- Volume 162, Issue 5, 2016

Coevolution between bacteriophages (phages) and their prey is the result of mutualistic interactions. Here, we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa and that selection of resistant bacterial mutants is favoured by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10− 5 for single phage infection and 10− 6 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered, presumably due to continuous evolutionary pressure.

The cellular proteolytic machinery orchestrates protein turnover and regulates several key biological processes. This study addresses the roles of Lon, a major ATP-dependent protease, in modulating the responses of Escherichia coli strain MG1655 to low and high amounts of sodium salicyclate (NaSal), a widely used clinically relevant analgesic. NaSal affects several bacterial responses, including growth and resistance to multiple antibiotics. The loss of lon reduces growth in response to high, but not low, amounts of NaSal. From amongst a panel of Lon substrates, MarA was identified to be the downstream target of Lon. Thus, stabilization of MarA in the absence of lon lowers growth of the strain in the presence of higher amounts of NaSal. The steady-state transcript levels of marA and its target genes, acrA, acrB and tolC, are higher in the Δlon strain compared with the WT strain. Consequently, the resistance to antibiotics, e.g. tetracycline and nalidixic acid, is enhanced in Δlon in a marA-dependent manner. Furthermore, the target genes of MarA, i.e. acrB and tolC, are responsible for NaSal-mediated antibiotic resistance. Studies using atomic force microscopy demonstrated that ciprofloxacin led to greater cell filamentation, which is lower in the Δlon strain due to higher levels of MarA. Overall, this study delineates the roles of Lon protease, its substrate MarA and downstream targets of MarA, e.g. acrB and tolC, during NaSal-mediated growth reduction and antibiotic resistance. The implications of these observations in the adaptation of E. coli under different environmental conditions are discussed.

A hospitalized 73-year-old woman was infected with a Proteus mirabilis strain, 12 B-r, isolated from the place of injection of a blood catheter. Another strain, 12 B-k, recognized as Proteus genomospecies 5 or 6, was isolated from the patient's faeces, which was an example of a nosocomial infection rather than an auto-infection. Serological investigation using ELISA and Western blotting showed that strain 12 B-k from faeces belonged to the Proteus O2 serogroup. Strain 12 B-r from the wound displayed cross-reactions with several Proteus O serogroups due to common epitopes on the core or O-specific parts of the lipopolysaccharide. Studies of the isolated 12 B-r O-specific polysaccharide by NMR spectroscopy revealed its close structural similarity to that of Proteus O8. The only difference in 12 B-r was the presence of an additional GlcNAc-linked phosphoethanolamine residue, which creates a putative epitope responsible for the cross-reactivity with Pt. mirabilis O16. The new O-antigen form could appear as a result of adaptation of the bacterium to a changing environment. On the basis of the data obtained, we suggest division of the O8 serogroup into two subgroups: O8a for strains of various Proteus species that have been previously classified into the O8 serogroup, and O8a,b for Pt. mirabilis 12 B-r, where ‘a’ is a common epitope and ‘b’ is a phosphoethanolamine-associated epitope. These findings further confirm serological and structural heterogeneity of O antigens of Proteus strains isolated lately from patients in Poland.

Enterococcus faecalis is an opportunistic pathogen that ranks among the leading causes of biofilm-associated infections. We previously demonstrated that the endocarditis- and biofilm-associated pili (Ebp) of E. faecalis play a major role in biofilm formation, adherence to abiotic surfaces and experimental infections. In this study, derivatives of E. faecalis strain OG1 were engineered to further characterize functions of Ebp pili. Loss of pili resulted in a 36-fold decrease in the number of closely associated cells when OG1RFΔebpABC was mixed with OG1SSpΔebpABC, compared with mixing the Ebp+ parental strains. In addition, using the Ebp+ parental strains as donor and recipient, we found a statistically significant increase (280–360 %, P < 0.05) in the frequency of plasmid transfer versus using Ebp−  mutants in the conjugation experiments. These results demonstrate a previously unrecognized role of Ebp pili, namely, as important contributors to microscale cell aggregation and horizontal spread of genetic material.

Pseudomonas putida A (ATCC 12633), a degrader of cationic surfactants, releases outer membrane vesicles (OMVs) when grown with tetradecyltrimethylammonium bromide (TTAB) as the sole carbon, nitrogen and energy source. The OMVs exhibit a bilayer structure and were found to be composed of lipopolysaccharides, proteins and phospholipids (PLs) such as cardiolipin, phosphatidylcholine, phosphatidic acid and phosphatidylglycerol (PG). The OMVs showed a marked increase in the PG content, approximately 43 % higher than the amount registered in the parent cells from which the vesicles were derived. After growth of P. putida with TTAB, the amount of lipoprotein covalently cross-linked to the peptidoglycan showed a twofold decrease when compared with values found after growth without the surfactant [16 ± 2 and 28 ± 3 μg (mg cell envelope protein)− 1, respectively]. This decrease in the amount of lipoprotein can be related to areas of loss of contact between the outer membrane and the peptidoglycan and, therefore, to OMV production. In addition, due to its amphiphilic nature, TTAB can contribute to OMV biogenesis, through a physical mechanism, by induction of the curvature of the membrane. Taking into account that OVMs were produced when the cells were grown under external stress, caused by the surfactant, and that TTAB was detected in the vesicles [48 nmol TTAB (nmol PL)− 1], we concluded that this system of TTAB elimination is a mechanism that P. putida A (ATCC 12633) would utilize for alleviating stress caused by cationic surfactants.

This work reports the evolutionary relationships, amplified expression, functional characterization and purification of the putative allantoin transport protein, PucI, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from Microbacterium liquefaciens, and related proteins from bacteria, fungi and plants. Membrane topology predictions for PucI were consistent with 12 putative transmembrane-spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The pucI gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions determined for optimal amplified expression of the PucI(His6) protein in Escherichia coli to a level of about 5 % in inner membranes. Initial rates of inducible PucI-mediated uptake of 14C-allantoin into energized E. coli whole cells conformed to Michaelis–Menten kinetics with an apparent affinity (K mapp) of 24 ± 3 μM, therefore confirming that PucI is a medium-affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilized from inner membranes using n-dodecyl-β-d-maltoside and purified. The isolated protein contained a substantial proportion of α-helix secondary structure, consistent with the predictions, and a 3D model was therefore constructed on a template of the Mhp1 structure, which aided localization of the potential ligand binding site in PucI.

In Aspergillus nidulans, uptake rather than hydrolysis is the rate-limiting step of lactose catabolism. Deletion of the lactose permease A-encoding gene (lacpA) reduces the growth rate on lactose, while its overexpression enables faster growth than wild-type strains are capable of. We have identified a second physiologically relevant lactose transporter, LacpB. Glycerol-grown mycelia from mutants deleted for lacpB appear to take up only minute amounts of lactose during the first 60 h after a medium transfer, while mycelia of double lacpA/lacpB-deletant strains are unable to produce new biomass from lactose. Although transcription of both lacp genes was strongly induced by lactose, their inducer profiles differ markedly. lacpA but not lacpB expression was high in d-galactose cultures. However, lacpB responded strongly also to β-linked glucopyranose dimers cellobiose and sophorose, while these inducers of the cellulolytic system did not provoke any lacpA response. Nevertheless, lacpB transcript was induced to higher levels on cellobiose in strains that lack the lacpA gene than in a wild-type background. Indeed, cellobiose uptake was faster and biomass formation accelerated in lacpA deletants. In contrast, in lacpB knockout strains, growth rate and cellobiose uptake were considerably reduced relative to wild-type, indicating that the cellulose and lactose catabolic systems employ common elements. Nevertheless, our permease mutants still grew on cellobiose, which suggests that its uptake in A. nidulans prominently involves hitherto unknown transport systems.

In this study, we demonstrated that in distilled water, a nutrient-starved condition that elicits autophagy in Saccharomyces cerevisiae, an array of autophagy-deficient mutants are resistant to the fungicidal effects of amphotericin B. In addition, we found that a dansyl-labelled derivative of the antibiotic colocalized with disintegrated vacuoles throughout the cytoplasm in the amphotericin B-sensitive parental strain suspended in distilled water. In contrast, the dansyl-labelled derivative was not internalized in the Δatg18 strain, which is deficient in the formation of autophagosomes, a key early step in autophagy. However, the derivative accumulated without significant toxicity in structurally intact vacuoles in the Δvma1 mutant, which is deficient in the degradation of autophagic bodies, the final stage in autophagy. Our data support the idea that amphotericin B can utilize autophagy-dependent trafficking into the intra-vacuolar lumen, where it interacts with the luminal leaf of the membrane to cause structurally catastrophic effects.

Solar UVA radiation is one of the main environmental stress factors for Pseudomonas aeruginosa. Exposure to high UVA doses produces lethal effects by the action of the reactive oxygen species (ROS) it generates. P. aeruginosa has several enzymes, including KatA and KatB catalases, which provide detoxification of ROS. We have previously demonstrated that KatA is essential in defending P. aeruginosa against high UVA doses. In order to analyse the mechanisms involved in the adaptation of this micro-organism to UVA, we investigated the effect of exposure to low UVA doses on KatA and KatB activities, and the physiological consequences. Exposure to UVA induced total catalase activity; assays with non-denaturing polyacrylamide gels showed that both KatA and KatB activities were increased by radiation. This regulation occurred at the transcriptional level and depended, at least partly, on the increase in H2O2 levels. We demonstrated that exposure to low UVA produced a protective effect against subsequent lethal doses of UVA, sodium hypochlorite and H2O2. Protection against lethal UVA depends on katA, whilst protection against sodium hypochlorite depends on katB, demonstrating that different mechanisms are involved in the defence against these oxidative agents, although both genes can be involved in the global cellular response. Conversely, protection against lethal doses of H2O2 could depend on induction of both genes and/or (an)other defensive factor(s). A better understanding of the adaptive response of P. aeruginosa to UVA is relevant from an ecological standpoint and for improving disinfection strategies that employ UVA or solar irradiation.

Ciprofloxacin is a widely used antibiotic, in the class of quinolones, for treatment of Pseudomonas aeruginosa infections. The immediate response of P. aeruginosa to subinhibitory concentrations of ciprofloxacin has been investigated previously. However, the long-term phenotypic adaptation, which identifies the fitted phenotypes that have been selected during evolution with subinhibitory concentrations of ciprofloxacin, has not been studied. We chose an experimental evolution approach to investigate how exposure to subinhibitory concentrations of ciprofloxacin changes the evolution of P. aeruginosa populations compared to unexposed populations. Three replicate populations of P. aeruginosa PAO1 and its hypermutable mutant ΔmutS were cultured aerobically for approximately 940 generations by daily passages in LB medium with and without subinhibitory concentration of ciprofloxacin and aliquots of the bacterial populations were regularly sampled and kept at  − 80 °C for further investigations. We investigate here phenotypic changes between the ancestor (50 colonies) and evolved populations (120 colonies/strain). Decreased protease activity and swimming motility, higher levels of quorum-sensing signal molecules and occurrence of mutator subpopulations were observed in the ciprofloxacin-exposed populations compared to the ancestor and control populations. Transcriptomic analysis showed downregulation of the type III secretion system in evolved populations compared to the ancestor population and upregulation of denitrification genes in ciprofloxacin-evolved populations. In conclusion, the presence of antibiotics at subinhibitory concentration in the environment affects bacterial evolution and further studies are needed to obtain insight into the dynamics of the phenotypes and the mechanisms involved.

Among multiple interconnected pathways for l-Lysine catabolism in pseudomonads, it has been reported that Pseudomonas aeruginosa PAO1 employs the decarboxylase and the transaminase pathways. However, up until now, knowledge of several genes involved in operation and regulation of these pathways was still missing. Transcriptome analyses coupled with promoter activity measurements and growth phenotype analyses led us to identify new members in l-Lys and d-Lys catabolism and regulation, including gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for d-Lys catabolism, lysR-lysXE for putative l-Lys efflux and lysP for putative l-Lys uptake. The gcdHG operon encodes an acyl-CoA transferase (gcdG) and glutaryl-CoA dehydrogenase (gcdH) and is under the control of the transcriptional activator GcdR. Growth on l-Lys was enhanced in the mutants of lysX and lysE, supporting the operation of l-Lys efflux. The transcriptional activator LysR is responsible for l-Lys specific induction of lysXE and the PA4181-82 operon of unknown function. The putative operator sites of GcdR and LysR were deduced from serial deletions and comparative genomic sequence analyses, and the formation of nucleoprotein complexes was demonstrated with purified His-tagged GcdR and LysR. The amaAB operon encodes two enzymes to convert pipecolate to 2-aminoadipate. Induction of the amaAB operon by l-Lys, d-Lys and pipecolate requires a functional AmaR, supporting convergence of Lys catabolic pathways to pipecolate. Growth on pipecolate was retarded in the gcdG and gcdH mutants, suggesting the importance of glutarate in pipecolate and 2-aminoadipate utilization. Furthermore, this study indicated links in the control of interconnected networks of lysine and arginine catabolism in P. aeruginosa.

The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (Δ256–292 or Δ107–237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (Δ1–20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3Δ strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3Δ strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA.