pH Adaptation Drives Diverse Phenotypes in a Beneficial Bacterium-Host Mutualism

Abiotic variation can influence the evolution of specific phenotypes that contribute to the diversity of bacterial strains observed in the natural environment. Environmentally transmitted symbiotic bacteria are particularly vulnerable to abiotic fluctuations, given that they must accommodate the transition between the free-living state and the host's internal environment. This type of life history strategy can strongly influence the success of a symbiont, and whether adapting to changes outside the host will allow a greater capacity to survive in symbiosis with the host partner. One example of how environmental breadth is advantageous to the symbiosis is the beneficial association between Vibrio fischeri and sepiolid squids (Cephalopoda: Sepiolidae). Since Vibrio bacteria are environmentally transmitted, they are subject to a wide variety of abiotic variables prior to infecting juvenile squids and must be poised to survive in the host light organ. In order to better understand how a changing abiotic factor (e.g., pH) influences the diversification of symbionts and their eventual symbiotic competence, we used an experimental evolution approach to ascertain how pH adaptation affects symbiont fitness. Results show that low pH adapted Vibrio strains have more efficient colonization rates compared to their ancestral strains. In addition, growth rates had significant differences compared to ancestral strains (pH 6.5–6.8, and 7.2). Bioluminescence production (a marker for symbiont competence) of pH evolved strains also improved at pH 6.5–7.2. Results imply that the evolution and diversification of Vibrio strains adapted to low pH outside the squid improves fitness inside the squid by allowing a higher success rate for host colonization and symbiotic competence.

Structure of theDietziaMrp complex reveals molecular mechanism of this giant bacterial sodium proton pump

Multiple resistance and pH adaptation (Mrp) complexes are sophisticated cation/proton exchangers found in a vast variety of alkaliphilic and/or halophilic microorganisms, and are critical for their survival in highly challenging environments. This family of antiporters is likely to represent the ancestor of cation pumps found in many redox-driven transporter complexes, including the complex I of the respiratory chain. Here, we present the three-dimensional structure of the Mrp complex from aDietziasp. strain solved at 3.0-Å resolution using the single-particle cryoelectron microscopy method. Our structure-based mutagenesis and functional analyses suggest that the substrate translocation pathways for the driving substance protons and the substrate sodium ions are separated in two modules and that symmetry-restrained conformational change underlies the functional cycle of the transporter. Our findings shed light on mechanisms of redox-driven primary active transporters, and explain how driving substances of different electric charges may drive similar transport processes.

Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.

Computational Analysis of the Primary and Secondary Structure of Amidases in Relation to their pH Adaptation

Background: Amidases are ubiquitous enzymes and biological functions of these enzymes vary widely. They are considered to be synergistically involved in the synthesis of a wide variety of carboxylic acids, hydroxamic acids and hydrazides, which find applications in commodity chemicals synthesis, pharmaceuticals agrochemicals and wastewater treatments. Methods: They hydrolyse a wide variety of amides (short-chain aliphatic amides, mid-chain amides, arylamides, α-aminoamides and α-hydroxyamides) and can be grouped on the basis of their catalytic site and preferred substrate. Despite their economic importance, we lack knowledge as to how these amidases withstand elevated pH and temperature whereas others cannot. Results: The present study focuses on the statistical comparison between the acid-tolerant, alkali tolerant and neutrophilic organisms. In silico analysis of amidases of acid-tolerant, alkali tolerant and neutrophilic organisms revealed some striking trends as to how amino acid composition varies significantly. Statistical analysis of primary and secondary structure revealed amino acid trends in amidases of these three groups of bacteria. The abundance of isoleucine (Ile, I) in acid-tolerant and leucine (Leu, L) in alkali tolerant showed the aliphatic amino acid dominance in extreme conditions of pH in acidtolerant and alkali tolerant amidases. Conclusion: The present investigation insights physiochemical properties and dominance of some crucial amino acid residues in the primary and secondary structure of some amidases from acid-tolerant, alkali tolerant and neutrophilic microorganisms.

Acid Experimental Evolution of the Extremely Halophilic Archaeon Halobacterium sp. NRC-1 Selects Mutations Affecting Arginine Transport and Catabolism

Background Halobacterium sp. NRC-1 (NRC-1) is an extremely halophilic archaeon that is adapted to multiple stressors such as UV, ionizing radiation and arsenic exposure. We conducted experimental evolution of NRC-1 under acid stress. NRC-1 was serially cultured in CM+ medium modified by four conditions: optimal pH (pH 7.5), acid stress (pH 6.3), iron amendment (600 μM ferrous sulfate, pH 7.5), and acid plus iron (pH 6.3, with 600 μM ferrous sulfate). For each condition, four independent lineages of evolving populations were propagated. After 500 generations, 16 clones were isolated for phenotypic characterization and genomic sequencing. Results Genome sequences of all 16 clones revealed 378 mutations, of which 90% were haloarchaeal insertion sequences (ISH) and ISH-mediated large deletions. This proportion of ISH events in NRC-1 was five-fold greater than that reported for comparable evolution of E. coli. One acid-evolved clone had increased fitness compared to the ancestral strain when cultured at low pH. Seven of eight acid-evolved clones had a mutation within or upstream of arcD, which encodes an arginine-ornithine antiporter; no non-acid adapted strains had arcD mutations. Mutations also affected the arcR regulator of arginine catabolism, which protects bacteria from acid stress by release of ammonia. Two acid-adapted strains shared a common mutation in bop, which encodes the bacteriorhodopsin light-driven proton pump. Unrelated to pH, one NRC-1 minichromosome (megaplasmid) pNRC100 had increased copy number, and we observed several mutations that eliminate gas vesicles and arsenic resistance. Thus, in the haloarchaeon NRC-1, as in bacteria, pH adaptation was associated with genes involved in arginine catabolism and proton transport. Conclusions Our study is among the first to report experimental evolution with multiple resequenced genomes of an archaeon. Haloarchaea are polyextremophiles capable of growth under environmental conditions such as concentrated NaCl and desiccation, but little is known about pH stress. Halobacterium sp. NRC-1 (NRC-1) is considered a model organism for the feasibility of microbial life in iron-rich brine on Mars. Interesting parallels appear between the molecular basis of pH adaptation in NRC-1 and in bacteria, particularly the acid-responsive arginine-ornithine system found in oral streptococci.

Acid Experimental Evolution of the Extremely Halophilic Archaeon Halobacterium sp. NRC-1 Selects Mutations Affecting Arginine Transport and Catabolism

ABSTRACTBackgroundHalobacterium sp. NRC-1 (NRC-1) is an extremely halophilic archaeon that is adapted to multiple stressors such as UV, ionizing radiation and arsenic exposure. We conducted experimental evolution of NRC-1 under acid stress. NRC-1 was serially cultured in CM+ medium modified by four conditions: optimal pH (pH 7.5), acid stress (pH 6.3), iron amendment (600 μM ferrous sulfate, pH 7.5), and acid plus iron (pH 6.3, with 600 μM ferrous sulfate). For each condition, four independent lineages of evolving populations were propagated. After 500 generations, 16 clones were isolated for phenotypic characterization and genomic sequencing.ResultsGenome sequences of all 16 clones revealed 378 mutations, of which 90% were haloarchaeal insertion sequences (ISH) and ISH-mediated large deletions. This proportion of ISH events in NRC-1 was five-fold greater than that reported for comparable evolution of E. coli. One acid-evolved clone had increased fitness compared to the ancestral strain when cultured at low pH. Seven of eight acid-evolved clones had a mutation within or upstream of arcD, which encodes an arginine-ornithine antiporter; no non-acid adapted strains had arcD mutations. Mutations also affected the arcR regulator of arginine catabolism, which protects bacteria from acid stress by release of ammonia. Two acid-adapted strains shared a common mutation in bop, which encodes the bacteriorhodopsin light-driven proton pump. Unrelated to pH, one NRC-1 minichromosome (megaplasmid) pNRC100 had increased copy number, and we observed several mutations that eliminate gas vesicles and arsenic resistance. Thus, in the haloarchaeon NRC-1, as in bacteria, pH adaptation was associated with genes involved in arginine catabolism and proton transport.ConclusionsOur study is among the first to report experimental evolution with multiple resequenced genomes of an archaeon. Haloarchaea are polyextremophiles capable of growth under environmental conditions such as concentrated NaCl and desiccation, but little is known about pH stress. Halobacterium sp. NRC-1 (NRC-1) is considered a model organism for the feasibility of microbial life in iron-rich brine on Mars. Interesting parallels appear between the molecular basis of pH adaptation in NRC-1 and in bacteria, particularly the acid-responsive arginine-ornithine system found in oral streptococci.

Transcriptional Sequencing Uncovers Survival Mechanisms ofSalmonella entericaSerovar Enteritidis in Antibacterial Egg White

ABSTRACTThe survival mechanism ofSalmonella entericaserovar Enteritidis in antibacterial egg white is not fully understood. In our lab, an egg white-resistant strain,S.Enteritidis SJTUF 10978, was identified. Cell envelope damage and osmotic stress response (separation of cell wall and inner membrane as well as cytoplasmic shrinkage) of this strain surviving in egg white were identified through microscopic observation. RNA-Seq analysis of the transcriptome ofSalmonellasurvival in egg white showed that a considerable number of genes involved in DNA damage repair, alkaline pH adaptation, osmotic stress adaptation, envelope damage repair,Salmonellapathogenicity island 2 (SPI-2), iron absorption, and biotin synthesis were significantly upregulated (fold change ≥ 2) in egg white, indicating that these pathways or genes might be critical for bacterial survival. RNA-Seq results were confirmed by qRT-PCR, and the survival analysis of six gene deletion mutants confirmed their importance in the survival of bacteria in egg white. The importance of alkaline pH adaptation and envelope damage repair forSalmonellato survive in egg white were further confirmed by analysis ofnhaA,cpxR,waaH, andecodeletion mutants. According to the RNA-Seq results, we propose that alkaline pH adaptation might be the cause of bacterial osmotic stress phenotype and that the synergistic effect between alkaline pH and other inhibitory factors can enhance the bacteriostatic effect of egg white. Moreover,cpxRandsigEwere recognized as the central regulators that coordinate bacterial metabolism to adapt to envelope damage and alkaline pH.IMPORTANCESalmonella entericaserovar Enteritidis is a major foodborne pathogen that causes salmonellosis mainly through contaminated chicken eggs or egg products and has been a worldwide public health threat since 1980. Frequent outbreaks of this serotype through eggs correlate significantly with its exceptional survival ability in the antibacterial egg white. Research on the survival mechanism ofS.Enteritidis in egg white will help to further understand the complex and highly effective antibacterial mechanisms of egg white and lay the foundation for the development of safe and effective vaccines to prevent egg contamination by thisSalmonellaserotype. Key pathways and genes that were previously overlooked under bactericidal conditions were characterized as being induced in egg white, and synergistic effects between different antimicrobial factors appear to exist according to the gene expression changes. Our work provides new insights into the survival mechanism ofS.Enteritidis in egg white.

The acid tolerance response and pH adaptation of Enterococcus faecalis in extract of lime Citrus aurantiifolia from Aceh Indonesia

Background: The objective of the present study was to evaluate the acid tolerance response and pH adaptation whenEnterococcusfaecalisinteracted with extract of lime (Citrus aurantiifolia).Methods:We usedE. faecalisATCC 29212 and lime extract from Aceh, Indonesia. The microbe was analyzed for its pH adaptation, acid tolerance response, and adhesion assay using a light microscope with a magnification of x1000. Further, statistical tests were performed to analyze both correlation and significance of the acid tolerance and pH adaptation as well as the interaction activity.Results:E. faecaliswas able to adapt to a very acidic environment (pH 2.9), which was characterized by an increase in its pH (reaching 4.2) at all concentrations of the lime extract (p < 0.05).E. faecaliswas also able to provide acid tolerance response to lime extract based on spectrophotometric data (595 nm) (p < 0.05). Also, the interaction activity ofE. faecalisin different concentrations of lime extract was relatively stable within 6 up to 12 hours (p < 0.05), but it became unstable within 24–72 hours (p > 0.05) based on the mass profiles of its interaction activity.Conclusions:E. faecaliscan adapt to acidic environments (pH 2.9–4.2); it is also able to tolerate acid generated byCitrus aurantiifoliaextract, revealing a stable interaction in the first 6–12 hours.

The acid tolerance response and pH adaptation of Enterococcus faecalis in extract of lime Citrus aurantiifolia from Aceh Indonesia

Background:The objective of the present study was to evaluate the acid tolerance response and pH adaptation whenEnterococcusfaecalisinteracted with extract of lime (Citrus aurantiifolia).Methods:We usedE. faecalisATCC 29212 and lime extract from Aceh, Indonesia. Both materials were analyzed for their pH adaptation, acid tolerance response, adhesion assay, and mass profiles using a light microscope with a magnification of x1000. Further, statistical tests were performed to analyze both correlation and significance of the acid tolerance and pH adaptation also the interaction activity.Results:E. faecaliswas able to adapt to a very acidic environment (pH 2.9), which was characterized by an increase in its pH (reaching 4.2) at all concentrations of the lime extract (p < 0.05).E. faecaliswas also able to provide acid tolerance response to lime extract based on spectrophotometric data (595 nm) (p < 0.05). Also, the interaction activity ofE. faecalisand the lime extract was relatively stable within 6 up to 12 hours (p < 0.05), but it became unstable within 24–72 hours (p > 0.05) based on the mass profiles of its interaction activity.Conclusions:E. faecaliscan adapt to acidic environments (pH 2.9–4.2); it is also able to tolerate acid generated byCitrus aurantiifoliaextract, revealing a stable interaction in the first 6–12 hours.