Robin Ghosh

Refractile bodies (R-bodies) of gram-negative bacteria are large proteinaceous assemblies, rolled up in the form of an Archimedean spiral. They exhibit rapid rod-like reversible extension in the micrometer range when cued by chemical environmental triggers and have potential for synthetic biology and biochip applications. Initially described for the Paramecium endosymbionts Caedibacter taeniospiralis and Caedimonas varicaedens , R-bodies have since been discovered in many classes of Pseudomonadota, both in endosymbionts and in non-endosymbionts. However, despite the fact that the genetics and morphologies, as well as the unrolling kinetics of R-bodies from different species, show considerable diversity, no recent study has integrated these aspects into a single framework. The latter would be advantageous for the creation of an R-body biotechnology toolbox, where different properties determine the application area. Here, we have examined the R-bodies from six different Pseudomonadota, comprising both phylogenetically diverse endosymbionts and non-endosymbionts. Comparison of the morphologies of the rolled-up and unrolled forms, obtained using electron microscopy and high-quality images, to their corresponding genetic data indicates that extensive lateral gene transfer has occurred, which confounds a common framework based on these data. However, we have also studied the R-body extension and retraction kinetics using high frame-rate light microscopic video recordings, where we show for the first time that R-bodies can be classified into two classes, showing “fast burst” or “slow” acid-induced extension kinetics, respectively. We propose that this criterion may, in fact, be the most useful for the choice of an R-body tool for biotechnological purposes. IMPORTANCE: R-bodies are unique proteinaceous macromolecular structures capable of massive reversible extension in response to external environmental triggers without the input of chemical energy. They comprise only a few small polypeptides, which makes them potentially highly amenable to tuning via genetic engineering, as well as being exceptionally stable. These properties would be highly desirable in biotechnology and synthetic biology, as well as in biochip applications, where a controlled mechanical extensor might play an integral part in a nanoscale molecular machine. So far, only R-bodies from a single species, Caedibacter taeniospiralis , have been characterized extensively. However, in recent years, genomic information has revealed that a panoply of R-bodies are widely distributed among gram-negative phyla, although studies have generally not included morphological data. This study brings these two areas together to provide a holistic overview of the field and also reveals new insights into key dynamic aspects of R-body extension.

Outer membrane porins from Gram-negative bacteria are established vehicles for the production of vaccines. Typically, one or more of the extracellular loops of a porin are replaced by a peptide encoding a foreign epitope, and recombinant porin is then used as a vaccine. However, many host strains are potentially pathogenic, and also produce toxic lipopolysaccharide (LPS), both of which are undesirable for safety reasons. In contrast, the outer membrane porins from photosynthetic, purple bacteria have no known human pathology and produce only weakly toxic LPS. The purple bacterium Rhodospirillum rubrum is well-suited for large-scale biotechnology, and expresses a major porin, Por39, which is a candidate for a vaccine platform. Unfortunately, the atomic structure of Por39 could not be determined so far, and Por39 shows only a weak homology to other porins of known structure, making the assignment of external loops difficult. Here, we construct a knowledge-based model of Por39 using secondary structure constraints from both the low sequence homology to the 2POR porin from Rhodobacter capsulatus, for which the X-ray structure is known, as well as those obtained using secondary structure prediction packages. The secondary structure predictions were used to constrain a three-dimensional model created using the I-TASSER package. The modelling procedure was validated by predicting the structure of 2POR using the same strategy, but excluding the 2POR X-ray structure from the I-TASSER database. The final Por39 model allows three external loops to be defined precisely, and could also be used to obtain an initial model for the closely related Por41 using molecular modelling. These structures provide a good starting point for the insertion of epitopes with vaccine potential.

Biohydrogen production in small laboratory scale culture vessels is often difficult to perform and quantitate. One problem is that commonly used silicon tubing and improvised plastic connections used for constructing apparatus are cheap and easy to connect but are generally not robust for gases such as hydrogen. In addition, this type of apparatus presents significant safety concerns. Here, we demonstrate the construction of hydrogen-tight apparatus using a commercially available modular system, where plastic tubing and connections are made of explosion-proof dissipative plastic material. Using this system, we introduce a gas chromatograph calibration procedure, which can be easily performed without necessarily resorting to expensive commercial gas standards for the calibration of hydrogen gas concentrations. In this procedure, the amount of hydrogen produced by the reaction of sodium borohydride with water in a closed air-filled bottle is deduced from the observed decrease of the oxygen partial pressure, using the ideal gas law. Finally, the determined calibration coefficients and the gas-tight apparatus are used for the analysis of simultaneous oxygen consumption and hydrogen production of the purple photosynthetic bacterium, Rhodospirillum rubrum, during semi-aerobic growth in the dark.

High performance liquid chromatography (HPLC) is a frequently used technique in carotenoid research. So far, however, little attention has been paid to the fact that many of the organic solvents used in HPLC separation of highly apolar C₄₀ carotenoids impose a significant threat to both health (especially for women) and the general laboratory environment. Here, we developed a solvent combination capable of allowing high-resolution HPLC separation of the C₄₀ carotenoid, spirilloxanthin, and all of its biosynthetic precursors beginning with phytoene, using relatively safe, environmentally friendly solvents. We show that separation of spirilloxanthin and its precursors anhydrorhodovibrin and lycopene using modern ultra-high performance chromatography (UHPLC) poses particular problems for apolar carotenoid separation, due to the long residence times in the sample delivery system, which facilitates carotenoid aggregation. We resolved these problems by developing the solvent delivery combination acetone/acetonitrile/isopropanol/methanol (65/30/5/2 (v/v/v/v)), which allows excellent column separation using the safe isocratic solvent system methanol/tetrahydrofuran (98/2 (v/v)). We also demonstrate that the development strategy for optimizing a solvent system for carotenoid separation can be well-described by the use of the average dielectric constant of the total sample delivery solvent, and present a formal method for analysis of the efficiency of separation.

The Kulka resorcinol assay (Kulka, R.G., Biochemistry 1956, 63, 542–548) for ketoses has been widely used in the literature but suffers from two major disadvantages: (a) it employs large amounts of potentially harmful reagents for a general biology laboratory environment; and (b) in its original formulation, it is unsuited for modern high-throughput applications. Here, we have developed a modified Kulka assay, which contains a safer formulation, employing approx. 5.4 M HCl in 250 µL aliquots, and is suitable for use in high-throughput systems biology or enzymatic applications. The modified assay has been tested extensively for the measurement of two ketoses—fructose (a common substrate in cell growth experiments) and 1-deoxy-d-xylulose-5-phosphate (DXP), the product of the DXP-synthase reaction—which until now has only been assayable using time-consuming chromatographic methods or radioactivity. The Kulka microassay has a sensitivity of 0–250 nmol fructose or 0–500 nmol DXP. The assay is suitable for monitoring the consumption of fructose in bacterial growth experiments but is too insensitive to be used directly for the measurement of DXP in in vitro enzyme assays. However, we show that after concentration of the DXP-enzyme mix by butanol extraction, the Kulka resorcinol method can be used for enzyme assays.

The in vivo physiological role of the gene cobZ, which encodes precorrin-3B synthase, which catalyzes the initial porphyrin ring contraction step of cobalamin biosynthesis via the cob pathway, has been demonstrated here for the first time. Cobalamin is known to be essential for an early step of bacteriochlorophyll biosynthesis in anoxygenic purple bacteria. The cobZ (cobZRR ) gene of the purple bacterium Rhodospirillum rubrum was localized to a 23.5 kb insert of chromosomal DNA contained on the cosmid pSC4. pSC4 complemented several mutants of bacteriochlorophyll and carotenoid biosynthesis, due to the presence of the bchCX and crtCDEF genes at one end of the cosmid insert, flanking cobZRR . A second gene, citB/tcuB, immediately downstream of cobZRR , shows homologies to both a tricarballylate oxidoreductase (tcuB) and a gene (citB) involved in signal transduction during citrate uptake. CobZRR shows extensive homology to the N-terminal domain of the bifunctional CobZ from Rhodobacter capsulatus, and the R. rubrum citB/tcuB gene is homologous to the CobZ C-terminal domain. A mutant, SERGK25, containing a terminatorless kanamycin interposon inserted into cobZRR , could not grow by anaerobic photosynthesis, but grew normally under dark, aerobic and microaerophilic conditions with succinate and fructose as carbon sources. The anaerobic in vivo activity of CobZ indicates that it does not require oxygen as a substrate. The mutant excreted large amounts of protoporphyrin IX-monomethylester, a brown precursor of bacteriochlorophyll biosynthesis. The mutant was complemented either by the cobZRR gene in trans, or when exogenous cobalamin was added to the medium. A deletion mutant of tcuB/citB did not exhibit the cob phenotype. Thus, a role for tcuB/citB in cobalamin biosynthesis could not be confirmed.

A simple, rapid, and inexpensive extraction method for carotenoids and other non-polar compounds present in phototrophic bacteria has been developed. The method, which has been extensively tested on the phototrophic purple non-sulphur bacterium Rhodospirillum rubrum, is suitable for extracting large numbers of samples, which is common in systems biology studies, and yields material suitable for subsequent analysis using HPLC and mass spectroscopy. The procedure is particularly suitable for carotenoids and other terpenoids, including quinones, bacteriochlorophyll a and bacteriopheophytin a, and is also useful for the analysis of polar phospholipids. The extraction procedure requires only a single step extraction with a hexane/methanol/water mixture, followed by HPLC using a Spherisorb C18 column, with a mobile phase consisting of acetone-water and a non-linear gradient of 50%–100% acetone. The method was employed for examining the carotenoid composition observed during microaerophilic growth of R. rubrum strains, and was able to determine 18 carotenoids, 4 isoprenoid-quinones, bacteriochlorophyll a and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The analytical procedure was used to examine the dynamics of carotenoid biosynthesis in the major and minor pathways operating simultaneously in a carotenoid biosynthesis mutant of R. rubrum.

Background: Cognitive impairment is common amongst acute geriatric hospital admissions but detection is often poor and this is associated with worse outcomes. The four-question abbreviated mental test (AMT4) has previously been promoted nationally in the acute setting as a succinct assessment tool. However, a recent national dementia Commissioning for Quality and Innovation (CQUIN) goal recommends a single screening question followed by the tenquestion abbreviated mental test (AMT10). We aimed to evaluate the negative predictive value of the AMT4 within the acute setting by comparing it to three other validated tools. Methods: We identified 100 acute medical admissions (>60 years old) with a negative AMT4 and administered the AMT10, six-item cognitive impairment test (6CIT) and confusion assessment method (CAM) within 24 hours of admission. Results: Forty-six percent of the participants scored positively on at least one of the additional tests despite a negative AMT4. Forty-four patients had a positive 6CIT, 23 had a positive AMT10 and six had a positive CAM. Using the 6CIT as a diagnostic standard, tests of short-term memory had the greatest sensitivity and specificity for cognitive impairment. Conclusions: Nearly half of the participants had signs of cognitive impairment despite a negative AMT4. Consequently, there is a risk of under diagnosis with potentially serious consequences for morbidity and mortality. Tests of shortterm memory were strongly associated with cognitive impairment. We propose the addition of such a test in order to increase the sensitivity of the AMT4 without compromising its brevity and utility in the acute setting.