Biology subjects

Newman, D. K.

Publications and source records attributed to Newman, D. K..

4 recordsLinked to original sources

bioRxiv2018

Extended hopanoid lipids promote bacterial motility, surface attachment, and root nodule development in the Bradyrhizobium diazoefficiens-Aeschynomene afraspera symbiosis

Hopanoids are steroid-like bacterial lipids that enhance membrane rigidity and promote bacterial growth under diverse stresses. Hopanoid biosynthesis genes are conserved in nitrogen-fixing plant symbionts, and we previously found that the extended (C35) class of hopanoids in Bradyrhizobium diazoefficiens are required for efficient symbiotic nitrogen fixation in the tropical legume host Aeschynomene afraspera. Here we demonstrate that the nitrogen fixation defect conferred by extended loss can fully be explained by a reduction in root nodule sizes rather than per-bacteroid nitrogen fixation levels. Using a single-nodule tracking approach to track A. afraspera nodule development, we provide a quantitative model of root nodule development in this host, uncovering both the baseline growth parameters for wild-type nodules and a surprising heterogeneity of extended hopanoid mutant developmental phenotypes. These phenotypes include a delay in root nodule initiation and presence of a subpopulation of nodules with slow growth rates and low final volumes, which are correlated with reduced motility and surface attachment in vitro and lower bacteroid densities in planta, respectively. This work provides a quantitative reference point for understanding the phenotypic diversity of ineffective symbionts in A. afraspera and identifies specific developmental stages affected by extended hopanoid loss for future mechanistic work.

microbiology↗

bioRxiv2018

The dormancy specific regulator, SutA, is an intrinsically-disordered protein that modulates transcription initiation in Pseudomonas aeruginosa

Though bacteria in nature are often nutritionally limited and growing slowly, most of our understanding of core cellular processes such as transcription comes from studies in a handful of model organisms doubling rapidly under nutrient-replete conditions. We previously identified a small protein of unknown function, called SutA, in a global screen of proteins synthesized in Pseudomonas aeruginosa under growth arrest (Babin BM, et al. (2016) SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa. PNAS 113(5):E597-605). SutA binds RNA polymerase (RNAP), causing widespread changes in gene expression, including upregulation of the ribosomal RNA (rRNA) genes. Here, using biochemical and structural methods, we examine how SutA interacts with RNAP and the functional consequences of these interactions. We show that SutA consists of a central -helix with unstructured N- and C-terminal tails, and binds to the {beta}1 domain of RNAP. It activates transcription from the P. aeruginosa rrn promoter by both the housekeeping sigma factor holoenzyme (E{sigma}70) and the general stress response sigma factor holoenzyme (E{sigma}S) in vitro, and its N-terminal tail is required for activation in both holoenzyme contexts. However, we find that the interaction between SutA and each holoenzyme is distinct, with the SutA C-terminal tail and an acidic loop unique to {sigma}70 playing the determining roles in these differences. Our results add SutA to a growing list of transcription regulators that use their intrinsically disordered regions to remodel transcription complexes.\n\nSIGNIFICANCELittle is known about how bacteria regulate their activities during periods of dormancy, yet growth arrest dominates bacterial existence in most environments and is directly relevant to the problem of physiological antibiotic tolerance. Though much is known about transcription in the model organism, Escherichia coli, even there, our understanding of gene expression during dormancy is incomplete. Here we explore how transcription under growth arrest is modulated in Pseudomonas aeruginosa by the small acidic protein, SutA. We show that SutA binds to RNA polymerase and controls transcription by a mechanism that is distinct from other known regulators. Our work underscores the potential for fundamental, mechanistic discovery in this important and understudied realm of bacterial physiology.

molecular biology↗

bioRxiv2018

Refining the application of microbial lipids as tracers of Staphylococcus aureus growth rates in cystic fibrosis sputum

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effect of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. Microbial community composition was measured by NanoString DNA analysis and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two different anteiso fatty acids (a-C15:0 and a-C17:0) using gas chromatography-mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae and hence their isotopic enrichment was interpreted as a growth proxy for S. aureus. Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients, thus deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small non-microbial background source detected in some patient samples, a-C15:0 fatty acid still appear to be a relatively robust proxy for CF pathogens, revealing a median generation time of ~1.5 days, similar to prior observations. Contrary to our expectations, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two best practices for application of stable isotope probing in CF sputum: (1) parallel determination of microbial community composition in CF sputum using culture-independent tools, and (2) analysis of samples with a minimum a-C15:0 concentration of 0.1 weight percent of saturated fatty acids.\n\nIMPORTANCEIn chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop an new workflow for the application of stable isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

microbiology↗

bioRxiv2018

Towards measuring growth rates of pathogens during infections by D2O-labeling lipidomics

RATIONALEMicrobial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids.\n\nMETHODSLipid extracts of D2O-treated E. coli cultures were measured on LC-ESI-MS instruments equipped with TOF and Orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio GC-MS. We then develop an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we apply D2O-labeling lipidomics to clinical samples from CF patients with chronic lung infections.\n\nRESULTSLipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum, indicates an average generation time of the pathogen on the order of one cell doubling per day.\n\nCONCLUSIONSThis study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2O.

biochemistry↗