Examining Epistatic and Environmental Effects Using an Alternative Formaldehyde Oxidation Pathway in Methtylobacterium Extorquens

Epistasis is the interaction of genes, particularly how the presence of a gene locus modifies the phenotype of other loci. Previous research in the lab studied the epistatic interactions of beneficial mutations that arose in an evolved isolate of Methylobacterium extorquens, a bacteria that has the ability to utilize one-carbon compounds as the sole source of energy. Four mutations were identified following the replacement of the methanopterin-dependent pathway of M. extorquens’ with a glutathione (GSH)-dependent pathway from Paracoccus denitrificans. The mutations were combined in all viable permutations on a genetic background that only contained the GSH-dependent pathway. We tested the fitness of each strain against the ancestral evolved isolate under two minimal media growth conditions: Hypho in flasks, the condition under which the original study that identified the mutations was conducted, and MPIPES, a more complete media, in sealed Balch tubes in which future experimental work will be performed. We expect to see similar epistatic interactions with diminishing returns as the original study found, suggesting minimal effect of these media conditions on epistasis. By better understanding gene-gene interactions in this system, we hope to lead to models capable of predicting adaptive routes through fitness landscapes underlying M. extorquens physiology

Global Transcriptional Response of <i>Methylorubrum extorquens</i> to Formaldehyde Stress Expands the Role of EfgA and Is Distinct from Antibiotic Translational Inhibition

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

Intracellular Concentrations of Borrelia burgdorferi Cyclic Di-AMP Are Not Changed by Altered Expression of the CdaA Synthase.

The second messenger nucleotide cyclic diadenylate monophosphate (c-di-AMP) has been identified in several species of Gram positive bacteria and Chlamydia trachomatis. This molecule has been associated with bacterial cell division, cell wall biosynthesis and phosphate metabolism, and with induction of type I interferon responses by host cells. We demonstrate that B. burgdorferi produces a c-di-AMP synthase, which we designated CdaA. Both CdaA and c-di-AMP levels are very low in cultured B. burgdorferi, and no conditions were identified under which cdaA mRNA was differentially expressed. A mutant B. burgdorferi was produced that expresses high levels of CdaA, yet steady state borrelial c-di-AMP levels did not change, apparently due to degradation by the native DhhP phosphodiesterase. The function(s) of c-di-AMP in the Lyme disease spirochete remains enigmatic

Reciprocal control of motility and biofilm formation by the PdhS2 two-component sensor kinase of Agrobacterium tumefaciens.

A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1 and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study, the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue implicated in phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 may function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA, which is inhibited by DivK. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, most likely functioning through CtrA to separately control motility and regulate the levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting the production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through the addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment

Homeostasis of Second Messenger Cyclic-di-AMP Is Critical for Cyanobacterial Fitness and Acclimation to Abiotic Stress

Second messengers are intracellular molecules regulated by external stimuli known as first messengers that are used for rapid organismal responses to dynamic environmental changes. Cyclic di-AMP (c-di-AMP) is a relatively newly discovered second messenger implicated in cell wall homeostasis in many pathogenic bacteria. C-di-AMP is synthesized from ATP by diadenylyl cyclases (DAC) and degraded by specific c-di-AMP phosphodiesterases (PDE). C-di-AMP DACs and PDEs are present in all sequenced cyanobacteria, suggesting roles for c-di-AMP in the physiology and/or development of these organisms. Despite conservation of these genes across numerous cyanobacteria, the functional roles of c-di-AMP in cyanobacteria have not been well-investigated. In a unique feature of cyanobacteria, phylogenetic analysis indicated that the broadly conserved DAC, related to CdaA/DacA, is always co-associated in an operon with genes critical for controlling cell wall synthesis. To investigate phenotypes regulated by c-di-AMP in cyanobacteria, we overexpressed native DAC (sll0505) and c-di-AMP PDE (slr0104) genes in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) to increase and decrease intracellular c-di-AMP levels, respectively. DAC- and PDE-overexpression strains, showed abnormal aggregation phenotypes, suggesting functional roles for regulating c-di-AMP homeostasis in vivo. As c-di-AMP may be implicated in osmotic responses in cyanobacteria, we tested whether sorbitol and NaCl stresses impacted expression of sll0505 and slr0104 or intracellular c-di-AMP levels in Synechocystis. Additionally, to determine the range of cyanobacteria in which c-di-AMP may function, we assessed c-di-AMP levels in two unicellular cyanobacteria, i.e., Synechocystis and Synechococcus elongatus PCC 7942, and two filamentous cyanobacteria, i.e., Fremyella diplosiphon and Anabaena sp. PCC 7120. C-di-AMP levels responded differently to abiotic stress signals in distinct cyanobacteria strains, whereas salt stress uniformly impacted another second messenger cyclic di-GMP in cyanobacteria. Together, these results suggest regulation of c-di-AMP homeostasis in cyanobacteria and implicate a role for the second messenger in maintaining cellular fitness in response to abiotic stress

<i>B</i>. <i>burgdorferi</i> CdaA synthesizes c-di-AMP.

<p>Representative mass spectrometric analysis of cytoplasmic extract from IPTG-induced <i>E</i>. <i>coli</i> strain CRS-0, which expresses <i>B</i>. <i>burgdorferi</i> CdaA from a chimeric plasmid. The identity of the peak at 3.35 min was not determined.</p

Alignment of the predicted amino acid sequences of <i>B</i>. <i>burgdorferi</i> CdaA and closely-related ci-di-AMP synthases of other bacteria.

<p>The two regions of conserved residues that constitute the DAC domain are boxed in blue. Residues found in all 5 proteins are indicated by an asterisk (*), residues in 4 proteins by a colon (:), and those in 3 proteins by a period (.). Enzyme sequences are identified as: Bb, <i>B</i>. <i>burgdorferi</i> CdaA; Bs, <i>Bacillus subtilis</i> CdaA (formerly YbbP); Lm, <i>Listeria monocytogenes</i> CdaA/DacA; Sa, <i>Staphylococcus aureus</i> DacA; and Ct, <i>C</i>. <i>trachomatis</i> DacA.</p

Effects of hyper-expressing CdaA in <i>B</i>. <i>burgdorferi</i>.

<p><b>A.</b> Measurements of <i>B</i>. <i>burgdorferi</i> cytoplasmic c-di-AMP levels in samples of uninduced and induced AG1. Bacteria were cultured to mid-exponential phase (approximately 10⁷ bacteria/ml), divided equally divided into two tubes, then <i>cdaA</i> transcription was induced by addition of 0.5 μg/ml (final concentration) anhydrotetracycline (ATc) to one tube, and both were incubated for 24h at 35°C. Equal volumes of borrelial cell extracts were analyzed. <b>B.</b> Immunoblot analyses of KS50 and AG1, without and with inclusion of 0.5 μg/ml anhydrotetracycline (ATc) inducer (- and +, respectively). Membranes were probed with antibodies directed against CdaA or the constitutively-expressed FlaB subunit of the flagella. Wild-type and uninduced AG1 bacteria produced substantially less CdaA than did induced AG1, and the immunoblot signal was not detectable for those strains/conditions at the illustrated exposure. Analyses of mRNA levels also indicated that <i>cdaA</i> is expressed at low levels by uninduced AG1 (data not shown). <b>C and D.</b> Q-RT-PCR analyses of the effects of CdaA hyperexpression on transcription of select <i>B</i>. <i>burgdorferi</i> mRNAs. Transcript fold changes are shown as the difference between uninduced and induced cultures for both strains KS50 and AG1, relative to control <i>flaB</i> or <i>recA</i>, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125440#pone.0125440.ref030" target="_blank">30</a>]. Multiple t tests were performed for each strain and examined transcript. Only the differences in levels of <i>cdaA</i> transcripts in induced cultures of AG1 were significant (indicated by **, p = 0.0012 when compared with <i>flaB</i>, and p = 0.0023 when compared with <i>recA</i>).</p