The funders had changing over time in response to growth conditions. We characterized this phenomenon using bulk liquid culture experiments, colony growth tracking, flow cytometry, single-cell timelapse microscopy, transcriptomics, and genome resequencing. Finally, we used mathematical modeling to better understand the processes by which cells change phenotype, and found evidence for both stochastic, bidirectional phenotypic diversification and responsive, directed phenotypic shifts, depending on the growth substrate and the presence of toxin.

The potency and indiscriminate nature of formaldehyde reactivity upon biological molecules make it a universal stressor. However, some organisms such as Methylorubrum extorquens possess means to rapidly and effectively mitigate formaldehyde-induced damage. EfgA is a recently identified formaldehyde sensor predicted to halt translation in response to elevated formaldehyde as a means to protect cells. Herein, we investigate growth and changes in gene expression to understand how M. extorquens responds to formaldehyde with and without the EfgA-formaldehyde-mediated translational response, and how this mechanism compares to antibiotic-mediated translation inhibition. These distinct mechanisms of translation inhibition have notable differences: they each involve different specific players and in addition, formaldehyde also acts as a general, multi-target stressor and a potential carbon source. We present findings demonstrating that in addition to its characterized impact on translation, functional EfgA allows for a rapid and robust transcriptional response to formaldehyde and that removal of EfgA leads to heightened proteotoxic and genotoxic stress in the presence of increased formaldehyde levels. We also found that many downstream consequences of translation inhibition were shared by EfgA-formaldehyde- and kanamycin-mediated translation inhibition. Our work uncovered additional layers of regulatory control enacted by functional EfgA upon experiencing formaldehyde stress, and further demonstrated the importance this protein plays at both transcriptional and translational levels in this model methylotroph.

A Gram-stain-negative, cream-pigmented, motile, extremely halophilic archaeon, designated strain IC38 T , was isolated from a saline mud sample taken from a hypersaline lake, Aran-Bidgol, in Iran. The strain required at least 2.5 M NaCl for growth. However, MgCl 2 was not required. Optimal growth occurred with 4.3 M NaCl and 0.2 M MgCl 2 . The optimum pH and temperature for growth were pH 7.0 and 35 6C, respectively, and strain IC38 T was able to grow over a pH range of 6.5-9.0, and a temperature range of 25-45 6C. Analysis of the 16S rRNA gene sequence revealed that strain IC38 T clustered with the two species of the genus Halovivax, Halovivax asiaticus EJ-46 T and Halovivax ruber XH-70 T , with sequence similarities of 96.4 % and 96.1 %, respectively. The similarities between the rpoB9 gene of the novel strain and Halovivax asiaticus and Halovivax ruber were 90.7 % and 90.3 %, respectively. The polar lipid pattern of strain IC38 T consisted of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Three unidentified glycolipids and two minor phospholipids were also observed. The DNA G+C content of strain IC38 T was 62.6 mol%. On the basis of the phylogenetic analysis, as well as the biochemical and physiological characteristics, the new isolate is suggested to be a representative of a novel species of the genus Halovivax, for which the name Halovivax limisalsi sp. nov. is proposed. The type strain of Halovivax limisalsi is IC38 T (5IBRC-M 10022 T 5KCTC 4051 T ).The genus Halovivax was proposed by Castillo et al. (2006) to accommodate an extremely halophilic archaeon, which was isolated from a saline lake, Ejinor, in Inner Mongolia, PR China. At the time of writing, the genus comprises two species with validly published names, Halovivax asiaticus (the type species of the genus) and Halovivax ruber (Castillo et al., 2007), which are pale-pink and red strains, respectively. Members of this genus are extremely halophilic, strictly aerobic and they do not contain phosphatidylglycerol sulphate among their polar lipids (Castillo et al., 2006(Castillo et al., , 2007. Here we describe the isolation and polyphasic characterization of a novel, cream-pigmented, extremely halophilic archaeon isolated from a mud sample taken from a hypersaline lake, Aran-Bidgol, in Iran and propose it to represent a novel species of the genus Halovivax.Strain IC38 T was isolated from a mud sample [pH 7.5; salinity 22 % (w/v)] taken from Aran-Bidgol, a hypersaline lake in Iran (35 u 709 N 51 u 399 E). We sampled the hypersaline mud (up to 40 cm in depth), collecting it in sterile plastic containers and then kept samples in the dark at an environmental temperature for four hours until it was analysed in the laboratory. The environmental sample was inoculated in modified growth medium (MGM) with 23 % (w/v) total salt concentration (Dyall-Smith, 2009

Acknowledgments 44We are grateful to Eric Bruger, Sergey Stolyar, Nicholas Shevalier, and Joshua Wirtz for 45 assistance in conducting M. extorquens experiments; to Dipti Nayak for advice and 46 resources facilitating those experiments; to Craig Miller for advice on analyzing colony 47 New, Samuel Hunter, and Matt Fagnan. Juan E. Abrahante of the University of 55 Minnesota Informatics Institute (UMII) assisted with RNA-Seq data analysis. IBEST is 56 supported in part by NIH COBRE grant P30GM103324. Funding for this work came 57 from an Army Research Office MURI sub-award to CJM (W911NF-12-1-0390), a CMCI 58Abstract 63 64 While microbiologists often make the simplifying assumption that genotype determines 65 phenotype in a given environment, it is becoming increasingly apparent that phenotypic 66 heterogeneity (in which one genotype generates multiple phenotypes simultaneously 67 even in a uniform environment) is common in many microbial populations. The 68 importance of phenotypic heterogeneity has been demonstrated in a number of model 69 systems involving binary phenotypic states (e.g., growth/non-growth); however, less is 70 known about systems involving phenotype distributions that are continuous across an 71 environmental gradient, and how those distributions change when the environment 72 changes. Here, we describe a novel instance of phenotypic diversity in tolerance to a 73 metabolic toxin within wild-type populations of Methylobacterium extorquens, a 74 ubiquitous phyllosphere methylotroph capable of growing on the methanol periodically 75 released from plant leaves. The first intermediate in methanol metabolism is 76 formaldehyde, a potent cellular toxin that is lethal in high concentrations. We have found 77 that at moderate concentrations, formaldehyde tolerance in M. extorquens is 78 heterogeneous, with a cell's minimum tolerance level ranging between 0 mM and 8 mM. 79 Tolerant cells have a distinct gene expression profile from non-tolerant cells. This form 80of heterogeneity is continuous in terms of threshold (the formaldehyde concentration 81 where growth ceases), yet binary in outcome (at a given formaldehyde concentration, 82 cells either grow normally or die, with no intermediate phenotype), and it is not 83 associated with any detectable genetic mutations. Moreover, tolerance distributions 84 within the population are dynamic, changing over time in response to growth conditions. 85We characterized this phenomenon using bulk liquid culture experiments, colony growth 86 tracking, flow cytometry, single-cell time-lapse microscopy, transcriptomics, and 87 genome resequencing. Finally, we used mathematical modeling to better understand 88 the processes by which cells change phenotype, and found evidence for both 89 stochastic, bidirectional phenotypic diversification and responsive, directed phenotypic 90 shifts, depending on the growth substrate and the presence of toxin. 91 92 Author Summary 93Scientists tend to appreciate microbes for their simplicity and predictability: a population 94 of genet...

The potency and indiscriminate nature of formaldehyde reactivity upon biological molecules make it a universal stressor. However, some organisms such as Methylorubrum extorquens possess means to rapidly and effectively mitigate formaldehyde-induced damage. EfgA is a recently identified formaldehyde sensor predicted to halt translation in response to elevated formaldehyde as a means to protect cells. Herein, we investigate growth and changes in gene expression to understand how M. extorquens responds to formaldehyde with and without the EfgA-formaldehyde-mediated translational response, and how this mechanism compares to antibiotic-mediated translation inhibition. These distinct mechanisms of translation inhibition have notable differences: they each involve different specific players and in addition, formaldehyde also acts as a general, multi-target stressor and a potential carbon source. We present findings demonstrating that in addition to its characterized impact on translation, functional EfgA allows for a rapid and robust transcriptional response to formaldehyde and that removal of EfgA leads to heightened proteotoxic and genotoxic stress in the presence of increased formaldehyde levels. We also found that many downstream consequences of translation inhibition were shared by EfgA-formaldehyde- and kanamycin-mediated translation inhibition. Our work uncovered additional layers of regulatory control enacted by functional EfgA upon experiencing formaldehyde stress, and further demonstrated the importance this protein plays at both transcriptional and translational levels in this model methylotroph.