Frances Chu

Do mycobacteria produce endospores

The genus Mycobacterium, which is a member of the high G+C group of Gram-positive bacteria, includes important pathogens, such as M. tuberculosis and M. leprae. A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette–Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. We also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. We conclude that it is unlikely that Mycobacterium is capable of endospore formation.

Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense

ABSTRACT Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.

XoxF Acts as the Predominant Methanol Dehydrogenase in the Type I Methanotroph Methylomicrobium buryatense

UNLABELLED Many methylotrophic taxa harbor two distinct methanol dehydrogenase (MDH) systems for oxidizing methanol to formaldehyde: the well-studied calcium-dependent MxaFI type and the more recently discovered lanthanide-containing XoxF type. MxaFI has traditionally been accepted as the major functional MDH in bacteria that contain both enzymes. However, in this study, we present evidence that, in a type I methanotroph, Methylomicrobium buryatense, XoxF is likely the primary functional MDH in the environment. The addition of lanthanides increases xoxF expression and greatly reduces mxa expression, even under conditions in which calcium concentrations are almost 100-fold higher than lanthanide concentrations. Mutations in genes encoding the MDH enzymes validate our finding that XoxF is the major functional MDH, as XoxF mutants grow more poorly than MxaFI mutants under unfavorable culturing conditions. In addition, mutant and transcriptional analyses demonstrate that the lanthanide-dependent MDH switch operating in methanotrophs is mediated in part by the orphan response regulator MxaB, whose gene transcription is itself lanthanide responsive. IMPORTANCE Aerobic methanotrophs, bacteria that oxidize methane for carbon and energy, require a methanol dehydrogenase enzyme to convert methanol into formaldehyde. The calcium-dependent enzyme MxaFI has been thought to primarily carry out methanol oxidation in methanotrophs. Recently, it was discovered that XoxF, a lanthanide-containing enzyme present in most methanotrophs, can also oxidize methanol. In a methanotroph with both MxaFI and XoxF, we demonstrate that lanthanides transcriptionally control genes encoding the two methanol dehydrogenases, in part by controlling expression of the response regulator MxaB. Lanthanides are abundant in the Earths crust, and we demonstrate that micromolar amounts of lanthanides are sufficient to suppress MxaFI expression. Thus, we present evidence that XoxF acts as the predominant methanol dehydrogenase in a methanotroph.

Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions

Significance Secreted pore-forming toxins are a common feature of bacterial virulence. Mycobacterium tuberculosis, the agent of human tuberculosis, has been reported to possess a pore-forming toxin called 6 kDa early secretory antigenic target (ESAT-6) that is secreted through a specialized secretion system called ESX-1 (ESAT-6 secretion system 1). We report here experiments showing that ESAT-6 does not lyse cells; the lytic activity previously attributed to this secreted protein is due to contaminating detergent in the recombinant protein preparations. Whereas the ESX-1 secretion system does lyse host cell membranes, we find this lysis is dependent on bacterial cell contact with the host membrane and results in tears in the membrane without any pore formation. Understanding the mechanism of this lysis may provide clues to how mycobacteria cause disease. Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1–secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1–dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1–mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1–mediated lysis.

Bioreactor performance parameters for an industrially-promising methanotroph Methylomicrobium buryatense 5GB1

BackgroundMethane is a feedstock of interest for the future, both from natural gas and from renewable biogas sources. Methanotrophic bacteria have the potential to enable commercial methane bioconversion to value-added products such as fuels and chemicals. A strain of interest for such applications is Methylomicrobium buryatense 5GB1, due to its robust growth characteristics. However, to take advantage of the potential of this methanotroph, it is important to generate comprehensive bioreactor-based datasets for different growth conditions to compare bioprocess parameters.ResultsDatasets of growth parameters, gas utilization rates, and products (total biomass, extracted fatty acids, glycogen, excreted acids) were obtained for cultures of M. buryatense 5GB1 grown in continuous culture under methane limitation and O2 limitation conditions. Additionally, experiments were performed involving unrestricted batch growth conditions with both methane and methanol as substrate. All four growth conditions show significant differences. The most notable changes are the high glycogen content and high formate excretion for cells grown on methanol (batch), and high O2:CH4 utilization ratio for cells grown under methane limitation.ConclusionsThe results presented here represent the most comprehensive published bioreactor datasets for a gamma-proteobacterial methanotroph. This information shows that metabolism by M. buryatense 5GB1 differs significantly for each of the four conditions tested. O2 limitation resulted in the lowest relative O2 demand and fed-batch growth on methane the highest. Future studies are needed to understand the metabolic basis of these differences. However, these results suggest that both batch and continuous culture conditions have specific advantages, depending on the product of interest.

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

ABSTRACT Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.

MxaY regulates the lanthanide-mediated methanol dehydrogenase switch in Methylomicrobium buryatense

Many methylotrophs, microorganisms that consume carbon compounds lacking carbon–carbon bonds, use two different systems to oxidize methanol for energy production and biomass accumulation. The MxaFI methanol dehydrogenase (MDH) contains calcium in its active site, while the XoxF enzyme contains a lanthanide in its active site. The genes encoding the MDH enzymes are differentially regulated by the presence of lanthanides. In this study, we found that the histidine kinase MxaY controls the lanthanide-mediated switch in Methylomicrobium buryatense 5GB1C. MxaY controls the transcription of genes encoding MxaFI and XoxF at least partially by controlling the transcript levels of the orphan response regulator MxaB. We identify a constitutively active version of MxaY, and identify the mutated residue that may be involved in lanthanide sensing. Lastly, we find evidence to suggest that tight control of active MDH production is required for wild-type growth rates.

Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C

The bacteria that grow on methane aerobically (methanotrophs) support populations of non-methanotrophs in the natural environment by excreting methane-derived carbon. One group of excreted compounds are short-chain organic acids, generated in highest abundance when cultures are grown under O2-starvation. We examined this O2-starvation condition in the methanotroph Methylomicrobium buryatense 5GB1. The M. buryatense 5GB1 genome contains homologs for all enzymes necessary for a fermentative metabolism, and we hypothesize that a metabolic switch to fermentation can be induced by low-O2 conditions. Under prolonged O2-starvation in a closed vial, this methanotroph increases the amount of acetate excreted about 10-fold, but the formate, lactate, and succinate excreted do not respond to this culture condition. In bioreactor cultures, the amount of each excreted product is similar across a range of growth rates and limiting substrates, including O2-limitation. A set of mutants were generated in genes predicted to be involved in generating or regulating excretion of these compounds and tested for growth defects, and changes in excretion products. The phenotypes and associated metabolic flux modeling suggested that in M. buryatense 5GB1, formate and acetate are excreted in response to redox imbalance. Our results indicate that even under O2-starvation conditions, M. buryatense 5GB1 maintains a metabolic state representing a combination of fermentation and respiration metabolism.

Using Quality Improvement Tools to Redefine a Clinical Librarianship Program: a Case Study

ABSTRACT University of Washington (UW) Medicine is a large hospital system in Seattle, Washington. Historically, the UW Health Sciences Library (HSL) has had one clinical librarian to support the organization. However, after the clinical librarian retired in 2015, 1.5 FTE clinical librarians were hired to support UW Medicine. This article details process improvement strategies being implemented at UWHSL to align and modernize the library’s clinical librarianship program given the significant changes occurring in healthcare. This case study will examine the use of qualitative research techniques for gathering information and the use of Value Engineering tools to analyze results.