene from its own promoter. An example of BM11L-nmv1 com-plementation is shown in Fig. 1C.

The frequency of new mutations arising in the ompR gene was analyzed in anadditional 110 B. multivorans BM11-derived NMVs obtained under prolonged stationaryphase for 14 days at 37°C (30 variants), 42°C (37 variants), and 7 days under subinhibi-tory concentration of ciprofloxacin (6 variants), amikacin (8 variants), and 1% NaCl (29variants). The nonmucoid morphotype of all variants, except one, was restored byexpressing in trans the parental ompR gene. Ten NMVs derived from isolates BM6, BM7,and BM9 under prolonged stationary phase for 21 days also restored the mucoidphenotype by expressing in trans the ompR gene. This shows that mutations accumu-late in the ompR gene independently of the stress conditions applied and that in thetested isolates, the ompR locus is the most frequent target of mutation when themucoid-to-nonmucoid switch takes place.

We assessed the stability of the nonmucoid morphotype of the NMVs by incubatingcells in salts medium supplemented with D-mannitol at 37°C for three passages, 2 dayseach. Only one variant, BM11-nmv9, produced mucoid colonies at a frequency of 1.3%

FIG 3 Mutations in the ompR gene (BMD20_RS11675) lead to short truncated proteins or affect thereceiver or the DNA-binding domains of the protein. Mutations are organized by the site in OmpR wherethey had occurred. Group I (G-I) mutations map at the DNA-binding domain, group II (G-II) mutationsmap at the receiver domain, and group III (G-III) includes mutations that give rise to short truncatedproteins. NMVs BM11L-nmv4, BM11L-nmv5, and BM11L-nmv8 (not shown) mapped to group G-III. Theeffect of each mutation in the production of exopolysaccharide in YEM agar is also depicted.

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(Fig. 1D). The genome sequence of one of these mucoid revertant colonies, BM11-nmv9r, showed an additional nonsynonymous mutation in the ompR gene (D13V) (Fig.3; Table 1). This aspartate residue is most likely involved in the coordination of theMg2� ion required for the phosphorylation of D57, and it might have suppressed thenegative effect of the L53P substitution. This ompR suppressor mutant BM11-nmv9r didnot produce as much EPS as the original BM11 ancestor (6.4 � 1.6 versus 12.7 � 0.5g/liter), indicating that the second ompR mutation only partially restored function.

Expression of bce genes is dependent on the ompR locus. One source of themucoid phenotype is the production of the cepacian polysaccharide, and so NMVs donot produce cepacian. We conducted reverse transcription-quantitative PCR (qRT-PCR)of the bceB and bceE genes from cluster bce-I and bceQ from cluster bce-II (3) inancestors and NMVs from each of the three ompR mutant groups. Reduced expressionof bceB and bceE was observed in all NMVs with ompR mutations but not in BM11L-nmv2 carrying the bceF mutation (Table 2). The expression of bceB and that of bceEwere not significantly different when the mucoid BM11-nmv9r was compared to BM11,suggesting that the partial recovery of EPS production by BM11-nmv9r is not due toinefficient expression of bce-I cluster genes. The expression of bceQ was slightlydecreased in the NMVs with ompR mutations. Interestingly, BM11-nmv9r had signifi-cantly lower bceQ expression than BM11, which could explain the partial recovery ofEPS production in this revertant. These results confirm that the nonmucoid morphotypeof the NMVs is most likely due to the decreased expression of the bce genes throughdirect or indirect regulation by the OmpR regulator. Additionally, the regulation of bceexpression seems to be dependent on intact OmpR phosphorylation and DNA-bindingdomains.

Mutations in ompR gene increase tolerance to osmotic stress. To assess whetherthe OmpR regulator was responsive to different medium osmolarities, ancestors andNMVs were grown in LB with 17, 86, or 426 mM NaCl. All 14 NMVs were tested, but forclarity, only the results from a few NMVs of each group (G-I, G-II, and G-III) are shown.No significant differences between the doubling times of the NMVs and their respectiveancestors nor between the two salt concentrations were observed in LB with 17 and 86mM NaCl (Table 3). The doubling times of isolate BM11L and derived NMVs weresignificantly lower than the ones for the other isolates and respective NMVs, irrespec-tive of the NaCl concentration (Table 3). This effect was most likely caused by the pointmutation in the gene BMD20_RS04295 encoding the beta subunit of acetyl coenzymeA (acetyl-CoA) carboxylase present in this BM11L isolate, leading to an A204D substi-tution. Under high-salt conditions (426 mM NaCl), the growth of all isolates was slowed,but with the exception of BM11L-nmv2 mutated in bceF and not ompR, NMVs grow 15%to 30% faster (P � 0.05) than their respective ancestors (Fig. 4A to C and Table 3). Also,NMVs with ompR mutations reached a higher biomass in stationary phase (Fig. 4A to C).The enhanced growth of NMVs BM11-nmv9 and BM11-nmv9r relative to that of theBM11 ancestor under high-salt conditions was confirmed by CFU counts (P � 0.05) (Fig.4B). In conclusion, under high osmotic conditions, the ompR mutations increased thegrowth rate and biomass yield in stationary phase, while no significant differences wereobserved under low osmotic conditions.

TABLE 2 Comparison of bce expression in ancestors and NMVs cultured in SM medium as determined by quantitative real-time PCR

Strain comparison

Fold change � SDa

bceB (BMD20_RS03545) bceE (BMD20_RS03560) bceQ (BMD20_RS04345)

BM6-nmv1 vs BM6 �8.9 � 3.2 A �40.7 � 13.4 B �2.8 � 1.9 NSBM11-nmv9 vs BM11 �5.3 � 1.6 B �23.0 � 10.7 B �1.6 � 1.3 NSBM11-nmv9 vs BM11-nmv9r �6.0 � 2.3 A �15.9 � 5.2 A 13.1 � 4.3 ABM11-nmv9r vs BM11 �1.0 � 0.3 NS �1.6 � 0.5 NS �8.3 � 2.6 ABM11L-nmv1 vs BM11L �9.6 � 1.0 C �85.6 � 52.2 B �2.1 � 1.3 NSBM11L-nmv2 vs BM11L �1.0 � 0.8 NS �1.2 � 0.8 NS �1.0 � 0.3 NSaThe fold change in expression (NMV versus ancestor) was calculated by using the 2�ΔΔCT formula. The reciprocal function was applied to values between 0 and 1,with the minus symbol denoting downregulation. Uppercase letters indicated statistical differences (by Student’s two-sample t tests) of expression of each gene inthe NMVs relative to that in the respective ancestor. NS, not statistically significant; A, P � 0.05; B, P � 0.01; C, P � 0.001.

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To understand whether these differences in growth behavior under a high saltconcentration could be attributed to the altered expression of outer membrane pro-teins, we analyzed this cellular fraction under low (17 and 86 mM) and high (426 mM)NaCl concentrations for BM11L and BM11L-nmv1. While the NMV produced a greaterband intensity overall, these differences are exaggerated under the high salt concen-tration (Fig. 4D). The protein bands labeled 1 to 4 appear to be augmented in the NMVouter membrane, and protein band 5 appears to be decreased. Overall, the outermembrane protein profile of this NMV is distinct from that of the ancestor, and that islikely to contribute to its growth advantage under conditions of high osmotic stress.

NMVs grow poorly in the presence of the disaccharide D-trehalose as a singlecarbon source. To account for possible metabolic differences between mucoid isolatesand NMVs, a metabolic fingerprint test was used to compare the abilities of BM11L andBM11L-nmv1 to metabolize 95 different carbon sources. A striking difference in theability for the NMV to metabolize D-trehalose was observed, and this pattern held forall NMVs with the ompR mutations (doubling time of 14 to 30 h versus 3 to 4 h forancestors) (Table 3).

Mutations in ompR increase the susceptibility to antimicrobial compounds. AsOmpR might regulate a set of genes which affect the bacterial cell wall, we tested theinhibitory effect of the beta-lactams aztreonam (Fig. 5A) and piperacillin (see Fig. S1A).All NMVs harboring the ompR mutation, as well as mucoid BM11-nmv9r, were moresensitive to the tested antibiotics (P � 0.001) than their ancestors. The NMV generatedby a bceF mutation (BM11L-nmv2) had no significant difference in susceptibility. TheMICs for both antibiotics against these genotypes were measured by Etest. AncestorMICs were generally 32 �g/ml for both drugs, whereas the MICs for the NMVs causedby ompR mutations ranged between 1 to 8 �g/ml for aztreonam and 1.5 to 7 �g/ml topiperacillin (Fig. 5B). Thus, the OmpR regulator plays an important role in antibioticresistance.

As two of the genes downstream of ompR encode putative alkyl hydroperoxidereductases (Fig. 1E), we tested the susceptibilities of exponential and stationary-phasegrown cells against hydrogen peroxide and cumene hydroperoxide. Despite the highsensitivities to these oxidative stress compounds, no significant differences wereobserved between the NMVs and the parental strains (data not shown). To determinewhether envelope integrity was compromised in the NMVs, outer membrane perme-ability was determined by using an N-phenyl-1-naphthylamine (NPN) uptake assay. Anintact outer membrane is a permeability barrier preventing access of hydrophobiccompounds such as NPN. If damaged, it enables the entry of NPN to the lipid bilayerwith a strong increase in fluorescence. In this experiment, we detected low fluorescence

TABLE 3 Doubling times of the strains under study grown in the indicated liquid mediaat 37°C with agitation

Strain

Doubling time (min � SD)a

LB with NaCl (mM)M63 plus 0.2%trehalose17 86 426

BM6 92.6 � 4.5 98.3 � 2.6 169.1 � 9.2 156.3 � 12.0BM6-nmv1 94.6 � 5.9 NS 98.4 � 2.6 NS 140.1 � 8.5 B 1018.1 � 245.8 CBM7 87.1 � 4.0 94.4 � 2.7 168.3 � 3.8 231.3 � 24.0BM7-nmv1 95.5 � 1.4 NS 88.0 � 3.3 NS 144.0 � 7.1 A 823.8 � 83.2 CBM11 84.4 � 10.1 87.9 � 3.0 155.7 � 13.3 163.0 � 2.5BM11-nmv9 88.6 � 10.1NS 89.0 � 3.7 NS 131.6 � 7.1 B 1092.6 � 117.6 CBM11-nmv9r 85.2 � 11.0 NS 89.1 � 1.5 NS 135.9 � 4.4 B 497.0 � 18.5 BBM11L 68.1 � 4.2 72.6 � 5.7 119.2 � 6.2 193.5 � 3.5BM11L-nmv1 79.1 � 8.1 NS 77.0 � 7.2 NS 95.5 � 10.1 A 1176.8 � 282.8 CBM11L-nmv7 75.0 � 8.8 NS 70.8 � 4.4 NS 90.3 � 6.1 B 1823.2 � 500.4 CBM11L-nmv2 77.9 � 0.7 NS 74.8 � 5.1 NS 116.8 � 7.0 NS 202.6 � 22.2 NSaUppercase letters indicated statistical differences (by one-way ANOVAs with Tukey’s mean comparisons) inthe doubling time of the NMVs relative to that in the respective ancestor. NS, not statistically significant; A,P � 0.05; B, P � 0.01; C, P � 0.001.

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levels for both the ancestors and NMVs, indicative of no major change in outermembrane permeability. Nevertheless, the NPN uptake factor was 10% to 30% higherfor most of the NMVs, suggesting a slightly higher permeability (Fig. S1B).

OmpR regulates motility, biofilm formation, aggregation, and adhesion toepithelial cells. Swimming and swarming motilities were assayed, and the results aredepicted in Fig. 6A and B, respectively. All variants carrying the ompR mutation showedincreased swimming and swarming motilities compared to those of the respectiveancestors (P � 0.001). In accordance with the higher growth rate, BM11L and the NMVderivatives had significantly higher motilities than BM11 and respective NMVs. Swim-ming and swarming motilities of BM11L-nmv2 containing a mutation in bceF werelower than those of the parental strain, in line with previous data for the Burkholderiacontaminans bceF mutant (22).

The ability of cells incubated in LB to produce biofilms on abiotic surfaces variedamong strain backgrounds. While NMVs derived from parental BM9, BM6, and BM11isolates had decreased biofilm formation, BM7-nmv1 did not differ significantly from itsancestor, and BM11L-nmv1 (and other variants derived from BM11L) produced morebiofilms (Fig. 7A). High osmotic stress conditions (LB with 426 mM NaCl) reducedbiofilm formation by the mucoid ancestors (with exception of BM11L) relative to thatfor LB (Fig. 7B). Contrastingly, NMVs with ompR mutations produced more biofilm.Biofilm competition assays between ancestors and variants grown under a high salt

FIG 4 Mutations in the ompR gene increase cell tolerance to high salt concentration and changes in the outermembrane protein profile. (A to C) Growth curves as determined by measurement of the optical density at 640 nm(OD640) and CFU (B) of cultures grown in LB supplemented with 426 mM NaCl at 37°C. Data are the means of resultsfrom three independent experiments (�SD). CFU of BM11-nmv9 and BM11-nmv9r at 9, 12, and 24 h weresignificantly different from the ancestor (P � 0.05 by Tukey’s honestly significant difference [HSD] multiple-comparison test). (D) Outer membrane protein extracts from cells grown in the indicated salt concentrationseparated by 12% SDS-PAGE and stained with Coomassie. Arrows indicate proteins with increased (1, 2, 3, and 4)and decreased (5) amounts in BM11L-nmv1 compared to that of the ancestor BM11L, at least for the highest saltconcentration. Lane M shows protein markers, with sizes on the left.

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concentration show that if the NMV is at proportion of 1:1 (or higher) with the ancestor,the amount of biofilm is equivalent to that of the pure NMV culture (Fig. 7C to E).Overall, these results show that OmpR is important for the regulation of biofilmformation in an osmotic-stress-dependent manner.

The osmotic stress that likely prevails in the CF airway also promotes the aggrega-tion and adherence of NMVs mediated by mutations in ompR. BM11L and BM11L-nmv1produce aggregates under low-salt conditions (Fig. 8A and B) that increase in size athigh-salt conditions (Fig. 8C and D), particularly in the case of the NMV. The adhesionby the ancestors and NMVs to CFBE41o� bronchial epithelial cells at high-salt condi-tions was generally reduced relative to that of those grown under low-salt conditions(not shown), but NMVs adhered significantly better than their ancestors (Fig. 8E).

OmpR is implicated in virulence in Galleria mellonella. Mucoid ancestors andNMVs were tested for virulence in G. mellonella. The survival curves showed virulenceattenuation for BM6-nmv1 and BM11-nmv9, while BM7-nmv1 and the mucoid BM11-nmv9r were as virulent as their parental strains (Fig. 9A to C). BM11L-nmv1 causes lowermortality than its ancestor 2 days postinfection, but this difference is attenuated by day3 postinfection (Fig. 9D). BM11L-nmv2 was the most attenuated variant, confirmingprevious data on the attenuation of the B. contaminans IST408 bceF mutant (22).

OmpR has a role in the transcriptional control of metabolism, stress response,and cell wall biogenesis-related genes. Expression profiling was performed using

FIG 5 NMVs with ompR mutations are more sensitive to antibiotics. (A) Susceptibility to aztreonamdetermined at 37°C after 24 h of incubation by measuring the diameter of cell growth inhibition. NMVswith the ompR mutation differed significantly from their ancestors. ***, P � 0.001 by Tukey’s HSDmultiple-comparison test. (B) MICs of aztreonam (ATM) and piperacillin (PIP) as determined by Etest.

FIG 6 NMVs with ompR mutations have increased swimming and swarming motilities. Swimmingmotility assayed in SM medium with 0.3% of agar plates incubated at 37°C for 24 h (A) and swarmingmotility assayed in Broomfield medium with 0.6% of agar plates incubated at 37°C for 48 h (B) weresignificantly higher for the NMVs with ompR mutations and significantly lower for the NMV BM11L-nmv2with the bceF gene mutation compared to those of the ancestors. ***, P � 0.001 by Tukey’s HSDmultiple-comparison test.

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ancestor BM11L and NMV BM11L-nmv1 (with a single ompR frameshift mutationproducing a premature stop) grown until early stationary phase (see Fig. S2). Acomparison of the expression values of BM11L-nmv1 versus BM11L using a lowerconfidence interval cutoff for the fold change of 1.2 (false discovery rate [FDR], 2.5%)

FIG 7 Biofilm formation by the NMVs with ompR mutations is enhanced in the presence of high saltconcentration. (A and B) Biofilm formation in LB with 86 mM (A) and 426 mM (B) NaCl as measured bythe crystal violet staining method. Biofilm formation by the NMVs with ompR mutations after a 48-hincubation is significantly different from the ancestors. *, P � 0.05; **, P � 0.01; ***, P � 0.001 by Tukey’sHSD multiple-comparison test. (C to E) Competition for biofilm formation by ancestors and NMVscarrying ompR mutations grown in the presence of 426 mM NaCl. The percentages inoculated forancestor/NMV are 100:0, 75:25 (3:1), 50:50 (1:1), 25:75 (1:3), and 0:100.

FIG 8 High salt concentration favors self-aggregation and adhesion to CF epithelial cells by the variants with ompRmutations. (A to D) Aggregates of B. multivorans BM11L and BM11L-nmv1 grown in LB supplemented with 86 mM NaCl(A and B, respectively) or 426 mM NaCl (C and D, respectively) for 24 h. (E) Adhesion to CF lung epithelial cells by B.multivorans mucoid ancestors and NMVs with ompR gene mutations grown with 426 mM NaCl, using an MOI of 10.NMVs with the ompR mutation differed significantly from their ancestors. ***, P � 0.001 by Tukey’s HSD multiple-comparison test.

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showed 156 genes with increased expression and 545 genes with decreased expression(the complete list of differentially expressed genes is shown in Table S2). Genesencoding proteins involved in nutrient uptake and outer membrane composition wereamong the most upregulated, and those encoding proteins involved in protein syn-thesis, metabolism, stress response, and cell envelope composition were among themost downregulated. These observations suggest that the loss of OmpR functionmodifies the metabolism and cell envelope in a manner consistent with starvationtolerance.

Modifications in the cell envelope are evident by the differential expression inBM11L-nmv1 of several genes directly involved in the biogenesis of this structure.These include nine genes encoding outer membrane porins (OMPs), two of themdownregulated and six upregulated (Table 4; see also Table S2). Other genes related tothe cell envelope that were downregulated encode surface antigens, several lipopro-teins, including WbxY and WaaA involved in the hydroxylation and transfer of thelipopolysaccharide component 3-deoxy-D-manno-octulosonic-acid (Kdo), respectively,and UDP-N-acetylglucosamine pyrophosphorylase GlmU, responsible for the synthesisof UDP-N-acetylglucosamine, an essential cytoplasmic metabolite situated at a branchpoint between the biosynthesis of peptidoglycan and lipopolysaccharide. Implicated inthe membrane composition, we found several downregulated genes involved in thebiosynthesis and transport of fatty acid and phospholipids, terpenoids, hopanoids, andthe lipid carrier undecaprenyl diphosphate synthase (uppS product) (Table 4; Table S2).In addition, genes BMD20_RS05735 and BMD20_RS05770, involved in the biosynthesisof an incompletely characterized polysaccharide of the biofilm matrix as reported forother Burkholderia (23), were upregulated. These data confirm that OmpR mediatesbacterial envelope changes modifying surface properties relevant for thriving undercertain stressful conditions.

A lower metabolic flow in BM11L-nmv1 is suggested by the differential expressionof genes whose products have metabolic or nutrient transport functions. Several genes

FIG 9 Survival of Galleria mellonella larvae inoculated with the NMVs is generally increased. Four groupsof 10 larvae were inoculated with the mucoid ancestor or the NMVs. The control experiment withoutbacteria is also represented. Larvae were injected with approximately 1 � 107 cells, and survival rateswere determined for 3 days. *, P � 0.05 versus the respective ancestors.

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TABLE 4 Selection of genes differentially expressed in the nonmucoid BM11L-nmv1 compared to that in the ancestor, BM11L, groupedby functional categories

Functional category and locus tag for B.multivorans D2095 (BM11) Gene name Description LB-FCa

Outer membrane proteinsBMD20_RS00485 17-kDa surface antigen �1.5BMD20_RS04380 Porin 1.4BMD20_RS07985 Porin �2.4BMD20_RS09515 Porin 1.4BMD20_RS13265 comL Competence lipoprotein ComL �1.6BMD20_RS13470 ompW OmpW family protein �2.7BMD20_RS15330 Porin 2.4BMD20_RS19290 Porin 1.3BMD20_RS21535 Porin 1.9BMD20_RS21730 Porin 1.4

LPS and peptidoglycan biosynthesisBMD20_RS17655 waaA 3-Deoxy-D-manno-octulosonic-acid transferase �1.3BMD20_RS17660 wbxY 3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) hydroxylase �2.8BMD20_RS21665 glmU UDP-N-acetylglucosamine pyrophosphorylase �1.3

Lipid metabolismBMD20_RS15840 mdcA Malonate decarboxylase, subunit alpha �2.1BMD20_RS15835 mdcC Malonate decarboxylase, subunit delta �1.3BMD20_RS15830 mdcD Malonate decarboxylase, subunit beta �1.4BMD20_RS15825 mdcE Malonate decarboxylase, subunit gamma �2.1BMD20_RS16270 hpnJ Hopanoid biosynthesis-associated radical SAM protein �1.7BMD20_RS23640 cfa Cyclopropane-fatty-acyl-phospholipid synthase �2.3BMD20_RS28855 mlaF ABC transporter maintaining membrane lipid asymmetry �1.2BMD20_RS28865 mlaD ABC transporter maintaining membrane lipid asymmetry �1.3

Central metabolismBMD20_RS00595 Cytochrome bd ubiquinol oxidase subunit I �1.9BMD20_RS00760 GCN5-related I-acetyltransferase �4.1BMD20_RS04045 Cytochrome B561 �2.2BMD20_RS05325 ach1 Succinate CoA transferase �2.5BMD20_RS06760 pckA Phosphoenolpyruvate carboxykinase �1.7BMD20_RS16695 fumC Fumarate hydratase �1.9BMD20_RS18360 Nitric oxide dioxygenase �2.0BMD20_RS22500 cydB Cytochrome bd ubiquinol oxidase, subunit II �1.9BMD20_RS22505 cydA Cytochrome bd ubiquinol oxidase, subunit I �2.0BMD20_RS25620 cbb Oxidoreductase alpha (molybdopterin) subunit �1.8

Nitrogen metabolismBMD20_RS06925 nos Molybdopterin oxidoreductase 4.2BMD20_RS06930 nirD Nitrite reductase [NAD(P)H], small subunit 4.8BMD20_RS06935 nirB Nitrite reductase [NAD(P)H], large subunit 3.9BMD20_RS06940 Nitrate/nitrite transporter 2.5BMD20_RS17595 urtA Urea ABC transporter, urea-binding protein 2.5BMD20_RS17600 urtB Urea ABC transporter, permease protein UrtB 2.0BMD20_RS17605 urtC Urea ABC transporter, permease protein UrtC 2.6BMD20_RS17610 urtD Urea ABC transporter, ATP-binding protein UrtD 2.3BMD20_RS17615 urtE Urea ABC transporter, ATP-binding protein UrtE 2.0BMD20_RS22640 rpoN RNA polymerase factor sigma-54 �1.6BMD20_RS23985 livH Inner-membrane translocator 1.8BMD20_RS23990 livM Inner-membrane translocator 1.8BMD20_RS23995 livG ABC transporter related 1.6BMD20_RS24000 livF ABC transporter related 1.8BMD20_RS25315 Allantoate amidohydrolase 4.3BMD20_RS25320 Putative oxidoreductase 1.6BMD20_RS25325 pyrD Dihydropyrimidine dehydrogenase 2.6BMD20_RS25485 narG Nitrate reductase, alpha subunit �1.9BMD20_RS25490 narH Nitrate reductase, beta subunit �1.9BMD20_RS25495 narJ Nitrate reductase molybdenum cofactor assembly chaperone �1.7BMD20_RS25500 narI Respiratory nitrate reductase, gamma subunit �1.7BMD20_RS28365 ggt Gamma-glutamyltransferase 2.1

DNA replication and RNA metabolismBMD20_RS01480 iciA Chromosome replication initiation inhibitor protein �1.4

(Continued on next page)

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TABLE 4 (Continued)

Functional category and locus tag B.multivorans D2095 (BM11) Gene name Description LB-FCa

BMD20_RS13260 Helicase c2 �1.8BMD20_RS14580 hfq2 RNA chaperone Hfq �2.0BMD20_RS17170 rpoZ DNA-directed RNA polymerase, subunit omega �1.4BMD20_RS18225 mutL DNA mismatch repair protein �1.4BMD20_RS20175 hip Histone family protein DNA-binding protein �1.6BMD20_RS28460 nusG Transcription antitermination protein NusG �1.5BMD20_RS28490 rpoC DNA-directed RNA polymerase, subunit beta �1.4

Stress responseBMD20_RS07675 UspA domain-containing protein �1.6BMD20_RS11665 ahpC Peroxiredoxin �3.1BMD20_RS12995 clpB ATP-dependent chaperone ClpB �1.5BMD20_RS15710 ftsH ATP-dependent metalloprotease FtsH �1.5BMD20_RS16505 rpoE RNA polymerase sigma factor RpoE �1.5BMD20_RS17580 pcm Protein-L-isoaspartate (D-aspartate) O-methyltransferase �1.6BMD20_RS18075 Peroxidase �1.8BMD20_RS18555 Glutathione S-transferase domain-containing protein �2.3BMD20_RS22005 dsbA DSBA oxidoreductase �1.4BMD20_RS22245 dsbE Redoxin domain-containing protein �1.7BMD20_RS25475 UspA domain-containing protein �6.6BMD20_RS25505 ppiC PpiC-type peptidyl-prolyl cis-trans isomerase �1.7BMD20_RS25625 clpA ATPase �2.2BMD20_RS25640 Dyp-type peroxidase family protein �3.3BMD20_RS29320 dsbC Protein-disulfide isomerase-like protein �1.6

Iron metabolismBMD20_RS00435 TonB-dependent hemoglobin/transferrin family receptor �5.4BMD20_RS00440 hmuS Hemin-degrading family protein �2.8BMD20_RS10255 Fe2� transport protein �2.6BMD20_RS10260 Periplasmic lipoprotein involved in iron transport �1.8BMD20_RS10275 Hemin uptake protein �3.0BMD20_RS10295 exbD Biopolymer transport protein ExbD/TolR �2.6BMD20_RS10300 exbB MotA/TolQ/ExbB proton channel �1.9BMD20_RS10305 tonB TonB family protein �2.5BMD20_RS10310 bfd Bacterioferritin associated �1.9BMD20_RS10650 iscR BadM/Rrf2 family transcriptional regulator 1.6BMD20_RS10655 iscS Cysteine desulfurase IscS 1.7BMD20_RS10660 iscU Scaffold protein 1.7BMD20_RS10665 iscA Iron-sulfur cluster assembly protein IscA 1.6BMD20_RS13925 orbL Putative ornibactin biosynthesis protein �4.2BMD20_RS13930 orbF Ornibactin synthetase F �2.1BMD20_RS13935 orbA TonB-dependent siderophore receptor �3.1BMD20_RS13940 pvdA Lysine/ornithine N-monooxygenase �7.4BMD20_RS13945 orbK Ornibactin biosynthesis protein �2.9BMD20_RS13960 orbE Ornibactin biosynthesis ABC transport protein PvdE �4.2BMD20_RS13980 orbC Putative iron transport-related ATP-binding protein �2.1BMD20_RS13985 orbG Taurine catabolism dioxygenase TauD/TfdA �3.8BMD20_RS13990 orbH MbtH domain-containing protein �8.4BMD20_RS13995 orbS Extracytoplasmic-function sigma-70 factor PvdS �1.6BMD20_RS15320 fecI ECF subfamily RNA polymerase sigma factor FecI �1.4

Utilization of alternative carbon sourcesBMD20_RS09555 proX Substrate-binding region of ABC-type glycine betaine transporter 2.2BMD20_RS09560 Oxidoreductase FAD-binding subunit 1.7BMD20_RS09565 Rieske (2Fe-2S) domain-containing protein 2.3BMD20_RS09580 dgcB (Fe-S)-binding protein 2.1BMD20_RS09585 dgcA NADH:flavin oxidoreductase/NADH oxidase 2.7BMD20_RS09590 4-Vinyl reductase 4VR 2.7BMD20_RS09595 Membrane dipeptidase 2.5BMD20_RS09600 glyA Serine hydroxymethyltransferase 2.5BMD20_RS09790 soxA Sarcosine oxidase alpha subunit family protein 2.1BMD20_RS09800 soxB Sarcosine oxidase beta subunit family protein 1.8BMD20_RS11550 Urate catabolism protein 1.7BMD20_RS11560 alc Allantoicase 1.6

aLB-FC, lower bound of fold change; list of genes with �1.6-fold increased or decreased expression and belonging to the selected functional categories. A few geneswith lower expression values but considered relevant in their functional category were also included. Genes with �1.6-fold increase/decrease whose function isunknown were not included.

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involved in the pentose phosphate pathway, glycolysis/gluconeogenesis, citrate cycle,and oxidative phosphorylation were downregulated (Table S2). Reduced expression ofRNA polymerase factor �54 (RpoN) led possibly to the decreased expression of severalgenes involved in nitrogen metabolism, as evidenced for genes involved in purine andpyrimidine metabolism and amino acids biosynthesis (Table S2). In line with thisapparent decrease of metabolic flow, 48 genes encoding ribosomal proteins, 40 genesinvolved in transcription, and 9 genes involved in cell division were downregulated.

Another upregulated gene is fliA, encoding the flagellar biosynthesis sigma factor.Consequently, other genes involved in the synthesis of flagella, such as fliD, fliE, fliM,flgB, and flhB (Table S2), were also upregulated, in line with the increased swimmingand swarming motilities of NMVs shown in Fig. 6.

A major category of differentially expressed genes is implicated in stress response.Here, we observed the downregulation of rpoE, encoding the RNA polymerase sigmafactor responsible for adjusting the cell envelope under stress conditions, and severalof its putative regulon members, such as the ones encoding oxidoreductases/disulfideisomerases DsbA, DsbC, DsbD, and DsbE, the peptidyl-prolyl cis-trans isomerases SurA,PpiB, and PpiC, the alkyl hydroperoxidases AhpD and AhpC, the ATP-dependentchaperones ClpA and ClpB, the ATP-dependent metalloprotease FtsH, the chaperoninGroEL, and others (Table 4; Table S2). In line with the decreased expression of genesinvolved in stress response, we also observed the downregulation of genes related tothe utilization of iron, a compound that can be an important source of oxidative stress.Therefore, BM11L-nmv1 cells showed decreased expression of the fecI gene encodinga regulator of iron homeostasis and consequently the downregulation of many genesinvolved in heme/hemin and siderophore biosynthesis and transport/uptake. In con-trast to the downregulation of genes involved in iron metabolism, BM11L-nmv1showed an upregulation of several genes involved in the repair of Fe-S clusters(iscRSUA), suggesting a preference for repair instead of de novo synthesis.

In conclusion, the expression data link the regulation of carbon and nitrogenmetabolism to the expression of cell envelope components and suggest the existenceof a complex regulatory network with the interplay between OmpR and severaltranscriptional regulators. In the absence of a functional OmpR regulator, cells enter astate characterized by a modified cell envelope and lower metabolic flow, which isprobably beneficial under particular environmental stressors such as high osmolarity,but renders cells more susceptible to antimicrobial compounds.

DISCUSSION

Bacteria often encounter many different environments, ranging from soil, aquatic,and human made to hosts, where nutrient availability and the presence of stresses canbe variable. Therefore, bacteria must be able to adapt in order to survive. Under moreextreme and sustained periods of stress, the inherent plasticity of bacterial populationsmay be insufficient to maintain fitness, which selects for mutants with superior growthor resistance (potentially at the expense of plasticity) and may result in rapid evolution.In the present study, we demonstrated that B. multivorans clinical isolates undergo aconversion from a mucoid to a nonmucoid colony morphotype when incubated undervaried stressful conditions. The genomes of the NMVs revealed mutations in the samelocus (the ompR gene) but were randomly distributed and of different natures (shortindels, point mutations). This observation, together with the fact that no significantreversibility to the mucoid morphotype was detected under the tested conditions,suggests that these morphotypes are not produced by a phase variation mechanismbut rather by strong selection for a particular set of traits that these mutations provide.In line with this work, B. cenocepacia switches between wrinkly small-colony variants(biofilm specialists with mutations in wsp genes) and smooth-colony variants, which ismediated by heritable changes that occurred in the same or other wsp genes, inBcen2424_1436 encoding a two-component response regulator, or in genes related topolysaccharide synthesis, depending on the evolutionary history of each biofilm spe-cialist (24).

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The involvement of OmpR homologues in regulating polysaccharide biosynthesishas been demonstrated in other bacteria. One such example is the regulation of genesresponsible for the expression under osmotic conditions of a major virulence determi-nant called Vi polysaccharide capsular antigen in Salmonella enterica serovar Typhi byEnvZ/OmpR (25). Also, in the phytopathogen Dickeya dadantii, glucose backbonesubstitutions of periplasmic glucans are osmolarity dependent and the expression ofthe succinyl transferase OpgC required for succinylation is directly controlled by bothEnvZ/OmpR and RcsCBD phosphorelay systems (26). Amylovoran biosynthesis in Er-winia amylovora has been shown to be negatively regulated by EnvZ/OmpR andGrrS/GrrA, since its production was significantly higher when both regulatory systemswere inactivated, although the mechanism is still unknown (27). In this work, we haveidentified a new regulator of cepacian biosynthesis by showing that mutations in theompR gene cause a loss of EPS production. We also demonstrated the recovery of thisproperty by in trans complementation with the native gene. Additionally, qRT-PCR dataconfirmed the downregulation of bce genes in the tested NMVs. Whether OmpR bindsdirectly to promoter regions within the bce gene clusters is unknown. What is certainis that cepacian biosynthesis regulation involves a complex regulatory network, which,in addition to OmpR, also comprises the sigma factor �54 (RpoN) (5), the responseregulator NtrC (6), the RNA chaperone Hfq (28), and the lactate dehydrogenase LdhA(29).

A considerable body of research indicates that OmpR is involved in the control ofvarious cellular processes in organisms such as E. coli, Shigella, Salmonella, and Yersinia,among several others (30–33). This regulator participates in the regulation of targetgenes in response to changes in osmolarity, pH, and temperature and is involved invirulence through the regulation of cellular adhesion, motility, and biofilm formation(34–36). Moreover, a systematic transcriptome analysis of all two-component regulatorysystems in E. coli has demonstrated that the OmpR/EnvZ system also controls themetabolism of amino acids and nutrient transport (37). Here, we show that OmpR fromB. multivorans regulates the expression of more than 700 genes affecting several traits,including at least eight OMP-encoding genes. Additionally, an analysis of outer mem-brane protein fractions of cells grown in different salt concentrations also showeddifferences in protein abundance between the ancestor and the NMV. Despite theunknown identity of these proteins, our results highlight the importance of OmpR inadjusting outer membrane composition. The response to medium osmolarity is thehallmark of OmpR from enterobacteria where OmpC (the narrow porin) levels increasein high osmolarity media, while those of OmpF (the wider porin) decrease (38). Thisalteration in membrane protein composition may limit the diffusion of harmful com-pounds of cells growing in adverse environments, including mammalian hosts. Anotherrelevant group of genes is mlaBDF, encoding an ABC-transporter that appears toremove phospholipids from the outer leaflet of the outer membrane, leaving only LPS(39). These genes were upregulated in the presence of OmpR, reinforcing its involve-ment in maintaining the integrity of the outer membrane as a protective barrier.

In addition to osmolarity, the E. coli OmpR/EnvZ two-component regulatory systemalso regulates biofilm formation. A mutation in the ompR gene causing an L43Rsubstitution in the protein stimulates laboratory strains of E. coli to grow as a biofilmcommunity rather than in a planktonic state. This mutated OmpR protein binds at thepromoter of the csgD gene encoding a transcription regulator, which in turn activatesthe transcription of the csgBA operon responsible for the synthesis of curli, extracellularstructures involved in bacterial adhesion (40). Although biofilm formation by E. coli isinhibited by increasing the osmolarity in the growth medium, the above-mentionedompR mutation counteracts adhesion inhibition by high medium osmolarity through astrong increase of the initial adhesion of bacteria to an abiotic surface (40). A possiblerole for B. cenocepacia OmpR in biofilm formation has been identified during experi-mental evolution studies (41). The authors of those studies found three OmpR muta-tions (E98G and E98D mapping at the receiver domain and K173E mapping at theDNA-binding domain) at frequencies of 3% to 5% in two long-term biofilm-evolved

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populations. In B. multivorans, OmpR appears to be required for biofilm formationunder a low medium osmolarity, since the ancestors in general produced more biofilmthan the NMVs. Nevertheless, a high medium osmolarity favored biofilm formation byall ompR mutants. Additionally, competition between ancestors and nonmucoid ompRmutants grown in high osmolarity for biofilm formation showed a competitive advan-tage of the mutants over the ancestors. Although this result can be affected by theincreased growth rate of the NMVs in high salt concentrations, we cannot exclude thatstructures such as fimbriae, curli, and pili can be involved in the initial adhesion steps,as shown for E. coli.

Motility due to the expression of flagella is also under the control of OmpR indifferent microorganisms. This regulation acts on the expression of the flhDC operonencoding the transcriptional activator that binds to the operons responsible for flagel-lum synthesis. OmpR has been reported to negatively regulate the expression of flhDCin E. coli and Xenorhabdus nematophila but positively regulate this operon in Yersiniapseudotuberculosis and Yersinia enterocolitica (42–45). B. multivorans OmpR negativelyregulates motility, as shown by the hypermotile phenotype of the ompR mutants.Accordingly, several genes involved in flagellum biosynthesis and regulation wereupregulated in the ompR mutant.

The laboratory selection imposed on mucoid ancestors to generate the NMVs maystrongly resemble the conditions faced by Bcc bacteria within CF lungs, characterizedby high osmolarity, oxidative stress, oxygen gradients, and nutrient limitation. There-fore, it is very likely that NMVs with ompR mutations evolve in the CF airways. Asevidence, our previous study of B. multivorans sequential isolates from a CF chroniclung infection revealed the fixation of a mutation near the start codon of ompR (18).Furthermore, this mutation increased the transcription of this gene in several isolatesrelative to that in the ones which lacked any mutation (18). Comparative genomics ofsequential isolates from CF chronic lung infection of B. cenocepacia also identifiedmutations in the ompR locus in some isolates (19), confirming its importance as aselection target in vivo. Despite being an in vivo selection target, OmpR is likelygoverning global regulatory changes that influence adaptation to particular environ-ments, and its activity might be mostly exerted at the gene expression level.

The NMVs generated in this work were more sensitive to antibiotics and were lesscapable of establishing acute infections in G. mellonella. This observation differs fromthe findings of Zlosnik and coauthors (15), in which the nonmucoid phenotype posi-tively correlated with the rate of decline in lung function in CF patients. Thesedifferences might result from the contrasting demands of acute and chronic infections,with NMVs being deficient in the former but superior in the latter. Alternatively, clinicalNMVs may have acquired several other mutations that influence pathogenesis beyondthe single mutations in OmpR we report here (18, 19, 46). Either way, the fact that theNMV phenotype evolves repeatedly during chronic CF infections of Bcc stronglysuggests that it is either directly or indirectly selected for its adaptive benefit.

In conclusion, this study provides evidence that mutations in OmpR experiencepositive selection during the adaptation of Bcc to chronic infections of the CF airway,and these selective forces can be recapitulated in the laboratory. OmpR is a majorregulator of many traits related to cell envelope composition and central metabolism,in which loss-of-function mutants enable greater tolerance and growth under stressconditions but are costly for fitness under other conditions.

MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in

this study are described in Table S1 in the supplemental material. Burkholderia and E. coli strainswere routinely grown in Lennox broth (LB) at 37°C, supplemented with antibiotics when appropriate.Antibiotics were used at the following concentrations for B. multivorans: chloramphenicol, 200�g/ml; gentamicin, 20 �g/ml; for E. coli concentrations were as follows: chloramphenicol, 25 �g/ml;gentamicin, 10 �g/ml. Growth in liquid medium was analyzed in triplicates by the measurement ofthe optical density at 640 nm (OD640) over time. Depending on the phenotypic test, Burkholderiastrains were grown in SM (12.5 g/liter Na2HPO4, 3.0 g/liter KH2PO4, 1.0 g/liter K2SO4, 1.0 g/liter NaCl,0.2 g/liter MgSO4·7H2O, 0.001 g/liter CaCl2·2H2O, 0.001 g/liter FeSO4·7H2O, 1.0 g/liter Casamino

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Acids, 1.0 g/liter yeast extract, 20 g/liter D-mannitol), YEM (0.5 g/liter yeast extract, 4 g/literD-mannitol, 2% agar), or M63 minimal medium supplemented with a 0.2% carbon source (47). To testgrowth in the presence of different carbon sources, each well of the Biolog GN microplate (Biolog,USA) was inoculated with 150 �l of bacterial suspension in saline solution with an OD640 of 0.2.Microplates were incubated at 37°C for 24 h and read at 600 nm in a VersaMax tunable microplatereader (Molecular Devices, USA).

For gene expression profiling (qRT-PCR and microarrays), cultures of ancestor isolates and nonmu-coid variants were grown overnight in SM medium and were diluted to an initial OD640 of 0.1 in the samemedium. Triplicate 250-ml Erlenmeyer flasks containing 100 ml medium were cultured at 37°C with 250rpm agitation for 17 h.

Isolation of B. multivorans nonmucoid variants. Triplicates of B. multivorans cultures were inoc-ulated in 3 ml of SM medium (OD640 of 0.1) and maintained statically at 42°C for 21 days. Aliquots wereremoved and the cultures were serially diluted, spread on the surfaces of YEM agar plates, and incubatedat 37°C for 2 days. The plates were then examined to recover nonmucoid colonies. When other stressors(195 �g/ml amikacin, 10 �g/ml ciprofloxacin, 10% NaCl) were applied, the experimental procedure torecover NMV was similar. To evaluate the in vitro stability of the nonmucoid morphotype, each NMV wasgrown statically for 2 days at 37°C in SM medium and passaged 3 times. At the end of the incubationtime, cultures were serially diluted and plated on YEM to determine if mucoid colonies were present.

DNA manipulation techniques. Genomic DNA was extracted from B. multivorans using the DNeasyblood and tissue kit (Qiagen, Germany) according to the manufacturer’s instructions. Plasmid DNAisolation and purification, DNA restriction, DNA amplification by PCR, agarose gel electrophoresis, and E.coli transformation were carried out using standard procedures (47).

Whole-genome sequence determination and in silico mutation analysis. DNA samples wereprocessed according to Illumina instructions and sequenced using an Illumina HiSeq 2500 at BaseClearB.V. (Netherlands) (for BM11L-nmv1) and an Illumina MiSeq system at Instituto Gulbenkian da Ciência(Portugal) for the remaining variants. All the raw paired-end reads from B. multivorans nonmucoidvariants were filtered on the basis of Phred quality scores, with adapter contamination and ambiguousnucleotides trimmed using the fastq-mcf tool (48). Filtered paired-end data sets (see Table S3) weremapped against the reference draft genome sequence of B. multivorans D2095 (20) using Bowtie2 v2.2.4(49) and SAMtools v0.1.19 (50), and the detection of mutations was carried out using the Breseq v0.26.1pipeline (51). Predicted mutations, including single nucleotide polymorphism (SNP) and indel mutations,were carefully inspected for coverage, mutation frequency, and presence in forward and reverse readsusing Geneious v6.1.8 (52).

Cloning of ompR gene and mutant complementation. A 1,334-bp fragment containing the ompRgene and the upstream promoter region was amplified from B. multivorans BM11 genomic DNA usingTaq DNA polymerase, with the forward and reverse ompR primers (see Table S4). The KpnI-BamHIfragment containing ompR and the upstream region was then cloned into pBBR1MCS digested with thesame enzymes to generate pLM014-5. This plasmid was mobilized from E. coli to B. multivorans NMVs bytriparental conjugation using the helper plasmid pRK2013. Transconjugants were selected on YEM agarplates containing 200 �g/ml of chloramphenicol and 20 �g/ml of gentamicin.

RNA extraction and quantitative real-time RT-PCR. Cells from three independent cultures wereresuspended in RNAprotect bacteria reagent, and total RNA extraction was carried out using the RNeasyMinikit (Qiagen) with DNase treatment, according to the recommendation of the manufacturer. For thereverse transcriptional step, 1 �g of total RNA from B. multivorans ancestors and the NMVs, derived fromthree independent samples, was used. cDNA was synthesized using TaqMan reverse transcriptionreagents (Applied Biosystems, USA) according to the manufacturer’s instructions. RT-PCR amplificationmixtures were diluted to use 400 ng of template cDNA, 2� SYBR green PCR master mix, and 0.4 mMreverse and forward primers for each gene (Table S4) in a total volume of 25 �l. The reactions wereperformed with a thermocycler from Applied Biosystems (model 7500). The expression ratio of the targetgenes relative to the reference gene proC, which showed no variation in transcriptional abundance underthe conditions tested, was determined. The relative fold change in target gene expression was calculatedusing the formula 2�ΔΔCT (53). Experiments were repeated three times.

Processing of RNA samples and microarrays data analysis. Cells from three independent culturesof B. multivorans BM11L and BM11L-nmv1 were resuspended in RNAprotect bacteria reagent, and totalRNA extraction was carried out as described above. The concentration and purity of total RNA sampleswere determined using a NanoDrop ND-1000 UV-visible spectrophotometer, and RNA integrity waschecked on an Agilent 2100 Bioanalyzer using an RNA Nano assay (Agilent Technologies, USA). RNA wasprocessed for use on Affymetrix custom dual-species Burkholderia arrays (Bcc1sa520656F) as previouslydescribed (54). Scanned arrays were analyzed with Affymetrix Expression Console software to ensure thatall quality parameters were in the recommended range. The subsequent analysis was carried out withDNA-Chip Analyzer 2008. The arrays were normalized to a baseline array with median CEL intensity byapplying an invariant set normalization method (55). Normalized CEL intensities of the arrays were usedto obtain model-based gene expression indices. Replicate data (triplicates) for each strain were weightedgene-wise by using the inverse squared standard errors as weights. All the genes compared wereconsidered to be differentially expressed if the 90% lower confidence bound of the fold change betweenthe experiment and baseline was greater than 1.2, resulting in 701 differentially expressed transcriptswith a median false discovery rate (FDR) of 2.5%. The lower confidence bound criterion meant that wewere 90% confident that the fold change was a value between the lower confidence bound and avariable upper confidence bound. Li and Wong have shown that the lower confidence bound is a

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conservative estimate of the fold change and therefore more reliable as a ranking statistic for changesin gene expression (55).

In silico analysis of nucleotide and amino acid sequences. The algorithm BLAST (56) was used tocompare sequences of the deduced OmpR amino acids to database sequences available at the NationalCenter for Biotechnology Information (NCBI) or the Burkholderia Genome Database (http://www.burkholderia.com/bgd) (57). Alignments were performed using the program ClustalW (58).

Determination of growth rates. Cultures were grown in LB with 17, 86, and 426 mM NaCl or in M63medium with 0.2% trehalose at 37°C with 200 rpm orbital agitation, and the OD640 was measured for 24h. The doubling time was calculated from the growth rate of the exponential growth phase. For LB with17 and 86 mM NaCl, the OD values used to estimate the growth rate were from 0.15 to 0.9, for LB with426 mM NaCl, the values were 0.15 to 0.7, and for M63 with trehalose, the values were 0.2 to 1.0 for theancestors and 0.1 to 0.15 for the NMVs, except BM11-nmv9r, which was 0.1 to 0.35. Three independentexperiments with two replicates each were performed.

Exopolysaccharide quantification. The amount of EPS was assessed based on the dry weight of theethanol-precipitated polysaccharide recovered from triplicates of 100-ml culture samples of the differentstrains grown in liquid SM medium over 6 days at 37°C with 250 rpm of orbital agitation, as previouslydescribed (54).

Analysis of outer membrane proteins. Outer membrane proteins were isolated from B. multivoransaccording to a modified version of the Sarkosyl insolubility protocol of Carlone and coworkers (59). Thewashed bacterial pellet, containing approximately 2 � 1011 cells, was suspended in 5 ml 10 mM Tris-HCl(pH 8.0) and broken by ten 30-s pulses of sonication (50% amplitude) in an ice bath, with 60-s intervalsfor cooling. Unbroken cells were removed by centrifugation at 10,000 � g for 10 min. Sarkosyl (N-laurylsarcosinate, sodium salt) and EDTA were added to the supernatant to final concentrations of 2% (wt/vol)and 10 mM, respectively. The mixture was incubated at room temperature for 1 h and then centrifugedat 100,000 � g for 1 h. The outer membrane (OM) pellets were washed twice by suspending in distilledwater and centrifuging at 100,000 � g for 1 h. The washed OM pellet was extracted with 1 ml sterilewater for 1 h at room temperature. Proteins (�40 �g) were boiled and separated in a 12% SDS-polyacrylamide gel and visualized by Coomassie staining.

Antimicrobial susceptibility and zone inhibition assays. Antimicrobial susceptibility was based onthe agar disk diffusion method (60) against piperacillin (100 �g) and aztreonam (30 �g). The disks,obtained from Becton Dickinson, were applied to the surfaces of Mueller-Hinton agar plates (DifcoLaboratories) that had been previously inoculated with 100-�l aliquots prepared from cultures grownovernight in SM at 37°C with agitation and diluted to a standardized culture OD640 of 0.1. The diametersof growth inhibition were measured after 24 h of incubation at 37°C. The standardized bacterialsuspensions used for plating on Mueller-Hinton agar plates were also used for Etest strips withaztreonam and piperacillin (0.016 to 256 �g/m; bioMérieux) and incubated for 24 h at 37°C. The MICswere read directly from the test strip at the point where the elliptical zone of inhibition intersected theMIC scale on the strip.

For zone inhibition assays, bacteria were grown overnight in SM medium at 37°C, and 100 �l of aculture with an OD640 of 1.0 was spread on SM plates. Sterile paper disks 6 mm in diameter were placedon the agar surface. A total of 20 �l of cumene hydroperoxide (CHP; 10% [vol/vol]) and H2O2 (30%[vol/vol]) was pipetted onto separate disks. The plates were incubated for 24 h at 37°C, and the zonegrowth inhibition was measured. The results are the mean values from at least three independentdeterminations of growth inhibition diameters.

The outer membrane permeability of parental isolates and NMVs was determined using theN-phenyl-1-naphthylamine (NPN) uptake assay (61). To this end, bacteria were resuspended in 5 mMHEPES buffer (pH 7.2) to an OD640 of 0.2. One hundred-microliter portions of the bacterial suspensionswere mixed with 95 �l of the HEPES buffer and 5 �l of a 0.5 mM NPN solution in acetone. Two controlsfor each strain analyzed were performed; the first contained 100 �l of the HEPES buffer and 100 �l of thebacterial suspension, whereas the second contained 95 �l of the HEPES buffer and 5 �l of the NPNsolution. The intensity of fluorescence was measured with a fluorescence spectrophotometer (VarianCary Eclipse; Agilent) for 10 min using excitation and emission wavelengths of 355 nm and 535 nm,respectively. To standardize the data, viable cells from the bacterial suspensions were counted in theplate assay. The data are reported as NPN uptake factor (ratio of background-subtracted fluorescencevalues to that of the buffer value, adjusted to the number of CFU) and are the means from twoindependent experiments with three biological repetitions for each strain.

Motility assays. For motility estimation, 5 �l of overnight SM bacterial cultures was inoculated onthe agar surfaces of swimming and swarming plates and incubated statically at 37°C for 24 h followedby colony diameter determination. Swimming plates were prepared with SM medium with 0.3% (wt/vol)Noble agar (Difco), while swarming plates were prepared in Broomfield medium (0.04% [wt/vol] tryptone,0.01% yeast extract [wt/vol], 0.0067% [wt/vol] CaCl2) with 0.6% (wt/vol) of Bacto agar. Three separateexperiments, each containing four technical replicates, were performed.

Biofilm formation on abiotic surfaces. Biofilm assays were performed as previously described (11).Overnight liquid cultures of the different B. multivorans strains were diluted to a standardized cultureOD640 of 0.1 in LB or LB supplemented with 426 mM NaCl, and 200 �l of these cell suspensions was usedto inoculate the wells of a 96-well polystyrene microtiter plate (Greiner Bio-One, Austria). The plates wereincubated at 37°C for 48 h without agitation. The biofilm was stained with crystal violet solution, followedby dye solubilization with ethanol and the measurement of the solution’s OD590 using a microplatereader. The results are the mean values from at least five repeats from three independent experiments.

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Host cell attachment. B. multivorans isolates were analyzed for adhesion to the bronchial epithelialcell line CFBE41o�, derived from a patient homozygous for the cystic fibrosis transmembrane conduc-tance regulator F508del mutation. Bacterial strains were grown overnight in LB, after which 200 �l ofthose cultures was grown in LB with 426 mM NaCl for 4 h and then used to infect epithelial cells at amultiplicity of infection (MOI) of 10 (10 bacterial cells to 1 epithelial cell). Bacteria were applied to a24-well plate previously seeded with CFBE41o� cells in minimal essential medium-supplemented medium(62) and the plates were centrifuged at 700 � g for 5 min. The plates were then incubated for 30 min at 37°Cin an atmosphere of 5% CO2. Afterwards, each well was washed three times with phosphate-buffered saline(PBS) to remove unbound bacteria, and the cells were lysed with lysis buffer (0.01 M PBS, 10 mM EDTA, 0.25%[vol/vol] Triton X-100, pH 7.4) for 20 min at 4°C. Serial dilutions were plated on LB agar, and adhesion wasquantified by determining CFU counts after 48 h of incubation at 37°C. Duplicates with each strain wereperformed per assay, and the results presented were obtained from three independent experiments. Theresults are shown as the percentage of adhesion, which was calculated as the number of CFU recovereddivided by the number of CFU applied to the epithelial cells multiplied by 100.

Microscopy analysis. B. multivorans BM11L and BM11L-nmv1 were grown for 24 h in LB with 86 mMor 426 mM NaCl at 37°C with 200 rpm agitation. To visualize the self-aggregation of cells grown in LBwith 86 mM NaCl, images were acquired on a Zeiss Axioplan microscope equipped with a PhotometricsCoolSNAP fx camera, using a 10� 0.3-numerical aperture (NA) objective and controlled with softwareMetaMorph version 4.6r9 (Fig. 8A and B). Images of macroscopic cell aggregates of strains grown in LBwith 426 mM NaCl were made with a Pentax *ist DS digital camera (Fig. 8C and D).

Virulence determination in Galleria mellonella. Killing assays were performed as previouslydescribed (54). Larvae were injected with 1 � 107 CFU diluted in 10 mM MgSO4 with 1.2 mg/mlampicillin, and the survival rate was evaluated 3 days postinfection. As a negative control, 10 mM MgSO4

with 1.2 mg/ml ampicillin was used. This assay was repeated four times with 10 larvae each.Statistical analysis. All quantitative data were obtained from at least three independent assays with

two biological replicates. Error propagation was used to calculate the standard errors, and Student’stwo-sample t tests or one-way analyses of variance (ANOVAs) with Tukey post hoc analyses (OriginPro 9.0;OriginLab Corporation) were performed to assess statistical significance. Differences were consideredstatistically significant if the P value was lower than 0.05.

Accession number(s). The DNA sequence reads for assemblies of the several NMV genomes areavailable in the NCBI SRA under accession number SRP136601. Alignment and SNP call files are infigshare (https://figshare.com/s/6cdfa12a1ff981c56953). Microarray data were deposited in the GeneExpression Omnibus (GEO) repository at NCBI under accession number GSE111467.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/JB.00216-18.

SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.SUPPLEMENTAL FILE 2, XLSX file, 0.1 MB.

ACKNOWLEDGMENTSThis work was supported by Fundação para a Ciência e a Tecnologia, Portugal

(projects PTDC/QUI-BIQ/118260/2010 and UID/BIO/04565/2013 and a postdoctoralgrant [SFRH/BPD/86475/2012] to I.N.S.), and by Programa Operacional Regional deLisboa 2020 (LISBOA-01-0145-FEDER-007317). V.S.C. is supported by the Cystic FibrosisFoundation Research Development Program award to the University of Pittsburgh.

Microarrays and bacterial genome sequencing were processed at the Gene Expres-sion Unit of Instituto Gulbenkian de Ciência.

The funders had no role in study design, data collection and interpretation, or thedecision to submit the work for publication.

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